ABSTRACT Title of Document: DIVERSITY OF CONJUGATING REN ALGAE; PHYLOGENETIC STUDIES OF A SPECIES-RICH EUKARYOTIC LINEAGE John David Hal, Ph.D., 2007 Directed By: Dr. Charles F. Delwiche Cel Biology and Molecular Genetics This thesis used molecular phylogenetic techniques to investigate diversity in the conjugating gren algae. The conjugating gren algae are closely related to land plants and evolution within the group provides a good analogy of how evolution may have proceded in the lineage that gave rise to land plants. I developed a dataset of the genes coxII, psaA and rbcL with 109 taxa to determine phylogenetic relationships of the families and genera. I found that the order Zygnematales is not monophyletic and that Spirogyra was the first to branch. The order Desmidiales is monophyletic if one includes the genus Roya. The family Peniaceae is not monophyletic. The genera Cosmarium, Cylindrocystis, Mesotaenium, Penium, Spondylosium, Staurodesmus and in later studies Desmidium and Hyalotheca were found to be paraphyletic or polyphyletic. Investigation of cel division syndromes among filamentous Desmidiaceae revealed greater diversity than was previously reported. Notable among these discoveries is that Spondylosium pulchrum displays the Desmidium-type cel division, Spondylosium pulchelum the Cosmarium-type, and Spondylosium tetragonum the newly described Teilingia-type cel division. The relationship among the syndromes was infered from phylogenetic analysis of the species that revealed a single lineage comprising filamentous and colonial species and multiple modes of cel division. This suggests that even the fundamental proces of cel division can be highly modified. Results from this study also resulted in the taxonomic resurrection of the genus Didymoprium, as wel as the creation of the new genus Isthmocatena and the combinations Didymoprium grevilei, Desmidium pulchrum, and Isthmocatena pulchela. Investigations of the Gonatozygaceae revealed unexpected diversity in Gonatozygon brebisonii and G. kinahani. Structural measurements were sufficient to distinguish among strains of Gonatozygon species except for Gonatozygon brebisonii. We have probably underestimated genetic and species diversity in this family. In contrast, the structuraly distinct species Triploceras gracile, was found to be closely related to Micrasterias. This relationship was confirmed by sequencing and phylogenetic analysis of the nuclear encoded EF1?, EIF4 and TUA. The results of this study indicate that Triploceras is probably actualy bilateraly symmetric, although it has been treated as a radialy symmetric species. DIVERSITY OF CONJUGATING REN ALGAE; PHYLOGENETIC STUDIES OF A SPECIES-RICH EUKARYOTIC LINEAGE By John David Hal Disertation submited to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfilment of the requirements for the degre of Doctor of Philosophy 2007 Advisory Commite: Dr. Charles F. Delwiche, Chair Profesor Elisabeth Gantt Profesor Charles Miter Dr. Richard M. cCourt Dr. Maile C. Nel ? Copyright by John David Hal 2007 i Acknowledgements The author wishes to thank those who have contributed to this research. Many people have given me materials and advice during the course of this study. I would like to thank the members of my commitee for discusion of various aspects of this work. In particular Dr. Richard McCourt has engaged me in a number of valuable discusions on the systematics of charophyte green algae. I would like to thank Dr. Monika Engels of the SVCK for sending strains for investigation free of cost. Also, many of the strains were isolated from aterial sent to the author by Ms. Michelle (Shelly) Haube and many other coleagues around the world. Dr. Ken Karol, Dr. Tsetso Bachvaroff, Dr. Malin Kerr and Dr. M. Virginia Sanchez- Puerta all provided valuable training and discusion. Virginia?s encouragement and assistance was particularly motivating. Professor Tod Cooke has provided valuable advice and mentoring in nearly all aspects of my research and graduate career. His wilingness to discus science has deepened my understanding of science, the scientific method, and the origin of land plants. Dr. Paul Silva has led me to a new appreciation for systematics and careful scholarship. I would like to especially thank Dr. Susan Carty who first introduced me to the algae and whose constant suport and guidance has been absolutely critical in the successful completion of this process. It was a project undertaken in her lab as an undergraduate student that convinced me to study algae and complete a graduate degree. Finally, I would like to acknowledge the contribution of my family, in particular my parents for encouraging me to continue my education. This research was suported in part by NSF grants DEB-97817, MCB-0523719 and by a USDA- CSRES graduate training fellowship grant 205-3842015761 to JDH. ii Table of Contents CHAPTER 1. GENERAL INTRODUCTION 1 EARLY LAND PLANT EVOLUTION...........................................................1 CHAROPHYTE GREN ALGAE..............................................................2 BIOLOGY AND SYSTEMATICS OF THE ZYGNEMATOPHYCEAE.....................................5 UNDERSTANDING DIVERSITY OF THE CONJUGATING REEN ALGAE...............................10 FIGURE 1.1 14 CHAPTER 2. IN THE SHADOW OF GIANTS; SYSTEMATICS OF THE CHAROPHYTE GREN ALGAE 15 ABSTRACT............................................................................15 INTRODUCTION........................................................................15 Historical perspective................................................................16 Why should one study the systematics of charophytes?.....................................19 Which gren algae belong to the Charophyta?............................................20 GENERAL SYSTEMATIC STUDIES OF CHAROPHYTES............................................2 SYSTEMATICS OF THE CHAROPHYTE LINEAGES...............................................24 Mesostigma........................................................................24 Chlorokybus atmophyticus............................................................25 Klebsormidiophyceae................................................................26 Zygnematophyceae..................................................................28 Coleochaetophyceae.................................................................30 Charophyceae sensu stricto (the stoneworts).............................................32 FUTURE DIRECTIONS AND THE ROLE OF GENOMICS IN CHAROPHYTE SYSTEMATICS..................3 Insights from published genomic data...................................................3 Future systematic investigations........................................................35 CHAPTER 3. PHYLOGENY OF THE CONJUGATING REN ALGAE BASED ON CHLOROPLAST AND MITOCHONDRIAL NUCLEOTIDE SEQUENCE DATA 40 ABSTRACT............................................................................40 INTRODUCTION........................................................................41 MATERIALS AND METHODS..............................................................4 Strains.............................................................................4 DNA extraction and fragment amplification..............................................49 Phylogenetic analyses................................................................49 RESULTS.............................................................................51 Taxa..............................................................................51 Multigene phylogeny.................................................................52 Individual gene analyses..............................................................5 Permutations of dataset...............................................................56 Topology tests......................................................................57 DISCUSION...........................................................................59 Taxa..............................................................................59 Multigene phylogeny.................................................................61 CHAPTER 4. PATERNS OF CEL DIVISION IN THE FILAMENTOUS DESMIDIACEAE, CLOSE GREN ALGAL RELATIVES OF LAND PLANTS 71 ABSTRACT............................................................................71 iv INTRODUCTION........................................................................71 MATERIALS AND METHODS..............................................................75 Terminology........................................................................75 Culture conditions...................................................................75 Molecular phylogenetic analysis.......................................................7 Microscopy.........................................................................78 RESULTS.............................................................................78 Cros wals.........................................................................78 Teilingia granulata..................................................................79 Spondylosium tetragonum.............................................................79 Spondylosium pulchelum.............................................................80 Hyalotheca disiliens and H. mucosa....................................................80 Groenbladia taylori.................................................................80 Bambusina boreri...................................................................81 Onychonema laeve var. micracanthum Nordst. and O. filiforme (Ehr.) Roy & Biset............81 Desmidium aptogonum Br?b., D. aptogonum var. ehrenbergi K?tz., Desmidium baileyi (Ralfs) Nordst., and D. swartzi (Agardh) Agardh................................................82 Spondylosium pulchrum (Bailey) Archer.................................................82 Micrasterias foliacea Bailey, Phymatodocis, Heimansia and Cosmocladium...................83 Molecular phylogeny.................................................................83 DISCUSION...........................................................................85 CHAPTER 5. SYSTEMATIC REVISION OF SOME FILAMENTOUS DESMIDIACEAE (ZYGNEMATOPHYCEAE, CHAROPHYTA) 98 ABSTRACT............................................................................98 INTRODUCTION........................................................................98 MATERIALS AND METHODS.............................................................10 RESULTS............................................................................105 DISCUSION..........................................................................106 SYSTEMATIC REVISIONS................................................................109 CHAPTER 6. INVESTIGATING DIVERSITY AMONG STRUCTURALY SIMPLE DESMIDS, THE GONATOZYGACEAE (DESIDIALES, ZYGNEMATOPHYCEAE) 119 ABSTRACT...........................................................................19 INTRODUCTION.......................................................................19 MATERIALS AND METHODS.............................................................121 RESULTS............................................................................127 Phylogenetic relationships...........................................................127 Structural investigation..............................................................128 DISCUSION..........................................................................129 CHAPTER 7. INVESTIGATION OF THE EVOLUTIONARY HISTORY OF TRIPLOCERAS GRACILE (DESMIDIACEAE). 142 ABSTRACT...........................................................................142 INTRODUCTION.......................................................................143 MATERIALS AND METHODS..............................................................145 RESULTS............................................................................151 DISCUSION 152 CHAPTER 8. CONCLUSIONS 169 EXPERIMENTAL CONCLUSIONS...........................................................169 v TOWARDS AN UNDERSTANDING OF DIVERSITY IN THE CONJUGATING REN ALGAE AND ITS APLICATION TO THE ORIGIN OF LAND PLANTS..........................................................171 LITERATURE CITED 175 vi List of Tables 2.1 CLASIFICATION OF CHAROPHYTA BASED ON LEWIS AND MCOURT (204) 18 3.1 STRAINS INVESTIGATED AND GENBANK NUMBERS???????????? 45 3.2 PRIMERS USED FOR PCR AMPLIFICATION OF FRAGMENTS 48 3.3 CONSTRAINTS USED IN AU TEST???????????????????? 58 4.1 STRAINS INVESTIGATED AND GENBANK NUMBERS 76 4.2 PUBLISHED IMAGES OF CEL DIVISION IN DESIDIACEAE??????? 91 5.1 STRAINS INVESTIGATED??????????????? ?.. 103 5.2 LIST OF NAMES AND SYNONYMS ????..??? 16 6.1 STRAINS INVESTIGATED AND GENBANK NUMBERS?????? 124 6.2 STRUCTURAL CHARACTERISTICS OF SOME Gonatozygon SP. ????.. 126 7.1 STRAINS INVESTIGATED AND GENBANK NUMBERS????????? 148 7.2 PCR PRIMERS AND CONDITIONS???????????...... 149 vii List of Figures 1.1 ML PHYLOGENY OF CHAROPHYTES BASED ON rbcL???????.......... 14 2.1 PHYLOGENY OF CHAROPHYTES SHOWING SPECIES RICHNES?????. 38 2.2 LIGHT MICROGRAPHS OF REPRESENTATIVES OF THE MAJOR LINEAGES OF THE CHAROPHYTA......................................................... 39 3.1 PHYLOGENY OF ZYGNEMATOPHYCEAE BASED ON rbcL, psaA ND coxII..... 69 3.2 PHYLOGENY OF DESMIDIACEAE BASED ON rbcL, psaA ND coxII?????.. 70 4.1 PHYLOGENY OF FILAENTOUS DESMIDIACEAE BASED ON rbcL AND coxII? 92 4.2 VARIOUS STAGES OF CEL DIVISION IN FILAMENTOUS DESMIDIACEAE 94 4.3 MODEL OF CEL DIVISION????????????????????..,.. 96 4.4 MODEL CLADOGRAM SHOWING DISTRIBUTION OF CEL DIVISION SYNDROMES??????????????????????????????. 97 5.1 PHYLOGENY OF FILAMENTOUS DESMIDIACEAE ?.. 17 5.2 STRUCTURAL CHARACTERISTICS OF SPECIES INVESTIGATED?????? 18 6.1 PHYLOGENY OF GONATOZYGACEAE BASED ON rbcL, psaA ND coxII . 135 6.2 LIGHT MICROGRAPHS SHOWING HABIT OF Gonatozygon AND Roya????. 137 6.3 LIGHT ICROGRAPHS SHOING CEL WALS OF Gonatozygon AND Roya?. 139 6.4 COMPARISON OF WIDTH IN STRAINS OF Gonatozygon kinahani???????. 141 6.5 COPARISON OF IDTH IN STRAINS OF Gonatozygon brebisoni ?.. 141 6.6 COMPARISON OF LENGTH IN STRAINS OF Gonatozygon kinahani?????? 141 6.7 COPARISON OF LENGTH IN STRAINS OF Gonatozygon brebisoni 141 7.1 LIGHT MICROGRAPH OF Triploceras gracile???????????????.. 158 7.2 GENEALOGIES BASED ON rSU??????? ?.. 160 7.3 GENEALOGIES BASED ON EF1 ?? ??????????????. 162 7.4 GENEALOGIES BASED ON EIF4???????? ?.... 164 7.5 GENEALOGIES BASED ON TUA? ???????????.... 16 7.6 MODELS OF POTENTIALY CONFOUNDING EVOLUTIONARY EVENTS ?. 168 1 Chapter 1. General Introduction Early land plant evolution The colonization of land by plants was one of the most important events in the history of modern life on Earth. Before the advent of land plants, photosynthesis was likely restricted to aquatic and damp terestrial habitats. Land plants, like their aquatic counterparts, consume carbon dioxide, fix and sequester the carbon in complex organic molecules, and release oxygen into the atmosphere. Today, terestrial plants acount for 56.4% of total net primary production (Field et al., 1998). Plants not only contributed to the total production of photosynthate on Earth, but were also responsible for building soils and their succes generated new habitats for other organisms. It is easy to speculate on the evolutionary developments that gave rise to the modern land flora once these organisms were established on land, but the nature of the photosynthetic pioneers remains obscure. What characteristics ? celular, sexual, cytoplasmic and genetic ? may have favored their succes? In animals, evolution of the modern fauna is comparatively wel documented in the fossil record. Early land plants, apparently, had few degradation resistant parts and are not wel represented in the fossil record. Some of the oldest fossil plants suggest that the origin of the land flora is at the latest 475 mya (Welman et al., 2003). These fossils resemble extant mosses. Of course, the colonization of land may have occurred long before these plants were fossilized. It is now widely acepted that land plants evolved from one lineage of gren algae, commonly known as the charophytes (Matox and Stewart, 1984; Huss and Kranz, 1995; Kranz et al., 1995; Lewis and McCourt, 2004; McCourt et al., 2004). 2 Charophyte gren algae There are at least six major lineages of extant charophyte gren algae (Lewis and McCourt, 2004), which wil be here discussed as clases of the division Charophyta. These include the Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Zygnematophyceae (conjugating gren algae), Coleochaetophyceae and the Charophyceae sensu stricto (stoneworts). Systematic relationships, fossil record and general characteristics of these lineages are discussed in Chapter 2. Charophytes are united by a number of cytological characteristics. Most of the structural evidence for the relatednes of charophyte algae and land plants is present in the motile cels. Chara and Coleochaete produce motile sperm while Coleochaete, Klebsormidium and Chlorokybus produce flagelate zoospores. Mesostigma viride is itself a biflagelate cel. These flagelate cels al have a subapical insertion of two isomorphic flagela (Picket-Heaps and Marchant, 1972; Matox and Stewart, 1984; Melkonian, 1989). These flagela are rooted with a characteristic asociated multilayer structure (MLS) at the base. The MLS is a dense aray of lamelar structures that atach to the basal bodies of the flagelar apparatus. While most charophytes have a single MLS, Mesostigma viride has two MLSs (Rogers et al., 1981; Melkonian, 1989). Although the MLS in gren algae is unique to the charophytes, a structure similar to a MLS has been found in other unrelated organisms. The conjugating gren algae are conspicuously without a flagelate stage in the life cycle and their inclusion in the charophyte gren algae was based on the presence of a persistent mitotic spindle (Picket-Heaps and Marchant, 1972; Matox and Stewart, 1984). Since the recognition of charophytes as a lineage unto themselves, other characteristics have been found which sem to unite the group or some members of the 3 group with land plants. Among the charophytes investigated, al sem to have celulose synthesizing rosetes, circles of celulose synthetase protein complexes, as opposed to linear arays of synthetase protein complexes found in most chlorophyte gren algae (Domozych et al., 1980; Hotchkis and Brown, 1987; Okuda and Brown, 1992; Tsekos, 1999). Diferent arangement of the synthetase complexes translates to diferent numbers of celulose microfibers and diferently shaped celulose fibers in the cel wals. Arguably, slight diferences in cel wal composition could correlate to diferences in cel-to-cel communication, structural strength, or other factors that may have given charophyte gren algae an advantage in colonizing terestrial habitat. In addition to celulose synthesizing rosetes, some charophytes also have an unique variant of cel division that involves a microtubular aray caled a phragmoplast (Matox and Stewart, 1984; Cook et al., 1998; Cook, 2004b). The phragmoplast forms at the time of cytokinesis and sems to be involved with the deposition of primary wal in a cel plate. In the case of Chara, Coleochaete and land plants, the cel plate is deposited from the center of the cel toward the periphery of the cel (Cook et al., 1998; Cook, 2004b). Al other charophytes divide by the centripetal encroachment of a division septum. In this case, the cel wal forms from the periphery of the cel toward the center without the asociation of a highly organized microtubular aray (Bech-Hansen and Fowke, 1972; Floyd et al., 1972; Picket-Heaps, 1972; Rogers et al., 1980; Matox and Stewart, 1984). This would sem to set Chara and Coleochaete apart as close relatives of land plants, however, some conjugating gren algae also use a phragmoplast-like microfilament aray (Fowke and Picket-Heaps, 1969a; Fowke and Picket-Heaps, 1969b; Picket-Heaps and Marchant, 1972). This has only been shown in Spirogyra, and was 4 later demonstrated to be functionaly and compositionaly diferent from a phragmoplast (Sawitzky and Grolig, 1995). Stil, its presence in one of the earliest diverging lineages of conjugating gren algae suggests that the phragmoplast may have been lost in the conjugating gren algae. Alternatively, the actin-based microfilaments may have preceded the microtubule aray used in plant phargmoplasts. Besides structural evidence, a number of phylogenetic studies suggest that the charophytes are closely related to land plants. Thre lineages, individualy or in combination, have been proposed as the closest relative of land plants: Charophyceae, Coleochaetophyceae and Zygnematophyceae (Manhart and Palmer, 1990; Huss and Kranz, 1995; Kranz et al., 1995; Bhatacharya and Medlin, 1998; Nickrent et al., 2000; Karol et al., 2001; Turmel et al., 2003, 2006). Any of these hypotheses would be consistent with the structural evidence available and, at the present, there is litle consensus among the competing hypotheses, however only these thre lineages sem to be likely candidates based on presently available data. Of these thre lineages, one exhibits a degre of taxonomic and structural diversity comparable to that of land plants: the conjugating gren algae (Zygnematophyceae). The extant Charophyceae are the remnants of a once more diverse lineage of macrophytes (Feist and Grambast-Fesard, 1991). The thalus of extant Charophyceae consists of a series of branched nodes and internodes. The internodes are single, large, multi-nucleate cels. The nodes, however, are made of many smaler cels with some being specialized cels. The branches atach in whorls at the nodes. Charophyceans have rhizome-like shoots that penetrate sediments as wel as rhizoids and 5 specialized reproductive structures, oogonia and antheridia. Extant Characeae are al oogamous, but may be hetero- or homothalic (Wood and Imahori, 1965). The Coleochaetophyceae are branched epiphytic filaments. These organisms have a specialized hair cel that extends above the cel into the surrounding medium. Coleochaetophytes produce both zoospores and flagelate sperm (Delwiche et al., 2002). Sexual reproduction is oogamous, and the oogonia may be completely or only partialy surrounded by jacket cels caled cortical cels (Szyma?ska, 1988; Delwiche et al., 2002; Szyma?ska, 2003). There are fewer than thirty known species of Coleochaetophyceae and only a couple hundred species of Charophyceae (Chapter 2). These numbers are dwarfed by the estimated 350,000 land plant species that have been described in the literature. Biology and systematics of the Zygnematophyceae The conjugating gren algae, unlike other charophyte lineages, include several thousand extant species (Hoshaw et al., 1990; Gerath, 1993). These organisms are not just taxonomicaly diverse, but they are also phyleticaly diverse. A phylogenetic analysis of just the gene rbcL from sequences of charophyte gren algae and land plants reveals that the branch lengths within the conjugating gren algae are both far greater than those within the Charophyceae and Coleochaetophyceae, but also that the family Desmidiaceae shows almost as much sequence divergence as angiosperms (Figure 5.1). The fundamental biological diferences among conjugating gren algae defy generalizations. The group includes organisms that are unicelular, colonial, and filamentous. Conjugating gren algae can be found in nearly every freshwater habitat on every 6 continent. They can be found on glaciers, in thermal pools, in acidic rivers and alkaline streams (Brook, 1981). Members of the group are often considered biological indicators of environmental conditions. For example, Mougeotia is sometimes an indicator of acidification, and Spirogyra an indicator of eutrophication. One group of zygnematophytes, the desmids, has been used more extensively in estimation of the geochemical trophic state and general water quality of freshwater habitats (Jarnefelt, 1952; Rawson, 1956; Brook, 1965; Vinebrooke and Graham, 1997; Coesel, 2001). Zygnematophytes demonstrate a number of diferent sexual reproductive syndromes including oogamy, heterogamy and isogamy. They may be homothalic or heterothalic (Brandham and Godward, 1965). The presence of such fundamental diferences among conjugating gren algae makes them an excelent model system for understanding the basis of these syndromes in structuraly simple organisms. A number of cytological diferences exist among the conjugating gren algae. The most striking aspect of these organisms is their spectacular chloroplasts. These organeles are often very large and have complex shapes. The spiraling chloroplasts of Spirogyra, the flat chloroplasts of Mougeotia, which twist and turn in response to light stimulus, and the stelate chloroplasts of Zygnema are among the most recognizable chloroplasts of al gren algae. Spirogyra is not the only familiar conjugating gren alga, however. The desmids (order Desmidiales) are also frequently studied in introductory biology courses. The desmids are best known for their exceptionaly intricate and decorated cel wals, however their chloroplasts are equaly ornate. The appearance of the cel wals by light microscopy does not demonstrate the complexity of these structures. Even apparently similar organisms may have cel wals 7 that are structuraly very diferent from one another. These diferences are the basis of the order and family-level clasification. One order, the Zygnematales, is characterized by a homogenous cel wal that is structuraly similar to that of land plants, Coleochaete and Chara (Mix, 1972; Domozych et al., 1980; Hotchkis et al., 1989). The cel wal consists of an extracelular primary celulosic cel wal and a secondary wal that is deposited inside the primary wal. In some Zygnematales, there is also an extramural matrix, consisting partialy of pectins and glycoproteins (Mix, 1972; Domozych et al., 1980; Hotchkis et al., 1989). In the order Desmidiales, the diferences in cel wal characteristics define the families (Mix, 1972). In the Closteriaceae, Gonatozygaceae and Peniaceae, al of the ornamentation of the mature cel wal is actualy present in the extramural matrix. Briefly, the extramural matrix consists of variously shaped fibrils that are aranged in a mesh-like network (Peniaceae and Gonatozygaceae) or more orderly lines (Closteriaceae) (Mix, 1966, 1969, 1972). Kouwets and Coesel (1984), found diferences betwen the cel wals of Penium and Gonatozygon to be so trivial that they proposed merging those taxa into a single family, the Peniaceae. The family Desmidiaceae, which contains the majority of desmid species, has a very diferent cel wal structure. In the Desmidiaceae, there are several diferent syndromes of cel division (investigated in Chapter 3) but there are general characteristics of the cel wal that are present in most taxa. In the case of Desmidiaceae, the primary wal has comparatively litle ornamentation. During development of the cel wal, the secondary wal, with the characteristic ornamentation, is deposited inside the primary cel wal. Once mature, the primary wal is completely or partialy shed leaving only the secondary wal along most 8 of the cel. Sometimes an extramural matrix is deposited outside the secondary wal. These cel wals also have a more complex kind of pore than is found in the other families of desmids. In the Closteriaceae, Gonatozygaceae and Peniaceae, the pores are actualy pits in the extramural matrix, which sometimes extend into the primary wal, but never penetrate the secondary wal. In the Desmidaceae, however, the pores completely traverse the secondary wal and sometimes have a complex asociated protein apparatus (Mix, 1972). Systematic treatment of the genera is heterogenous across the conjugating gren algae. Among the Zygnematales, concepts of the genera are based on growth form (unicelular versus filamentous) and the shape of the chloroplast (Czurda, 1932; Transeau, 1951; Prescott et al., 1972; Kadlubowska, 1984). Among the Desmidiales, most generic concepts are based on diferences in cel shape: presence or absence of proceses, general shape of the cel, presence of apical lobes or incisions, presence, nature and distribution of ornamentation, etc. The importance of most of these characteristics has been directly contested in the literature resulting in the merging or spliting of groups of species into larger or smaler genera (Prescott et al., 1975; Comp?re, 1976; Palamar-Mordvintseva, 1976; Prescott et al., 1977; Prescott et al., 1981; Prescott et al., 1982; Croasdale et al., 1983; Compere, 1996; Palamar-Mordvintseva, 2003, 2005). At the moment, the general trend sems to be in favor of separating species with fundamentaly diferent structural characteristics into diferent genera. For example, the genus Haplotaenium was recently separated from Pleurotaenium when it was found that one group of species lacked a terminal vacuole, has axile as opposed to parietal chloroplasts as wel as a diferent distribution of pores on the cel wal (Bando, 9 1988). Similarly, the genus Tortitaenia was split from Spirotaenia based on the diference in chloroplast distribution (axile versus parietal) (Brook, 1998). These are just two of many examples. Characters used to identify species vary among the zygnematophyte lineages. Among the Zygnematales, species delimitation hinges on diferences in chloroplast distribution in the cel as wel as the characteristics of the zygospore wals (Czurda, 1932; Transeau, 1951; Kadlubowska, 1984; Rundina, 1998). This makes identification of field material dificult because sexual cels are infrequently encountered during most of the year. Among the Desmidiales, species are based on diferences in cel shape and ornamentation, however both are known to be plastic within species. Taxonomy of Desmidiales is complicated by a long history of the use of subspecies, varieties and forms. For many desmid species, this means that there may be dozens of subspecific names asociated with the specific epithet (West and West, 1904; Prescott et al., 1972; Prescott et al., 1975, 1977; Prescott et al., 1981; Prescott et al., 1982; Croasdale et al., 1983). The basic concept of a species also difers among genera and investigators. In many cases, the biological species concept is inappropriate because the species is asexual, or not testable because it is not known how to grow the organism, induce sexual reproduction or induce germination of the resulting spores. In some groups, species are thought to exist in breding complexes (ie. species complex) (Denboh et al., 2003a). In others, it is thought that many of the organisms described as varieties are very likely biological species. 10 Understanding diversity of the conjugating gren algae Given this bewildering diversity and inconsistent taxonomic treatment, how is one to interpret previous cytological and developmental studies or make any inferences about evolution within the conjugating gren algae, much les about the lineage that gave rise to land plants? Investigation of these characteristics in a systematic and phylogenetic context would make it possible to both order the known information and would indicate where information about the organisms is lacking. This was the underlying motivation of this thesis. At the initiation of this disertation work, very litle was known about the phylogenetic relationships of conjugating gren algae. Studies at that time had been based on very few taxa and only a single gene, rbcL (Park et al., 1996; McCourt et al., 2000). These phylogenies were not wel supported statisticaly and lacked many of the genera and species for which structural information was known. To addres this, I first completed a phylogenetic study of the conjugating gren algae using multiple genes and then investigated some of the gaps in our knowledge of the basic biology of these organisms with an aim to beter understand their diversity and evolution. Diversity has many definitions. When discussing ?diversity,? most scientists are refering specificaly to species or taxonomic diversity, sometimes caled biodiversity. Often, scientists are focused on the number and identity of species present in a particular location. There are other kinds of diversity, however, which may be pertinent to questions of evolution. Some other possible measures of diversity include phyletic, ontogenetic, structural, ecological and physiological diversity. In the case of conjugating gren algae, structural diversity is closely related to taxonomic diversity because diferentiation of species relies almost exclusively on structural characteristics. 11 Structural and taxonomic diversity are not necesarily directly related to other measures of diversity. Based on structural characteristics, some genera of conjugating gren algae contain over 1,000 species, but our interpretation of their evolution would be very diferent if they were found by molecular phylogenetic investigation to al be part of a very recent radiation with very short shalow branches as opposed to a very deeply branching clade, or if species in this genus were polyphyletic with respect to other genera. In this hypothetical genus with a thousand species, the phyletic diversity may be congruous with the taxonomy of the group but a phylogenetic analysis can reveal diferences among lineages that are relevant to our understanding of evolution of the group. Similarly organisms that have similar gross structural characteristics may have diferent physiological responses to environmental stres, diferent developmental pathways or diferent environmental niches. Each measure provides diferent information about the group being studied. Much energy has been spent on structural diversity of the cel wals of conjugating gren algae and significantly les emphasis has been placed on the characteristics of the cytoplasm including the chloroplast. Additionaly, much information on the ecological diversity of the conjugating gren algae exists, although the interactions betwen species and their environment on a physiological and biochemical level remains obscure. In this work, I investigated not only taxonomic diversity, but phyletic diversity, ontogenetic diversity and structural diversity in a molecular systematic context. By doing so, I hoped to increase our understanding of the diversity of the conjugating gren algae and provide some insight into how diferent characteristics evolved in the conjugating gren algae and, possibly in the lineage that gave rise to land plants. 