ABSTRACT 
 
 
 
 
Title of Document: PATTERNS OF SONG AND PREFERENCE 
VARIATION IN LAUPALA CERASINA AND 
THEIR EVOLUTIONARY IMPLICATIONS 
  
 Jaime Leigh Grace, Ph.D., 2010 
  
Directed By: Professor Kerry L. Shaw, Department of Biology 
 
 
Closely related species often differ conspicuously in secondary sexual characters, 
many of which may be shaped by sexual selection. Changes in these characters may 
directly influence speciation, since females use them to assess males and to choose a 
mate. I investigate the hypothesis that in closely related populations, divergence in 
preference for sexually selected characters may promote sexual isolation through 
assortative mating. Ultimately, if reproductive isolation is complete, this process of 
divergent sexual selection through preference divergence may result in the formation 
of new species. I have investigated the relationship between divergence in the pulse 
rate of male song and female preference for pulse rates in a group of Hawaiian 
crickets, Laupala cerasina. Within a population, males sing at a characteristic pulse 
rate, but males from different populations sing with significantly different pulse rates. 
The degree of population divergence is almost an order of magnitude less than that 
which distinguishes closely related species of Laupala. Using phonotaxis trials, I 
found that females show acoustic preferences for males whose pulse rates fall nearest 
  
to the population mean. Across populations, male pulse rates and female preferences 
are significantly correlated, suggesting that they are coevolving. I have demonstrated 
that females discriminate among songs whose pulse rates vary within the range of 
pulse rates found among neighboring populations. Furthermore, in choice trials, 
females prefer the songs of males from the same population to those from 
neighboring populations. These females also exhibit significant assortative mating 
based on their acoustic preferences. Divergence in female acoustic preference among 
populations thus provides a mechanism for generating and maintaining biodiversity 
through divergence in sexually selected characters used in mate choice. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
PATTERNS OF SONG AND PREFERENCE VARIATION IN LAUPALA 
CERASINA AND THEIR EVOLUTIONARY IMPLICATIONS 
 
 
 
By 
 
 
Jaime Leigh Grace Your Full Name as it appears in University Records. 
 
 
 
 
 
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 
2010 
 
 
 
 
 
 
 
 
 
 
Advisory Committee: 
Professor Kerry L. Shaw, Chair 
Professor Gerald S. Wilkinson 
Professor Gerald Borgia 
Professor David J. Hawthorne 
Professor David D. Yager 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
? Copyright by 
Jaime Leigh Grace 
2010 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ii 
 
Preface 
This dissertation contains three chapters. Chapters I, II, and III are presented in 
manuscript form, with abstract, introduction, methods, results, and discussion, 
followed by tables and figures. A single bibliography section occurs at the end for 
references cited throughout the dissertation. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 iii 
 
Acknowledgements 
I would like to thank my dissertation advisor, Kerry Shaw, for all of the support she 
has provided me throughout my time as her student. She has made me an immensely 
more mature and thoughtful scientist, and I appreciate her sharing her scientific 
worldview with me. Because of her, I think in terms of hypotheses, and refrain from 
leaping to conclusions that are not supported by the data. I would also like to thank 
my committee members for their support and help throughout the years: David 
Hawthorne, Gerry Borgia, Jerry Wilkinson, and David Yager. Their encouragement 
helped me to believe in the project and my ability to complete it. I would also like to 
thank Karen Carleton and Doug Gill for stimulating and helpful discussions of 
various aspects of my research. Patty Shields also provided encouragement and 
support. Lois Reid was fundamental in making sure everything happened as it should 
and was a miraculous problem solver from the first semester through the last, and 
countless times in between. 
 Completing a dissertation is an immense amount of work from conception, 
through data collection, analysis, and writing a cohesive product. Numerous people 
assisted at various times throughout the project. My time in the cricket room would 
have been quite dismal without the support I received from Jenna Jadin, Tagide 
deCarvalho, Dan Fergus, and Gina Conte, all of whom assisted with cricket care at 
some point in time. Additionally, Kerry Shaw, Sky Lesnick, Sean Mullen, Pat 
Danley, Tagide deCarvalho, and Dan Fergus collected and brought back from Hawaii 
many of the crickets that I used in my experiments at Maryland. Throughout the 
arduous time of writing both my proposal and my dissertation, a number of people 
 
 iv 
 
assisted with ideas and commented on numerous drafts and research presentations, 
including Tami Mendelson, Pat Danley, Dan Fergus, Tagide deCarvalho, Jenna Jadin, 
Sky Lesnick, Sean Mullen, Chris Wiley, Chris Ellison, Kevin Oh, Elizabeth Turnell, 
Sheila Reynolds, Jean-Fran?ois Savard, Jason Keagy, Linda Cendes, Julie Byrd, Joan 
West, Maria Murray, Gang Chen, Peter Thompson, Jennifer Siani, Paula Rodgers, 
James Sikes, and various other members of the BEES, BIOL, and ENTM graduate 
programs. In addition to their technical and intellectual assistance, they provided a 
sense of community and camaraderie throughout my time in College Park. Sky 
Lesnick made the basic topographic maps of Hawaii that I used in making my figures 
of population locations. I received statistical advice, including code for use with SAS 
and/or R from Jim Booth, Estelle Russek-Cohen, and Frank Siewerdt. Farzad Sanami 
and Wan Chan provided computer and technical support. Lastly, I would like to thank 
members of my family, particularly Steve and Carole Fenwick for their unconditional 
love, their emotional and occasional financial support, and for always believing in 
me. 
 This research was funded by an NSF Predoctoral Fellowship, University of 
Maryland Department of Biology Block Fellowship, HHMI Teaching and Learning 
Fellowship, Center for Comparative Evolutionary Biology of Hearing Fellowship, 
Department of Biology and Eugenie Clark summer research fellowships, Chesapeake 
Bay Fund, Ann G. Wylie Dissertation Improvement Award, and Jacob K. Goldhaber 
and College of Chemical and Life Sciences Conference Travel Awards. 
 
 v 
 
Table of Contents 
 
 
Preface........................................................................................................................... ii
 Acknowledgements...................................................................................................... iii
 Table of Contents.......................................................................................................... v
 List of Tables ............................................................................................................... vi
 List of Figures ............................................................................................................. vii
 Chapter I: Female preference covaries with male acoustic signal in the geographically 
widespread Hawaiian cricket, Laupala cerasina .......................................................... 1
 ABSTRACT.............................................................................................................. 1
 INTRODUCTION .................................................................................................... 2
 METHODS ............................................................................................................... 5
 RESULTS ............................................................................................................... 11
 DISCUSSION......................................................................................................... 17
 TABLES ................................................................................................................. 26
 FIGURE LEGENDS............................................................................................... 30
 FIGURES................................................................................................................ 32
 Chapter II: Rapid evolution of divergent female acoustic preferences facilitates sexual 
isolation in a Hawaiian cricket, Laupala cerasina...................................................... 40
 ABSTRACT............................................................................................................ 40
 INTRODUCTION .................................................................................................. 41
 METHODS ............................................................................................................. 44
 RESULTS ............................................................................................................... 49
 DISCUSSION......................................................................................................... 50
 TABLES ................................................................................................................. 56
 FIGURE LEGENDS............................................................................................... 58
 FIGURES................................................................................................................ 60
 CHAPTER III: Divergent female acoustic preference facilitates sexual isolation 
through assortative mating in the Hawaiian cricket Laupala ..................................... 66
 ABSTRACT............................................................................................................ 66
 INTRODUCTION .................................................................................................. 67
 METHODS ............................................................................................................. 71
 RESULTS ............................................................................................................... 77
 DISCUSSION......................................................................................................... 80
 TABLES ................................................................................................................. 85
 FIGURE LEGENDS............................................................................................... 87
 FIGURES................................................................................................................ 89
 Bibliography ............................................................................................................... 93
  
 
 
 
 
 
 vi 
 
List of Tables 
 
CHAPTER I 
 
Table 1. Geographic variation in male song characters, including pulse rate, pulse 
duration, and carrier frequency (page 26). 
 
Table 2. Correlations among male song characters and with temperature (page 27). 
 
Table 3. Geographic variation in female preference for pulse rate (page 28). 
 
Table 4. Wild-caught and G2 comparisons of song and preference (page 29). 
 
CHAPTER II 
 
Table 1. Population variation in male song characters (page 56). 
 
Table 2. Population variation in female preference for pulse rate (page 57). 
 
CHAPTER III 
 
Table 1. Average pulse rate, pulse duration, and carrier frequency values for male 
songs used in phonotaxis trials (page 85). 
 
Table 2. Comparison of male song characters between contrasted populations/species 
(page 86). 
 
 vii 
 
List of Figures 
CHAPTER I 
 
Figure 1. Topographic map of the Big Island of Hawaii showing collecting locations 
of 13 populations of L. cerasina and their approximate coordinates (page 32). 
 
Figure 2. Geographic variation in male pulse rate, by year (page 33). 
 
Figure 3. Geographic variation in male carrier frequency, by year (page 34). 
 
Figure 4. Geographic variation in male pulse duration, by year (page 35). 
 
Figure 5. Comparison of male pulse rate and female pulse rate preference across 
generations (page 36). 
 
Figure 6. Geographic variation in female pulse rate preference, by year (page 37). 
 
Figure 7. Correlation between female pulse rate preference and male pulse rate, by 
year (page 38). 
 
Figure 8. Covariation between female pulse rate preference and male pulse rate across 
all 13 populations of L. cerasina (page 39). 
 
CHAPTER II 
 
Figure 1. Topographic map of the Big Island of Hawaii showing collecting locations 
and pairwise planned comparisons of eight populations of L. cerasina (page 60). 
 
Figure 2. Schematic of arena used in phonotaxis trials (page 61). 
 
Figure 3. Geographic variation in male song characters (page 62). 
 
Figure 4. Geographic variation in female phonotaxis responses (page 64). 
 
Figure 5. Geographic variation in mean female pulse rate preference (page 65). 
 
CHAPTER III 
 
Figure 1. Map of Hawaii showing the six populations of L. cerasina and one 
population of L. eukolea used for intraspecific and interspecific mate choice trials 
(page 89). 
 
 
 viii 
 
Figure 2. Schematic of the experimental setup, illustrating the phonotaxis arena   
(page 90). 
 
Figure 3. Female phonotaxis preference in intraspecific and interspecific choice trials 
(page 91). 
 
Figure 4. Mating success in homotypic and heterotypic mating trials (page 92). 
 
 
 1 
 
Chapter I: Female preference covaries with male acoustic 
signal in the geographically widespread Hawaiian cricket, 
Laupala cerasina 
ABSTRACT 
 
Sexual selection is a powerful evolutionary force shaping phenotypes involved in 
mate choice, and can cause phenotypic shifts resulting in (or reinforcing) 
population divergence and speciation when such shifts reduce mating 
probabilities among divergent populations. In the Hawaiian cricket genus 
Laupala, pulse rate of male calling song is a conspicuous mating signal that 
differs among species, and may behave as a speciation phenotype. Females 
prefer pulse rates that roughly correspond to the male population mean, and 
signal and receiver values appear to be coupled across species. Populations of L. 
cerasina show subtle but significant differences in pulse rate across the species? 
range. This study documents the degree of differentiation among populations in 
three features of calling song: pulse rate, pulse duration, and carrier frequency. 
All three show significant population differentiation, with pulse rate showing the 
greatest heterogeneity; however, a Mantel test found no relationship between 
geographic distance and pulse rate divergence, indicating a simple model of 
greater divergence with increasing distance is insufficient to explain the 
observed pattern of differentiation. Female preference functions for pulse rate 
also show significant differentiation among populations. Furthermore, the 
 
 2 
 
estimates of pulse rate preference are highly correlated with mean pulse rates 
across populations. This pattern of correlated divergence between sender and 
receiver suggests that sexual selection on song actively maintains population 
differentiation, and is consistent with the hypothesis that sexual selection may 
have promoted speciation in this genus. The correlated divergence in a sexually 
selected trait and preference among populations has the potential to reduce gene 
flow among divergent populations that are sexually isolated, which may 
ultimately result in speciation. 
 
INTRODUCTION 
Many closely related species differ conspicuously in secondary sexual signals and 
preferences of the mate recognition system (Darwin 1871; West-Eberhard 1983; 
Ritchie 2007). The processes by which these patterns arise are of intense interest 
because of their direct bearing on the origins of species. While the processes causing 
the evolution of such phenotypes occur within populations, the consequential 
divergence among populations can have significant repercussions for communication 
between populations (Lande 1981; Endler 1992). Evolved mating signals within a 
population may result in sexual isolation from other populations whose mating 
signals have remained the same or evolved differently (Kirkpatrick 1982; Lande 
1982). If signals and preferences covary among populations, assortative mating could 
result in reduced mating probabilities and depressed gene flow between populations 
(Panhuis et al. 2001; Uyeda et al. 2009). 
 
