ABSTRACT 
 
 
Title of Dissertation:  CONSERVATION GENETICS OF THE ENDANGERED 
HAWAIIAN PETREL (PTERODROMA 
SANDWICHENSIS) ACROSS SPACE AND TIME 
 
 Andreanna Jo Welch, Doctor of Philosophy, 2011 
 
Dissertation directed by: Dr. Robert C. Fleischer 
Center Head, Center for Conservation and Evolutionary 
Genetics, Smithsonian Conservation Biology Institute, 
National Zoological Park 
 
Dr. David J. Hawthorne 
Associate Professor, Department of Entomology, Behavior, 
Ecology, Evolution and Systematics Program, University of 
Maryland 
 
 
 The endemic Hawaiian Petrel (Pterodroma sandwichensis) is a long-lived pelagic 
seabird. Although endangered, subfossil evidence indicates that it was formerly more 
abundant in the past. In addition to a wider distribution on islands where the petrel 
currently breeds, two colonies, one on Oahu and one on Molokai, have been extirpated 
since humans colonized the Islands. Despite this, little is known about this species. Here I 
use conservation genetic and ancient DNA techniques to investigate the taxonomic status 
and population dynamics of the Hawaiian petrel. Investigation of the timing and 
magnitude of divergence between the Hawaiian petrel and its sister species, the 
Galapagos petrel (P. phaeopygia), revealed that these taxa diverged approximately 
550,000 years ago. In a phylogenetic tree constructed from mitochondrial data Galapagos 
and Hawaiian petrels were reciprocally monophyletic, however, trees from the nuclear 
data set were unresolved. Low estimates of gene flow between taxa indicate that 
incomplete lineage sorting is causing the difference in resolution between data sets and 
that Galapagos and Hawaiian petrels are likely separate species. In addition to the 
mitochondrial and nuclear intron data sets, I developed a suite of 20 polymorphic 
microsatellite loci: I developed 10 specifically for the Hawaiian petrel, and characterized 
an 10 additional previously reported loci in this species. Using these three data sets I 
investigated the pattern of gene flow and divergence between modern, historical, and 
ancient populations of the Hawaiian petrel. The mitochondrial data set showed strong 
levels of differentiation between modern populations. The two nuclear data sets also 
revealed significant population structure, although it was weaker. Mitochondrial DNA 
sequences obtained from subfossil bones indicate that populations were significantly 
differentiated in the past, although there was low divergence between the extirpated Oahu 
and Molokai populations and modern birds from Lanai, suggesting that perhaps as 
colonies dwindled individuals dispersed to that island. Investigation of the effective 
population size indicates that no significant change has occurred on Hawaii or Maui. It 
appears that the long generation time of this species may have allowed it to escape a 
genetic bottleneck after the arrival of humans in the Hawaiian Islands. 
 
 
 
 
 
CONSERVATION GENETICS OF THE ENDANGERED HAWAIIAN PETREL 
(PTERODROMA SANDWICHENSIS) ACROSS SPACE AND TIME 
 
 
by 
 
Andreanna Jo Welch 
 
 
 
 
Dissertation submitted to the Faculty of the Graduate School of the  
University of Maryland, College Park in partial fulfillment 
of the requirements for the degree of 
Doctor of Philosophy 
2011 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Advisory Committee: 
 
Professor David J. Hawthorne, Chair 
Dr. Robert C. Fleischer, Co-Advisor 
Dr. Jonathon D. Ballou 
Professor Michael P. Cummings 
Dr. Helen F. James 
Professor Charles Mitter 
 
 
 
 
 
 
 
 
? Copyright by 
 
Andreanna J. Welch 
 
2011 
 
 
 ii 
ACKNOWLEDGEMENTS 
 
I have benefited from the guidance, help, and support of a great number of people while 
working on this research. The list below is by no means exhaustive, and I appreciate 
everyone who has been there for me in one way or another over the years. 
 
I am especially grateful to my advisor, Rob Fleischer. His kindness and generosity are 
rare traits in academia. He gave me the freedom to grow as a scientist while at the same 
time keeping me from going too far afield. I would also like to thank Helen James who 
has been a mentor in so many different ways. I am also indebted to Michael Cummings. 
He has given me many great opportunities, which have helped make this  - and my future 
research endeavors - a success. Dave Hawthorne, Charlie Mitter, and Jon Ballou have 
challenged and encouraged me, and ensured that I received the best graduate training 
possible. Sara Via and Matt Hare were also instrumental in earlier stages of my graduate 
career. 
 
Anne Wiley has been a great friend and compatriot during our work on the Hawaiian 
petrel. I appreciate her helpful insights, and enjoyed trading many long emails (and 
conference calls, for that matter) with her. Peggy Ostrom?s enthusiastic spirit have proven 
invaluable. I am also thankful to Megan Spitzer, who patiently worked with me over 
draws and draws of beautifully organized Hawaiian petrel bones. Allison Yoshida and 
Colleen Cotton both provided valuable assistance in the lab. 
 
I have collaborated with so many great people who are associated with Hawaiian petrel 
research and conservation. Nick Holmes, Jay Penniman, Fern Duvall, Seth Judge, Darcy 
Hu, Josh Adams, Keith Swindle, David Duffy, and David Anderson all helped me obtain 
samples that were critical to this project. I also had quite a few long discussions about 
Hawaiian petrel biology with many of these folks, in addition to David Ainley and 
Cathleen Hodges. I am also thankful to Vicki Friesen, Francisco Cruz-Delgado, and Jos? 
Gonz?lez for access to Galapagos petrel samples. Hawaii Volcanoes National Park, the 
Hawaii Experimental Tropical Forest, and Galapagos National Park also provided access 
to samples. I thank Gary Nunn for preliminary work investigating the taxonomic status of 
Galapagos and Hawaiian petrels. 
 
I am also indebted to many people and museums for my work with ancient and historic 
samples. At the Smithsonian?s National Museum of Natural History, I thank James Dean 
for permission to sample bones and museum specimens. Christina Gebhard provided 
much appreciated assistance in preparing voucher specimens of Hawaiian petrels. At the 
Bernice P. Bishop Museum I would like to thank the indomitable Karla Kishinami and 
Lydia Garetano. I would also like to thank Kimball Garrett at the Los Angeles County 
Museum of Natural History and Sharon Birks at the Burke Museum of Natural History 
and Culture. 
 
I am also especially grateful to all of my friends. They have patiently answered my 
questions, discussed Hawaiian petrels ad nauseum, and they have just been there for me 
through thick and thin. I discussed much of this work with Frank Hailer, and he provided 
 iii 
many valuable comments and insights. I thank Joan West and Julie Byrd who were great 
friends right from the beginning. I am also indebted to Gwen Shlichta, Maria Murray and 
Pete Thompson, who have taken me under their wing. Paul Zwiers, Jon Beadell, Emily 
Latch, Heather Lerner, and Nancy Rotzel have been invaluable. I would like to thank 
Akito Kawahara, Kelsey Glennon, Adam Bazinet and Daniel Ayers who have also put up 
with me over the years. I would also like to thank all of my other friends in the Behavior, 
Ecology, Evolution and Systematics Program, in the Biology and Entomology Programs, 
and at the Center for Conservation and Evolutionary Genetics. You have been great. 
 
Lois Reid, Sarah Hankerson, Abby Kula and folks in the BEES Office have always gone 
out of their way to help me, and without them I would have gotten lost in a sea of 
paperwork. 
 
Finally, I am grateful to my family. My husband, Quentin Welch, has always been there 
for me and has suffered through many late dinners and months alone at home with the 
dogs while I have been away on fieldwork. My parents, Louis and Arlene King, have 
endowed me with determination, which has been one of my greatest assets in conducting 
research. My grandmother, Leota King, taught me to keep everything in perspective. 
 
Funding for this work was generously provided by the Behavior, Ecology and Evolution 
and Systematics Program here at the University of Maryland, the Smithsonian Office of 
Fellowships, the Smithsonian?s Walcott Fund, the Center for Conservation and 
Evolutionary Genetics, and the National Science Foundation (grant # DEB-0745604 to P. 
Ostrom, R. Fleischer and H. James).  
 iv 
TABLE OF CONTENTS 
 
 
ACKNOWLEDGEMENTS        ii 
 
LIST OF TABLES         vi 
 
LIST OF FIGURES         viii 
 
 
INTRODUCTION         1 
 
 
CHAPTER I 
Mitochondrial and nuclear DNA sequences reveal recent divergence in  
morphologically indistinguishable petrels      7 
 
Abstract          7 
Introduction         9 
Methods          11 
Results          20 
Discusion          26 
Tables          33 
Figures          37 
 
 
CHAPTER II 
Polymorphic microsatellite markers for the endangered Hawaiian petrel  
(Pterodroma sandwichensis)        42 
 
Abstract          42 
Main Text          43 
Tables          48 
 
 
CHAPTER III 
Population divergence and gene flow in an endangered and highly mobile 
seabird           52 
 
Abstract          52 
Introduction         54 
Methods          56 
Results          64 
Discusion          69 
Tables          77 
Figures          80 
 v 
 
 
CHAPTER IV 
Ancient DNA reveals resilience despite the threat of extinction: three thousand  
years of population genetic history in the endemic Hawaiian petrel  
(Pterodroma sandwichensis)        84 
 
Abstract          84 
Introduction         86 
Methods          90 
Results          100 
Discussion          108 
Tables          120 
Figures          126 
 
 
APPENDIX A  
Museum sample and voucher information.       132 
 
APPENDIX B 
Mitochondrial and nuclear DNA sequence primer information   135 
 
APPENDIX C 
Supplementary tables and figures for Chapter I     136 
 
APPENDIX D 
Supplementary tables and figures for Chapter III     143 
 
APPENDIX E 
Supplementary tables and figures for Chapter IV     148 
 
 
REFERENCES         158 
 
 vi 
LIST OF TABLES 
 
 
CHAPTER I 
Mitochondrial and nuclear DNA sequences reveal recent divergence in morphologically 
indistinguishable petrels 
 
Table 1. Summary of taxa sampled, collecting locality and sample sizes 
 
Table 2. Genealogical sorting index and associated p-values by locus for Galapagos and 
Hawaiian petrels. 
 
Table 3. Migration estimates for Hawaiian petrels from the program Migrate 
 
Table 4. Migration estimates for Galapagos petrels from the program Migrate 
 
 
 
CHAPTER II 
Polymorphic microsatellite markers for the endangered Hawaiian petrel  
(Pterodroma sandwichensis) 
 
Table 1. Characterization of 10 microsatellite loci isolated from the Hawaiian petrel  
 
Table 2. Characterization of 10 previously developed microsatellite loci in the Hawaiian 
petrel 
 
 
 
CHAPTER III 
Population divergence and gene flow in an endangered and highly mobile seabird 
 
Table 1. Sample sizes for Hawaiian petrels (Pterodroma sandwichensis) obtained from 
all islands where this species is currently, or was historically, known to breed. 
 
Table 2. Population differentiation of historic and modern Hawaiian petrels for 
mitochondrial and nuclear DNA sequence data sets 
 
Table 3. Population differentiation of modern Hawaiian petrels for the microsatellite data 
set 
 
 
 
 vii 
CHAPTER IV 
Ancient DNA reveals resilience in the face of extinction: three thousand years of 
population genetic history in the endemic Hawaiian petrel (Pterodroma sandwichensis) 
 
Table 1. Number of Hawaiian petrel samples, by island and time period, for which DNA 
sequences were successfully obtained. 
 
Table 2. Pairwise F
ST
 and associated p-values for Cytochrome b sequences obtained from 
ancient, historic, and modern Hawaiian petrels, representing samples from all islands 
where they have been known to breed.  
 
Table 3. Pairwise F
ST
 and associated p-values for sequences from ancient Hawaiian 
petrels on Hawaii, Maui, and Oahu.  
 
Table 4. Pairwise F
ST
 and associated p-values for sequences from petrels from different 
areas on the island of Hawaii.  
 
Table 5. Haplotype and nucleotide diversity, and their 95% confidence intervals, by time 
period for samples obtained on the island of Hawaii. 
 
Table 6. Haplotype and nucleotide diversity, and their 95% confidence intervals, by time 
period for samples obtained on the island of Maui. 
 viii 
LIST OF FIGURES 
 
 
CHAPTER I 
Mitochondrial and nuclear DNA sequences reveal recent divergence in morphologically 
indistinguishable petrels 
 
Figure 1. Map of the Pacific Ocean and the Hawaiian and Galapagos archipelagoes 
 
Figure 2. Bayesian majority rule consensus tree for the mitochondrial Cytb gene. 
 
Figure 3. Bayesian majority rule consensus tree obtained for the Lam intron as well as the 
partitioned analysis of three nuclear introns. 
 
Figure 4. Marginal posterior distributions from the Isolation with Migration program for 
migration rates and divergence time. 
 
Figure 5. Comparison of divergence time estimates from the Isolation with Migration 
program and BEAST for the mitochondrial and nuclear intron data sets. 
 
 
 
CHAPTER III 
Population divergence and gene flow in an endangered and highly mobile seabird 
 
Figure 1. Map of the main Hawaiian Islands with approximate locations of modern 
(Hawaii, Maui, Lanai, and Kauai) and historically known (Molokai) breeding colonies 
shaded. 
 
Figure 2. Haplotype network for 322 modern and historic Hawaiian petrel mitochondrial 
Cytochrome b sequences. 
 
Figure 3. Genetic ancestry of Hawaiian petrels as estimated by the program STRUCTURE 
from the microsatellite data set using the admixed model with correlated allele 
frequencies and sampling location as a prior.  
 
Figure 4.  Graph of ?K statistic (Evanno et al. 2005) for STRUCTURE runs with the 
number of clusters (K) set between 1 and 8.  
 
 
 
 ix 
CHAPTER IV 
Ancient DNA reveals resilience in the face of extinction: three thousand years of 
population genetic history in the endemic Hawaiian petrel (Pterodroma sandwichensis) 
 
Figure 1. Map of the main Hawaiian Islands with approximate locations of modern 
(Hawaii, Maui, Lanai, and Kauai) and historically known (Molokai) breeding colonies 
shaded, as well as collection localities of sub-fossil bones.  
 
Figure 2. Haplotype network depicting relationships between Cytochrome b sequences 
obtained for 417 ancient, historical, and modern Hawaiian petrels from all islands where 
the birds are currently known, or have been known in the past, to breed.  
 
Figure 3. Temporal trend in the effective population size of the Hawaiian petrel on 
Hawaii and Maui. 
 
Figure 4. BOTTLESIM simulations of change in the expected heterozygosity of the 
Hawaiian petrel as a result of a decline to a constant effective population size ranging 
from 10 to 1000 individuals at 150 years in the past. 
 
Figure 5. BOTTLESIM simulations of the impact of life history characteristics on the 
change in the expected heterozygosity as a result of a decline to a constant effective 
population size of 100 individuals at 150 years in the past.  
 
Figure 6. Relative contribution of modern and extirpated Hawaiian petrel populations to 
overall genetic diversity and allelic richness.  
 
 
 1 
INTRODUCTION 
 
 
Humans have greatly impacted the Earth, leading to a biodiversity crisis (Pechmann and 
Wilbur 1994; Ceballos and Ehrlich 2002; Ehrlich and Wilson 1991; Myers and Knoll 
2001; Pimm et al. 2006). Anthropogenic impacts include direct exploitation (Brashares et 
al. 2004; Milner-Gulland et al. 2003), habitat fragmentation or destruction (Debinski and 
Holt 2000; Brook et al. 2003), introduction of invasive species (Mooney and Cleland 
2001), and introduction of exotic pathogens (Daszak et al. 2000; Kilpatrick et al. 2006). 
Thousands of species have likely gone extinct (Pimm et al. 2006), but even those that 
remain have been greatly impacted. The range of many extant species has been reduced, 
often due to population extirpations, and as a result, many are increasingly threatened by 
extinction (Hughes et al. 1997). 
 
There are several reasons to conserve biodiversity including ecosystem services (Luck et 
al. 2003; Naeem et al. 1994) and economic benefits (Goodwin 1996; Kirkby et al. 2010), 
as well as both aesthetic and ethical reasons (Frankham et al. 2002). An understanding of 
genetics can aid in conservation efforts in many ways (Frankham 2005). For example, it 
can be used to identify species or populations of conservation concern, including 
identification of cryptic species, resolution of taxonomic uncertainty, and delineation of 
evolutionarily significant units (Yoder et al. 2000; Moritz 1994). Genetics can also be 
used to investigate population connectivity and isolation (Foote et al. 2011; Funk et al. 
2005), as well as to investigate changes in genetic diversity and trends in effective 
population size (Lipp? et al. 2006; Tessier et al. 1997) 
 
 2 
At the species level, genetics can inform us about the process of evolution. If two 
populations become separated (either by extrinsic or intrinsic factors) they may diverge to 
the extent where they become reproductively isolated, and therefore be considered 
separate species (Coyne and Orr 2004). The genome of the organisms will reflect these 
changes, but the signal of divergence may accumulate at different rates (Hudson and 
Coyne 2002; Avise et al. 1983). For example, in most vertebrates, due to the haploid 
nature and the maternal inheritance mode of mitochondrial DNA, it will have a shorter 
coalescence time than that of nuclear DNA (Palumbi et al. 2001), and hence will often be 
a relatively ?leading indicator? of divergence (Zink and Barrowclough 2008). If not taken 
into account, this process of lineage sorting can make it difficult to discern taxonomic 
relationships (Weisrock et al. 2010). Still, genetic techniques have been successfully used 
to identify taxa that may warrant conservation efforts, but which might otherwise be 
overlooked (Hailer et al. 2011). 
 
If two populations are not isolated, and instead they exchange migrants, then they may 
remain a single species. In this situation it can be useful to gain a better understanding of 
the patterns of gene flow and isolation between populations. This is particularly true for 
endangered species that may be isolated due to anthropogenic effects (Schwartz et al. 
2005).  Knowledge of population connectivity can also be important for reserve design 
(Palumbi 2003) and the selection of individuals for translocations (Schultz et al. 2011). 
Finally, investigations of gene flow can also reveal hybridization of endangered species 
with more common species, which can be a serious problem (Weigel et al. 2003). 
 
 3 
Within populations, genetics can be used to investigate changes in population size. When 
species undergo a severe decline in number, or bottleneck, they may also exhibit a 
decrease in genetic diversity (Taylor et al. 1994) and this lower level of genetic diversity 
may increase the extinction risk (Frankham 2005). Molecular markers can also be used to 
investigate the effective population size of a species. The effective population size of a 
species is the size of an ideal population that would lose genetic diversity (i.e. through 
genetic drift) at the same rate as the population of interest (Frankham et al. 2002). This 
concept becomes important when individuals in a population do not contribute equally to 
the gene pool. This is often the case in real populations and can be caused, for example, 
by variation in reproductive success or unequal sex ratios (Allendorf and Luikart 2007). 
Changes in effective population size can also be informative for conservation, such as the 
impact of translocated individuals or supportive breeding programs (Tessier et al. 1997). 
 
Combining genetic techniques with the use of historical, archeological, and 
paleontological specimens can lead to additional insights important for conservation 
(Leonard 2008). Through the use of ancient DNA, extinct species can be studied to 
determine phylogenetic relationships with extant species. For example, ancient DNA has 
been used to investigate a radiation of geese in the Hawaiian Islands. This radiation 
included a giant, flightless form which is not known from historical records (Paxinos et 
al. 2002b). From ancient samples it is also possible to learn about past levels of gene flow 
and changes in population size, for example, in response to anthropogenic impacts or 
climate change (Campos et al. 2010). While special care is required to work with ancient 
 4 
DNA (Axelsson et al. 2008), it has the ability to give insights that cannot be obtained 
with modern DNA alone (Ramakrishnan et al. 2005; Leonard 2008). 
 
The Hawaiian Islands have a rich subfossil history, including 39 species of land birds that 
are not known from historical records. Many of these went extinct after the colonization 
of the Islands by humans (Olson and James 1982b). Of the species that persisted, many 
have undergone a range contraction and a reduction in the number of populations, and 
many are endangered. The Hawaiian petrel (Pterodroma sandwichensis) is one such 
species. 
 
The Hawaiian petrel is a long-lived pelagic seabird. It is endemic to the Hawaiian Islands 
and today it breeds on four of the main islands: Hawaii, Maui, Lanai, and Kauai (Simons 
and Hodges 1998). However, in the past, it was more widely distributed. Subfossil 
evidence indicates that it was formerly more wide spread on the islands where it is 
currently known to breed, with colonies in the past occurring just above sea level, 
whereas today colonies are found only at high elevation (Athens et al. 1991). A large 
colony was also present in the lowlands of Oahu, but there are no historical records 
documenting that Hawaiian petrels there (Olson and James 1982a). There was also a 
large colony on the island of Molokai, with birds so numerous that they were said to 
?darken the sky? (Munro 1955), however recent surveys have failed to locate a 
substantial number of birds breeding there (Simons and Hodges 1998). 
 
 5 
It appears that humans have had great impacts on this species, both directly and 
indirectly. Historic records indicate that Hawaiian petrel chicks were considered a 
delicacy for the Hawaiian chiefs (Bryan 1908; Henshaw 1902). There is also evidence 
that the adults were consumed by Hawaiians on trips to acquire resources from high 
elevation sites (Athens et al. 1991; Hu et al. 2001). Humans also introduced exotic 
mammalian predators, such as rats, cats, and mongoose, which have been and continue to 
be a serious threat to their persistence (Carlile et al. 2003; Bryan 1908; Simons 1984). In 
addition to these factors, grazing by introduced ungulates and the introduction of invasive 
plants have led to habitat destruction (Simons and Hodges 1998). Today, at sea estimates 
indicate that there are between 11,000 and 34,000 Hawaiian petrels remaining, however 
given that the distribution of these birds was formerly much larger, in addition to high 
levels of predation by introduced mammals observed today, the number of Hawaiian 
petrels may have been much larger in the past. 
 
Despite all of this, little is actually known about the Hawaiian petrel. As with most other 
procellariiform species, it spends the majority of its life at sea, but little is known about 
it?s non-breeding distribution (Simons and Hodges 1998). When this species returns to 
land, it breeds in remote and inaccessible colonies, often nesting in burrows that are 
greater than 2 m long, making it difficult to study it directly (Simons 1985). And in 
addition to all of this, Hawaiian petrels return to land nocturnally (Simons and Hodges 
1998). Even the taxonomic status of this species remains somewhat uncertain. For many 
years the Hawaiian petrel was considered a subspecies of the dark-rumped petrel, along 
with the Galapagos petrel. However, in 2002 these two were split based on behavioral, 
 6 
biochemical, and subtle morphological differences. This split has not been investigated 
further. 
 
The goal of this work is to use conservation genetics and ancient DNA techniques to 
investigate the taxonomic standing and population dynamics of the endangered Hawaiian 
petrel. In the first chapter I use mitochondrial and nuclear DNA sequences to investigate 
if Hawaiian and Galapagos petrels represent separate species. In the second chapter I 
describe the development of 10 microsatellite loci specifically for the Hawaiian petrel, as 
well as characterization of 10 previously reported loci in the Hawaiian petrel. In the third 
chapter, I combine these microsatellite loci with mitochondrial and nuclear intron 
sequences to investigate the patterns of gene flow and isolation in contemporary 
populations, as well as a population on Molokai that may have been extirpated in the last 
100 years. Finally, in the fourth chapter I utilize ancient DNA from subfossil Hawaiian 
petrel bones to examine temporal trends in genetic diversity and gene flow as humans 
colonized the Hawaiian Islands. 
 
 7 
 
CHAPTER I 
 
 
Mitochondrial and nuclear DNA sequences reveal recent divergence in 
morphologically indistinguishable petrels* 
 
 
Often during the process of divergence genetic markers will only gradually obtain the 
signal of isolation. Studies of recently diverged taxa utilizing both mitochondrial and 
nuclear data sets may therefore yield gene trees with differing levels of phylogenetic 
signal due to differences in coalescence times. However, several factors can lead to this 
same pattern, and it is important to distinguish between them in order to gain a better 
understanding of the process of divergence and the factors driving it. Here we employ 
three nuclear intron loci in addition to the mitochondrial Cytochrome b gene to 
investigate the magnitude and timing of divergence between two endangered and nearly 
indistinguishable petrel taxa: the Galapagos and Hawaiian petrels (Pterodroma 
phaeopygia and P. sandwichensis). Phylogenetic analyses indicated reciprocal 
monophyly between these two taxa for the mitochondrial data set, but trees derived from 
the nuclear introns were unresolved. Coalescent analyses revealed effectively no 
migration between Galapagos and Hawaiian petrels over the last 100,000 generations and 
that they diverged relatively recently, approximately 550,000 years ago, coincident with a 
time of intense ecological change in both the Galapagos and Hawaiian archipelagoes. 
This indicates that recent divergence and incomplete lineage sorting are causing the 
difference in the strength of the phylogenetic signal of each data set, instead of 
insufficient variability or ongoing male-biased dispersal. Further coalescent analyses 
show that gene flow is low even between islands within each archipelago suggesting that 
 8 
divergence may be continuing at a local scale. Accurately identifying recently isolated 
taxa is becoming increasingly important as many clearly recognizable species are already 
threatened by extinction. 
 