12 This thesis contains five chapters based on newly collected data (Chapter 3-7) and one chapter that reviews charophyte systematics (Chapter 2). Chapter 3 presents my findings in a phylogenetic study of the conjugating gren algae based on thre organelar genes. This study was designed to provide phylogenetic context for as many of the genera as could be obtained from culture collections and isolations from the wild. Based on these data, a number of observations were made and thre lineages were investigated in greater detail. The first is the filamentous Desmidiaceae (Chapters 4 and 5). It was found that some genera were polyphyletic and that most filamentous desmids formed a single lineage within the family. Because of the known diferences in cel division, I investigated cel division paterns in greater detail in the filamentous Desmidiaceae (Chapter 4) and then investigated other aspects of their structure including cel and chloroplast shape. These characteristics were used in a systematic treatment of the filamentous genera where I propose moving two species to diferent genera and created one new generic name (Chapter 5). The second case study investigated cryptic diversity in the Gonatozygaceae. Previous phylogenetic studies suggested a close relationship betwen Gonatozygon (Desmidiales) and Roya (Zygnematales). I also found in Chapter 2, a surprising degre of sequence diversity among structuraly similar strains of Gonatozygon. To further investigate this, I increased taxon sampling in the Gonatozygaceae and investigated structural characteristics among the strains. The Gonatozygaceae are an excelent model for microbial diversity because, like many other unicelular organisms, they are structuraly simple. Chapter 6 investigates the phyletic diversity within the family in an 13 atempt to determine if the structural simplicity of the cels results in an underestimation of the species diversity. If so, I hoped to determine which characteristics distinguished lineages so that it might be possible to predict to which clade (or species) structuraly similar organisms from the wild may belong. The third case study investigated the phylogenetic position of Triploceras gracile. This genus is unique among the Desmidiaceae because some portions of the cel are radialy symmetric while others are bilateraly symmetric. This character is often used in systematics of conjugating gren algae and such transitions are considered important evolutionary events in the evolution of animals. In chapter 2, I found that Triploceras was embedded in a clade of Micrasterias. This placement was inconsistent with that reported by Gontcharov et al. (2003) based on SU data. This raised the possibility of a complex evolutionary history. To investigate the evolutionary history of Triploceras, I sampled thre, independent nuclear genes from several species of Desmidiaceae. The results of this investigation are presented in Chapter 7. Desmidiales Desmidiaceae Zygnematales Zygnematophyceae Charophyceae Coleochaetophyceae Embryophyceae Angiosperms 0.1 substitutions/site Figure 1.1 Maximum likelihood phylogeny of charophytes based on rbcL. Branchlengths are proportional. Note that the branchlengths in the family Desmidiaceae are comparable to those of all angio- sperms (dashed boxes). Basal angiosperms and monocots were included in the analysis. 15 Chapter 2. In the shadow of giants; systematics of the charophyte gren algae Abstract Charophyte gren algae are those organisms most closely related to land plants. The group has at least five major lineages the Charophyceae, Coleochaetophyceae, Zygnematophyceae, Klebsormidiophyceae, Chlorokybophyceae and probably the Mesostigmatophyceae. These organisms are briefly introduced and their relative phylogenetic position discussed. Current systematic understanding of the groups is discussed as wel as the potential role of genomic studies in the systematics of charophyte gren algae. Genomic studies are beginning to elucidate the order of ancient branching events in the lineage, however, greater molecular and broader taxon sampling wil be required to resolve some relationships. In addition to deep nodes, molecular phylogenetic investigations of populations and species of al the lineages are wanting. Continued investigation and greater sampling wil provide more insight into the evolution of these organisms and early land plant evolution. Introduction Gren algae are one of the most diverse groups of organisms on earth both structuraly and in terms of number of described species. They occupy almost every habitat and are the algal relatives of the one of the most species-rich lineages, the land plants. Although land plants evolved from gren algae, there are major diversifications in the gren algae that preceded the invasion of land and radiation of embryophytes. One major distinction is betwen the Chlorophyta and the Charophyta (Charophyceae sensu 16 Matox and Stewart, 1984). Land plants are members of the Charophyta and their closest algal relatives are here caled the charophytes. The charophytes are only a smal part of total gren algal diversity, but their evolutionary history gives direct insight into the evolution of plants. Nomenclature used here follows Lewis and McCourt (2004; se Table 2.1). In this paper, ?charophyte? refers to those algae most closely related to land plants (not just the Charophyceae sensu stricto). When refering to Charophyceae sensu stricto, I use either Characeae or their common name, stoneworts. Historical perspective The charophytes, in the sense discussed here, have been recognized as a group only since the 1980s. They include their namesake Characeae (Charophyceae sensu stricto) and a collection of semingly disparate lineages: the Coleochaetophyceae, the conjugating gren algae (Zygnematophyceae), the Klebsormidiophyceae, Chlorokybus atmophyticus Geitler (Chlorokybophyceae) and, probably, Mesostigma (Mesostigmatophyceae). Some charophytes are quite large, such as members of the Characeae, and have been known for several hundreds of years: Linnaeus named some species of Chara, which had previously been described as aquatic forms of Equisetum (Wood and Imahori, 1965); others, such as Spirogyra, were among the first algae discovered by means of Van Leuwenhoek?s microscopes in the late 17 th century (Leuwenhoek, 1674). Even the relationship betwen gren algae and land plants had long been supposed as implied by discussions of microscopic vegetables (Ingenhousz, 1779). Bower (1908) even described the plant-like characteristics of many algae, including some today recognized as charophytes. It was not until the later part of the 20 th century that science and technology (in this case 17 electron microscopy) converged to provide evidence for the relationships among gren algae and their afinity to land plants. The groups currently thought to belong to the charophyte lineage, with the exception of the polemic Mesostigma viride Lauterborn, were recognized by Matox and Stewart (1984) in their systematic treatment of the gren algae based on comparative cytology. Charophyte systematics, however, did not begin in the 20 th century. Members of these lineages were known in the 19 th century and earlier. Two groups in particular, the Characeae and the conjugating gren algae (Zygnematophyceae), include several hundreds or thousands of named species, respectively. Both of these have a long history of independent systematic investigation and several monographs are dedicated to their taxonomy and distribution. Pringsheim (1860) investigated Coleochaete and contemporary authors have criticaly studied this genus and described new species (Szyma?ska, 1989; Cimino and Delwiche, 2002; Delwiche et al., 2002). Al the charophytes are commonly (and historicaly) included in local florulas, though rarely as a group unto themselves. Even today, valuable systematic data are often published as part of local or regional florulas. Systematic investigation, therefore, proceds on many fronts: higher-level clasification of the lineages and families as wel as the population and species levels and molecular, genomic, cytological and morphological methods are used. 18 Table 2.1. Clasification of the Charophyta based on Lewis and McCourt (2004) Kingdom Viridiplantae Division Chlorophyta Division Charophyta Clas Mesostigmatophyeae sensu stricto Clas Chlorokybophyceae Clas Klebsormidiophyceae Clas Zygnematophyceae Order Desmidiales Order Zygnematales Clas Coleochaetophyceae Clas Charophyceae sensu G. M. Smith Clas Embryophyceae 1 1 This sufix is derived from the corect placement of embryophytes among charophytes, however, some embryophytologists may prefer a diferent rank or sufix. 19 Why should one study the systematics of charophytes? The charophyte gren algae hold a unique phylogenetic position as the closest extant relatives of terestrial plants (in fact, embryophytes could be more correctly treated as a specialized charophyte lineage). Understanding of relationships within and among these lineages continues to provide insight into the evolution of land plants and their occupation of terestrial habitats. Charophytes are also of systematic interest because they inhabit environments that are greatly afected by humans. Charophytes are primarily freshwater organisms (although a few occur in brackish pools) and many charophytes are most common in oligotrophic waters. These habitats are particularly impacted by human activities. There is a great need for monitoring and investigation of freshwater biodiversity, as estimates of the rate of extinction among freshwater organisms is very high (Leidy and Moyle, 1998; Watanabe, 2005). Few data are available on the conservation status of most charophycean taxa. Available data on Characeae and conjugating gren algae indicate that local extinctions have occurred and global extinction may be likely. Few countries (including the United States) have biotic inventories that would reveal local extinctions, so the real loss of global charophyte biodiversity is unknown. Those countries with biotic inventories indicate that many charophyte taxa are threatened, endangered or already localy extinct (Krause, 1984; Siemi?ska, 1986; Stewart and Church, 1992; Adam, 2004; N?meth, 2005; Watanabe, 2005). Charophyte systematics must continue with some haste if we are to record the global diversity and natural distribution of these important organisms. 20 Which gren algae belong to the Charophyta? The charophytes constitute one of the two primary lineages of gren algae and are distinguished from their relatives, the chlorophytes, by a number of distinct if not imediately obvious features. Matox and Stewart (1984) separated the charophyte algae from other gren algae based on the presence of a multilayer structure at the base of the two flagela which insert subapicaly on the asymmetric flagelate cels. It is now known that multilayer structures occur in other groups of algae besides the charophytes, but the subapical insertion of two similar flagela is distinctive to the group. The Zygnematophyceae lack flagela and were included in the group because of their persistent mitotic spindle (Matox and Stewart, 1984). Other characteristics were later discovered that indicated that the conjugating gren algae are closely related to other charophytes, but these are not diagnostic of al charophytes. It is important to note that the charophytes are only a monophyletic group if land plants are considered among their ranks (Bhatacharya et al., 1998; Karol et al., 2001). The clasification provided by Lewis and McCourt (2004) asigns clas status to the major lineages of charophytes, including the embryophytes (land plants). Higher- level nomenclature is stil a source of disagrement among systematists, as are the relationships among the charophyte lineages. However, most authors do agre that the charophyte algae are diferent from other gren algae (Chlorophyta), that they are closely related to land plants, and that most of the taxa proposed by Matox and Stewart (1984) belong to this group. The inclusion of Mesostigma viride is les certain but sems very likely (se discussion below). The placement of embryophytes as a monophyletic lineage, deeply embedded within the charophytes, is not controversial, but the nomenclatural implications of this biological fact are highly so. 21 The number of charophyte species reported in the literature is not known with certainty. The best estimates are: Mesostigmatophyceae, 2; Chlorokybophyceae, 1 (Geitler, 1942b); Klebsormidiophyceae, 19 (Etl and G?rtner, 1995; Lokhorst, 1996); Zygnematophyceae, 4,000-13,000 (Gerath, 1993; Hoshaw et al. 1990); Coleochaetophyceae, 22 (Thompson, 1969; Szyma?ska, 2003); Charophyceae, 395 (Wood and Imahori, 1965). These estimates should be sen as minimum, provisional, good faith estimates taken from the literature, and should be interpreted with caution. Species estimates among charophytes are dificult: inclusion of certain genera in the group is not certain and estimates are strongly biased by the treatment of varieties, particularly among the stoneworts and conjugating gren algae. The total diversity of al other charophytes fals wel within the uncertainty of estimates for the conjugating gren algae alone. That said, estimates for the number of conjugating gren algae range from about 3000 desmids (Gerath, 1993) and 800 filamentous Zygnematales (Kadlubowska, 1984) to 10,000 -12,000 placoderm desmids excluding the filamentous and unicelular Zygnematales (Hoshaw et al., 1990). Diferences in these estimates are due in part to the inacesibility of the relevant literature: no one is certain how many taxa have been described, much les how many are synonymous. Another major factor in the variability of estimates is the treatment of varieties. Taxonomists of the Desmidiaceae (Zygnematophyceae) continue to use subspecies, varieties and forms, a practice abandoned in many other algal groups. This means that any one species name can have as many as several dozen subspecific taxa asociated with it. The relationship betwen a desmid ?species? and a biological species is not clear. If the varieties of conjugating 22 gren algae were treated as species, their number would likely approach the 12,000 species estimate of Hoshaw et al. (1990). Estimates of the number of species of charophytes are consequently subject to considerable interpretation. It might be expected that with the number of papers dedicated to, in particular, the taxonomy of the conjugating gren algae, there would be a clear understanding of their diversity or at least the number of species on earth. Unfortunately, information on their numbers and distribution is limited by the geographic location of investigators, acesibility of study sites, and time available for investigation. A limitation unique to the charophytes, to a greater or leser degre, includes a tendency for some charophyte species to be overlooked in general floristic studies: Coleochaete grows atached to substrata, such as rocks and aquatic plants, which are frequently not collected; many desmids are benthic and do not appear in plankton studies; and smaler species of conjugating gren algae and Klebsormidium are often mistaken for chlorophytes or xanthophytes in floristic surveys. As with many microscopic taxa, apparent distributions may be primarily a function of the geography of the investigators. It is also important to note that estimates only reflect the number of described taxa; the actual number of living charophytes in the world could be much greater. Vast portions of the world have not been investigated and new species are frequently described from even the best studied regions (e.g. Coesel, 2002; Szymanska, 2003). General systematic studies of charophytes Since the time the charophytes were formaly recognized as a distinct group of gren algae, few studies have investigated the relationships among these disparate 23 lineages. Many of these studies have focused on two important questions: which taxon is most closely related to land plants and how might the ancestor of the charophytes (and possibly al gren algae) have appeared? If these questions could be confidently answered, one could use the characteristics of the extant charophytes to make inferences about the evolution of land plants and charophyte algae. Systematic investigation, particularly molecular systematics, is necesary because the fossil record of these early diverging lineages is very poor; only the stoneworts (Charophyceae sensu stricto) have a wel documented fossil record (Feist and Grambast-Fesard, 1990). Few molecular systematic investigations have focused on the relationships within the charophyte lineages. Nearly every lineage of charophytes, or combination of lineages has been proposed as the most closely related to land plants at one time or another (Huss and Kranz, 1995; Kranz et al., 1995; Karol et al., 2001; Turmel et al., 2002c; Turmel et al., 2002a). However, most studies have very limited molecular or taxonomic sampling or both. Lemieux et al. (2000), based on a dataset of 53 genes and eight taxa, recovered Mesostigma viride at the base of the Viridiplantae clade. Other comparable phylogenies based on fewer genes support the placement of Mesostigma as the basal most lineage of the Charophyta (Bhatacharya et al., 1998; Karol et al., 2001). In a study using 72 mitochondrial genes and six taxa, Turmel et al. (2003) found a sister relationship betwen stoneworts and embryophytes. One molecular study (Karol et al., 2001) exhibits both substantial molecular sampling (4 genes) and a broad taxon sampling (40 taxa). This study found the stoneworts sister to land plants and the remaining lineages to be a paraphyletic asemblage with Mesostigma the earliest diverging member of the group 24 (Figure 5.2.1). The relationship betwen stoneworts and land plants was strongly supported, as was the placement of Mesostigma as the basal most charophyte, although the relationships among the other lineages of the charophytes received les support (Karol et al., 2001). It is worth noting, however, that several studies (particularly those using rDNA data) have found the stoneworts to be very distantly related to the land plants and, generaly, suggest that the lineage most closely related to land plants is one or an asemblage of the other charophyte clases (Huss and Kranz, 1995; Kranz et al., 1995; Turmel et al., 2002c; Peterson et al., 2003). One study based on 76 chloroplast genes found the conjugating gren algae (Zygnematophyceae) to be the sister lineage to land plants with stoneworts being only distantly related (Turmel et al., 2006). Evolution of the charophyte lineages is not completely understood. Systematics of the charophyte lineages Mesostigma The earliest diverging lineage of the Charophyta may be Mesostigma viride Lauterborn (Figure 2.2A). This organism was originaly clasified among the prasinophyte gren algae (scaly unicelular flagelates). On the basis of root configuration, Melkonian (1989) noted the afinity of M. viride to the charophytes. This was confirmed by analyses of actin sequences (Bhatacharya et al., 1998) that indicated Mesostigma is the basal most lineage of the Charophyta. Mesostigma viride is unicelular and covered in minute scales. Cels are circular and compresed with a concave inner surface (Melkonian, 1989). Unlike other charophytes, Mesostigma viride has two multilayer structures (MLSs) and an eyespot (Rogers et al., 1981). Mesostigma viride is known only from freshwater habitats, and has been infrequently reported from the wild. 25 Although its phylogenetic position is not known with certainty, a number of studies suggest that Mesostigma viride is either very ancient or very divergent (probably both) compared to other gren algae: it has an unusual compliment of photosynthetic pigments (Yoshii et al., 2003; Stabenau and Winkler, 2005) and Stabenau and Winkler (2005) suggest that the microbodies found in Mesostigma may be ancestral to leaf peroxisomes and glyoxysomes since it has enzymes normaly asociated with both. Some molecular phylogenetic studies place Mesostigma at the base of the Charophyta (Bhatacharya et al., 1998; Marin and Melkonian, 1999; Karol et al., 2001) while others find it to be sister to both the charophytes and chlorophytes (Lemieux et al., 2000; Turmel et al., 2002c; Turmel et al., 2002a). Evidence in favor of including Mesostigma in the Charophyta is mounting: Mesostigma, the other charophytes and land plants sem to share a unique duplication of the GapA /GapB gene (Peterson et al., 2006). Regardles of its position, Mesostigma is critical to our understanding of evolution of the gren algae. At least one other species of Mesostigma has been described (M. grande Korshikov), but this species is not available in culture and, consequently, has not been thoroughly investigated. Chlorokybus atmophyticus Chlorokybus atmophyticus Geitler is the sole known representative of its lineage, the Chlorokybophyceae. The species was discovered growing among mosses in a park in Vienna, Austria (Geitler, 1942b). It is unique among the charophyte algae, with the possible exception of some conjugating gren algae, in its sarcinoid growth habit, i.e. it grows as smal packets of cels enveloped in a common mucilaginous matrix (Figure 26 2.2B). Its cel division, as wel as production of autospores and swarmers, has been documented (Geitler, 1942a; Rieth, 1972). Sexual reproduction has not been reported. Zoospores of C. atmophyticus have a single multilayer structure and two subapical flagela that are covered in scales (Rieth, 1972; Rogers et al., 1980), a structure that is consistent with other flagelate charophyte cels. Although its distribution is unknown, it has been found in subaerial habitats in Europe and Russia (Geitler, 1942b; Rieth, 1972; Etl and G?rtner, 1995). Many molecular studies place Chlorokybus atmophyticus near the base of the Charophyta (Bhatacharya and Medlin, 1998; Karol et al., 2001). Phylogenetic analyses of 18S rDNA and chloroplast rbcL sequences suggests that Spirotaenia (normaly considered to be a member of the Zygnematophyceae) may form a monophyletic group with Chlorokybus (Gontcharov and Melkonian, 2004). This relationship is certainly unexpected since Spirotaenia is known to lack flagela and to conjugate during sexual reproduction, characteristics typical of the conjugating gren algae. The analysis showed only moderate statistical support for the group and could not rule out other placements of Spirotaenia. However, those data did reject a sister relationship betwen Spirotaenia and the conjugating gren algae. The relationships of Chlorokybus and Spirotaenia to other charophyte algae certainly merit further investigation. Klebsormidiophyceae The Klebsormidiophyceae, particularly Klebsormidium spp. (Figure 2.2C), are among the most ubiquitous charophytes. The two genera most commonly included in the group (Klebsormidium and Entransia) are typicaly unbranched filamentous algae that 27 reproduce by fragmentation of the filaments and the release of (presumably) biflagelate zoospores (Lockhorst, 1996; Cook, 2004a). Although structures consistent with zoosporangia were observed in Entransia fimbriata Hughes, no flagelate cels were observed (Cook, 2004). Klebsormidium typicaly has a single parietal chloroplast that partly encircles the cel with a single, embedded pyrenoid. Filaments are sometimes atached by holdfasts or interupted by ?H? pieces (Lockhorst, 1996). Entransia has one or two parietal chloroplasts that are deeply, iregularly lobed (se Cook, 2004, for discussion of morphology). Inclusion of other genera, such as Stichococcus and Raphidonema remains uncertain; some species of Stichococcus have been transfered to Klebsormidium (Etl and G?rtner, 1995) while others clearly belong to the Chlorophyta (Lewis and Lewis, 2005). The genus name Klebsormidium was created to acommodate Hormidium sensu Klebs. The taxon Hormidium was abandoned because of synonymy and general confusion (Silva et al., 1972). Since that time, a total of about seventen species have been described or transfered to the genus. Taxonomy of the genus is based on morphological characters, such as: filament width, cel wal surface characteristics, and chloroplast shape (Lokhorst, 1996). Klebsormidium species are common in freshwater habitats but also occur in many subaerial habitats including desert crusts, urban wals, and freshwater seps (Lewis and Fletchner, 2002; Johansen et al., 2004). Entransia is clearly distinct from Klebsormidium, sems to be rare and occurs infrequently in ponds, bogs, or seps (Hughes, 1948; Prescott, 1966; Cook, 2004a). Entransia was originaly thought to belong to the conjugating gren algae (Zygnematophyceae), although it is not known to conjugate. Only one of the two species 28 has been investigated, E. fimbriata, which is thought to reproduce by zoospores (Cook, 2004a). Sexual reproduction has not been reported. Molecular phylogenetic investigations by McCourt et al. (2000) placed E. fimbriata outside the conjugating gren algae and Karol et al. (2001) found it to be most closely related to Klebsormidium, a relationship supported by ultrastructural data (Cook, 2004a). Very few Klebsormidium isolates have been studied using molecular systematic methods (Novis, 2006) and more work certainly remains. Zygnematophyceae The Zygnematophyceae are the most species-rich clade of the Charophyta (excepting land plants). They are commonly refered to as the conjugating gren algae because of their unusual mode of sexual reproduction by fusion of non-flagelate gametes. Zygnematophytes may be unicelular, colonial, or filamentous, depending on the species. Historicaly the filamentous species with smooth wals (Zygnemataceae) and the unicelular forms were treated separately, although they were thought to be closely related to one another long before the evolutionary proces was understood (Ralfs, 1848). The current family-level clasification of the conjugating gren algae is based primarily on diferences in cel wal structure (Mix, 1972). The clas is often divided into two orders, the Zygnematales and the Desmidiales, with two and four families, respectively. The species number some thousands, with the majority belonging to the Desmidiaceae (Gerath, 1993). The placement of the Zygnematophyceae within the Charophyta is unclear. Most molecular studies place these algae sister to a clade with Coleochaetophyceae, stoneworts 29 and land plants, or as part of a lineage sister to land plants and including other charophyte clases. Consistent with the hypothesis that the conjugating gren algae are more closely related to land plants than Klebsormidium and Chlorokybus is the presence of a phragmoplast-like microtubule aray in some Zygnematophyceae (Fowke and Picket- Heaps, 1969a). Additionaly, the Zygnematophyceae, Coleochaetophyceae, Characeae and land plants share similar celulose synthesizing rosetes (Tsekos, 1999). However, one study using many chloroplast genes (but relatively few taxa) places the conjugating gren algae sister to land plants (Turmel et al., 2006). Molecular phylogenetic studies suggest that the two traditional families of the Zygnematales, the Zygnemataceae and the Mesotaeniaceae, are not monophyletic with respect to one another (McCourt et al., 1995; McCourt et al., 2000; Gontcharov et al., 2003). It also appears that many of the genera in the traditional family Mesotaeniaceae may not be monophyletic (Gontcharov et al., 2003, 2004). The order Zygnematales may not be monophyletic but rather consist of two lineages in paraphyly, one containing Spirogyra and Sirogonium and the other containing most of the remaining Zygnematales (McCourt et al., 2000; Gontcharov et al., 2003). The lineage that best corresponds to the clasical Desmidiales (Figure 2.2D) almost certainly includes organisms formerly clasified among the Mesotaeniaceae (Park et al., 1996; McCourt et al., 2000; Gontcharov et al., 2003). One study (Gontcharov and Melkonian, 2004) indicates that at least one genus clasified among the conjugating gren algae, Spirotaenia, may not be part of the main line of zygnematophyte evolution. Taxonomy within the Zygnematophyceae has relied heavily on general morphology and fine structure, particularly cel wal structures (Desmidiaceae), or spore 30 wal ornamentation (Zygnemataceae). Molecular phylogenetic methods may prove valuable for infrageneric phylogeny within the group, as indicated by the few studies that investigated species relationships (Denboh et al., 2001; Le, 2001; Nam and Le, 2001; Gontcharov and Melkonian, 2005). However, most of these studies did not test dificult relationships and diferent molecules and methods may be necesary for investigating closely related species. Coleochaetophyceae The Coleochaetophyceae are branched, filamentous epiphytes. Four genera, Coleochaete, Chaetosphaeridium, Chaetotheke and Awadhiela, are thought to belong to this group (Bourely, 1990; Delwiche et al., 2002). The organisms may be epiphytic, endophytic, or loosely atached to submerged vascular plants, Characeae or other suitable substrate, and are occasionaly found fre-floating. Sexual reproduction is oogamous in al species for which sexual reproduction has been described. Zoospores, meiospores and sperm are biflagelate with an MLS and lateral, subapical insertion of the flagela. Thali are composed of compact or loosely branched filaments. Chaetosphaeridium is occasionaly reported as unicelular, though this is true only in early developmental stages and mature plants form filaments (Thompson, 1969) albeit often with widely spaced cels. About 16 species of Coleochaete have been described (Szyma?ska, 2003), and at least 4 species of Chaetosphaeridium (Thompson, 1969). A single species of Awahdiela is known but it is extremely rare and its phylogenetic placement largely speculative. Chaetotheke is more common, but is dificult to recognize and it has received litle study. A number of other genera have been clasified among the 31 Coleochaetophyceae. Although many of these taxa can probably be refered to the Chlorophyta, others may legitimately belong to this lineage (Printz, 1964; Bourrely, 1990). The relationship betwen Coleochaete (Figure 2.2E) and Chaetosphaeridium has been asumed for some time, on the basis of their unusual sheathed hairs and similar chloroplast structure. Some phylogenetic analyses of 18S rDNA sequences indicate that the two may not be closely related, and place Chaetosphaeridium in a monophyletic group with Mesostigma viride at the base of the charophyte tre (Marin and Melkonian, 1999). My own analyses indicate that rDNA data provide weak support for such a placement, but analyses of rbcL, atpB and nad5 consistently show the Coleochaetophyceae as a monophyletic group albeit often with modest bootstrap support (Karol et al., 2001; Cimino and Delwiche, 2002; Delwiche et al., 2002). This position is consistent with morphological and cytological characteristics (Karol et al., 2001; Delwiche et al., 2002). The phylogenetic placement of Awahdiela and Chaetotheke is unknown, although the filament structure, hairs and chloroplasts are similar to those of Coleochaete and Chaetosphaeridium. The most recent treatment of Chaetosphaeridium is that of Thompson (1969), which is not a full monograph. The sparse information on Chaetosphaeridium is probably not because the organism is rare, but because it is easily overlooked and more dificult to isolate than other charophytes. Coleochaete has been the subject of more comprehensive systematic studies. The first monograph listed four species (Pringsheim, 1860) and a number of studies have revised and added to the work (Printz, 1964; Szyma?ska, 1989; Delwiche et al., 2002; Szyma?ska, 2003). Several new species were 32 recently described including a previously unrecognized group characterized by incomplete envelopment of the zygote following fertilization, or incomplete ?cortication? (Szyma?ska, 1988, 1989). Studies of endophytic strains akin to C. nitelarum Jost suggest that much of Coleochaete diversity remains undescribed (Cimino and Delwiche, 2002). Certainly further investigation wil reveal stil more species and, very likely, a greater structural diversity than is currently recognized. Charophyceae sensu stricto (the stoneworts) The stoneworts, or Charophyceae sensu stricto, are the most plant-like in appearance among the charophyte algae: they are macroscopic gren algae with whorls of branches at nodes. Thali may be monoecious or dioecious, depending on the species (Wood and Imahori, 1965; Corilion, 1972). Al species are oogamous with motile sperm produced in complex antheridia. Oogonia and antheridia are surrounded by sterile jacket cels. Fertilized eggs (zygotes) develop a thick covering of sporopollenin. Zygotes and thali may become impregnated with calcium carbonate (Wood and Imahori, 1965). These sporopollenin encrusted spores (caled gyrogonites) are wel preserved in the fossil record, which extends back in exces of 380 hundred milion years (Feist and Grambast- Fesard, 1991). Six genera in a single family represent the extant Characeae. The two most common genera are Chara (Figure 2.2F) and Nitela. Fosil structural diversity is greater than extant diversity, however, and many families are known only from the fossil record. Taxonomy within the group has been greatly afected by Wood and Imahori (1965) who produced the most recent global monograph of the Characeae. 33 In their monograph, Wood and Imahori (1965) divide the family Characeae into two tribes, the Chareae (Chara, Lamprothamnium, Nitelopsis, and Lychnothamnus) and the Niteleae (Nitela and Tolypela). Both sem to be monophyletic (Sanders et al., 2003), or the Nitelae may be paraphyletic (McCourt et al., 1996; McCourt et al., 1999). The genera, although represented by very few species in most analyses, sem to be monophyletic with the possible exception of Chara, which may include Lamprothamnium, based on 18S rDNA sequences and a broad taxon sampling within Chara (Meiers et al., 1999). Systematic investigation within the two largest genera, Chara and Nitela, is wanting. Molecular investigations of Nitela subgenus Tiefallenia suggest that some sections may be artificial and that mesospore membrane fine structure may be a valuable taxonomic characteristic, at least in subgenus Tiefallenia (Sakayama et al., 2004b; Sakayama et al., 2004a; Sakayama et al., 2005). Although Wood and Imahori (1965) reduced many described species to varieties, they also provided a list of the ?species? described at that time, which they termed ?microspecies.? The molecular investigations as wel as other morphological studies sem to favor the ?microspecies? concept (Wood and Imahori, 1965), particularly in the genus Chara (Corilion, 1972; Krause, 1997). Regardles of the treatment of varieties, there are probably a few hundred described, extant stoneworts. Future directions and the role of genomics in charophyte systematics Insights from published genomic data Some charophyte organelar genomic data have been published, including the complete chloroplast genome of Zygnema, Staurastrum (Turmel et al., 2005), Chaetosphaeridium (Turmel et al., 2002a), Chara (Turmel et al., 2006) and Mesostigma 34 (Lemieux et al., 2000), as wel as the mitochondrial genome of Chara (Turmel et al., 2003), Chaetosphaeridium (Turmel et al., 2002a) and Mesostigma (Turmel et al., 2002b). These studies provide insight into the evolution of chloroplast and mitochondrial genomes. In particular, complete organelar genomes provide information about gene content, gene order and trends in evolution of these genomes. The value of these sequences for systematic investigation wil increase as more genome-scale data are collected from other charophyte algae. As of the beginning of 2006, no charophyte nuclear genome has been published (excluding embryophytes). Besides organelar genomes, two expresed sequence tag (EST) surveys have been published. The first investigated the expresed mRNAs of members of the Closterium peracerosum-stigosum-litorale complex (Sekimoto et al., 2003) and related studies identified a pheromone that induces sexual cel division (Fukumoto et al., 2003a; Tsuchikane et al., 2003). The second survey sequenced more than 10,000 ESTs from Mesostigma viride and recovered transcripts of genes important for celular proceses such as translation and transcription, signaling and metabolism, though the majority have unknown functions (Simon et al., 2006). Such studies may provide the phylogenetic data necesary to resolve the branching order of the charophytes. However, it is important to remember the value of this information outside pure systematics. Genomic studies provide valuable information about the biology of these organisms and how their ancestors may have evolved to give rise to the complex metabolic pathways and gene families found in land plants. This information is critical to our understanding and interpretation of evolution within the charophytes as wel as the origin and evolution of land plants. 