 3 
 
 While interspecific differences in mate recognition phenotypes appear to 
make critical contributions to reproductive isolation among species, determining the 
origins and evolutionary dynamics of these differences must involve the study of 
intraspecific variation in signals and preferences (H?rdling and Karlsson 2009; Uy et 
al. 2009). The functional interdependence of signal and preference suggests that a 
preference function (often expressed by females) can describe the selective pressure 
on signals (often expressed by males) due to female choice (Wagner 1998). The 
degree of overlap between signal and preference variation is an important factor in 
predicting the evolutionary potential and trajectory of signal-preference evolution 
(Ritchie 1996). For example, it is often hypothesized that a mate recognition system 
with strong stabilizing selection will tend toward extensive trait-preference matching 
within and covariance across populations (Butlin and Ritchie 1989). In addition, the 
genetic architecture of these traits can constrain, or promote, the tempo and 
coevolutionary precision of trait and preference that ultimately leads to interspecific 
differences. For example, theory suggests that details of the genetic architecture of 
diverging sexual traits can influence the likelihood and rate of speciation (Dieckmann 
and Doebeli 1999; Kondrashov and Kondrashov 1999; Verzijden et al. 2005; Hall and 
Kirkpatrick 2006; Hayashi et al. 2007). 
 Elucidation of the potential pathways by which interspecific differences in 
mate recognition evolve requires systems that exhibit intraspecific variation in these 
same traits. While numerous studies are beginning to examine geographic variation in 
mating signals and preferences (Simmons et al. 2001; Simmons 2004; Prohl et al. 
2006; Prohl et al. 2007; Reynolds and Fitzpatrick 2007; Ryan et al. 2007) particularly 
 
 4 
 
valuable would be studies that simultaneously document 1) variation in both signals 
and preferences for those signals among populations, 2) a genetic basis to covariation 
between trait and preference, and 3) interspecific variation in these same traits, 
enabling mechanistic and evolutionary studies of different stages of the speciation 
process (Panhuis et al. 2001; Boake 2002). Intraspecific variation in such systems can 
yield temporally fine-grained observations of trait differentiation, promising insights 
into 1) how interspecific differences accumulate from intraspecific differentiation and 
2) the relationship between female preference functions and the degree to which 
signal and preference co-evolve or become decoupled during divergence. 
 The Hawaiian cricket genus Laupala has undergone rapid speciation 
(Mendelson and Shaw 2005), resulting in 38 closely related species that differ 
conspicuously in acoustic aspects of sexual communication, but that are otherwise 
cryptic (Otte 1994). Males of the different species of Laupala produce structurally 
similar songs that vary primarily in pulse rate, and species occurring in sympatry can 
always be distinguished by pulse rate (Shaw 1999; Parsons and Shaw 2001; 
Mendelson and Shaw 2002; Shaw 2002; Mendelson and Shaw 2005). Furthermore, 
females preferentially respond to the pulse rate produced by males from their own 
species (Shaw 2000b; Shaw and Herlihy 2000; Mendelson and Shaw 2002). Genetic 
studies of species differences in Laupala show that both pulse rate and preference 
variation is due to relatively small, additive allelic effects (Shaw et al. 2007; Shaw 
and Lesnick 2009). Furthermore, loci underlying both song and preference 
differences between species show genomic linkage (Shaw and Lesnick 2009; Wiley 
and Shaw 2010). These observations between species suggest the hypothesis that 
 
 5 
 
divergent evolution of song and preference within species occurs through small, 
coordinated changes in signal and preference. 
 We examine this hypothesis in a study of Laupala cerasina, a widely 
distributed species endemic to the Big Island of Hawaii. Field recordings have 
suggested extensive pulse rate differentiation across the range of L. cerasina (Otte 
1994), indicating that L. cerasina populations have diverged in the same character 
that varies conspicuously among species. The current study provides statistical 
support for the extensive pulse rate variation in L. cerasina and tests the hypothesis 
that male sexual signals and female preferences for those signals covary across the 
range of the species. The hypothesis that differences among populations in male song 
and female preference have a genetic basis is tested using a common garden 
experiment. The results are discussed with respect to the mode of change in both male 
and female sides of the acoustic communication system and in the context of 
geographic structure among populations. 
 
METHODS 
Cricket subjects and rearing conditions 
Subadult L. cerasina crickets were collected from thirteen locations (Pololu Valley, 
Muliwai Plateau, Waimea Reservoir, Kalopa Park, Laupahoehoe, Akaka Falls, 
Eucalyptus Toe, Kaiwiki, Ola?a Flume, Glenwood, Wright Road, Naulu Trail, and 
Kaiholena), hereafter referred to as populations, from the Big Island of Hawaii in July 
2004, 2005, and 2006 (Figure 1). In the laboratory at the University of Maryland, 
individuals were reared to maturity under a 12:12 light: dark cycle in a temperature-
 
 6 
 
controlled environment maintained at 20?C, reflecting field conditions. Two to three 
wild caught individuals of the same sex were reared together in medical specimen 
cups, with a moistened kimwipe for water and humidity. Fresh cricket chow (Fluker?s 
Farms, LA, USA) was provided twice per week (2004, part of 2005) or cricket chow 
treated with the anti-fungal agent methyl paraben (Tegosept, Fisher Scientific) once 
per week (part of 2005, 2006). First laboratory generation crickets (G1) were collected 
as eggs from naturally inseminated females caught in the wild or paired with a wild 
caught male in the laboratory. Second laboratory generation crickets (G2) were 
generated through random pairing of non-sibling males and females from the same 
population. 
 
Male song measurements 
Single mature males were recorded with a Sony Professional Walkman (model WM-
 D6) and Sony condenser microphone from a clear plastic cup fitted with a screen top 
in a temperature-controlled room maintained at 20?C. A female from the same 
population was placed with males that failed to sing spontaneously (males produce 
only one type of song in Laupala). Songs were digitized and analyzed using Raven 
version 1.2.1 (Bioacoustics Research Program, Cornell University Lab of 
Ornithology). From each song, five non-consecutive measurements were taken of 
pulse period, pulse duration, and carrier frequency. Pulse period, measured as the 
time between the beginning of one pulse and the beginning of the next pulse, was 
transformed to calculate the pulse rate (the inverse of the pulse period). Pulse duration 
 
 7 
 
is the time elapsed between the beginning and end of a single pulse. Finally, carrier 
frequency is calculated as the dominant frequency of the pulse. 
 Male song data were analyzed using SAS version 8.2 for Windows (SAS 
Institute, Cary, NC). The five measurements of pulse rate, pulse duration, and carrier 
frequency were averaged for each male to get a single measurement per individual. 
The coefficient of variation (CV) was calculated for each male by dividing the 
standard deviation by the mean and multiplying by 100 to get a percent. A low CV 
suggests that using mean values from individuals is appropriate for subsequent 
analysis. An analysis of variance was performed to test the hypothesis that song 
variables show significant heterogeneity among populations. Additionally, a 
correlation analysis was performed using pulse rate, pulse duration, carrier frequency, 
and temperature to test for independence of song characters. Each song value was 
adjusted to account for population differences by subtracting the value for each trait 
from the population mean for each individual, and using those values (the residuals) 
in the analysis. 
 To determine whether geographic variation in song has a genetic component, 
individuals from the 2005 collections at Glenwood Road, Kalopa Park, Laupahoehoe, 
Muliwai Plateau, Naulu Trail, and Waimea Reservoir were subjected to a common-
 garden study in the laboratory for two generations. Then the songs of the resulting 
progeny of outbred pairs were recorded under the same experimental conditions as 
the wild-caught generation. Measurements of pulse rate, pulse duration, and carrier 
frequency were conducted in the G2 males using the same procedures as in the wild-
 caught males, and the results were compared across generations. The mean song 
 
 8 
 
values measured from the G2 progeny were used in a correlation analysis with the 
means from the parental (wild-caught) generation. 
To test for a relationship between geographic distance and pulse rate 
divergence, a Mantel test was performed using XLSTAT in Microsoft Excel. 
Geographic distances were calculated using the ruler tool in Google Earth, with 
collecting locations estimated within one second of the recorded coordinates listed in 
Figure 1. Song divergence was measured as the absolute value of the difference 
between the mean male pulse rates for each population pair. 
 
Female acoustic preference measurements 
Phonotaxis methods were identical to those in Shaw and Herlihy (2000). Briefly, a 
circular test arena (47 cm radius) covered in fiberglass screen was housed in a 
temperature-controlled acoustic chamber (Acoustic Systems) for phonotaxis trials. 
Two 8.5 cm speakers (Radio Shack model 40-1218) placed 180? apart just outside the 
arena broadcast synthesized songs offered in pairs from a PC computer using custom 
software. From each speaker, a pulsed, sinusoidal tone was played back through a 16-
 bit digital/analogue converter (Tucker-Davis Technologies, Gainesville, FL). The 
pulse amplitude envelope had a rise time of 10 ms and a fall time of 30 ms and the 
acoustic output was filtered at 10 kHz using a Krohn-Hite filter (model 3322) to avoid 
aliasing. Sound pressure levels were equalized to 90 dB on a 4.0-pulses per second 
(pps) tone monitored with a Bruel and Kjaer sound pressure level meter (type 2230, 
fast-root-mean-square setting). 
 
 9 
 
 To conduct a trial, a female was placed beneath a plastic cup in the center of 
the arena. Pulse duration and carrier frequency were held constant at 40 ms and 5.0 
kHz, respectively, while pulse rate varied depending on the trial. Each female was 
used in a series of six different trials (2.0 versus 2.5 pulses s-1, 2.2 versus 2.7 pulses  
s-1, 2.4 versus 2.9 pulses s-1, 2.6 versus 3.1 pulses s-1, 2.8 versus 3.3 pulses s-1, and 3.0 
versus 3.5 pulses s-1) in randomized order, two per day, over the course of three days. 
These pulse rate values more than span the natural range of variation observed in 
male calling song across L. cerasina populations (Otte 1994). Paired songs were 
broadcast for five minutes before the cup was remotely raised, allowing the female 
access to the arena. If the female approached within 10 cm of one of the speakers 
within the trial period, the pulse rate choice was recorded and the trial ended. If the 
female did not approach either speaker after five minutes, the trial was ended and no 
choice was recorded. Only sexually mature, virgin females 20 days past the final molt 
were used in trials. In each trial, the speakers were randomly assigned a pulse rate. 
 In general, females responded to the faster pulse rate at the slow end of the 
trial series and to the slower pulse rate at the fast end of the trial series. We conducted 
an analysis to estimate the pulse rate corresponding to the inflection point, or point of 
indifference, where the proportion of females responding to the faster (or slower) 
pulse rate equaled 0.5 in a given population. Responses from a given population were 
analyzed simultaneously using logistic regression with the statistical software 
package R. In order to determine a standard error for these estimated mean 
preferences, the preference data sets (preference functions) of females within a given 
population were sampled with replacement to generate 1000 bootstrap replicates per 
 
 10 
 
population, maintaining the original sample size for each replicate. For each bootstrap 
replicate, the pulse rate corresponding to the inflection point was calculated, 
providing a pulse rate preference estimate for that replicate sample. The mean 
preference estimate and its standard deviation (i.e. standard error) were calculated for 
each of the 1000 bootstrapped replicates. 
 To determine whether there is a genetic component to geographic variation in 
female acoustic preference, individuals from the 2005 collections at Glenwood Road 
and Naulu Trail were reared in a common garden study for two generations (as with 
males; see above). The resulting G2 progeny were used in the same experimental 
design as the wild-caught individuals. Logistic regressions of phonotaxis response 
were compared across generations within populations (to test whether population 
preferences change across generations), and between populations within generations 
(to test whether differences persist across generations between populations) using 
Wald chi-square tests (Grace and Shaw 2004). 
 
Song-preference comparisons 
To test for population-level correlations between song and preference, a Spearman 
rank correlation test was performed using the mean pulse rate and preference 
estimates for each population for each year separately. We also performed a global 
correlation by combining all of the data for each population and performing a single 
correlational analysis across all 13 populations. 
 For the two populations (Glenwood Road and Naulu Trail) where song and 
preference data were available for wild-caught and G2 generations, we tested the 
 
 11 
 
hypothesis of no difference between pulse rate and pulse rate preference between 
populations in each generation. 
 
RESULTS 
Male song variation 
 
Wild-caught generation: Temperature varied little throughout the course of the 
experiment (average recording temperature = 20.09 ? 0.17), and as expected, there 
was no relationship between temperature and any of the measured song characters 
(Pearson r = -0.01 to -0.05, p = 0.29-0.84). Analyses were conducted separately for 
each collection year, and results were compiled separately. In total, songs were 
recorded from 328 males. Pulse rates were significantly heterogeneous among 
populations of L. cerasina in all years sampled (2004: F(5,103)=24.07, p<0.0001; 2005: 
F(6,101)=42.97, p<0.0001; 2006: F(6,104)=32.13, p<0.0001). In 2004, 7 out of 15 
pairwise comparisons were significantly different (p<0.05) following Sidak 
correction for multiple comparisons; in 2005, 16 out of 21 pairwise comparisons were 
significantly different; and in 2006, 11 out of 21 were significantly different (Figure 
2; Table 1). Carrier frequency was also significantly heterogeneous among 
populations in all years (2004: F(5,103)=7.02, p<0.0001; 2005: F(6,101)=3.62, p=0.0027; 
2006: F(6,104)=4.27, p=0.0007), but with fewer significant pairwise comparisons (5 out 
of 15 in 2004, 1 out of 21 in 2005, and 2 out of 21 in 2006; Figure 3; Table 1). Pulse 
duration was significantly heterogeneous among populations in 2004 (F(5,103)=6.12, 
p<0.0001), but not in 2005 or 2006 (2005: F(6,101)=1.84, p=0.099; 2006: F(6,104)=0.37, 
p=0.89). In 2004, 4 out of 15 pairwise comparisons were significantly different 
 
 12 
 
(Figure 4; Table 1). The pulse rates for Glenwood Road males sampled in 2004 were 
significantly slower than males sampled in 2005, as was true for Wright Road in these 
same years (Table 1; Figure 2). Wright Road males also had a significantly lower 
carrier frequency in 2004 than 2005 (Table 1; Figure 3). No other year effects were 
found within populations. A correlation analysis of pulse rate, pulse duration, carrier 
frequency and recording temperature found one significant negative correlation 
between carrier frequency and pulse duration, with all other relationships non-
 significant (Table 2). There was no support for a relationship between geographic 
distance and pulse rate divergence across populations (Mantel test: 50,000 
permutations; Pearson r = -0.168, p = 0.346). 
 Laboratory generation: Songs were analyzed from 284 males in the G2 
generation. The six populations bred for two generations in the lab (G2 males) were 
significantly heterogeneous for pulse rate (F(5,278)=67.84, p<0.0001) and carrier 
frequency (F(5,278)=3.67, p=0.0031), but not pulse duration F(5,278)=1.69, p=0.136). 
Fewer pairwise comparisons were significant for pulse rate (9 out of 15), but all of the 
comparisons that were significant in the G2 generation were also significant in the 
parental generation. For carrier frequency, 2 out of 15 pairwise comparisons were 
significant, including the pair that was different in the parental generation. Population 
means derived from wild-caught and G2 males were significantly correlated across 
the six populations in pulse rate (Pearson r=0.972, p=0.0012) and carrier frequency 
(Pearson r=0.899, p=0.0149), but not pulse duration (Pearson r=0.699, p=0.51). 
 The pulse rate means of wild-caught males from Glenwood Road and Naulu 
Trail collected in 2005 were among those that differed significantly (t(29)=6.19, 
 
 13 
 
p<0.0001). While we found no significant differences in pulse rate between the 
parental and G2 generations within each population (Glenwood t(56)=-0.93, p=0.36; 
Naulu t(51)=-0.66, p=0.51; Figure 5), the pulse rates of G2 songs did differ between the 
two populations (t(78)=10.71, p<0.0001; Figure 5). From the analysis of wild-caught 
males, the two populations also differed in carrier frequency (t(29)=-2.12, p=0.043), 
but not pulse duration (t(29)=-0.30, p=0.77). However, the difference in carrier 
frequency did not persist across generations between these populations (G2 male 
analysis: t(78)=-1.30, p=0.20). 
 