*A. J. Welch, A. A. Yoshida, and R. C. Fleischer. 2011. Molecular Ecology 20 (7) : 1364 
? 1377.
 9 
INTRODUCTION 
  
Genetic divergence can arise between populations very quickly, for example through 
chromosomal rearrangements, founder effects, or changes in ploidy (Navarro and Barton 
2003; Soltis and Soltis 1999; Carson and Templeton 1984), but in many cases it can be a 
long process. Often when two taxa become isolated genetic markers will only gradually 
move to equilibrium - first giving a polyphyletic, then paraphyletic, and eventually a 
reciprocally monophyletic phylogenetic signal. The length of time required for lineages 
to sort out depends on the effective population size and coalescence time of the loci 
utilized (Avise 2004). For example, since mitochondrial loci have an effective population 
size one quarter that of nuclear loci, mitochondrial loci will have a shorter coalescence 
time (Palumbi et al. 2001). During the divergence process this can lead to gene trees with 
different levels of support for the same clade. For example, Hudson and Coyne (2002) 
point out that in the simplest theoretical case, where two populations diverge in the 
absence of gene flow and where population sizes are equal, it is expected that a single 
mitochondrial locus and a single nuclear locus will give different phylogenetic signals for 
approximately 6N
e
 generations (where N
e
 represents the effective population size), 
because of shared ancestral polymorphism alone.  
 
Empirically, when gene trees with differing levels of phylogenetic support are obtained 
from multilocus, multigenomic data sets it can be difficult to determine the underlying 
causes (Maddison 1997; Maddison and Knowles 2006).  The pattern of recent divergence 
and incomplete lineage sorting can be difficult to distinguish from ongoing male-biased 
gene flow or even from a lack of signal in nuclear gene sequences (Avise et al. 1983; 
 10 
Degnan and Rosenberg 2009).  Such situations may be encountered with increasing 
frequency as continual technological developments make it easier to obtain both 
mitochondrial and nuclear gene sequences for non-model organisms (Harismendy et al. 
2009). In the case of gene trees where clades have different levels of phylogenetic 
support, methods that implement coalescent theory can be particularly helpful as they 
permit estimation of parameters such as gene flow and divergence time, and allow 
assessment of the uncertainty of these estimates (Kuhner 2009; Nielsen and Wakeley 
2001). Disentangling the factors behind the observed difference in resolution can have 
important consequences for our understanding of the divergence process and the factors 
driving it, as well as for delimiting species boundaries in taxa threatened by extinction 
(Weisrock et al. 2010). 
 
In this study we investigated evidence for recent and ongoing divergence between the 
endangered Galapagos and Hawaiian petrels (Pterodroma phaeopygia and P. 
sandwichensis, respectively). Traditionally these taxa have been considered a single 
species, likely as a result of their striking morphological similarity (Brooke 2004). Both 
taxa are medium sized (43 cm in length) and have black and white dichromatic plumage 
making them nearly indistinguishable, particularly at sea (Spear et al. 1995; Brooke 2004; 
Tomkins and Milne 1991; Simons 1985; Force et al. 2007). Seabirds, such as these 
petrels, are highly mobile and have few apparent physical barriers to dispersal. Yet many 
species exhibit strong philopatry and therefore have the potential to become genetically 
distinct over short geographical distances (Milot et al. 2008). A study of thirteen 
allozyme loci from blood samples found that eleven were fixed for the same allele in both 
 11 
Hawaiian and Galapagos petrels, one locus was polymorphic in Galapagos petrels, and a 
single locus displayed a fixed difference between the two taxa (Browne et al. 1997). 
Based on these biochemical data as well as variation in breeding phenology and song, 
these taxa have recently been elevated to species status (Banks et al. 2002), but this split 
has not been investigated further. This is especially important as both of these taxa are 
increasingly threatened by extinction (Brooke 2004; Simons and Hodges 1998). 
 
To gain a better understanding of the extent of genetic differentiation between the 
endangered and morphologically similar Galapagos and Hawaiian petrels, we sequenced 
the entire mitochondrial Cytochrome b gene and three nuclear intron loci. We employed 
both phylogenetic and coalescent-based approaches to examine evidence for recent 
divergence versus ongoing male-biased gene flow. Finally, we investigated migration 
between populations within each species to explore the potential for divergence at a local 
scale. 
 
MATERIALS AND METHODS 
 
Samples 
 
We sampled a total of twenty-eight Hawaiian petrels, which included seven individuals 
from each of the four main Hawaiian Islands where they are currently known to breed 
(Figure 1, Table 1). Historically there was also a large colony on the island of Molokai, 
but it is now feared that this colony has been extirpated (BirdLife International 2009). 
 12 
Samples included blood taken from grounded fledgling birds that were subsequently 
rehabilitated and released, as well as tissue, feather, and bone from depredated carcasses 
collected opportunistically during the breeding season from 1990 to 2008. Hawaiian 
petrels nest in burrows and are particularly vulnerable to exotic mammalian predators 
during this time. Non-breeding birds are known to fly over the colonies, but rarely come 
to ground and depart early in the breeding season (Simons and Hodges 1998), therefore 
samples are most likely to come from breeders or their offspring. Voucher information 
for Hawaiian petrel specimens can be found in Appendix A. A subset of the Galapagos 
petrel samples previously collected by Friesen et al. (2006) were utilized in this study. 
We obtained a total of thirty-five samples, which included seven individuals from each of 
the five Galapagos Islands that make up the entire breeding range of this bird (Figure 1, 
Table 1). For outgroup taxa, six blood samples each were obtained from the Atlantic 
petrel (Pterodroma incerta) and Soft-plumaged petrel (P. mollis) on Gough Island. 
Additionally, we obtained a tissue sample from the Bernice P. Bishop Museum 
(accession number BPBM184828) of one Murphy?s petrel (P. ultima), which was 
originally collected at sea (Appendix A). These outgroup taxa represented both closely 
(P. ultima) and more distantly related (P. incerta and P. mollis) Pterodroma petrels 
(Nunn and Stanley 1998; Lawrence et al. 2009). 
 
Molecular characterization 
 
Total genomic DNA was extracted from Hawaiian petrel and outgroup samples using 
either the DNeasy tissue kit (Qiagen) for tissue and blood or a phenol/chloroform 
 13 
method, developed for use with ancient and degraded DNA, for bone and feather samples 
(Horv?th et al. 2005; Fleischer et al. 2000). DNA was previously extracted from 
Galapagos petrel samples (Friesen et al. 2006). The entire mitochondrial Cytochrome b 
(Cytb) gene was amplified using overlapping primers (Appendix B) designed in Primer3 
(Rozen and Skaletsky 2000). A simulation study by McCormack et al. (2009) 
demonstrated a plateau in accuracy for sampling efforts beyond three loci and nine gene 
copies when utilizing coalescent-based approaches to investigate the relationships of 
recently diverged taxa. Therefore, three nuclear introns were amplified: ?-enolase intron 
8 (Enol), lamin A intron 3 (Lam), and ribosomal protein 40 intron 5 (RP40) (Friesen et 
al. 1999; Friesen et al. 1997).  For samples with degraded DNA, shorter overlapping 
primer sets were developed (Appendix B). Polymerase chain reactions were carried out in 
a total volume of 15 uL, utilizing 1x PCR Gold Buffer (Applied Biosystems), 2.5 to 4 
mM MgCl
2
, 0.2 mM each dNTP, 1.2 mg/mL BSA, 0.5 ?M of each primer, 1 unit 
AmpliTaqGold Polymerase (Applied Biosystems) and 1 to 2 uL of DNA extract. 
Amplifications took place under the following thermocycle profile: an initial denaturation 
step at 94
0
C for 8 minutes, and then 35 cycles of 92
0
C for 30 sec, a primer specific 
annealing temperature between 50
 0
C and 60
0
C for 30 sec, and an extension step at 72
0
C 
ranging from 30 sec to 1 min proportional to the length of the PCR product, followed by 
a final 7 minute extension at 72
0
C.  
 
Excess primers in the PCR product were degraded using a 1/10 dilution of ExoSAP-IT 
(USB) before cycle sequencing in both directions using the Big Dye Terminator v. 3.1 
Cycle-Sequencing Kit (Applied Biosystems). Sequencing reactions were purified in 
 14 
Sephadex (G-50 fine) columns and electrophoresed in a 3130xl Genetic Analyzer 
(Applied Biosystems). Sequences were assembled in Sequencher v. 4.8 (Gene Codes 
Corporation) and visually inspected. Since some analyses conducted in this study (e.g. 
the Isolation with Migration program) required known phase for nuclear sequences, 
gametic phase was determined probabilistically using the program PHASE (Stephens and 
Donnelly 2003; Stephens et al. 2001). For individuals with resulting phase probabilities < 
80% (Harrigan et al. 2008), haplotypes were determined via cloning using the TOPO TA 
cloning kit for sequencing (Invitrogen) and direct sequencing of more than five colonies. 
Phased haplotypic data were used for all nuclear analyses (i.e. each individual was 
represented by two sequences). Sequences for most loci were aligned in Sequencher, 
however the RP40 intron locus contained three short gaps and was aligned using 
ClustalX v2.0.12 (Larkin et al. 2007; Thompson et al. 1997). Gaps were retained in the 
data set and handled according to the default settings for each program. For example, 
gaps were treated as missing data in phylogenetic analyses, and discarded by the Isolation 
with Migration program. 
 
Phylogenetic analyses 
 
To investigate the magnitude of divergence between Galapagos and Hawaiian petrels, 
gene trees were constructed. First, however, Cytb sequences were translated in DAMBE 
(Xia and Xie 2001) and examined for the presence of stop codons and other indicators 
that they were nuclear copies (Sorenson and Fleischer 1996; Sorenson and Quinn 1998). 
Once the mitochondrial origin of the sequences was confirmed, the best-fit model of 
 15 
nucleotide substitution for mitochondrial and nuclear DNA sequences was examined 
using jModelTest 0.1.1 (Posada 2008; Guindon and Gascuel 2003) with the Akaike 
information criterion.  
 
Phylogenetic reconstruction was conducted with both maximum likelihood and Bayesian 
inference using Garli 0.96 (Zwickl 2006) and MrBayes 3.1.2 (Altekar et al. 2004; 
Ronquist and Huelsenbeck 2003), respectively. The HKY + I substitution model was 
utilized for both mitochondrial and nuclear data sets and analyses were conducted for 
each locus individually. Three additional partitioned Bayesian analyses were also carried 
out with all parameters except topology and branch lengths unlinked. First, the Cytb 
sequences were partitioned by codon position using the HKY, HKY + I, and HKY + G 
models for first, second, and third codon positions, respectively. Bayes factors were 
estimated in Tracer 1.4.1 to assess support for the codon-partitioned model over the 
unpartitioned model (Drummond and Rambaut 2007; Suchard et al. 2001; Kass and 
Raftery 1995). Second, a partitioned analysis was conducted on a data set that included 
all nuclear intron loci. Third, a partitioned analysis of the combined mitochondrial and 
nuclear data sets was performed. Finally, a maximum likelihood analysis was carried out 
for the three nuclear intron loci concatenated into a single data set. For maximum 
likelihood analyses, multiple runs were performed with random and stepwise addition, 
and support was assessed via 1000 nonparametric bootstrap replicates. Bayesian runs 
were conducted for between 10 x 10
6
 and 50 x 10
6
 generations each with four Markov 
chains and the default heating scheme. Output was recorded so that 10,000 data points 
were saved for each run and the first 10% were discarded as burn-in. Stationarity was 
 16 
investigated by plotting ?lnL across generations in Tracer 1.4.1 (Drummond and 
Rambaut 2007) and examining the effective sample size (ESS), which was greater than 
500 for all estimated parameters.  Convergence was assessed through comparison of 
multiple independent runs.  
 
Genealogical sorting index 
 
A measure known as the genealogical sorting index (gsi, Cummings et al. 2008) has 
recently been developed to quantify lineage divergence in cases where taxa do not form 
monophyletic groups during phylogenetic reconstruction. It measures divergence on a 
continuous scale from 0 to 1, where 1 represents the qualitative state of monophyly. To 
account for the uncertainty in the relationship between Galapagos and Hawaiian petrels in 
each nuclear intron phylogeny, the ensemble gsi statistic for each locus was calculated on 
the ~7,100 trees that represent the 95% credible set found in MrBayes. Significance of 
this statistic was determined through 10,000 permutations. For comparison, gsi was also 
calculated for an additional, previously published seabird mitochondrial DNA sequence 
data set investigating speciation in populations of band-rumped storm petrels breeding 
sympatrically in the Galapagos Islands during different seasons (Smith and Friesen 
2007).  
 
Inter-specific gene flow and divergence time 
 
 17 
To investigate the timing and magnitude of divergence between Galapagos and Hawaiian 
petrels in a coalescent framework, the Isolation with Migration, (IMa; Hey & Nielsen 
2007; Nielsen & Wakeley 2001) was utilized. IMa assumes no recombination within loci, 
and this assumption was tested using the difference of sums of squares method (DSS), a 
sliding window technique, implemented in TOPALi v 2.5 (McGuire and Wright 2000; 
Milne et al. 2009). Window size was set to 70 bp with a 10 bp increment, and statistical 
significance was determined through 100 parametric bootstraps. Initial IMa runs were 
conducted using extremely wide priors to establish the appropriate values for further 
analyses (Won and Hey 2005). Multiple independent final runs were conducted for 
between 5 x 10
6
 and 2 x 10
8
 generations both with and without heating. The first 10% of 
the genealogies were discarded as burn-in. Runs were considered to have reached 
stationarity after examination of plots of marginal posterior probability for each 
parameter and when the ESS for each run was > 200 (Won and Hey 2005).  
 
Parameter estimates from IMa are compounded by the mutation rate, ?, and generation 
time. Therefore, to translate parameter estimates into values that are more easily 
interpretable, a mutation rate and generation time were applied. For the Cytb gene the 
mutation rate was determined by multiplying the sequence length (Appendix A) by an 
estimate of mutation rate for procellariiform seabirds of 1.89 x 10
-8
 substitutions/site/year 
(Weir and Schluter 2008). For the nuclear intron data set, sequence length for each locus 
was multiplied by a mutation rate of 3.6 x 10
-9
 substitutions/site/year (Axelsson et al. 
2004; Carling and Brumfield 2008) and then the geometric mean of these three individual 
mutation rates was determined. The substitution rate of Axelsson et al. (2004) was 
 18 
utilized since it was derived from birds and estimated from a broad sampling of 33 
autosomal loci.  To translate parameter estimates into demographic terms, generation 
time was also required.  Unfortunately, little is known about generation time for these 
long-lived taxa. The average age at first breeding for procellariiform seabirds is roughly 
correlated with body size, and birds of similar size to Galapagos and Hawaiian petrels 
reach sexual maturity by approximately 5 or 6 years of age (Simons 1984; Warham 
1990). Some albatross species are known to begin breeding regularly within 3 years after 
sexual maturity with the latest beginning to breed by about 12 years of age. While age at 
first breeding does not necessarily equal generation time, it seems reasonable that 
generation time for petrels of this size is between 6 and 12 years, therefore we calculated 
demographic estimates using generation times of both 6 and 12 years. Interpretation of 
results was the same independent of which estimate was utilized, therefore results for a 
generation time of 6 years are presented. 
 
The IMa model also assumes that in the past there was one ancestral population that split 
into two daughter populations. This generally corresponds to the divergence between 
Galapagos and Hawaiian petrels, however, based on the literature (Friesen et al. 2006) 
and topology of the inferred mitochondrial gene tree, there is some evidence for 
population genetic differentiation within each taxon. Given these departures from the 
assumptions, we also estimated divergence time in BEAST v 1.5.2 (Drummond and 
Rambaut 2007). Outgroups were used to root trees constructed in BEAST, therefore 
analyses were conducted with a Yule tree prior. For Cytb data, a single haplotype from 
each island was utilized for the Galapagos and Hawaiian petrels, in addition to a 
 19 
sequence from Pterodroma ultima, and sequences from P. inexpectata, and P. externa 
downloaded from the GenBank database (Accession numbers U74346 and U74339, 
respectively). However, for the intron data set only sequences for P. ultima in addition to 
Galapagos and Hawaiian petrels were available. Divergence dates were estimated using 
both a strict and relaxed clock model (Drummond et al. 2006) with the substitution rate 
fixed as indicated above. Multiple independent runs were completed for 10 x 10
6
 
generations, with the first 10% discarded as burn-in. Stationarity and convergence were 
assessed as described for phylogenetic analyses. Bayes factors were estimated to assess 
support for the relaxed clock model. 
 
Intra-specific gene flow 
 
To investigate the potential for divergence within each taxon at a local scale, gene flow 
between islands in each archipelago was examined using a coalescent approach as 
employed in the program MIGRATE v3.0.3 (Beerli 2006; Beerli and Felsenstein 1999). 
Coalescent based approaches for estimating intra-specific gene flow have higher 
statistical power than summary statistics, are robust, and allow investigation of 
confidence in estimates (Kuhner 2009). Also, they have been found to be accurate with 
small sample sizes. A simulation study by Felsenstein (2006) found the optimal number 
of haplotypes for some coalescent analyses to be eight, or possibly even fewer, per locus. 
 
Migration rates were estimated from the data set of three nuclear introns.  As mentioned 
above, gametic phase was determined for nuclear sequences and so each individual was 
 20 
represented by two sequences in these analyses (i.e. N = 14 gene sequences per locus per 
population, for a total of 64 and 56 gene sequences for Galapagos and Hawaiian petrels, 
respectively). The Bayesian mode was employed with the transition/transversion ratio = 
4.0 (as estimated in Garli), uniform priors, and a single long chain with three replicates. 
A static heating scheme with parameters set to 1.0, 1.5, 3.0, and 10.0 was utilized to 
increase the effectiveness of the search. The chain was run for 20 x10
6
 generations with 
samples recorded every 1000
th
 generation, after discarding the first 10% as burn-in. 
Stationarity was examined and convergence was assessed from multiple independent runs 
initiated with different random seeds. In MIGRATE estimates are compounded by the 
mutation rate, ?, (i.e. M = m/?), and as such, they were scaled by the appropriate 
mutation rate from above.  
 
RESULTS 
 
We sequenced 73 individuals for all 1143 base pairs of the mitochondrial Cytochrome b 
gene and for 741 total base pairs from three nuclear introns. Three Galapagos petrel 
samples (two from Santa Cruz and one from Santiago) were discarded from all analyses 
due to missing data. All sequences were deposited in the GenBank database under 
accession numbers HQ420313-HQ420813. The Cytb data were examined to verify 
mitochondrial origin of sequences. No insertions or deletions were detected, and after 
translation no nonsense or stop codons were found. The ratio of transitions to 
transversions was 19:1 in Galapagos petrels and 12:1 in Hawaiian petrels, and the 
 21 
majority of substitutions occurred in the third codon position. This evidence indicates that 
a mitochondrial origin of the Cytb sequences is likely.  
 
Analyses conducted in Arlequin v.3.1 (Excoffier et al. 2005) on the Cytb and nuclear data 
sets revealed that they contained approximately the same number of variable sites. Fifty-
one polymorphic sites were found for the mitochondrial data set while the combined data 
set of all three nuclear intron loci exhibited 56 total variable sites (10 in Enol, 24 in Lam, 
and 22 in RP40). Within Galapagos and Hawaiian petrels, 14 and 16 haplotypes were 
found for the Cytb gene, respectively, with no haplotypes shared. Additionally, there 
were 16 fixed differences found between the two taxa (Appendix C, Supplementary 
Table 1). For nuclear introns, 8, 21, and 17 haplotypes were discovered for Enol, Lam, 
and RP40, respectively, with 2, 1, and 8 haplotypes shared between taxa (Appendix C, 
Supplementary Tables 2 - 4). There were zero fixed differences found for nuclear 
sequences, but haplotype frequencies differed substantially (Appendix C, Supplementary 
Tables 2 - 4). 
 
Phylogenetic Reconstructions 
 
The gene tree produced from the mitochondrial data set showed strong support for the 
reciprocally monophyletic relationship of Galapagos and Hawaiian petrels, however gene 
trees constructed from the nuclear intron loci were unresolved. The total evidence data set 
yielded the same topology as the mitochondrial tree, but this could be biased by the 
relatively stronger signal in the mtDNA data. For analysis of the Cytb sequences Bayes 
 22 
factors showed strong support for the codon-partitioned model over the unpartitioned 
model (2ln Bayes Factors = 298.688). The topologies of the Bayesian and maximum 
likelihood Cytb trees were congruent, and indicated that Galapagos and Hawaiian petrels 
were each monophyletic with moderate to high support (Figure 2). Within the Galapagos 
petrels, individuals from the same island did not group together. Within Hawaiian petrels, 
however, there were two clades with high support that mostly contained individuals from 
Lanai and Kauai, respectively, while individuals from Hawaii and Maui failed to form 
distinct clades.  
 
The gene trees constructed for each nuclear intron showed a polyphyletic relationship 
between Galapagos and Hawaiian petrels. In two of the three intron trees (Enol and 
RP40) the outgroups, even the most distantly related species from the mitochondrial tree, 
were polyphyletic with Hawaiian and Galapagos petrels. However, in the tree 
reconstructed from the Lam intron sequences the distantly related Atlantic and soft-
plumaged petrels formed a separate clade with high support, while Murphy?s petrel 
remained polyphyletic with the ingroups (Figure 3). The tree reconstructed from the 
partitioned analysis of all intron loci, which included comparatively the same total 
number of variable sites as the Cytb locus (56 versus 51, respectively), nearly twice the 
number of variable sites in Galapagos petrels (25 versus 13), and approximately the same 
number of variable sites in Hawaiian petrels (17 versus 20) yielded the same topology as 
that obtained for the Lam intron, with high support for a clade containing Galapagos, 
Hawaiian, and Murphy?s petrels (Figure 3). For nuclear loci the genealogical sorting 
index for Galapagos and Hawaiian petrels ranged from 0.1785 to 0.2783 and was highly 
 23 
significant in all cases (p < 0.0004, Table 2). These values are also similar to those 
determined for the mitochondrial control region of band-rumped storm petrels (gsi = 
0.2630 and 0.3527, p < 0.0004) that may be undergoing sympatric speciation (Friesen et 
al. 2007b). Despite the unresolved topology of the nuclear gene trees, the gsi analysis 
indicated the presence of weak, but statistically significant phylogenetic signal in the 
nuclear intron data set. Overall, phylogenetic signal was strong in the mitochondrial data 
set, but apparently weak in the nuclear intron data set. 
 
 Inter-specific gene flow and divergence time 
 
To determine if the weak phylogenetic signal in the nuclear intron data set was due to 
incomplete lineage sorting or ongoing gene flow, as well as to investigate the timing of 
divergence of these two taxa, additional analyses were carried out utilizing the IMa 
program (Hey and Nielsen 2004). For both the Cytb and nuclear intron loci, plots of the 
marginal posterior probability for the migration parameters between the Galapagos and 
Hawaiian petrels were unambiguous and very narrow (Figure 4A, C). For three of the 
four migration parameters the peak in the posterior probability occurred in the smallest 
interval measured in the analysis (from 0 to 5.67 x 10
-10
 migrants per generation). While 
it is theoretically possible that given a finer resolution a nonzero peak may be found in a 
smaller interval, these estimates were considered effectively zero (Won and Hey 2005). 
The remaining migration parameter, the number of Hawaiian petrels (HAPE) per 
generation that migrated into the Galapagos petrel (GAPE) population estimated from the 
 24 
intron data, had a wider HPD (highest posterior density), but was still effectively zero 
(M
HAPE
?
GAPE 
=2.86x10
-6
 migrants per generation, 90% HPD: 4.99x10
-7
 to 1.34x10
-5
).  
 
The divergence time estimated by IMa from the nuclear intron data indicated that these 
two taxa split approximately 570,700 years ago (90% HPD: 159,800 to 45,658,200 years 
ago; Figure 4B and Figure 5). A distinct peak occurred in the posterior distribution, 
however the tail hovered near zero for divergence times beyond 2,000,000 years resulting 
in the broad HPD interval (Figure 4). Hence, obtaining smaller HPD intervals may 
require sequencing a larger number of loci (Hey 2005). The mitochondrial data specified 
nearly the same time frame for the split (545,300 years ago, Figure 5), however the 
posterior distribution failed to converge to zero after a peak in the beginning of the 
distribution (Figure 4D). When taxa have reached the state of reciprocal monophyly at a 
locus, as the Galapagos and Hawaiian petrels have for the Cytb gene, there is little or no 
information remaining regarding the divergence process and therefore estimating the 
timing of divergence can be especially difficult (Edwards and Beerli 2000).  
 
Since the potential presence of population structure within the Hawaiian petrels, as 
identified by the mitochondrial DNA phylogeny, may violate assumptions of the IMa 
model, divergence time was also estimated in the program BEAST.  Divergence time 
estimates from BEAST were similar to those found in IMa but with narrower HPD 
intervals (Figure 5). Investigation of Bayes factors indicated little support for the relaxed 
clock over the strict clock model (2ln Bayes Factors = -1.116 and -2.736 for Cytb and 
intron data, respectively). For the strict molecular clock model and the Cytb data set and 
 25 
the split between Galapagos and Hawaiian petrels was estimated to have occurred 
588,600 years ago (95% HPD: 382,300 to 810,800 years ago) and for the intron data set, 
the split was estimated to have occurred 1,376,000 years ago (95% HPD: 683,900 to 
2,170,200). Despite somewhat different point estimates obtained from each data set, the 
95% HPD intervals overlap, indicating that the estimated dates do not differ significantly. 
Additionally, point estimates were similar between the BEAST and IMa analyses. This 
strengthens confidence in the divergence time estimates and indicates that IMa analyses 
were robust to slight model misspecifications, as demonstrated by Strasburg and 
Rieseberg (2010). 
 
Intra-specific analyses 
 
To investigate the potential for divergence of each taxon on a local scale, migration rates 
were estimated for Galapagos and Hawaiian petrels within their respective archipelagoes. 
Posterior distributions for demographic estimates from MIGRATE were narrow and 
unimodal for 31 of 32 parameters investigated. The only exception was for migration of 
Galapagos petrels from the island of Isabela to Floreana, for which the posterior 
distribution was narrow yet bimodal. Therefore this estimate should be interpreted 
cautiously. However the effective sample size for all estimates was greater than 100,000, 
which is one rough indication that the analyses were run for a sufficient length. Results 
indicated low levels of gene flow among islands within each archipelago (Tables 3 and 
4). Estimates were roughly symmetric between islands and ranged from 0.007 to 0.848 
and 0.092 to 0.802 migrants per generation for Galapagos and Hawaiian petrels, 
 26 
respectively. Additionally, the 95% confidence intervals for more than half of the 
migration estimates for each taxon overlapped with zero. 
 