35 Future systematic investigations As noted above, the branching order of the deeper nodes (clases) remains uncertain. Molecular phylogenetic analyses and continued cytological observations may provide the data needed to answer these questions. Many published molecular datasets are very limited in character or taxon sampling, both of which afect inference of relationships. However, molecular investigations of currently available taxa may not be enough to resolve the branching order of the charophyte tre. Evolution within the lineages wil remain uncertain until more taxa are available for investigation. Within the Zygnematophyceae, for instance, the known structural diversity has yet to be probed. Relationships among orders, families and genera of the conjugating gren algae remain poorly resolved. Fewer than half of the known genera of Zygnematophyceae are available for molecular investigation. Molecular phylogenetic analyses of the Klebsomidiophyceae remain in their early stages and many relationships remain unclear in the Coleochaetophyceae and Characeae. Future studies may depend on a broader sampling of taxa as wel as more sizable molecular datasets. None of the charophyte lineages has been comprehensively surveyed by molecular methods. Species relationships remain poorly understood in al but the Coleochaetophyceae. Published work on the Characeae and Zygnematophyceae has only begun to addres species relationships using molecular methods, and population level studies remain few. Although a number of outstanding morphological, mating and AFLP analyses have been published (Grifin and Proctor, 1964; Grant and Proctor, 1972; 36 Meiers et al., 1999; Mannschreck et al., 2002), very few studies have addresed the structure of charophyte populations using molecular sequence techniques. How populations interact as wel as the distribution of charophyte algae are generaly unknown. Even though there are scatered reports for Characeae and Zygnematophyceae, litle is known about global population status and local surveys suggest that many populations may be severely presured (Krause, 1984; Siemi?ska, 1986; Stewart and Church, 1992; N?meth, 2005; Watanabe, 2005). Besides the ordering and documentation of the known species, systematists are also concerned with the discovery of new species. A number of extensive floristic studies exist, but as is often the case for widespread and taxonomicaly dificult taxa, these are strongly biased by geographic location of investigators and acesibility of study sites. The physicaly smaler species in particular have been poorly documented. It is dificult to imagine that Chlorokybus and Klebsormidium are represented by the relatively few described species particularly since Klebsormidium thrives in a wide range of habitats. A great many species are likely to be hidden in such unlikely places as university fountains and garden wals, as was the case for Chlorokybus (Geitler, 1942b). Another strong bias in current charophyte systematics is the ?microbial bias.? Except for the Characeae, nearly al charophytes used in molecular phylogenetic studies have been cultured from the wild. This almost certainly introduces biases in the investigation analogous to those encountered when surveying Bacteria and Archea by culturing. As a complement to traditional culture-based methods, molecular sequence data provide an independent means of investigating diversity that eliminates the culture bias, though it introduces others. Molecular environmental studies of even wel 37 characterized habitats have received a lot of atention because many new lineages have been discovered (L?pez-Garc?a et al., 2001; Venter et al., 2004). Relating these sequences to the organisms from which they came, however, is dificult. Not just new species, but new kinds of organisms with potentialy diferent life histories and metabolic pathways may yet be found. Systematic investigation of the charophyte gren algae wil continue to provide insight into the diversity and evolution of these exceptional organisms. Future studies wil, hopefully, embrace new techniques and technologies and use them to answer fundamental systematic questions. Much remains to be investigated at al levels and in al charophyte lineages. Land Plants ~300,000 C har oph y c eae ~395 C oleo chaet oph y c eae ~22 Z y gnema t oph y c eae ~4-12,000 K lebsor midioph y c eae ~19 C hlor ok yb oph y c eae 1 M esostigma t oph y c eae 2 Figure 2.1. Phylogeny of charophytes showing species richness. Branching order of the lineages of Charophyta based on Karol et al. 2001. Line width at top of diagram is approximately proportional to the number of described species for the group. Charo- phyceae and the land plants are the only lineages for which there are ancient fossils. These fossils indicate that the Charophyceae were probably more diverse in the past than they are at present. For the other lineages of charophyte algae, their prehistoric diversity is almost completely unknown. A C E B D F Figure 2.2. Light micrographs of representatives of the major lineages of the Charophyta. A. Mesostigma viride , scale bar = 20 um; B. Chlor okybus atmophyticus , scalebar = 10um; C. Klebsormidium flaccidum , scale bar = 25 um; Micrasterias r otata , scale bar = 50 um; E. Coleochaete pulvinata , scalebar = 25 um; F . Chara tomentosa , scale bar = 5 mm. 40 Chapter 3. Phylogeny of the conjugating gren algae based on chloroplast and mitochondrial nucleotide sequence data Abstract The conjugating gren algae are a lineage of charophyte gren algae known for their structural diversity and unusual mode of sexual reproduction, conjugation. These algae are ubiquitous in freshwater environments, where they are often important primary producers, but few studies have investigated evolutionary relationships in a molecular systematic context. A 109 taxon dataset consisting of thre gene fragments (two from the chloroplast and one from the mitochondrial genome) was used to estimate the phylogeny of the genera of the conjugating gren algae. Maximum Likelihood, Parsimony and Bayesian Inference were used to estimate relationships from the 4047 alignable nucleotides. This study confirmed polyphyly of the Zygnemataceae and Mesotaeniaceae with respect to one another. The Peniaceae were found to be paraphyletic, and two genera traditionaly clasified among the Zygnematales appear to belong to the lineage that gave rise to the Desmidiales. Six genera, Euastrum, Cosmarium, Cylindrocystis, Mesotaenium, Spondylosium and Staurodesmus, were found to be polyphyletic. These findings have important implications for the evolution of structural characteristics in the group, and wil require some taxonomic changes. More work wil be required to delineate lineages of Zygnematales in particular, and to identify structural synapomorphies for some of the newly identified clades. 41 Introduction Charophyte gren algae constitute one of the two major lineages of gren algae and include those most closely related to land plants. The charophyte algae are, therefore, critical to our understanding of evolution of land plants and their invasion of the terestrial environment. They are also prominent members of the freshwater microflora, and in some systems make a major contribution to primary productivity. There are six primary clades of charophyte algae (Lewis and McCourt, 2004). Each of these lineages is thought to be ancient, and yet many are represented by very few (1-25) known species. However, one lineage, the conjugating gren algae (Zygnematophyceae), contains the majority of charophyte taxonomic diversity, with several thousand named species. These organisms are remarkable among gren algae for their diversity of cel wal structure and ornamentation. Additionaly, members of this group have been important to the understanding of plant proceses including cel wal morphogenesis and phytochrome signaling (Bock and Haupt 1961, Haupt 1959, 1960, Haupt and Bock 1962, Meindl 1993, Walczak et al. 1990, Winands and Wagner 1996). The conjugating gren algae are among the most common of al freshwater algae, and members of the group can be found in a number of extreme habitats including acid bogs, alkaline streams, desert crusts, and snow. Although widely distributed, the group?s diversity is poorly understood, and many phylogenetic relationships remain untested with molecular methods. The conjugating gren algae (zygnematophytes) include the traditional Zygnemataceae as wel as the ?sacoderm? and ?placoderm? desmids (Mix 1972). These comprise a species-rich and structuraly heterogeneous group clearly embedded within the charophyte gren algae, and united by their unique mode of sexual reproduction, 42 conjugation, and a complete absence of a flagelate stage in the lifecycle. Many species are dificult to identify, especialy the smalest taxa, and some species are diferentiated based on infrequently observed sexual reproductive characteristics. Taxonomic concepts of the families and orders within the conjugating gren algae are based primarily on ultrastructural characteristics. Mix (1972) clasified the conjugating gren algae in two orders and six families based on the structure of their cel wals. The order Zygnematales included conjugating gren algae whose wals are smooth and whose primary wal is indehiscent. Species in this order were clasified in two families, the Mesotaeniaceae (unicelular organisms) and the Zygnemataceae (filamentous organisms). The order Desmidiales included the remaining conjugating gren algae whose wals are interupted by sutures, pores, or both. The Desmidiales were clasified in two suborders and four families: the family Desmidiaceae in the suborder Desmidiineae and the Closteriaceae, Gonatozygaceae and Peniaceae in the suborder Archidesmidiineae (Mix 1972). Zygnematales sem to be homogenous with respect to the construction of their cel wals, but their asignment to families based on growth habit (unicelular vs. filamentous) is artificial (Park et al. 1996, McCourt et al. 1995, McCourt et al. 2000, Gontcharov et al. 2003, 2004). Each of the families of the Desmidiales was characterized by a unique cel wal construction (Mix 1972) While Mix?s (1972) publication is the most recent widely acepted clasification of the conjugating gren algae, there have been a number of revisions proposed. Kouwets and Coesel (1984) proposed that the Gonatozygaceae be merged into the Peniaceae. Various authors have proposed the abandonment of certain higher groups because molecular data suggest that they are not monophyletic; these include the 43 Mesotaeniaceae and Zygnemataceae sensu stricto (McCourt et al. 1995, Park et al. 1996, McCourt et al. 2000, Gerath 2003, Gontcharov et al. 2003, 2004) and the Archidesmidiineae (McCourt et al. 2000, Gontcharov et al. 2004). The few published molecular phylogenetic studies, based on nuclear large-subunit ribosomal data, the chloroplast gene rbcL, or both, have confirmed the monophyly of the group (McCourt et al. 2000, Gontcharov et al. 2003, 2004), with the possible exception of Spirotaenia (Gontcharov and Melkonian 2004). These studies investigated the relationships of the families and genera, but the genera were in most cases represented by only one or a very few exemplar taxa. A relatively wide sampling of species within select genera was presented by Gontcharov et al. (2004), and Gontcharov and Melkonian (2005) used non-coding ITS1-ITS2 to test the relationships among Staurastrum-like species. Drummond et al. (2005) investigated the relationships among species of Spirogyra and Sirogonium and found these two genera to be distinct and each monophyletic, with the exception of Spirogyra maxima UTEX 2495, which was found to be embedded in a clade of Sirogonium. Despite this progres, many generic relationships in the conjugating gren algae remain untested or poorly resolved. The present study used a multi-gene dataset, including two newly developed molecular markers, and dense taxon representation to test the concepts of families and genera of the conjugating gren algae. Because many genera are species rich and structuraly diverse, it was not practical to exhaustively test the monophyly of al the genera, but the relatively fine-grained taxon sampling made it possible to identify some non-monophyletic genera, and confirm the likely monophyly of others. 44 Materials and Methods Strains Strains used in this study were obtained from public culture collections or isolated from the wild (se Table 3.1). Strains were maintained in liquid media; either Guilard?s Woods Hole Medium, Bold?s Basal Medium (Nichols 1973), or Bold?s Basal Medium enriched with 20 mL?L -1 of soil extract. Cels grew in 25 mL flasks in a Percival growth chamber (Percival, Pery, IA) with a light: dark cycle of 16:8 hrs under Sylvania Cool White fluorescent lamps (Danvers, MA). Cels were observed at time of acesioning and extraction for contamination. Al strains were identified morphologicaly with light microscopy; in several cases the diagnosis was diferent from the name atached to the strain in culture collection records (Table 3.1). Corrected names are provided in Table 3.1, with the culture name in brackets. Other sampled taxa have been asigned revised names as proposed by other authors. Synonomy is provided in parentheses in Table 3.1. 45 Table 3.1 Strains investigated and GenBank numbers Taxon Strain rbcL psaA coxII Zygnematophyceae Desmidiales Desmidiaceae Actinotaenium (Penium) silvae-nigrae (Rabanus) Kouwets and Coesel SVCK 295 EF371323 EF37123 EF37116 Actinotaenium cucurbita (Br?bison) Teiling ex Ruzicka & Pouzar JH0383 ----- EF37173 EF37106 Actinotaenium curcurbitinum (Biset) Teiling ACOI 901 EF371279 EF37172 EF371065 Bambusina boreri (Ralfs) Cleve JH019 EF371283 EF3717 EF371070 Bambusina boreri (Ralfs) Cleve JH0125 EF371284 EF37178 EF371071 Cosmarium botrytis (Meneghini) Ralfs UTEX 301 EF37128 EF37182 EF371075 Cosmarium sp. JH041 EF371290 EF37184 EF37107 Cosmarium pseudoconatum Nordstedt JH0264 EF371280 EF37174 EF371067 Cosmarium elanosporum Archer JH01 EF371289 EF37183 EF371076 Cosmocladium saxonicum Hilse ACOI 95 EF371292 EF37186 EF371079 Desmidium aptogonum var. ehrenbergi K?tzing JH0184 EF371298 EF37192 EF371085 Desmidium baileyi (Ralfs) Nordstedt JH028 EF37129 EF37193 EF371086 Desmidium swartzi Agardh ex Ralfs JH042 EF371297 EF37191 EF371084 Euastrum intermedium Gay JH0159 EF371302 EF37196 EF371089 Euastrum crasum var. michiganense Prescot JH018 EF37130 EF37194 EF371087 Euastrum verucosum Ehrenberg ex Ralfs JH068 EF371301 EF37195 EF37108 Haplotaenium (Pleurotaenium) minutum (Ralfs) Bando SVCK 302 EF371326 EF37127 EF37120 Heimansia (Cosmocladium) pusilum (Hilse) Coesel SVCK 428 EF371291 EF37185 EF371078 Hyalotheca disiliens Br?bison ex Ralfs SAG 384-2 AF20349 EF371202 EF371095 Hyalotheca mucosa (Mertens) Ehrenberg ex Ralfs JH05 EF371305 EF371203 EF371096 Hyalotheca mucosa (Mertens) Ehrenberg ex Ralfs JH063 EF371306 EF371204 EF371097 Micrasterias foliacea Bailey NIES 297 EF37131 EF371209 EF37102 Micrasterias radiata Hasal JH064 EF371313 EF37121 EF37104 Micrasterias rotata (Grevile) Ralfs UTEX 1941 EF371312 EF371210 EF37103 Onychonema laeve var. micracanthum Nordstedt JH0198 EF371318 EF371218 EF3711 Onychonema laeve var. micracanthum Nordstedt JH026 EF371319 EF371219 EF37112 Phymatodocis nordstedtiana Wole SAG 47.89 AJ53962 EF37125 EF37118 Phymatodocis nordstedtiana ole JH0164 EF371325 EF37126 EF37119 Pleurotaenium baculoides (Roy & Biset) Playfair JH008 EF371327 EF37128 EF37121 Pleurotaenium constrictum (Bailey) Wod JH0135 EF371328 EF37129 EF3712 Pleurotaenium ehrenbergi f. columelare Ir?n?e- Marie JH031 EF371329 EF371230 EF37123 Spondylosium tetragonum West JH0175 EF371304 ----- ----- Spondylosium tetragonum est JH0281 EF37136 EF371239 ----- Spondylosium tetragonum West [Groenbladia undulata] SVCK 40 AF203498 EF371201 EF371094 Spondylosium pulchelum Archer SVCK 365 AF203505 EF371245 EF37137 Spondylosium pulchrum (Bailey ex Ralfs) Archer JH0269 EF371341 EF371246 EF37138 Staurastrum tetracerum (K?tzing) Ralfs [Arthrodesmus sp.] UTC 348 EF371282 EF37176 EF371069 46 Staurastrum arctiscon (Ehrenberg) Lundel JH014 EF371343 EF371248 EF37140 Staurastrum arctiscon (Ehrenberg) Lundel JH070 EF371346 EF371251 EF37143 Staurastrum pseudosuecicum Prescot and Scot JH010 EF371342 EF371247 EF37139 Staurastrum polytrichum f. biseriatum Kaiser JH015 EF37134 EF371249 EF37141 Staurastrum gladiosum Turner JH0132 EF371347 EF371252 EF3714 Staurstrum brevispinum Br?bison ex Ralfs JH0180 EF371349 EF371254 EF37146 Staurastrum polymorphum Br?bison JH053 EF371345 EF371250 EF37142 Staurodesmus convergens (Ehrenberg) Teiling [Arthrodesmus sp.] UTEX 2508 EF371281 EF37175 EF371068 Staurodesmus extensus (Anders.) Teiling JH0386 EF371317 EF371217 EF37110 Staurodesmus mamilatus (Nordstedt) Teiling JH090 EF371348 EF371253 EF37145 Teilingia (Sphaerozosma) granulata (Roy and Biset) Bourely SAG 39.83 EF37135 EF371237 EF37130 Teilingia granulata (Roy and Biset) Bourely JH0140 EF371351 EF37125 EF37148 Teilingia granulata (Roy and Biset) Bourely SAG 25.8 EF371350 EF371256 EF37147 Teilingia granulata [Sphaerozosma sp.] (Roy and Biset) Bourely UTC 284 AF203504 EF371238 EF37131 Tetmemorus brebisoni (Meneghini) Ralfs SVCK 409 EF371352 EF371257 EF37149 Tetmemorus laevis (K?tzing) Ralfs SVCK 27 EF371353 EF371258 EF37150 Triploceras gracile Bailey SAG 24.82 EF371354 EF371259 EF37151 Xanthidium antilopaeum (Br?b. Ex Meneghini) K?tzing JH0261 EF37135 EF371261 EF37153 Xanthidium hastiferum Turner JH054 EF378638 EF371260 EF37152 Peniaceae Penium cf. didymocarpum Lundel JH0212 EF371316 EF371216 EF37109 Penium cylindrus (Ehrenberg) Br?bison ex Ralfs ACOI 780 EF371320 EF37120 EF37113 Penium argaritaceum (Ehrenberg) Br?bison ex Ralfs UTEX 60 EF371321 EF37121 EF37114 Penium argaritaceum (Ehrenberg) Br?bison ex Ralfs ACOI 30 EF37132 EF37122 EF37115 Penium spirostriolatum Barker SVCK 205 EF371324 EF37124 EF37117 Closteriaceae Closterium acerosum (Schrank) Ehrenberg ex Ralfs UTEX 1075 EF371285 EF37179 EF371072 Closterium ehrenbergi var. malinvernianum (De Notaris) Rabenhorst JH013 EF371286 EF37180 EF371073 Closterium libelula Focke JH021 EF371287 EF37181 EF371074 Spinoclosterium cuspidatum (Bailey) Hirano NIES 325 AJ53965 EF371240 EF37132 Gonatozygaceae Gonatozygon pilosum Wole ACOI 1096 EF371303 EF37120 EF371093 Gonatozygon kinahani (Archer) Rabenhorst ACOI 350 AJ53945 EF37198 EF371091 Gonatozygon brebisoni var. laeve (Hilse) West & West JH03 EF37132 EF37123 EF37126 Genicularia spirotaeniae de Bary SAG 54.86 AJ53946 EF37197 EF371090 Gonatozygon monotaenium de Bary UTEX 1253 U71438 EF3719 EF371092 Zygnematales Mesotaeniaceae Cylindrocystis brebisoni Meneghini UTEX 1259 EF371293 EF37187 EF371080 47 Cylindrocystis crasa de Bary ACOI 310 EF371294 EF3718 EF371081 Cylindrocystis sp. UTEX 1925 EF371295 EF37189 EF371082 Cylindrocystis sp. JH038 EF371296 EF37190 EF371083 Mesotaenium caldariorum (Lagerheim) Hansgirg UTEX 41 EF371307 EF371205 EF371098 Mesotaenium endlicharianum N?geli ACOI 451 EF371308 EF371206 EF37109 Mesotaenium kramstai Lemerman UTEX 1024 EF371309 EF371207 EF3710 Mesotaenium sp. JH031 EF371310 EF371208 EF37101 Netrium digitus (Ehrenberg) Itzigson & Rothe UTEX 561 U38698 EF371214 EF37107 Netrium digitus var. lamelosum (Br?bison) Gr?nblad UTEX 125 EF371315 EF371215 EF37108 Roya anglica West UTEX 934 AJ53963 EF371231 EF37124 Roya obtusa (Br?bison) West & West SAG 168.80 EF37131 EF371232 EF37125 Zygnemataceae Mougeotia sp. UTEX 758 AF408252 EF371212 EF37105 Mougeotia sp. JH040 EF371314 EF371213 EF37106 Mougeotia sp. JH0304 EF371364 EF371270 EF37163 Sirogonium elanosporum (Randhawa) Transeau ARL 70 L13484 EF371234 EF37127 Sirogonium sp. JH082 DQ015929 EF371236 EF37129 Sirogonium sticticum (Engler) K?tzing UTEX 1985 DQ015924 EF371235 EF37128 Spirogyra grevileana (Hasal) K?tzing UTEX 47 DQ015938 EF371243 EF37135 Spirogyra condensata (Vaucher) K?tzing UTEX 174 DQ015936 EF371242 EF37134 Spirogyra gracilis (Hasal) K?tzing UTEX 1743 DQ015937 EF371241 EF3713 Spirogyra maxima (Hasal) Witrock UTEX 2495 DQ15941 EF37124 EF37136 Zygnema circumcarinatum Czurda UTEX 42 EF371356 ----- EF37154 Zygnema cylindricum Transeau SAG 689-2 EF371357 EF371262 EF3715 Zygnema peliosporum Witrock UTEX 45 U38701 EF371263 EF37156 Zygnema sp. JH039 EF371360 EF371265 EF37158 Zygnema sp. JH087 EF371362 EF371267 EF37160 Zygnema sp. JH049 EF371361 EF37126 EF37159 Zygnema sp. JH007 EF371359 EF371264 EF37157 Zygnemopsis minuta Randhawa ACOI 60 EF371363 EF371268 EF37161 Zygogonium tunetanum Gauthier-Li?vre UTC 136 AF203509 EF371269 EF37162 Outgroup Coleocheatales Coleochaete scutata Br?bison SAG 3.9 AY082324.1 EF371273 EF3716 Coleochaete nitelarum Jost UTEX 1261 AY082325.1 EF371275 EF37168 Coleochaete divergens Pringsheim 30d1 AY082324.1 EF371272 EF37165 Coleochaete pulvinata Braun ex K?tzing 57b6 AY082307.1 EF371274 EF37167 Coleochaete sieminskiana Szymanska 10d1 AF408791.1 EF37127 EF37170 Chaetosphaeridium globosum (Nordstedt) Klebahn SAG 26.98 AF408792.1 EF371276 EF37169 Chlorokybales Chlorokybus atmophyiticus Geitler UTEX AF40805.1 EF371271 EF37164 48 2591 Mesostignematales Mesostigma viride Lauterborn SAG 50-1 AF40806.1 EF371278 EF37171 Notes: ACOI, Coimbra Collection of Algae; NIES, National Institute for Environmental Studies; SAG, Samlung von Algenkulturen der Universit?t G?ttingen; SVCK, Samlung von Conjugaten-Kulturen; UTC, University of Toronto Culture Collection of Algae and Cyanobacteria; UTEX, Culture Collection of Algae at University of Texas. Vouchers of strains beginning with JH are available from the authors. Sequences generated for this study are in bold. Table 3.2. Primers used for PCR amplification of fragments psaA 2100R GCATCACCAGAG psaA IF TCTTGCTCATGATC psaA 569F GGCTGTCATCAATCATGTGTC coxII 5'ZYG GCGTGTATGTACATGCA coxII 3'G CAGCTGCTCAAGCAAGTGA 49 DNA extraction and fragment amplification DNA was extracted using the Nucleon Phytopure DNA extraction kit (Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer?s protocol with an additional chloroform separation. Thre genes were chosen (chloroplast ribulose-1, 5- bisphosphate carboxylase/oxygenase large subunit (rbcL) and photosystem I P700 chlorophyll A apoprotein A1 (psaA) and mitochondrial cytochrome oxidase subunit II (coxII) to infer the phylogenetic relationships. Portions of the chloroplast gene rbcL were amplified using published primers (McCourt et al. 2000). Other gene fragments were amplified by PCR using the primers listed in Table 3.2. Al genes were amplified on a Whatman Biometra (G?ttingen, Germany) T-Gradient thermal cycler. These primers, and other taxon specific internal primers, were used to sequence the fragments of the rbcL, psaA and coxII genes, on an Applied Biosystems AB 3100 or AB 3730 capilary sequencer using Big Dye Terminator sequencing chemistry (Applied Biosystems, Foster City, California). Fragments were sequenced in both directions and asembled using Sequencher 4.2 (GeneCodes, Ann Arbor, MI). Phylogenetic analyses Sequences were aligned using MacClade 4.07 (Maddison and Maddison 2000), and phylogenetic analyses performed in PAUP* version 4b.10 (Swofford 2003), phyML (Guindon and Gascuel 2003) and MrBayes 3.1 (Huelsenbeck and Ronquist 2000). Portions of the alignment were excluded from the analyses because of indels or mising data: positions 1753-1758, 1969-1992, 2953-2958, and 3226-3228 of the multi-gene 50 alignment. Genes were aligned individualy and analyzed under Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) (data not shown). Homogeneity of base frequency among taxa was tested using a X 2 test as implemented in in PAUP*. Each of the genes was found to be homogenous among taxa (data not shown). Estimated topologies were generaly consistent among the thre genes, although none confidently resolved deeper nodes (data not shown). Gene fragments were concatenated into a single dataset. This concatenated dataset was analyzed with and without third codon position. Phylogenetic tres were estimated under the MP and ML criteria in PAUP* as wel as using Bayesian Inference (BI) in MrBayes. Non-parametric bootstrap (BS) under MP and ML and posterior probability (P) in BI were used as estimates of support for taxon bipartitions. Ten random sequence addition replications and tre bisection-reconnection (TBR) branch swapping were used during the heuristic search under the Parsimony criterion; support was estimated from 500 BS replications with TBR branch swapping and ten random addition sequence repetitions. Under the Maximum Likelihood criterion I searched for the best tre using thre random addition sequence replicates. Support was estimated using 100 BS replicates with a single random addition sequence and TBR branch swapping. I used the GTR + I + ? model, with al parameters estimated from the data as recommended by Mrodeltest 2.2 (Nylander 2004). BS analyses were performed in PhyML. Two independent analyses using Metropolis-coupled Markov Chain Monte Carlo (MCMCMC) methods in MrBayes were used with four chains sampled for four milion generations to estimate topology, parameter values, and the posterior probabilities of the 51 taxon bipartitions. The chains were sampled every one hundred generations and the first two thousand samples discarded as burn-in. Both analyses converged on similar topologies. Individual genes were analyzed similarly but with 100 MP and ML BS pseudoreplicates and two milion generations under BI. Because the best tres were inconsistent with current taxonomic concepts, a number of alternative topologies were explicitly tested. To reduce computation time, the 109 taxon dataset was sampled to include 55 taxa, including al Zygnematales and a subset of the Desmidiaceae. Tre files were edited manualy to constrain regions of interest in the tre. These files were then used as constraints and the ML tre given those constraints were estimated. To save time when estimating the most likely alternative tres, the outgroup genus Coleochaete was constrained as monophyletic, as were Zygnema spp.. The positions of al other taxa were alowed to vary. These topologies were tested using the Approximately Unbiased (AU) test (Shimodaira, 2002), in Consel v. 0.1h (Shimodaira and Hasegawa, 2001). Results Taxa When preparing for DNA extraction, it was found that several strains from culture collections were contaminated or misidentified. Contaminated strains were excluded from the study and those misidentified, when possible, were asigned to the correct genus and species. This resulted in the absence in this analysis of some genera and several strains that were included in previous studies. No strain consistent with the morphological description of Sphaerozosma or Groenbladia was available for this study 52 (se discussion). Arthrodesmus sp. UTC 348 is here identified as Staurastrum tetracerum. Multigene phylogeny This study included more taxa (109) and more nucleotide characters (4047) than any previous analysis of the conjugating gren algae. In broad terms the analyses were consistent with those based on rbcL and SU rDNA. Use of multiple genes from two organelar genomes permited relatively confident inference of branching order of the major lineages of the Desmidiales. Early branching events in the Zygnematales remains poorly resolved. Topologies from analysis of individual genes were consistent with that of the combined dataset although no individual gene tre resolved deeper nodes with confidence (data not shown). By combining the genes into a single dataset, I wase able to more confidently estimate many relationships among the taxa. Although this study was not designed to test the monophyly of the conjugating gren algae and excluded some taxa that are important to that question (i.e. Klebsormidiales and Spirotaenia spp.), the conjugating gren algae included in this study formed a monophyletic clade with high support (100/100/1.0; MP/ML/P). Spirogyra and Sirogonium spp. formed a monophyletic clade (100/100/1.0) that was the sister taxon to the remainder of the conjugating gren algae, diverging before the separation of the Desmidiales and the other Zygnematales, however Spirogyra was not monophyletic with respect to Sirogonium, even with the asumption that Spirogyra maxima is a Sirogonium sp. (Figure 3.1). However, Spirogyra and Sirogonium together formed a monophyletic group (100/100/1.0). 53 A second major lineage of zygnematalean taxa, including most of the remaining genera of the Zygnematales, except Netrium spp. and Roya spp., was resolved, but with litle support (-/73/1.0). Within this lineage, several strains of Zygnema form a wel- supported (100/100/1.0) monophyletic clade. One species, Zygnema circumcarinatum shares 99.7% sequence identity with Zygogonium tunetanum. The significance of this relationship is unclear because neither strain could be confidently identified (se discussion). Although some strains of Mesotaenium sem to be very divergent, one strain, M. caldariorum, was consistently resolved as sister to Mougeotia (97/100/1.0). Additionaly, a clade containing both unicelular and filamentous taxa, including Zygnemopsis minuta, Mesotaenium kramstai and two strains of Cylindrocystis, was found (100/100/1.0). This clade appears to be equivalent to the MZC clade as described by Gontcharov et al. (2004). Netrium digitus, clasicaly part of the Zygnematales, was found to be sister to taxa traditionaly clasified among the Desmidiales. Within the lineage most consistent with the concept of the Desmidiales, most families were monophyletic: Gonatozygaceae (100/100/1.0), Closteriaceae (100/100/1.0) and Desmidiaceae (85/90/1.0) (Figure 3.1). Penium formed two paraphyletic lineages sister to the Desmidiaceae; one containing the taxa P. cylindricum and P. cf. didymocarpum, and the other P. margaritaceum and P. spirostriolatum. Monophyly of the Peniaceae was tested explicitly and found to be inconsistent with the present data based on an AU test (p = 3e -3 ). Within the Gonatozygaceae, Genicularia spirotaenia was always embedded among species of Gonatozygon, usualy sister to G. pilosum (100/99/1.0) (Figure 3.1). Placement of Roya spp., which may or may not be part of the Gonatozygaceae (Gontcharov et al. 2004), 54 remains unclear but are supported as members of a lineage including most Desmidiales (95/100/1.0). When included in likelihood analyses of al nucleotide positions, Roya spp. appeared sister to a clade of Gonatozygon spp. (-/-/0.91). In every analysis, Phymatodocis nordstedtiana Wolle was sister to al other Desmidiaceae (Figure 3.2). When using al characters, Actinotaenium spp. formed a monophyletic clade diverging after P. nordstedtiana but before al other Desmidiaceae (Figure 3.2). These remaining Desmidiaceae comprised two large monophyletic groups: one contained the filamentous and colonial genera (except Phymatodocis), and the other contained al unicelular taxa (except Actinotaenium spp.). This topology contrasts with some other published phylogenies (se discussion). Among the filamentous desmids, two lineages were resolved, one containing Spondylosium tetragonum, Teilingia granulata, and Cosmocladium saxonicum (100/100/1.0), and the other containing Spondylosium pulchelum, S. pulchrum, Desmidium spp, Bambusina borreri, Hyalotheca spp., Heimansia pusilum and Onychonema leave var. micracanthum (99/100/1.0) (Figure 3.2). Among unicelular Desmidiaceae, Haplotaenium minutum (previously Pleurotaenium minutum) did not group with Pleurotaenium spp., supporting the taxonomic separation of these two taxa based on ultrastructural characteristics (Figure 3.2). Triploceras gracile was found embedded among species of Micrasterias with high support (99/100/1.0). This result was unexpected because these two taxa are structuraly disimilar. Although most analytical methods resulted in similar topologies, some inferences were specific to a particular dataset, analytical method or model parameters. ML analysis resolved Haplotaenium minutum sister to other unicelular taxa, but the MP 55 analysis found this species embedded among Staurastrum spp. with moderate (BS = 78) support. The MP tre also showed Mesotaenium endlicherianum and M. sp. JH0031 sister to Netrium. While testing monophyly of genera was not the central intent of this study, the data refute the monophyly of some genera. In particular, Cylindrocystis spp., and Mesotaenium spp. occurred in two diferent clades, as did Cosmarium spp., Euastrum spp. Spondylosium spp. and Staurodesmus spp.. These results appear to be robust because they were found regardles of analytical method or dataset. Individual gene analyses Before concatenation, individual genes were analyzed separately to determine their utility for phylogenetic investigation and their combinability. Because of the number of taxa included, the short alignments from individual genes were generaly poorly resolved and the deeper nodes received litle statistical support. Some diferences among the datasets were discovered. The gene rbcL (base frequency A=0.28486 C=0.18748 G=0.22378 T=0.30388; length=1353 bp) had 656 variable sites, of which 568 were parsimony informative. The topology of the most likely tre was similar to that of the combined analysis. The major diference was that Mesotaenium enlicherianum was found outgroup to al other conjugating gren algae, but without support for the placement (-/-/0.65), and Penium was present in thre paraphyletic lineages, as opposed to the two found in the analysis of the concatenated dataset (data not shown). In the rbcL analysis, Roya was found sister to Gonatozygon, although this relationship was weakly supported (-/-/0.80). 56 The gene psaA (base frequency A=0.318557 C=0.124769 G=0.084047 T=0.472627; length=2118bp) was nearly twice as long as the rbcL sequence fragment and included two indels that were excluded from analyses. Of the 2073 included characters, 1052 were variable and 947 were parsimony informative. The topology of the most likely tre was very similar to that of the combined analysis, the diference being that Penium was placed into thre rather than two paraphyletic lineages with low statistical support (64/66/0.94 for the sister relationship betwen Penium cylindrus and the Desmidiaceae). Additionaly, Roya spp were found sister to the clade containing Closteriaceae, Peniaceae and Desmidiaceae (69/72/-). The mitochondrial gene coxII (base frequency A=0.309707 C=0.131077 G=0.126975 T=0.432241; length=615bp) was the shortest gene included and had a single indel region, which was excluded from analyses. Of the 609 included nucleotides, 370 were variable and 334 were parsimony informative. The most likely tre estimated from this dataset was unlike that of the other two genes in that the deeper nodes were entirely unresolved including the separation from the outgroup taxa Coleochaete and Chaetosphaeridium. However, the midlevel relations were resolved with generaly high bootstrap support (data not shown). Some species relationships, particularly among the Desmidiaceae, could not be distinguished based on their coxII sequence. Although the deeper nodes were unresolved, the relationships that were resolved were consistent with the topologies found in the psaA, rbcL and combined analyses. Permutations of dataset When third codon positions were removed from the combined dataset, support at 57 deeper nodes declined, and relationships among genera of the Desmidiaceae were almost entirely unresolved. In ML analyses Spirogyra and Sirogonium were part of a monophyletic clade including most zygnematalean taxa that excluded Netrium and Mesotaenium endlicherianum. Mesotaenium endlicherianum was sister to al other conjugating gren algae, and did not group with other Mesotaenium spp.. One noteworthy result of the exclusion of third position was that the filamentous lineage of Desmidiaceae, as previously described, was dismantled: the lineage containing Teilingia and Spondylosium tetragonum was sister to Actinotaenium, albeit with litle statistical support (-/-/1.0). Topology tests Many of the relationships found are inconsistent with traditional clasification. To test specific hypotheses of monophyly ? particularly those posed by traditional clasifications ? constraint analyses were performed under the Likelihood criterion. The constrained nodes are listed in Table 3.3. These tres and the site-wise likelihoods were used to test if tres consistent with particular phylogenetic hypotheses were significantly worse than the best tre using the AU test. Thre of the tested topologies were rejected by the data based on the AU test: monophyletic Penium (and Peniaceae) (AU= 3e -4 ), monophyletic Mougeotia and Mesotaenium (AU= 0.002), and monophyletic Cylindrocystis (AU= 3e -8 ) (Table 3.3). 