Female preference variation 
 
Wild-caught generation: In total, 269 wild-caught females were run in preference 
trials. Typically, females responded to the faster song of the pair at the slow end of 
the trial range, and to the slower song at the fast end of the trial range. Population 
means and standard errors within populations were estimated using the bootstrap 
analysis described above. After applying logistic regression to the bootstrap 
replicates, it became apparent that estimates of pulse rate preference scaled with pulse 
rate means from their respective populations (see below). However, preference 
estimates from slower singing populations were associated with substantially larger 
95% confidence intervals when based on all six acoustic choice trials. This likely 
occurred because the trials at the fast end of the range were beyond the normal 
discrimination range of females from slower populations. To improve estimates, the 
means and standard errors were recalculated using the first four trials only, the first 
five trials only, and all six trials, for those populations where male pulse rates were 
 
 14 
 
less than 2.5 pulses per second (pps), between 2.5 and 2.8 pps, and over 2.8 pps, 
respectively. Of the 17 groups that had recalculated estimates, 14 showed a reduced 
standard error. With this systematic adjustment, standard errors dropped by 35% and 
were similar across populations (Table 3). Population preference means were 
considered significantly different when the mean of one population was excluded 
from the 95% confidence interval (=1.96SE) of a second population. Note that this 
allows for three categories of relationship: when the means of both populations are 
within the other?s confidence intervals (NS), when one population?s mean is excluded 
from the other?s confidence interval, but the other mean is included within the 
confidence interval of the first (1-way), and when both means are reciprocally 
excluded from each other?s confidence intervals (2-way). 
 Populations were analyzed separately by year to mirror the analysis of song 
variation, although no two-way significant year effects were detected in female 
preference estimates (see below). As with song, statistically significant differences in 
pulse rate preference among populations were detected in all three years of the study. 
In 2004, 9 out of 15 pairwise comparisons were significantly different, with 2 
comparisons showing one-way significance and 7 comparisons showing two-way 
significance (Figure 6). In 2005, 15 out of 21 pairwise comparisons were significantly 
different, with 5 comparisons showing one-way significance and 10 comparisons 
showing two-way significance (Figure 6). In 2006, 16 out of 21 pairwise comparisons 
were significantly different, with 2 comparisons showing one-way significance and 
14 comparisons showing two-way significance (Figure 6).  There was one significant 
year effect on preference: at Ola?a Flume, our analysis revealed a one-way significant 
 
 15 
 
difference between 2004 and 2006, with the 2004 estimate (2.61) falling just outside 
the 95% confidence interval of the 2006 estimate (2.62-2.76). 
 Laboratory generation: From the G2 generation (derived from the 2005 wild-
 caught generation), a total of 16 females from Glenwood Road and 16 females from 
Naulu Trail were run in preference trials. Consistent with the hypothesis that pulse 
rate preferences breed true and thus have a genetic basis, a Wald chi-square test for 
logistic regression showed no significant difference between the parental and G2 
generations within the Naulu Trail or Glenwood Road populations, regardless of 
whether trials 1-4, 1-5, or all trials 1-6 were considered (Trials 1-4: Glenwood: Wald 
?2 (1)=1.63, p=0.20; Naulu: Wald ?
 2 
(1)=0.10, p=0.75). Analyses using 1-4 trial data 
revealed significant differences between Glenwood and Naulu populations in both the 
wild-caught and G2 generations (wild-caught: Wald ?
 2 
(1)=5.38, p=0.0204; G2: Wald 
?2 (1)=5.79, p=0.0161; Figure 5). Consistent with our observation that the fifth and 
sixth phonotaxis trials inflated the standard error estimates for populations with slow 
songs and preferences (like Naulu), we found no significant differences between these 
two populations when data from the fifth trial or all six trials were included. 
 The bootstrap method for estimating preference and 95% confidence intervals 
(above) revealed a two-way significant difference between Naulu and Glenwood in 
the wild-caught generation (Table 4). When applied to the G2 generation, the analysis 
resulted in a one-way significant difference, with the Naulu estimated preference 
mean falling outside the 95% confidence interval of Glenwood, but the Glenwood 
estimated preference mean falling just inside the Naulu 95% confidence region (Table 
4). This was true when data from trials 1-4 and 1-5 were used for Naulu and 
 
 16 
 
Glenwood, respectively (consistent with our inclusion criteria outlined above) or 
when only trials 1-4 were used for both populations. 
 The 95% binomial confidence intervals were also calculated for wild-caught 
and G2 generations for each pulse rate trial separately. Females from Naulu and 
Glenwood in both generations responded significantly differently only in trial 2 (trial 
midpoint pulse rate = 2.45 pps), corresponding to the pulse rate range (2.2 versus 2.7) 
that distinguishes males of these two populations (Figure 5). Collectively, these 
results suggest that the observed differences in phonotaxis preference among 
populations persist across generations, demonstrating a genetic basis to population 
variation. 
 
Song-preference covariation 
For 2005 and 2006, pulse rate and preference were significantly correlated (2005: n=7 
populations, Spearman r=0.964, p=0.0005; 2006: n=7 populations, Spearman 
r=0.929, p=0.0025; Figure 7).  Although there was a qualitatively similar trend, there 
was no significant relationship between song and preference in 2004 (n=6, Spearman 
r=0.148, p=0.67; Figure 7). A global analysis combining data across years for each 
population found a significant correlation between the mean estimated female 
preference and the mean male pulse rate across all thirteen populations (n=13, 
Spearman r=0.934, p<0.0001; Figure 8).  Due to significant year effects, only 2004 
data (yielding the largest sample) were used from Glenwood Road and Wright Road 
populations in the global analysis. 
 
 
 17 
 
DISCUSSION 
Recent, rapid radiations present ideal opportunities to study species that have 
diverged in relatively few characters, facilitating focus on evolutionary processes 
relevant to speciation. If such radiations harbor species that exhibit intraspecific 
differentiation as well, this provides the opportunity to examine the multiple temporal 
stages of differentiation that Mayr (1963) viewed as essential to understanding the 
mechanisms of speciation. When the divergent characters in question are involved in 
sexual communication and mate recognition, the populations are predisposed toward 
the evolution of sexual isolation, likely the earliest form of reproductive isolation to 
evolve in many taxa (Lande 1981; West-Eberhard 1983). In the genus Laupala, the 
pulse rate of the male calling song is a conspicuous character that varies among 
otherwise cryptic species, suggesting that this trait may be a speciation phenotype, 
i.e., a trait whose divergence reduces gene flow among incipient species (Shaw 2001; 
Shaw and Danley 2003). If this is true, then populations of L. cerasina that have 
diverged in pulse rate may be incipient species, provided that pulse rate preference 
has evolved in a correlated fashion with pulse rate. 
 
Male song variation 
We found that the mean pulse rates across the range of L. cerasina are heterogeneous 
to a statistically significant degree. Whereas Otte (1994) presented qualitatively 
similar results from field-recorded L. cerasina, the variation demonstrated here is 
observed among populations of wild-caught crickets brought to the laboratory where 
temperature conditions were monitored and controlled. Because temporal components 
 
 18 
 
of song in orthopterans are notoriously sensitive to temperature variation (Gerhardt 
and Huber 2002; Grace and Shaw 2004), any demonstration that pulse rates are 
statistically different among populations requires measurement at constant 
temperatures. Two populations, Glenwood Road and Wright Road, showed a 
significant year effect on pulse rate between 2004 and 2005. Because pulse rate can 
be measured very precisely, it is possible that these differences are due to subtle 
temperature differences at the field sites prior to their collection. The environmental 
temperature experienced during development can have a lasting effect on adult song 
phenotypes (Grace and Shaw 2004), so any temperature differences experienced at 
those locations between 2004 and 2005 could have contributed to the differences in 
pulse rate observed under laboratory conditions.  
 Following two generations of laboratory rearing (G2 generation), we showed 
that the pulse rate heterogeneity among populations persisted, and that the pulse rate 
means of wild-caught and G2 lab-reared males were highly correlated. The mean 
pulse rate difference between wild-caught males from Naulu Trail and Glenwood 
Road, the two populations examined for preference variation in the G2 generation, 
were among those that remained significantly different in the G2 generation.  These 
second lab generation observations allow us to rule out the possibility that a maternal 
by environment effects account for the difference between populations.  Instead, these 
findings implicate a genetic contribution to the pulse rate variation among natural 
populations of L. cerasina. 
 To a lesser degree, significant variation was observed in carrier frequency and 
pulse duration among wild-caught L. cerasina populations, although only carrier 
 
 19 
 
frequency differences remained significantly heterogeneous in the G2 generation. 
However, fewer pairwise significant differences in carrier frequency were found 
among populations, and the significant difference in carrier frequency between Naulu 
and Glenwood wild-caught males did not breed true in the G2 generation. Thus, male 
condition, natal environment and/or maternal effects may play a larger role in both 
pulse duration and carrier frequency variation. In a previous study, Shaw and Herlihy 
(2000) found that pulse rate and carrier frequency showed relatively low levels of 
intra-male variability, whereas pulse duration exhibited relatively high intra-male 
variability. Nevertheless, a negative correlation was found between carrier frequency 
and pulse duration, suggesting a constraint to the independent evolution of each.  
Previous work showed that females express unimodal preference functions for carrier 
frequency but open-ended preferences for pulse duration (Shaw and Herlihy 2000). 
These patterns match those found in frogs (Gerhardt 1991; Gerhardt and Huber 2002) 
and are consistent with the suggestion that variation is influenced by environment or 
male condition (although this hypothesis was not explicitly tested).  Both low carrier 
frequency and long pulse duration have been associated with large body size in other 
crickets (Brown et al. 1996; Simmons and Ritchie 1996). 
 
Female preference variation 
The phonotaxis data indicate that females across the range of L. cerasina express 
unimodal preference functions for pulse rate, but females differed by population as to 
which pulse rates they are most attracted. Unlike the pulse rate estimates for male 
song, there was no effect of year on estimates of female pulse rate preference. In 
 
 20 
 
general, variability in the standard errors associated with mean pulse rate preference 
estimates were higher than for mean pulse rates, likely due to either the nature of the 
measurement or expression of preference. Nonetheless, we found statistically 
significant differences in pulse rate preference among populations of wild-caught 
females as measured under controlled temperature conditions in the laboratory. 
Furthermore, the significant difference in mean pulse rate preference between wild-
 caught Naulu and Glenwood females persisted in the G2 generation. As with pulse 
rate, these findings demonstrate a genetic contribution to the variation in preference 
for pulse rate among natural populations of L. cerasina. 
  
Song-preference covariation 
Pulse rate variation was strongly predicted by pulse rate preference across the range 
of L. cerasina (Figures 7, 8). This finding provides support for the hypothesis that the 
divergent evolution of song and preference among populations is characterized by 
small, parallel changes to both trait and preference characters. Additionally, we found 
a high degree of synchrony between trait and preference means across populations 
(Figure 8). Phenotypic correlations such as these could be explained by genetic 
correlations (through linkage disequilibrium or genetic coupling), pleiotropy (where a 
shared genetic element similarly affects both song and preference to the same 
degree), or environmentally based divergence, including genetic x environment 
interactions. The persistence of song-preference covariation across generations in a 
common garden environment indicates that environmental differences among 
populations is not sufficient to explain variation, and suggests that there is a genetic 
 
 21 
 
basis to divergence. Although the genetic architecture of song and preference 
variation has not been examined in L. cerasina, these findings are consistent with a 
genetic correlation between trait and preference within populations underlying the 
correlated phenotypic divergence of these characters among populations. In a closely 
related pair of congeners, L. kohalensis and L. paranigra, song and preference 
variation colocalized to a common QTL (Shaw and Lesnick 2009), and multiple QTL 
that were associated with song variation also predicted preference variation (Wiley 
and Shaw 2010), suggesting that linkage disequilibrium and/or genetic coupling may 
keep song and preference characters matched during population divergence. 
 There was one deviation from this pattern of correlated divergence. At Kalopa 
Park, females appear to prefer songs that are faster than the population average. This 
is likely a statistical anomaly produced from the experimental design. At the faster 
end of the range of preference trials, the choices of pulse rates presented to females 
(2.8 vs 3.3 pps; 3.0 vs 3.5 pps) lie far outside the range of male song variation (2.19-
 2.47 pps). Females were less responsive, and exhibited no significant preference for 
either song. When the final trial was removed, the mean preference decreased from 
2.61 to 2.55, and the standard error was cut in half. When the final two trials were 
removed, preference dropped to 2.51 and the standard error decreased another 25%. 
While this estimate is still faster than the average male song at Kalopa Park, it is 
likely a more accurate representation of the true preference. Kalopa Park males sang 
more slowly than preliminary research had suggested, and a better experimental 
design for these females would have included slower song choices. Despite the 
coarseness of the method for estimating female preference across a wide spectrum of 
 
 22 
 
variation, the results are remarkably consistent overall, suggesting that the striking 
pattern of song and preference covariation is a real biological phenomenon, and 
strong enough to overcome the limitations of the methods for measuring it. 
 