DISCUSSION 
 
Phylogenetic analysis of the Cytb gene demonstrated a reciprocally monophyletic 
relationship for Galapagos and Hawaiian petrels, indicating that these two taxa have 
diverged to a great extent (Figure 2). However, gene trees constructed from three nuclear 
intron loci were unresolved, depicting a polyphyletic relationship for these taxa, as well 
as the clearly distinct Murphy?s petrel (Figure 3). This observed lack of resolution could 
result from insufficient variability in the nuclear data, ongoing male-biased gene flow, or 
a recent divergence with incomplete lineage sorting.  
 
A comparison of the mitochondrial and nuclear data sets suggests that low variability or 
small sample size is not the culprit: the mitochondrial data set contained 51 variable sites, 
and the combined data set for three nuclear introns contained 56 variable sites.  Further, if 
the observed polyphyly was due to insufficient variability in the data set then further 
population based analyses, such as estimates of migration obtained from IMa, should also 
be inconclusive, and this was not the case (Figure 4). Simulation studies of sampling 
design have suggested that when utilizing coalescent based approaches a data set 
consisting of three loci with three to nine gene copies per taxon recovers the relationships 
between recently diverged species with high accuracy (McCormack et al. 2009; 
Maddison and Knowles 2006). Here we sampled three nuclear loci with 14 gene copies 
 27 
each. Additionally, a population level signal of divergence between taxa was apparent 
from haplotype networks constructed for the nuclear intron data sets (Appendix C, 
Supplementary Figures 1 - 3). Finally, the genealogical sorting index indicated the 
presence of weak, but statistically significant phylogenetic signal. Therefore, even though 
there was weak phylogenetic signal in the nuclear intron data set, there was still a strong 
signal of divergence at the population level.   
 
The differing levels of support between gene trees could be caused by ongoing male-
biased gene flow (Degnan and Rosenberg 2009). There are few obvious physical barriers 
to dispersal in seabirds (Friesen et al. 2007a) and in some species dispersal is common 
(Inchausti and Weimerskirch 2002). In some cases sex-biased dispersal has been 
documented (Young et al. 2009; Young 2010). However, for Galapagos and Hawaiian 
petrels, Isolation with Migration analyses indicate there is effectively no gene flow 
between taxa (Figure 4). Therefore, it is most likely that recent divergence and 
incomplete lineage sorting is causing the differences in the strength of the phylogenetic 
signal between the nuclear intron and mitochondrial gene trees. The mitochondrial gene 
sequences have acquired the signal of divergence between Galapagos and Hawaiian 
petrels, but the nuclear intron sequences are comparatively lagging as phylogenetic 
indicators of this divergence. Even when more than thirty nuclear loci were utilized to 
investigate recently diverged taxa, none demonstrated reciprocal monophyly (Lee and 
Edwards 2008).  
 
 28 
Using the simplified scenario outlined by Hudson and Coyne (2002), along with an 
estimate of effective population size, the time required to reach the state of reciprocal 
monophyly at nuclear and mitochondrial loci can be estimated. Unfortunately, population 
size is difficult to estimate in procellariid seabirds such as the Galapagos and Hawaiian 
petrels. Current census population estimates for both taxa range between 10,000 and 
30,000 individuals (Spear et al. 1995; Day and Cooper 1995). Census size often does not 
equal effective population size due to factors such as variance in reproductive success. 
However, life history characteristics of petrels, such as overlapping generations and a 
monogamous mating system, should act to decrease variance among individuals (Nunney 
1995).  Given the difficulties of estimating population size in these taxa, and taking into 
account that populations sizes were much larger in the recent past before humans 
colonized the islands (Athens 2002), we optimistically assume an effective population 
size of 20,000 individuals (equal to the average estimated census size). With this estimate 
the state of reciprocal monophyly would be achieved at a single mitochondrial locus in 
just 264,000 years, while it would take three nuclear intron loci between 1,044,000 and 
1,416,000 years to reach the same state. Divergence time estimates for Galapagos and 
Hawaiian petrels from both BEAST and IMa pointed to a split occurring approximately 
550,000 years ago. This is longer than the time required for reciprocal monophyly to be 
obtained at a mitochondrial locus, but well within the time frame where incomplete 
lineage sorting is predicted for nuclear loci. 
 
The estimated divergence time of 550,000 years ago roughly corresponds to a period of 
intense ecological change in the Hawaiian and Galapagos archipelagos. Several of the 
 29 
main islands in both archipelagoes formed during this period. For instance, Hawaii first 
appeared around 0.5 million years ago and the oldest rocks from Maui-Nui (which 
includes the Hawaiian islands of Maui, Lanai, Molokai and Kahoolawe) date to between 
1.6 to 1.9 million years ago (Carson and Clague 1995). In the Galapagos, the islands of 
Isabela, Santiago, Santa Cruz, and Floreana all appeared between approximately 0.5 and 
2.3 million years ago (Bailey 1976; Parent et al. 2008). This timing also coincides with 
the Pleistocene and petrels may have been impacted by changes in sea levels and 
productivity (Lea et al. 2000; Nunn 1998; Smith et al. 2007). Therefore periods of 
intense volcanism or climate change may have acted as a catalyst in initiating the 
divergence of these taxa. Regardless of the causes, reciprocal monophyly in the 
mitochondrial gene tree and evidence for a lack of gene flow from the nuclear intron data 
set indicate that Galapagos and Hawaiian petrels have progressed substantially in the 
process of divergence. 
 
It is not only difficult to detect recently diverged taxa, it can also be difficult to determine 
when these taxa have diverged to the extent that they should be considered distinct 
species (Harrison 1998; de Queiroz 2007; Sites and Marshall 2004). In the past, 
taxonomic decisions have relied strongly on morphological evidence. In 1942, Ernst 
Mayr described the biological species concept stating that distinct species should not 
interbreed, or if they do they should not produce viable, fertile offspring (Mayr 1942). 
The phylogenetic species concept, on the other hand, emphasizes fixed differences and/or 
the formation monophyletic groups on gene trees (Coyne and Orr 2004). These species 
concepts are often difficult to apply to taxa early in the process of divergence. Such taxa 
 30 
are often morphologically very similar, and depending on the extent of divergence, fixed 
genetic differences and phylogenetic resolution may also be lacking. Finally, for many 
species it may be impossible to directly test for reproductive isolation. In these cases, 
perhaps the evolutionary species concept is most appropriate. Under this definition, 
lineages that are independently evolving and that have their own evolutionary fate are 
considered distinct species (Wiley 1976).  
 
Galapagos and Hawaiian petrels are just one example of the difficulty of identifying 
species boundaries for taxa that are in the process of divergence. These birds are difficult 
to distinguish, particularly at sea where they spend the majority of their lives (Spear et al. 
1995; Brooke 2004; Tomkins and Milne 1991; Simons 1985). Their complicated life 
history makes directly testing for reproductive isolation nearly impossible. However, 
evidence of effectively no gene flow between Galapagos and Hawaiian petrels over the 
last 100,000 generations, despite their striking mobility, may give some limited, indirect 
evidence for reproductive isolation. The presence of sixteen fixed differences in 
mitochondrial DNA sequences, and reciprocal monophyly in the Cytb gene tree give 
some support for species status under the phylogenetic species concept, but the nuclear 
data set lacked resolution. While this is likely due to recent divergence and incomplete 
lineage sorting, the phylogenetic species concept does not take such factors into account. 
Given all of the evidence mentioned above it does appear that Galapagos and Hawaiian 
petrels are evolving independently and therefore could be considered separate species 
under the evolutionary species concept. This supports the recent taxonomic revision 
elevating Galapagos and Hawaiian petrels to the level of distinct species. 
 31 
 
Investigations of migration within each species indicated that divergence may be 
occurring at the local geographic scale of islands within each archipelago. While sample 
sizes for population analyses were limited, there was sufficient signal to detect population 
differentiation. Additionally, low estimates of migration between populations of 
Galapagos and Hawaiian petrels demonstrated here are concordant with the literature. 
Using microsatellite markers Friesen et al. (2006) found evidence of strong population 
structure among Galapagos petrels breeding on different islands. Also, banding studies of 
both Galapagos and Hawaiian petrels found high levels of philopatry over multiple years, 
and even between multiple colonies on the same island (Simons 1985; Simons and 
Hodges 1998; Tomkins and Milne 1991). Further analyses with additional samples from 
each island will strengthen these conclusions.  
 
Accurately identifying cryptic and recently diverged taxa can have important 
conservation implications (Bickford et al. 2007). In the case of seabirds, spuriously 
grouping taxa could be especially tragic as they are increasingly threatened, both at land 
and at sea. At the breeding grounds, for instance, seabirds must contend with predation 
by introduced mammals such as rats and cats, construction of obstacles such as power 
lines and wind turbines, and light pollution that leads to high juvenile mortality during 
fledging (Brooke 2004; Rayner et al. 2007; Cruz-Delgado et al. 2010). At sea, petrel 
mortality on fishing gear can be high (Tuck et al. 2001). For white-chinned petrels 
(Procellaria aequinoctialis) alone, up to 80,000 individuals are killed each year as a 
result of by-catch (Techow et al. 2009). It is important to gain a better understanding of 
 32 
the distribution of cryptic diversity as taxa that are already threatened by extinction may 
in fact represent multiple species that have even smaller population sizes, even more 
restricted ranges (Simons 1985), and that may require even more rigorous intervention to 
ensure their continued existence (Bickford et al. 2007).  
 33 
TABLES 
 
 
Table 1. Summary of taxa sampled, collecting locality, and sample size (N) 
 
Taxon Location N 
Pterodroma phaeopygia Galapagos Islands 35 
 Floreana 7 
 Isabela 7 
 San Crist?bal 7 
 Sana Cruz 7 
 Santiago 7 
Pterodroma sandwichensis Hawaiian Islands 28 
 Hawaii 7 
 Maui 7 
 Lanai 7 
 Kauai 7 
Pterodroma incerta Gough Island 6 
Pterodroma mollis Gough Island 6 
Pterodroma ultima At sea 1 
 
 34 
Table 2. Genealogical sorting index and associated p-values by locus for Galapagos and 
Hawaiian petrels. 
 
 
Galapagos Petrels Hawaiian Petrels 
Locus gsi
T
 p gsi
T
 p 
Cytb 1.0000 0.00001 1.0000 0.00001 
Enol 0.2297 0.00001 0.2573 0.00001 
Lam 0.2783 0.00001 0.2353 0.00001 
RP40 0.1785 0.00040 0.1832 0.00001 
 35 
Table 3. Migration estimates for Hawaiian petrels from the program Migrate. Values in 
bold are the estimated number of migrants per generation from the population on the left 
into the population at the top of the table. 95% confidences intervals are in parentheses 
below. 
 
 
 Hawaii Maui Lanai Kauai 
Hawaii - 
0.802 
(0.631-0.946) 
0.171 
(0.026-0.289) 
0.145 
(0.000-0.263) 
Maui 
0.145 
(0.000-0.263) 
- 
0.145 
(0.000-0.263) 
0.118 
(0.000-0.263) 
Lanai 
0.197 
(0.000-0.368) 
0.302 
(0.105-0.447) 
- 
0.171 
(0.000-0.342) 
Kauai 
0.118 
(0.000-0.237) 
0.092 
(0.000-0.210) 
0.171 
(0.026-0.342) 
- 
 
 36 
Table 4. Migration estimates for Galapagos petrels from the program Migrate. Values in 
bold are the estimated number of migrants per generation from the population on the left 
into the population at the top of the table. 95% confidences intervals are in parentheses 
below. 
 
 
 Santiago Isabela Santa Cruz Floreana 
San 
Crist?bal 
Santiago - 
0.243 
(0.105-0.355) 
0.007 
(0.000-0.171) 
0.072 
(0.000-0.210) 
0.099 
(0.000-0.197) 
Isabela 
0.204 
(0.039-0.381) 
- 
0.848 
(0.657-1.025) 
0.099 
(0.000-0.210) 
0.151 
(0.013-0.276) 
Santa 
Cruz 
0.112 
(0.000-0.223) 
0.112 
(0.000-0.223) 
- 
0.230 
(0.000-0.342) 
0.112 
(0.000-0.210) 
Floreana 
0.059 
(0.000-0.171) 
0.177 
(0.053-0.302) 
0.007 
(0.000-0.223) 
- 
0.204 
(0.079-0.315) 
San 
Crist?bal 
0.059 
(0.000-0.171) 
0.269 
(0.131-0.407) 
0.112 
(0.000-0.250) 
0.151 
(0.013-0.276) 
- 
 
 37 
 
FIGURES 
 
 
Figure 1 Map of the Pacific Ocean (a) depicting Hawaiian (light gray) and Galapagos 
(medium gray) petrel breeding distribution and foraging range (Brooke 2004). Maps of 
the Hawaiian (b) and Galapagos archipelagoes (c) showing islands where Hawaiian and 
Galapagos petrels are known to breed and where samples were obtained (light gray and 
medium gray, respectively). 
 
 38 
Figure 2. Bayesian majority rule consensus tree for the mitochondrial Cytb gene.  A total 
evidence data set comprised of the Cytb gene and three nuclear introns resulted in a 
congruent topology, but lacked taxa for which no intron data was available (dashed 
branches). Numbers above the branch are posterior probabilities and maximum likelihood 
bootstrap support for the Cytb tree while those below the branch are posterior 
probabilities for the total evidence tree. Dashes indicate no substantial support. GAPE = 
Galapagos petrel, HAPE = Hawaiian petrel, MRPH = Murphy?s petrel, SPP = soft-
plumaged petrel, ATP = Atlantic petrel. Letters in parenthesis indicate island of origin for 
a haplotype: S = Santiago, Sc=Santa Cruz, F=Floreana, I=Isabela, C=San Cristobal, 
L=Lanai, K=Kauai, M=Maui, and H=Hawaii.  
  
 39 
Figure 3. Bayesian majority rule consensus tree obtained for the Lam intron as well as 
the partitioned analysis of all three nuclear introns. Numbers above the branch are 
posterior probability and maximum likelihood bootstrap support for the Lam locus, below 
the branch is the posterior probability for the combined intron data set. Brackets and 
shading indicate location of taxa on the tree, with the darker, overlapping blocks where 
multiple species occur within a single clade. MRPH = Murphy?s petrel, GAPE = 
Galapagos petrel, HAPE = Hawaiian petrel, SPP = soft-plumaged petrel, ATP = Atlantic 
petrel. 
 
 
40
 
Figure 4.
 Marginal posterior distributions from
 
the Isolation with Migration program
 (IMa)
 for migration rates and divergence tim
e
 
(years b
efore presen
t). (a) and (b
) sh
ow re
sults f
o
r a data set com
posed of three nuc
le
ar in
tron lo
c
i; (
c) and (d)
 display resu
lts f
o
r th
e 
m
itochondrial 
Cytb 
gene. 
 
 
 41 
Figure 5. Comparison of divergence time estimates, in years before present, from IMa 
and BEAST for the mitochondrial and nuclear intron data sets. Black bars indicate the 90 
and 95% highest posterior density (HPD) for estimates from IMa and BEAST, 
respectively. The posterior distribution for the divergence time parameter estimated in 
IMa from the Cytb data set failed to converge. Therefore confidence in this estimate 
cannot be assessed. 
 
 
 
 
 
 42 
CHAPTER II 
 
 
Polymorphic microsatellite markers for the endangered Hawaiian petrel 
(Pterodroma sandwichensis)* 
 
 
The endemic and endangered Hawaiian petrel (Pterodroma sandwichensis) breeds on 
four of the six main Hawaiian Islands, however the levels of gene flow between these 
islands remain unclear. We describe 10 novel polymorphic microsatellite loci isolated 
from this species, and characterize 10 additional previously published primer sets in 40 
individuals from the island of Maui. Loci exhibited between 3 and 14 alleles (mean 6.85), 
and observed heterozygosity ranged from 0.150 to 0.825 (mean 0.540). Four loci showed 
evidence for null alleles, although only one locus was found to have significant deviation 
from Hardy-Weinberg equilibrium after Bonferonni correction. Two primer pairs showed 
significant gametic disequilibrium. The loci described here should provide a useful 
toolset for investigations of population connectivity and parentage. 
 
*A. J. Welch and R. C. Fleischer. 2011. Conservation Genetics Resources, In press. 
 
 43 
Seabirds face a multitude of threats, both on land and at sea. As a result many species are 
in danger of extinction (Brooke 2004). The endemic Hawaiian petrel, Pterodroma 
sandwichensis, was apparently abundant prior to human colonization of the Hawaiian 
Islands (Athens et al. 1991; Olson and James 1982a), but direct exploitation by humans, 
predation by introduced mammals, and habitat destruction have led to population declines 
and this species is now considered endangered (Simons and Hodges 1998). Several 
Hawaiian petrel colonies have been or are currently in danger of being extirpated: a 
colony on the island of Oahu was eliminated prehistorically, a formerly large colony on 
Molokai has not recently been relocated, and colonies on the Big Island and western 
Maui appear to be in peril (Simons and Hodges 1998; Birdlife-International 2010). A 
mitochondrial DNA sequence dataset suggests that gene flow may be low between petrel 
colonies on different islands (Welch et al. 2011), however additional information from 
biparentally-inherited markers is necessary to gain a better understanding of the patterns 
of gene flow and the impacts of population extirpation. Here were describe development 
of 10 novel polymorphic microsatellite markers and the characterization of 10 additional 
loci originally isolated in other species.  
 
Microsatellite enriched genetic libraries were constructed following Hamilton et al. 
(1999). Briefly, genomic DNA was extracted from tissue samples of one male and one 
female Hawaiian petrel obtained from the University of Washington?s Burke Museum 
(accession numbers 55605 and 55576) using the Qiagen DNEasy tissue kit. DNA was 
digested using the restriction enzymes HaeIII, XmnI, and NheI (New England Bioloabs) 
and fragments approximately 200 ? 1000 bp in size were ligated to SNX linkers and 
 44 
amplified using SNX primers. These fragments were then hybridized to (CA)
10
, 
(AAAG)
5
, (AAAC)
5
, or (AGAGG)
5
 biotinylated oligos and any remaining unhybridized 
fragments were removed using Dynabeads M-280 Streptavidin magnetic beads 
(Invitrogen). Fragments were amplified again, ligated into pBluescript II SK + plasmids, 
and then transformed into ultracompetent XL-10 Gold E. coli (Stratagene). 
 
Positive clones were PCR screened using a combination of three primers: T7 (5?-
GTAATACGACTCACTATAGGGC-3?), T3 (5?-AATTAAC- CCTCACTAAAGGG-3?), 
and the appropriate oligo from the enrichment step above. Clones that appeared as smears 
instead of distinct bands on agarose gels were then amplified using only the T7 and T3 
primers, cycle sequenced using Big Dye Terminator v. 3.1, and electrophoresed on a 
3130 Genetic Analyzer (Applied Biosystems). The resulting sequences were examined in 
Sequencher v. 4.9 (Gene Codes).  Approximately 1400 colonies were screened. A total of 
65 unique clone sequences contained sufficient flanking region for primer design and had 
? 7 dinucleotide repeats or ? 5 tetra- or pentanucleotide repeats. However, 11 of these 
(17%) apparently contained an artifact consisting of the oligo used for hybridization 
ligated to a 9 bp remnant of the SNX linker.  
 
Primers were designed for the remaining 54 clones using Primer3 (Rozen and Skaletsky 
2000) with forward primers including an M13 tail (5?-TGTAAAACGACGGCCAGT-3?). 
One sequence, Ptero06 (Table 1), contained an (AAGG)
13
 motif as well as an upstream, 
interrupted (TG)
18 
motif. Primers were designed for each region independently, and both 
produced multiple alleles, but results for only the (AAGG) motif are shown below. In 
 45 
addition, we screened 37 previously published primer sets for cross amplification in the 
Hawaiian petrel, including all loci from Brown and Jordan (2009), Bried et al. (2008), 
Gonzalez et al. (2009), and Techow and O?Ryan (2004), as well as the individual loci 
RBG18, RBG29, Dp?01, and DC16, which were found to be polymorphic in the closely 
related Galapagos petrel, Pterodroma phaeopygia (Given et al. 2002; Dawson et al. 1997; 
Burg 1999; Friesen et al. 2006). Some previously published primer sets were redesigned 
(indicated by M following primer name in Table 2) to amplify shorter fragments. Primer 
sets were initially screened for polymorphism using a total of 15 individuals from the 
islands of Hawaii, Maui, Lanai, and Kauai. 
 
Amplifications were carried out following the three-primer protocol of Schuelke (2000), 
in which a fluorescently labeled M13 (5?-FAM-M13-3?) primer was utilized in addition 
to the reverse and the M13-tailed forward primers. Amplifications were conducted in 15 
?L reactions with 1x colorless GoTaq Flexi buffer, a primer-specific concentration of 
MgCl
2 
(Tables 1 and 2), 0.2 mM each dNTP, 1.2 mg/mL BSA, 0.14 ?M M13-tailed 
forward primer, 0.4 ?M reverse and fluorescently labeled M13 primers, 1 unit GoTaq 
Flexi DNA polymerase (Promega) and 1.5 ?L DNA extract. The thermocycle profile 
began with a denaturation step for 2 min at 95
o
C, followed by 35 cycles of 95
o
C for 30 s, 
a primer-specific annealing temperature (Tables 1 and 2) for 30 s, 72
o
C for 30 s, and a 
final step at 72
o
C for 30 min. Products were electrophoresed on a 3130xl Genetic 
Analyzer (Applied Biosystems) and alleles were called manually in GeneMapper v. 4.1 
(Applied Biosystems). For loci that were found to be polymorphic, fluorescently labeled 
forward primers (without the M13 tail) were obtained and 40 individuals from the island 
 46 
of Maui were amplified. Previously published loci that were found to be polymorphic in 
the Hawaiian petrel were sequenced, as described above, to examine homology to the 
original loci. The program MICRO-CHECKER v. 2.2.3 (van Oosterhout et al. 2004) was 
used to test for the presence of large-allele drop-out and null alleles. The program 
GENEPOP v. 4.0 (Rousset 2008) was used to test for departures from Hardy-Weinberg 
expectations and for linkage disequilibrium.  
 
Of the 54 Hawaiian petrel (HAPE) specific primer pairs, 15 were monomorphic, 29 did 
not amplify or produced irresolvable stutter, and 10 produced two or more alleles in the 
initial screen. Of the 37 previously published primer pairs, 18 produced two or fewer 
alleles, 9 did not amplify, and 10 were polymorphic in initial screens. Therefore, 20 loci 
were genotyped in the larger set of 40 individuals (Tables 1 and 2). Two HAPE-specific 
primer pairs exhibited allele sizes inconsistent with the repeat motif. Sequencing of these 
alleles revealed a 38 bp deletion in the flanking region of Ptero10, and 2 bp and 5 bp 
deletions in Ptero07. None of the loci appear to be sex-linked and there was no 
indications of large-allele dropout. Four loci (Ptero06, Ptero10, Parm05, and PuffPM2) 
exhibited an apparent excess of homozygotes across all allele classes (van Oosterhout 
null allele frequencies were 0.215, 0.143, 0.150, and 0.154, respectively). The underlying 
causes of the observed homozygote excess remain unclear, but could potentially include 
the presence of additional undetected insertion/deletions or inbreeding within the Maui 
population. Despite the potential presence of null alleles, only Ptero06 showed 
significant deviations from Hardy-Weinberg equilibrium after Bonferroni correction for 
multiple tests (Tables 1 and 2). Two pairs of loci were found to be in linkage 
 47 
disequilibrium after Bonferroni correction: Parm01 and RBG29, and Parm01 and 
Ptero03 (p < 0.0001 for both). Overall, the loci developed and characterized here should 
provide a useful toolset for investigations of population connectivity and parentage in the 
endangered Hawaiian petrel.  
 