58 Table 3.3 Constraints used in AU test Constraint AU KH SH Monophyletic Cylindrocystis 0.00003 0.00 0.00 Monophyletic Gonatozygon and Roya 0.496 0.402 0.959 onophyletic Mougeotia and Mesotaenium excluding M. kramstai 0.089 0.08 0.285 Monophyletic Mougeotia and Mesotaenium including M. kramstai 0.02 0.01 0.05 Monophyletic Penium 3E-04 0.02 0.139 Monophyletic Spirogyra 0.592 0.482 0.953 Monophyletic Zygnema 0.617 0.518 0.967 Monophyletic Zygnematales incuding Netrium 0.404 0.391 0.831 Monophyletic Zygnematales w/o Netrium 0.30 0.29 0.939 onophyletic Zygnematales without Netrium or Spirogyra 0.517 0.376 0.970 59 Discusion Taxa This study aimed to include al available genera of conjugating gren algae and to broadly sample the structural diversity within these genera. Because of the great morphological diversity within the conjugating gren algae, this is a chalenging task. It was also complicated by some of the chalenges inherent in maintaining large culture collections. Five strains from culture collections were contaminated, and four others were asigned names inconsistent with the species description. In most cases misidentified taxa had been asigned to the wrong genus. Consequently it was necesary to carefully identify the organisms prior to DNA extraction. This should not be interpreted as a criticism of culture collections, which often have to struggle with the complexities of maintaining a bewildering range of organisms on a minimal budget, but is a caution to al who make use of such culture collections. In this case, using the original strain designation would have strongly afected my interpretation of the relationships among the genera and caused us to eroneous conclude that many genera were not monophyletic. There are several possible explanations for these misidentified strains, each of which caries its own implications. It may be that cultures were inadvertently mixed or contaminated by other strains in the collection during maintenance. It is also possible that the strains were misidentified at the time of submision. Without aces to voucher material from the original isolates of many of these strains, it not feasible to determine the source of eror with certainty. This emphasizes the importance of vouchering and identifying material at the time of analysis, even if it is available in culture collections. 60 Although eforts were made to identify every strain, it was not possible to do so with several of the filamentous Zygnematales. Taxonomy among these taxa is based on gametangial and zygospore characteristics. Most of these strains could not be induced to go through a sexual cycle. In particular, Zygogonium tunetanum was found to difer very litle in primary sequence from Zygnema circumcarinatum in al genes sampled. The identity of neither strain could be verified, and the identification used here follows that of the culture collection. Zygnema and Zygogonium have been considered synonymous (Czurda 1932), but some authors separate the genera based on the shape of the chloroplasts (Transeau 1951). Both strains in this study have chloroplasts consistent with the genus Zygnema. More strains of Zygogonium, preferably freshly isolated and matched with vouchered field material, wil be needed to establish the relationship betwen these genera. No strains consistent with Sphaerozosma or Groenbladia were included in this study. Although several strains in culture collections are listed as Sphaerozosma, these strains are more consistent with Teilingia: the cels are relatively rectangular, lack apical proceses and often have granules at the angles of the hemicels. Spondylosium tetragonum [Groenbladia undulata] SVCK 440 can be easily distinguished from Groenbladia both by the shape of the cel (which is more constricted than in G. undulata) and by the shape of the chloroplast. Groenbladia spp. have one or two axile taeniform chloroplasts while strain SVCK 440 has a lobed chloroplast similar to other species of Spondylosium. Additionaly, Staurastrum tetracerum [Arthrodesmus sp.] UTC 348 cels have smal proceses at the angles that are inconsistent with Arthrodesmus (now Octacanthium or Staurodesmus). 61 Multigene phylogeny Because individual gene phylogenies were not incongruent with one another, the thre genes were concatenated into a single dataset of over 4000 bp. This is a large dataset and two of the thre markers have not previously been used for this lineage (coxII and psaA) although they have been used to estimate relationships in other algal groups. Single gene phylogenies, while topologicaly similar, had poor bootstrap proportions and low Bayesian posterior probabilities for several key branches. By combining these datasets, I was able to increase support for the resulting topology. Combining the datasets, while improving support across most of the tre compared to individual gene analyses, did not result in full support for al nodes in the phylogeny. In particular, the deepest branches were not highly supported. In several cases, relationships with high posterior probabilities were not supported in bootstrap analysis. This may be because posterior probabilities tend to be somewhat of an overestimate for support (Douady et al., 2003; Simons et al., 2004; Lewis et al., 2005). In the coxII dataset, members of the Gonatozygaceae and Roya spp. were found to be somewhat divergent, however their positions were not strongly supported in bootstrap analyses (data not shown). Organisms clasified in the Zygnematales, such as Roya, are characterized by smooth cel wals with an indehiscent primary wal. This character sems to be pleisiomorphic, and is found in several distinct lineages. Historicaly, the filamentous (Zynemataceae) and unicelular (Mesotaeniaceae) species with smooth wals were clasified separately. Members of these two families are polyphyletic (McCourt et al. 1995, Gontcharov et al. 2003), and unicelular and filamentous organisms interdigitate 62 in the phylogeny (Figure 3.1). These data suggest a number of transitions in growth form among these organisms. Asuming that the molecular phylogeny is corect, new morphological characters wil need to found to permit morphological clasification within the Zygnematales. Chloroplast shape is one possibility (McCourt et al. 1995). Several lineages have similar chloroplast shapes: Mesotaenium and Mougeotia; Zygnema and Zygogonium; Cylindrocystis and Zygnemopsis; and Spirogyra and Sirogonium. The consistent placement of Spirogyra and Sirogonium as the basal-most lineage of the conjugating gren algae is important and implies a unique status for these organisms. Unlike most conjugating gren algae, which with a few exceptions have chloroplasts based in the center of the cel, Spirogyra and Sirogonium have parietal chloroplasts. A second lineage where chloroplast shape appears to be informative is in the Desmidiales, including Netrium. In this clade, al the basal lineages are characterized by ridged central chloroplasts. Exceptions include apparently derived characteristics, such as the reduced lobes of Gonatozygon and the highly lobed and disected chloroplasts of some Desmidiaceae. It also true that some species of Spirotaenia have a similar chloroplast, but placement of these taxa in the conjugating gren algae is uncertain (Gontcharov & Melkonian 2004). The placement of Spirogyra and Sirogonium as one of the earliest diverging lineages of conjugating gren algae was observed in 18S rDNA analyses, but in that study their position was unresolved (Gontcharov et al. 2003). Spirogyra is not monophyletic with respect to Sirogonium, which difers from one previous study (Drummond et al. 2005), but is consistent with others (Gontcharov et al. 2003, Kim et al. 63 2006). The monophyly of Spirogyra, with the caveat that Spirogyra maxima is a Sirogonium, could not be rejected by these data based on the AU test (p = 0.592). It is not clear what is responsible for the diferent phylogenies discovered in diferent studies, but taxon sampling for Spirogyra and Sirogonium did vary widely among the studies. The remaining Zygnematales (except Netrium spp. and Roya spp.) form a monophyletic clade with varying support (-/73/1.0). Two single-genus lineages are consistently found, one containing Mesotaenium endlicherianum and M. sp. JH0031 and the other containing Cylindrocystis crasa Bary and C. brebisonii Meneghini, although species of both Mesotaenium and Cylindrocystis occur in other clades as wel (Figure 3.1). The monophyly of Cylindrocystis could be rejected by my data based on the results of an AU test (p = 3e -8 ). A grouping of Mougeotia and al Mesotaenium strains was also rejected (p = 0.002). Besides the Spirogyra clade, thre strongly supported zygnematalean clades were resolved. The first contains Zygnema spp and Zygogonium tunetanum (100/100/1.0). The second lineage includes Mougeotia spp. and Mesotaenium caldariorum (97/100/1.0). A relationship betwen Mougeotia and Mesotaenium would be predicted based on their structural and cytological characteristics: both have axile taeniform chloroplasts and both have similar cel wal structure (Brook 1982). The third lineage contains a mix of taxa including the filamentous Zygnemopsis minuta and the unicelular Cylindrocystis sp. UTEX, C. sp. JH0038 and Mesotaenium kramstai (100/100/1.0). Similar relationships were resolved using 18S rDNA and rbcL sequences (Gontcharov et al. 2004). Although these sem unlikely at first, it may be that there are characteristics that unite these taxa that have been overlooked. Additionaly, the Zygnematales are not monophyletic in analyses that use al codon 64 positions, but are monophyletic (with the exclusion of Roya spp., Netrium spp., and Mesotaenium endlicherianum) when the third codon-positions are removed, but with litle support (-/51/0.96). Monophyly of the Zygnematales could not be rejected by these data based on the AU test (p = 0.404) regardles of the exclusion of Netrium and Roya spp. (p = 0.330) or Netrium, Roya and Spirogyra (p = 0.517). The Zygnematales clearly have a complex evolutionary history in their own right, but the majority of described species of conjugating gren algae belongs to the Desmidiales. This later order is monophyletic with high support (95/100/1.0) (Figure 3.1). The outgroup to the Desmidiales and distinct from other Zygnematales, albeit with low BS support (-/-/0.99), is a lineage containing Netrium digitus and N. digitus var. lamelosum. Within the Desmidiales, the branching order of the families is generaly wel supported, although the placement of Roya is uncertain, and the families are monophyletic, with the exception of the Peniaceae. In this clade, the first lineage to branch includes species of Gonatozygon and Genicularia spirotaenia SAG 54.86. Genicularia spirotaenia is sister to Gonatozygon pilosum (100/99/1.0). Previous studies have suggested that this relationship necesitates the abandonment of the genus Genicularia (Gontcharov et al. 2003). The genus Genicularia is characterized by parietal spiraling chloroplasts (Bary 1848). However, this strain has what appears to be an axile chloroplast that is either complete or reticulate (Hal unpublished data), a condition not previously reported for the Gonatozygaceae. It is not clear if this strain is typical of G. spirotaenia or is an atypical strain of Gonatozygon; twisted axile chloroplasts has been reported for several species of Gonatozygon (Bary 1848), and further sampling of 65 Gonatozygon wil be needed to clarify the situation. The Peniaceae, comprising solely the genus Penium, is the only desmidialean family that appears to be non-monophyletic (Gontcharov et al. 2004; this study). In this study, these taxa form two paraphyletic clades. The first contains Penium margaritaceum and P. spirostriolatum; the second P. cylindrus and P. cf. didymocarpum. Both P. margaritaceum and P. spirostriolatum are elongate cels with pseudo-girdle bands (reminiscent of the Closteriaceae) and have thickened outer wals with linear ornamentation. The other lineage contains two species with les elongate cels and les conspicuous ornamentation. These results are consistent with those found by Gontcharov et al. (2004). The diagnosis of Penium spp. here is tentative, as I did not perform the TEM needed for unequivocal identification. These taxa warant further morphological study and denser taxon sampling. The Desmidiaceae were relatively poorly resolved, but several wel-supported clades were consistently found. These include early-branching lineages of Phymatodocis nordstedtiana (85/90/1.0) and Actinotaenium spp. (89/90/1.0), a clade of filamentous or colonial species, and a clade of primarily unicelular species. Actinotaenium spp. are found among the basal-most clades in the Desmidiaceae. One species, Actinotaenium silvae-nigrae, was recently moved from Penium based on its cel wal structure (Kouwets and Coesel 1984), a decision supported by the present data. Most unicelular species appear as a single lineage and most filamentous taxa in a second lineage. In the former, some Euastrum species and Tetmemorus form a monophyletic group (97/99/1.0). These genera share a characteristic apical incision, but Euastrum spp. are generaly somewhat more compresed. E. verucosum is not part of this clade and is 66 found deeply embedded in a clade of Cosmarium spp.. Euastrum verucosum, unlike other species of Euastrum, has a very shalow apical incision and its general shape has made its placement in any particular genus uncertain, although most authors have asigned it to Euastrum. The placement of this species among Cosmarium spp. suggests that many characteristics used to asign organisms to particular genera (such as the presence of lobes or an apical incision) may be homoplastic. For the moment, it would sem ost appropriate to refer this taxon to Cosmarium verucosum (Ehrenberg) Meneghini. The position of Triploceras gracile among Micrasterias species is surprising. This relationship was moderately strongly supported in al analyses (99/100/1.0). Micrasterias species are much compresed and have highly disected lateral lobes, while Triploceras gracile is elongate and radialy symmetric at the isthmus. Explanations for this would include derivation of the genus Triploceras from a Micrasterias-like ancestor, more complex evolutionary histories such as organelar capture, lineage sorting or hybridization, and analytical artifact (although there is no particular reason to suspect such an artifact). The Staurastrum species included in this study were found to be monophyletic. The species included span much of the known structural diversity of Staurastrum, from the spiny S. polytrichum f. biseratum to the long-procesed S. arctiscon. This lineage does not include the monospinous species previously asigned to Staurastrum that could be asigned to Staurodesmus. Teiling (1948, 1965) asigned al monospinous species (those with a single spine at each angle of the hemicel), regardles of shape, to the genus Staurodesmus, including organisms with only mucrae. This study suggests that previous 67 taxonomic treatments do not acurately reflect the evolutionary history of these subtly diverse organisms. This analysis suggests that a number of desmid genera may not be monophyletic. Cosmarium, Staurodesmus, Spondylosium and Euastrum are found to be polyphyletic as suggested by previous studies (Gontcharov et al 2003, 2004; Gontcharov & Melkonian 2005). This study included only strains that could be identified with confidence. This excludes many published sequences because of the lack of adequate voucher information. If al related sequences available in GenBank were included, many additional genera would be non-monophyletic, but the significance of this observation is unclear. In this analysis, most filamentous and colonial forms are found in a single clade with moderate support (83/84/0.97). Within this clade, there are two clear lineages, one containing Cosmocladium saxonicum, Spondylosium tetragonum and Teilingia granulata (100/100/1.0), and the other containing species of Spondylosium, Desmidium, Hyalotheca, Bambusina, Onychonema and Heimansia (99/100/1.0). These results support the separation of Heimansia from Cosmocladium (Coesel 1993) and suggest that the genus Spondylosium, as currently circumscribed, is untenable. Species of Spondylosium are resolved as thre separate lineages. Spondylosium pulchrum is found sister to Desmidium spp., while S. pulchelum is an independent lineage sister to one of the two main clades of filamentous Desmidiaceae. Thre strains here identified as S. tetragonum are found sister to Teilingia granulata. Previous analyses (Gontcharov et al. 2003) also found the genus Desmidium to be polyphyletic; this analysis did not include the same taxa as that study, and the strains here included form a monophyletic group sister to Spondylosium pulchrum (Figure 3.2). 68 In sum, this study used DNA sequences from multiple organelar loci to estimate the phylogeny of the conjugating gren algae. These analyses suggest that these algae have a complex evolutionary history and that some systematic characteristics may be pleisiomorphic such as smooth indehiscent wals. Other characteristics wil have to be determined before we can realy understand the evolution of this group or predict to which lineage many organisms (particularly among the ?zygnematales?) belong. I suggest that cytological characteristics such as chloroplast shape and location may be useful characteristics. Careful observation of the organisms is necesary not only for the purposes of discovering structural or cytological synapomorphies of the clades resolved and described, but also to be certain that the data going into the phylogenetic analysis are as acurate as possible. 0.05 substitutions/site 68/82/* */*/* */*/* 98/92/* 87/*/* */*/* */*/* */*/* */86/0.99 */*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/ * */98/* -/73/* -/50/* -/81/* -/62/* 97/*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* 91/99/* -/96/* 85/90/* 98/96/* */*/* */99/* 95/*/* -/-/0.99 -/-/0.99 -/-/0.91 -/50/- */*/* 96/94/* */*/* 96/96/* 89/67/0.97 89/ 94/* Zygnematales Desmidiales Desmidiaceae Figure 2 Peniaceae Closteriaceae Gonatozygaceae outgroup Penium cf. didymocarpum Penium cylindrus Penium margaritaceum UTEX 600 Penium margaritaceum ACOI 330 Penium spirostriolatum Closterium acerosum Closterium ehrenbergii var. malinvernianum Closterium libellula Spinoclosterium cuspidatum Genicularia spirotaenia Gonatozygon pilosum Gonatozygon monotaenium Gonatozygon brebissonii var. laeve Gonatozygon kinahanii Netrium digitus Roya anglica Roya obtusa Netrium digitus var. lamellosum Cylindrocystis brebissonii Cylindrocystis crassa Zygnema circumcarinatum Zygogonium tunetanum Zygnema cylindrus Zygnema sp. JH0007 Zygnema peliosporum Zygnema sp. JH0039 Zygnema sp. JH0087 Zygnema sp. JH0049 Sirogonium melanosporum Sirogonium sp. JH0082 Spirogyra maxima Sirogonium sticticum Spirogyra gracilis Spirogyra grevilleana Spirogyra condensata Coleochaete divergens Coleochaete scutata Coleochaete nitellarum Coleochaete pulvinata Coleochaete sieminskiana Chaetosphaeridium globosum Chlorokybus atmophyticus Mesostigma viride Mesotaenium endlicherianum Mesotaenium sp. JH0031 Cylindrocystis sp. UTEX1925 Zygnemopsis minuta Mesotaenium kramstai Cylindrocystis sp. JH0038 Mesotaenium caldariorum Mougeotia sp. UTEX758 Mougeotia sp. JH0040 Mougeotia sp. JH0304 Figure 3.1. Phylogeny of the Zygnematophyceae based on rbcL, psaA and coxIII. Tree based on the most likely tree when analyzing 3 genes with all codon positions included. Numbers above branches are ML and MP BS values and PP, respectively. Dashes indicate values less than 50% (BS) or .50 (PP). An asterisk indicates support values of 100 (BS) or 1.0 (PP). */*/* */*/* 97/*/* 85/90/* 89/90/* 93/93/* */*/* */*/* */*/* 99/*/* */*/* */*/* */*/* */*/* -/-/- 94/96/* 83/84/.97 99/*/* 55/90/* 89/91/* 77/91/* */*/* */*/* */*/* */*/* 97/99/* -/ -/0.69 -/-/0.81 66/74/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* */*/* 87/ 89/* 73/64/* */*/* */*/* 86/91/* 99/*/* -/-/0.88 -/-/0.75 66/-/0.99 80/88/* 89/85/0.96 -/-/0.66 68/-/* -/-/0.70 -/89/* -/88/* */*/* 97/*/* Desmidiaceae filamentous Bambusina borreri Bambusina borreri Hyalotheca dissiliens Hyalotheca mucosa Hyalotheca mucosa Onychonema laeve var. micracanthum Onychonema laeve var. micracanthum Desmidium swartzii Desmidium aptogonum var. ehrenbergii Desmidium baileyi Spondylosium pulchrum Heimansia pussillum Spondylosium pulchellum Cosmocladium saxonicum Spondylosium tetragonum SVCK 440 Spondylosium tetragonum JH0175 Spondylosium tetragonum JH0281 Teilingia granulata SAG 39.83 Teilingia granulata JH0140 Teilingia granulata UTCC 284 Teilingia granulata SAG 25.88 Actinotaenium curcurbitinum Actinotaenium curcurbita Actinotaenium silvae-nigrae Cosmarium melanosporum Staurodesmus extensus Phymatodocis nordstedtiana SAG 47.89 Phymatodocis nordstedtiana JH0164 Cosmarium pseudoconnatum Cosmarium botrytis Cosmarium sp. JH0041 Euastrum verrucosum Pleurotaenium baculoides Pleurotaenium ehrenbergii f. columellare Pleurotaenium constrictum Xanthidium hastiferum Xanthidium antilopaeum Staurastrum tetracerum Staurastrum arctiscon JH0014 Staurastrum arctiscon JH0070 Staurastrum pseudosuecicum Staurastrum polytrichum f. biseriatum Staurastrum gladiosum Staurastrum brevispinum Staurastrum polymorphum Micrasterias foliacea Micrasterias rotata Micrasterias radiata Triploceras gracile Haplotaenium minutum Euastrum humerosum var. affine Euastrum crassum var. michiganense Tetmemorus brebissonii Staurodesmus convergens Staurodesmus mamillatus 0.05 substitutions/site Tetmemorus laevis Figure 3.2. Phylogeny of Desmidiaceae based on rbcL, psaA and coxIII. Enlarged portion of ML tree from figure 1 showing the phylogeny of the families of the Desmi- diaceae based on the most likely tree when analyzing 3 genes with all codon positions included. Numbers above branches are ML and MP BS values and PP, respectively. Dashes indicate values less than 50% (BS) or .50 (PP). An asterisk indicates support values of 100 (BS) or 1.0 (PP). 71 Chapter 4. Patterns of cel division in the filamentous Desmidiaceae, close gren algal relatives of land plants Abstract Paterns of cel division and cross wal formation vary among the charophyte gren algae, the closest living relatives of land plants. The conjugating gren algae are highly diverse and are known to display substantial variation in mode of cel division, but the details of these cel division paterns and their phylogenetic distribution remain poorly understood. I report here a study of cross wal development in filamentous Desmidiaceae (conjugating gren algae) using light and fluorescence microscopy. Al strains investigated showed centripetal encroachment of a septum, but with several distinctly diferent developmental paterns. In some cases cel wal formation is delayed with respect to the Cosmarium-type of cel division and the cross wal modified considerably after deposition in a manner specific to particular clades of filamentous desmids. These characteristics were mapped on a phylogeny estimated from a dataset of two organelar genes, and the evolutionary implications of the character state distribution were evaluated. The data suggest a complex history of evolution of cel division in this lineage, and also imply that Desmidium and Spondylosium are polyphyletic. These results suggest that cel division and the mature cross wals may be useful taxonomic characters for identifying filamentous conjugating gren algae. Introduction Cel division is a fundamental proces critical to the proliferation of al cels. In plants and many algae, once nuclei, organeles and other cytoplasmic contents separate, 72 cels must undergo cytokinesis as wel as the simultaneous construction of a new extracelular wal. Land plants share a single mode of cel division involving the use of a phragmoplast, but their close relatives, the charophyte gren algae, show diferences in their modes of cel division. The nature of cel wal deposition plays an important role in determining the shape of the cel. In unicelular organisms this determines the structure of the organism, and changes in the proces of cel division are considered important evolutionary steps in the evolution of complex celular organization (Graham et al., 2000). In Coleochaete, for example, the ability to control the plane of cel wal deposition alows the filaments to bifurcate, and leads to the branched thalus characteristic of the genus. Transitions from unicelular to filamentous forms have occurred many times in the evolution of the conjugating gren algae (Zygnematophyceae) (McCourt et al., 1995; Gontcharov et al., 2003). Their close relationship to land plants makes the lineage an exceptional model for studying the proces of cel division and morphogenesis and provides direct insight into the evolution of multicelularity in the lineage that gave rise to land plants (Karol et al., 2001; Turmel, Otis, and Lemieux, 2006). Many algae lack a cel wal, but al charophyte algae (except Mesostigma, which has scales), have a celulosic cel wal that must form during division or subsequent development (Graham and Wilcox, 2000). Among charophytes, cel wal formation proceds by centifugal growth of a cel plate (land plants, Charales and Coleochaetales) (Cook, Graham, and Lavin, 1998; Cook, 2004) or by centripetal encroachment of a peripheral septum (Zygnematophyceae, Klebsormidiophyceae, and Chlorokybus) (Graham et al., 2000). The conjugating gren algae (Zygnematophyceae), however, are 73 known to have several diferent variations of centripetal cel division (Brook, 1981). The diversity of paterns of cel division among the conjugating gren algae is far greater than that found among land plants, but has received litle investigation. Many conjugating gren algae undergo cel division as Klebsormidium and Chlorokybus do: via centripetal encroachment of a peripheral septum. Many constricted species - that is, those that are divided into semicels connected by a narow cytoplasmic isthmus - exhibit the ?Cosmarium-type? cel division in which the division septum forms soon after mitosis. The two new semicels then expand and the secondary wal is not deposited until the semicel is nearly fully formed (Picket-Heaps, 1972; Meindl, 1993). In the Desmidiaceae, one of four families of conjugating gren algae commonly refered to as desmids, the primary wal is then shed in its entirety or in fragments leaving only the secondary wal in mature cels. Although the Cosmarium-type of cel division is common among constricted desmids it is not the only means of division for constricted cels. In Onychonema laeve Nordstedt, a filamentous desmid with constricted cels, a common vesicle forms betwen the semicels (termed a division vesicle). This vesicle enlarges to nearly the size of an adult semicel before a division septum forms (Krupp and Lang, 1985b). Once the wal forms, the semicels continue to grow and take the shape of their parent cel, in this case forming apical proceses and lateral spines. The filamentous habit is maintained by a fragment of primary wal that is shared by adjacent cels, however, most of the primary wal is jetisoned as in other Desmidiaceae (Krupp and Lang, 1985a), Perhaps the most remarkable variation of centripetal cel division is represented by Bambusina borreri (Ralfs) Cleve. In this alga, after the new cel wal forms (during 74 cytokinesis), a cylinder of primary and secondary cel wal is deposited in the center of the cel. This results in what appears to be folds (replications) in the cross wal (se Figure 4.2K, L). As the cels elongate, these folds turn out and flaten, which results in a nearly full-size cel with a complete wal (Gerath, 1973; Krupp and Lang, 1985b). This kind of cel division was thought to be typical of many filamentous desmid genera: Bambusina, Desmidium, Streptonema and Haplozyga (Krupp and Lang, 1985a). Stil other forms of cel division are known among the filamentous desmids. Hyalotheca, for example, shows simple centripetal cel division more characteristic of the Zygnematales than the Desmidiaceae (Acton, 1916; Krupp, 1980). Furthermore, cel division has not been explicitly studied in a number of filamentous genera and the mode of cel division is often infered from the presence of cross wals of a particular shape, or cel division is asumed to be the same as that of superficialy similar unicelular and filamentous species. Cel division paterns in al previously studied species are variations of centripetal cel division common to other charophyte and chlorophyte algae. In the course of a molecular survey of the conjugating gren algae and extensive field study, I observed undocumented variation in cel division among filamentous Desmidiaceae. To understand the phylogenetic and developmental significance of this variation, I undertook a detailed study of cel division in these taxa, and place that diversity in the context of molecular phylogenetic data. In addition to reporting characteristics of cel division for a number of genera and species, I also explored evidence concerning the evolution of centripetal cel division within the Zygnematophyceae as infered from chloroplast and mitochondrial gene phylogenies. 75 Materials and Methods Terminology For the purpose of this paper, ?cel division? refers to the entire proces of division from pre-mitotic elongation of cels, through chloroplast division, mitosis, cytokinesis and the deposition of primary and secondary wal material. Chloroplast and nuclear division are refered to separately. A ?cross wal? refers not only to the primary wal or the position of wal deposition, but rather to the wal, sometimes with both primary and secondary material, that is deposited before a final stage of elongation in filamentous conjugating gren algae. I follow previous authors in using terminology that paralels wal terminology for plants (Gerath, 1973; Krupp and Lang, 1985b). The first wal deposited during cytokinesis is refered to as the primary wal and the subsequent thickened layer deposited inside the first wal as a secondary wal. Culture conditions Strains investigated were requested from public culture collections or isolated from the wild (Table 4.1). Al strains were grown in Guilard?s Woods Hole medium (Nichols 1973) in a Percival growth chamber (Percival, Pery, IA) under Sylvania Cool White fluorescent lamps (Danvers, MA) and iradiance of 30 microeinsteins on a 16:8 hr light:dark cycle. at 15? C. Cels were observed at several times throughout the day with many cross wals visible several hours after the lights came on. Dividing cels could be found at any time of day. A list of strains used and acesion information can be found in Table 4.1. Strains that were misidentified in culture collections are indicated in brackets. Synonymy is indicated with parentheses. 76 Table 4.1. Strains investigated and Genbank numbers Taxon Strain rbcL coxII Bambusina boreri JH0125 EF371284 EF371071 Closterium acerosum UTEX 1075 EF371285 EF371072 Closterium ehrenbergi var. malinvernianum JH013 EF371286 EF371073 Closterium libelula JH021 EF371287 EF371074 Cosmocladium saxonicum ACOI 95 EF371292 EF371079 Desmidium aptogonum SVCK 108 EF463091 EF463086 Desmidium aptogonum var. ehrenbergi JH0184 EF371298 EF371085 Desmidium baileyi JH028 EF37129 EF371086 Desmidium grevilei (cylindricum) SVCK 13 EF463090 EF463085 Desmidium grevili JH096 EF463092 EF46308 Desmidium swartzi JH042 EF371297 EF371084 Gonatozygon monotaenium UTEX 1253 U71438 EF371092 Gonatozygon pilosum ACOI 1096 EF371303 EF371093 Groenbladia taylori JH039 EF463093 EF463084 Heimansia pusila SVCK EF371291 EF371078 Hyalotheca disiliens SAG 384-2 AF20349 EF371095 Hyalotheca mucosa JH05 EF371305 EF371096 Micrasterias foliaceae NIES 297 EF37131 EF37102 Micrasterias radiata JH064 EF371313 EF37104 Micrasterias rotata UTEX 1941 EF371312 EF37103 Onychonema laeve var. micracanthum JH0198 EF371318 EF3711 Onychonema moniliforme JH0420 EF463094 EF463089 Penium argaritaceum UTEX 60 EF371321 EF37114 Penium spirostriolatum SVCK 205 EF371324 EF37117 Phymatodocis nordstedtiana SAG 47.89 AJ53962_1 EF37118 Phymatodocis nordstedtiana JH0164 EF371325 EF37119 Spondylosium pulchelum SVCK 365 AF203505 EF37137 Spondylosium pulchelum JH0368 EF463096 EF463087 Spondylosium pulchrum JH0269 EF371341 EF37138 Spondylosium tetragonum SVCK 40 AF203498 EF371094 Staurastrum arctiscon JH014 EF371343 EF37140 Staurastrum polytrichum f. biseriatum JH015 EF37134 EF37141 Teilingia granulata SAG 39.83 EF37135 EF37130 Teilingia granulata UTC 284 AF203504 EF37131 Teilingia granulata [Spondylosium planum] SVCK 418 EF463095 EF463083 Teilingia granulata [Spondylosium secedens] SVCK 24 EF463097 ----- Notes: ACOI, Coimbra Collection of Algae; NIES, National Institute for Environmental Studies; SAG, Samlung von Algenkulturen der Universit?t G?ttingen; SVCK, Samlung von Conjugaten-Kulturen; UTEX, Culture Collection of Algae at University of Texas; UTC, University of Toronto Culture Collection of Algae and Cyanobacteria. Vouchers of strains beginning with JH are available from the authors. Bold type sequences were generated specificaly for this study. 77 Molecular phylogenetic analysis DNA was extracted using the Nucleon Phytopure DNA extraction kit (Amersham Pharmacia Biotech, Piscataway, NJ). The chloroplast gene rbcL and mitochondrial coxII were amplified by PCR using published primers (Chapter 3) and sequenced using Big Dye Terminator sequencing technology on an Applied Biosystems 3100 capilary sequencer (Applied Biosystems, Foster City, California). Fragments were sequenced in both directions and asembled using Sequencher 4.2 (GeneCodes, Ann Arbor, MI). Sequences were aligned in MacClade 4.7 (Maddison and Maddison, 2000). Phylogenetic analyses were performed in PAUP* (Swofford, 2003). Under the Parsimony criterion (MP), best tres were searched for heuristicaly with ten random taxon addition sequences and tre-bisection-reconnection (TBR) branch swapping. Support for taxon bipartitions was estimated using the non-parametric bootstrap (BS), with 500 pseudoreplicates and 10 random taxon addition sequences in each pseudoreplicate. Under the Maximum Likelihood criterion (ML) I used the GTR+I+G model of sequence evolution with parameters estimated from the data. One hundred bootstrap replicates were run with thre random taxon addition sequences per replicate. Two analyses in MrBayes (BI), each using four chains sampled for four milion generations, sampled every one hundred generations, were used to estimate the topology and posterior probabilities (P) of the taxon bipartitions. The first two thousand tres were discarded as burnin. 