Causes of differentiation 
While this study was not designed to address the causes of divergence, it provides 
data that can be considered with regard to evolutionary causes. Divergent sexual 
selection and reproductive character displacement have both been proposed as 
hypotheses to explain the diversification of Laupala (Otte 1989; Otte 1994; 
Mendelson and Shaw 2005).  
 If reproductive character displacement influences population divergence in 
song, there should be evidence that males have shifted their pulse rates to maximize 
the difference from sympatric congeners (Hoskin and Higgie 2010). The congeners 
that live sympatrically with L. cerasina are part of a distinct morphological and 
genetic lineage of the Laupala radiation (the pacifica group) and are not close 
relatives (Otte 1994; Shaw 2002), despite extensive sharing of mitochondrial DNA 
haplotypes in zones of sympatry, indicating hybridization may have occurred in the 
past (Shaw 2002). However, if reproductive character displacement has occurred, it is 
not readily apparent. For example, the population at Pololu Valley is known to co-
 occur with a fast-singing species (L. kohalensis, pulse rate ~4.0 pps) (Shaw 1996), but 
the pulse rate of L. cerasina males here is intermediate (~2.6 pps) and nearby males at 
Waimea Reservoir, where apparently no congeners occur, have a somewhat slower 
song (~2.5 pps). In addition, while much of the range of L. cerasina is shared with L. 
 
 23 
 
pruna (~2.0 pps) (Shaw 1999), this includes not only the populations where the 
fastest L. cerasina songs are documented (Eucalyptus Toe, Akaka Falls, Kaiwiki), but 
also populations where songs are intermediate (Ola?a Flume, Laupahoehoe, 
Glenwood Road) and slow (Naulu). However, the pulse rates of L. pruna are also 
geographically heterogeneous (Otte 1994), and may shift in concert with L. cerasina 
across their collective ranges. The question of whether reproductive character 
displacement influences diversification within L. cerasina deserves further attention. 
 Sexual selection could influence population divergence in song through either 
genetic correlations between trait and preference (Lande 1981), sensory drive using 
pre-existing biases in female perception (Boughman 2002), or sexual conflict 
(Gavrilets and Hayashi 2005). If sexual selection influences population divergence 
through genetic correlations, then variation in pulse rate should be correlated with 
variation in female preference for pulse rate, and this correlation should be found at 
every stage of the speciation process (Panhuis et al. 2001). In contrast, a model based 
on sensory drive or sexual conflict may not consistently find correlations between 
trait and preference across disparate populations, due to the inherent mismatch 
between trait and preference that drives evolution in those models. This study found 
that song and preference are significantly correlated across thirteen geographically 
disparate populations of L. cerasina, representing nearly the entire range of the 
species. However, these data represent a current snapshot in time and in the 
continuum of population divergence, and can only support the conclusion that sexual 
selection on song likely maintains the divergence that currently exists. Divergence of 
sexual traits among populations could be driven by a multitude of sexual selection or 
 
 24 
 
reinforcement processes. For example, while the Fisher process (Lande 1981), good 
genes (Iwasa and Pomiankowski 1994), sensory drive (Boughman 2002), or sexual 
conflict (Gavrilets and Hayashi 2005) models suggest powerful mechanisms for the 
evolution of signals and preferences, each can operate with multiple preference 
functions, varying degrees of matching, and alternative genetic architectures of trait 
and preference. For these reasons, and because signal and preference are often 
expressed in multiple sensory modalities (Candolin 2003; Papke et al. 2007), the mate 
recognition system is a complex trait (Boake 2002; Chenoweth and Blows 2006; 
Mackay and Anholt 2007). Yet, even within a single sensory modality, we have an 
insufficient understanding of how and why signals and preferences diverge, obscuring 
our view of the speciation process. While the finding of a significant correlation 
among trait and preference suggests that sexual selection is involved in maintaining 
population differences, it would be difficult to disentangle the ultimate cause of the 
initial divergence from the consequential effect of that divergence among isolated 
populations. 
 
Conclusions 
In L. cerasina, there is statistically significant variation among populations in male 
acoustic traits, variation among populations in female acoustic preferences, and 
evidence for a genetic basis of the variation among populations. The evidence 
presented here supports the hypothesis that acoustic preference covaries with song. 
Taken together, these results indicate the potential for a direct role for these traits in 
the evolution of reproductive isolation.  
 
 25 
 
 These experiments have characterized a pattern of song differentiation in a 
species that is part of a rapidly diversifying clade of insects. This pattern of 
divergence within species is similar to the pattern of divergence among species, 
although to a lesser degree, which makes an investigation of the consequences of 
population divergence potentially useful for understanding the mechanisms that may 
influence speciation in the group. This study finds evidence for divergence in female 
preference for pulse rate among populations and shows that females prefer songs 
similar to those produced by males from the same population. The correlated 
divergence of song and preference among populations has the potential to reduce 
gene flow across the species and could promote speciation, if the differences among 
populations in song and preference are sufficient to cause sexual isolation through 
assortative mating. 
 
 26 
 
TABLES 
 
Table 1. Geographic variation in male song. Mean male pulse rate (PR), pulse 
duration (PD), and carrier frequency (CF) with one standard error (SE) for all 13 
populations, for each year sampled. Populations are ordered from north to south. 
Coefficient of variation (CV) shown for each measurement is averaged across all 
individuals. 
 
Population Year N PR (SE) PD (SE) CF (SE) 
Pololu Valley 2005 5 2.58 (0.037) 39.1 (2.65) 4780 (47) 
Muliwai Plateau 2004 11 2.55 (0.026) 40.6 (1.65) 4840 (62) 
Muliwai Plateau 2005 20 2.62 (0.019) 42.7 (0.90) 4840 (40) 
Muliwai Plateau 2006 28 2.59 (0.020) 41.0 (1.38) 4980 (40) 
Waimea Reservoir 2004 23 2.49 (0.017) 44.5 (0.88) 4530 (44) 
Kalopa Park 2004 24 2.33 (0.015) 46.0 (0.68) 4600 (33) 
Laupahoehoe 2005 18 2.74 (0.026) 44.3 (1.64) 4730 (36) 
Laupahoehoe 2006 16 2.65 (0.023) 41.3 (1.78) 4730 (61) 
Akaka Falls 2006 21 2.95 (0.024) 39.2 (1.16) 4910 (40) 
Eucalyptus Toe 2005 19 2.91 (0.020) 40.3 (1.17) 4900 (47) 
Eucalyptus Toe 2006 12 2.91 (0.025) 39.6 (1.51) 4870 (57) 
Kaiwiki 2006 5 2.80 (0.046) 40.4 (1.54) 4700 (112) 
Ola'a Flume 2004 9 2.61 (0.035) 40.9 (1.65) 4690 (102) 
Ola'a Flume 2006 14 2.70 (0.022) 38.9 (1.37) 4750 (64) 
Glenwood Road 2004 25 2.53 (0.024) 40.0 (0.97) 4590 (28) 
Glenwood Road 2005 18 2.63 (0.020) 39.7 (1.16) 4740 (48) 
Naulu Trail 2005 13 2.44 (0.023) 40.2 (1.28) 4880 (36) 
Wright Road 2004 17 2.61 (0.022) 40.0 (1.60) 4420 (45) 
Wright Road 2005 15 2.75 (0.019) 40.3 (1.69) 4680 (44) 
Kaiholena 2006 15 2.73 (0.033) 40.3 (1.71) 4780 (32) 
Coefficient of 
Variation (CV)    0.69% 4.35% 0.55% 
 
 
 
 
 27 
 
Table 2. Correlations among song characters in wild-caught males. The correlation 
coefficients from a Pearson correlation analysis using pulse rate, pulse duration, 
carrier frequency, and recording temperature are shown below, adjusted to account 
for population differences. Following a Bonferroni correction for multiple 
comparisons, only one correlation, between pulse duration and carrier frequency, 
showed a significant (negative) relationship (in bold), with a p<0.0001. All other 
relationships were non-significant (p>0.05). 
 
 
Pulse  
Rate 
Pulse 
Duration 
Carrier 
Frequency 
Pulse  
Rate ---   
Pulse 
Duration 0.053 ---  
Carrier 
Frequency 0.119 -0.213 --- 
 
Temperature 0.074 -0.054 0.013 
 
 
 
 
 
 28 
 
Table 3. Geographic variation in female preference for pulse rate. Mean female 
preference plus one standard error (SE) for all 13 populations, for each year sampled. 
The trials used in calculating the estimate are also shown for each population and year 
(see text). Populations are ordered from north to south. Average male pulse rates (PR) 
are reported here again for reference. 
 
Population Year N PR preference (SE) Trials used Male PR 
Pololu Valley 2005 10 2.48 (0.040) 1-5 2.58 
Muliwai Plateau 2004 16 2.56 (0.050) 1-5 2.55 
Muliwai Plateau 2005 6 2.52 (0.070) 1-5 2.62 
Muliwai Plateau 2006 20 2.58 (0.047) 1-5 2.59 
Waimea Reservoir 2004 19 2.56 (0.063) 1-4 2.49 
Kalopa Park 2004 18 2.51 (0.033) 1-4 2.33 
Laupahoehoe 2005 8 2.77 (0.081) 1-5 2.74 
Laupahoehoe 2006 25 2.73 (0.044) 1-5 2.65 
Akaka Falls 2006 11 2.88 (0.069) 1-6 2.95 
Eucalyptus Toe 2005 9 2.91 (0.105) 1-6 2.91 
Eucalyptus Toe 2006 14 2.90 (0.060) 1-6 2.91 
Kaiwiki 2006 9 2.85 (0.040) 1-5 2.80 
Ola'a Flume 2004 10 2.61 (0.044) 1-5 2.61 
Ola'a Flume 2006 8 2.69 (0.035) 1-5 2.70 
Glenwood Road 2004 21 2.63 (0.029) 1-5 2.53 
Glenwood Road 2005 7 2.64 (0.030) 1-5 2.63 
Naulu Trail 2005 11 2.40 (0.063) 1-4 2.44 
Wright Road 2004 17 2.75 (0.029) 1-5 2.61 
Wright Road 2005 12 2.77 (0.157) 1-5 2.75 
Kaiholena 2006 18 2.78 (0.035) 1-5 2.73 
 
 
 
 29 
 
Table 4. Wild-caught and G2 comparisons. Female mean pulse rate preference (PR 
pref) estimates and male mean pulse rates, both measured in pulses per second (pps) 
are compared across generations for two populations of L. cerasina. 
 
Population Generation PR pref (pps) 
Mean (SE, N) 
95% CI 
(pps) 
Pulse Rate 
Mean (SE, N) 
Naulu 
Trail 
2005  
(wild-caught) 
 
2.40 (0.063, 11) 
 
2.28-2.52 
 
2.44 (0.023, 13) 
Glenwood 
Road 
2005  
(wild-caught) 
 
2.64 (0.030, 7) 
 
2.58-2.70 2.63 (0.020, 18) 
Naulu 
Trail 
G2  
(lab-reared) 
 
2.36 (0.112, 16) 
 
2.14-2.58 
 
2.43 (0.011, 40) 
Glenwood 
Road 
G2  
(lab-reared) 
 
2.56 (0.054, 16) 
 
2.45-2.67 
 
2.61 (0.013, 40) 
 
 
 
 
 30 
 
FIGURE LEGENDS 
 
Figure 1. Topographic map of the Big Island of Hawaii, showing the approximate 
locations of the thirteen collecting sites where Laupala cerasina was sampled, along 
with their estimated coordinates. 
 
Figure 2. Geographic variation in the pulse rate of male song, by year. Mean male 
pulse rates (plus one standard error) are reported for each population, arranged 
geographically from north to south, with each collecting year reported separately. 
 
Figure 3. Geographic variation in the carrier frequency of male song, by year. Mean 
male carrier frequencies (plus one standard error) are reported for each population, 
arranged geographically from north to south, with each collecting year reported 
separately. 
 
Figure 4. Geographic variation in the pulse duration of male song, by year. Mean 
male pulse durations (plus one standard error) are reported for each population, 
arranged geographically from north to south, with each collecting year reported 
separately. 
 
Figure 5. Comparison of male pulse rate and female preference for pulse rate across 
generations. The histograms on the left show male pulse rate from second-generation 
(G2) laboratory-reared individuals (bottom) and their wild-caught ancestors (top) 
 
 31 
 
from Naulu and Glenwood. The figures on the right show female phonotaxis results 
from G2 individuals (bottom) and their wild-caught ancestors (top) from Naulu and 
Glenwood. The y-axis shows the proportion of females responding to the faster pulse 
rate in a two-choice trial, along with the 95% confidence intervals. The dashed line at 
0.5 indicates no preference for a given trial; populations whose confidence intervals 
exclude 0.5 show significant preference for one of the two pulse rates in the trial. 
Trials in which the confidence intervals of one population exclude the mean 
preference of the other have significantly different pulse rate preferences for that trial. 
 
Figure 6. Geographic variation in female pulse rate preference, by year. Mean female 
pulse rate preferences (plus one standard error) are reported for each population, 
arranged geographically from north to south, with each collecting year reported 
separately. 
 
Figure 7. Correlation between female preference and male song, by year. Female 
pulse rate preference means were plotted against male pulse rate means for all 
populations of L. cerasina, across all years sampled. Lines show the slope of the 
linear regression (trend line) for each year. 
 