48
 
T
ABLES
 
 Table 1.
 Characterization of 10 novel m
i
crosatellite 
loci isolated from
 the Hawaiian petrel (
Pterodroma sandwichensis
) 
 Loc
u
s 
H
O
 
GenB
a
n
k # 
Clone 
Prim
er seque
nces (5? to 
3?) 
Repeat m
o
tif 
Dye 
Size (b
p) 
Mg
Cl
2
 
(m
M
) 
T
a
 
(
o
C)
 
N
A
 
H
E
 
HW 
(p) 
Ptero01 
27
.2
8 
F:
 GAAAAC
AACTCCCCC
ACAAC 
(CA)
7
 
Hex 16
3
-
1
6
7 
2.
5 
53 
3 
0.
20
0 
1.
00
0 
H
Q
91
821
8
  
R:
 TCCGTC
AGACCT
GCTGTAT
G
 
 
 
 
 
 
 
0.187 
 
Ptero02 
27
.3
6 
F:
 A
A
G
C
G
C
TTCA
CTGGAG
GA
 
(
C
A
)
9
 Fam
 
13
1
-
1
4
1 
3.
0 
53 
5 
0.
55
0 
0.
42
5 
H
Q
91
821
9
  
R:
 TGACCTGTGT
GCCTTC
ATTC 
 
 
 
 
 
 
0.547 
 
Ptero03 
27
.7
2 
F:
 T
G
T
G
T
A
CAGC
AT
GTG
C
TTG
AG 
(CA)
9
 Fam
 
13
0
-
1
4
2 
3.
0 
53 
7 
0.
82
5 
0.
89
2 
H
Q
91
822
0
  
R:
 GCT
G
AAT
GGC
AGTTTC
TTCC 
 
 
 
 
 
 
0.775 
 
Ptero04 
27
.9
6 
F: 
T
G
CATTGTTTCTGTCC
A
AACTC (CA)
13
 Hex 
14
6
-
1
6
0 
3.
0 
53 
8 
0.
77
5 
0.
75
2 
H
Q
91
822
1
  
R:
 G
G
CTG
G
AAT
GC
ATA
G
TACC
AAC 
 
 
 
 
 
 
0.
73
4 
 
Ptero05 
2
5
.153 
F:
 T
G
CT
GTC
GGCT
G
GGT
T
A
C 
(A
AG
)
2
AGG
(A
AG
)
3
 Hex 
20
6
-
2
1
5 
2.
5 
53 
3 
0.
15
0 
1.
00
0 
H
Q
91
822
2
  
R:
 C
T
GCCTGCCTTCCTGAAAC 
 
 
 
 
 
 
0
.
1
4
2
 
 
Ptero06 
2
7
.134 
F:
 G
TTCCATG
G
C
G
A
TGAA
GG
 
(
A
A
GG)
13
 Hex 
14
5
-1
7
7 
3.
0 
53 
9 
0.
42
5 
<0.
00
0
1* 
H
Q
91
822
3
  
R:
 CTGA
AAA
TGG
C
A
T
CCA
AA
CG
 
 
 
 
 
 
 
0
.
8
01
 
 
Ptero07 
28
.4
2 
F:
 TTAA
AAATCG
GTCCA
ATA
G
TCG
 
?(
AAA
G)
8
 Fam
 
25
3
-
2
8
9 
2.
5 
53 
8 
0.
82
5 
0.
69
0 
H
Q
91
822
4
  
R:
 GC
ACA
G
AGT
TG
ACC
G
TG
TT
G 
 
 
 
 
 
 
0.
81
8 
 
Ptero08 
28
.5
0 
F:
 G
CA
CCTGCTG
GTGA
TGA
G
T
C 
?(
AAG
G)
8
 Hex 
16
2
-
2
2
6 
2.
5 
53 
10 
0.
70 
0.
00
5 
H
Q
91
822
5
  
R:
 AG
GGA
AA
AGG
AA
CCA
T
CCAG
 
 
 
 
 
 
 
0
.
8
2
3
 
 
Ptero09 
28
.8
5 
F:
 G
C
AA
ATACCA
G
T
CTTCCA
AA
GG
 
(
A
A
GG)
8
? Hex 
21
2
-2
3
6 
2.
5 
53 
7 
0.
77
5 
0.
90
2 
H
Q
91
822
6
  
R:
 TTTAAGATAAAGATGTTTGAGAAC
C
AC 
 
 
 
 
 
 
0.671 
 
Ptero10 
25
.0
6 
F:
 A
G
C
TG
TGTG
GG
CTTAATTG
C 
(
T
AGG
A)
9
? Fam
 
20
5
-2
9
0 
2.
0 
56 
8 
0.
57
5 
0.
01
3 
H
Q
91
822
7
  
R:
 
ACCCTAC
TCTGTGGC
ACCTC 
    
   
(TAGGA)
7
 
  
 
 
 
0.
81
0 
 
Mean  
 
 
  
 
 
6.
8 
0.
58
0 
 
 
49
 
Table 1 (Continued) MgCl
2
: Final 
magnesiu
m
 chloride concentration (m
M), T
a
: Annealing tem
p
er
ature (
o
C), N
A
: Num
b
er of alle
les in a sa
m
p
l
e
 of 40 Hawaiian petrels from
 
Maui, H
O
: Observed heterozy
gosity
, H
E
: 
Expected heterozy
g
o
sit
y
, 
HW: p value for test of dep
a
rture fro
m
 Hardy
-
Wein
berg expectation
s
, * Significa
nt 
deviation fro
m
 
Hardy
-
Weinberg equili
b
r
ium
 after Bo
nferroni correc
tion. Mean n
u
m
b
er of alleles 
and observed heterozygosit
y
 shown
 
on the last line. 
 
50
 
Table 2.
 Characterization of 10 previously developed 
m
i
crosatellite lo
ci in Hawaiian petre
l
 (
Pterodroma sandwichensis
)  
 
Loc
u
s 
Ori
g
inal re
peat
 m
o
tif 
H
O
 
GenB
a
n
k # 
Prim
er seque
nces (5? to 
3?) 
Rep
eat m
o
tif in
 
HAPE 
Dy
e 
Size (b
p) 
Mg
Cl
2
 
(m
M
) 
T
a
 
(
o
C)
N
A
 
H
E
 
HW
 
(p)
 
Ref
 
Parm
01M 
F:
 A
GGG
CAG
AAG
CTCGTCTA
TC 
(
CA
)
13
 Fam
 
18
9
-2
1
9 
3.
0 
53 
13 
0.
67
5 
0.
77
3 
a 
H
Q
91
821
4
 
R:
 AGTGCCCTCTTCCCCATAAC 
(CA)
13
  
 
 
 
 
0.
76
0 
 
 
Parm
02M 
F:
 T
G
TT
AT
G
T
CCTGCG
G
A
T
GA
G 
(CA)
6
TA(CA)
9
 Hex 
16
8
-1
8
0 
3.
0 
53 
6 
0.
22
5 
1.
00
0 
a 
H
Q
91
821
5
 
R:
 TGAA
TCCA
GGG
TGAGTCA
GG
 
(
CA
)
7
TA(CA)
8
  
 
 
 
 
0.
21
0 
 
 
Parm
03M 
F:
 A
T
TG
CTCA
GGG
AGG
TG
CTG
 
(
C
A
)
5
TA(CA)
11
 Fam
 
14
9
-1
6
7 
2.
5 
53 
9 
0.
60
0 
0.
06
8 
a 
H
Q
91
821
6
 
R:
 TT
GGT
TGAAGGTCTGT
G
TGG 
(CA)
11
  
 
 
 
 
0.
63
7 
 
 
Parm
05M 
F:
 CTC
AGTGCCAATTC
GTACCC 
(CA)
11
 Hex 
93
-
99 
3.
0 
53 
4 
0.
30
0 
0.
00
3 
a 
H
Q
91
821
7
 
R:
 A
A
T
G
CA
AG
GCT
GAT
GCTA
AT
G 
(CA)
10
  
 
 
 
 
0.
46
4 
 
 
Cd2 
F:
 C
AAC
AAC
TCACCCTGC
CTTT 
(TG)
11
(CG
)
2
(TG
)
2
 Fam
 
18
9
-
1
9
3 
3.
0 
55 
3 
0.
15
4 
0.
31
7 
b 
H
Q
91
821
1
 
R:
 G
C
TCCTTTCTGA
AG
CTG
T
GG
 
(
T
G)
6
CG(TG)
2
  
 
 
 
 
0.
18
9 
 
 
Cd10 
F:
 G
GTG
TG
TG
GGG
AAA
GA
AA
CA
 
(
T
G)
9
 Fam
 
18
7
-
2
0
1 
2.
0 
54 
7 
0.
65
0 
0.
35
9 
b 
H
Q
91
821
2
 
R:
 TCTGTTC
AGGGGCT
G
TCCT 
(TG)
10
  
 
 
 
 
0.
62
7 
 
 
PuffPM2 
F:
 AA
A
GAT
G
C
TTA
AG
GA
T
A
CAT
A
ACT
G
C 
(GT
)
16
 Fam
 
18
3
-1
8
9 
3.
0 
55 
3 
0.
37
5 
0.
00
4 
c 
H
Q
91
822
8
 
R:
 TT
GTT
G
G
C
AA
AA
GT
G
A
TA
A
G
G
TT 
(GT
)
13
  
 
 
 
 
0.
58
9 
 
 
Paequ3 
F:
 TG
TG
GG
TG
CAG
T
A
GAG
CA
 
(
G
A
)
19
 Hex 
21
2
-
2
2
0 
3.
0 
53 
5 
0.
42
5 
0.
13
8 
d 
H
Q
91
821
3
 
R:
 C
A
ATA
A
G
A
A
GATC
A
GCA
G
A
A
C
A
GAC 
(G
A)
10
  
 
 
 
 
0.
47
4 
 
 
RBG18M 
F:
 TT
GTTCC
TTGGCTTCT
A
TCTC 
(GT
)
11
 Fam
 
98
-
10
8 
3.
0 
53 
5 
0.
82
5 
0.
07
3 
e 
H
Q
91
822
9
 
R:
 CG
CTG
AAG
A
T
G
T
AG
AG
GGA
AA
 
(
G
T)
9
  
 
 
 
 
0.
61
7 
 
 
RBG29 
F:
 CCT
AGCT
TTTTGGACT
CAGT 
(GT
)
13
 Fam
 
12
1
-1
7
1 
3.
0 
53 
14 
0.
77
5 
0.
95
4 
e 
H
Q
91
823
0  
R:
 GT
G
G
TG
T
T
GCA
A
T
A
G
GAT
TC 
(GT
)
14
  
 
 
 
 
0.
78
3 
 
 
Mean 
 
 
  
 
 
6.
9 
0.
50
0 
 
 
  
 
51
 
Table 2 (Continued) MgCl
2
: Final 
magnesiu
m
 chloride concentration (m
M), T
a
: Annealing tem
p
er
ature (
o
C), N
A
: Num
b
er of alle
les in a sa
m
p
l
e
 of 40 Hawaiian petrels from
 
Maui, H
O
: Observed heterozy
gosity
, H
E
: 
Expected heterozy
g
o
sit
y
, 
HW: p value for test of dep
a
rture fro
m
 Hardy
-
Wein
berg expectation
s
, * Significa
nt 
deviation fro
m
 
Hardy
-
Weinberg equili
b
r
ium
 after Bo
nferroni correction. 
a
 Brow
n and Jordan 
(2009),
b
 Brie
d et al. (2008), 
c
 Gonzalez et al. (2009), 
d
 
Techow and O?R
y
an (20
0
4
), 
e
 Given et 
al. (2002) Me
an num
ber of alleles and observed heterozy
g
o
sit
y
 sho
w
n on the last
 line. 
  
 52 
CHAPTER III 
 
 
Population divergence and gene flow in an endangered and highly 
mobile seabird* 
 
 
The endemic Hawaiian petrel (Pterodroma sandwichensis) is a highly vagile seabird that 
was formerly distributed more widely across the Hawaiian Islands. Today it is considered 
endangered, and known to breed only on the islands of Hawaii, Maui, Lanai, and Kauai. 
Historical records indicate that a large population formerly bred on Molokai, but it is 
feared that this population has recently been extirpated. Given the great dispersal 
potential of petrels it remains unclear if populations are isolated from one another or if 
there is substantial gene flow between them. We sampled petrels from across their current 
range, as well as individuals collected on Molokai in the early 1900?s. We sequenced 524 
bp of mitochondrial DNA, 741 bp from three nuclear introns, and genotyped 18 
microsatellite loci in order to investigate patterns of divergence and gene flow in the 
Hawaiian petrel. Mitochondrial DNA demonstrates significant, strong population genetic 
structure among all modern populations. Low differentiation was found between historic 
samples from Molokai and modern birds from Lanai. Weak, but significant structure was 
also detected in both nuclear data sets. Migration estimates were low, and a Bayesian 
clustering analysis revealed at least two genetic populations, one of which primarily 
consisted of birds from Hawaii and Lanai and the other consisted primarily of birds from 
Maui, with Kauai individuals representing a mixture. Evidence for significant population 
structure, in addition to ecological differentiation, indicates that distinct management 
 53 
units may exist in this species. Conservation efforts should be considered on an island-to-
island basis to preserve genetic diversity in Hawaiian petrels. 
 
*A. J. Welch, R. C. Fleischer, H. F. James, A. E. Wiley, M. P. Ostrom, J. Adams, F. 
Duvall, N. Holmes, D. Hu, J. Penniman, and K. A. Swindle. For submission to Heredity. 
 
 54 
INTRODUCTION 
 
Populations are a fundamental unit in evolutionary biology and exist in a delicate balance 
between isolation and gene flow (Allendorf and Luikart 2007). Gene flow can decrease 
inbreeding (Madsen et al. 1999; Westemeier et al. 1998) and prevent losses of genetic 
diversity (Pinsky et al. 2010), but it can also lead to population genetic homogenization 
and outbreeding depression (Frankham 1995a). Populations are also of central 
importance for the persistence of species (IUCN 2001; Redford et al. 2011) and 
population loss may be an indicator increased threat of extinction (Ceballos and Ehrlich 
2002). Indeed, it has been argued that changes in the size or number of populations may 
be a more immediate indicator of the long-term impacts of anthropogenic influences on 
species persistence than estimates of broad-scale extinction rates (Balmford et al. 2003). 
 
Populations of many seabird species have declined in number or disappeared as a result 
of anthropogenic influences including habitat destruction, the introduction of exotic 
mammalian predators, and industrial fishing (Brooke 2004). Declines of seabirds could 
potentially have broad ecological consequences as they are top marine predators and play 
an important role in transferring marine nutrients to terrestrial ecosystems on oceanic 
islands (Fukami et al. 2006; Croll et al. 2005). However, information regarding seabird 
population size and dispersal, which is important for conservation management decisions, 
can be difficult to obtain for some species (Shirihai et al. 2009). Factors such as remote 
and inaccessible breeding locations, delayed maturity, high dispersal potential, and a 
pelagic lifestyle, can make direct observation difficult (Brooke 2004). Fortunately, 
 55 
genetic methods have great potential power to accurately assess patterns of differentiation 
and dispersal without direct observation or manipulation of live birds (Friesen et al. 
2007a). 
 
The Hawaiian petrel (Pterodroma sandwichensis) is an endangered pelagic seabird that 
breeds exclusively in the Hawaiian Islands (Simons and Hodges 1998). It is known to 
forage long distances away from the colony during the breeding season, ranging from the 
equator to the Aleutian Islands (Spear et al. 1995; Adams et al. 2006; Simons and Hodges 
1998). Today, the Hawaiian petrel is currently known to breed on the islands of Hawaii, 
Maui, Lanai, and Kauai, however colonies on Hawaii appear to be dwindling (Birdlife-
International 2010). Based on subfossil evidence, it appears that this species was 
previously much more wide spread, both on islands where it currently breeds (e.g. at 
lower elevations than where modern colonies are located) as well as on two additional 
islands where it has apparently been extirpated (Simons and Hodges 1998; Olson and 
James 1982a). A large colony was formerly present in the lowlands of Oahu, but this 
species has not been recorded as breeding there in historical times (i.e. since the 
discovery of the Islands by Captain Cook in 1778; Olson and James 1998). Additionally, 
historical records (Munro 1955) indicate that there was a breeding population on the 
island of Molokai with petrels so abundant that they darkened the sky. However recent 
surveys have been unsuccessful in locating a substantial colony on that island (Simons 
and Hodges 1998). Fortunately, study skin specimens from this colony do survive in 
museum collections. 
 
 56 
Little information is available about migration among Hawaiian petrel populations. These 
birds are difficult to study as they spend most of their life at sea and return to land 
nocturnally to breed. Additionally, they currently nest in burrows in high elevation 
colonies which are often located in remote and very rugged terrain (Simons and Hodges 
1998). Banding studies in a colony on Maui have shown that adults are highly 
philopatric, returning to the same burrow to breed for multiple years (Simons 1985). 
However, Hawaiian petrels exhibit delayed maturity and don?t begin breeding until 
approximately six years of age (Simons 1984), so it remains unclear if they demonstrate 
natal philopatry. Genetic information on dispersal is also limited. Browne et al. (1997) 
utilized allozymes to study genetic variation, but only in a single colony on Maui. 
Recently Welch et al. (2011) demonstrated that population structure may be present 
within this species, but a more extensive analysis is needed. 
 
We have conducted a comprehensive, range wide population genetic study of the 
Hawaiian petrel. We have collected samples from all islands where these birds are 
currently, or were historically, known to breed and developed three genetic datasets, 
including both nuclear intron and mitochondrial DNA sequences as well as genotypes 
from 18 microsatellite loci, to investigate population divergence and gene flow among 
extant and potentially extirpated populations of this species. 
 
 
METHODS 
 
 57 
Samples 
 
A total of 322 Hawaiian petrel samples were obtained from across the current and 
historical breeding range of this species (Table 1, Figure 1). Modern samples (N = 293) 
consisting of blood, tissue, bone, and feather were collected opportunistically from 
carcasses of birds depredated in breeding colonies on Hawaii, Maui, Lanai, and Kauai 
between 1990 and 2010, or from birds that were handled during conservation 
management procedures, such as rehabilitation after attraction to artificial lights and 
subsequent downing. Carcasses and downed birds were assumed to be breeding on that 
island (or the offspring of birds breeding on that island) since non-breeders depart early 
in the breeding season (Simons and Hodges 1998). Blood samples of chicks previously 
collected from Haleakala National Park, Maui, were also obtained (Browne et al. 1997). 
Toe pads were sampled from historical Hawaiian petrel museum specimens (N = 28) 
originally collected on Molokai between 1907 and 1914 and deposited at the Bernice P. 
Bishop Museum (BPBM) and the Los Angeles County Natural History Museum  
(Appendix A). It is possible that genetic differentiation could occur between different 
colonies on the same island (Friesen et al. 2007b), however sample sizes are not 
sufficient to address those questions here, and samples were grouped according to island 
for all analyses. 
 
Molecular Techniques 
 
 58 
Genomic DNA was extracted from blood and tissue samples using the DNEasy tissue kit 
(Qiagen). DNA was obtained from bone, feather, and toe pad samples via 
phenol/chloroform extraction and centrifugal dialysis (Fleischer et al. 2000). All 
extractions for historical samples were performed in a physically separate, dedicated 
ancient DNA laboratory, and a sample from a different species was extracted in between 
each Hawaiian petrel sample to detect cross-contamination. Multiple extraction and 
negative PCR controls were also used to detect contamination.  
 
Three data sets were generated for this study. First, a 524 bp fragment of the 
mitochondrial Cytochrome b gene was amplified using the primers CytbL and CytbR9 
according to Welch et al. (2011). Second, a set of three nuclear intron loci were 
sequenced, including ?-Enolase intron 8, Lamin A intron 3, and Ribosomal Protein 40 
intron 5 (Friesen et al. 1999; Friesen et al. 1997). For both sequence data sets Primer3 
(Rozen and Skaletsky 2000) was used to design small, overlapping fragments for 
amplification with historic and degraded samples (Appendix B). Third, a set of eighteen 
polymorphic microsatellite loci were amplified (Welch and Fleischer 2011). For historic 
samples a minimum of two independent amplifications were conducted for each 
mitochondrial and nuclear intron sequence primer set. For microsatellites, loci were 
amplified and assayed between three and five times per individual (Taberlet et al. 1996).  
 
Polymerase chain reactions were carried out in 15 ?L (for modern) or 25 ?L (for 
historical samples) total volumes. Reactions consisted of 1x colorless GoTaq Flexi buffer 
(Promega) or PCR Gold Buffer (Applied Biosystems, ABI), 2.0 ? 4.0 mM MgCl
2
, 0.2 
 59 
mM each dNTP, 1.2 mg/mL BSA, 0.5 ?M each primer, 1 unit of Promega GoTaq Flexi 
or AmpliTaq Gold DNA polymerase, and 1 ? 3 ?L DNA extract. Thermocycle profiles 
consisted of a denaturation step of either 95
o
C for 2 min (for GoTaq Flexi) or 94
o
C for 8 
min (for AmpliTaq Gold), followed by 35 ? 45 cycles of 95
o
C for 30 sec, a primer 
specific annealing temperature for 30 seconds, 72
o
C for 30 ? 45 seconds, proportional to 
the length of the fragment, and a final 72
o
C extension step for either 7 minutes for 
sequences, or 30 min for microsatellites. For sequencing, PCR products were cleaned up 
using a 1:10 dilution of ExoSAP-IT (USB), cycle-sequenced in both directions using the 
Big Dye Terminator v3.1 Cycle-Sequencing kit (ABI), and then purified through 
Sephadex G-50 fine columns. All fragments were electrophoresed on an ABI 3130xL 
Genetic Analyzer. Sequences were assembled, aligned and visually inspected in 
SEQUENCHER v 4.9, and genotypes were called manually in GENEMAPPER v 4.1. 
 
Data analysis 
 
Prior to any analyses, the program GENECAP (Wilberg and Dreher 2004) was utilized to 
identify any individuals that may have inadvertently been sampled multiple times (e.g. 
individuals banded during rehabilitation and later depredated in the breeding colony). The 
probability of identity (Sib P[ID]) was calculated from modern microsatellite genotypes 
and individuals that were found to match with p < 0.05 were deemed to be duplicates. 
When duplicates were detected one of the pair was discarded from further analyses.  
 
Sequences 
 60 
 
Mitochondrial DNA sequences were investigated to determine whether they could 
represent a nuclear copy. Sequences were characterized using MACCLADE v 4.08 
(Maddison and Maddison 2008) and translated in DAMBE v 5.1.2 (Xia and Xie 2001). 
 To visualize relationships among haplotypes at the population level a statistical 
parsimony network for Cytochrome b sequences was constructed using TCS v. 1.21 
(Clement et al. 2000) with a 95% connection limit. For both mitochondrial and nuclear 
sequence data sets, pairwise F
ST
 was calculated in ARLEQUIN v 3.1 (Excoffier et al. 2005) 
from a Kimura two parameter distance matrix. This substitution model was selected using 
JMODELTEST (Posada 2008) and the Akaike information criterion. Statistical significance 
of F
ST
 values was determined through 1000 permutations with the sequential Bonferroni 
correction (Rice 1989) applied for multiple tests. ARLEQUIN was also used to determine if 
there was a correlation between geographic and genetic distances. Mantel tests were 
conducted using Slatkin?s linearized F
ST
 and a matrix of distances between each of the 
islands, with significance determined through 1000 permutations. 
 
Although traditional frequency-based statistics are simple to calculate, they often rely on 
very simplistic assumptions such as equal population sizes and symmetrical gene flow 
(Beerli and Felsenstein 2001; Kuhner 2009). Coalescent-based analyses also have 
underlying assumptions (for example, some programs assume that population dynamics 
have been stable over many generations) and because of the stochastic nature of the 
coalescent they are best utilized with multiple loci. However, these analyses allow more 
realistic models to be utilized and can be more powerful (Kuhner 2009). Therefore, to 
 61 
estimate migration of Hawaiian petrels between islands, we also utilized the coalescent-
based program MIGRATE v 3.2.6 (Beerli 2006; Beerli and Felsenstein 2001). We used the 
Bayesian mode with uniform priors and substitution model parameters set to values 
estimated in JMODELTEST: the transition/transversion ratio = 14.0 for mitochondrial data 
and 8.0, 3.0, 8.0 for the nuclear introns Enol, Lam, and RP40, respectively, with rate 
heterogeneity for the mitochondrial locus modeled by a gamma distribution with alpha = 
0.083.  Three simultaneous replicate analyses were run with a single long chain of 20 
million steps, of which the first 10% were discarded as burn-in. A static heating scheme 
with four chains was utilized to increase searching effectiveness, and heating parameters 
were set to 1.0, 1.2, 3.0, and 6.0.  Convergence was assessed through examination of 
results from independent runs, and the effective sample sizes (ESS) for all parameters 
were greater than 1000. Effective population size (?) and gene flow (m) estimates from 
MIGRATE are compounded by the mutation rate (i.e. ? = xNe? and m = M/?, where x is a 
scalar dependent on the ploidy and inheritance mode of the locus). Therefore, to avoid 
making an assumption about mutation rates (particularly for the microsatellite data set), 
the effective number of migrants per generation (NeM/x) was calculated.  
 
Microsatellites 
 
The microsatellite data were screened for the presence of large-allele dropout and null 
alleles using the program MICROCHECKER v. 2.2.3 (van Oosterhout et al. 2004). Null 
alleles arise from genotyping artifacts, leading to an excess of homozygotes. However 
inbreeding, which can be an important issue for insular populations of endemic species 
 62 
(Frankham 1998), results in the same pattern. If null alleles are present in the data set in 
high frequencies they can bias estimates of population differentiation, such as F
ST
. 
Therefore, we used the program INEST (Chybicki and Burczyk 2009) to simultaneously 
estimate the inbreeding coefficient and null allele frequency. The data were also checked 
for departure from Hardy-Weinberg expectations and the presence of gametic 
disequilibrium using the program GENEPOP v 4.0 (Rousset 2008). The program CONVERT 
(Glaubitz 2004) was utilized to create infiles for further population genetic analyses. 
Simulations were conducted in POWSIM (Ryman and Palm 2006) to determine if the 
microsatellite data set contained sufficient power to detect low levels of population 
genetic differentiation. In the simulated data sets the effective population size was set to 
1000 and divergence time to 10, so that the overall F
ST
 of the simulated populations was 
0.005. A total of 100 simulations were performed, with sample sizes from the simulated 
populations drawn corresponding to those utilized here, with significance determined 
through Fisher?s exact test. 
 
To investigate levels of differentiation between Hawaiian petrels breeding on different 
islands, we calculated an estimate of F
ST
, G?
ST
, and D for the microsatellite data set. F
ST
 
was originally derived for biallelic data and depends on the variation of the loci utilized. 
In the case of highly polymorphic markers, such as microsatellites, F
ST
 may therefore 
underestimate genetic differentiation (Meirmans and Hedrick 2011). Several alternative 
measures have been suggested, including G?
ST
 and D. Both of these correct for maximum 
possible differentiation (Jost 2008; Hedrick 2005b) but differ in respect to the aspect of 
genetic diversity examined: G?
ST
 examines heterozygosity whereas D takes into account 
 63 
the effective number of alleles (Meirmans and Hedrick 2011). An estimate of D was 
calculated using the package DEMETICS (Gerlach et al. 2010) implemented in R 
(R_Development_Core_Team 2009) and G?
ST
 was calculated using DEMETICS in 
conjunction with the program RECODEDATA (Meirmans 2006). The unbiased estimator 
was utilized for both statistics and significance p-values were estimated using 100 
bootstrap replicates (Gerlach et al. 2010). As mentioned above, both of these statistics 
rely on assumptions like symmetric gene flow. Therefore, gene flow was also estimated 
using the program MIGRATE, as described above except that the Brownian motion 
approximation of the stepwise substitution model was utilized instead. 
 