78 Microscopy Living cels were observed at various stages of cel division using a Zeis Axioskop microscope (Zeis, Germany). Image data were recorded as digital micrographs using an AxioCam HRc CD camera (Zeis, Germany). Many cels at diferent stages of cel division were observed, and when possible a single cel was followed through a division cycle. In most cases, cross wals were visible using diferential interference contrast (DIC) microscopy, however, some stages were recorded by staining the cels with the celulose-specific fluorochrome Calcofluor. When staining, live cels were first harvested by gentle centrifugation and fixed with 3% glutaraldehyde for 20 minutes. Cels were alowed to setle and rinsed once in Guilard?s Wood?s Hole medium, and then stained for one hour with 1% Calcofluor. Cels were destained by thre ten minutes rinses in medium and viewed on the same microscope using an HBO 50 Mercury arc lamp (excitation near 395) and a long-pas emision filter of 470nm. Results Cross walls Diferences in timing of celular events, such as cel wal deposition, among species were observed. Many diferences in cross wal structure were only apparent in the fully mature cross wal, that is, the stage of development just before the cels elongated to form ature semicels. Al cross wals formed by the centripetal encroachment of a peripheral septum. Complex features of the cross wal formed after cytokinesis and the deposition of primary wal material. Calcofluor efectively stained secondary wal material, however, the first wals deposited were not always visible when stained with Calcofluor, even if easily visualized with DIC. This was particularly 79 noticeable in Hyalotheca spp., Spondylosium pulchelum Archer, Teilingia granulata (Roy & Biset) Bourrely and Spondylosium tetragonum West. Teilingia granulata Teilingia cels are somewhat rectangular and the semicels are connected across a narow isthmus. In strains of T. granulata, following chloroplast division (or simultaneous with it), the cels elongated and formed a narow connection about the width of the isthmus (Figure 4.2N). A cross wal then formed across this vesicle and the resulting semicels further elongated and inflated until the miror image of the parent semicel was fully developed. Thus I observed a number of cels that were elongate without a cross wal (Figure 4.3 J3) and elongate cels with a separating wal (Figure 4.3 J4). Spondylosium tetragonum Cels of S. tetragonum are nearly rectangular and have a broad isthmus. Consequently, the elongate cels were les obvious than in Teilingia granulata. Early stages of cel division were not observed in this species, however a number of elongate cels were observed (Figure 4.3 K4). Cels elongated very litle before depositing a cross wal. Later stages were consistent with division similar to that of Teilingia granulata (Figure 4.3 K5, K6). Because of the smal size and variability in cel length, key characteristics of cel division were at the limit of the techniques used here and confident interpretation of events would require more study. 80 Spondylosium pulchelum S. pulchelum forms filaments composed of cels that are markedly constricted, compresed, and have a trapezoidal shape. Both strains observed underwent cel division via the Cosmarium-type cel division (se Figure 4.3I). Semicels were observed to disasociate slightly at the isthmus and formed a cross wal at the isthmus very early in cel division (Figure 4.2M). The resulting semicels expanded as a papilum-like protrusion that inflated to form a large hemisphere, ultimately taking the shape of a trapezoidal hemicel. Portions of the primary wal dehisced. Hyalotheca disiliens and H. mucosa Hyalotheca cels are cylindrical and very subtly constricted in the mid region. In both species cels elongated very litle before a wal was deposited. Encroachment of a peripheral septum was observed. This cross wal was iregular initialy, but became linear as it thickened. The daughter cels then further elongated and pores appeared. Groenbladia taylori Cels of G. taylori are cylindrical and not noticeably constricted. When G. taylori elongated before cytokinesis, it was apparent in some Calcofluor stained cels that new al material was deposited at the isthmus (Figure 4.2O). Encroachment of a peripheral septum was not observed but cross wals and subsequent elongation were consistent with the hypothesis that G. taylori underwent simple centripetal cel division much like Hyalotheca spp. and probably very similarly to Spondylosium tetragonum. 81 Bambusina borreri Bambusina cels are cylindrical, often inflated in the midregion and almost always longer than broad. In B. borreri, cels elongated slightly before cytokinesis. A plane cross wal was deposited at the median. A cylinder of primary and secondary wal material formed in the center of the cel (Figure 4.2K). This cylinder then unfolded and turned out as the cel elongated (Figure 4.2L). Only the center portion, which is the point of connection betwen adjacent cels, remained intact. Se Gerath (1973) for details. Desmidium grevilei (K?tz.) Bary Cels of D. grevilei are cylindrical, broader than long, and connected along the entire apex. Early in cel division, cels of D. grevilei elongated, sometimes resulting in what appeared to be a shared vesicle (Figure 4.3 E4). The cel first divided by a cross wal and then a cylinder of cel wal material deposited in the center (Figure 4.2E, F). These folds were about 2 or 3 ?m in amplitude compared to the nearly 5 ?m folds found in B. borreri. Onychonema laeve var. micracanthum Nordst. and O. filiforme (Ehr.) Roy & Biset Semicels of Onychonema are reniform and connected by a narow isthmus. Before cytokinesis, cels of Onychonema form a large shared vesicle (division vesicle) betwen the semicels (Krupp and Lang, 1985a; this study). A division septum then divided the vesicle somewhat asymmetricaly (Figure 4.2I, J). Once separated, the new daughter semicels rounded out at the angles and continued to expand. After the wal was deposited, the apical proceses characteristic of Onychonema formed. This is the first description of cel division in Onychonema filiforme. 82 Desmidium aptogonum Br?b., D. aptogonum var. ehrenbergii K?tz., Desmidium baileyi (Ralfs) Nordst., and D. swartzi (Agardh) Agardh Cels of these species are angular in apical view, broader than long and connected by apical proceses leaving open space betwen adjacent cels. Cels first elongated forming a shared vesicle at the isthmus (Figure 4.2C, D, G, H and Figure 4.3, C3). This vesicle was then divided by a plane cross wal. Unlike Bambusina, a cylinder of cel wal material was deposited at each angle of the semicel at the location where the cels would be connected by apical proceses rather than in the center of the cel (Figure 4.2 C, D, G, H). The number of such cylinders correlated to the number of apical proceses, found in the cel: the biradiate Desmidium aptogonum var. ehrenbergii had two; the triradiate D. baileyi had thre and quadriradiate forms of Desmidium had four (observed, but none included in this study). The size of these cylinders was approximately proportional to the length of the apical proceses of the mature cels. Desmidium baileyi, which has long proces (>5?m), had the largest fold (also >5?m). These cylinders were apparent when the cels were stained with Calcofluor but dificult to se in untreated cels. Desmidium swartzi, which has very short proceses, also had very short replicate folds, each fold being betwen 1 and 2?m in length. This made visualization with Calcofluor fluorescence dificult. Using DIC, the folds were visible but obscured by cytoplasmic contents in living cels. Spondylosium pulchrum (Bailey) Archer Cels of S. pulchrum are very deeply constricted and have a smal apical proces that is the point of connection betwen adjacent cels. Cel division in S. pulchrum 83 difered from that of other Spondylosium species investigated. In this species, the cels initialy elongated and formed a division vesicle similar to that found in Onychonema. A cross wal then divided the vesicle and a smal cylinder of cel wal material was deposited in the center of the cross wal. The replicate fold was very smal and appeared, as in Desmidium swartzi, as a smal bubble on the cross wal (Figure 4.2 B). This mode of cel division was most similar to that of Desmidium baileyi, difering primarily in the scale of the features. Micrasterias foliacea Bailey, Phymatodocis, Heimansia and Cosmocladium Cel division paterns of two filamentous and two colonial desmids included in the phylogeny were not investigated. These were Phymatodocis, Micrasterias foliacea, Heimansia and Cosmocladium. These are al thought to use the Cosmarium-type cel division (se Spondylosium pulchelum in this study). My observations were consistent with this hypothesis but insufficient to confirm it. Only division in Micrasterias foliacea has been studied in detail (Lorch and Engels, 1979) and was found to be of the Cosmarium-type. Molecular phylogeny Phylogenetic analyses were performed on a dataset containing fragments of the chloroplast gene rbcL and the mitochondrial gene coxII. Outgroup species were selected from Chapter 3 and species within the filamentous clade were added to this analysis. Many other strains of filamentous Desmidiaceae were screned for diferences in cross wals. 84 With the exception of Phymatodocis and Micrasterias foliacea, a single lineage of filamentous and colonial species was resolved with moderate statistical support (Figure 4.1) (90, 98, 1.0; MP, ML and P). Within this clade, two lineages were resolved: one containing Teilingia granulata, Spondylosium tetragonum, and Cosmocladium saxonicum Bary; the other containing the remaining filamentous or colonial species (Figure 4.1). In the first lineage, Cosmocladium, a colonial desmid connected by delicate cel wal strands, diverged first. Spondylosium tetragonum was sister to a clade of Teilingia granulata. Teilingia strains showed some sequence and structural diversity (se discussion). The second lineage included the remaining filamentous and colonial forms. Spondylosium pulchelum was the first to branch and was represented by two strains that were structuraly very similar. Heimansia pusila (Hilse) Coesel separated from other strains with moderate support (83/85/1.0). A lineage containing Desmidium aptogonum, D. swartzi, D. aptogonum var. ehrenbergii, and D. baileyi as wel as Spondylosium pulchrum was resolved with strong support (100/100/1.0). Placement of Onychonema spp. was les supported, but the two species included, O. laeve var. micracanthum and O. fililiforme, were strongly supported as monophyletic (100/100/1.0). Two species of Hyalotheca were also monophyletic with strong support (100/100/1.0). The strains of Hyalotheca were part of a lineage that included Groenbladia taylori (but not Spondylosium tetragonum = ?Groenbladia undulata?), as wel as Bambusina borreri, Desmidium grevili and D. cylindricum. Desmidium was, therefore, polyphyletic, and the cylindrical forms were sister to Bambusina while the angular forms (mostly triangular in apical view) were sister to Spondylosium pulchrum. 85 Discusion Several modes of cel division are wel characterized among the conjugating gren algae (Brook, 1981). Of the two orders of conjugating gren algae, the Zygnematales and the Desmidiales, this diversity is concentrated among the later. The filamentous Zygnematales, use a simple mode of centripetal cel division involving the encroachment of a peripheral septum. In one species of Spirogyra, cel division also involves a cytoskeletal aray similar to a phragmoplast (Fowke and Picket-Heaps, 1969a; Fowke and Picket-Heaps, 1969b). Among the Desmidiales, the most common form of cel division is the Cosmarium-type. The filamentous Desmidiales demonstrate stil more diversity in their modes of cel division and these modes are compared to the Cosmarium- type for convenience. Some filamentous Desmidiales use the Cosmarium-type cel division. Spondylosium pulchelum, Cosmocladium saxonicum, Heimansia pusila and the distantly related Micrasterias foliaceae and Phymatodocis nordstedtiana al use the Cosmarium- type cel division. Teilingia granulata and Spondylosium tetragonum delay primary wal deposition until after and initial phase of elongation. This results in the formation of a vesicle in Teilingia and elongate cels in Spondylosium tetragonum. These ?vesicles? do not inflate which distinguishes them from similar structures in Onychonema and Spondylosium pulchrum. Among the remaining filamentous desmids, there are four unique modes of cel division. Cel division in Groenbladia taylori and Hyalotheca is similar to that of Spondylosium tetragonum. In these species, cels elongate without forming an inflated 86 division vesicle. In Onychonema, deposition of primary wal material is delayed until after an initial phase of elongation (Krupp & Lang, 1985b; Figure 4.2I). The vesicle that results inflates to nearly the size of a mature semicel before it is divide transversely by a wal. The apical proceses begin to form soon after cytokinesis, but are not apparent on the mature cross wal. In the previously discussed species, diferences in their cel division are related to the timing and degre of celular proceses. Al delay deposition of cel wal material with respect to the Cosmarium-type. The inflated vesicle of Onychonema is similar to the early developing semicels of Cosmarium in that the vesicle consists of mostly primary wal material (Krupp et Lang, 1985b). The cylinders of cel wal material deposited on the mature cross wals of Desmidium and Bambusina share no known homologues among gren algae. A similar structure is found in some Spirogyra spp., however, in that organism the cylinders are present on the mature cels and are not part of a transitory developmental phase as in Desmidium and Bambusina. Convergence of two lineages of desmids on this similar mechanism would be surprising and the apparent distribution may be the result of losses rather than convergence. Nevertheles, these lineages are separated, phylogeneticaly, by organisms with other modes of cel division. Additionaly, the two lineages difer somewhat in the placement of the cylinders on the mature cross wal. In the case of Bambusina borreri and Desmidium grevilei, a single cylinder is found in the center of the cel. In Spondylosium pulchrum and Desmidium baileyi, as wel as other species of Desmidium, the cylinders are positioned at the site of future apical proceses and vary in number as a function of the number of apical proceses. 87 These observations are supported by dozens of published images of cels in the proces of division. A table of some of these images is provided (Table 4.2). Only two images appear to be inconsistent with my results. In one case the image may depict a cel that has almost completed cel division (F?rster, 1974). In the other case (F?rster, 1964), there is no obvious explanation for the discrepancy except to say that the diagnostic characteristics in that species would be very smal and the previous report was based on fixed material and, most likely, a single or very few cels. Other images showing a flat division plate in Desmidium and Bambusina would sem to be inconsistent with my results, however, in these species a flat cross wal is initialy deposited and the characteristic cylinders of cel wal material develop subsequently. Therefore these images are not in conflict with my results. When first observed, the shared vesicle found in Teilingia was thought to be the product of a failure of cytokinesis. This is a common mutation in constricted desmids that gives rise to ?giant? cels. These mutant cels are maintained through succesive divisions, though it is possible for them to give rise to typical-shaped cels (Brook, 1981). In this case, cels were followed through a division cycle and found to separate normaly. Most filamentous Desmidiaceae were resolved in a single lineage in this analysis, however, another study based on 18S rDNA sequences found two clades of filamentous desmids (Gontcharov et al., 2003). These two clades are probably analogous to the clades described here (Figure 4.1). The two clades contain organisms with similar modes of cel division. However, even though cel division in Teilingia granulata is similar to cel division in both Onychonema and Groenbladia taylori, this is very likely an 88 independently derived state. These species were resolved in separate clades among organisms with other modes of cel division (Figure 4.4). Basal nodes in the second lineage of filamentous desmids were not strongly supported (-/52/1.0) and the relationships betwen Heimansia pusila, Spondylosium pulchelum and the lineage containing Hyalotheca and Desmidium swartzi were weakly supported (Figure 4.1). In this clade, Onychonema exhibits a unique mode of cel division (Figure 4.4). The presence of a replicate fold on the mature cross wal unites Spondylosium pulchrum with several angular species of Desmidium, which are monophyletic (100/100/1.0). Desmidium grevilei and Bambusina borreri formed a monophyletic clade with strong support (89/98/1.0) and shared a form of cel division involving the deposition of a single cylinder of wal material following cytokinesis (Figure 4.2 E, F, K, L). Desmidium is, therefore, polyphyletic. Desmidium spp. with an exactly similar cross wal shared a common ancestor with Bambusina borreri, while other species of Desmidium did not. These two groups of Desmidium are clearly distinct phylogeneticaly and structuraly (Gontcharov et al. 2003; this study). Two previous studies found that species of Spondylosium were polyphyletic (Gontcharov et al., 2003, 2004). Part of this confusion resulted from the misidentification of two strains of Teilingia (previously reported as S. planum, and S. secedens) (Chapter 3). A polyphyletic Spondylosium was also found in this study. I believe that the strains are now correctly identified and the apparent phylogenetic relationships of the organisms are supported by the observation that the thre lineages exhibit thre diferent kinds of cel division (Figure 4.4). The clade of Teilingia granulata shows more sequence diversity than one might expect. In addition to this sequence diversity there were also 89 structural diferences betwen the strains. Strains difered in cel sizes and the degre of constriction (data not shown). Within each strain, some cels lacked the apical granules characteristic of the genus. Although several modes of cel division were known for conjugating gren algae, the species investigated in this study are unique in that they are filamentous representatives of a species rich and mostly unicelular family, the Desmidiaceae. Acordingly, cel division in this lineage brings some evidence to bear on the evolution of multicelularity in charophyte gren algae. There are few desmid lineages that contain filamentous species. Phymatodocis is one such organism and sems to represent an entirely independent evolution of the filamentous state. Micrasterias foliacea also forms filaments, however, in this species the filaments are held together by the interlocking of apical proceses, not by means of a shared primary wal (Lorch and Engels, 1979). It is also true that this ?filamentous? lineage contains organisms which are beter described as colonial, namely Cosmocladium saxonicum and Heimansia pusila. These species have stil other means of connecting cels to one another (Gerath, 1970). It is not clear why there are so few transitions from unicelular to colonial and filamentous forms among the desmids. In a related group, the Zygnematales, organisms have made the transition betwen unicelular and filamentous forms several times (McCourt et al., 1995; McCourt et al., 2000; Gontcharov, Marin, and Melkonian, 2003, 2004; Chapter 3). These results provide some indication of the plasticity possible in the proces of cel division. While the cels in these filaments most likely do not share cytoplasmic connections, the cels, at the very least cooperate with one another to form filaments of diverse architecture. The diferences in cel division are the product of not only changes 90 in the timing and order of celular events, but also the evolution of novel structures. This demonstrates that variation is possible in proceses as complex and critical as cel division. It is clear that there is much diversity of cel division among the filamentous conjugating gren algae, but the exact details of these diferences remain obscure. I investigated cross wals as structural characteristics and to some degre their formation, but other aspects of cel division are important for understanding the evolution of these cross wals such as timing of chloroplast and nuclear division, and the fate of the primary wal. Al of these require more in-depth study of each species and probably the employment of diferent methods such as TEM. This study is only the beginning of such an investigation. Future work may reveal more information that wil be useful in determining the evolutionary history of cel division in this unique group of organisms. 91 T a bl e 4. 2 . P ub l i s he d i m a ge s of c e l l di vi s i on i n D e s m i da c e a e T a x o n C i t a t i o n T y p e C o n s i s t e n t N o t e B a m b u s i n a b o r r e r i * G e r r a t h 1 9 7 5 B a m b u s i n a Y e s B a m b u s i n a b o r r e r i * H a u p t f l e i s c h 1 8 8 8 B a m b u s i n a Y e s B a m b u s i n a b o r r e r i * K r u p p & L a n g 1 9 8 5 b B a m b u s i n a Y e s D e s m i d i u m a p t o g o n u m v a r . a c u t i u s F ? r s t e r 1 9 6 4 , p l . 3 6 , f i g . 2 B a m b u s i n a N o D e s m i d i u m b a i l e y i F ? r s t e r 1 9 7 4 , p l . 3 6 , f i g . 5 B a m b u s i n a N o l a t e s t a g e ? D e s m i d i u m b a i l e y i G e r r a t h 2 0 0 3 , f i g . 5 0 D e s m i d i u m Y e s D e s m i d i u m b a i l e y i H a u p t f l e i s c h 1 8 8 8 D e s m i d i u m Y e s D e s m i d i u m b a i l e y i S c o t t & P r e s c o t t 1 9 6 1 , p l . 6 2 , f i g . 8 , 1 0 D e s m i d i u m Y e s D e s m i d i u m b a i l e y i S c o t t e t a l . 1 9 6 5 , f . 2 4 7 D e s m i d i u m Y e s D e s m i d i u m b a i l e y i S m i t h 1 9 2 0 , p l . 8 8 , f i g . 7 D e s m i d i u m Y e s D e s m i d i u m g r e v i l l e i * C o u c h & R i c e 1 9 4 8 , f i g . 1 1 B a m b u s i n a Y e s D e s m i d i u m g r e v i l l e i S m i t h 1 9 2 0 , p l . 8 8 , f i g . 1 B a m b u s i n a Y e s l a t e s t a g e D e s m i d i u m g r e v i l l e i T e l l & D o m i t r o v i c 1 9 9 2 , p l . 2 , f i g . 1 3 b B a m b u s i n a Y e s l a t e s t a g e D e s m i d i u m s w a r t z i i * H a u p t f l e i s c h 1 8 8 8 D e s m i d i u m Y e s H y a l o t h e c a d i s s i l i e n s * A c t o n 1 9 1 6 H y a l o t h e c a Y e s H y a l o t h e c a m u c o s a * H a u p t f l e i s c h 1 8 8 8 H y a l o t h e c a Y e s O n y c h o n e m a l a e v e * K r u p p & L a n g 1 9 8 5 a O n y c h o n e m a Y e s O n y c h o n e m a l a e v e v a r . s u m a t r a n a S c o t t & P r e s c o t t 1 9 6 1 , p l . 6 1 , f i g . 1 O n y c h o n e m a Y e s S p o n d y l o s i u m p u l c h e l l u m n o n e S p o n d y l o s i u m p u l c h r u m n o n e S p o n d y l o s i u m t e t r a g o n u m n o n e T e i l i n g i a g r a n u l a t a n o n e T e i l i n g i a s p i n u l o s a P a l a m a r - M o r d v i n s e v a 2 0 0 5 , p l . 1 4 9 , f i g . 1 2 T e i l i n g i a Y e s T e i l i n g i a w a l l i c h i i [ a s S p h a e r o z o s m a w a l l i c h i i ] T u r n e r 1 8 9 2 , p l . 1 8 , f i g . 1 T e i l i n g i a Y e s * T he s e s t udi e s s pe c i f i c a l l y m e nt i on t he c r os s w a l l s i n t he publ i c a t i on T ype ? t ype of c e l l di vi s i on c ons i s t e nt w i t h pub l i s he d i m a ge C ons i s t e nt ? w he t he r or not t he i m a ge i s c ons i s t e nt w i t h m y f i ndi ngs N ot e ? c om m e nt on pos s i bl e s our c e of i nc ons i s t e nc y Desmidium grevillei SVCK 113 Desmidium grevillei JH0096 Bambusina borreri Hyalotheca mucosa Hyalotheca dissiliens Groenbladia taylori Onychonema laeve var. micracanthum Onychonema filiforme Desmidium aptogonum Desmidium swartzii Desmidium aptogonum var. ehrenbergii Desmidium baileyi Spondylosium pulchrum Heimansia pusilla Spondylosium pulchellum JH0368 Spondylosium pulchellum SVCK 365 Teilingia granulata SAG 39.83 Teilingia granulata SVCK 24 Teilingia granulata SVCK 418 Teilingia granulata UTCC 284 Spondylosium tetragonum Cosmocladium saxonicum Micrasterias rotata Micrasterias radiata Micrasterias foliacea Staurastrum arctiscon Staurastrum polytrichum Phymatodocis nordstediana JH0164 Phymatodocis nordstedtiana SAG 47.89 Penium margaritaceum Penium spirostriolatum Closterium ehrenbergii var. malinvernianum Closterium acerosum Closterium libellula Gonatozygon monotaenium Gonatozygon pilosum 0.01 substitutions/site */*/* */*/* 64/65/.86 54/ 59/.75 84/81/* -/-/.67 64/*/* */*/* */*/* */*/* */*/* */*/* */*/* 86/84/* 99/*/* 98/ 99/* 56/80/.97 96/97/* 90/94/* */*/* */*/* 90/95/* 64/84/* -/-/.55 -/-/- */*/* */*/* 78/79/* */99/* */*/* */*/* 97/76/.83 99/97/* filamentous clade I filamentous clade II Desmidiaceae outgroup Figure 4.1. Phylogeny of the filamentous Desmidiaceae based on rbcL and coxIII gene sequences. Topology based on the ML tree. Numbers above branches are bootstrap values from Parsimony and Maximum Likelihood and Posterior Probabili- ties from Bayesian Inference. Bootstrap values of 100 and posterior probabilities of 1.0 are indicated by an asterisk (*). 93 Figure 4.2. A-O. Various stages of division in filamentous Desmidiaceae. A. Light micrograph of early vesicle formation in Spondylosium pulchrum. Chloroplast has divided, but no wal material has been deposited. B. Fluorescence micrograph of Calcofluor stained S. pulchrum showing development of lobes and the presence of a smal cylinder of cel wal material in the center of the cel. C. Light micrograph of Desmidium swartzi showing the smal cylinders of cel wal material near the angles of the cel. D. Fluorescence micrograph of D. swartzi showing cylinder of material at angles of cel, new al deposition. Red is autofluorescence from the chloroplast. E. Light micrograph of D. grevili showing a late stage of cel division with folds at the angles, visible as highly refractive spots on the cel plate. F. Fluorescent micrograph of Calcofluor stained D. grevili. Late stage showing mature cel plate. G. Light micrograph of D. baileyi fixed in the final stages of cel division. Edges of cel have pulled apart and cylinders of cel wal material are clearly visible at the angles. H. Fluorescence micrograph of an earlier stage in cel division of D. baileyi, before the edges of the cels have pulled apart. I. Light micrograph of early stage of cel division in Onychonema filiforme. Division vesicle is fully formed and the primary wal has been deposited across the vesicle. J. Fluorescence micrograph of early stage cel wal deposition in O. filiforme. K. Light micrograph of cel division in Bambusina borreri showing folds of cel wal material. Upper cel in a slightly earlier stage than the lower which has already pulled apart at the edges and is beginning to elongate. L. Fluorescence micrograph of B. borreri showing a late stage of cel division. Cylinder of cel wal material is fully formed and the cels have begun to move apart turning the folds inside out. M. Light micrograph of cel division in Spondylosium pulchelum. Septum forms very early in division and the daughter hemicels form from the isthmus of their parent hemicel. Two cels are in this stage of division. N. Light micrograph of cel division in Teilingia granulata. Vegetative stage cels visible at the bottom of the micrograph. Cels elongate at the isthmus forming a rectangular vesicle that is later divided by a septum. Thre cels in the center of the micrograph have formed wals across this vesicle. O. Fluorescence micrograph of Groenbladia taylori showing, regions of new al deposition along edges of cel as wel as the position of the primary wal across the dividing cel. A C E B D F G I K H J L M N O 95 Figure 4.3. Model of cel division in eleven diferent species of filamentous Desmidiaceae. Row 1 shows the cels in apical view and is scaled to show the relative size of the organisms. Spondylosium tetragonum is, on average, about 5 ?m wide and S. pulchrum nearly 60 ?m wide. Other rows are scaled arbitrarily, but consistently within the column, to show the features of cel division plates. Dashed lines show the plane of primary wal deposition, and where new al has clearly been deposited in Groenbladia undulata and Hyalotheca disiliens. Besides these, no distinction is made in this figure betwen primary and secondary cel wals. Row 2 shows a normal vegetative cel; row 3 shows cels at the end of the predivision elongation; row 4 shows the plane of cel division and aspect of the cels at the time of primary cel wal deposition; row 5 shows the mature cel plates; row 6 shows the post cytokinesis elongation of the cels; row 7 shows the juxtaposition of the resulting cels in a filament. S p ond ylosium pulchr um D esmidium sw ar tzii D esmidium baile yi 1 A B C D E F G H I J K 2 3 4 5 6 7 On y chonema filif or me H y alothec a dissiliens S p ond ylosium pulchellum T eilingia gr anula ta S p ond ylosium t etr agonum D esmidium gr e villii B ambusina b or r er i G r o enbladia ta ylor i D ela y ed 1? w all dep osition D ela y ed 1? w all dep osition R eplic a t e f old R eplic a t e f old Heimansia pusillaHyalotheca dissiliens Gr oenbladia taylorii Bambusina borr eri Desmidium gr evillii Onychonema moniliforme Spondylosium pulchrum Desmidium swartzii Spondylosium pulchellum Cosmocladium saxonicum Spondylosium tetragonum T eilingia granulata C? C C T T DDOBBHH Figure 4.4 Model cladogram showing distribution of cell division syndromes. Model based on the Maximum Likelihood topology with representative taxa. A diagnostic stage of cell division is shown on the right. Letters refer to the general type of cell division: B is the Bambusina -type, C is the Cosmarium -type, D is the Desmidium -type, H is the Hyalotheca -type, O is the Onychonema -type and T is the T eilingia -type. Characteristics shared by a lineage, such as a delay in primary wall deposition, are indicated on the model. Branches that are not strongly supported in the ML phylogeny are dashed. 98 Chapter 5. Systematic revision of some filamentous Desmidiaceae (Zygnematophyceae, Charophyta) Abstract Molecular phylogenetic and ontogenetic investigations of filamentous Desmidiaceae (Zygnematophyceae) indicated that some genera in this family are not monophyletic but that the discovered clades share structural and developmental synapomorphies. To bring the taxonomy, phylogeny and structure of these organisms into synchrony I propose a number of taxonomic changes. I have emended the genus Desmidium, and resurrected the genus Didymoprium. I also propose moving two species of Spondylosium to other genera, one to Desmidium pulchrum and the other to the newly created genus Isthmocatena. The characteristics of these newly circumscribed taxa are discussed as wel as the systematic value of various characteristics for predicting relatednes in the filamentous Desmidiaceae. Introduction Desmids are a group of mostly unicelular microalgae that are known for their structural diversity. Many thousands of species are thought to inhabit freshwater and semi-terestrial habitats on every continent. Although overshadowed by their unicelular relatives, a number of filamentous species exist, including the namesake Desmidium. Most filamentous genera contain fewer than fifty species (Gerath, 1993). However, these genera are structuraly diverse, being distinguished by the size and shape of their proceses, the presence or absence of granules, and the distribution of pores (Croasdale et al., 1983). Previous investigations discovered some diferences in the 99 proces of cel division as wel (Hauptfleisch, 1888; Gerath, 1973; Krupp and Lang, 1985a) (Chapter 4). In molecular phylogenetic analyses most of the genera were found to form a monophyletic group to the exclusion of genera of unicelular taxa and the filamentous Phymatodocis nordstedtiana (Gontcharov et al., 2003) (Chapter 4). Previous studies included few representatives of each genus and, with the exception of Chapter 4, did not addres the correlative structural diferences and synapomorphies of the groups found. Most unicelular Desmidaceae use the Cosmarium-type cel division (Picket- Heaps, 1972), as do some filamentous and colonial species (Spondylosium pulchelum, and probably Heimansia pusila and Cosmocladium saxonicum). This type of cel division involves the deposition of primary wal material before a stage of substantial celular elongation. The new als then expand to form the other half of the cel and secondary wal is deposited when the cel is almost full sized, at which point the primary wal is dehisced (Picket-Heaps, 1972). Other filamentous species use simple centripetal cel division (e.g., Hyalotheca), centripetal cel division with delayed formation of the separating wal (e.g., Teilingia and Spondylosium tetragonum), or some combination of a division vesicle and the deposition of cylinders of cel wal material (e.g., Onychonema, Desmidium and Bambusina) (Chapter 4). Studies of cel division indicate that the desmids are in fact structuraly and developmentaly diverse. Because of this structural diversity, taxonomy of desmids is particularly dificult. Desmidologists maintain a complex clasification of varieties and forms. Characteristics of varieties are thought to be heritable and these varieties are, generaly, thought to be incapable of interbreding. Characteristics of formae are thought 100 not to be heritable but may be induced by environmental factors. Disagrement among authors as to which characteristics are heritable and which are not as wel as which characteristics are sufficient for species diferentiation has led to a great deal of shuffling among the diferent ranks and, consequently, to a high degre of synonymy. An additional peculiarity of desmid taxonomy is that nomenclature of desmids has a starting point (Ralfs, 1848) later than that of most other algae, which is Linneaeus?s ?Species Plantarum? (1753). It was thought that Ralfs acounted for every desmid taxon reported at that time. As a result, al names of desmids published before 1848, including those published by Ralfs, were devalidated (Art. 13.1e Vienna Code, McNeil et al., 2006). In this study, we increased taxon sampling among the filamentous Desmidiaceae, compared to previous studies, to determine the phylogenetic relationships of as many species as possible. Because of previous studies, we hypothesized that the molecular phylogenetic relationships would be inconsistent with the traditional clasification. In order to beter predict relationships of unsampled species, we collected structural data including chloroplast shape and modes of cel division for the investigated taxa. The taxonomic implications are discussed and, in some cases, changes to the existing nomenclature are proposed. Materials and Methods Strains used in this study were requested from culture collections or isolated from the wild. A list of strains, their collection information and GenBank acesion numbers is provided in Table 5.1. Al strains were maintained in Guilard?s Woods Hole Medium or Bold?s Basal Medium (Nichols, 1973) enriched with 20 mL soil extract per liter of medium and kept in a growth chamber at 18? C under fluorescent lights with 30 ?E flux. 101 Strains were identified using Croasdale et al. (1983). DNA extraction and PCR amplification followed previously reported methods (Chapter 3). Fragments of the chloroplast rbcL and mitochondrial cox II genes were analyzed separately and then combined into a single dataset of 1962 characters and 60 taxa. Under the Maximum Parsimony criterion (MP), 100 random addition sequences were used in a heuristic search for the shortest tre in Paup* v. 4b10 (Swofford, 2003). Bootstrap support (BS) was estimated from 500 pseudoreplicates with 10 random addition sequences per pseudoreplicate. Under the Likelihood criterion (ML), the GTR+I+G model was used with ten random addition sequence replicates and TBR branch swapping in the heuristic search. One hundred bootstrap pseudoreplicates were generated in PhyML (Guindon and Gascuel, 2003) with the GTR model, 6 rate categories, and the invariant sites and gama distribution estimated from the data. Bayesian Inference (BI) was also employed as implemented in MrBayes v. 3.0b4 (Ronquist and Huelsenbeck, 2003). Four chains run for four milion generation (sampled every 100) with the first 2501 tres discarded as burnin were used to estimate the posterior probabilities (P). Two independent BI analyses converged on the same topology. In addition to molecular analysis, strains were investigated microscopicaly. Observations were made by DIC light microscopy on a Zeis Axioskop compound microscope (Zeis, Germany). Digital photomicrographs of the species were recorded using a Zeis AxioCam LCD camera (Zeis, Germany). To observe cel wal structure, cels were fixed in 3.5% glutaraldehyde for 1 hour and then dehydrated in an ethanol series. To observe timing of cel wal deposition, cels were fixed and then rinsed thre times in tap water, stained with 1% Calcofluor White for one hour and then rinsed twice 102 in tap water. Stained cels were observed on the same microscope but were iluminated with a broad-band mercury arc lamp. 103 T a bl e 5. 