Figure 8. Covariation between female preference for pulse rate and male pulse rate 
across all populations of L. cerasina. Preference and pulse rate means were averaged 
across all years for which there were data. The error bars shown are the 95% 
confidence intervals for each estimate. 
 
 32 
 
FIGURES 
Figure 1. 
 
 
 
Population Latitude (N) Longitude (W) 
Pololu Valley 20 deg 10' 155 deg 46' 
Muliwai Plateau 20 deg 7' 155 deg 37' 
Waimea Reservoir 20 deg 3' 155 deg 38' 
Kalopa Park 20 deg 2' 155 deg 27' 
Laupahoehoe 19 deg 57' 155 deg 16' 
Akaka Falls 19 deg 51' 155 deg 10' 
Eucalyptus Toe 19 deg 48' 155 deg 9' 
Kaiwiki 19 deg 46' 155 deg 10' 
Ola?a Flume 19 deg 42' 155 deg 11' 
Glenwood Rd 19 deg 28' 155 deg 8' 
Wright Rd 19 deg 26'45" 155 deg 14'30? 
Naulu Trail 19 deg 21'30" 155 deg 10? 
Kaiholena 19 deg 10'30" 155 deg 35' 
 
 
 
 33 
 
 
Figure 2. 
 
 
 
 
 
 
 
 34 
 
Figure 3. 
 
 
 
 
 
 
 
 
 35 
 
Figure 4. 
 
 
 
 
  
 
 36 
 
Figure 5. 
 
 
 
 
 
 
 37 
 
Figure 6. 
 
 
 
 
 
 38 
 
Figure 7. 
 
 
 
 
 
 
 
 
 
 
 39 
 
Figure 8. 
 
 
 
 
 
 40 
 
Chapter II: Rapid evolution of divergent female acoustic 
preferences facilitates sexual isolation in a Hawaiian cricket, 
Laupala cerasina 
ABSTRACT 
Sexual selection may cause the rapid evolution of sexually preferred characters 
within populations, since the most attractive individuals experience a direct 
increase in fitness through increased mating success. Consequently, isolated 
populations that experience divergence in their preferences for sexually selected 
traits may experience rapid evolution of those traits, resulting in population 
differentiation in a sexually selected character. In order for speciation by sexual 
selection to occur, females in diverging populations must discriminate among 
sexual signals that are in the process of divergence, and show preference for 
sexual signals representative of their own population over others. Previous 
research has documented that populations of the Hawaiian cricket Laupala 
cerasina show significant differentiation in the pulse rate of male song, a sexually 
selected character. However, nearby populations, whose songs tend to be less 
differentiated, have greater opportunities for migration and gene exchange, and 
this gene flow is reduced if migrants from sexually diverged populations 
experience reduced mating success. The current study investigates whether 
females can discriminate between songs whose differences are typical of those 
separating neighboring populations. Females from eight populations of L. 
cerasina were given the opportunity to choose between pairs of songs that 
 
 41 
 
differed by only 0.2 pulses per second (pps), consistent with the divergence 
typical of adjacent populations. Not only did females discriminate among songs 
differing by 0.2 pps, but mean preferences also closely matched the mean pulse 
rates measured for males from the same population. Divergence in preference 
was observed only between populations that also differed in song. The correlated 
divergence of song and preference could be a mechanism that promotes 
assortative mating among populations, thereby reducing gene flow, and may 
contribute to the speciation process in Laupala.  
 
INTRODUCTION 
More than a century ago, Darwin (1871) first observed that secondary sexual 
characters diverge rapidly among closely related species, suggesting that sexual 
selection is an important mechanism influencing evolution within species. Much 
more recently, divergent sexual selection among populations has been proposed as a 
mechanism that could drive speciation (West-Eberhard 1983; Panhuis et al. 2001; 
Coyne and Orr 2004; Ritchie 2007). Since sexual selection within species is widely 
reported (Andersson 1994), its action within isolated populations may lead to 
population divergence across a broader geographic range (Lande 1982; Endler 1992). 
Indeed, because isolated populations experience no selection pressure to maintain 
reproductive compatibility, the divergence of geographically isolated populations is 
possibly inevitable (Mayr 1963; West-Eberhard 1983; Turelli et al. 2001).  
While divergence through natural selection (Schluter 2009; Schluter and 
Conte 2009; van Doorn et al. 2009) or the evolution of genetic incompatibilities 
 
 42 
 
(Pryke and Griffith 2009) as by-products may collaborate, there are several reasons to 
suspect that sexual selection actively facilitates speciation. First, reproductive 
isolation is more likely to result from a divergence in traits directly involved in mate 
choice, such as sexually selected characters (West-Eberhard 1983; Panhuis et al. 
2001). Second, in several taxonomic groups, behavioral (and especially sexual) 
isolation appears to evolve at a faster rate than hybrid incompatibilities (Coyne and 
Orr 1997; Mendelson 2003; Alexander and Breden 2004). Third, comparative studies 
using various proxies for the intensity of sexual selection have indicated that 
phylogenetic clades with higher levels of sexual selection (and sexual conflict) tend to 
be species-rich (Barraclough et al. 1995; Mitra et al. 1996; Arnqvist et al. 2000). 
Lastly, the rapid evolution of sexual signaling is a key feature associated with 
diversification in two systems demonstrating accelerated rates of speciation (cichlids, 
Danley and Kocher 2001; crickets, Mendelson and Shaw 2005).  
Conclusive demonstration of sexual selection-driven speciation requires 
detailed studies of specific populations in the process of divergence. Many species 
exhibit minor inter-population differentiation in male sexual signals (Andersson 
1994; Gerhardt and Huber 2002); however, geographic variation in sexual signals 
may not always reflect a correlated divergence in female preference for those signals 
(Hill 1994; Zuk et al. 2006; van der Sluijs et al. 2010).  Under most circumstances, 
coevolution of signal and preference are necessary to bring about the assortative 
mating that can close the reproductive boundary between incipient species (Lande 
1981; Kirkpatrick 1982).  Furthermore, the significance of population differences in 
male sexual signals must be assessed relative to whether or not females discriminate 
 
 43 
 
differences in these male signals, a necessary prerequisite to skewed male mating 
success (Schluter and Price 1993). Several studies of various frog species have found 
evidence that divergence in male acoustic characters among populations may have 
been the result of divergent sexual selection (Prohl et al. 2006; Boul et al. 2007); 
however, these studies have not addressed differences in the shape of female 
preference functions in populations in the process of divergence. In contrast, many 
studies of divergent sexual selection in crickets do indicate divergent preference 
functions (Gray and Cade 2000; Shaw 2000b); however, these studies document 
differences at the interspecific level. There seems to be a lack of information about 
the shape of female preference functions among populations in the early stages of 
divergence, when these differences have the greatest potential to either accelerate or 
hinder the rate of population divergence by impacting the degree of gene flow.  
The Hawaiian cricket genus Laupala makes an ideal candidate for studying 
questions of sexual selection and speciation. The 38 constituent species evolved 
recently and are morphologically cryptic (Otte 1994; Shaw 2000a; Mendelson and 
Shaw 2005), yet distinctive in pulse rate of the male calling song, a sexually selected 
trait (Shaw and Herlihy 2000; Mendelson and Shaw 2002). Male song is easily 
quantified and synthesized (Otte 1994; Shaw 1996) and females readily respond to 
these synthesized songs, exhibiting preferences for songs with pulse rates at or near 
the male population mean (Shaw 2000b; Shaw and Herlihy 2000). Both pulse rate and 
preference variation have a genetic basis (Shaw et al. 2007; Shaw and Lesnick 2009; 
Wiley and Shaw 2010; Oh et al. in prep).  L. cerasina show differentiation in pulse 
rate (Otte 1994) and preference across its geographic range (Chapter 1), providing an 
 
 44 
 
opportunity to investigate the importance of this variation female mating behavior. 
This study examines whether females can discriminate among slight differences in 
the songs that characterize adjacent populations (~ 0.2 pps). If female preference 
divergence influences male song evolution, females from neighboring populations are 
expected to exhibit biased phonotactic responses to songs that differ by this order of 
magnitude. Furthermore, when neighboring populations are compared, females 
should express divergent preferences, thus favoring songs representative of 
homotypic over heterotypic males. 
 
METHODS 
Overview 
Laupala cerasina were collected from eight locations on the Big Island of Hawaii in 
July 2005 (Figure 1), hereafter referred to as populations. The collecting sites cover 
the majority of the range of L. cerasina, both geographically and phenotypically, and 
included the following paired comparisons: A. Muliwai Plateau (MP) vs. Waimea 
Reservoir (WM) (7.55 km apart), B. Kalopa Park (KP) vs. Laupahoehoe (LP) (21.26 
km apart), C. Eucalyptus Toe (ET) vs. Kaiwiki (KW) (4.08 km apart), and D. 
Glenwood Road (GR) vs. Naulu Trail (NT) (12.46 km apart). In a previous study of 
wild-caught individuals, two of these population pairs showed significant divergence 
in song (B and D) and two did not (A and C) (Chapter 1). 
 
 
 45 
 
Rearing conditions 
In the laboratory at the University of Maryland, individuals were reared under a 12:12 
light/dark cycle in a temperature-controlled room maintained at 20?C. Small nymphs 
were reared in 1-quart glass jars with no more than 20 individuals per jar, and larger 
nymphs and adults were kept in plastic specimen cups, with no more than three per 
cup, separated by sex. Individuals were provided with moistened kimwipes for 
moisture and fresh cricket chow (Fluker Farms, Louisiana) treated with methyl 
paraben (Tegosept, Fisher Scientific) once per week. Females collected from Hawaii 
as adults were housed individually to allow collection of eggs produced from natural 
matings prior to capture. All other individuals were reared to maturity and then 
randomly paired with a mate from the same population to generate offspring. Eggs 
collected from wild-inseminated adult females and through laboratory matings were 
reared to maturity under the same conditions as the parents. These lab-reared 
offspring were used in the experiments described below. 
 
Quantifying song differentiation 
To record songs, males were placed beneath clear plastic cups fitted with mesh covers 
in a temperature-controlled room maintained at 20?C. Upon singing, males were 
recorded onto cassette tapes using a Sony Professional Walkman (Model WM-D6) 
and Sony condenser microphone. A female from the same population was introduced 
if the male did not sing spontaneously. The songs of 260 males were recorded and 
digitized using Raven 1.2 (Cornell University, Ithaca, NY). Five non-consecutive 
measurements per male were made for pulse period, pulse duration, and carrier 
 
 46 
 
frequency. These were averaged to obtain a single value for each male for each 
character. Pulse period, measured as the time between the beginning of one pulse and 
the beginning of the next pulse, is transformed to calculate pulse rate (the inverse of 
the pulse period), which is the primary song feature that differs among species of 
Laupala. Pulse duration is measured as the time between the beginning and end of a 
single pulse. Carrier frequency is measured as the dominant frequency of a single 
pulse. These are the same measurements that have previously been reported for wild-
 caught L. cerasina individuals (Chapter 1). Planned comparisons were conducted 
between the songs of males from neighboring populations (population pairs A-D) 
using t-tests. 
 
Quantifying female discrimination 
Virgin females approximately 20-25 days past the final molt were used in phonotaxis 
trials. Females of this age show a high level of responsiveness to synthesized cricket 
songs (Chapter 1). Since several adjacent populations of L. cerasina express a song 
divergence of around 0.2 pulses per second, our experimental design tested for this 
level of discrimination. To test for female discrimination behavior, six trials were 
conducted which differed in the pulse rate choices presented to the female: 2.0 vs. 
2.2, 2.2 vs. 2.4, 2.4 vs. 2.6, 2.6 vs. 2.8, 2.8 vs. 3.0, and 3.0 vs. 3.2 pps. These trials 
span the natural range of variation for L. cerasina songs and song preferences 
(Chapter 1). All parameters other than pulse rate of the synthetic songs were held 
constant at values that approximate the natural parameters found in Laupala species: 
 
 47 
 
pulse duration, 40 ms and carrier frequency, 5 kHz (Shaw and Herlihy 2000). A total 
of 233 females were used, once in each trial (six times total per female).  
Phonotaxis trials were conducted in a circular arena (radius 47 cm) housed in 
a temperature-regulated (20?C), acoustically buffered sound chamber (Acoustic 
systems) illuminated by red gel-filtered lights. Two speakers (Radio Shack 40-1218) 
broadcast programmable, digitally synthesized, pulsed sinusoidal tones created using 
custom-designed software (T.G. Forrest, Univ. of North Carolina, Asheville) from 
opposite ends of the arena. A 16-bit digital/analog converter (Tucker-Davis 
Technologies) and 10 kHz filter (Krohn-Hite 3322) processed the acoustic output. A 
digital attenuator (Bruel and Kjaer 4155) was used to monitor and equalize sound 
pressure levels between speakers. 
For each trial, playbacks occurred for an acclimation period of five minutes, 
during which time a female was restrained beneath a plastic cup in the center of the 
arena. Once the cup was raised, she received an additional five minutes to respond to 
the acoustic stimuli. A 10-cm score zone was designated around each speaker (Figure 
2). Once a female entered the score zone associated with a speaker, the trial was 
ended. She received a score of one for approaching the faster song, or zero for the 
slower song. If the female did not approach either score zone after five minutes, no 
score was recorded for that trial. A random number generator was used to determine 
the order of presentation and the speaker assigned to play the faster/slower song. 
Females were used in two trials per day over three consecutive days to complete the 
series of six trials. 
 