The number of genetic populations was investigated using the Bayesian clustering 
program STRUCTURE v. 2.3.3 (Pritchard et al. 2000; Falush et al. 2003). This program 
clusters individuals according to their multilocus genotype. We conducted analyses for 1 
? 8 population clusters (K) using the admixture ancestry model and the correlated allele 
frequency model, both with and without sampling location information included as part 
of the prior (Hubisz et al. 2009). The model including sampling location information has 
been found to be sensitive to weak population structure, but yet unbiased when no 
structure is exists. Five independent replicates were performed, and runs were conducted 
for 3 million generations with the first 10% discarded as burn-in. Results from 
STRUCTURE were input into the program STRUCTURE HARVESTER (Earl 2009), to 
calculate the ad-hoc ?K statistic suggested by Evanno (2005) that takes into account the 
change in the log probability of the data between increasing numbers of clusters. 
 
 64 
 
RESULTS 
 
Tests for duplicate modern samples from GENECAP revealed three individuals (p <0.05) 
that were each sampled twice: first as a chick or downed fledgling, and then later as a 
carcass (e.g. a set of wings and tail or a clusters of feathers) collected on the same island. 
The initial sample was retained for further analyses and the other discarded from the data 
set. Therefore three samples, one from Maui and two from Kauai, were removed from the 
data set. 
 
Mitochondrial sequences 
 
Mitochondrial DNA sequences were obtained for a total of 322 modern and historical 
Hawaiian petrel samples (Table 1, Figure 2). The success rate for historical samples from 
Molokai was 100% with a mean combined sequence length of 479 bp. A total of 35 
haplotypes were found with 11, 18, 7, and 9 haplotypes from Hawaii, Maui, Lanai, and 
Kauai respectively. There were no gaps present in the alignment, and after translation no 
nonsense or stop codons were detected. The majority (~72%) of substitutions occurred in 
the third codon position and 34 out of 35 were transitions. This evidence indicates that a 
mitochondrial, and not nuclear, origin of the sequences is likely.  
 
Overall, mitochondrial DNA sequences revealed strong differentiation between petrels 
breeding on different islands (global F
ST
 = 0.425, p < 0.001). Pairwise F
ST
 ranged from 
 65 
between 0.037 and 0.633 (Table 2) and was significant in 9 of 10 comparisons. The 
highest F
ST
 occurred between the islands of Lanai and Kauai (F
ST
 = 0.633), but 
differentiation was also high between Hawaii and Lanai (F
ST
 = 0.405), as well as between 
Maui and Kauai (F
ST
 = 0.574). F
ST
 was not significantly different from zero between 
Lanai and Molokai after sequential Bonferroni correction, and divergence was relatively 
low, but significant, between Hawaii and Maui. Mantel tests indicated there was no 
significant relationship between isolation and geographic distance (p = 0.15). 
 
Results from MIGRATE concurred with estimates of F
ST
, showing low migration between 
all pairs of populations. In analyses including only modern samples from the islands of 
Hawaii, Maui, Lanai, and Kauai, posterior distributions were unimodal and narrow. In 
analyses including modern samples as well as historic samples from Molokai, all 
posterior distributions were unimodal, however the migration parameters of from Hawaii 
into Maui, from Lanai into Molokai, and from Kauai into Molokai were relatively wide. 
However, all analyses showed that migration was very low. The highest migration 
estimate obtained was 2 migrants per 1000 generations (from Hawaii into Maui), but the 
average migration rate was approximately 1 individual per 1000 generations (m = 
0.00072 migrants/generation). For 13 out of 20 migration parameters the 95% confidence 
interval included a migration rate of 0. 
 
 
Nuclear intron sequences 
 
 66 
Three nuclear introns were sequenced for each of 164 individuals, for a combined total of 
741 bp (Table 1). Sufficient nuclear data could not be obtained for historical Molokai 
samples, and so they were not included in further analyses. Sixteen variable sites were 
discovered in the remaining four populations. There were 14 transitions and 2 
transversions. A total of 25 haplotypes were found: 3 for the Enol locus, 8 for Lam, and 
13 for RP40.  
 
Nuclear sequences revealed a somewhat different pattern of differentiation than that 
obtained from mitochondrial sequences. Overall, significant differentiation was found 
(global F
ST
 = 0.066, p < 0.001), but it was lower than that for mitochondrial sequences, 
with F
ST
 estimated from the intron data set ranging from 0 ? 0.145 (Table 2). The highest 
pairwise F
ST
 occurred between Maui and Lanai, however there was no significant 
differentiation between birds from Hawaii and Lanai or between Maui and Kauai. Mantel 
tests suggested that there was no relationship between isolation and distance (p = 0.76). 
Results from MIGRATE again concurred with estimates of F
ST
. Posterior distributions 
were narrow and unimodal. Migration estimates were low, approximately 1 migrants per 
1000 generations or less (e.g. m = 0.0009). The 95% confidence intervals for all 
parameters ranged from 0 to a maximum of 0.06 migrants/generation. The highest 
migration rate occurred from Maui into Kauai (m = 0.0009 migrants/generation), with 
slightly lower rates between Lanai and Hawaii (m = 0.0006 migrants/generation). 
 
Microsatellite data set 
 
 67 
A total of 232 individuals were genotyped for 18 microsatellite loci and there was an 
average of 6.75 alleles per locus. Expected heterozygosity ranged from 0.08 to 0.88, with 
an average expected heterozygosity for each population between 0.57 and 0.62. Results 
from MICROCHECKER indicated that no loci exhibited large-allele dropout, but two loci 
(Ptero06 and Ptero10) did display evidence for the presence of null alleles. Simultaneous 
estimation of the inbreeding coefficient and the null allele frequency from INEST 
indicated that inbreeding was low. Null allele frequencies ranged from 0.115 to 0.247 for 
Ptero06, and from 0.125 to 0.252 for Ptero10. The Ptero10 locus was previously found 
to contain a 38 base pair deletion in some individuals (Welch and Fleischer 2011), and 
additional undetected insertions or deletions could explain the relatively high incidence 
of null alleles. The cause of the observed excess of homozygotes for the Ptero06 locus 
remains unclear. Regardless, both loci were discarded from further analyses. No other 
loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction. 
Two loci (Parm01 and RBG29) were found to be in significant linkage disequilibrium 
after correction for multiple tests, therefore the Parm01 locus was also discarded and a 
total of 15 loci were utilized in further analyses. Despite the removal of three loci, 
simulations demonstrated that the microsatellite data set still contained sufficient power 
to detect very weak population structure. Population structure was detected with 100% 
accuracy for simulated populations with an F
ST
 of 0.005. Even when F
ST
 was decreased to 
0.0025, structure was correctly detected in 93% of simulations. 
 
The microsatellite data set revealed patterns of differentiation similar to those of the 
nuclear intron data set. As in the nuclear intron data set, there was weak but significant 
 68 
differentiation (global F
ST
 = 0.019, p < 0.001).  The highest levels of F
ST
 occurred 
between Lanai and Maui and between Kauai and Lanai (Table 3). Estimates of G?
ST
 and 
D were very similar, although D was slightly higher, with G?
ST
 ranging from 0.015 to 
0.057 and D ranging from 0.022 to 0.060. Both were higher than F
ST
, as expected (Table 
3). Estimates of G?
ST
 or D were marginally non-significant after sequential Bonferroni 
correction. Mantel tests also found no significant relationship between isolation and 
distance (p = 0.94). Migrate results demonstrated similar patterns. Point estimates of most 
migration rates were low, with 9 of 12 parameters showing a rate of about 1 or 2 migrants 
per 100 generations (e.g. m = 0.014 migrants/generation) or less. However migration 
rates from Hawaii into Kauai and from Lanai into Kauai were slightly higher (m = 0.71 
migrants/generation), and rates from Maui into Kauai were higher yet, with an estimate 
of about 8 individuals per generation. Overall, 95% confidence intervals were much 
broader than for previous analyses and ranged from 0 to about 5 migrants per generation 
for most parameters. 
 
Population structure was also investigated using a Bayesian clustering analysis. Under the 
model including sampling location information as part of the prior, runs with the number 
of clusters (K) set to 2 (Figure 3) grouped individuals from Hawaii and Lanai separately 
from individuals on Maui, similar to the nuclear intron data set. Kauai birds appeared to 
be an admixture of the two groups. With K = 3, Hawaii and Lanai were still grouped 
together, but Kauai was separated from that group as well as from Maui. Finally, with K 
= 4, birds from each island formed separate groups, however, individuals from Hawaii 
still contained some admixture with the Lanai population. Under the criteria of Evanno et 
 69 
al. (2005), the grouping K = 2 received the highest support (Figure 4) as ?K falls 
substantially and tends to level off for values beyond this. Additionally, even though it 
isn?t possible to assess support for the situation where K = 1 using the criteria of Evanno 
et al., the log probability of the data under this model is worse compared to models where 
K = 2 (e.g. -8780 vs. -8660) and above. In the analysis without sampling information, the 
grouping of K = 3 received the highest support under the Evanno criteria, however the 
maximum log probability of the data occurred for K = 1. 
 
 
DISCUSSION 
 
We conducted a population genetic study of the endangered Hawaiian petrel using three 
data sets: sequences of the mitochondrial Cytochrome b gene, sequences of three nuclear 
intron loci, and genotypes from 15 microsatellite loci. Analyses of the mitochondrial data 
set revealed strong population genetic structure between nearly all populations.  No 
significant divergence was found between museum specimens from a potentially 
extirpated colony on Molokai and modern birds breeding on Lanai. Nuclear sequences 
and microsatellites revealed significant, but relatively lower signal of divergence among 
populations. Gene flow appeared to be highest between individuals from Hawaii and 
Lanai and between individuals from Maui and Kauai. The highest levels of differentiation 
were found between Lanai and Maui. 
 
 70 
Mitochondrial DNA results show that contemporary populations of petrels on the islands 
of Hawaii, Maui, Lanai, and Kauai are significantly isolated from one another, with 
females rarely dispersing to breed. Pair formation in procellariiform birds requires 
multiple years and is thought to occur primarily on the breeding grounds (Warham 1990). 
Although it has not been directly observed, it is hypothesized that copulation of Hawaiian 
petrels takes place at the breeding grounds as well, likely within the burrow (Simons 
1985). If birds spend the non-breeding season in population-specific areas then they may 
return together to the same breeding colony, which could in turn lead to population 
differentiation (Burg and Croxall 2001). This may be the case for Hawaiian petrels, as the 
stable isotope composition of flight feathers grown during the non-breeding season 
differs significantly between some islands (Wiley et al. 2011). Strong philopatry could 
also lead to differentiation between birds on different islands (Friesen et al. 2007a), and 
procellariiform seabirds in general are known to be highly philopatric (Brooke 2004; 
Friesen et al. 2006; Smith et al. 2007; Fisher 1976). Recent work also demonstrates that 
vocalizations of Hawaiian petrels are unique on each island (Judge 2011), suggesting 
they may be philopatric as well. 
 
Significant differentiation was found between petrels sampled on Hawaii and Maui, 
although F
ST
 appeared to be lower than that found between other islands, suggesting that 
low levels of gene flow may be occurring. This could potentially be due to individuals 
dispersing from the dwindling population on Hawaii to Maui. Colonies on Maui and 
Hawaii both occur at high elevation with little precipitation and scrubby vegetation (Hu 
et al. 2001; Brandt et al. 1995), so dispersing birds would find more similar habitat on 
 71 
these islands than on Lanai or Kauai, which differ strikingly in both elevation and rainfall 
(Ainley et al. 1997). Simons (1985) suggested that the eggs of birds breeding on Maui, 
which exhibit reduced shell thickness and functional pore area, may be specifically 
adapted to prevent water loss during incubation at high elevations. Such local adaptation 
could potentially limit gene flow between high and low elevation colonies but allow gene 
flow between high elevation sites. In addition to significant differences in vocalization, 
Judge (2011) found that adults from Maui were significantly larger than adults from 
Hawaii in both wing chord and tarsus length. While this could result from several factors, 
such as diet and distances to foraging locations, it may also indicate that contemporary 
gene flow is minimal. 
 
Investigation of a potentially extirpated population on the island of Molokai revealed 
significant differentiation from all of the modern populations except for Lanai. By the 
early 1900?s the number of Hawaiian petrels breeding on Molokai had apparently already 
begun declining, likely due to the introduction of mongoose to the island (Bryan 1908). 
During a 1907 collecting trip W. A. Bryan found a female mongoose with five young 
living in a former petrel burrow (Bryan 1908). The situation on Lanai around this time 
was probably also less than ideal for petrels. Foraging by introduced ungulates led to 
severe habitat degradation as early as 1870, and continued foraging impeded natural 
recovery until 1911 when goats were removed from the island (Munro 2007). There are 
few, if any, records of breeding Hawaiian petrels (on any island) between about 1914 and 
1948, and this species was feared extinct (Baldwin and Hubbard 1948). Therefore, the 
recent discovery of a large colony on Lanai (Birdlife-International 2010), just a handful 
 72 
of generations later, is somewhat unexpected given that these long-lived petrels lay a 
single egg each year and exhibit delayed maturity. Failure to relocate a formerly large 
colony on Molokai coupled with no significant divergence between birds from Molokai 
and Lanai, may indicate that petrels from Molokai dispersed to Lanai as the colony 
dwindled, or at least that there was a significant level of gene flow between the two 
islands prior to the decline of the Molokai population.  
 
Dispersal can be substantial in seabirds. It has been hypothesized that adults may disperse 
if reproductive success becomes low (Friesen et al. 2007a). Social attraction is important 
for many seabirds and therefore individuals may disperse from dwindling colonies 
(Parker et al. 2007). Zador et al. (2009) describes dispersal of groups of up to 256 
common murre (Uria aalge) up to 30 m away from other nesting sites, although dispersal 
of < 10 individuals was more common over the 11 year study. Laysan albatrosses, which 
have been found to be highly philopatric, have re-colonized several previously extirpated 
colonies, as well as expanded their range to new sites, within the last 35 years (Young 
2010).  This seeming inconsistency between strong philopatry and high dispersal ability 
has been termed the ?seabird paradox? (Milot et al. 2008). There are several potential 
explanations. Dispersal may occur most frequently in young birds scouting potential 
burrow locations (including formerly extirpated colonies) who then remain philopatric to 
their chosen location in subsequent years (Brooke 2004).  From the mitochondrial data 
examined here, it appears that contemporary female Hawaiian petrels predominantly 
exhibit strong natal philopatry, although in the past birds may have dispersed from the 
failing Molokai colony to Lanai.  
 73 
 
Compared to the mitochondrial data, nuclear sequences and microsatellites revealed a 
slightly different pattern. Overall population genetic structure was weaker than that found 
from the mitochondrial data set. The Bayesian clustering analyses suggested the presence 
of at least two modern genetic populations. Recently the models for the Bayesian 
clustering method used in STRUCTURE were extended so that sampling locations could be 
included as prior information, resulting in increased power to detect weak structure 
(Hubisz et al. 2009). Despite this increased sensitivity, models were not found to be 
biased in cases where population structure was absent (Hubisz et al. 2009). Here the 
presence of population structure found in the STRUCTURE analysis is corroborated by 
other analyses of the microsatellite data set (e.g. F
ST
 and MIGRATE) as well as by analyses 
of the nuclear intron data set. Although estimates of G?
ST
 and D were higher than F
ST
, 
they were marginally non-significant. 
 
Both the nuclear intron and microsatellite data sets indicated that differentiation was low 
between individuals from Hawaii and Lanai and between individuals from Kauai and 
Maui. On the other hand, isolation appeared to be high between birds sampled on Maui 
and Lanai. These patterns of gene flow do not seem to be linked to distance, considering 
that colonies on Maui and Lanai are just ~75 km apart, whereas colonies on Hawaii and 
Lanai are separated by approximately 225 km, and colonies on Maui and Kauai are about 
350 km apart. Additionally, as mentioned above, birds dispersing between Hawaii and 
Lanai or between Maui and Kauai would find very different habitat than in their natal 
colony (Brandt et al. 1995). Wind patterns are an important factor in the flight of seabirds 
 74 
(Spear and Ainley 1997) and could affect dispersal, but it remains unclear if wind 
direction or speed differ substantially between islands in a manner consistent with these 
patterns. Interestingly, Wiley et al (In prep) conducted a study on the foraging ecology of 
the Hawaiian petrel using primary feathers (which are grown in the non-breeding season) 
and found that adults from Hawaii and Lanai had a very similar stable carbon and 
nitrogen isotope composition, whereas individuals from Maui and Kauai both differed  
significantly in nitrogen isotope values. Friesen et al. (2007a) have suggested that 
population specific foraging ranges or non-breeding distributions may act to reduce gene 
flow, and that could be one mechanism operating in the Hawaiian petrel. 
 
While mitochondrial and nuclear data sets differed subtly in the pattern of differentiation 
between populations (i.e. four apparent contemporary genetic populations vs. two), the 
differences in magnitude of divergence were also conspicuous. This could be due to 
male-biased dispersal, which has been documented in some seabirds (Young 2010). 
However, evidence for low overall gene flow was present in both nuclear intron and 
microsatellite data sets, as shown by significant F
ST
 and by low levels of gene flow found 
in MIGRATE analyses. Sex-biased dispersal in birds can be difficult to study using 
molecular markers alone because in birds females are the heterogametic sex. In addition 
to sex-biased dispersal the differences in the strength of signal could be due to different 
levels of variability among the data sets. Mitochondrial sequences yielded 31 haplotypes 
from the 4 modern populations, whereas the nuclear intron data set contained a slightly 
lower number of 25 haplotypes, and this may account for some difference. However, 
power was high in the microsatellite data set, with simulations showing that even very 
 75 
weak population genetic structure could be accurately detected. Microsatellite allele size 
homoplasy can also be problematic in some cases, particularly when population sizes are 
large and the mutation rate is high, but Estoup et al. (2002) point out that in general it 
does not pose a significant problem for most studies. Observations of differing levels of 
differentiation between mitochondrial and nuclear data sets can also be due to recent 
divergence. It appears that the Hawaiian petrel and its sister species, the Galapagos petrel 
(P. phaeopygia), diverged approximately 550,000 years ago (Welch et al. 2011) and that 
nuclear intron sequences for each species are still undergoing the process of lineage 
sorting. In such cases mitochondrial DNA, with its smaller effective population size, may 
act as a leading indicator of population divergence with nuclear DNA relatively lagging 
(Zink and Barrowclough 2008). 
 
Genetic differentiation has implications for management of endangered populations. 
While the criteria for delineating evolutionarily significant units has been hotly debated 
(Fraser and Bernatchez 2001), it appears that distinct management units may exist within 
the Hawaiian petrel. Here we demonstrate that populations of Hawaiian petrels breeding 
on different islands are genetically isolated to some degree, although not to the extent 
where outbreeding depression would be a major issue (Frankham et al. 2011). In addition 
to significant levels of genetic differentiation, some populations demonstrate foraging 
segregation during the non-breeding season and could be ecologically distinct (Wiley et 
al. 2011). Finally, management concerns and challenges are unique on each island. For 
example, goats have been successfully removed from Lanai, but are still present on the 
islands of Kauai and Hawaii and may cause significant habitat degradation. Additionally, 
 76 
the sources of predation vary on each island and can be substantial. On Kauai and Lanai 
predation by cats can be high, and on Maui and Hawaii introduced mongoose pose an 
additional threat (Hodges and Nagata 2001; Carlile et al. 2003). Therefore we propose 
that conservation measures for this species should be considered on an island-to-island 
basis.  
 
Seabirds play an important role in both marine and terrestrial ecosystems (Croll et al. 
2005), so it is important to gain a better understanding of their population dynamics and 
dispersal patterns. Here we demonstrate that birds breeding on one island may be 
genetically isolated from conspecifics breeding on other islands less than 100 km away. 
Many seabirds are increasingly threatened by extinction, and identifying isolated 
populations can be especially important for developing conservation management plans. 
Additionally, even in species that are relatively common, distinct evolutionary lineages 
may go unnoticed without further investigation (Hailer et al. 2011). 
 77 
TABLES 
 
 
Table 1. Sample sizes for Hawaiian petrels (Pterodroma sandwichensis) obtained from 
all islands where this species is currently, or was historically, known to breed. 
 
Sample Size 
Island 
Time 
period 
mtDNA nuDNA Micros 
Hawaii Modern 71 51 48 
Maui Modern 122 54 114 
Lanai Modern 38 25 28 
Molokai Historic 28 0 0 
Kauai Modern 63 34 42 
Total 
 
322 164 232 
 
 
 
 
 
 78 
Table 2. Population differentiation of historic and modern Hawaiian petrels. Pairwise Fst 
values for the Cytb gene are below the diagonal, while those for a data set of sequences 
from three nuclear introns are above. *Indicates the estimate is significantly different 
from zero after sequential Bonferroni correction, 
?
indicates significance before 
correction but not after, and 
?

indicates p-values  > 0.05.
NA = not available. 
 
 
 Hawaii Maui Lanai Molokai Kauai 
Hawaii 
- 0.092
*
 0.060
?
 NA 0.064
?
 
Maui 0.068
*
 - 0.095
*
 NA -0.030
?
 
Lanai 
0.405
*
 0.543
*
 - NA 0.145
*
 
Molokai 0.226
*
 0.404
*
 0.037
?
 - NA 
Kauai 0.511
*
 0.574
*
 0.633
*
 0.424
*
 - 
 
 
 
 79 
Table 3. Population differentiation for the microsatellite data set in modern Hawaiian 
petrels. Pairwise F
ST
 is shown below the diagonal and G?
ST
 is shown above. *Indicates 
the estimate is significantly different from zero after sequential Bonferroni correction, 
?
indicates significance before correction but not after, and 
?

indicates p-values  > 0.05.
 
 
 
Hawaii Maui Lanai Kauai 
Hawaii 
- 0.028
?
 0.030
?
 0.021
?
 
Maui 0.016
*
 - 0.057
?
 0.015
?
 
Lanai 0.021
*
 0.033
*
 - 0.046
?
 
Kauai 
0.010
*
 0.012
*
 0.027
*
 - 
 
 
 80 
 
FIGURES 
 
 
Figure 1. Map of the main Hawaiian Islands with approximate locations of modern 
(Hawaii, Maui, Lanai, and Kauai) and historically known (Molokai) breeding colonies 
shaded. After Simons and Hodges (1998). Hawaiian petrels were formerly known to have 
a wider distribution on these islands, and there was an additional colony on the island of 
Oahu, however it was extirpated prior to the arrival of Captain Cook in 1778. 
 
 
 
 
 81 
Figure 2. Haplotype network for 319 modern and historic Hawaiian petrel mitochondrial 
Cytochrome b sequences. The sizes of the circles are proportional to the haplotype 
frequency, and lines indicate the number of substitutions between haplotypes.  
 
 
 
 82 
 
Figure 3. Genetic ancestry of Hawaiian petrels as estimated by the program STRUCTURE 
from the microsatellite data set using the admixed model with correlated allele 
frequencies and sampling location as a prior. Top K=2, Middle K=3, Bottom K=4. 
 
 
 
 83 
Figure 4. Graph of ?K statistic (Evanno et al. 2005) for STRUCTURE runs with the 
number of clusters (K) set between 1 and 8. This statistic takes into account the change in 
the log probability of the data as K increases. 
 84 
CHAPTER IV 
 
 
Ancient DNA reveals resilience despite the threat of extinction: three 
thousand years of population genetic history in the endemic Hawaiian 
petrel* 
 
 
Humans have had great impacts on Pacific Islands. In the Hawaiian Islands human 
colonization is associated with the extinction of nearly 50% of the terrestrial avifauna and 
a decline in the population size and range of many species, including several seabird 
species. The endemic Hawaiian petrel has escaped extinction, but colonies on two islands 
have been extirpated and populations on remaining islands have decreased. We obtained 
mitochondrial DNA sequences from 100 subfossil bones, 28 museum specimens, and 289 
modern samples to investigate patterns of gene flow and temporal changes in the genetic 
diversity of this endangered species. Overall, differentiation was high between modern 
birds from Hawaii, Maui, Lanai, and Kauai, as well as between ancient samples from 
Hawaii, Maui, and Oahu. However, gene flow was substantial between the extirpated 
colonies on Oahu and Molokai and modern birds from the island of Lanai. No significant 
reductions in genetic diversity occurred over the three thousand year period, despite fears 
early in the last century that this species may have gone extinct. Modeling suggests that 
given current census size estimates, the decline was probably not as severe as previously 
thought. Simulations show that even a decline to a stable effective population size of 100 
individuals would result in the loss of only 5% of the expected heterozygosity over a 150-
year period. Simulations also show that in closed populations high levels of genetic 
diversity may be retained due to the long generation time of this species. Therefore, it 
appears that a pattern of dispersal from declining colonies, in addition to long generation 
 85 
time, may have allowed the Hawaiian petrel to escape a severe genetic bottleneck after 
the arrival of humans in the Hawaiian Islands. 
 
*A. J. Welch, A. E. Wiley, H. F. James, M. P. Ostrom, T. W. Stafford, J. Southon, and R. 
C. Fleischer. For submission to Molecular Biology and Evolution. 
 