1 S t r a i ns i nve s t i ga t e d T a x o n S t r a i n r b c L c o x I I I L o c a t i o n B a m b u s i n a b o r r e r i J H 0 1 2 5 E F 3 7 1 2 8 4 E F 3 7 1 0 7 1 L a k e T o m o h a w k , W I B a m b u s i n a b o r r e r i J H 0 1 9 9 O x b o w L a k e , W I C l o s t e r i u m a c e r o s u m U T E X 1 0 7 5 E F 3 7 1 2 8 5 E F 3 7 1 0 7 2 I N C l o s t e r i u m e h r e n b e r g i i v a r . m a l i n v e r n i a n u m J H 0 0 1 3 E F 3 7 1 2 8 6 E F 3 7 1 0 7 3 L a k e A r t e m i s i a , M D C l o s t e r i u m l i b e l l u l a J H 0 0 2 1 E F 3 7 1 2 8 7 E F 3 7 1 0 7 4 J y m e B o g , W I D e s m i d i u m a p t o g o n u m J H 0 3 8 5 C a r o l i n e C o . , M D D e s m i d i u m a p t o g o n u m J H 0 3 8 7 C a r o l i n e C o . , M D D e s m i d i u m a p t o g o n u m S V C K 1 0 8 F i n l a n d D e s m i d i u m a p t o g o n u m v a r . e h r e n b e r g i i J H 0 1 8 4 E F 3 7 1 2 9 8 E F 3 7 1 0 8 5 L a k e T o m o h a w k , W I D e s m i d i u m a p t o g o n u m v a r . e h r e n b e r g i i J H 0 1 8 8 - - - - - - - - - - L a k e T o m o h a w k , W I D e s m i d i u m b a i l e y i J H 0 1 5 5 B i r d L a k e R o a d B o g , W I D e s m i d i u m b a i l e y i i J H 0 2 2 8 E F 3 7 1 2 9 9 E F 3 7 1 0 8 6 S p e n c e r L a k e , W I D e s m i d i u m g r e v i l l e i [ c y l i n d r i c u m ] S V C K 1 1 3 F i n l a n d D e s m i d i u m g r e v i l l e i J H 0 0 9 4 W a k e C o . , N C D e s m i d i u m g r e v i l l e i J H 0 0 9 6 W a s h i n g t o n C o . , O H D e s m i d i u m p s e u d o s t r e p t o n e m a J H 0 4 8 2 T h a i l a n d D e s m i d i u m p s e u d o s t r e p t o n e m a J H 0 5 1 3 T h a i l a n d D e s m i d i u m s w a r t z i i v a r . a m b l y o d o n J H 0 0 4 2 E F 3 7 1 2 9 7 E F 3 7 1 0 8 4 L a k e A r t e m i s i a , M D D e s m i d i u m s w a r t z i i v a r . a m b l y o d o n J H 0 1 1 2 J y m e B o g , W I D e s m i d i u m s w a r t z i i v a r . a m b l y o d o n J H 0 1 2 1 B u g L a k e , W I D e s m i d i u m s w a r t z i i v a r . a m b l y o d o n J H 0 1 3 6 H e m l o c k L a k e , W I D e s m i d i u m s w a r t z i i v a r . a m b l y o d o n J H 0 1 9 5 O x b o w L a k e , W I D e s m i d i u m s w a r t z i i v a r . s w a r t z i i J H 0 1 2 2 B u g L a k e , W I D e s m i d i u m s w a r t z i i v a r . s w a r t z i i J H 0 1 5 0 B i r d L a k e R o a d B o g , W I D e s m i d i u m s w a r t z i i v a r . s w a r t z i i J H 0 2 3 1 O n e i d a C o . W I G o n a t o z y g o n m o n o t a e n i u m U T E X 1 2 5 3 U 7 1 4 3 8 E F 3 7 1 0 9 2 M N G o n a t o z y g o n p i l o s u m A C O I 1 0 9 6 E F 3 7 1 3 0 3 E F 3 7 1 0 9 3 S e r r a d a E s t r e l a , P o r t u g a l G r o e n b l a d i a t a y l o r i i J H 0 3 3 9 S p e n c e r L a k e , W I H e i m a n s i a p u s i l l a S V C K 4 2 8 E F 3 7 1 2 9 1 E F 3 7 1 0 7 8 D e m i n g P o n d , M N H y a l o t h e c a d i s s i l i e n s J H 0 1 8 7 - - - - - - - - - - L a k e T o m o h a w k , W I H y a l o t h e c a d i s s i l i e n s S A G 3 8 4 - 2 A F 2 0 3 4 9 9 E F 3 7 1 0 9 5 ? H y a l o t h e c a m u c o s a J H 0 0 0 3 T u c k e r C o . , W V H y a l o t h e c a m u c o s a J H 0 0 5 5 E F 3 7 1 3 0 5 E F 3 7 1 0 9 6 W a s h i n g t o n C o . , O H H y a l o t h e c a m u c o s a J H 0 0 6 3 W a s h i n g t o n C o . , O H H y a l o t h e c a m u c o s a J H 0 4 1 5 C a r o l i n e C o . , M D 104 M i c r a s t e r i a s f o l i a c e a e N I E S 2 9 7 E F 3 7 1 3 1 1 E F 3 7 1 1 0 2 H i r o s h i m a , J a p a n M i c r a s t e r i a s r a d i a t a J H 0 0 6 4 E F 3 7 1 3 1 3 E F 3 7 1 1 0 4 W a s h i n g t o n C o . , O H M i c r a s t e r i a s r o t a t a U T E X 1 9 4 1 E F 3 7 1 3 1 2 E F 3 7 1 1 0 3 B e a v e r L a k e , B C , C a n a d a O n y c h o n e m a l a e v e v a r . m i c r a c a n t h u m J H 0 1 9 8 E F 3 7 1 3 1 8 E F 3 7 1 1 1 1 O x b o w L a k e , W I O n y c h o n e m a l a e v e v a r . m i c r a c a n t h u m J H 0 2 6 6 B i r d L a k e R o a d B o g , W I O n y c h o n e m a m o n i l i f o r m i s J H 0 4 2 0 - - - - - - - - - - C a r o l i n e C o . , M D P e n i u m m a r g a r i t a c e u m U T E X 6 0 0 E F 3 7 1 3 2 1 E F 3 7 1 1 1 4 ? P e n i u m s p i r o s t r i o l a t u m S V C K 2 0 5 E F 3 7 1 3 2 4 E F 3 7 1 1 1 7 H a m b u r g , G e r m a n y P h y m a t o d o c i s n o r d s t e d t i a n a J H 0 1 6 4 E F 3 7 1 3 2 5 E F 3 7 1 1 1 9 B i r d L a k e R o a d B o g , W I P h y m a t o d o c i s n o r d s t e d t i a n a S A G 4 7 . 8 9 A J 5 5 3 9 6 2 _ 1 E F 3 7 1 1 1 8 N a c o g d o c h e s , T X S p o n d y l o s i u m p u l c h e l l u m J H 0 3 6 8 S p e n c e r L a k e , W I S p o n d y l o s i u m p u l c h e l l u m S V C K 3 6 5 A F 2 0 3 5 0 5 E F 3 7 1 1 3 7 I r e l a n d S p o n d y l o s i u m p u l c h r u m J H 0 2 6 9 E F 3 7 1 3 4 1 E F 3 7 1 1 3 8 B i r d L a k e R o a d B o g , W I S p o n d y l o s i u m t e t r a g o n u m S V C K 4 4 0 A F 2 0 3 4 9 8 E F 3 7 1 0 9 4 D e m i n g P o n d , M N S t a u r a s t r u m a r c t i s c o n J H 0 0 1 4 E F 3 7 1 3 4 3 E F 3 7 1 1 4 0 L a k e A r t e m i s i a , M D S t a u r a s t r u m p o l y t r i c h u m f . b i s e r i a t u m J H 0 0 1 5 E F 3 7 1 3 4 4 E F 3 7 1 1 4 1 L a k e A r t e m i s i a , M D T e i l i n g i a g r a n u l a t a J H 0 1 4 0 E F 3 7 1 3 5 1 E F 3 7 1 1 4 8 H e m l o c k L a k e , W I T e i l i n g i a g r a n u l a t a S A G 2 5 . 8 8 - - - - - - - - - - M i r a , P o r t u g a l T e i l i n g i a g r a n u l a t a S A G 3 9 . 8 3 E F 3 7 1 3 3 5 E F 3 7 1 1 3 0 N e u s t i f t , A u s t r i a T e i l i n g i a g r a n u l a t a U T C C 2 8 4 A F 2 0 3 5 0 4 E F 3 7 1 1 3 1 B a b y L a k e , O N , C a n a d a T e i l i n g i a g r a n u l a t a [ S p o n d y l o s i u m p l a n u m ] S V C K 4 1 8 - - - - - - - - - - L a k e M u c u b a j , V e n e z u e l a T e i l i n g i a g r a n u l a t a [ S p o n d y l o s i u m s e c e d e n s ] S V C K 2 4 - - - - - - - - - - H u s u m , G e r m a n y N ot e s : A C O I , C oi m br a C ol l e c t i on of A l ga e ; N I E S , N a t i ona l I ns t i t ut e f or E nvi r onm e nt a l S t ud i e s ; S A G , S a m m l ung von A l ge nkul t u r e n de r U ni ve r s i t ? t G ?t t i nge n; S V C K , S a m m l ung v on C onj uga t e n - K ul t ur e n; U T E X , C ul t u r e C ol l e c t i on o f A l ga e a t U ni ve r s i t y of T e xa s ; U T C C , U ni ve r s i t y of T o r ont o C ul t u r e C ol l e c t i on o f A l ga e a nd C ya noba c t e r i a . V ouc he r s of s t r a i ns be gi nni ng w i t h J H a r e a va i l a bl e f r om t he a ut hor s . T a xa i n b r a c ke t s w e r e i nc or r e c t l y i de nt i f i e d i n c ul t ur e c ol l e c t i ons . 105 Results Likelihood analysis of the cox II fragment revealed a single lineage of filamentous and colonial species (87/92/.96; MP, ML, P). As in previous analyses (Gontcharov et al., 2003; Chapter 3), the filamentous species were split into two major clades, one containing Teilingia granulata, Spondylosium tetragonum, Cosmocladium saxonicum and two strains of Hyalotheca mucosa (analogous to Figure 5.1); and the other containing the remaining filamentous species. Spondylosium spp. were found in thre diferent strongly supported clades. Hyalotheca spp. were also found in two diferent clades, one sister to Groenbladia taylori and the other resolved separately. Desmidium grevilei was sister to Bambusina borreri and resolved separate from other Desmidium spp.. Relationships among species of Desmidium were generaly poorly resolved, although clades of D. baileyi (100/100/1.0), D. pseudostreptonema (99/100/1.0) and D. swartzi var. swartzi (83/97/1.0) were resolved with high support. Desmidium aptogonum, D. aptogonum var. ehrenbergii, and D. swartzi var. amblyodon were found to share similar primary sequences (Figure 1). Likelihood analysis of the rbcL dataset recovered a similar topology, although the (Teilingia, Cosmocladium) clade was sister to a clade of unicelular desmids with very litle support (-/-/-). Additionaly, the relative positions of Heimansia pusila and Spondylosium pulchelum were reversed with respect to the cox II and 2-gene dataset (Figure 5.1). The clade of Desmidium aptogonum, D. aptogonum var. ehrenbergii and D. swartzi var. amblyodon showed some unexpected strongly supported pairings including: D. aptogonum JH0387 with D. swartzi var. amblyodon JH0042 and JH0136 (96/97/1.0) 106 and D. swartzi var. ambylodon JH0112 with D. aptogonum JH0385. The gene fragments from D. aptogonum JH0385 and JH0387 were amplified and sequenced a second time and found to be correct. In spite of the strongly supported diferences among Desmidium spp., most relationships were congruous betwen datasets, so the two genes were concatenated into a single dataset and analyzed again. The estimated phylogeny was most similar to the cox II and the areas of conflict betwen the cox II and rbcL datasets were not strongly supported (i.e. the placement of Heimansia pusila (-/-/.71) and the relationships among strains in the Desmidium aptogonum, D. swartzi var. amblyodon clade (Figure 5.1). Based on this dataset, the filamentous forms are monophyletic with moderate support (67/84/1.0) and the two major lineages received stronger support than in the single-gene analyses. Results of the morphological investigation are summarized in Figure 5.2. Discusion Phylogenetic analyses often disagre with traditional systematic treatments. In the case of the conjugating gren algae, previous systematic treatment relied almost exclusively on structural characteristics of the cel wals (Croasdale et al., 1983), although a few treatments considered chloroplast shape (Carter, 1919b, 1919a; Teiling, 1952). My analysis combined observations on the cel shape, chloroplast and cel division characteristics of the organisms with a molecular phylogenetic approach and revealed congruence betwen the molecular phylogeny and the biology and morphology of the organisms. 107 Strains of Spondylosium were discovered in thre distinct lineages (Figure 5.1). This result is supported morphologicaly by the fact that these organisms have diferent modes of cel division (Chapter 4) (Figure 5.2). I recognize thre developmentaly disimilar organisms hat were previously placed in a single genus. These species share a number of characteristics with their sister taxa (Figure 5.2). Spondylosium tetragonum undergoes cel division much like Teilingia granulata and can be structuraly almost indistinguishable under certain environmental conditions. Spondylosium pulchrum, shares a mode of cel division very similar to that of Desmidium swartzi. Spondylosium pulchelum utilizes the Cosmarium-type cel division that is, most likely, plesiomorphic. Desmidium sensu lato is not monophyletic: D. grevilei is sister to Bambusina borreri. The phylogeny is partialy consistent with Ralfs? (1848) systematic treatment, in which Desmidium grevilei and Bambusina borreri were grouped in Didymoprium. Ralfs (1848) noted not only the diferences in cel shape, but also the diferences in the chloroplast shape, stating that in Desmidium (ie. D. swartzi), the number of lobes of the chloroplast corresponded to the number of angles of the cel, while in Didymoprium (which is oval, not angular, in apical view), this was not the case. Based on these data, that asertion is acurate. Desmidium swartzi and its kin can be distinguished from D. grevilei and Bambusina spp. by the shape of the chloroplast: Desmidium swartzi having bifurcate lobed chloroplasts where each lobe extends from a central mas into the angles of the cel, and Desmidium grevilei having an iregular, radiating lobed chloroplast, often with four lobes but sometimes more and never bifurcating, much like Bambusina borreri. In fact, the chloroplasts of D. grevilei are very much like those of Hyalotheca. These 108 two genera can be dificult to distinguish in field specimens, but careful observation of cel division should provide ample evidence of their identity. The discovery of two lineages of Hyalotheca spp. was initialy dismised as contamination, but repeated extractions resulted in identical sequences. Isolation of a second strain with similar phylogenetic afinities from a geographicaly diferent location suggests that this is not a contaminant but a biological or analytical artifact. I cannot, however, offer any quantitative structural diferences betwen these strains of Hyalotheca mucosa and other strains alied to H. disiliens. (Figure 5.1). Further investigation may reveal structural, physiological or developmental disparities. The fact that relationships among thre varieties of Desmidium (D. aptogonum, D. aptogonum var. ehrenbergii and D. swartzi var. amblyodon), were unresolved was not unexpected. In single gene datasets, most of the strains in this group shared very similar primary sequences and the greatest diference betwen any two of these strains in the combined dataset was 22 nucleotides (about 1%). It may be that these strains represent a species complex capable of interbreding, but it may also be that the molecular markers used are too conserved to resolve these relationships. The molecular markers used are often employed to estimate relationships among families and genera of angiosperms (Bel et al., 2005; Yoo et al., 2006). The relationships are not resolved and the apparent paterns are likely the result of lineage sorting. Other, faster evolving molecules would have to be sampled in order to test this hypothesis. In the case of filamentous Desmidiaceae, overal similarity of the organisms, particularly among Desmidium spp. and Spondylosium spp., has masked the underlying biological diferences. Cel division is one of the most important celular functions and 109 species with thre diferent kinds of cel division were asigned to Spondylosium. While gross structural characteristics may be sufficient for identification, developmental diferences may be hidden just below the intricately ornamented wals. Basic developmental and cytological characteristics of most species of conjugating gren algae are unknown. Authors rarely record chloroplast shape or pyrenoid position, much les aspects of cel division. Future systematic investigations would benefit greatly from careful observation of the organisms. By combining careful morphological observation and molecular phylogenetic data, it is possible to achieve a greater understanding of systematic relationships among these complex organisms. Systematic Revisions Evidence from my study and previous studies suggests that thre currently recognized genera are polyphyletic, resulting in nomenclatural ambiguities. Because of the strong agrement betwen the molecular phylogeny and the morphology of certain groups of filamentous Desmidiaceae, I venture to propose changes to an already dificult nomenclature. The proposed revisions center on the clasification of thre organisms: Desmidium grevilei, Spondylosium pulchrum, and S. pulchelum. It has been shown that Desmidium grevilei is only distantly related phylogeneticaly to other Desmidium species (Gontcharov et al., 2004; Figure 5.1). Desmidium swartzi was designated the type of the generic name Desmidium by N?geli (1849) so the generic name resides with the clade containing that species. The phylogenetic relationship betwen Bambusina borreri and Desmidium grevilei gives some taxonomic leway in the generic asignment. One could 110 reunite Desmidium grevilei and Bambusina borreri, thus reconstituting Didymoprium as circumscribed by Ralfs (1848). However, only a single species of Bambusina and Desmidium grevilei were available for morphological and molecular phylogenetic analysis and it would be premature to speculate on the phylogenetic placement of other species. In light of this uncertainty, I choose to resurrect the generic name Didymoprium for D. grevilei and maintain Bambusina as a separate genus. Further revision may be required as strains of Bambusina and Didymoprium are found and investigated. Spondylosium pulchrum was originaly described as a species Sphaerozosma, but was later asigned to Spondylosium because of the absence of apical proceses typical of the genus Sphaerozosma. The species is, however, rather unlike any other species of Spondylosium: it is much larger and uses the Desmidium-type cel division (Chapter 4) (Figure 5.2). Because its mode of cel division and chloroplast shape agre with Desmidium species, and its phylogenetic placement within the Desmidium clade is strongly supported (100/100/1.0), I propose to move this taxon to the genus Desmidium, as Desmidium pulchrum (Bailey ex Ralfs) J. D. Hal comb. nov. Although the exact phylogenetic position of Spondylosium puchelum is not strongly supported (-/70/*), it is clearly distinct from al other species of the genus for which I have molecular data. I have no molecular data for S. depresum, the lectotype of the generic name designated by Croasdale et al. (1983). This species is not known from North America is rarely reported from Europe. I have not sen any material that could be designated as the type material for that species. One of the first published images of the species showed a short filament with a single dividing cel (Pritchard, 1861, pl. II, fig. 9). This figure shows a simple wal across an elongated isthmus indistinguishable from 111 that which has been reported for Teilingia granulata and S. tetragonum (Figure 5.2). While S. depresum cannot conclusively be asigned to any group, the figure is inconsistent with the mode of cel division reported for Desmidium pulchrum (Bailey ex Ralfs) J. D. Hal comb. nov. (Spondylosium pulchrum) and Spondylosium pulchelum (Chapter 4) (Figure 5.2). Because the existing figures of Spondylosium depresum indicate that it has a mode of cel division unlike that of S. pulchelum, I propose to asign a diferent generic name to Spondylosium pulchelum, Isthmocatena pulchela (Archer) J. D. Hal comb nov. This new genus difers from the genus Spondylosium in its mode of cel division, general shape of the cel and the deep constriction at the isthmus. It difers from the structuraly similar cels of Euastrum and Cosmarium in that it forms filaments. It is unknown if other species currently asigned to Spondylosium are closely related to this species, but it sems very likely that some species of Cosmarium or Euastrum may be. These taxonomic changes resolve phylogenetic incongruence with the systematic treatment in the case of Desmidium and Spondylosium, however Hyalotheca mucosa was also shown to be polyphyletic. I defer taxonomic changes in this case until I have some indication of the structural diferences betwen the groups (if there are any), and to which group the name Hyalotheca should be asigned. Croasdale et al. (1983) designated Hyalotheca mucosa as the type of the generic name. Strains identified as H. mucosa were resolved in two diferent clades. It is possible that the apparent phylogenetic placement of Hyalotheca strains is an artifact of contamination, model mispecification or some more complex evolutionary proces such as organelar capture. The cultures appear to be fre of contamination, and a similar relationship was reported by Gontcharov 112 et al. (2003) for a diferent strain of Hyalotheca, creating an interesting if dificult conundrum. I had hoped to designate physical specimens as lectotypes for the thre species here treated. Suitable material was not found. If specimens identified by original authors were found, it would be appropriate to designate these as additional lectotypes. Although I am hesitant to designate any of my strains, or those from culture collections as epitypes because none are from the type localities, I believe al strains treated here are corectly identified and their structural characteristics are consistent with the names asigned. Desmidium Agardh ex Ralfs Cels form filaments connected by apical proceses; normaly broader than long; in apical view, cels oval, triangular, or multiangular; in apical view, chloroplasts lobed and bifurcate with one lobe extending into each angle of the cel; cel division involves formation of a division vesicle as wel as the deposition of a cylinder of cel wal material at each apical proces before a final period of elongation. Genus difers from Didymoprium and Bambusina in that the cels connect by apical proceses, that the number and position of division cylinders correlates to the apical proceses, and that the chloroplast is both bifurcate and the lobes extend into the angles of the cels. Desmidium pulchrum (Bailey ex Ralfs) J. D. Hall comb. nov. Basionym = Sphaerozosma pulchrum Bailey ex Ralfs 1848 Brit. Desmid.: 209 Synonym = Spondylosium pulchrum (Bailey ex Ralfs) W. Archer 1861: 724 Lectotype here designated = Ralfs 1848 Brit. Desm.: 209, pl. 35, fig. 2a, 2b Type location = West Point, NY; Princeton, NJ Comment. - Excludes Sphaerozosma pulchrum var. planum Wolle (1892 Desmids U.S.: 29, pl. 60, fig. 3,4) and Spondylosium pulchrum var. planum (Wolle) W. West & G. S. 113 West (1896 Trans. Lin. Soc. London, Bot. 5:231). Material held at BM (slide 11798) that was sent to Ralfs by Bailey was in too poor condition to designate as a type specimen, leaving only the image drawn by Ralfs as a possible lectotype. Didymoprium K?tz. ex Ralfs Cels form filaments connected along the apex; cels broader than long; in apical view, cels circular or somewhat eliptic, often with two smal protrusions on opposite sides of the cel; in apical view, chloroplasts lobed (usualy four or more) without bifurcations, chloroplast radiate from a central mas; cel division involving deposition of a single central cylinder of cel wal material (much like Bambusina borreri) without an inflated division vesicle. Difers from Bambusina in that the cels are broader than long and from Hyalotheca in that cel division involves the deposition of a cylinder of cel wal material. Type species designated here = Didymoprium grevilei Ralfs 1848 Brit. Desm.: 57 Didymoprium grevilei Ralfs 1848 Synonym = Desmidium cylindricum Grev. 1827 Scott. Crypt. Fl. 5; Pl. 293 - devalidated Synonym = Arthrodesmus cylindricus Menegh. 1840 Linnea 14: 204 - devalidated Synonym = Desmidium compresum Corda 1840 Alman. Carlsbad 10: 203 - devalidated Synonym = Didymoprium grevili K?tz. 1843 Phyc. Generalis: 166 (?) - ilegitimate Synonym = Didymoprium cylindricum (Grev.) Ralfs 1845 Ann. & Mag. Nat. Hist. 16: 10 - devalidated Synonym = Hyalotheca grevili Br?b. 1846 ? devalidated Type here designated = Ralfs 1848 Brit. Desm. 57, pl. 2, fig. a-k Type location here designated = Prussia; Prague; Carlsbad; Reichenberg; Falaise; New York, Rhode Island, USA 114 Comment: This species was described by Grevile (1827) as Desmidium cylindricum. K?tzing (1843) later moved it to his new genus Didymoprium, but in so doing unnecesarily changed the epithet to grevilei [grevili]. Ralfs (1845) restored the original epithet, naming the species Didymoprium cylindricum (Grev.) Ralfs, but in his starting-point monograph (Ralfs, 1848), he adopted K?tzing?s epithet. Because this monograph is the start date for desmid taxonomy, and K?tzing?s previous combination was invalid, Ralfs (1848) is the appropriate authority for the taxon Didymoprium grevilei. Atempts to locate Grevile?s materials were unsuccesful and I here designate the image from Ralfs (1848) as the lectotype of this species. Isthmocatena J. D. Hall gen. nov. Diagnosis: Genus novum Desmidiacearum. Celulae plerumque catenaformes, raro solae; sinus profundus; semicelulae trapeziformes compreses; ad regionem isthmum paululum inflatae; a vertice visae elipticae; divisio a Cosmarium similis. Cels forming short filaments, sometimes unicelular; individual cels deeply constricted, hemicels trapeziform and compresed; somewhat lobed near the isthmus; ovoid in apical view; surface finely punctate; chloroplast 4-lobed with the lobes extending from a mas above the isthmus toward the apex of the cel appearing furcated in face and apical view; cel division via the Cosmarium-type cel division. Etymology. Name refers to the median constriction found in these cels, and their tendency to form short filaments. Type species: Isthmocatena pulchela (Archer) J. D. Hal comb. nov. 115 Isthmocatena pulchela (Archer) J. D. Hall comb. nov. Basionym = Sphaerozosma pulchelum W. Archer 1858 Nat. Hist. Rev. 5 (Proc.): 253, pl. XI: fig. 7 Synonym = Spondylosium pulchelum (W. Archer) W. Archer 1861 History of Infusoria: 724 Synonym = Sphaerozosma secedens var. pulchelum (W. Archer) Hansgirg 1888 Prodr. Alg. Bohmen 1: 170 Type location here designated = Near Dublin, Ireland Lectotype here designated = Archer 1858 Nat. Hist. Rev. 5 (Proc.): 253, pl. XI: fig. 7 as Sphaerozosma pulchelum W. Archer Comment. ? I was not able to locate material from Archer?s collections that corresponded to this taxon. I designate the original drawing as the lectotype. Bambusina borreri (Ralfs) Cleve 1864 ?fvers. Forh. Kongl. Svenska Vetenskaps-Akad. 20: 496 This taxon should be added to the list of conserved taxa as it is the type for the conserved genus Bambusina Cleve 1864 l.c. 116 Table 5.2 List of names and synonyms Arthrodesmus Ehrenb. ex Ralfs A. cylindricus (Grev.) Menegh. (devalidated) Bambusina K?tz. B. boreri (Ralfs) Cleve Cosmarium Corda ex Ralfs Cosmocladium Br?b. C. saxonicum de Bary Desmidium C. Agardh ex Ralfs D. aptogonum (Ralfs) W. Archer D. aptogonum var. ehrenbergi K?tz. D. baileyi (Ralfs) Nordst. D. compressum Corda (devalidated) D. cylindricum Grev. (devalidated) D. grevilei Ralfs D. pseudostreptonema West & G.S. West D. pulchrum (Bailey ex Ralfs) J.D. Hall D. swartzi Ralfs D. swartzi var. amblyodon (Itzigs.) Rabenh. Didymoprium K?tz. ex Ralfs D. cylindricum (Grev.) Ralfs (devalidated) D. grevilei K?tz. ex Ralfs Euastrum Ehrenb. ex Ralfs Groenbladia Teiling G. taylori A.M. Scot & Gr?nblad Heimansia Coesel H. pusila (Hilse) Coesel Hyalotheca K?tz. ex Ralfs H. disiliens Ralfs H. grevilei Br?b. (devalidated) H. mucosa Ralfs Isthmocatena J.D. Hall I. pulchella (W. Archer) J.D. Hall Onychonema G.C. Wall. Phymatodocis Nordstedt Phymatodocis nordstedtiana Wole Sphaerozosma Corda ex Ralfs S. pulchrum Bailey ex Ralfs S. pulchrum var. planum Wole Spondylosium Br?b. ex K?tz. S. depressum Br?b. ex K?tz. S. pulchellum (W. Archer) W. Archer S. pulchrum (Bailey ex Ralfs) W. Archer S. pulchrum var. planum (Wole) West & G.S. West S. tetragonum West Teilingia Bourr. Teilingia granulata (J. Roy & Biset) Bourr. D. aptogonum JH0385 D. aptogonum JH0387 D. aptogonum SVCK 108 D. swartzii var. amblyodon JH0121 D. swartzii var. amblyodon JH0195 D. swartzii var. amblyodon JH0042 D. swartzii var. amblyodon JH0112 D. swartzii var. amblyodon JH0136 D. aptogonum var. ehrenbergii JH0184 D. aptogonum var. ehrenbergii JH0188 D. swartzii var. swartzii JH0122 D. swartzii var. swartzii JH0150 D. swartzii var. swartzii JH0231 D. pseudostreptonema JH0482 D. pseudostreptonema JH0513 D. baileyi JH0155 D. baileyi JH0228 Sp. pulchrum JH0269 D. grevillei JH0094 D. grevillei JH0096 D. grevillei SVCK 113 B. borreri JH0125 B. borreri JH0199 H. mucosa JH0055 H. mucosa JH0063 H. dissiliens JH0187 H. dissiliens SAG 384-2 Groenbladia taylorii O. laeve var. micracanthum JH0198 O. laeve var. micracanthum JH0266 O. filiforme Heimansia pusilla Sp.pulchellum SVCK 365 Sp. pulchellum JH0368 T. granulata SAG 39.83 T. granulata JH0140 T. granulata SVCK 418 T. granulata SVCK 24 T. granulata SAG 25.88 T. granulata UTCC 284 Sp. tetragonum SVCK 440 Sp. tetragonum JH0175 Sp. tetragonum JH0281 C. saxonicum H. mucosa JH0003 H. mucosa JH0415 Micrasterias rotata Micrasterias radiata Micrasterias foliacea Staurastrum arctiscon Staurastrum polytrichum Phymatodocis nordstedtiana SAG 47.89 Phymatodocis nordstedtiana JH0164 Penium margaritaceum Penium spirostriolatum Closterium acerosum Closterium libellula Closterium ehrenbergii var. malinvernianum Gonatozygon monotaenium Gonatozygon pilosum 0.01 substitutions/site 67/83/* -/-/- -/63/.98 */*/* */*/* */ */* 88/95 /* -/75/.98 */*/* */*/* */*/* */*/* */*/* * /*/* */*/* */*/* 99 /89/* -/72/.90 */*/* 72/95/* 71/73/* 88/97/* 95/95/* 72/75/.97 */96/* */*/* */*/* */*/* */*/* -/66/.55 99/98/* 99/*/* 89/99/* 67/84/* */96/* 98/98/* 96/98/* 91/ 94 /* 88/92/* 76/69/.98 77/88/* -/-/.71 -/-/.72 -/-/.52 65/80/* */*/* */*/* */*/* */*/* */*/* */ */* 57/56/.67 */*/* 82/90/* Figure 5.1. Phylogeny of the filamentous Desmidiaceae based on the ML tree from the rbcL and cox III combined analysis. Numbers above the branches are MP and ML BS and BI PP, respectively. Support of 100 (BS) and 1.0 (PP) are indicated with an aster- isk and support of less than 50 (BS) or less than 0.50 (PP) are indicated with a dash. Hyalotheca dissiliens Gr oenbladia taylorii Onychonema laeve Desmidium gr evillei Bambusina borr eri Spondylosium pulchrum Desmidium swartzii Heimansia pusilla Spondylosium pulchellum T eilingia granulata Spondylosium tetragonum Cosmocladium saxonicum Hyalotheca mucosa RadialSymmetry Shape Circular Circular Oval Circular Circular Oval Angular Oval Oval Oval Oval Oval Circular Division V esicle Delay Plastid Connection Apex Apex* Apex Apex* Apex Apex* Apex* Apex* Pore thread Apex*Process Process Primary** Lobed, Radial Lobed, Radial Lobed, RadialT aeniform H- Lobed Lobed, Bifurcate Lobed, Bifurcate H-Lobed (?) H-Lobed H-Lobed H-Lobed H-Lobed (?) Lobed, Radial Y es Y es Y es Y es Y es Y es Y es No No Y es Y es No Y es No No Y es No Y es Y es No No No No No No Hyalotheca Hyalotheca Onychonema Bambusina Bambusina Desmidium Desmidium Cosmarium Cosmarium T eilingia T eilingia Cosmarium Hyalotheca Radial Radial Radial Biradiate Radial Biradiate Biradiate Biradiate Biradiate Biradiate RadialBiradiate No Figure 5.2 Structural characteristics of species investigated. Shape = cell shape in apical view . Cells which are circular are generally subcircular or broadly oval, angular species may be 2 to 4-angled de pending on the species; Symmetry = symmetry of cell contents in apical view; Division = division type as described by Hall et al. 200 7a; V esicle = division involves formation of an inflated division vesicle; Delay = delay in deposition of primary wall material with r espect to the Cosmarium -type cell division; Plastid = plastid shape, H-lobed means that the chloroplast lobes extend from a centr al mass forming something like an ?H? in any view , (?) indicates uncertainty; Connection = point of connection between adjacent cel ls, all species so far investigated are held together by a shared primary wall of some form. *Consistent with light microscopical investigation but not confirme d by TEM **Connected by loosened primary walls (Coesel, 1993) 119 Chapter 6. Investigating diversity among structurally simple desmids, the Gonatozygaceae (Desmidiales, Zygnematophyceae) Abstract The family Gonatozygaceae is the structuraly simplest family of desmids. We investigated the phyletic and structural diversity in this family to determine if their structural simplicity masks their overal diversity. We found that phylogeneticaly distinct lineages of Gonatozygon kinahanii and G. brebisonii are structuraly similar. In the case of Gonatozygon kinahanii, these lineages can be distinguished based on traditional taxonomic characteristics, however this was not the case for G. brebisonii. We also found that Genicularia spirotaenia was embedded in Gonatozygon and the two available strains have diferent structural characteristics. Implications for the systematics and identification of these ecological indicator species are discussed. Introduction Before molecular sequence methods were commonly used, most microorganisms were asigned to species based on their physical appearance in the light microscope or their metabolic capabilities (bacteria and some fungi). Investigation of molecular sequence data suggests that many microorganisms have been overlooked by these methods (L?pez-Garc?a et al., 2001; Venter et al., 2004). Studies utilizing environmental sequencing methods have detected these novel sequences, but the organisms from which the sequences are derived are entirely unknown. In other cases, strains of known organisms that are structuraly similar show an unexpected degre of sequence diversity (Ciniglia et al., 2004). Investigations of molecular sequence diversity are stil in their 120 infancy and correlation betwen these sequences and the organisms from which they are derived has only just begun. Such correlation is necesary if we are to understand the limits of these diferent measures of diversity. In many cases, organisms that initialy sem structuraly indistinguishable are found to have cryptic peculiarities, sometimes subtle and other times profound. Among the eukaryotic algae, the diatoms and the desmids are known for their complexity of unicelular form. However, even among the many thousands of named desmids there are some that are structuraly simple. One family in particular, the Gonatozygaceae (Desmidiales, Zygnematophyceae), includes species that are cylindrical and without the proceses and lobes characteristic of other desmids. Only about 13 morphological species have ben described in the genera Gonatozygon and Genicularia. Other desmid families contain many more species: Closteriaceae, 140; Desmidiaceae, 3,000 (Gerath, 1993). Relationships among very few species belonging to the Gonatozygaceae have been investigated using molecular phylogenetic markers (Park et al., 1995). The paucity of structural characteristics makes identification of species dificult. Species in this family are, however, considered positive indicators of water quality (Coesel, 2001). Previous studies of cel wal structure and molecular phylogeny indicate that the Gonatozygaceae are an ancient lineage of the Desmidiales (Ruzicka, 1970; Mix, 1972; Park et al., 1996; McCourt et al., 2000; Gontcharov, Marin, and Melkonian, 2003) and that the zygnematalean genus Roya may be alied to this family (Park et al., 1996; McCourt et al., 2000; Gontcharov et al., 2003). Gonatozygon and Roya have similar axile chloroplasts, however, the wals of Roya are completely without pores or sutures 121 (Mix, 1972). Species of Gonatozygon, the most species-rich genus in the family, are diferentiated based on features of the cel wal, shape of the apex of the cels and the cel dimensions (Prescott et al., 1972). R??i?ka (1970) concluded that cel size was a poor taxonomic character because the dimensions of many of the varieties were highly variable and sometimes overlapping. Other diagnostic features of the cel shape vary: shape of the apex, the presence or absence of cel wal ornamentation. While this variability can be dificult to ases in wild populations because the variants would be asigned to diferent species, it is frequently observed in clonal cultures (Hal, personal observation). With so few structural features and so much variation within those observable features, asesment of identity and diversity becomes dificult. Molecular sequence data are not obscured by structural variability and can sometimes reveal phyletic diversity among structuraly simple or homogenous organisms. I hypothesize that because systematic treatments depend heavily on structural characteristics, the smal number of species of Gonatozygaceae may be an artifact of the structural simplicity of the cels. To test this hypothesis, I collected sequence data from several genes in 16 strains of Gonatozygon, Genicularia, and Roya including several representatives of each species. I also measured many observable characteristics of the cels including cel wal features, and cel dimensions. Materials and Methods Strains were isolated from the wild or requested from public culture collections (Table 6.1). These strains were maintained in either Guilards Woods Hole Medium, or 122 Bold?s Basal Medium enriched with soil water (Nichols, 1973) in a growth chamber at 18? C with cool white fluorescent lights at 30 ?E. Cultures were verified and checked for contamination at time of acesioning and when DNA was extracted. Cels were observed in living condition as wel as after several treatments for light and scanning electron microscopy (SEM). Cels were treated to remove extracelular materials and expose the surface of the cel wal. A six-hour treatment of 1% hypochlorite was found to remove the mucilage and bacteria from the surface without noticeably damaging the cel wal. After treatment, cels were rinsed thre times in medium to remove the hypochlorite. Morphological measurements were obtained from approximately 100 individual cels from strains investigated (Table 6.2). Al characters traditionaly used in systematic treatments were measured. Size measurements were rounded to the nearest hundredth of a micrometer. Width measurements for figure 6.4 and 6.5 were rounded to the nearest micrometer. Length measurements for figures 6.6 and 6.7 were binned into twenty- micrometer groups. For SEM, cels were first fixed in glutaraldehyde and then dehydrated in an ethanol series. The dehydrated cels were dried in a critical point dryer and mounted with double sided sticky tape and coated with Gold Paladium. Cels were observed at ~5-10 kv on a Hitachi S-4700 FESEM. DNA was extracted using the Nucleon Phytopure DNA extraction kit and the manufacturer?s protocol (Amersham, MA). Thre genes, rbcL, psaA and cox II were amplified from the total DNA using published primers (Chapter 3). Sequences were edited in Sequencher 4.0 (GeneCodes) and aligned by eye in MacClade v. 6.0 (Maddison 123 and Maddison, 2000). Outgroup sequences were downloaded from GenBank (Table 6.1). Each gene was analyzed individualy and then concatenated into a single dataset with 4,047 nucleotides. The concatenated dataset was analyzed under the Parsimony (MP) and Maximum Likelihood (ML) criteria in Paup* (Swofford, 2003), PhyML (Guindon and Gascuel, 2003) and under Bayesian Inference (BI) in MrBayes (Ronquist and Huelsenbeck, 2003). MP analysis was performed using a heuristic search with 100 random taxon addition sequence replicates and TBR branch swapping. Five hundred bootstrap pseudoreplicates were used to estimate the support for taxon bipartitions. An optimal ML tre was discovered in Paup* from a heuristic search with thre random taxon addition sequence replicates, and TBR branch swapping under the GTR+I+G model with al parameters estimated from the data. Bootstrap analysis was performed in PhyML using the GTR+I+G model with the proportion of invariant sites and the alpha parameter estimated from the data and empirical base frequencies. Markov chains were employed in MrBayes for 4M generations using the GTR+I+G model with the first 2501 tres discarded as burnin. The same topology was recovered in two independent BI analyses. 