 48 
 
To determine whether females discriminate between songs differing by 0.2 
pulses per second, the proportion of females that approached the faster song of a 
given pair was compared to the null expectation under a model of random choice, 
where the probability of choosing either song is 0.5. The 95% confidence intervals for 
each proportion were calculated from the binomial distribution. If these confidence 
intervals excluded 0.5, the population showed a significant response bias towards 
either the fast or the slow song of a given trial. 
To determine whether neighboring populations differ in their song 
preferences, female preference data were further analyzed using the statistical 
program R. To estimate the mean female preference for a population, the entire set of 
female phonotaxis data is analyzed simultaneously using logistic regression. The 
point at which females are equally likely to choose either the fast or the slow song 
(probability of 0.5) was determined for each population, providing an estimate of the 
mean pulse rate preference (Shaw and Herlihy 2000; Grace and Shaw 2004; Chapter 
1). Standard errors for this estimate were calculated for each population by bootstrap 
analysis, which randomly resampled the data with replacement, while maintaining the 
original sample size, for 1000 replicates (Chapter 1). For each replicate, the mean 
pulse rate preference was estimated, allowing a calculation of the standard deviation 
of the means (i.e., the standard error). Planned comparisons between neighboring 
population pairs (shown in Figure 1) were also performed using the Wald ?2 test 
(Grace and Shaw 2004).  
 
 
 49 
 
RESULTS 
Male song variation 
Populations that showed evidence of pulse rate differentiation in the wild-caught 
generation (KP-LP and GR-NT; Chapter 1) also showed significant pulse rate 
differentiation in this experiment (KP-LP: t=-12.21, p<0.0001; GR-NT t=8.36, 
p<0.0001; ET-KW: t=1.68, p=0.095; MP-WM: t=0.10, p=0.92; Figure 3; Table 1). 
Additionally, two pairs (MP-WM and KP-LP) showed significant differences in 
carrier frequency (MP-WM: t=2.80, p=0.055; KP-LP: t=2.23, p=0.0267; GR-NT: t=-
 1.06, p=0.29; ET-KW: t=-0.31, p=0.76; Figure 3; Table 1), but none were 
differentiated in pulse duration (GR-NT: t=1.91, p=0.057; MP-WM: t=1.40, p=0.16; 
ET-KW: t=-0.57, p=0.57; KP-LP: t=-0.25, p=0.80; Figure 3; Table 1). 
 
Female discrimination 
For each population, females showed biased responses (differing significantly from 
0.5) in at least two of the six trials (average: 4; range: 2 to 6; Figure 4). At the low 
end of the series of trials, females respond preferentially to the faster song of the pair, 
and at the high end, they tend to respond to the slower song (Figure 4). For each 
population, this resulted in a single transition of preference from the faster to the 
slower song, allowing an estimation of the inflection point where preference for the 
faster song equals that of the slower song, which provides an estimation of the mean 
preference. 
 
 
 50 
 
Female preference variation 
Using the results from all six trials together allows estimation of the mean preference 
for each population. A significant logistic regression is possible only when sufficient 
signal exists in the data to allow a single transition from a preference for faster songs 
to a preference for slower songs across the whole range of phonotaxis trials, which 
appears as a sigmoidal pattern in the data (Figure 4). All of the populations showed 
this pattern, which allowed mean preferences to be estimated using logistic regression 
(Table 2). Standard errors were calculated using the bootstrapping procedure outlined 
above. When preferences were compared across populations, two of the four planned 
comparisons (KP-LP and GR-NT) showed significant differentiation in female 
preference functions (KP-LP: Wald ?2=12.12, p=0.0005; GR-NT: Wald ?2=11.49, 
p=0.0007; Figure 4; Table 2). These were the same populations that showed 
differentiation in pulse rate (Figure 3; Table 1). Populations that did not show 
significant pulse rate divergence also did not show divergence in preference (MP-
 WM: Wald ?2=0.57, p=0.45; ET-KW: Wald ?2=0.10, p=0.75; Figure 4; Table 2).  
 
DISCUSSION 
One of the biggest weaknesses in our understanding of the evolution of reproductive 
isolation occurs at the interface between population divergence and speciation. At 
some point along this continuum, divergent selection among populations results in 
reproductive isolation among species, but the factors that facilitate this transition are 
relatively poorly understood. If divergent sexual selection among populations drives 
speciation through sexual isolation and assortative mating, it must be shown that 
 
 51 
 
females from the diverging populations have different preferences. If divergent 
preferences are to have an impact on gene flow, females must be able to discriminate 
between the signals of local males (homotypic) and those from divergent populations 
(heterotypic). Furthermore, females must exhibit clear preferences for homotypic 
male characteristics and discriminate against heterotypics even at these early stages of 
trait divergence. 
 In Laupala cerasina, when neighboring populations exhibit small but 
significant differences in pulse rate, females display preferential phonotaxis toward 
songs more similar to males from the same local population than males from adjacent 
populations. These data indicate that females across all populations could 
discriminate the pulse rate differences in the trials and, further, that they demonstrated 
a choice based on the perceived difference between them. This suggests both that 
females can discriminate between the songs and that populations exhibit significant 
preferences for one song over another in a given pair. This study reveals that even 
small differences in song and preference are biologically meaningful and may be 
sufficient to initiate a pattern of assortative mating preferences across populations 
whose songs and preferences are divergent. These results demonstrate how female 
acoustic preference within a population can result in sexual isolation between 
populations.  
This study is the first to demonstrate the precision with which females are able 
to discriminate among divergent pulse rates in Laupala, and to provide an estimate as 
to what magnitude of change is sufficient to induce a pattern of acoustic preference 
divergence among populations. Divergent phonotaxis preferences were found in two 
 
 52 
 
independent population pairs, KP-LP and GR-NT. These same pairs also differed in 
the pulse rate of the male song. Population pairs that did not show significant song 
divergence, MP-WM and ET-KW, likewise, did not show divergence in female 
preference. Based on these results, the degree of sexual isolation is expected to be 
greatest between populations having the greatest divergence in sexual communication 
traits, regardless of physical proximity. However, it is worth noting that the two 
population pairs that showed the least divergence in song and preference also 
happened to be separated by the shortest distances. Although there is no evidence for 
isolation by distance with respect to divergent pulse rates (Chapter 1), such a pattern 
may be more evident over the shorter distances involved in this study. The correlated 
divergence of trait and preference among populations (Chapter 1) is a potential 
signature of speciation by sexual selection (Lande 1981), but not necessarily 
predicted by other models of speciation, such as ecological speciation (Hill 1994; Zuk 
et al. 2006). 
A QTL (quantitative trait locus) study in the closely related species pair 
Laupala paranigra and L. kohalensis has provided evidence that some of the same 
loci or closely linked loci may be involved in phenotypic divergence of both song and 
preference (Shaw and Lesnick 2009). Furthermore, the average effect size of QTL 
alleles that differ between species underlying male song was found to be around 0.2 
pps (Shaw et al. 2007), which is the same magnitude of differentiation used to 
examine female preference functions and discrimination. If the effect sizes of the 
QTL differences identified are due to single mutations, our results here suggest that 
females are able to discriminate the effects of such mutations to single QTL. 
 
 53 
 
However, we have yet to determine whether common QTL underlie song divergence 
in L. cerasina. A similar genetic architecture in L. cerasina would suggest that 
physical linkage and/or pleiotropy is responsible for maintaining the behavioral 
coupling of song and preference that we have observed. If song and preference were 
genetically coupled, this would provide a powerful mechanism for explaining their 
correlated divergence among populations (as shown here and in Chapter 1). 
Alternatively, genetic correlations could be maintained through the joint inheritance 
of song and preference alleles that occurs when females with strong preferences 
choose males with preferred traits, passing on the genes for both trait and preference 
to the next generation, as described by Fisher (1930) and modeled by Lande (1981). 
Since both song and preference are likely polygenic traits (Shaw 1996, 2000b; Wiley 
and Shaw 2010), both physical linkage and selection-generated linkage 
disequilibrium may contribute to the observed pattern of covariation. 
In other organisms where sexual selection acts on traits used in sexual 
communication, few studies have been able to demonstrate female preference for 
local signals over foreign ones. In the Amazonian frog Physalaemus petersi, females 
prefer the songs of local males to those of foreign males, and song divergence is 
correlated with reduced gene flow (Boul et al. 2007). However, a separate study in 
the closely related congener P. pustulosus found that females prefer the calls of local 
males over those of foreign conspecifics only one-third of the time in choice trials 
(Pfennig and Ryan 2007). All of these studies were conducted under field conditions 
in the natural environment, so the additive genetic component of these phenotypic 
differences may be difficult to disentangle from environmental and developmental 
 
 54 
 
differences. The use of laboratory-born individuals in this experiment provides 
additional evidence that the observed differences among populations of Laupala have 
a genetic basis. The environmental variation that might contribute to total phenotypic 
variation is reduced, since these individuals were born and raised entirely under 
laboratory conditions, and individuals experienced the same common garden 
environment throughout their entire lives.  
Comparisons between different populations of L. cerasina showed that 
divergence in the pulse rate of male song predicts a correlated divergence in female 
preference, consistent with previous analyses using wild-caught individuals (Chapter 
1). This study provides further evidence of a genetic basis to covariation, since 
individuals were bred and reared in a common garden environment. Furthermore, 
females could resolve differences in pulse rate approximating the differences between 
neighboring populations, setting the stage for assortative mating. This sexual isolation 
has the potential to reduce gene flow, since rare male migrants to a new population 
may have difficulty attracting a mate. This preference for local mates is astonishing, 
given the relatively minor divergence in pulse rates between neighboring populations 
(~0.2 pps), compared to that observed between L. cerasina and its sister species (~1.0 
to 1.5 pps). In all other characters that have been measured, L. cerasina populations 
are considered to be a single species, with little population-specific variation (Otte 
1994; Mendelson et al. 2004; Mendelson and Shaw 2005). The fact that populations 
could be reproductively isolated based on divergence in a single character would 
provide evidence that acoustic preference for song contributes to the divergence 
among populations. However, phonotaxis preference is only the first opportunity for 
 
 55 
 
sexual selection to influence mate choice, and further studies are needed to 
specifically address the hypothesis that divergent phonotaxis preference predicts 
assortative mating. In addition, the influence of variation in pulse rate relative to other 
male song characters (e.g., pulse duration, carrier frequency) has not been assessed in 
Laupala, but could have major consequences for female preference evolution 
(Gerhardt 2008; Gerhardt and Brooks 2009). The finding that females can 
discriminate among songs so precisely goes a long way towards demonstrating that 
these subtle differences in song are maintained by sexual selection, and ultimately, 
that sexual selection may be driving speciation in this group. 
 
 
 
 56 
 
TABLES 
Table 1. Population variation in mean pulse rate (in pulses per second), pulse 
duration (in milliseconds), and carrier frequency (in Hertz) of male songs, plus one 
standard error (SE). 
 
Population N Pulse Rate (SE) Pulse Duration (SE) Carrier Frequency (SE) 
MP 33 2.604 (0.019) 40.45 (1.03) 4834 (27) 
WM 33 2.602 (0.016) 38.19 (1.07) 4725 (29) 
KP 46 2.421 (0.014) 40.67 (0.96) 4835 (26) 
LP 33 2.695 (0.015) 41.04 (1.23) 4755 (26) 
ET 35 2.903 (0.016) 38.47 (0.99) 4846 (19) 
KW 15 2.852 (0.030) 39.63 (1.34) 4861 (48) 
GR 33 2.683 (0.018) 36.42 (1.21) 4755 (26) 
NT 32 2.479 (0.017) 33.30 (1.39) 4797 (30) 
 
 
 
 57 
 
Table 2. Population variation in estimated mean female preference for pulse rate (in 
pulses per second) plus one standard error (SE). 
 
Population N Preference (SE) 
MP 30 2.439 (0.123) 
WM 30 2.541 (0.054) 
KP 28 2.325 (0.097) 
LP 32 2.693 (0.091) 
ET 28 2.830 (0.057) 
KW 25 2.851 (0.096) 
GR 30 2.673 (0.065) 
NT 30 2.354 (0.099) 
 
 
 
 58 
 
FIGURE LEGENDS 
 
Figure 1. Topographic map of the Big Island of Hawaii showing collecting locations 
of eight populations of L. cerasina. Boxes indicate pairwise planned comparisons 
between neighboring populations.  
 
Figure 2. Schematic of arena used in phonotaxis trials. An acoustic preference is 
recorded if the female enters the score zone immediately adjacent to one of the two 
speakers. 
 
Figure 3. a) Geographic variation in pulse rate of male song, organized from north to 
south. Mean pulse rate in pulses per second (pps) plus one standard error are plotted 
for each population. b) Geographic variation in carrier frequency of male song, 
organized from north to south. Mean carrier frequency in Hertz (Hz) plus one 
standard error are plotted for each population. c) Geographic variation in pulse 
duration, organized from north to south. Mean pulse duration of male song in 
milliseconds (ms) plus one standard error are plotted for each population. 
 
Figure 4. Geographic variation in female phonotaxis responses. The y-axis shows the 
proportion of females responding to the faster pulse rate in a two-choice trial, along 
with the 95% confidence intervals. The dashed line at 0.5 indicates no preference for 
a given trial; populations whose confidence intervals for a given trial exclude 0.5 
show significant preference for one of the two pulse rates in the trial. Trials in which 
 
 59 
 
the confidence intervals of one population exclude the mean preference of the other 
population have significantly different preferences for that trial. Plots are organized 
according to planned population comparisons: a) Muliwai Plateau and Waimea 
Reservoir b) Kalopa Park and Laupahoehoe c) Eucalyptus Toe and Kaiwiki d) 
Glenwood Road and Naulu Trail 
 
Figure 5. Geographic variation in mean female pulse rate preference, organized from 
north to south. Mean pulse rate preferences in pulses per second (pps) plus 1 standard 
error are plotted for each population.  
 
 60 
 
FIGURES 
Figure 1.  
 
 
 
 
 61 
 
Figure 2. 
 