 86 
INTRODUCTION 
 
There is little debate that humans have had a great impact on global biodiversity (Ehrlich 
and Wilson 1991; Myers and Knoll 2001). Human colonization of the islands of the 
Pacific Ocean was no exception (Steadman 1995). The area known as Eastern Polynesia, 
which includes the Cook Islands, Society Islands, the Hawaiian Islands, as well as Easter 
Island and New Zealand (Steadman 2006), was one of the last habitable areas to be 
colonized by humans (Wilmshurst et al. 2011). Despite this relatively more recent arrival, 
there has been great debate about the exact chronology of colonization of Hawaii (Kirch 
and Kahn 2007). It appears most likely that the Hawaiian Islands were colonized some 
time between 1200 and 800 years ago (Wilmshurst et al. 2011; Kirch 2000). The earliest 
known settlements are located on Oahu, Molokai, and Hawaii in areas that would have 
had plentiful resources. Over time, however, humans colonized even the most marginal 
of habitats and population sizes may have exceeded 200,000 individuals prior to contact 
with Europeans (Kirch 1985). It appears, though, that even before human abundance 
reached maximum levels there may have been large ecological impacts (Athens et al. 
2002). 
 
The Hawaiian Islands are well known for their endemic avifauna (Carson and Clague 
1995). The fossil record appears to indicate that the background extinction rate of birds in 
the Pacific was low prior to human colonization (Steadman 2006). In Hawaii, extinction 
rates increased dramatically coincident with the arrival of humans (Olson and James 
1982b). These declines have often been associated with the introduction of exotic species, 
habitat destruction or modification, and direct exploitation (Blackburn et al. 2004; 
 87 
Milberg and Tyrberg 1993; Athens et al. 1991; Athens et al. 2002). Extinctions primarily 
consisted of flightless species of geese and rails, although many other species also went 
extinct, including several raptors, crows, and more than a dozen honeycreepers (Olson 
and James 1982b). One seabird species, Pterodroma jugabilis, also disappeared during 
this time (Olson and James 1982a). Another wave of extinction occurred after European 
contact (Pratt 1994). In addition to extinction, populations of many species declined in 
number or experienced range contractions (Olson and James 1982a). Today many of the 
remaining native and endemic species of Hawaiian birds are threatened by extinction and 
have been listed under the US Endangered Species Act. 
 
Severe population declines can have impacts on the genetic diversity of a species 
(Frankham et al. 2002). Declines in population size can lead to inbreeding, which can 
decrease survival and reduce reproduction. Additionally, decreased genetic diversity may 
limit evolutionary potential. All of these factors may in turn increase the risk of 
extinction (Frankham 2005). The amount of genetic diversity lost as a species passes 
through a decline depends on the length of time and effective population size during the 
decline: H
t
 = H
0
 (1 ? 1/(2N
e
))
t
, where H
0
 and H
t
 represent heterozygosity in the past and 
the present, t represents time, and N
e
 represents the effective population size (i.e. the size 
of an ideal population that would lose genetic diversity at the same rate as the population 
of interest) (Allendorf and Luikart 2007). Ancient changes in population size can be 
difficult to detect because populations will have had an opportunity to recover and 
mutations may have accumulated in the intervening time. Fortunately, ancient DNA 
techniques allow utilization of temporally spaced samples, which increases power, and 
 88 
can allow us to look back in time to gain a better understanding of the evolutionary 
history of a species (Ramakrishnan et al. 2005). 
 
Several studies have demonstrated the power of this approach empirically. Using ancient 
DNA, Shapiro et al. (2004) investigated if temporal population dynamics of bison were 
associated with the presence of humans, and Campos (2010) investigated if changes in 
genetic diversity of musk ox resulted from climatic fluctuations. Another ancient DNA 
study, of penguins in New Zealand, showed that one endangered species actually 
underwent a range expansion after the extinction of a conspecific (Boessenkool et al. 
2009). Ancient DNA has also been utilized to investigate temporal changes in genetic 
diversity of Hawaiian species. The endangered Hawaiian goose, or nene, was found to 
have undergone a severe prehistoric bottleneck (Paxinos et al. 2002a), coincident with 
human arrival in the Islands. It remains unclear how genetic diversity has changed over 
time for other bird species that have survived through this period. 
 
The endemic Hawaiian petrel (Pterodroma sandwichensis) is a long-lived pelagic 
seabird. Subfossil bones of this species have been found on the islands of Hawaii, Maui, 
Lanai, Molokai, Oahu, and Kauai, but their range is considerably smaller today (Figure 
1). The lowlands of Oahu were apparently the home of a large petrel colony, but 
historical records of it are lacking, indicating that it may have been extirpated prior to the 
arrival of Europeans. Historical accounts tell of a large colony on the island of Molokai 
with birds so plentiful that they ?darkened the sky? (Munro 1955), however recent survey 
trips have failed to locate a substantial colony on this island (Simons and Hodges 1998). 
 89 
Of the four remaining islands where the Hawaiian petrel currently breeds (Hawaii, Maui, 
Lanai, and Kauai) it appears that the birds have undergone a range contraction. Formerly 
on these islands bones were found over a range of elevations, from the coast up to the 
slopes of the highest volcanoes (Athens et al. 1991; Olson and James 1982a).  
 
Many factors may have contributed to the decline of the Hawaiian petrel, including 
predation by both humans and introduced mammals such as rats, cats, and mongoose. In 
Hawaii several lines of evidence indicate direct exploitation of petrels by humans. 
Historical accounts indicate that Hawaiian petrel chicks were considered a delicacy and 
were reserved for the social elite (Henshaw 1902; Bryan 1908). At some sites on the 
island of Hawaii approximately 70% of bird bones found in an archeological context 
belonged to Hawaiian petrels (Athens et al. 1991), and in a modern breeding colony on 
Mauna Loa, 19 of 41 burrows discovered during a survey occurred in pits that were 
apparently modified by humans in prehistoric or early historic times (Hu et al. 2001). In 
addition to humans, cats, rats and mongoose have been significant sources of mortality 
for the Hawaiian petrel (Simons and Hodges 1998; Henshaw 1902; Bryan 1908; Hu et al. 
2001) Due to these factors, and an apparent lack of sightings, ornithologists in the mid-
1900?s feared that this species was extinct (Baldwin and Hubbard 1948). Since that time 
the Hawaiian petrel has been rediscovered, and recent census population estimates now 
range from a total of 11,000 to 34,000 individuals with potentially 3,750 to 4,500 
breeding pairs (Spear et al. 1995). Census sizes on each island are not precisely known, 
but it is likely that today there are several thousand birds each on Maui, Lanai, and Kauai. 
 90 
On Hawaii populations appear to be declining and there may be fewer than 500 birds 
remaining (Birdlife-International 2011). 
 
Here we investigate the temporal population dynamics of the endangered Hawaiian 
petrel. We obtained mitochondrial Cytochrome b sequences from 100 ancient, 28 historic, 
and 289 modern Hawaiian petrel samples collected from all islands where this bird has 
been known to breed, including a prehistorically extirpated colony on Oahu and a 
potentially historically extirpated colony on Molokai. We examined patterns of gene flow 
and divergence, as well as changes in population size over the last 3000 years as humans 
colonized the Hawaiian Islands and became increasingly abundant. 
 
 
METHODS 
 
Samples and dating 
 
A total of 512 Hawaiian petrel samples were obtained for this study, representing 
approximately 3500 years of the population history of this species (Table 1). A total of 
289 samples were obtained opportunistically from modern petrels on Hawaii, Maui, 
Lanai, and Kauai. Bone, feather, and tissue samples were collected from carcasses of 
birds that had been depredated in breeding colonies or that died as a result of grounding 
(i.e. after attraction of fledglings to artificial light sources). Blood was sampled from 
grounded birds that were rehabilitated and subsequently released. We also obtained blood 
 91 
samples that were previously acquired from chicks captured in burrows on Maui (Browne 
et al. 1997). Since non-breeders depart early in the season (Simons and Hodges 1998), we 
assume that birds found on a given island represent breeders or their offspring. Because a 
large colony on Molokai may have been recently extirpated contemporary samples were 
unavailable; therefore we sampled toe pads of 28 Hawaiian petrel museum specimens 
that were collected on Molokai between 1907 and 1914 and deposited at the Bernice P. 
Bishop Museum and at the Los Angeles County Museum of Natural History (Appendix 
A). Finally, 195 ancient Hawaiian petrel bone samples were acquired from the 
Smithsonian?s National Museum on Natural History, the Bernice P. Bishop Museum 
(Appendix A), or collected in the field at both archeological and paleontological sites (see 
for example Olson and James 1982a; Figure 1). In sites where skeletal remains were 
dissociated, a single element was sampled (e.g. a right humerus) to prevent duplication. 
 
Radiocarbon dates of bones were obtained through accelerator mass spectrometry using a 
protocol modified from Stafford et al. (1988). As described by Wiley et al. (In prep), 
XAD-treated, gelatinized bone collagen samples were combusted to CO
2
, graphitized, 
and dated at the W. M. Keck Carbon Cycle Accelerator Mass Spectrometry Lab, at 
University of California, Irvine. Since Hawaiian petrels forage at sea they obtain all of 
their carbon from the ocean. Mixing of carbon in the ocean and the atmosphere is not 
immediate, and upwelling can lead to mixing of ocean layers of differing ages, so carbon 
dates can be biased by several hundred years. We applied the global marine reservoir 
correction (Reimer et al. 2004) in addition to a regional correction of 54 years developed 
specifically for the Hawaiian petrel (James et al. In prep). Radiocarbon dates were 
 92 
calibrated using the program CALIB v. 6.0 (Stuvier and Reimer 1993) and are presented 
as calendar years before present. Radiocarbon dates were obtained for all bones found in 
a paleontological context. For bones found in an archeological context, which generally 
accumulate over a shorter period of time, a series of dates was obtained and remaining 
samples were assigned the average age. 
 
Molecular Techniques 
 
Genomic DNA was extracted from blood and tissue samples using the DNEasy tissue kit 
(Qiagen) and from bone, feather, and toe pad samples via phenol/chloroform extraction 
and centrifugal dialysis (Fleischer et al. 2000). Stringent protocols were maintained to 
prevent contamination of ancient samples. All extractions for ancient and historical 
samples were performed in a physically separate, dedicated ancient DNA laboratory and 
work was conducted moving up a concentration gradient of PCR products. For 45% of 
the ancient and 100% of the historical samples, a sample from a different species was 
extracted in between each Hawaiian petrel sample to detect cross-contamination. For the 
remaining ancient samples Hawaiian petrel bones from different islands were alternated 
whenever possible. Multiple extraction and negative reagent controls were also used to 
detect contamination. Initial mitochondrial DNA sequencing results indicated the 
presence of cytosine deamination artifacts in ancient samples. Therefore 50 ?L aliquots 
of all ancient DNA extracts from bone were incubated with 1X uracil-DNA-glycosylase 
buffer and 1 unit uracil-DNA glycosylase at 37
o
C for 10 minutes, followed by incubation 
with 1 unit uracil glycosylase inhibitor (New England Biolabs) at 37
o
C for 10 min, and a 
 93 
final incubation for 10 min at 95
o
C. This treatment should destroy uracil residues that 
result in miscoding legions (Hofreiter et al. 2001). 
 
We amplified a 524 bp portion of the 5? variable region of the mitochondrial Cytochrome 
b gene. In well-preserved modern samples this region was amplified using the primers 
CytbL and CytbR9 (Welch et al. 2011). Many population genetic studies utilize the 
mitochondrial control region instead, but this region has been duplicated in 
procellariiform seabirds (Abbott et al. 2005). Cytochrome b has been widely use for 
seabirds studies and has high levels of variation (Welch et al. In Prep; Nunn and Stanley 
1998; Zino et al. 2008; Techow et al. 2009). For ancient, historical, and degraded modern 
samples we amplified seven short (< 150 bp) overlapping fragments (Welch et al. In 
Prep). For ancient and historical samples, each fragment was amplified at least twice. 
Polymerase chain reaction (PCR) and sequencing were carried out as in Welch et al. (In 
Prep). Briefly, PCR was conducted with 15 ?L total reaction volumes, 1 unit AmpliTaq 
Gold DNA polymerase (Applied Biosystems), 1- 2 ?L DNA template, and 35 cycles for 
modern samples and 25 ?L total reaction volumes, 1 unit Amplitaq Gold DNA 
polymerase, 2 - 4 ?L DNA template, and 45 cycles for ancient and historical samples. 
We also attempted to amplify nuclear intron and microsatellite loci (Welch et al. In Prep), 
but success was low, even for historical samples, and so those data were not utilized in 
further analyses. All fragments were electrophoresed in an ABI 3130 xL Genetic 
Analyzer (Applied Biosystems) and sequences were aligned and visually inspected in 
SEQUENCHER v 4.9 (GeneCodes).  
 
 94 
Data analysis 
 
Sequences were characterized in MACCLADE v. 4.08 (Maddison and Maddison 2008) and 
translated in DAMBE v. 1.5.2 (Xia and Xie 2001) to examine the potential for presence of 
nuclear copies in the mitochondrial data set (Sorenson and Fleischer 1996). The program 
JMODELTEST v. 0.1 (Posada 2008) was utilized to select the best fitting substitution 
model: the HKY model of nucleotide substitution with rate heterogeneity modeled by a 
gamma distribution. To depict the relationship between haplotypes a statistical parsimony 
network was created in TCS v. 1.21 with a 95% connection limit. 
 
Gene flow and population divergence 
 
Rates of gene flow and levels of differentiation were investigated between birds sampled 
on each island, including between modern samples and those from the extirpated colony 
on Oahu and the potentially extirpated colony on Molokai. It is also possible that the 
philopatric nature of procellariiform seabirds could lead to population differentiation on a 
local scale (Smith et al. 2007). To investigate this further we examined gene flow and 
differentiation between four groups of samples from the largest island of Hawaii: modern 
birds that breed on the slopes of Mauna Loa in Hawaii Volcanoes National Park 
(HAVO), ancient birds that were discovered in an archeological context at a site known 
as Fireplough Cave, ancient birds that were discovered in a paleontological context in the 
Pu?u Wa?a Wa?a region on the Kona side of the island, and finally a group that consisted 
 95 
of birds from a mixture of the remaining sites that died during approximately the same 
time frame (100 ? 400 years ago) (Figure 1).  
 
We estimated pairwise F
ST
 values in ARELQUIN v. 3.1 (Excoffier et al. 2005) from a 
Kimura two-parameter distance matrix. Depaulis et al. (2009) pointed out that there could 
be bias in summary statistics, such as F
ST
, calculated from heterochronous (i.e. ancient 
DNA) data sets. However, bias should be small if sampling occurs over a relatively short 
time as compared to evolutionary time. Here, corrected and uncorrected estimates of F
ST
 
differed by at most 0.002, and so uncorrected estimates are shown. Significance of p-
values was determined after sequential Bonferroni correction for multiple tests (Rice 
1989). We also estimated migration rates using the program MIGRATE v. 3.2.7 (Beerli 
2006; Beerli and Felsenstein 2001). We employed the Bayesian mode with uniform 
priors. The transition/transversion ratio was set to 14.0, and rate heterogeneity was 
modeled by a gamma distribution with alpha set to 0.083, as determined from 
JMODELTEST. Analyses were conducted both with and without the use of sampling dates, 
and both analyses produced very similar results with 95% confidence intervals 
overlapping. Three simultaneous replicates were performed, each for 20 million 
generations, with the first 10% discarded as burn-in. A four-chain heating scheme was 
used to increase the effectiveness of the search, with heating parameters set to 1.0, 1.2, 
3.0 and 6.0. Convergence was assessed through multiple independent runs started from 
different random number seeds. Effective sample sizes for all runs were > 1000. 
Migration estimates from the program MIGRATE are compounded with the mutation rate 
 96 
(i.e. m = M/?), so migration rates were scaled by an estimate of the mutation rate 
obtained from BEAST analyses (1.0 x 10
-7
, see below). 
 
Temporal changes in effective population size  
 
We also investigated changes in effective population size (N
e
) and genetic diversity over 
time to determine the impact of the decline in Hawaiian petrel population sizes. A 
coalescent Bayesian skyride analysis was conducted in BEAST v.1.6.1 (Drummond and 
Rambaut 2007; Minin et al. 2008). The Bayesian skyride is similar to a Bayesian skyline 
analysis but requires relatively weaker prior assumptions about population history. In the 
skyline analysis, the number of points where the effective population size is allowed to 
change, which is related to smoothness of the population size trajectory, must be set a 
priori. In the skyride analysis this is determined from the data by penalizing changes 
between coalescent intervals. This can be done in a ?time-aware? fashion by assuming 
that changes in successive intervals are smaller than changes in more distant intervals 
(Minin et al. 2008; Ho and Shapiro 2011). 
 
A Bayesian skyride analysis was conducted separately for samples collected on Hawaii 
and Maui. A maximum likelihood tree was generated in the program GARLI v. 0.96b 
(Zwickl 2006) to provide a starting tree for subsequent analyses. The HKY substitution 
model was implemented with rate heterogeneity modeled by a gamma distribution as 
determined from JMODELTEST. The 95% confidence interval of the radiocarbon date was 
utilized as a prior on the sampling time for ancient samples. Analyses were performed for 
 97 
1.5 x 10
8
 generations sampling every 2,000 generations. Convergence was assessed 
through multiple independent runs, and effective sample sizes were examined in Tracer v. 
1.5 (Drummond and Rambaut 2007). Since post-mortem damage of ancient DNA could 
potentially artificially inflate genetic diversity in the past (Axelsson et al. 2008), in 
addition to uracil-DNA-glycosylase treatment,  we performed analyses in BEAST both 
with and without a model of DNA damage (Ho et al. 2007). Finally, we performed a 
BEAST analysis under the coalescent constant population size model. Support for various 
models was compared using Bayes factors, as estimated in Tracer (Suchard et al. 2001; 
Kass and Raftery 1995). 
 
In addition to coalescent-based estimation of changes in effective population size, we 
utilized the ?temporal alleles? approach to estimate the variance effective population size. 
This method estimates the harmonic mean of N
e
 in the time period between the sampling 
points by assuming that changes in allele frequencies are due to genetic drift (Luikart et 
al. 2010). Samples from Hawaii were binned by age and any sequences with greater than 
15% missing data were excluded from the analysis. We also excluded samples from the 
oldest time bin (i.e. older than 1000 years) because of the relatively low sample size. 
Estimates were obtained using the program TM3.1 (Berthier et al. 2002) assuming a 
maximum N
e
 of 100,000 with 20,000 iterations. For comparison, nucleotide diversity and 
gene diversity were calculated for the same samples in ARLEQUIN. 
 
Modeling and Simulations 
 
 98 
We utilized modeling and simulations to investigate the severity of the decline of the 
Hawaiian petrel as well as the effect of generation time on retention of genetic diversity. 
First, we used population models to investigate if, given the long generation time of the 
Hawaiian petrel, this species could have actually been on the brink of extinction in the 
early and mid-1900?s, as previously suggested. We did this by modeling the minimum 
population growth rate necessary for Hawaiian petrels during this time and comparing it 
to estimates obtained from the literature. To our knowledge the last major record of the 
Hawaiian petrel from the early 1900?s is the collection of 26 birds in breeding colonies 
on Molokai by W. A. Bryan in 1914 (Banko 1980; Bryan 1908). After this no breeding 
colonies were known until 1964 when a colony of between 150 and 300 birds was 
discovered in Haleakala National Park, Maui (Banko 1980). In 1995, Spear et al. 
estimated that there were 11,000 to 34,000 individuals based on at-sea sightings. 
 
Since Hawaiian petrels have a long life span, delayed sexual maturity, and low annual 
fecundity, they have a very low population growth rate. To estimate the minimum growth 
rate necessary we used the simple exponential growth equation N
t
 = N
0
e
rt
 (where N 
represents population size, r represents growth rate, and t represents time). In this model 
individuals can begin reproducing in the first year. While this extreme scenario is 
unlikely to actually occur it represents the absolute minimum growth rate required during 
this time period. Therefore, if growth rates obtained from the literature (i.e. based on 
observations of Hawaiian petrels in the field) are far below this level, then there would be 
evidence that Hawaiian petrels may not have been as near extinction as previously 
thought. We tested various values for each parameter of the model (Appendix E, 
 99 
Supplementary Table 1). We also used this equation, along with an estimate of the 
maximum population growth rate for Hawaiian petrels of between 0.005 and 0.008 
(Simons 1984), to obtain a range of estimates of the number of individuals that may have 
actually been present in the past (Appendix E, Supplementary Table 2). 
 
Next, we used simulations to investigate the severity of a decline needed to result in a 
decrease in genetic diversity of the Hawaiian petrel, given its life history characteristics. 
Assuming a life span of 36 years, with 100% overlapping generations, an age of first 
reproduction of six years, and an equal sex ratio (Simons 1984), we simulated various 
declines in the effective population size using the program BOTTLESIM (Kuo and Janzen 
2003) and examined the resulting change in genetic diversity in terms of expected  
heterozygosity and allelic richness. One hundred simulations were run using haplotype 
frequencies observed in the ancient Hawaii data set. 
 
A second set of simulations was used to explore the effect of life history (i.e. longevity 
and age at first reproduction) on the retention of genetic diversity. Assuming that a 
species starts with the level of genetic variation observed in the ancient Hawaiian petrel 
samples collected on Hawaii, and that there is no gene flow or selection, we used 
BOTTLESIM to simulate different life history strategies and examined the effect (in 
expected heterozygosity and allelic richness) of a decline 150 years ago to a stable 
effective population size of 250 individuals. The life history strategies investigated 
included that of the Hawaiian petrel (as described above), a range of theoretical values, 
and several life histories similar to those of other animals that live in Hawaii or for which 
 100 
ancient DNA has been used to investigate changes in genetic diversity over time 
(Appendix E, Supplementary Table 3). These included life histories similar to elephants 
(Loxodonta africana, and, by proxy, mammoths), musk ox (Ovibos moschatus), bison 
(Bison bison), nene (Branta sandvicensis), and a Hawaiian honeycreeper, the Palila 
(Loxioides bailleui;) (Laursen and Bekoff 1978; Meagher 1986; Lent 1988; Banko et al. 
1999; Banko et al. 2002; Debruyne et al. 2008). 
 
Population contribution to overall genetic diversity 
 
Finally, we investigated the contribution of each Hawaiian petrel population to overall 
genetic diversity. Total genetic diversity can be partitioned into a diversity component 
(i.e. the probability that two alleles of a gene drawn at random are different) and a 
richness component (i.e. the number of alleles) and compared across populations. Each of 
these components can be further broken down into two subcomponents: diversity within a 
population and the divergence between that population and others (Petit et al. 1998). We 
used the program CONTRIB v. 1.02 to calculate the contribution of modern and extirpated 
populations to overall genetic diversity. Since allelic richness can be sensitive to sample 
size, samples sizes were rarefied to 12, which represents the smallest samples size (Oahu) 
in the study. 
 
 
RESULTS  
 
 101 
 
Mitochondrial DNA sequences were obtained from a total of 417 Hawaiian petrel 
samples: 100 ancient, 28 historical, and 289 modern birds (Table 1). The success rate for 
ancient bones from Hawaii was about 76% (77/101), and ages ranged from 112 to 3228 
years, although the mean age was 467 years (Figure 3; Appendix E, Supplementary Table 
4). The success rate for ancient samples from Maui was 38% (9/24), with samples 
ranging from 555 to 3435 years old and a mean age of 1317 years (Figure 3). Success 
was 18% (12/66) for Oahu, with samples ranging from 615 to 1863 years old and an 
average age of 1310 years (Appendix E, Supplementary Figure 1). Relatively fewer 
bones were available from the islands of Lanai and Molokai and success was low: 1/5 for 
Lanai and 1/3 for Molokai. No bones were available from Kauai for destructive analyses. 
The success rate was 100% for historical museum specimens collected on Molokai (N = 
28) approximately 100 years ago. The sequence length was 524 bp for modern samples, 
and the average combined sequence length was approximately 480 bp and 400 bp for 
historic and ancient samples, respectively. No instances of Hawaiian petrel contamination 
were found in either extraction, blank, or alternate species control samples. Additionally, 
Bayes factors indicate no support for BEAST analyses that included an age-dependent 
model of DNA damage over analyses without (2 ln Bayes factor = -4.70). 
 
A total of 47 haplotypes were found from 51 variable sites. Of these, 21% occurred in the 
first codon position, 4% occurred in the second, and 75% occurred in the third. Ninety-
one percent of the variable sites exhibited transitions. There were no gaps in the 
alignment and after translation no nonsense or stop codons were found. This evidence 
 102 
indicates that a mitochondrial, and not nuclear, origin of the sequences is likely. A total 
of 18 haplotypes were found in birds from Hawaii, 20 from Maui, 8 from Lanai, 9 from 
Molokai, 6 from Oahu, and 10 from Kauai (Figure 2). A total of 34 haplotypes were 
found in modern and historical samples, and 21 were found in ancient samples with 8 
shared between time periods. 
 
Gene flow and population divergence 
 
Using ancient DNA we investigated patterns of gene flow and divergence between extant 
and extirpated populations of the Hawaiian petrel. We also examined these patterns 
through time. Overall, differentiation was high (Table 2). In the analysis with modern 
Hawaiian petrels, as well as birds sampled from the two extirpated populations, 12 out of 
15 pairwise F
ST
 values were significantly greater than zero. Of these twelve, 11 had an 
F
ST
 of about 0.20 or greater, with a maximum F
ST
 of 0.633 observed between Kauai and 
Lanai. There was no significant differentiation between modern individuals from Lanai 
and individuals from the extirpated populations on Oahu and Molokai. In the past, 
individuals from Hawaii, Maui, and Oahu were also significantly differentiated (Table 3). 
F
ST
 ranged between 0.229 and 0.437 with the highest level found between Hawaii and 
Oahu. Results from Migrate were consistent with those obtained from estimation of F
ST
 
(data not shown). 
 