124 T a bl e 6. 1 S t r a i ns i nve s t i ga t e d a nd G e nba nk num be r s T a x o n A u t h o r i t y S t r a i n r b c L p s a A c o x I I I Z y g n e m a t o p h y c e a e D e s m i d i a l e s D e s m i d i a c e a e B a m b u s i n a b o r r e r i ( R a l f s ) C l e v e J H 0 1 9 9 E F 3 7 1 2 8 3 E F 3 7 1 1 7 7 E F 3 7 1 0 7 0 C o s m a r i u m b o t r y t i s ( M e n e g h i n i ) R a l f s U T E X 3 0 1 E F 3 7 1 2 8 8 E F 3 7 1 1 8 2 E F 3 7 1 0 7 5 D e s m i d i u m s w a r t z i i A g a r d h e x R a l f s ? J H 0 0 4 2 E F 3 7 1 2 9 7 E F 3 7 1 1 9 1 E F 3 7 1 0 8 4 H a p l o t a e n i u m ( P l e u r o t a e n i u m ) m i n u t u m ( R a l f s ) B a n d o S V C K 3 0 2 E F 3 7 1 3 2 6 E F 3 7 1 2 2 7 E F 3 7 1 1 2 0 H e i m a n s i a ( C o s m o c l a d i u m ) p u s i l l u m ( H i l s e ) C o e s e l S V C K 4 2 8 E F 3 7 1 2 9 1 E F 3 7 1 1 8 5 E F 3 7 1 0 7 8 M i c r a s t e r i a s r o t a t a ( G r e v i l l e ) R a l f s U T E X 1 9 4 1 E F 3 7 1 3 1 2 E F 3 7 1 2 1 0 E F 3 7 1 1 0 3 O n y c h o n e m a l a e v e v a r . m i c r a c a n t h u m N o r d s t e d t J H 0 1 9 8 E F 3 7 1 3 1 8 E F 3 7 1 2 1 8 E F 3 7 1 1 1 1 P h y m a t o d o c i s n o r d s t e d t i a n a W o l l e S A G 4 7 . 8 9 A J 5 5 3 9 6 2 _ 1 E F 3 7 1 2 2 5 E F 3 7 1 1 1 8 S t a u r o d e s m u s c o n v e r g e n s [ A r t h r o d e s m u s s p . ] ( E h r e n b e r g ) T e i l i n g U T E X 2 5 0 8 E F 3 7 1 2 8 1 E F 3 7 1 1 7 5 E F 3 7 1 0 6 8 P e n i a c e a e P e n i u m c f . d i d y m o c a r p u m L u n d e l l J H 0 2 1 2 E F 3 7 1 3 1 6 E F 3 7 1 2 1 6 E F 3 7 1 1 0 9 P e n i u m c y l i n d r u s ( E h r e n b e r g ) B r ? b i s s o n e x R a l f s A C O I 7 8 0 E F 3 7 1 3 2 0 E F 3 7 1 2 2 0 E F 3 7 1 1 1 3 P e n i u m m a r g a r i t a c e u m ( E h r e n b e r g ) B r ? b i s s o n e x R a l f s U T E X 6 0 0 E F 3 7 1 3 2 1 E F 3 7 1 2 2 1 E F 3 7 1 1 1 4 P e n i u m s p i r o s t r i o l a t u m B a r k e r S V C K 2 0 5 E F 3 7 1 3 2 4 E F 3 7 1 2 2 4 E F 3 7 1 1 1 7 C l o s t e r i a c e a e C l o s t e r i u m a c e r o s u m ( S c h r a n k ) E h r e n b e r g e x R a l f s U T E X 1 0 7 5 E F 3 7 1 2 8 5 E F 3 7 1 1 7 9 E F 3 7 1 0 7 2 C l o s t e r i u m e h r e n b e r g i i v a r . m a l i n v e r n i a n u m ( D e N o t a r i s ) R a b e n h o r s t J H 0 0 1 3 E F 3 7 1 2 8 6 E F 3 7 1 1 8 0 E F 3 7 1 0 7 3 C l o s t e r i u m l i b e l l u l a F o c k e J H 0 0 2 1 E F 3 7 1 2 8 7 E F 3 7 1 1 8 1 E F 3 7 1 0 7 4 S p i n o c l o s t e r i u m c u s p i d a t u m ( B a i l e y ) H i r a n o N I E S 3 2 5 S C U 5 5 3 9 6 5 E F 3 7 1 2 4 0 E F 3 7 1 1 3 2 G o n a t o z y g a c e a e G e n i c u l a r i a s p i r o t a e n i a e d e B a r y S A G 5 4 . 8 6 G S P 5 5 3 9 4 6 E F 3 7 1 1 9 7 E F 3 7 1 0 9 0 G o n a t o z y g o n b r e b i s s o n i i d e B a r y S V C K 2 1 0 G o n a t o z y g o n b r e b i s s o n i i d e B a r y J H 0 3 7 5 G o n a t o z y g o n b r e b i s s o n i i d e B a r y J H 0 3 7 7 G o n a t o z y g o n b r e b i s s o n i i v a r . l a e v e ( H i l s e ) W e s t & W e s t J H 0 0 3 3 E F 3 7 1 3 3 2 E F 3 7 1 2 3 3 E F 3 7 1 1 2 6 G o n a t o z y g o n k i n a h a n i i ( A r c h e r ) R a b e n h o r s t A C O I 3 5 0 G K 1 5 5 3 9 4 5 E F 3 7 1 1 9 8 E F 3 7 1 0 9 1 G o n a t o z y g o n k i n a h a n i i ( A r c h e r ) R a b e n h o r s t U T E X 2 4 7 1 125 G o n a t o z y g o n k i n a h a n i i v a r . m a j u s T a y l o r J H 0 3 5 6 G o n a t o z y g o n m o n o t a e n i u m d e B a r y U T E X 1 2 5 3 U 7 1 4 3 8 E F 3 7 1 1 9 9 E F 3 7 1 0 9 2 G o n a t o z y g o n m o n o t a e n i u m d e B a r y S V C K 1 0 8 G o n a t o z y g o n m o n o t a e n i u m d e B a r y J H 0 2 8 2 G o n a t o z y g o n p i l o s u m W o l l e A C O I 1 0 9 6 E F 3 7 1 3 0 3 E F 3 7 1 2 0 0 E F 3 7 1 0 9 3 G o n a t o z y g o n p i l o s u m W o l l e S V C K 6 4 Z y g n e m a t a l e s M e s o t a e n i a c e a e C y l i n d r o c y s t i s b r e b i s s o n i i M e n e g h i n i U T E X 1 2 5 9 E F 3 7 1 2 9 3 E F 3 7 1 1 8 7 E F 3 7 1 0 8 0 M e s o t a e n i u m s p . J H 0 0 3 1 E F 3 7 1 3 1 0 E F 3 7 1 2 0 8 E F 3 7 1 1 0 1 N e t r i u m d i g i t u s ( E h r e n b e r g ) I t z i g s o n & R o t h e U T E X 5 6 1 U 3 8 6 9 8 E F 3 7 1 2 1 4 E F 3 7 1 1 0 7 R o y a a n g l i c a W e s t U T E X 9 3 4 A J 5 5 3 9 6 3 E F 3 7 1 2 3 1 E F 3 7 1 1 2 4 " R o y a a n g l i c a " W e s t ? U 3 8 6 9 4 - - - - - - - - - - - - - - - - - - - - R o y a o b t u s a ( B r ? b i s s o n ) W e s t & W e s t S A G 1 6 8 . 8 0 E F 3 7 1 3 3 1 E F 3 7 1 2 3 2 E F 3 7 1 1 2 5 R o y a o b t u s a v a r . m o n t a n u m W e s t J H 0 3 5 7 Z y g n e m a t a c e a e M o u g e o t i a s p . U T E X 7 5 8 A F 4 0 8 2 5 2 E F 3 7 1 2 1 2 E F 3 7 1 1 0 5 S p i r o g y r a m a x i m a ( H a s s a l l ) W i t t r o c k U T E X 2 4 9 5 D Q 1 5 9 4 1 E F 3 7 1 2 4 4 E F 3 7 1 1 3 6 Z y g n e m a c y l i n d r i c u m T r a n s e a u S A G 6 8 9 - 2 E F 3 7 1 3 5 7 E F 3 7 1 2 6 2 E F 3 7 1 1 5 5 Z y g n e m o p s i s m i n u t a R a n d h a w a A C O I 6 0 E F 3 7 1 3 6 3 E F 3 7 1 2 6 8 E F 3 7 1 1 6 1 Z y g o g o n i u m t u n e t a n u m G a u t h i e r - L i ? v r e U T C C 1 3 6 A F 2 0 3 5 0 9 E F 3 7 1 2 6 9 E F 3 7 1 1 6 2 O u t g r o u p C o l e o c h e a t a l e s C o l e o c h a e t e s c u t a t a B r ? b i s s o n S A G 3 . 9 A Y 0 8 2 3 2 4 . 1 E F 3 7 1 2 7 3 E F 3 7 1 1 6 6 C o l e o c h a e t e n i t e l l a r u m J o s t U T E X 1 2 6 1 A Y 0 8 2 3 2 5 . 1 E F 3 7 1 2 7 5 E F 3 7 1 1 6 8 C o l e o c h a e t e d i v e r g e n s P r i n g s h e i m 3 0 0 d 1 A Y 0 8 2 3 2 4 . 1 E F 3 7 1 2 7 2 E F 3 7 1 1 6 5 C o l e o c h a e t e p u l v i n a t a B r a u n e x K ? t z i n g 5 7 b 6 A Y 0 8 2 3 0 7 . 1 E F 3 7 1 2 7 4 E F 3 7 1 1 6 7 C o l e o c h a e t e s i e m i n s k i a n a S z y m a n s k a 1 0 d 1 A F 4 0 8 7 9 1 . 1 E F 3 7 1 2 7 7 E F 3 7 1 1 7 0 126 T a bl e 6. 2 S t r uc t u r a l c ha r a c t e r i s t i c s of s om e G onat oz y gon s pp. T a xon L e ngt h ( ? m ) W i dt h ( ? m ) A pe x ( ? m ) P yr e noi ds L : W G . br e bi s oni i J H 0375 119. 92 ? 23. 38 6. 32 ? 0. 65 4. 03 ? 0. 46 5. 89 ? 1. 93 19. 16 ? 0. 02 G . br e bi s s oni i S V C K 114. 87 ? 32. 97 6. 63 ? 0. 89 4. 43 ? 0. 51 5. 78 ? 1. 85 17. 60 ? 5. 41 G . k i nahani i va r . m aj us J H 0356 291. 90 ? 68. 30 19. 22 ? 1. 74 N A N A 11. 66 ? 2. 98 15. 21 ? 0. 01 G . k i nahani i A C O I 180. 92 ? 33. 48 12. 16 ? 1. 65 11. 32 ? 1. 55 6. 67 ? 1. 61 15. 15 ? 0. 02 G . k i nahani i U T E X 2495 526. 73 ? 162. 13 14. 12 ? 1. 79 13. 83 ? 1. 84 13. 64 ? 4. 37 37. 61 ? 0. 01 127 Results Phylogenetic relationships In the combined ML analysis, Gonatozygon and Genicularia were recovered as a monophyletic group with very strong support (99/100/1.0; MP/ML/P) (Figure 6.1). Most Roya spp. were found as monophyletic with strong support (100/100/1.0), however, the placement of the Roya clade was not supported (-/-/-). The ML tre found in individual gene analyses showed Roya sister to the Gonatozygaceae and Peniaceae (rbcL, cox II) or sister to the Peniaceae and Desmidiaceae (psaA), but not as a monophyletic group with the Gonatozygaceae (data not shown). In the combined analysis, the nine most parsimonious tres difered in the placement of Roya (never monophyletic with Gonatozygaceae), the placement of Netrium digitus and relationships among the Desmidiaceae (data not shown). When al thre genes were concatenated, likelihood analyses resolved Roya as sister to a clade of Desmidiales excluding Gonatozygaceae with litle support (Figure 6.1). Roya spp. were found to be monophyletic and Roya obtusa and its variety montana were sibling taxa (Figure 6.1). The earliest branching member of the Gonatozygaceae, was Gonatozygon kinahanii var. majus. This strain was paraphyletic to the typical G. kinahanii UTEX 2471, ACOI 350 (Figure 6.1). Additionaly, G. brebisonii SVCK 210 was found to be paraphyletic with respect to other isolates of that species as wel as the variety laeve. The dubious strain ?Roya anglica U38694? appeared sister to a clade of G. monotaenium and G. pilosum. Genicularia spirotaenia was sister to two strains of Gonatozygon pilosum with moderate support (76/68/0.93), and included in a larger group with G. monotaenium with high support (100/100/1.0). 128 Structural investigation Celular characteristics of living and treated specimens were investigated by light microscopy. Most strains exhibited structural characteristics consistent with published work. The genus Gonatozygon is characterized by flat chloroplasts, however, al strains investigated with the exception of varieties of G. kinahanii, exhibited a slight ridge down the center of the chloroplast (Figure 6.2a, c). Cels sometimes exhibited several large ridges that extended the length of the chloroplast and appeared similar to the plastids of Penium spp., this was particularly true of G. brebisonii var. laeve (Figure 6.2b). Most species exhibited a series of pyrenoids aranged linearly down the center of the chloroplast (Figure 6.2a, c, f), however, G. kinahanii var. majus had pyrenoids aranged somewhat iregularly in the chloroplast (Figure 6.2d) as was reported by Taylor (1934). In living specimens, the cel wal of G. brebisonii var. laeve appeared smooth (data not shown). A strain designated Genicularia spirotaenia SAG 54.86was also included in the study. The cel wal features of this strain were similar to Gonatozygon monotaenium. The chloroplast appeared to be either entire or a somewhat reticulate network (data not shown). A second strain, Genicularia spirotaenia SVCK 329, was obtained. This strain difered from the included strain at only two nucleotide positions (data not shown). In this strain, however, the chloroplast appeared to be two long chloroplasts (or possibly lobes of a single chloroplast) that joined somewhere near the mid region. Although mostly parietal, the chloroplast crossed the center of the cel (data not shown). Investigation of the cel wals of the Gonatozygaceae revealed a number of previously unidentified characteristics. Cel wals of Roya spp. appeared to be completely without pores or granules (Figure 6.3g). This condition was also true of 129 Gonatozygon kinahanii and G. kinahanii var. majus (Figure 6.3d). G. monotaenium had smal granules (Figure 6.3a). These granules were very short and sometimes acute, appearing as very short spines in living specimens (Figure 6.2a). Granules in G. monotaenium are unlike the spines found in G. pilosum, which are only slightly longer, but normaly atenuated into short spines of iregular length (Figure 6.3b). Gonatozygon monotaenium JH0282, difered from the other strains in that the granules were larger and iregularly shaped (visible at the end of the cel in Figure 6.3A). These granules could perhaps be beter refered to as verucae. Al strains of G. brebisonii had granulate cel wals (Figure 6.2c, 6.3c). However, the granules of G. brebisonii var. laeve were so fine that the cel wals appeared smooth in living cels (Figure 6.3e). Additionaly, treatment with hypochlorite revealed transverse sutures in G. brebisonii and G. brebisonii var. laeve (Figure 6.3e, 6.3f). In cases where taxa could not be distinguished using structural characteristics, cel size was investigated. Length and width measurements were made in G. kinahanii and G. brebisonii. Cel width was sufficient to distinguish betwen the G. kinahanii var. kinahanii and G. kinahanii var. majus, (Figure 6.4) but the width distributions in G. brebisonii were most overlapping (Figure 6.5) and the two strains had a similar mean width (Table 6.2). Strains could not be distinguished based on cel length (Figure 6.6, 6.7). Cel length was much more variable than cel width, particularly in G. kinahanii UTEX 2471. Discusion The Gonatozygaceae is the most structuraly simple family of desmids. 130 To test the correlation betwen structural and genetic diversity, I considered the structural characteristics of the strains in the context of a molecular phylogeny. This combined approach revealed more structural diversity than was previously reported and helped distinguish at least one previously unrecognized lineage of Gonatozygon brebisonii. Based on these genes, every strain had unique primary sequence with the exception of two strains of G. brebisonii (JH0375, JH0377) collected from the same pond on the same day and two strains of G. pilosum (ACOI 1096 and SVCK 64) from Portugal and Norway, respectively. While JH0375 and JH0377 are very likely to be vegetative clones of one another, the strains of G. pilosum are les likely to be. Shared primary sequence, in this case, does not necesarily indicate that the organisms are of the same clonal strain. The genes used to estimate the phylogeny are very conservative and may not be informative at the species or subspecies level. The phylogeny does indicate a surprising degre of genetic divergence among strains of G. brebisonii and among strains of G. kinahanii. This finding supports the hypothesis that the structural simplicity of the group may have resulted in an underestimation of their genetic (and probably species) diversity. Placement of the Gonatozygaceae among the conjugating gren algae is not controversial. Both structural investigation and molecular phylogenetic analyses are consistent with the family being an early branching member of the Desmidiales (Ruzicka, 1970; Mix, 1972; Park et al., 1996; McCourt et al., 2000; Gontcharov et al., 2003). Placement of Roya is les certain. Gontcharov et al. (2003) found Roya sister to al Desmidiales or part of a clade including Netrium digitus. My analyses indicate that Roya is part of the Desmidiales to the exclusion of Netrium digitus and the best ML tre shows 131 that Roya may be more closely related to other Desmidiales than the Gonatozygaceae (Figure 6.1). However, no one placement is strongly supported. The GenBank acesion ?Roya anglica? U38694 is unlikely to be derived from a Roya spp. and is certainly not from the strain of Roya anglica investigated (as indicated by the information in GenBank). This was previously reported by Gontcharov et al. (2004). Low bootstrap support for the placement of Roya could be the result of biases in the molecular sequence data, biases induced by poor taxon sampling or model mispecification, or could represent a genuine biological phenomenon (e.g., rapid radiation). Bootstrap analyses under the parsimony criterion did not support the placement of Roya spp. (data not shown) indicating that model mispecification is an unlikely cause of the ambiguity. While I cannot eliminate the possibility of bias in the genes selected, previous studies also failed to confidently place Roya (Gontcharov et al., 2003). Perhaps greater taxon sampling among the basal lineages of the Desmidiales wil provide beter support for the placement of Roya. Notwithstanding, Roya spp. are structuraly similar to Gonatozygon. They have an axile taeniform chloroplast and their cel wal is smooth like that of G. kinahanii. There may be, however, structural or compositional diferences in the cel wals. Gonatozygon was clasified among the Desmidiales because of its cel wal characteristics, namely the presence of an extracelular electron dense matrix that harbors pores, spines and granules (Mix 1972). Roya spp. were investigated by light microscopy and SEM and found to lack granules and pores (data not shown). Among the Gonatozygaceae, phylogenetic analysis revealed a group containing Gonatozygon pilosum, G. monotaenium and Genicularia spirotaenia as wel as 132 paraphyletic asemblages of Gonatozygon brebisonii and G. kinahanii (Figure 6.1). The identity of the Genicularia spirotaenia strain is uncertain as its structural characteristics do not exactly match the description of that species. Interestingly, there is as much nucleotide diference among strains of Gonatozygon monotaenium as betwen Genicularia spirotaenia and Gonatozygon pilosum (p-dist = 0.0368 vs 0.0283). Perhaps G. monotaenium JH0282 represents a diferent variety or a diferent species, a hypothesis supported by the diference in the shape of the granules (Table 6.2). Two lineages of G. brebisonii were resolved. Cels of these strains were structuraly similar and basic celular measurements revealed no quantitative diferences (Table 6.2). The diference in the mean length (115 ?m vs. 120 ?m) would probably not be diagnostic in wild material. It is dificult to distinguish these strains structuraly, however, they could be distinguished based on nucleotide sequence diferences. Gonatozygon brebisonii is one of the most common species of the genus and its characteristics are particularly variable (Ruzicka, 1970). It may be that G. brebisonii as currently circumscribed includes a number of species. These data also suggest that at least some of these species may be structuraly indistinguishable. Many more strains of G. brebisonii would be needed to test this hypothesis. Two strains used in this study were inconsistent with the name asigned to them in public culture collections. Gonatozygon pilosum SVCK 64 was asigned to G. aculeatum in the culture collection, however, the strain has hairs very similar to G. pilosum ACOI 1096 (Figure 6.3b) and not like those of G. aculeatum (Figure 6.2e). While it is possible for the hairs of G. pilosum to be aculeate, G. aculeatum is characterized by a combination of spine shape and size inconsistent with those of strain 133 SVCK 64. Moreover, this strain was unusual in that fewer than 10% of the cels displayed the expected structural features. Cels taken from the same culture vesel had mostly smooth wals, but a few cels showed both smooth and pilose sections of wal on the same cel indicating that this is a peculiarity of the strain and not a case of a mixture of two diferent strains. The second strain, G. kinahanii UTEX 2471, was labeled Mougeotia sp. Paradoxicaly, these two genera are sometimes dificult to distinguish, yet are only very distantly related (McCourt et al., 2000; Gontcharov et al., 2003) (Chapter 3). In this case, the strain can be identified as G. kinahanii because adjacent cels in the filament do not share any wals. This is also true of other strains of G. kinahanii investigated, and inconsistent with the structure of filaments of Mougeotia. Because of the similarity of G. kinahanii and Mougeotia one must consider the posibility that G. kinahanii (or species structuraly similar to it) is more common than is reported. It is possible that a number of species currently asigned to Mougeotia belong to Gonatozygon. If correct, such confusion would require a revaluation of the use of Mougeotia and Gonatozygon as indicators of water quality. In general, structural characteristics were discovered which correlated wel with the molecular signatures of the strains investigated. In most cases, these characteristics were sufficient to distinguish taxa. The resolved relationships can also be reconciled with the taxonomy of the strains, except in the case of G. kinahanii var. major. This species is found to be paraphyletic to the typical variety and could probably be best treated as a species unto itself. The other two varieties, G. brebisonii var. laeve and Roya obtusa var. montana, showed some sequence divergence from their respective typical varieties, 134 but their phylogenetic positions are not inconsistent with their nomenclature and no changes are proposed. Al characteristics, including cel dimensions, have to be employed to distinguish some strains of Gonatozygon. Only in the case of G. brebisonii are the structural characteristics insufficient to distinguish among the resolved clades. In the case of the Gonatozygaceae, it is clear that there has been an underestimate of the phyletic and probably species diversity. It is not clear why there is so litle structural diference betwen distantly related species. Perhaps the shape is highly constrained by genetic predisposition or, perhaps, there are environmental or physiological constraints on the cel shape. If other structuraly simple organisms are as phyleticaly diverse as the Gonatozygaceae, there may be many more species than are currently recognized. Roya obtusa var. montanum Roya obtusa Roya anglica Closterium libellula Closterium ehrenbergii var. malinvernianum Closterium acerosum Spinoclosterium cuspidatum Penium margaritaceum Penium spirostriolatum Micrasterias rotata Haplotaenium minutum Cosmarium botrytis Staurodesmus convergens Desmidium swartzii Bambusina borreri Onychonema laeve var. micrcanthum Heimansia pusilla Phymatodocis nordstedtiana Penium didymocarpum Gonatozygon kinahani JH0356 Gonatozygon brebissonii JH0375 Gonatozygon brebissonii JH0377 Gonatozygon brebissonii var. laeve Gonatozygon brebissonii SVCK 210 Genicularia spirotaenia Gonatozygon pilosum ACOI 1096 Gonatozygon pilosum SVCK 64 Gonatozygon monotaenium UTEX 1253 Gonatozygon monotaenium SVCK 108 Gonatozygon monotaenium JH0282 Gonatozygon kinahani ACOI 350 Gonatozygon kinahani UTEX 2471 Zygnemopsis minuta Zygogonium tunetanum Zygema cylindrus Mesotaenium sp. JH0031 Cylindrocystis brebissonii Mougeotia sp. UTEX 758 Netrium digitus Spirogyra maxima Coleochaete divergens Coleochaete scutata Coleochaete nitellarum Coleochaete pulvinata Coleochaete sieminskiana 0.05 substitutions/site Penium cylindrus Roya anglica U38694 */*/* */98/* */99/* 63/81/* 63/64/* 51/-/.97 -/-/.64 -/-/- 71/-/* 89/87/.92 */*/* */*/* */*/* */*/* */*/* 79/68/.93 95/97/* 98/98 /* 73/86/* -/65/.99 */*/**/*/* */*/* */*/* 88/98/* 99/*/* 67/74/* */*/* */*/* */*/* */*/* 58/79/* 92/*/* */*/* 51/80/* -/-/.79 51/93/* -/72/* -/-/.81 98/96/* */98/* -/-/- 92/82/* 99/*/* Figure 6.1. Inferred phylogeny of Gonatozygaceae. Maximum likelihood tree from analysis of the genes rbcL, psaA and coxIII. Numbers above branches are MP and ML BS and Bayesian PP, respectively. Values of 100 BS and 1.0 PP are indicated with and asterisk (*). Values less than 50 BS and 0.50 PP are indicated with a dash (-). 136 Figure 6.2. Light micrographs showing habit of Gonatozygon and Roya. A. Gonatozygon monotaenium; B. G. brebisonii var. laeve; C. G. brebisonii; D. G. kinahanii var. majus; E. G. aculeatum; F. Roya obtusa var. montanum. Scale bars are 25 micrometers long. A C E D F B 138 Figure 6.3. Light micrographs showing cel wals of Gonatozygon and Roya. Cels treated with hypochlorite to show surface ornamentation. A. G. monotaenium; B. G. pilosum; C. G. brebisonii; D. G. kinahanii var. majus; E. G. brebisonii var. laeve; F. G. brebisonii var. laeve; G. Roya anglica. Scale bars are 25 micrometers long. A C E D F G 140 Figure 6.4. Comparison of width of Gonatozygon kinahanii strains from about 100 randomly sampled cels. Figure 6.5. Comparison of width of Gonatozygon brebisonii strains from about 100 randomly sampled cels. Figure 6.6 Comparison of length of Gonatozygon kinahanii strains from about 100 randomly sampled cels. Figure 6.7. Comparison of length of Gonatozygon brebisonii strains from about 100 randomly sampled cels. G. brebissoniiG. kinahani G. brebissoniiG. kinahani Length (um) Number of cells Length (um) Number of cells Width (um) Number of cells Width (um) Number of cells SVCK 210 ACOI 350 UTEX 2471 JH0356 ACOI 350 UTEX 2471 JH0356 JH0375 SVCK 210 JH0375 100 1 3 0 5 10 15 20 25 30 40 50 60 70 80 90 100 35 4 75 9 11 10 13 16 19 22 25 28 50 0 5 10 15 25 20 30 35 40 75 100 125 150 175 200 225 250 0 5 10 15 20 25 30 300 500 700 900 1100 5 Figure 6.4 Figure 6.5 Figure 6.6 Figure 6.7 142 Chapter 7. Investigation of the evolutionary history of Triploceras gracile (Desmidiaceae). Abstract Shifts in celular organization are thought to be important events in the evolution of complex forms. Among conjugating gren algae, shifts from radial to bilateral symmetry are thought to be few and organisms are often asigned to genera based, in part, on the degre of symmetry they display. Triploceras gracile is unusual because the middle portions of the cel wal display radial symmetry while the apex is bilateraly symmetric. Previous analysis of the rSU suggested that Triploceras was closely related to Pleurotaenium, which is bilateraly symmetric. Analysis of chloroplast and mitochondrial genes indicated that Triploceras was actualy closely related to the bilateraly symmetric Micrasterias. To resolve the apparent contradiction in phylogenetic placement, we sequenced the nuclear genes EF1 ?, EIF4 and TUA and analyzed the fragments using phylogenetic methods. We found no strongly supported contradictions betwen phylogenies from these nuclear markers and the chloroplast and mitochondrial markers suggesting that Triploceras is closely related to Micrasterias and there has been no confounding evolutionary events such as hybrid speciation, horizontal gene transfer or lineage sorting. We propose that Triploceras is actualy a highly modified bilateraly symmetric organism closely related to Micrasterias. . 143 Introduction Triploceras is a unicelular member of a relatively derived lineage of conjugating gren algae, the Desmidiaceae (Gontcharov et al., 2003) (Chapter 3). This species is elongate with a wel-defined constriction at the isthmus, whorls of spinous proceses and a spinous apical lobe (Bailey, 1851). It is unlike most other desmids in that the isthmal part of the cel is radialy symmetric in apical view hile the apex is bilateraly symmetric (Figure 7.1). There are no obvious afinities betwen this genus and any other desmid genus although many authors have alied it with other genera that are elongate and have an apical lobe, such as Triplastrum, Ichthyodontum and Ichthyocercus (Gauthier-Lievre, 1960; Prescott, Croasdale, and Vinyard, 1975; Palamar-Mordvintseva, 2003). The conjugating gren algae (Zygnematophyceae) are closely related to land plants (Matox and Stewart, 1984; McCourt et al., 2000; Karol et al., 2001; Turmel et al., 2006; Hal and Delwiche, 2007)(Chapter 2). One recent phylogenetic analysis based on 76 chloroplast gene sequences from nine charophytes (including land plants) suggested that the conjugating gren algae are sister to land plants (Turmel et al., 2006). The conjugating gren algae are the most species-rich lineage of charophyte gren algae (Chapter 2) and evolution within the group provides direct insight into the evolution of the lineage that gave rise to extant charophytes and land plants. One major transition in the evolution of land plants was from unicelular to multicelular organization or the thalus (Graham et al., 2000). The increase in organismal complexity was probably preceded by a number of changes in celular 144 organization including an increase in asymmetry of the cel. In animals, the transition from radial to bilateral symmetry preceded a great radiation of form. The conjugating gren algae is one lineage that contains organisms that are both radialy and bilateraly symmetric. Among the conjugating gren algae, transitions in symmetry are thought to be rare and species with radial and bilateral symmetry are treated as separate genera (Brook, 1981). In this lineage, bilateraly symmetric organisms are thought to have evolved from radialy symmetric ancestors (Teiling, 1952; Prescott et al., 1972; Palamar-Mordvintseva, 2003). However, many genera, including Triploceras, were not placed in a phylogenetic context in these studies. It is not known, therefore how many transitions betwen radial and bilateral symmetry there have been. It is also not clear if Triploceras should be considered radialy or bilateraly symmetric. Previous phylogenetic analyses indicated that Triploceras was either closely related to Pleurotaenium (Gontcharov et al., 2003) (elongate radialy symmetric species without either apical proceses or whorls of proceses) or Micrasterias (Chapter 3). Micrasterias species are circular or subcircular in lateral view and dorsi-ventraly compresed in apical view. This fundamental dichotomy of growth form, radialy symmetric versus bilateraly symmetric, would suggest that the organisms are not closely related. The strong support for a relationship betwen these two genera based on chloroplast and mitochondrial gene data (Chapter 3) contrasts sharply with that of the SU topology (Gontcharov et al., 2003) and indicates that there may be a more complex evolutionary history of the genus Triploceras than was previously suspected. Potentialy confounding events include lineage sorting, horizontal gene transfer and hybrid speciation. 145 There are no definitive reports of inter-generic hybridization in the conjugating gren algae. A number of reports of interspecific hybridizations exist including evidence for species complexes in Closterium (Denboh et al., 2003b) and Pleurotaenium (Ling and Tyler, 1974). In the case of Pleurotaenium, however, the resulting zygotes were not demonstrated to be viable. Additionaly, there are a number of reports of interspecific hybridization reported from the wild including many in the genus Spirogyra (Transeau, 1951). There have been no reports of horizontal gene transfer or lineage sorting in the conjugating gren algae. To investigate the evolutionary history of the enigmatic Triploceras gracile, we sequenced thre nuclear encoded genes from Triploceras, Micrasterias, Pleurotaenium and other related Desmidiaceae to determine the phylogenetic relationships. Our goal was to determine if the relationship betwen Triploceras and Micrasterias represents a transition in symmetry and if there has been a confounding event in the evolutionary history of Triploceras. Materials and methods Unialgal strains were requested from public culture collections or isolated from the wild (Table 7.1). These strains were maintained in Guilards Woods Hole Medium (Nichols, 1973). DNA was extracted using the Phytopure DNA Extraction kit (Amersham, San Francisco, CA) following the manufacturer?s protocol with a second chloroform extraction. The smal subunit (SU) of the nuclear ribosomal DNA (rDNA) was amplified and sequenced using previously published primers and protocols (Marin et al., 1998; 146 Gontcharov et al., 2003). Portions of the nuclear encoded eukaryotic translation elongation factor 1 alpha (hereafter EF1 ?) were amplified from genomic DNA using the nested primers and protocol indicated in Table 7.2. Al PCR was performed using 35 cycles of a denaturation at 95? C for 30 seconds, variable annealing temperature for 30 seconds followed by a variable extension time (Table 7.2) at 72? C using Taq polymerase. EF1 ? and eukaryotic initiation factor 4 (EIF4) primers were designed by the author. Primers for the amplification of alpha tubulin (TUA) were taken from Kim et al. (2006). Atempts to amplify EIF4, TUA and other nuclear gene fragments from whole DNA extractions were partialy or entirely unsuccesful. In order to obtain these data, RNA was extracted from actively growing cultures using the RNAqueus mini kit (Ambion, Austin, TX) following the manufacturer?s instructions. cDNA was synthesized using the Acuscript Kit (Stratagene, La Jolla, CA) following the manufacturer?s protocol with oligo dT primers. One microliter of the cDNA was used as a template in subsequent PCR of the EIF4 and TUA using the primers and protocol indicated in Table 2. Even using cDNA as a template, multiple bands were often encountered, particularly when amplifying TUA, and the fragments were ligated into the pGem T-easy vector (Promega, Madison, WI). Following PCR, products were precipitated using 20% polyethylene glycol (PEG), rinsed in 70% ethanol and resuspended in picopure water. These products were ligated into the pGem vector at 4? C over night (16 hours) and were then cloned into 15 ?L of competent E. coli. SOC broth was added to 1 mililiter. These bacteria were grown at 37? C for two hours in a shaking water bath, then 200 ?L of SOC with bacteria were plated onto 2% agar LB plates with ampicilin. These were alowed to air dry for ten minutes and placed upside down in a growth chamber at 37? C overnight 147 (16 hours). Betwen eight and forty-eight of the resulting bacterial colonies were picked and the inserted fragments were amplified off the vector using the primers in Table 2, and an extension time corresponding to the cloned gene fragment. Multiple clones were sequenced and included in analyses. 