 
Speaker A 
Speaker B 
Score 
Zone A Score 
Zone B 
 
 62 
 
Figure 3. 
 
 a) 
 
 
 
b) 
 
 
 
 
 63 
 
c) 
 
 
 
 
 64 
 
Figure 4. 
 
 
a)      b) 
 
 
c)      d) 
 
   
 
 65 
 
 
Figure 5.  
 
 
 
 
 
 
   
 
 66 
 
CHAPTER III: Divergent female acoustic preference 
facilitates sexual isolation through assortative mating in the 
Hawaiian cricket Laupala 
ABSTRACT 
Closely related species often differ in secondary sexual characters, prompting 
the hypothesis that sexual selection may promote speciation; however, strong 
evidence supporting this hypothesis remains elusive. Crickets in the genus 
Laupala are morphologically and ecologically cryptic, but have diverged in a 
secondary sexual character, pulse rate of male song. In phonotaxis trials, females 
prefer the pulse rates of conspecific males. It is hypothesized that divergent 
phonotaxis preferences promote sexual isolation between closely related species, 
and that divergent sexual selection may also promote isolation among 
populations within a species. The mate choice preferences of two closely related 
sister species, L. cerasina and L. eukolea, were examined, as well as the mate 
choice preferences of neighboring populations of L. cerasina. Females were 
presented the songs of two males: one from the same population and species 
(homotypic) and one from the contrasted population or species (heterotypic). 
Following phonotaxis, females were presented with either the male whose song 
they preferred or the alternate male. When females selected males on the basis of 
their phonotaxis preference, they showed significant assortative mate preference 
in intraspecific trials and complete assortative mate preference in interspecific 
trials. Females usually completed a 4-7 hour courtship culminating in 
 
 67 
 
insemination by the male with whom they were presented, whether it was the 
male they selected on the basis of song or the male that they did not select. The 
number of offspring produced did not differ among the different groups, 
although the rate of failure to produce any offspring was higher in the 
heterospecific pairs. These results support the hypothesis that divergent 
phonotaxis preference contributes to the sexual isolation observed among 
populations and species of Laupala. 
 
INTRODUCTION 
Sexual selection is invoked to explain the evolution of a wide variety of phenotypes 
associated with mate choice throughout the animal kingdom and beyond (Darwin 
1871; Andersson 1994; Andersson and Simmons 2006); however, its role in 
generating reproductive isolation is not fully understood (Ritchie 2007). Because 
sexual selection directly affects traits involved in sexual communication and mate 
recognition, population divergence in sexually selected traits and preferences has the 
potential to generate sexual isolation through assortative mate choice (Questiau 1999; 
Panhuis et al. 2001). This may occur when sexual selection within populations results 
in the strengthening of a correlation between preference and trait (Lande 1981, 1982), 
or when preferences have diverged among populations (Boughman 2002). Either of 
these processes would contribute to a reduction in gene flow among populations, 
since assortative mating would cause migrants to be less successful in obtaining 
mates from populations with divergent sexual signaling. Sexual selection, ultimately, 
is a process that occurs within populations, so any consideration of its potential to 
 
 68 
 
maintain population divergence must consider both the action of sexual selection 
within populations and the consequences of sexual selection across populations 
(Boughman 2001). Thus, it is imperative to address this question in populations that 
are still in the process of divergence, rather than those in which speciation is already 
complete. 
In order to demonstrate a causative role of sexual selection on species 
divergence, several requirements must be met (Panhuis et al. 2001). First, the trait (or 
traits) that differs among species should be sexually selected, and females must mate 
assortatively due to variation in trait preference. Assortative mating will establish a 
genetic correlation between trait and preference (Lande 1981), making interbreeding 
and gene flow increasingly unlikely as populations continue to diverge. Second, for a 
response to selection and evolutionary divergence to occur, the trait must have a 
genetic basis. The hypothesis that sexual selection is the primary driver of speciation 
would be bolstered by documenting that the traits involved in population divergence 
are the same as those underlying species differences (Boake 2002). Finally, little or 
no divergence in traits unrelated to mate choice provide ancillary evidence of the role 
of sexual selection, as divergence in other characters may suggest that other 
processes, such as natural selection, are the cause of divergence (Schluter 2001, 
2009). For example, an absence of postzygotic reproductive isolation would rule out 
the process of reinforcement (Marshall et al. 2002; Servedio and Noor 2003).  
Studies of sexual selection often rely on the assumption that female preference 
for a trait predicts mate choice, but this assumption is not always subjected to 
rigorous analysis (Walling et al. 2010). Particularly in phonotaxis trials, it is rarely 
 
 69 
 
tested whether positive phonotaxis predicts mating success. In Laupala, this 
assumption is not trivial, because a single mating bout will take several hours to 
complete, and sperm is transferred only at the end of this extended courtship (Shaw 
and Khine 2004; deCarvalho and Shaw 2005, 2010). Thus, there are many 
opportunities for additional factors to influence mating decisions beyond phonotaxis 
behavior. This would be true for all courtships involving long-range attraction 
(Leonard and Hedrick 2010), but particularly when the duration of courtship - 
following initial attraction but prior to insemination - is so prolonged. This makes it 
particularly important to test the underlying assumption that a phonotaxis preference 
is indicative of a mate choice preference. 
Several recent studies have begun to explicitly test the hypothesis that sexual 
selection drives speciation (Ritchie 2007); however, most of these studies do not meet 
the necessary standards to demonstrate an exclusive role of sexual selection in driving 
divergence. While collectively they make a compelling argument that sexual selection 
likely contributes to population divergence and reproductive isolation, individual 
studies rarely meet all the conditions necessary to demonstrate conclusively that 
sexual selection is the driving force of speciation (Panhuis et al. 2001). One notable 
exception are studies involving Amazonian frogs from the genus Physalaemus (Prohl 
et al. 2006; Boul et al. 2007; Ryan et al. 2007; Guerra and Ron 2008); however, even 
in these cases, more recent evidence indicates more ecological divergence than 
previously thought (Prohl et al. 2010). It is worth noting that very few closely related 
species groups differ only in sexually selected traits, and many examples of sexual 
selection driving speciation also involve ecological adaptations (Endler and Houde 
 
 70 
 
1995; Boughman et al. 2005; Kronforst et al. 2006; Cocroft et al. 2010). This is likely 
because once speciation by sexual selection has occurred, the newly formed, 
reproductively isolated species will acquire new adaptations post-speciation that 
obscure the signature of sexual selection.  
Speciation in the Hawaiian cricket genus Laupala may have occurred by the 
action of divergent sexual selection. The 38 described species are morphologically 
and ecologically cryptic, and are primarily differentiated using the pulse rate of male 
calling song (Otte 1994; Shaw 2000a; Mendelson and Shaw 2005), a sexually 
selected trait (Shaw 2000b; Shaw and Herlihy 2000; Mendelson and Shaw 2002). 
Previous studies of L. cerasina, a species showing intraspecific variability in pulse 
rate, showed significant differentiation in pulse rate and pulse rate preference 
(Chapter 1). Additional studies of phonotactic behavior in several populations of L. 
cerasina document that females from neighboring populations with slightly, but 
significantly differentiated songs are capable of distinguishing these small, 
interpopulational differences in pulse rate (Chapter 2), and furthermore, show an 
acoustic preference for pulse rates characteristic of their own population (Chapter 1; 
Chapter 2). Here, we investigate the hypothesis that females of L. cerasina from 
acoustically distinct populations mate assortatively based on their phonotactic 
preferences. If sexual selection on song maintains or accelerates population 
divergence, then female phonotactic preference across variant populations should be 
assortative. If phonotactic behavior is predictive of mate choice, then following 
phonotaxis, female mating success should be high. Song acts as a long-distance signal 
and may have less influence on mate choice at close range (Mendelson and Shaw 
 
 71 
 
2006), even though males use the same calling song during both long-distance and 
close-range courtship. If close-range signals have also diverged among populations or 
species, such as cuticular hydrocarbons (Mullen et al. 2007; Mullen et al. 2008), 
phonotactic behavior may not be predictive of mate choice once potential partners are 
in physical contact. In addition, postzygotic isolation among populations or species 
would indicate a need for further investigation of other potential divergence processes 
in Laupala speciation. We further provide a comparative context for the analysis of 
mate choice in L. cerasina through similar studies of its sister species, L. eukolea, 
which shows much greater divergence both genetically and phenotypically from L. 
cerasina than populations within L. cerasina (Mendelson et al. 2004). 
 
METHODS 
Overview 
Tests of assortative mating based on phonotaxis preference and staged encounters 
were performed between neighboring populations of Laupala cerasina (intraspecific) 
and between L. cerasina and its allopatric sister species, L. eukolea (interspecific). 
For the intraspecific comparisons, neighboring populations of L. cerasina were 
chosen, since they likely interact through chance migration events. Assortative mating 
in L. cerasina was assessed between two pairs of populations: (1) Kalopa Park (KP) 
and Laupahoehoe (LP) (distance: 21.3 km), and (2) Glenwood Road (GR) and Naulu 
Trail (NT) (distance: 12.5 km) (Chapter 2; Figure 1). Each population pair shows 
significant divergence in female pulse rate preference that is consistent with the 
divergence in male pulse rate: KP-LP mean pulse rates are 2.42 and 2.70 pulses per 
 
 72 
 
second (pps), respectively, with estimated preferences of 2.33 and 2.69 pps; GR-NT 
mean pulse rates are 2.68 and 2.48 pps, respectively, with estimated preferences of 
2.67 and 2.35 pps (Chapter 2). The same experimental design (see below) was used to 
quantify sexual isolation between L. cerasina, endemic to the Big Island of Hawaii, 
and the Maui endemic L. eukolea, its sister species. For this interspecific comparison, 
populations of L. cerasina from Muliwai Plateau (MP) and Waimea Reservoir (WM) 
were contrasted with the Ginger Camp (GC) population of L. eukolea (Figure 1). 
These two populations of L. cerasina have similar song and preference means: MP 
and WM mean pulse rates are both 2.60 pps and estimated preferences are 2.43 and 
2.54 pps, respectively (Chapter 2). These populations occur 7.5 km apart along the 
region of the Big Island nearest to Maui, where L. eukolea occurs (Figure 1). L. 
eukolea from Ginger Camp sing much faster than L. cerasina males, with pulse rate 
and estimated preference around 3.9-4.0 pps (Oh et al. in prep). 
 
Rearing conditions 
Crickets from the six populations of L. cerasina and one population of L. eukolea 
were collected in July 2005 and brought to the laboratory at the University of 
Maryland, College Park. Females collected as adults were housed individually to 
allow collection of eggs produced through natural inseminations prior to capture. All 
other individuals were reared to maturity and paired randomly with a mate from the 
same population to generate offspring. These lab-reared offspring were reared to 
adulthood and then randomly paired with a mate from the same population but 
 
 73 
 
different family (i.e., no inbreeding) to produce a second generation of laboratory-
 reared offspring for experimental trials.  
All crickets were maintained on a 12:12 light: dark cycle in a temperature-
 controlled room maintained at 20?C. Inseminated females were provided with 
moistened kimwipes as a substrate for oviposition. These kimwipes were collected, 
transferred to plastic specimen cups lined with additional moistened kimwipes, and 
monitored for hatching nymphs. Small nymphs were reared in one-quart glass jars 
with 15-20 individuals per jar, while larger nymphs and adults were housed in plastic 
specimen cups, no more than three crickets per cup, separated by gender and grouped 
according to approximate age. All crickets were provided moist kimwipes for water 
and humidity and fed weekly with cricket chow (Fluker Farms, LA) treated with 
methyl paraben (Tegosept, Fisher Scientific) to reduce mold. Crickets were checked 
twice weekly to determine approximate date of adult eclosion, and individuals were 
considered sexually mature approximately 20-25 days post-eclosion. 
 
Pre-trial song analysis 
To generate playback parameters for mating trials (see below), songs were recorded 
from the experimental males 1-7 days prior to the experiment using a Sony 
Professional Walkman (Model WM-D6) and Sony condenser microphone. To record 
a song, an individual male was placed beneath a clear plastic cup with screen top in a 
temperature-controlled room maintained at 20?C. Males that did not sing 
spontaneously were presented with a female from the same population and species 
until singing commenced (Laupala males produce only one type of song, regardless 
 
 74 
 
of social context). Approximately one minute of song was recorded from each male, 
after which males were returned to an individual cup with fresh water and food. If a 
female was introduced into the recording cup, she was removed before any mating 
activity occurred. Song recordings were digitized using Raven 1.2 (Cornell 
University, Ithaca, NY). For each male, five measurements were made of pulse 
period, pulse duration, and carrier frequency, using non-consecutive pulses (as in 
Chapter 1). Pulse period is measured as the time between the beginning of one pulse 
and the beginning of the next. Pulse duration is measured as the time between the 
beginning and end of a single pulse (duration of wing closure). Carrier frequency is 
the dominant frequency of a pulse. All three values were used, since all are known to 
be variable among males (Shaw and Herlihy 2000; Chapter 1). Only pulse rate has 
diverged among populations and among species, but pulse duration and carrier 
frequency differ among males to varying degrees. The average values for each song 
parameter were determined for each male and used in the experiment as described 
below. The two males used in each trial were paired randomly while matching for age 
similarity prior to song analysis, and each male was used only once.  
 