We also investigated the potential for genetic differentiation on a local scale on the island 
of Hawaii. Samples found in a paleontological context in the Pu?u Wa?a Wa?a region 
 103 
(ranging in age from 100-400 years) were significantly differentiated from all other 
groups of individuals, including modern birds sampled on the eastern side of the island at 
HAVO (F
ST
 > 0.350, Table 4). Birds found in an archeological context at Fireplough 
Cave (which was utilized by Hawaiians from about 400 to 500 years ago) were not 
significantly differentiated from any group but Pu?u Wa?a Wa?a. Geographically, 
Fireplough cave and the Pu?u Wa?a Wa?a site are relatively much closer to each other 
than to HAVO (Figure 1). Low, but significant, differentiation was found between the 
modern birds from HAVO and the combined group of samples from across the island 
(F
ST
 > 0.100, Table 4). Again, results from Migrate were consistent with those obtained 
from estimation of F
ST
 (data not shown). 
 
Temporal changes in effective population size 
 
The Bayesian skyride analysis for birds from Hawaii shows a population trend that has 
been relatively stable over time (Figure 3). Starting about 845 years ago and continuing 
toward the present a decrease can be seen. While the change appears small in Figure 3 
because of the log scale for the y-axis, it represents a change in effective population size 
of 41% over the last 800 years. The 95% confidence interval, however, is wide. The 
lower interval includes a scenario in which Hawaiian petrel populations decline by 76%, 
but the upper interval indicates that a constant population size, with no change, is also 
possible. Bayes factors indicate some support for the constant population size model over 
the skyride model (2 ln Bayes factor = 27.37), suggesting that while there may have been 
a decline in effective population size it did not significantly change genetic diversity.  
 104 
 
Samples were also combined into three different time bins (400 ? 1000 years old, 100 ? 
400 years old, and modern) and changes in genetic diversity were investigated using 
haplotype and nucleotide diversity. Haplotype diversity was significantly higher in the 
oldest time bin (Table 5, p < 0.05), however there was no significant difference between 
the 100 - 400 and the modern time bins. There was also no significant difference in 
nucleotide diversities between any time bins. Finally, we investigated changes in 
effective population size on Hawaii using the ?temporal alleles? approach, which 
estimates the harmonic mean of the effective population size in the period between two 
samples. The effective population size between the middle and older time bins was 
47,693 (95% Confidence interval (CI): 1415 to 97,397) and between the modern and 
middle time bin it was 954 (95% CI: 287 to 82,759). Confidence intervals for these 
estimates overlap, indicating no significant change over a period of about 1000 years.  
 
The Bayesian skyride analysis for birds from Maui showed an increase in effective 
population size over time until about 1100 years ago, after which it stabilized and 
remained constant. This could be due to a relatively low ancient sample size (N = 9) for 
this island, which would result in few coalescent events in the past. Bayes factors indicate 
no support for the skyride model over the constant size model (2 ln Bayes factor = -8.80). 
Since the ancient sample size was lower for Maui as compared to Hawaii, samples were 
divided into two time bins, modern and ancient ( > 500 years), and haplotype and 
nucleotide diversity were calculated. No significant changes were found (Table 6). Since 
 105 
there were only two time bins, change in effective population size using the temporal 
alleles approach was not investigated. 
 
Modeling and Simulations 
 
We used the exponential growth model to investigate if Hawaiian petrels could have 
actually been near extinction in the middle of the last century given their life history and 
recent estimates of census population size.  The smallest population growth rate found (to 
reach the minimum census population estimate of 11,000 individuals in 1995) was 0.03 
(Appendix E, Supplementary Table 1), assuming that Hawaiian petrels declined to a 
minimum census size of 1000 individuals and had 80 years to recover. Growth rates 
based on larger declines (e.g. to 50 or 200 individuals), larger population sizes in 1995 
(e.g. the average or maximum census size) or shorter times for recovery (e.g. 60 or 40 
years), resulted in minimum necessary growth rates between about 0.04 and 0.12. These 
rates are much higher than those described in the literature. Simons (1984) modeled 
petrel population dynamics based on observations of a colony on Maui. His models 
included a breeding frequency of 89%, annual adult survival of 93%, and annual juvenile 
survival of 80%. The maximum reproductive success that he observed during a three-year 
field study of this species was 72%, which yielded a growth rate of 0.005. He also 
reported a maximum reproductive success of 75% for similar seabird species, which 
would translate to a maximum growth rate for Hawaiian petrels of 0.008. Using growth 
rates from the literature it appears that there would have been at least 5,800 Hawaiian 
 106 
petrels in the 1900?s and very likely many more than that (Appendix E, Supplementary 
Table 2). 
 
We conducted simulations in order to determine the severity of a population decline that 
would be required to reduce genetic diversity in the Hawaiian petrel. Simulations were 
conducted in which a population declines from an effective population size of 50,000 at 
150 years in the past to current effective population size from 10 ? 1000 individuals. 
Simulations showed that declines to an effective population size of between 100 and 
1000 resulted in approximately 5% or less loss in expected heterozygosity (Figure 4). 
More severe declines, down to an N
e
 of 50, 25, and 10 resulted in a loss of 11%, 23%, 
and 44% of the expected heterozygosity, respectively. Allelic richness showed a similar 
pattern but with more severe reductions in the percentage of alleles retained (Appendix E, 
Supplementary Figure 2) 
 
To investigate what role longevity and generation time may play in maintaining genetic 
diversity, we simulated demographic changes in species with various life histories (i.e. 
longevity and age at first reproduction), ranging from a species that lives 50 years and 
begins breeding in the tenth year, to a species that lives 2 years and begins reproduction 
in the first year. Simulations, in which a population declines from 50,000 individuals 150 
years in the past to a stable number of 250 individuals, demonstrated that species 
exhibiting long life spans and delayed reproduction lost less than 8% of the expected 
heterozygosity, while species with shorter life spans that began breeding in the first year 
lost between 21% and 45% of their expected heterozygosity (Figure 5A). For 
 107 
comparison, we simulated the change in genetic diversity under life histories similar to 
those of species for which changes in genetic diversity have recently been investigated 
using ancient DNA or which also live in the Hawaiian islands and were also impacted by 
human colonization. Elephants (and by proxy mammoths), with the longest life span and 
latest age at first reproduction, demonstrated the least decline, losing about 3% of 
expected heterozygosity (Figure 5B). The palila, an endemic Hawaiian honeycreeper, 
experienced the largest loss of expected heterozygosity of about 16%. The bison and 
musk ox lost slightly less (13%), while the Hawaiian petrel and the Nene, the endemic 
Hawaiian goose, were intermediate loosing 6% and 8% respectively. Again, allelic 
richness showed a similar patter, but with more severe reductions (Appendix E, 
Supplementary Figure 3). 
 
Population contribution to overall genetic diversity 
 
An analysis of the contribution of each population to overall genetic diversity showed 
that birds from Maui and Kauai made the largest contribution to the genetic diversity 
component (0.17%) while birds from Maui and Lanai made the largest contribution to the 
allelic richness component (0.56% and 0.28%, respectively). Breaking each of these into 
subcomponents of diversity and differentiation allows for a more detailed investigation of 
these patterns (Figure 6). For the genetic diversity component of birds from Kauai, the 
diversity subcomponent showed levels below the average, but the differentiation 
subcomponent was very high, and the net difference between these two subcomponents 
lead to the comparatively high genetic diversity component overall. For the allelic 
 108 
richness component, the diversity and differentiation subcomponents effectively canceled 
each other out, leading to an average level of contribution to the allelic richness 
component overall. Therefore, for birds from Kauai the largest contribution appears to 
come from differentiation while diversity is about average. This is in contrast to the 
extirpated populations on Molokai and Oahu. These populations both had slightly higher 
than average genetic diversity, but low differentiation from other populations swamped 
this out. Birds from Lanai had a high relative contribution, but only to allelic richness, 
whereas birds from Maui had high contributions to both diversity and richness. Birds 
from Hawaii had lower contributions in both of these components than average. 
 
 
DISCUSSION 
 
Here we examined patterns of gene flow and differentiation, as well as changes in genetic 
diversity and effective population size, in an endangered seabird over the last 3000 years. 
In general differentiation was high between petrels sampled across most islands, both 
currently and in the past. However, low levels of F
ST
 were found between modern birds 
from Lanai and extirpated populations from Molokai and Oahu. Examination of effective 
population size over time on Hawaii and Maui revealed a relatively stable trend since 
human colonization of the Hawaiian Islands, although reductions of about 40% may have 
occurred on Hawaii. Modeling indicates that Hawaiian petrel population sizes 
approximately 80 years ago may have been as high as 7000 individuals and simulations 
 109 
demonstrate that long life span and generation time allow higher levels of genetic 
diversity to be retained over time. 
 
Gene flow and population divergence 
 
Investigation of differentiation between modern birds from Hawaii, Maui, Lanai, and 
Kauai indicates that isolation is strong between populations. This also appears to be the 
case in the past, as well. Significant F
ST
 was found between ancient individuals from 
Hawaii, Maui, and Oahu (F
ST
 > 0.229). Many seabirds, and especially procellariiform 
seabirds, are known to exhibit strong philopatry despite being highly mobile (Smith et al. 
2007; Friesen et al. 2006; Zino et al. 2008). The Hawaiian petrel is known to make 
foraging trips of greater than 10,000 km to the Gulf of Alaska, however on average it 
appears to disperse less than 300 km to breed. Differentiation may also have been 
stronger in the past than it is today. An F
ST
 of 0.372 was found between ancient samples 
from Maui and Hawaii, whereas an F
ST
 of 0.068 was found between modern birds on the 
same island. Ancient samples from Oahu also exhibited higher F
ST
 when compared to 
ancient samples from Hawaii (F
ST
 = 0.437) than with modern samples (F
ST
 = 0.192), 
although the pattern is reversed for individuals from Maui, with higher F
ST
 in 
comparisons with modern birds (F
ST
 = 0.349). While mitochondrial DNA sequences only 
reflect the movements of females, weak but significant differentiation has been found 
between modern birds from each island using data sets consisting of multilocus 
microsatellite genotypes and nuclear intron sequences (Welch et al. In Prep). 
 
 110 
It appears that population extirpation may cause Hawaiian petrels to deviate from their 
generally philopatric tendencies. While birds breeding today are significantly 
differentiated from one another, extirpated populations show low levels of divergence 
between them, as well as with modern birds from Lanai. Since the population on Oahu is 
known only from the fossil record it is likely that that population was extirpated first (the 
most recent radiocarbon dated petrel bone from Oahu in this study was 615 years old 
(95% CI: 546 ? 662 years). Social attraction may be important for seabird species (Parker 
et al. 2007; Danchin et al. 1998), and individuals from Oahu may have dispersed to either 
Lanai or Molokai as numbers in their own colony dwindled. Then later, as the number of 
birds on Molokai declined birds may have dispersed to Lanai. The ancient samples used 
here were collected from various caves around each island at elevations lower than where 
petrels are currently known to breed, and may potentially represent extirpated breeding 
colonies.  
 
Zador et al. (2009) noted that groups of common murre (Uria aalge) ranging in size from 
2 - 256 individuals formed new subcolonies up to 30 m away from other nesting areas. In 
procellariiform seabirds dispersal can be high. Laysan albatross have often been found to 
breed within 20 km of their hatching site, however, this species has undergone a recent 
range expansion, which includes the re-colonization of formerly extirpated sites (Young 
2010). A recent population genetic study demonstrated low differentiation among 
colonies (Young 2010; Young et al. 2009). Perhaps the lack of isolation could be a result 
of population contraction as birds dispersed away from declining populations. It also 
seems reasonable that given the long life span of these birds, which can be greater than 
 111 
60 years (Kaufman 2011), individuals re-colonizing a former site may have actually 
hatched there themselves (Parker et al. 2007). A similar process of displacement from 
extirpated colonies may be occurring in Hawaiian petrels. 
 
On a more local scale, colonies on the island of Hawaii may have been significantly 
differentiated from one another. Significant pairwise F
ST
 was found between individuals 
discovered in a paleontological context (i.e. without the presence of native Hawaiian 
artifacts) at the Pu?u Wa?a Wa?a site on western Hawaii and modern birds breeding on 
the eastern slopes of Mauna Loa. However, about 300 years separates these populations, 
and as such could have lead to changes in allele frequencies over time. It does provide 
limited evidence of the potential for fine-scale population differentiation, which is 
difficult to assess today as it appears that most islands now contain a single breeding 
colony. No significant differentiation was found between bones discovered in an 
archeological context at Fireplough cave and either bones from around the island 
(excluding the Pu?u Wa?a Wa?a site) or modern birds from Mauna Loa. This could 
indicate that Hawaiians were collecting birds from a large area and bringing them to 
Fireplough cave to process them, although it has been hypothesized that adult petrels 
were consumed opportunistically while parties hunted passerines for feathers or petrel 
chicks, which were a delicacy reserved for the social elite (Athens et al. 1991; Henshaw 
1902). Another possibility is that birds breeding near Fireplough cave dispersed to the 
eastern Mauna Loa area as the colony declined. If additional caves with ancient Hawaiian 
petrel bones are discovered near where petrels currently breed, they may provide 
additional information about the contraction of populations on each island. Also, 
 112 
discovery of additional Hawaiian petrel bones from Lanai and Molokai would be helpful 
in determining if gene flow occurred between islands in the past or if birds only dispersed 
as colonies dwindled. 
 
 
Temporal changes in effective population size and genetic diversity 
 
Investigation of changes in effective population size over time on the island of Hawaii 
suggests relative stasis over the past three thousand years, as Polynesians, and then 
Europeans colonized the islands. Humans have had broad ecological impacts on islands 
across the Pacific, including Hawaii. Humans have altered habitat, introduced exotic 
mammalian predators, and directly exploited plant and wildlife populations (Steadman 
2006; Athens et al. 1991; Athens et al. 2002). Coincident with human arrival in the 
Islands many species went extinct, including one seabird species, Pterodroma jugabilis 
(Olson and James 1982a; Olson and James 1982b). Even for species that didn?t go 
extinct, such as the nene or Hawaiian goose, declines in genetic diversity have been 
observed (Paxinos et al. 2002a). However, significant declines in the effective population 
size of the Hawaiian petrel have apparently not occurred, as demonstrated by both a 
Bayesian skyride analysis as well as the traditional temporal alleles approach. A similar 
pattern was found on Maui. While these findings are based only on mitochondrial DNA, 
it is unlikely that nuclear DNA would show a different pattern given that nuclear markers 
themselves have a higher effective population size than mitochondrial loci. 
 
 113 
The median posterior probability of the Bayesian analysis for the island of Hawaii 
showed a trend in which the effective population size decreased by about 40%. Even 
though this does not represent a significant change in the genetic diversity of the 
Hawaiian petrel, it may indicate that the census size in the past was larger than today. 
Since the effective population size of mitochondrial DNA is about one quarter that of 
nuclear DNA, due to its haploid nature and maternal inheritance pattern, the total 
effective population size of petrels on Hawaii when they were most abundant (just eight 
hundred years ago) could have been 1.5 million. Given the ratio of effective to census 
population size (N
e
/N
c
), an estimate of the total number of birds at that time could be 
obtained. While N
e
/N
c
 has been found to be about 0.10 on average (Frankham 1995b), 
overlapping generations and relatively low variance in reproductive success of the due to 
a monogamous breeding system, suggests that for the Hawaiian petrel N
e
/N
c
 may be 
closer to 0.50 (Hedrick 2005a; Nunney 1995). If these assumptions are true then the total 
population size on Hawaii may have been as high 3 million individuals. A decline of 40% 
would represent the loss of about 1.2 million individuals, but given the current population 
size estimates, the decline was likely to be much greater than that. Since Hawaiian petrels 
are top marine predators, and transfer nutrients from the ocean to the terrestrial ecosystem 
on the islands, a severe decline in numbers could have broad ecological impacts.  
 
The observed pattern of stasis in effective population size could be caused by low 
variability or small sample sizes in the data set. However, this does not appear to be the 
case. Mitochondrial DNA sequences contained 51 variable sites, from which 47 
haplotypes were discovered. Also, on Hawaii ancient samples (N = 77) outnumbered the 
 114 
modern samples (N = 71). However, due to the stochastic nature of the coalescent, 
additional loci may be beneficial (Kuhner 2009). This pattern of stasis could also be 
explained if gene flow brought genetic diversity from other islands. This also does not 
appear to be the case, either, as F
ST
 was found to be high between individuals from 
Hawaii, Maui and Oahu in the past and between modern individuals from all other 
islands, including the extirpated population on Molokai. Two additional explanations 
could lead to the observed pattern: The population decline of the Hawaiian petrel was not 
as severe as previously thought, and/or perhaps the long generation time of the bird is 
buffering it against loss of genetic diversity.  
 
To investigate if Hawaiian petrels may have been on the brink of extinction, as 
previously feared (Munro 1955; Simons and Hodges 1998; Baldwin and Hubbard 1948), 
we used the exponential growth equation to model the minimum required population 
growth rate. Assuming that the Hawaiian petrel was near extinction (i.e. 50 birds) in 
1915, the year after the last major museum collection, a growth rate of 0.067 would be 
required to reach the lower 95% confidence limit of the estimated census population size 
(Spear et al. 1995). This is an order of magnitude larger than highest measured growth 
rate (0.005) for a population of petrels studied on the island of Maui (where predator 
control programs were already in effect) and almost an order of magnitude larger than the 
growth rate (0.008) resulting from the best case scenario of 75% reproductive success 
proposed by Simons (1984). Therefore, it appears unlikely that the Hawaiian petrel was 
near extinction in the beginning of the last century. Using the growth rates of Simons 
(1984), and assuming exponential growth of Hawaiian populations, it is estimated that 
 115 
between 5,800 and 7,374 individuals remained, or likely even more. It is understandable 
that this species may have been overlooked. The Hawaiian petrel spends the majority of 
its life at sea, only coming to land to breed. Even then it returns to breeding colonies 
nocturnally and nests in underground burrow that are often greater than 2 m long. In 
addition extant colonies occur in rugged terrain and can be difficult to access (Simons 
and Hodges 1998). 
 
While Hawaiian petrel populations may not have declined to the brink of extinction, the 
distribution of subfossil bones suggest that they were more abundant in the past (Athens 
et al. 1991; Olson and James 1982a). Therefore it is possible that the long generation time 
of the Hawaiian petrel is buffering it against a significant loss of genetic diversity (Amos 
and Balmford 2001). We performed simulations to explore if, given the diversity 
observed in ancient Hawaiian petrels from Hawaii, different combinations of life span 
and age at first reproduction would result in different levels of genetic diversity being 
retained when held at an N
e
 of 100 for 150 years. Similar to other reports and consistent 
with population genetics theory (Amos and Balmford 2001; Hailer et al. 2006; Lipp? et 
al. 2006; Kuo and Janzen 2004), we found that species with a longer life span retained 
higher levels of expected heterozygosity as well as a larger number of alleles. For species 
with a life span > 20 years a maximum of 8% loss was observed, whereas species with a 
life span < 10 lost between 20 and 45% heterozygosity. This may mean that a loss of an 
entire generation of offspring may not be as detrimental for long-lived species, such as 
the Hawaiian petrel, as it would be for other species with shorter generation times. 
 
 116 
We compared the Hawaiian petrel with other species for which changes in genetic 
diversity have been investigated using ancient DNA (Paxinos et al. 2002a; Debruyne et 
al. 2008). Assuming that all of these species started with the same level of genetic 
diversity as the Hawaiian petrel, and assuming that they more or less fit the model 
equally well, elephants (and by proxy, mammoths, Debruyne et al. 2008) and Hawaiian 
petrels show the lowest losses of genetic diversity, whereas bison and musk ox show 
higher losses. Therefore, if all else were equal it might be expected that Hawaiian petrels 
and mammoths would show fewer changes in effective population size over time than 
either bison or musk ox. This is consistent with empirical findings (Campos et al. 2010). 
Nene should be intermediate between these groups, and changes in genetic diversity were 
found (Paxinos et al. 2002a). Also, for comparison we simulated the impact of a decline 
on the palila, an endemic Hawaiian honeycreeper. A large portion of honeycreeper 
species have gone extinct over the last 1500 years, since humans have arrived in the 
Hawaiian Islands (Olson and James 1982b), and many remaining species are endangered. 
The palila, with a relatively lower generation time, exhibited the largest decrease in 
simulations with a loss of 15% heterozygosity. Of course, in reality, many assumptions of 
these simulations may be violated. For example, even if generation time is very short, 
gene flow may prevent significant losses of genetic diversity. Also, life history is more 
complex than that modeled here, including biased sex ratios, non-random mating, 
multiple offspring per reproductive event, and so on. However, despite these violations 
simulations suggest that the generation time of an organism may mask temporal changes 
in genetic diversity, for example, in response to climate change or anthropogenic impacts, 
therefore careful interpretations of observed patterns are required. 
 117 
 
Given the relatively constant level of effective population size observed over time, we 
also simulated population declines of varying severity to explore how severe a decline 
would need to be before genetic diversity in the Hawaiian petrel to decrease. Simulations 
were conducted of a decline in effective population size from 50,000 to a size of 1000 
individuals or less. Results indicate that a very severe decline would be required to lose 
high levels of genetic diversity. Effective population size could decline to 50 individuals 
and remain there for 150 years and only 10% of the expected heterozygosity would be 
lost. Below this effects seemed to increase substantially, and a decline to an effective 
population size of 10 would result in a loss of 45% heterozygosity. However, in these 
drastic cases, long generation time would actually impede population growth, allowing 
the effects of the decline to perhaps persist longer than expected (Allendorf and Luikart 
2007). 
 
Conservation implications 
 
Overall, genetic diversity appears to be relatively stable in the Hawaiian petrel. An 
analysis of the net contribution of birds from different islands to the total genetic 
diversity suggests that populations on Maui and Kauai contribute the most to genetic 
diversity, and that Maui and Lanai contribute most to allelic richness. However, closer 
investigation shows that every population makes a contribution to genetic diversity in 
some respect, even if the net contribution appears to be about the average. For example, 
extirpated populations on Molokai and Oahu had somewhat higher than average genetic 
 118 
diversity but lower than average differentiation. So while loss of these populations would 
not reflect a large net loss, they could still be important, assuming that neutral genetic 
diversity is related to quantitative traits or fitness (Reed and Frankham 2001; Hansson 
and Westerberg 2002; Foerster et al. 2003; Reed and Frankham 2003). In addition to 
genetic diversity other aspects of populations are important to take into account when 
evaluating their contribution towards the continued existence of this species, including 
their ecology, census size, and distribution.  
 
It may be best for conservation management actions to focus on stabilizing population 
trends and increasing the population growth rate on each island, for example through 
continued predator control programs. Since genetic diversity is relatively high, inbreeding 
depression will probably not be a major issue for birds on most islands. However, the 
same life history traits that have lead to retention of genetic diversity in this species also 
prevent quick population recovery after a decline. If a severe decline occurs, slow 
demographic recovery could result in an extended population bottleneck and a longer 
period of time in which genetic diversity is vulnerable to erosion (Allendorf and Luikart 
2007). In these situations is may be beneficial to encourage gene flow, perhaps through 
translocation of individuals or the use social attraction (Parker et al. 2007). One such case 
may be occurring on the island of Hawaii where colonies appear to be dwindling. It 
appears that in the past Hawaiian petrels may have used dispersal as a mechanism to 
avoid extirpation. Habitat on Maui is similar to that on Hawaii, and individuals from 
Hawaii and Maui show relatively lower genetic differentiation in mitochondrial DNA. 
However, nuclear DNA indicates low differentiation with individuals from Lanai (Welch 
 119 
et al. In Prep). Given the levels of divergence observed here, outbreeding depression is 
not likely to be a major issue (Frankham et al. 2011), so increasing movements between 
these islands could be beneficial. 
 
Overall, it appears that a combination of long generation time and the ability to disperse 
in response to population extirpation have prevented declines in Hawaiian petrel 
population size and range from significantly decreasing genetic diversity. This could also 
account for the observation that while populations of many seabird species have been 
extirpated, relatively few species have gone extinct (Steadman 1995). The story of the 
Hawaiian petrel makes a case for some optimism despite the broad ecological and 
evolutionary impacts of humans on Pacific islands. 
 120 
TABLES 
 
 
Table 1.  Number of Hawaiian petrel samples, by island and time period, for which DNA 
sequences were successfully obtained. NA indicates samples were not available due to 
colony extirpation or lack of bones for destructive analyses. 
 
 
Time Period 
Island 
Ancient Historical/Modern 
Total 
Hawaii 77 71 148 
Maui 9 119 128 
Lanai 1 38 39 
Molokai 1 28 29 
Oahu 12 NA 12 
Kauai NA 61 61 
Total 100 317  
 
 
 
 121 
Table 2. Pairwise F
ST
 (below the diagonal) and associated p-values (above the diagonal) 
for Cytochrome b sequences obtained from ancient, historic, and modern Hawaiian 
petrels, representing samples from all islands where they have been known to breed. 
*Indicates significant and 
?
indicates non-significant comparisons after sequential 
Bonferroni correction. 
 
 Time Hawaii Maui Lanai Molokai Oahu Kauai 
Hawaii Modern - 0.000 0.000 0.000 0.000 0.000 
Maui Modern 0.068
*
 - 0.000 0.000 0.000 0.000 
Lanai Modern 0.405
*
 0.543
*
 - 0.072 0.099 0.000 
Molokai Historic 0.226
*
 0.404
*
 0.037
?
 - 0.432 0.000 
Oahu Ancient 0.192
*
 0.349
*
 0.074
?
 -0.004
?
 - 0.000 
Kauai Modern 0.511
*
 0.574
*
 0.633
*
 0.424
*
 0.538
*
 - 
 
 
 
 122 
Table 3. Pairwise F
ST
 (below the diagonal) and associated p-values (above the diagonal) 
for sequences from ancient Hawaiian petrels. *Indicates significant comparisons after 
sequential Bonferroni correction. 
 