148 Table 7.1 Strains investigated and Genbank numbers Taxon Strain GenBank Closterium peracerosum-strigosum-litorale NIES67, 68 Cosmarium botrytis UTEX 301 Cosmarium verucosum JH068 Euastrum crasum var. michiganense JH018 Micrasterias depauperata JH0364 icrasterias foliaceae NIES 297 Micrasterias laticeps JH0239 icrasterias muricata JH019 Micrasterias muricata JH0120 icrasterias muricata JH0179 Micrasterias pinatafida JH0106 icrasterias radiata JH064 Micrasterias radiata JH026 icrasterias rotata UTEX 1941 Micrasterias truncata JH017 Phymatodocis nordstedtiana SAG 47.89 Pleurotaenium baculoides JH008 Pleurotaenium constrictum JH0135 Triploceras gracile JH0215 Triploceras gracile SAG 24.82 Xanthidium hastiferum JH054 Actinotaenium cucurbita AY964132.1 Cosmarium costatum AY964126.1 Cosmarium norimbergense var. depresum AY964138.1 Cosmarium obsoletum AY964128.1 Cosmarium pseudoconatum AY964150.1 Cosmarium tenue AY964127.1 Desmidium swartzi AJ42813.1 Euastrum oblongum AJ428095.1 Euastrum pectinatum AJ54927.1 Euastrum pinatum AJ428096.1 Groenbladia neglecta AJ42819.1 Haplotaenium inutum AJ428090.1 Heimansia pusila AJ428125.1 Hyalotheca disiliens AJ428120.1 Micrasterias crux-melitensis AJ428097.1 icrasterias fimbriata AJ428098.1 Onychonema laeve AJ428127.1 Phymatodocis nordstedtiana AJ42812.1 Pleurotaenium ehrenbergi AJ428132.1 Pleurotaenium trabecula AJ428131.1 Staurastrum cristatum AJ42810.1 Staurastrum ophiura AJ428104.1 Staurastrum pingue AJ428109.1 Staurodesmus bulnheimi AJ42811.1 Staurodesmus convergens AY964143.1 Staurodesmus mucronatus AJ428103.1 Tetmemorus brebisoni AJ428091.1 149 Triploceras gracile AJ428089.1 Xanthidium antilopaeum AJ428093.1 Xanthidium armatum AJ428094.1 Xanthidium brebisoni AJ428092.1 Table 7.2 PCR primers and conditions Gene Primer Temp. Time 18S 18S A - CTGTGATYCTGCAGT 52 C 45 sec 18S B - CYGCAGTCACTACRG EF1 ? EF1F1 - GCTGAGATGACAGAG 52 C 45 sec EF1R2 - GCGACTCTCAGTAG EF1 ? nested F6 Mic - CACATTCTCATAGCGG 52 C 45 sec R4 ic - GCTGGTGCTGACAGCTCAG EIF4 EIF4F - CGCGCAGTGACTG 52 C 45 sec EIF4R - GTCTGCAGCATGCGCTCGTC TUA TUAF2/23 - CACATCGNCARGCGNRTCA 48 C 120 sec TUAR 1268/26 - GCYTCRGARAYTCNCYTCTCAT TUA nested TUAF58/25 - TGCTGGAGCTNTACTGCTNGAGCA 48 C 120 sec TUAR 1248/26 - TCTCATNCYTCNCNACRTACA pGEM vector T7 F - TATACGACTCACTATAGG 52 C variable M13 R - ACATGATACGCAG Notes: Primers in bold were designed by the author. Temp. refers to the annealing temperature of the PC reaction. Time refers to the length of the extension time in seconds. When amplifying from vector, the extension time depended on the length of the target fragment. 150 Gene fragments were aligned by eye in MacClade 4.0 (Maddison and Maddison, 2000). Introns, indels, and unalignable portions were excluded from the analysis (positions 1-211, 368-589, 629-676, 776-961, 986-1127 of EF1 ?; positions 181-336 of EIF4; and positions 85-87, 289-381, 588-678, 1315-1365 of TUA). Either Phymatodocis or Closterium were used to root the tres. Phylogenetic analyses were performed in Paup* v. 4 b.10 (Swofford, 2003) and MrBayes v. 3.1 (Huelsenbeck and Ronquist, 2000). Other taxa belonging to other lineages separating Micrasterias and Triploceras in the SU phylogeny were also included in the analysis. Under the Parsimony criterion (MP), the best tre was searched for heuristicaly with starting tres constructed by one hundred random taxon addition sequences and using tre bisection-reconnection (TBR) branch swapping. Support for taxon bipartitions was estimated from 500 bootstrap pseudoreplicates with ten random taxon addition sequences and TBR swapping. For likelihood analyses, the models and parameter setings were selected based on the output of Mr. Modeltest v.2 (Nylander, 2004): SU, GTR+I+G; EF1, SYM+G; EIF4, GTR+G; TUA, GTR+I+G. Under the likelihood criterion (ML), the best tre was calculated from ten random addition sequence replicates and TBR branch swapping. Support was estimated from 100 bootstrap replicates with thre random taxon addition sequences and TBR swapping. Under Bayesian Inference (BI), tre space was explored using a markov chain monte carlo method with four chains run for 4,000,000 generations sampled every 100 generations. The first 2501 tres, wel into the stationary phase in al datasets, were discarded as burnin. 151 Results Sequences from GenBank were concatenated into a dataset with only Desmidiales, mostly Desmidiaceae and the alignment was analyzed using al previously described methods. In this analysis, the position of Triploceras was not resolved (Figure 7.2), but its placement difered from that of the published SU topology. Direct sequencing of EF1 ? from the nuclear DNA, revealed two slightly diferent sequences in Triploceras. These sequences were identical to or difered by one base pair from those obtained for al Micrasterias species (Figure 7.3), resulting in two esentialy identical most parsimonious tres (Figure 7.3A, B). Likelihood analyses showed similar results difering only in the relative support for relationships among outgroup taxa (Figure 7.3C, D). Fragments of EIF4 were obtained by cloning PCR products amplified from either the total nuclear DNA or cDNA prepared from RNA (which, presumably, would lack introns). Parsimony analysis of this dataset found four equaly parsimonious tres that were mostly unresolved but showed a sister relationship betwen Triploceras and Micrasterias (Figure 7.4B, E) as wel as a relationship betwen Triploceras and Pleurotaenium (Figure 7.4A) among others (Figure 7.4D). No placement of Triploceras was statisticaly supported in parsimony analysis of EIF4. Under the likelihood criterion, only a relationship betwen Triploceras and Micrasterias muricata was discovered, although this relationship was not statisticaly supported in either ML (>50; Figure 7.4C) or BI (0.54; Figure 7.4F). Multiple acesions for each strain were included to show the variation induced, presumably, from cloning individual PCR products. Sequences of TUA were not available from Closterium, and it was not possible to amplify a fragment from Phymatodocis (the two taxa selected as outgroups), so these 152 tres are shown as unrooted cladograms. In parsimony analysis, sixty-thre most parsimonious tres were encountered. These difered only in the placement of the terminal taxa. Figure 7.5A shows one of these tres with proportionate branch lengths, while 7.5B shows the strict consensus of the sixty-thre most parsimonious tres (branch lengths not significant). Al tres showed a sister relationship betwen Micrasterias muricata and Triploceras as wel as a monophyletic Micrasterias that included Triploceras (Figure 7.5A, B). In the ML analysis, the internal nodes were not resolved and Triploceras appeared sister to Micrasterias foliacea in the most likely tre, but Micrasterias was not monophyletic (Figure 7.5C). Under Bayesian Inference, a monophyletic Micrasterias was present in the consensus tre (Figure 7.5D), and Triploceras appeared sister to Micrasterias foliacea with moderate support (0.89). Discusion The discovered relationship betwen Micrasterias and Triploceras was not expected. As previously noted, these organisms are very diferent structuraly and previous phylogenetic analysis of ribosomal SU found no relationship betwen these genera (Gontcharov et al., 2003). Atempts to reproduce those experiments resulted in a topology that was consistent with al other sampled markers (Figure 7.2) but inconsistent with the published SU topology (Gontcharov et al., 2003). It is likely that the placement of Triploceras discovered by Gontcharov et al. (2003) was the result of either a spurious sequence that was asigned to Triploceras, a peculiarity of their alignment, or taxon sampling. Gontcharov et al. (2003) did not include either of the species of Micrasterias that most often appeared sister to Triploceras: Micrasterias foliaceae and M. muricata. 153 If taken at face value, the results presented by Gontcharov et al. (2003) may indicate that there was some more complex evolutionary history that gave rise to the diferent gene tres. These include, but are not limited to, horizontal gene transfer, lineage sorting and hybridization (Linder and Rieseberg, 2004). It is possible to distinguish among these potentialy confounding factors and refute the hypothesis that one of those mechanisms is responsible for the apparent incongruence. In al thre cases, a past event wil result in a gene tre that is not a reflection of the species tre (Figure 7.6). In horizontal gene transfer, a gene or portion of a genome is transfered from one organism to another - potentialy distantly related - organism. Viruses and bacteria are often suspected as vector agents, but other biological proceses may be involved. When estimating phylogenies based on a single gene with a history of horizontal transfer, one would find an unexpected relationship betwen two organisms (Figure 7.6A). In general, horizontal gene transfers can be detected by investigating multiple loci and increasing taxon sampling to determine where the horizontaly transfered sequence originated. Lineage sorting can be more dificult to detect. In this case, the relationships of extant taxa are distorted by past sorting events. For example, if a lineage of organisms contained multiple aleles of a particular gene at some point in the past, but these aleles were lost over time, then the apparent relationship of extant taxa based on that gene would not be indicative of the species relationships, but rather - if that gene tre could be mapped onto a phylogenetic tre showing the true evolutionary history - it would indicate the history of alele or gene evolution and extinctions (Figure 7.6B). Again, lineage 154 sorting can often be detected by sampling multiple genes with independent evolutionary histories. Hybrid speciation is very diferent from the two preceding proceses. In its simplest form, hybridization involves the union of whole genomes from two diferent species to give rise to a new morphologicaly distinct species. Although rare in animals, this proces is comparatively common in plants and is considered an important evolutionary phenomenon (Hegarty and Hiscock, 2005). Hybrid organisms have some peculiar genomic features and the genes in the nuclear genome can have a number of fates. However, when sampled in a phylogenetic context, the nuclear genome wil often appear to be a mix of the two parental species (Nishimoto et al., 2003; Linder and Rieseberg, 2004; Hegarty and Hiscock, 2005; Kameyama et al., 2005). The general congruence among genes sampled in this study suggests that horizontal gene transfer is not responsible for the relationships. It is possible that the ribosomal genes were transfered from another organism, but this analysis of the rSU indicates a possible relationship betwen Triploceras and Micrasterias (Figure 7.2). This general congruence among the molecules also suggests that lineage sorting does not sem to explain the close relationship betwen Micrasterias and Triploceras. If lineage sorting were a factor, one would expect to find diferent relationships when analyzing diferent genes, which was not the case. The absence of incongruous tres also esentialy negates the hypothesis of a past hybridization event. To be certain, one would have to sample al the genes in the genomes of Micrasterias, Triploceras and some outgroup taxa. At the present, there is no compeling reason to do so. 155 In the published rbcL, psaA and coxII datasets (Chapter 3), as wel as the newly presented EF1 ? dataset, Triploceras and some Micrasterias species shared identical primary sequence (Figure 7.3). Most sampled genes indicated that species of Micrasterias and Triploceras had litle diference in primary sequence of the gene fragments, but there was also litle diference betwen primary sequence of coding regions in Micrasterias and outgroup taxa such as Euastrum and Cosmarium (Figures 7.4, 7.5). This made estimation of the relationships among the species particularly dificult in EIF4 and TUA datasets, as indicated by low bootstrap support and low posterior probabilities (Figures 7.4, 7.5). These genes are probably not sufficiently variable to resolve these relationships, and future studies investigating species-level relationships wil likely have to rely on noncoding sequence such as introns or microsatelites. However, in the one instance where intron sequences were available, the EF1 ? dataset, the intron sequence Micrasterias and other Desmidiaceae were not alignable (data not shown). The apparent relationship betwen Micrasterias and Triploceras may have been previously overlooked because of taxon sampling within Micrasterias. In my analyses, Triploceras was found at the base of the Micrasterias clade, usualy asociated with Micraterias foliacea or M. muricata. Neither of these taxa was included in previous SU studies (Gontcharov et al., 2003). These taxa are unique among Micrasterias species for diferent reasons. Micrasterias foliacea, although structuraly similar to other highly disected species of Micrasterias, forms long chains of cels held together by their apical proceses (Lorch and Engels, 1979). Micrasterias muricata, on the other hand, is structuraly unlike most other species of Micrasterias in that it lacks the compresed 156 lateral lobes so typical of Micrasterias and, instead, has long tubular proceses similar to many species of Staurastrum. The discovered phylogenetic relationships among Micrasterias species brings to light aspects of celular structure that had been previously overlooked. First, it is interesting that the filamentous M. foliacea is among the basal lineages of Micrasterias. Its position at the base of the clade suggests that the ancestor to extant Micrasterias species may have had many disected lobes as found in M. foliacea and many other Micrasterias species. Also, the close relationship betwen Micrasterias and Triploceras requires careful consideration of the use of symmetry as a systematic characteristic. Triploceras is often considered radialy symmetric (Brook, 1981), although its apical lobe is certainly bilateraly symmetric. If it is considered radialy symmetric, then we must conclude that transitions from radial to bilateral symmetry (or the reverse) have happened many times in the evolution of desmids and at least once in the Micrasterias lineage. If Triploceras is bilateraly symmetric, then it would sem that the portion of the cel that appears to be radialy symetric is in fact a highly modified bilateraly symmetric semicel. Acepting the possible ambiguity of radial symmetry, one must also consider the possibility that many other desmid genera which appear to be radialy symmetric are, in fact, bilateraly symmetric. This sems to be the case for a great many ?radialy? symmetric desmids. Filamentous desmids such as Didymoprium grevilei, Bambusina borreri, and Hyalotheca disiliens, often have lateral tubercles that indicate that the cel wal is not radial, but rather bilateral. In al cases, it is stil possible that the chloroplast (and possibly other cytoplasmic contents) may be aranged in radial symmetry, but the cel wal is not. 157 Under this asumption, the whorls of proceses in Triploceras could be interpreted as reduced, multidimensional lobes. The genetic basis for such a transition is unknown, but a potentialy interesting line of research that may prove to be important to our understanding or celular development and morphogenesis in desmids. 50 um Figure 7.1 Light micrograph of Triploceras gracile var. gracile 159 Figure 7.2. Genealogies based on rSU. A. is one of the 115 most parsimonious tres. Numbers above branches are bootstrap values. A dash indicates support les than 50. B. is the strict consensus of the 115 MP tres. C. Maximum Likelihood tre. F. Likelihood tre from Bayesian Inference. Numbers above branches are posterior probabilities. A dash represents support les than 0.50. Desmidium swartzii Onychonema laeve Heimansia pusilla Hyalotheca dissiliens Groenbladia neglecta Cosmarium tenue Staurodesmus mucronatus Actinotaenium cucurbita Euastrum pinnatum Euastrum oblongum Tetmemorus brebissonii Staurodesmus bulnheimii Staurodesmus convergens Euastrum pectinatum Xanthidium antilopaeum Xanthidium brebissonii Staurastrum ophiura Staurastrum pingue Staurastrum cristatum Micrasterias crux-melitensis Micrasterias fimbriata Triploceras gracile Cosmarium obsoletum Cosmarium pseudoconnatum Cosmarium costatum Xanthidium armatum Haplotaenium minutum Pleurotaenium ehrenbergii Pleurotaenium trabecula Phymatodocis nordstedtiana Cosmarium norimbergense Desmidium swartzii Actinotaenium cucurbita Onychonema laeve Heimansia pusilla Hyalotheca dissiliens Groenbladia neglecta Euastrum pinnatum Euastrum oblongum Cosmarium tenue Staurodesmus mucronatus Tetmemorus brebissonii Staurodesmus bulnheimii Staurodesmus convergens Euastrum pectinatum Xanthidium antilopaeum Xanthidium brebissonii Staurastrum ophiura Staurastrum pingue Staurastrum cristatum Micrasterias crux-melitensis Micrasterias fimbriata Triploceras gracile Cosmarium obsoletum Cosmarium pseudoconnatum Cosmarium costatum Xanthidium armatum Haplotaenium minutum Pleurotaenium ehrenbergii Pleurotaenium trabecula Phymatodocis nordstedtiana Cosmarium norimbergense 5 changes Desmidium swartzii Onychonema laeve Hyalotheca dissiliens Groenbladia neglecta Heimansia pusilla Tetmemorus brebissonii Staurodesmus bulnheimii Cosmarium tenue Staurodesmus mucronatus Actinotaenium cucurbita Euastrum pinnatum Euastrum oblongum Euastrum pectinatum Cosmarium norimbergense Staurastrum ophiura Staurastrum pingue Staurastrum cristatum Micrasterias crux-melitensis Micrasterias fimbriata Triploceras gracile Haplotaenium minutum Staurodesmus convergens Xanthidium armatum Xanthidium antilopaeum Xanthidium brebissonii Pleurotaenium ehrenbergii Pleurotaenium trabecula Cosmarium costatum Cosmarium obsoletum Cosmarium pseudoconnatum Phymatodocis nordstedtiana 60 69 - - - - - 100 100 99 - - - - - - - - - - 92 96 - - - - - - 60 86 58 - - - - - - - - - - - - - - 75 100 95 96 70 100 Desmidium swartzii Onychonema laeve Hyalotheca dissiliens Groenbladia neglecta Heimansia pusilla Tetmemorus brebissonii Staurodesmus bulnheimii Cosmarium tenue Staurodesmus mucronatus Actinotaenium cucurbita Euastrum pinnatum Euastrum oblongum Staurastrum ophiura Staurastrum pingue Staurastrum cristatum Haplotaenium minutum Staurodesmus convergens Euastrum pectinatum Cosmarium norimbergense Micrasterias crux-melitensis Micrasterias fimbriata Triploceras gracile Xanthidium antilopaeum Xanthidium brebissonii Xanthidium armatum Pleurotaenium ehrenbergii Pleurotaenium trabecula Cosmarium obsoletum Cosmarium pseudoconnatum Phymatodocis nordstedtiana Cosmarium costatum 0.90 1.0 1.0 1.0 0.81 0.96 0.530.57 0.65 0.97 0.99 0.99 0.94 0.94 0.79 0.95 0.73 0.73 0.78 A B C D - - - - 69 100 97 - - - - - - - - - - - - - - - - - 99 96 92 100 0.005 substitutions/site 0.01 substitutions/site 161 Figure 7.3. Genealogies based on EF1. A, B are the two most parsimonious tres. Numbers above branches are bootstrap values. A dash indicates support les than 50. C. Maximum Likelihood tre. D. Likelihood tre from Bayesian Inference. Numbers above branches are posterior probabilities. A dash represents support les than 0.50. Micrasterias muricata JH0120 Micrasterias depauperata JH0364 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Triploceras gracile SAG 24.82 Triploceras gracile SAG 24.82 Cosmarium botrytis UTEX 310 Cosmarium botrytis UTEX 310 Xanthidium hastiferum JH0054 Pleurotaenium baculoides JH0008 Euastrum crassum var. michiganense JH0018 Phymatodocis nordstedtiana SAG 47.89 5 changes Micrasterias muricata JH0120 Micrasterias depauperata JH0364 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Triploceras gracile SAG 24.82 Triploceras gracile SAG 24.82 Cosmarium botrytis UTEX 310 Cosmarium botrytis UTEX 310 Xanthidium hastiferum JH0054 Pleurotaenium baculoides JH0008 Euastrum crassum var. michiganense JH0018 Phymatodocis nordstedtiana SAG 47.89 5 changes 0.05 substitutions/site Micrasterias muricata JH0120 Micrasterias depauperata JH0364 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Triploceras gracile SAG 24.82 Triploceras gracile SAG 24.82 Cosmarium botrytis UTEX 310 Cosmarium botrytis UTEX 310 Xanthidium hastiferum JH0054 Pleurotaenium baculoides JH0008 Euastrum crassum var. michiganense JH0018 Phymatodocis nordstedtiana SAG 47.89 0.05 substitutions/site Micrasterias muricata JH0120 Micrasterias depauperata JH0364 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Triploceras gracile SAG 24.82 Triploceras gracile SAG 24.82 Cosmarium botrytis UTEX 310 Cosmarium botrytis UTEX 310 Xanthidium hastiferum JH0054 Pleurotaenium baculoides JH0008 Euastrum crassum var. michiganense JH0018 Phymatodocis nordstedtiana SAG 47.89 99 46 100 39 42 99 100 39 46 63 97 48 22 81 0.91 1.0 1.0 0.54 A B C D 163 Figure 7.4. Genealogies based on EIF4. A, B, D, and E are the four most parsimonious tres. Numbers above branches are bootstrap values. A dash indicates support les than 50. C. Maximum Likelihood tre. F. Likelihood tre from Bayesian Inference. Numbers above branches are posterior probabilities. A dash represents support les than 0.50. 10 changes Micrasterias truncata JH0017 Micrasterias truncata JH0017 Micrasterias depauperata JH0364 Micrasterias laticeps JH0239 Micrasterias laticeps JH0239 Micrasterias radiata JH0064 Micrasterias radiata JH0064 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Triploceras gracile JH0215 Cosmarium verrucosum JH0068 Cosmarium verrucosum JH0068 Closterium peracerosum-strigosum-littorale NIES 67 Micrasterias truncata JH0017 Micrasterias truncata JH0017 Micrasterias depauperata JH0364 Micrasterias laticeps JH0239 Micrasterias laticeps JH0239 Micrasterias radiata JH0064 Micrasterias radiata JH0064 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Triploceras gracile JH0215 Cosmarium verrucosum JH0068 Cosmarium verrucosum JH0068 Closterium peracerosum-strigosum-littorale NIES 67 Micrasterias truncata JH0017 Micrasterias truncata JH0017 Micrasterias depauperata JH0364 Micrasterias laticeps JH0239 Micrasterias laticeps JH0239 Micrasterias radiata JH0064 Micrasterias radiata JH0064 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Triploceras gracile JH0215 Cosmarium verrucosum JH0068 Cosmarium verrucosum JH0068 Closterium peracerosum-strigosum-littorale NIES 67 Micrasterias truncata JH0017 Micrasterias truncata JH0017 Micrasterias depauperata JH0364 Micrasterias laticeps JH0239 Micrasterias laticeps JH0239 Micrasterias radiata JH0064 Micrasterias radiata JH0064 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Triploceras gracile JH0215 Cosmarium verrucosum JH0068 Cosmarium verrucosum JH0068 Closterium peracerosum-strigosum-littorale NIES 67 Micrasterias truncata JH0017 Micrasterias truncata JH0017 Micrasterias depauperata JH0364 Micrasterias laticeps JH0239 Micrasterias laticeps JH0239 Micrasterias radiata JH0064 Micrasterias radiata JH0064 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Triploceras gracile JH0215 Cosmarium verrucosum JH0068 Cosmarium verrucosum JH0068 Closterium p-s-littorale NIES 67 Micrasterias truncata JH0017 Micrasterias truncata JH0017 Micrasterias depauperata JH0364 Micrasterias laticeps JH0239 Micrasterias laticeps JH0239 Micrasterias radiata JH0064 Micrasterias radiata JH0064 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Micrasterias muricata JH0119 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Pleurotaenium baculoides JH0008 Triploceras gracile JH0215 Cosmarium verrucosum JH0068 Cosmarium verrucosum JH0068 Closterium p-s-littorale NIES 67 10 changes 10 changes 10 changes 0.05 substitutions/site 0.05 substitutions/site 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.54 0.98 0.99 0.93 0.93 100 100 100 97 - - - 100 100 100 97 73 - 100 100 100 100 100 97 100 97 73 - - - - 100 100 86 91 75 86 100 100 96 - - 65 53 100 100 100 97 - - - 100 100 100 97 73 - 100 100 100 97 - - - 100 100 100 97 73 - A B C D E F 165 Figure 7.5. Genealogies based on TUA. A. One of 63 most parsimonious tres showing branchlengths. Numbers above branches are bootstrap values. A dash represents support les than 50. B. Strict consensus of 63 most parsimonious tres. C. Maximum Likelihood tre. D. Likelihood tre from Bayesian Inference. Numbers above branches are posterior probabilities. A dash represents support les than 0.50. Triploceras gracile JH0215 Micrasterias muricata JH0179 Micrasterias radiata 226 Micrasterias truncata JH0106 Micrasterias foliaceae NIES 297 Xanthidium hastiferum JH0054 Cosmarium verrucosum JH0068 Euastrum crassa var. michiganense JH0018 Pleurotaenium contrictum JH0135 Triploceras gracile JH0215 Micrasterias muricata JH0179 Micrasterias radiata 226 Micrasterias truncata JH0106 Micrasterias foliaceae NIES 297 Xanthidium hastiferum JH0054 Cosmarium verrucosum JH0068 Euastrum crassa var. michiganense JH0018 Pleurotaenium contrictum JH0135 10 changes Triploceras gracile JH0215 Micrasterias muricata JH0179 Micrasterias radiata 226 Micrasterias truncata JH0106 Micrasterias foliaceae NIES 297 Xanthidium hastiferum JH0054 Cosmarium verrucosum JH0068 Euastrum crassa var. michiganense JH0018 Pleurotaenium contrictum JH0135 0.01 substitutions per site Triploceras gracile JH0215 Micrasterias muricata JH0179 Micrasterias radiata 226 Micrasterias truncata JH0106 Micrasterias foliaceae NIES 297 Xanthidium hastiferum JH0054 Cosmarium verrucosum JH0068 Euastrum crassa var. michiganense JH0018 Pleurotaenium contrictum JH0135 0.05 substitutions per site 79 - 100 58 - - 100 74 1.0 0.83 0.55 1.00.89 -- - - A B C D 167 Figure 7.6. Model genealogies showing potentialy confounding evolutionary events. A B C D A A a B C D A C B D A B E C D A B C D A B E C D A B E C D Species tree A. Horizontal gene transfer B. Lineage sorting C. Hybridization Either Or Gene tree 169 Chapter 8. Conclusions Experimental conclusions In the completion of this work, two molecular markers for estimation of phylogenetic relationships among the conjugating gren algae were developed. Hundreds of new strains were isolated that wil be available for future investigations. This study of the phylogenetic relationships among conjugating gren algae revealed that our concepts of the families and genera in the Zygnematales requires substantial revision. Besides that the Zygnematales appears to be paraphyletic (Chapter 3), it is clear that the families and most of the unicelular genera of the Zygnematales are not monophyletic. These certainly merit more careful investigation using both cytological and molecular phylogenetic methods. Although most families in the Desmidiales are monophyletic, the family Peniaceae (and the genus Penium) is not supported as monophyletic based on the collected data (Chapter 3) and the desmid lineage almost certainly includes or is closely related to the zygnematalean genus Roya (Chapter 3, 5). These data support the use of cytological features as systematic characters in some cases. The separation of the genus Heimansia from Cosmocladium and Haplotaenium from Pleurotaenium, decisions made based on structural characteristics, was supported in these analyses (Chapter 3). These data also suggest that chloroplast position may be a useful systematic character as this characteristic sets apart major lineages within the Zygnematales and separates these organisms from basal desmids (Chapter 3). These data also indicate that the genera Mesotaenium, Cylindrocystis, Cosmarium, Staurodesmus, Penium, Hyalotheca, Desmidium and Spondylosium, are not monophyletic. 170 In some cases, these phylogenetic relationships can be correlated to diferences in ontogeny or celular structure. In the case of the filamentous Desmidiaceae, a suite of cel division characteristics as wel as chloroplast shape characterized the clades of the polyphyletic Desmidium and Spondylosium (Chapter 4, 5). Cel division in Desmidium pulchrum, Isthmocatena pulchela, Spondyosium tetragonum and Teilingia granulata was described for the first time. This led to the systematic reasignment of several taxa and the creation of a new generic name, Isthmocatena (Chapter 5). This implies that critical celular proceses such as cel division can vary and the diferences discovered were apparent changes in the timing and order of celular events as wel as the evolution of a novel structure. Sometimes the reason for phylogenetic ambiguity is les obvious. In the case of the Gonatozygaceae, organisms that were asigned to the same species based on their gross structural characteristics were phylogeneticaly distinct (Chapter 6). Careful atention to the structural characteristics was sufficient to distinguish most clades, but in the case of Gonatozygon brebisonii, it was not posible to distinguish among the diferent lineages even using EM techniques (Chapter 6). In this case the structural simplicity of organisms can mask their phylogenetic diversity. This poses a dificult chalenge when interpreting any study based on the morphological identity of structuraly simple microorganisms. The results of this study suggest that we may be underestimating phylogenetic and possibly species diversity in groups of structuraly simple organisms. However, one must be careful in the interpretation of certain characteristics. Investigaton of Triploceras suggests that even fundamental characteristics such as apparent symmetry may be variable among closely related organisms (Chapter 7). Never 171 before had a relationship betwen Micrasterias and Triploceras been proposed. Knowledge of this relationship forces us to think about the utility of symmetry as a taxonomic character, and encourages us to think more criticaly about the anatomy of these organisms. In this case, the relationship opens up a number of possible research avenues into the basic biology of these organisms, their morphogenesis and their evolution. Towards an understanding of diversity in the conjugating gren algae and its application to the origin of land plants In general, caution when interpreting the structure of microorganisms must be observed. Structural characteristics must be investigated in a systematic context if we are to understand their origin, evolution and systematic utility. While molecular phylogenetic investigations should continue, data collected in this work suggest that there is a wealth of systematic information hidden in the structure and ontogeny of microorganisms. Future investigations can use careful observation in a phylogenetic context to begin to understand the evolution of microorganisms. These data also suggest that both the developmental and phyletic diversity of conjugating gren algae are probably underestimated. While this poses some systematic and taxonomic chalenges, it also suggests that the conjugating gren algae may be a richer source of evolutionary information than was previously suspected. This work suggests that the conjugating gren algae would be excelent models for the study of celular morphogenesis, cel division, and cel wal synthesis. It is possible that there are also genetic and genomic characteristics shared betwen the conjugating gren algae and land plants that have contributed to their succes. 172 It is not clear which lineage of charophyte gren algae is most closely related to land plants. However, the Characeae, Coleochaetales and the conjugating gren algae are al possible candidates. Among these, the conjugating gren algae are the most structuraly and developmentaly diverse and can provide insight into the evolution of these characteristics that other lineages cannot. Shifts in body plan are considered important evolutionary changes in the history of complex forms such as land plants. The conjugating gren algae are al strongly axile, having a single main axis and cel division occurs in only one plane. However, al conjugating gren algae are capable of site-specific modification of the cel wal. Formation of conjugation tubes, gamete pores, and rhizoids as wel as secretion of extracelular polysacharides are carefully controlled. Most conjugating gren algae sem to have taken a diferent evolutionary path than Spirogyra, the earliest branching lineage of the conjugating gren algae (Chapter 2). In most Zygnematophytes, the cytoplasmic contents are based in the center of the cel, however, in Spirogyra (including Sirogonium) only the nucleus is central and the other cytoplasmic contents are appresed to the outer membrane by a large central vacuole. In this respect, Spirogyra is organized more like Coleochaete and Klebsormidium than most desmids, with the exception of the placement of the nucleus. In these later taxa, the nucleus is also peripheral. It is noteworthy that the earliest diverging lineages of zygnematophytes contain filamentous organisms. Because the most closely related charophyte algae are also filamentous, this is likely to be a plesiomorphic trait and the unicelular condition of some zygnematophytes may, therefore, be derived. 173 Early diverging lineages of zygnematophytes contain organisms that are structuraly diverse and developmentaly complex. Al conjugating gren algae have zygotic meiosis. However, many desmids are thought to be asexual, some are parthenogenic, and zygotes may result from various sexual strategies: anisogamy (oogamy?), isogamy, homothalism, heterothalism, etc. (Brook, 1981). We do not fully understand the proceses involved in sexual reproduction. It is known that at least one pheromone is involved in the induction of sexual cel division in Closterium (Fukumoto et al., 2003b). It is now possible to begin to understand the phylogenetic relationships among these organisms and speculate on their evolutionary significance. Even within the critical proces of cel division, this research has shown that many variations are possible. The discovered diferences were not only modifications of existing proceses, but involved the evolution of novel structures. Additionaly, these variations were found among some of the structuraly least complex members of the Desmidiaceae, suggesting that apparent structural diversity in interphase cels does not necesarily correlate to overal developmental complexity. Estimation of ancestral characteristics is never a trivial undertaking and diversity in the conjugating gren algae makes such asignments particularly dificult. What is clear is that the conjugating gren algae and probably their last common ancestor are structuraly, developmentaly, and reproductively complex. This implies that the ancestor of the zygnematophytes and the lineage that gave rise to land plants were probably also complex organisms. 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