 
 Time 
Hawaii Maui Oahu 
Hawaii Ancient - 0.000 0.000 
Maui Ancient 0.372
*
 - 0.040 
Oahu Ancient 0.437
*
 0.229
*
 - 
 
 
 123 
Table 4. Pairwise F
ST
 (below the diagonal) and associated p-values (above the diagonal) 
for sequences from petrels from different areas on the island of Hawaii. HAVO indicates 
individuals collected in Hawaii Volcanoes National Park on the eastern slope of Mauna 
Loa, PWW indicates bones of individuals from the Pu?u Wa?a Wa?a area, Fireplough 
indicates bones of individuals found in Fireplough Cave, and Other represents a grouping 
of individuals from all other sites from about the same time (see Figure 1) *Indicates 
significant and 
?

indicates non-significant comparisons after sequential Bonferroni 
correction. 
 
 
 
Time (YBP) HAVO PWW Other Fireplough 
HAVO  0 - 20 - 0.000 0.006 0.626 
PWW
a
 100 - 400 0.364
*
 - 0.000 0.000 
Other
b
 100 - 400 0.111
*
 0.431
*
 - 0.384 
Fireplough
c
 400 - 500 -0.026
?
 0.373
*
 -0.003
?
 - 
a
Paleontological site
 
b
Mix of archeological and paleontological sites 
c
Archeological site 
 124 
 
 
 
Table 5. Haplotype and nucleotide diversity, and their 95% confidence intervals, by time 
bin for samples obtained on Hawaii. 
 
 
Time period 
(YBP) 
N  Haps
Haplotype 
Diversity 
95% CI 
% Nt 
Diversity 
95% CI 
400 - 1000 11 9 0.964 0.862 ? 1.000 0.377 0.000 ? 0.913 
100 - 400 33 9 0.816 0.739 ? 0.894 0.368 0.000 ? 0.856 
Modern 70 9 0.772 0.720 ? 0.824 0.409 0.000 ? 0.919 
 
 125 
 
 
Table 6. Haplotype and nucleotide diversity, and their 95% confidence intervals, by time 
bin for samples obtained on Maui . 
 
 
Time period 
(YBP) 
N  Haps
Haplotype 
Diversity 
95% CI 
% Nt 
Diversity 
95% CI 
Ancient 9 7 0.917 0.733 ? 1.000 0.554 0.000 ? 1.289 
Modern 15 8 0.791 0.581 ? 1.000 0.384 0.000 ? 0.897 
 
 
 126 
FIGURES 
 
Figure 1. Map of the main Hawaiian Islands with approximate locations of modern 
(Hawaii, Maui, Lanai, and Kauai) and historically known (Molokai) breeding colonies 
shaded, as well as collection localities of sub-fossil bones. Approximate locations of 
archeological sites are marked with a triangle, paleontological sites are marked by circles. 
Specific sites on Hawaii are indicated, as well, for investigation of local population 
differentiation. 
 
 
 
 
 
 
 
 
127
 
Figure 2.
 Haplotype network depic
ting relationships between 
Cytochrome b
 sequences obtained for 417 ancient, historical, and 
modern Hawaiian petrels from
 all islands wh
ere the birds are currently kn
own, or have been known in the past, to breed. The siz
e of 
the circle is proportional to the hap
l
o
t
ype frequen
c
y, and dash
es indicate the presen
ce of m
u
ltip
le s
ubstitu
tion
s between hap
l
o
types.
 
    
 
128
 
Figure 3.
 Tem
poral trend in the effective populati
on size of the Hawaiian petrel. Upper plots 
show the Bayesian skyride of effective 
population sizes (thick black line is the 
m
e
dian posterior probability, thin black 
lines are the upper and 
lower 95% confidence
 
intervals) on Hawaii and Maui. Lower plots 
show num
ber of sam
ples and 95% confidence
 interval for their radiocarbon ages (ybp).
 
Shaded areas coincide with periods of co
lonization and expansion by humans. W
hite = pr
e-hum
an arrival, Light grey = Polynesian 
colonization and early population growth, Me
dium grey = Polynesian population expans
ion, Dark grey = European arrival and 
expansion.
 
   
 
 129 
Figure 4. BOTTLESIM simulations of change in the expected heterozygosity (H
e
) of the 
Hawaiian petrel as a result of a decline to a constant effective population size ranging 
from 10 to 1000 individuals at 150 years in the past. 
 
 
 
 130 
Figure 5. BOTTLESIM simulations of the impact of life history characteristics on the 
change in the expected heterozygosity (H
e
) as a result of a decline to a constant effective 
population size of 100 individuals at 150 years in the past. (A) The Hawaiian petrel 
compared to species with theoretical life histories (longevity, age at first reproduction); 
(B) The Hawaiian petrel as compared to species for which ancient DNA has been used to 
investigate temporal changes in genetic diversity or for species that may have been 
impacted by arrival of humans on the Hawaiian Islands.  
 
A. 
 
B. 
 
 131 
Figure 6. Relative contribution of modern and extirpated Hawaiian petrel populations to 
overall genetic diversity (GD) and allelic richness (R). Each of these has been further 
divided into diversity and differentiation subcomponents (see methods).  
 
 
 
 
 
 132 
APPENDIX A 
 
Museum specimen and voucher information 
 
 
Unless otherwise indicated all specimens are Hawaiian petrels. In some cases, multiple 
subfossil bones were assigned the same catalogue number, but only one element (e.g. left 
humerus) was sampled from each group to prevent duplicate sampling. The remaining 
subfossil bones have not been catalogued. Catalogue indicates the institution?s catalogue 
number, Year indicates year collected, and Type indicates type of specimen: S = study 
kin and B = bone/skeleton. 
 
Museum Catalogue Year Island Type Collector 
BPBM
1
 X150206 1976 Maui B Howarth et al. 
BPBM X157438 1979 Hawaii B Unknown 
BPBM X157513 1980 Kauai S Unknown 
BPBM 1992.166.1 1991 Hawaii B Giffen & Stone 
BPBM 1992.166.2 1992 Hawaii B Giffen & Stone 
BPBM 183557 1995 Kauai S Montgomery, S. 
BPBM
*
 184828 2005 At Sea S Unknown 
BPBM 4662 1907 Molokai S Bryan, W.A. 
BPBM 4663 1907 Molokai S Bryan, W.A. 
BPBM 4669 1907 Molokai S Bryan, W.A. 
BPBM 4671 1907 Molokai S Bryan, W.A. 
BPBM 4672 1907 Molokai S Bryan, W.A. 
BPBM 4673 1907 Molokai S Bryan, W.A. 
BPBM 4674 1907 Molokai S Bryan, W.A. 
BPBM 4675 1907 Molokai S Bryan, W.A. 
BPBM 4676 1907 Molokai S Bryan, W.A. 
BPBM 4677 1907 Molokai S Bryan, W.A. 
BPBM 4679 1907 Molokai S Bryan, W.A. 
BPBM 4680 1907 Molokai S Bryan, W.A. 
LACM
2
 20266 1914 Molokai S Bryan, W.A. 
LACM 20267 1914 Molokai S Bryan, W.A. 
LACM 20268 1914 Molokai S Bryan, W.A. 
LACM 20269 1914 Molokai S Bryan, W.A. 
LACM 20270 1914 Molokai S Bryan, W.A. 
LACM 20271 1914 Molokai S Bryan, W.A. 
 133 
LACM 20272 1914 Molokai S Bryan, W.A. 
LACM 20273 1914 Molokai S Bryan, W.A. 
LACM 20274 1914 Molokai S Bryan, W.A. 
LACM 20275 1914 Molokai S Bryan, W.A. 
LACM 20280 1914 Molokai S Bryan, W.A. 
LACM 20281 1914 Molokai S Bryan, W.A. 
LACM 20282 1914 Molokai S Bryan, W.A. 
LACM 20283 1914 Molokai S Bryan, W.A. 
LACM 20284 1914 Molokai S Bryan, W.A. 
LACM 20285 1914 Molokai S Bryan, W.A. 
USNM
3
 343310 1981 Oahu B Olson et al. 
USNM 391137 1980 Oahu B ARCH 
USNM 391317 1980 Oahu B Unknown 
USNM 391422 1980 Oahu B Unknown 
USNM 391726 1977 Oahu B Olson et al. 
USNM 392735 1980 Oahu B Unknown 
USNM 392780 1976 Oahu B Unspecified 
USNM 434261 1981 Oahu B Olson et al. 
USNM 434310 1981 Oahu B Olson et al. 
USNM 434349 1977 Oahu B Olson et al. 
USNM 434412 1977 Oahu B Olson et al. 
USNM 434417 1981 Oahu B Olson et al. 
USNM 434654 1981 Oahu B Olson et al. 
USNM 442732 1981 Oahu B Olson et al. 
USNM 442817 1981 Oahu B Olson et al. 
USNM 442916 1977 Oahu B Olson et al. 
USNM 442984 1977 Oahu B Olson et al. 
USNM 443027 1977 Oahu B Olson et al. 
USNM 443054 1977 Oahu B Olson et al. 
USNM 443178 1977 Oahu B Olson et al. 
USNM 447585 1981 Oahu B Olson et al. 
USNM 447815 1977 Oahu B Olson et al. 
USNM 447915 1977 Oahu B Olson et al. 
USNM 494137 1964 Maui S Unknown 
USNM 494138 1964 Maui S Unknown 
USNM 639494 2005 Kauai S Imberski, M. 
 134 
USNM 639495 2005 Kauai S Unknown 
USNM 639496 2006 Maui S Nohara, T. 
USNM 639497 2006 Kauai S Vercelli, J. 
USNM 639498 2002 Maui S Duvall, F. 
USNM 639500 2006 Kauai S Unknown 
USNM 639501 2006 Kauai S Vercelli, J. 
USNM 639502 1997 Kauai S Unknown 
USNM 639503 2005 Maui S Alexander, C. 
USNM 639504 2006 Maui S Medeiros, F. 
UWBM
4
 55576 1991 At Sea S Unknown 
UWBM 55605 1991 At Sea S Unknown 
*
Murphy?s petrel (Pterodroma ultima) 
1
Bernice P. Bishop Museum, Honolulu, HI 
2
Los Angeles County Museum of Natural History, Los Angeles, CA 
3
National Museum of Natural History, Smithsonian Institution, Washington, DC 
4
University of Washington Burke Museum of Natural History and Culture, Seattle, WA 
 135 
APPENDIX B 
 
Mitochondrial and nuclear DNA amplification and sequencing primer information 
 
Locus Primer Nucleotide sequence (5? ? 3?) 
Size 
(bp) 
Source 
Cytb  524  
 CytbL 
CCTCAAACATCTCTGCTTGAT 547 
a 
 CytbR9 
CGATACCGAGAGGGTTGT  
 
 aCytb1.1F 
CCCCTCAAACATCTCTGCTT 133 
a 
 aCytb1.1R 
AGCGACGGATGAGAAAGCTA  
 
 aCytb2F 
CGGCCTCCTACTAGCCATAC 121 
a 
 aCytb2R 
GGCTCCATTTGCATGTAGGT  
 
 aCybt3F 
CTCACACCTGCCGAAATGTA 108 
a 
 aCytb3R 
AGAATCCTCGTCCGATGTGT  
 
 aCytb4.1F 
CCTACACATCGGACGAGGAT 105 
a 
 aCytb4.1R 
CCTACGAAGGCAGTTGCTATG  
 
 aCytb5.2F 
CCCTCATAGCAACTGCCTTC 126 
a 
 aCytb5.2R 
CCCATTCTACGAGGGTTTGG  
 
 aCytb6F 
GAGGTGCTACAGTCATCACCAA 125 
a 
 aCytb6R 
GTGTAGGGCGAAGAATCGAG  
 
 aCytb7F 
CCCTACATTAACTCGATTCTTCG 121 
a 
 aCytb7R 
TCGATACCGAGAGGGTTGTT  
 
?-Enolase 
 
212  
 Intron 8 EnolL731 
TGGACTTCAAATCCCCCGATGATCCCAGC 270 
b 
 EnolH912 
CCAGGCACCCCAGTCTACCTGGTCAAA  
 
 aEnol1L 
GACTTCAAATCCCCCGATG 158 
a 
 aEnol1R 
ACRGAGGGAATGCACCTATC  
 
 aEnol2L 
AGGGAATCAGCACTGG 158 
a 
 aEnol2R 
AGGCACCCCAGTCTACCTG  
 
Lamin A  220  
 Intron 3 LamL724 
CCAAGAAGCAGCTGCAGGATGAGATGC 274 
b 
 LamH892 
CTGCCGCCCGTTGTCGATCTCCACCAG  
 
 aLam1L 
CGTTGTCGATCTCCACCAG 181 
a 
 aLam1R 
GAAGAACATTTACAGCGAGGTG  
 
 aLam2L 
GGGATGGGGACACAACAC 158 
a 
 aLam2R 
AGAAGCAGCTGCAGGATGAG  
 
Ribosomal Protein 
40 
 
309  
  Intron 5 RP40L 
GGGCCTGATGTGGTGGATGCTGGC 392 
c 
 RP40H 
GCTTTCTCAGCAGCAGCCTGCTC  
 
 aRP401L 
CTGGCTCGGGAGGTTCTG 180 
a 
 aRP401R 
CTGTCCTCAACAGCAAGCAC  
 
 aRP402L 
ATCTCCTGYTTCTGCTCCTG 221 
a 
 aRP402R 
TGCTCCTCCTTTTCGATCTG   
  
a
This study, 
b
Friesen et al. 1997, 
c
Friesen et al. 1999
 136 
APPENDIX C 
 
Supplementary tables and figures for Chapter I: 
 
Mitochondrial and nuclear DNA sequences reveal recent divergence in 
morphologically indistinguishable petrels 
 
 
Supplementary Table 1. Haplotypes, their frequencies, and the number of variable sites 
for the mitochondrial Cytochrome b gene in Galapagos and Hawaiian petrels 
 
Cytb Galapagos Hawaiian 
Haplotype petrels petrels 
1 0.06 - 
2 0.22 - 
3 0.28 - 
4 0.03 - 
5 0.03 - 
6 0.03 - 
7 0.03 - 
8 0.09 - 
9 0.03 - 
10 0.06 - 
11 0.03 - 
12 0.03 - 
13 0.03 - 
14 0.03 - 
15 - 0.07 
16 - 0.03 
17 - 0.07 
18 - 0.07 
19 - 0.07 
20 - 0.17 
21 - 0.03 
22 - 0.03 
23 - 0.03 
24 - 0.03 
25 - 0.17 
26 - 0.07 
27 - 0.03 
28 - 0.03 
29 - 0.03 
30 - 0.03 
# of variable sites 13 20 
 
 137 
Supplementary Table 2. Haplotypes, their frequencies, and the number of variable sites 
for the Enol nuclear intron locus in Galapagos and Hawaiian petrels 
 
Enol Galapagos Hawaiian 
Haplotype petrels petrels 
1 0.44 0.39 
2 0.44 - 
3 0.06 - 
4 0.02 - 
5 0.02 - 
6 0.03 0.04 
7 - 0.55 
8 - 0.02 
# of variable sites 6 3 
 138 
Supplementary Table 3. Haplotypes, their frequencies, and the number of variable sites 
for the Lam nuclear intron locus in Galapagos and Hawaiian petrels 
 
Lam Galapagos Hawaiian 
Haplotype petrels petrels 
1 0.06 - 
2 0.73 - 
3 0.02 - 
4 0.03 - 
5 0.03 - 
6 0.03 - 
7 0.02 - 
8 0.02 - 
9 0.02 - 
10 0.02 - 
11 0.03 0.11 
12 - 0.55 
13 - 0.02 
14 - 0.05 
15 - 0.09 
16 - 0.07 
17 - 0.02 
18 - 0.04 
19 - 0.02 
20 - 0.02 
21 - 0.02 
# of variable sites 12 8 
 
 139 
 
Supplementary Table 4. Haplotypes, their frequencies, and the number of variable sites 
for the RP40 nuclear intron locus in Galapagos and Hawaiian petrels 
 
RP40 Galapagos Hawaiian 
Haplotype petrels petrels 
1 0.12 - 
2 0.04 - 
3 0.05 0.07 
4 0.12 - 
5 0.05 - 
6 0.10 - 
7 0.19 0.45 
8 0.04 - 
9 0.06 - 
10 0.05 - 
11 0.03 0.04 
12 - 0.04 
13 0.03 0.33 
14 0.02 0.02 
15 0.03 0.02 
16 0.02 0.02 
17 0.03 0.02 
# of variable sites 7 6 
 
 140 
Supplementary Figure 1 Statistical parsimony haplotype network constructed in TCS 
(Clement et al. 2000) for the Enol nuclear intron data set. Circle size is proportional to 
haplotype frequency and color indicates the taxonomic origin for individuals possessing 
that sequence. The number of individuals is indicated for common haplotypes. Open 
circles indicate an inferred haplotype that was not sampled. HAPE = Hawaiian petrel, 
GAPE = Galapagos petrel, BPBM = Murphy?s petrel, ATP = Atlantic petrel, and SPP = 
soft-plumaged petrel. 
 
 
 
 
 
 141 
 
Supplementary Figure 2 Statistical parsimony haplotype network constructed in TCS 
(Clement et al. 2000) for the Lam nuclear intron data set. Circle size is proportional to 
haplotype frequency and color indicates the taxonomic origin for individuals possessing 
that sequence. The number of individuals is indicated for common haplotypes. Open 
circles indicate an inferred haplotype that was not sampled. HAPE = Hawaiian petrel, 
GAPE = Galapagos petrel, BPBM = Murphy?s petrel, ATP = Atlantic petrel, and SPP = 
soft-plumaged petrel.  
 
 
 
 
 142 
Supplementary Figure 3 Statistical parsimony haplotype network constructed in TCS 
(Clement et al. 2000) for the RP40 nuclear intron data set, with gaps treated as a fifth 
state. Circle size is proportional to haplotype frequency and color indicates the taxonomic 
origin for individuals possessing that sequence. The number of individuals is indicated 
for common haplotypes.  HAPE = Hawaiian petrel, GAPE = Galapagos petrel, BPBM = 
Murphy?s petrel, ATP = Atlantic petrel, and SPP = soft-plumaged petrel. With gaps 
treated as a fifth state, Atlantic and soft-plumaged petrel sequences did not join the 
network.  
 
 
 
 
 
 143 
APPENDIX D 
 
Supplementary tables and figures for Chapter III: 
 
Population divergence and gene flow in an endangered and highly mobile seabird 
 
 
Supplementary Table 1. Allele frequencies (%) for 15 microsatellite loci amplified in 
modern Hawaiian petrels. Continued on the next page. 
 
 
 
 144 
Supplementary Table 1 (cont.) 
 
 
 
 
 145 
Supplementary Table 2. Migration estimates from the mitochondrial data set obtained 
from the program MIGRATE. Values in bold are the estimated number of migrants per 
generation from the population on the left into the population at the top of the table. 95% 
confidences intervals are in parentheses below. 
 
 
 Hawaii Maui Lanai Kauai 
Hawaii - 
0.001 
(0.000-0.260) 
0.001 
(0.000-0.026) 
0.004 
(0.000-0.093) 
Maui 
0.003 
(0.000-0.685) 
- 
0.001 
(0.000-0.027) 
0.004 
(0.000-0.093) 
Lanai 
0.003 
(0.000-0.680) 
0.001 
(0.000-0.255) 
- 
0.004 
(0.000-0.095) 
Kauai 
0.003 
(0.000-0.675) 
0.001 
(0.000-0.253) 
0.001 
(0.000-0.027) 
- 
 
 
 
 
 
 
 
 
 
 146 
Supplementary Table 3. Migration estimates from the nuclear intron data set obtained 
from the program MIGRATE. Values in bold are the estimated number of migrants per 
generation from the population on the left into the population at the top of the table. 95% 
confidences intervals are in parentheses below. 
 
 
 Hawaii Maui Lanai Kauai 
Hawaii - 
0.001 
(0.000-0.002) 
0.004 
(0.003-0.006) 
0.001 
(0.000-0.002) 
Maui 
0.001 
(0.000-0.014) 
- 
0.001 
(0.000-0.002) 
0.001 
(0.000-0.002) 
Lanai 
0.001 
(0.000-0.011) 
0.001 
(0.000-0.002) 
- 
0.001 
(0.000-0.002) 
Kauai 
0.001 
(0.000-0.010) 
0.002 
(0.001-0.003) 
0.001 
(0.000-0.002) 
- 
 
 
 
 
 
 147 
Supplementary Table 4. Migration estimates from the microsatellite data set obtained 
from the program MIGRATE. Values in bold are the estimated number of migrants per 
generation from the population on the left into the population at the top of the table. 95% 
confidences intervals are in parentheses below. 
 
 
 Hawaii Maui Lanai Kauai 
Hawaii - 
0.013 
(0.000-3.763) 
0.001 
(0.000-0.011) 
0.715 
(0.000-214.6) 
Maui 
0.014 
(0.000-4.504) 
- 
0.001 
(0.000-0.014) 
7.870 
(0.000-327.7) 
Lanai 
0.014 
(0.000-4.056) 
0.013 
(0.000-3.687) 
- 
0.715 
(0.000-280.5) 
Kauai 
0.014 
(0.000-4.868) 
0.013 
(0.000-3.890) 
0.001 
(0.000-0.016) 
- 
 
 
 
 148 
APPENDIX E 
 
Supplementary tables and figures for Chapter IV:  
 
Ancient DNA reveals resilience despite the threat of extinction: three thousand 
years of population genetic history in the endemic Hawaiian petrel 
 
 
Supplementary Table 1. Minimum population growth rate required for Hawaiian petrels 
in the early 1900?s using various parameter values in the exponential growth model N
t 
= 
N
0
e
rt
. N is population size, t is time, and r is the growth rate. 
 
Parameter Varied N
0
 N
t
 t r = (ln (N
t
/N
0
))/t 
N
0
 50 11000 80 0.067 
 100 11000 80 0.059 
 200 11000 80 0.050 
 500 11000 80 0.039 
 1000 11000 80 0.030 
N
t
 100 11000 80 0.059 
 100 22500 80 0.068 
 100 34000 80 0.073 
 500 11000 80 0.039 
 500 22500 80 0.048 
 500 34000 80 0.053 
t 100 11000 80 0.059 
 100 11000 60 0.078 
 100 11000 40 0.118 
 500 11000 80 0.039 
 500 11000 60 0.052 
 500 11000 40 0.077 
 
 149 
Supplementary Table 2. Minimum census population sizes of Hawaiian petrels in the 
early 1900?s using various parameter values in the exponential growth model N
t 
= N
0
e
rt
. 
N is population size, t is time, and r is the growth rate. Estimates of r taken from Simons 
1984. 
 
Parameter Tested N
t
 t r N
0
 = N
t
/e
rt
 
r, N
t
 11000 80 0.008 5801 
 11000 80 0.005 7374 
 22500 80 0.008 11865 
 22500 80 0.005 15083 
 34000 80 0.008 17929 
 34000 80 0.005 22792 
r, t 11000 80 0.008 5801 
 11000 80 0.005 7374 
 11000 40 0.008 7988 
 11000 40 0.005 9006 
 34000 80 0.008 17929 
 34000 80 0.005 22792 
 34000 40 0.008 24690 
 34000 40 0.005 27837 
 150 
Supplementary Table 3. Life history parameter estimates used for simulation of the loss 
of genetic diversity after a decline to a constant effective population size of 100 
individuals at 150 years in the past. Simulations were conducted assuming 100% 
overlapping generations, that random mating occurred within each species, and that sex 
ratios were equal. Longevity of some species is not well known, therefore the age of the  
oldest known individual were used instead. 
 
 
Organism Scientific Name Longevity 
Age at First 
Reproduction 
African elephant Loxodonta africana 70 10 
Hawaiian petrel Pterodroma sandwichensis 36 6 
Musk ox Ovibos moschatus 20 3 
Bison Bison bison 15 3 
Nene Branta sandvicensis 28 3 
Palila Loxioides bailleui 13 2 
 
 151 
Supplementary Table 4. Information for all Hawaiian petrel bones that were 
radiocarbon dated for this study. Eleven bones from which DNA sequences were 
obtained were not radiocarbon dated: ten from Hawaii and one from Oahu. Location 
indicates cave where bone was discovered. 
 
 
 152 
Supplementary Table 4 (cont.) 
 
 
 
 153 
Supplementary Table 4 (cont.) 
 
 
 
 154 
Supplementary Table 4 (cont.) 
 
 
 155 
 
Supplementary Figure 1. Distribution of radiocarbon dates obtained for subfossil bones 
from the extirpated colony on Oahu that yielded ancient DNA sequences. 
 
 
 156 
Supplementary Figure 2. BOTTLESIM simulations of the change in the number of alleles 
in the Hawaiian petrel as a result of a decline to a constant effective population size 
ranging from 10 to 1000 individuals at 150 years in the past. 
 
 157 
Supplementary Figure 3. BOTTLESIM simulations of the impact of life history 
characteristics on the change in the number of alleles retained after a decline to a constant 
effective population size of 100 individuals at 150 years in the past. (A) The Hawaiian 
petrel compared to species with theoretical life histories (longevity, age at first 
reproduction); (B) The Hawaiian petrel as compared to species for which ancient DNA 
has been used to investigate temporal changes in genetic diversity or for species that may 
have been impacted by arrival of humans on the Hawaiian Islands.  
 
A.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
B. 
 
 
 
 
 
 
 
 158 
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