Trial conditions 
Song playback: Custom software (Tucker-Davis hardware/software system) was used 
to generate synthetic song that reflected the mean song characteristics of a given 
experimental male. The intensity of the speakers (Radio Shack model 40-1218) was 
calibrated to 80 dB on a 4.0 pulse/second, 5 kHz tone using a Bruel and Kjaer digital 
attenuator (type 4155) approximately once per week throughout the experiment to 
 
 75 
 
ensure speaker equality. Each trial was randomized using a binomial probability 
generator to determine which male was assigned to each speaker.  
Phase one (phonotaxis): Playback experiments were performed in a 
rectangular phonotaxis arena (dimensions: 3 feet x 1 ft X 1 ft) constructed of a balsa 
wood frame enclosed on all sides by dark mesh window screening (Figure 2) and 
located within an Acoustic Systems acoustically buffered sound chamber. Detachable 
mesh tubes were placed at either end of the arena, adjacent to each speaker. During a 
trial, the female was placed beneath a cup in the center of the arena. Synthetic songs 
from one male of each population (two total) were simultaneously broadcast from the 
two speakers. After a five-minute acclimation period, the cup was raised, allowing 
full access to the arena. If the female entered the mesh tube adjacent to one of the 
speakers, we regarded this as her acoustic preference, and she entered the next phase 
of the experiment. The duration of the phonotaxis period was recorded. If the female 
did not enter either mesh tube after 15 minutes, the trial was ended, and a second 
female was tested using the same parameters. However, once a female showed 
acoustic preference for one of the two songs, that pair of songs (and therefore, those 
two males) was never used again. Experimental males were kept outside the 
phonotaxis chamber during this portion of the trial, to prevent acoustical interference 
with the synthesized playbacks. 
Phase two (mate choice): In Laupala, courtship lasts for 4-7 hours and 
consists of the production and transfer of numerous spermatophores throughout the 
day (Shaw and Khine 2004). Once a female entered the mesh tube adjacent to a 
broadcasting speaker, that tube was carefully detached from the rest of the arena with 
 
 76 
 
the female inside. The male whose song was chosen, housed in a specimen cup, was 
introduced at one end of the tube, and the lid of a specimen cup closed the other end 
of the tube, forming the mating chamber. The male and female were observed for the 
entire day and the following courtship and mating behaviors were recorded: 
microspermatophore (micro) production, micro transfer, macrospermatophore 
(macro) production, macro transfer, and macro attachment duration. Only the macro 
contains sperm (Shaw and Khine 2004); thus, the successful transfer of a macro is 
used to define mating success. 
Control manipulation: In a subset of trials, the female was presented with the 
male whose song she did not choose. The male and female were observed and the 
same behavioral data listed above were collected. This allowed a comparison of the 
mating behaviors and reproductive output of females who were paired with their 
preferred male and those who were paired with the non-preferred male. 
 Post-trial conditions: Following mating, females were kept in isolation and 
provided with clean, moistened kimwipes as substrate for oviposition. Eggs were 
collected from all mated females until their natural death. The number of live 
offspring hatched from each mating was recorded for each female as a measure of 
fitness. Starting about four weeks following successful mating, egg cups were 
monitored for hatching nymphs. Every two to three days all nymphs were collected 
from each egg cup. When at least one month had passed with no new nymphs 
hatching, the egg cup was discarded. If no nymphs hatched from an egg cup, the cup 
continued to be monitored for at least three months or until at least four weeks 
following the death of the female, whichever was longer, and then discarded. 
 
 77 
 
RESULTS 
Song analysis 
The songs of males used in the choice trials were typical of their populations (Table 
1). Additionally, mean male song characters for a given population did not differ 
between the two female populations being tested in each pairwise set (p>0.05 for all 
within-population comparisons for pulse rate, pulse duration, and carrier frequency). 
For each female population tested, paired t-tests showed that the pulse rates from the 
two contrasted populations (or species) being offered were significantly different 
(Table 2). Conversely, pulse duration and carrier frequency did not differ 
significantly between the contrasted populations (or species), with one exception: in 
trials where L. eukolea (GC) females were tested with males from MP (L. cerasina) 
or GC (L. eukolea), carrier frequency was significantly higher for MP males (Table 
2). 
 
Phonotaxis preference 
A total of 162 phonotaxis trials ended with females expressing biased phonotaxis 
behavior. A global ?2 test indicated that females showed no preference for one side of 
the arena over another (?2(df=1)=1.58, p=0.2). In intraspecific trials, when populations 
are considered in pairs, females significantly preferred males from the same 
population (Fisher?s exact test: KP/LP, p=0.0011 (n=50); GR/NT, p=0.0031 (n=40). 
However, when females from each population are considered separately, only two of 
the four populations, KP (n=23) and NT (n=20) showed assortative mating 
preferences, where the 95% confidence interval of the proportion showing assortative 
 
 78 
 
preference excludes 50% (random preference), while the other two populations, LP 
(n=27) and GR (n=20) did not (Figure 3). In interspecific trials, both L. cerasina 
(n=38) and L. eukolea (n=34) females showed complete assortative phonotaxis 
preference, meaning females always preferred the songs of conspecifics over those of 
heterospecifics (Figure 3). 
In intraspecific trials, there was no significant heterogeneity among female 
populations in the phonotaxis response time, nor did it differ between homotypic and 
heterotypic responses. There was also no significant difference in the phonotaxis 
response time between L. cerasina and L. eukolea in interspecific trials. However, 
females responded significantly faster in interspecific trials when compared to 
intraspecific trials (average duration of phonotaxis: interspecific, 289 seconds; 
intraspecific, 382 seconds; t=2.9, p=0.0042). 
 
Mating success 
Mating success, defined by the successful transfer of a macrospermatophore, was 
high across all trial types (Figure 4). Across all 90 intraspecific trials, mating success 
was 82% for homotypic matings (n=67) and 87% for heterotypic matings (n=23). A 
G-test of heterogeneity indicated no difference in mating success between homotypic 
and heterotypic pairings, whether populations were considered separately 
(?2(df=7)=2.108, p=0.95) or combined (?2(df=1)=0.306, p=0.58). In 38 interspecific trials 
involving L. cerasina females, mating success was 86% for conspecific trials (n=28) 
and 80% for heterospecific trials (n=10). For the 34 L. eukolea females, mating 
success was 96% for conspecific trials (n=24) and 60% for heterospecific trials 
 
 79 
 
(n=10). We did not find evidence of mate discrimination; a G-test of heterogeneity 
failed to reject the null hypothesis of equal mating success among all four 
interspecific trial types (?2(df=3)=6.811, p=0.078). 
 
Hatching success 
Of the 74 intraspecific trials where a male transferred a macrospermatophore, 14 
(19%) produced no offspring. A G-test of heterogeneity indicated that these failures 
were randomly distributed across populations and across homotypic and heterotypic 
pairings (?2(df=7)=5.322, p=0.62). Considering only those matings where offspring 
were produced, there was no significant difference in offspring production between 
homotypic (mean=17.9, SD=12.1) and heterotypic (mean=19.8, SD=13.4) trials 
(?2(df=1)=0.1227, p=0.73), or between trials where females received the chosen 
(mean=21.5, SD=15.1) or the non-chosen (mean=18.0, SD=12.1) male 
(?2(df=1)=0.3957, p=0.53).  
Of the 61 interspecific trials where a male transferred a macrospermatophore, 
14 (23%) produced no offspring. Of those 14 trials failing to produce offspring, seven 
(50%) were male-manipulated trials where a female was paired with the male she did 
not choose (i.e., a heterospecific). A G-test of heterogeneity indicated that failure to 
produce offspring was significantly higher in heterospecific pairings (?2(df=3)=8.156, 
p=0.0429). Considering only those matings in which offspring were produced, a 
Kruskal-Wallis test found that the number of offspring produced from heterospecific 
(mean=28.3, SD=9.7) pairings did not differ from conspecific (mean=39.0, SD=16.2) 
pairings (?2(df=1)=2.90, p=0.0883). 
 
 80 
 
DISCUSSION 
If sexual selection can create and maintain population differentiation in traits 
associated with mate choice, then it has the power to cause reproductive isolation 
among populations that differ in these traits (West-Eberhard 1983; Panhuis et al. 
2001). However, in order to demonstrate that populations are sexually isolated, it 
must be shown that females mate assortatively on the basis of these divergent, 
sexually selected traits (Lande 1981; Walling et al. 2010).  
While previous research in Laupala has indicated that average preference 
functions have significantly diverged among neighboring populations, this is 
insufficient to demonstrate that sexual isolation has evolved. The current experiment 
used a combination of phonotaxis and staged mating encounters to demonstrate that 
females preferentially respond to songs produced by homotypic males. Furthermore, 
females exhibiting phonotaxis behavior have a high propensity to mate, as evidenced 
by the high percentage of successful courtships. While phonotaxis preferences were 
largely assortative, following phonotaxis, females generally engaged in courtship 
behavior with the male they were given, regardless of whether it was the same male 
they had initially selected based on song preference. This suggests that phonotaxis 
behavior is extremely important in establishing mating pairs in Laupala; thus, 
divergence in this phenotype will likely result in reduced gene flow across the 
divergent types. 
Consistent with the hypothesis that sexual selection on song within 
populations may cause sexual isolation among divergent populations, female 
phonotaxis preference favored homotypic males in both intraspecific trials, where the 
 
 81 
 
overall pattern was significantly assortative, and in interspecific trials, where 
preference was completely assortative. In intraspecific trials, females from two of the 
four populations deviated significantly from random choice; and when pairs were 
analyzed jointly, phonotaxis preference was significantly assortative. These results 
are consistent with the pattern expected under a model of sexual selection driving 
sexual isolation, since population divergence occurs in the same trait (pulse rate of 
male song) as species divergence. Also consistent with this model, the degree of 
sexual isolation is stronger between species than within species, where between-
 species trait divergence almost an order of magnitude greater than within-species 
divergence. 
Populations of L. cerasina used in this experiment inhabit nearby locations, 
making their interaction more probable. This experiment used songs based on 
measurements made from individual males, which provides a more realistic 
assessment of the likelihood of assortative mating in their natural habitat. The average 
song of a population does not provide a complete picture of the full range of 
variation, and in fact, the population pairs being contrasted showed a small degree of 
overlap in pulse rate measurements, with the fastest males from the slower population 
KP (NT) overlapping with the slowest males from the faster population LP (GR). 
Likewise, individual variation in pulse duration and carrier frequency was also 
incorporated into the experimental design, allowing the relative roles of these 
characters in determining mate choice to be evaluated. The use of multiple cues in 
mate assessment has been observed in several other systems (Candolin 2003; van 
Doorn and Weissing 2004; Leonard and Hedrick 2009), and it has been demonstrated 
 
 82 
 
in L. cerasina that females have acoustic preferences for long pulse duration and 
intermediate carrier frequency (Shaw and Herlihy 2000); however the interaction of 
multiple variable acoustic characters had been previously unknown. In KP and NT 
(the two slower populations), female phonotaxis preference departed significantly 
from random choice, with females favoring songs with the slower pulse rate, which 
corresponded to the homotypic male. However, in LP and GR (the two faster 
populations), the binomial confidence intervals for preference based on pulse rate did 
not exclude random choice (50%). In these cases, the proportion of females choosing 
males with the longer pulse duration in a given trial did deviate significantly from 
random choice, indicating that on balance, while females still more often chose the 
homotypic males, they also apparently also favored males whose songs had longer 
pulse durations. For LP females choosing KP males, in 8 out of 11 trials the KP males 
had longer pulse durations (8.4 ms longer on average); for GR females choosing NT 
males, in 4 out of 7 trials the NT males had longer pulse durations (4.75 ms longer on 
average). Additionally, for 10 out of 16 LP/LP trials and 12 out of 13 GR/GR trials, 
the females choosing homotypic males were also choosing the male with the longer 
pulse duration. This apparent preference for longer pulse duration was not seen in KP 
or NT females. However, since the components of song are not independent, it is 
difficult to conclude that pulse duration is more important than pulse rate in 
determining mate choice in these groups, and further work is needed to disentangle 
the multiple signals that females may receive from any given song. 
Mating success was high throughout the experiment, demonstrating that 
females that express phonotaxis behavior are likely to engage in courtship and 
 
 83 
 
copulation with the male they encounter. Given that the duration of courtship in 
Laupala is so prolonged, and that there are many opportunities for mutual mate 
assessment following initial contact but prior to sperm transfer, it is somewhat 
surprising that the initial female phonotaxis response resulted in such high rates of 
mating success. In control trials, where the male given to the female was the opposite 
male than the one she chose, no significant difference in mating success was 
observed, indicating that additional assessments made during the course of courtship 
had relatively little impact on the final outcome of the encounter. Even in 
interspecific trials, where all control trials were heterospecific since females never 
preferred heterospecific male song, females typically completed courtship and 
accepted a macrospermatophore from the heterospecific male. While this indicates 
that females are extremely selective when it comes to song preferences, it also 
indicates that once a female has selected a male based on song, she is unlikely to 
deviate from her initial choice. This suggests that female acoustic preferences are 
extremely important in determining male mating success, indicating sexual selection 
may be likely to influence song evolution through differential male mating success. 
In intraspecific crosses, there were no significant differences in offspring 
production among any group. This indicates that females are just as likely to produce 
offspring when they are mated with the preferred male or another male, and when 
they are mated with a homotypic or heterotypic male. There is no indication of any 
kind of postzygotic incompatibility among populations; however, offspring fertility 
was not assessed. In interspecific trials, a greater number of the heterotypic crosses 
failed to produce offspring compared to conspecific crosses. However, these results 
 
 84 
 
are confounded by the fact that the female was both being paired with the non-
 preferred male and the heterotypic male. Of the heterospecific pairs that did produce 
offspring, the numbers were similar to those produced in conspecific crosses. This 
suggests that the failure to produce offspring in a high proportion of trials is likely not 
due to postzygotic reproductive incompatibility, which would be expected to reduce 
the number of hybrids in all heterospecific crosses. While a failure rate of 50% is 
rather high, the sample size of 14 heterospecific crosses is still too low to make 
definitive conclusions about postzygotic isolation in this species pair, and the reasons 
for failure to produce offspring (fertilization failure, oviposition failure, egg 
inviability, etc.) are unknown. 
The process of speciation is likely ongoing in L. cerasina. Populations that 
have diverged in the same character (pulse rate) that is divergent among species show 
assortative mating on the basis of this divergence. This suggests that population 
divergence of female acoustic preference (and correlated divergence in male song) 
may reduce gene flow among neighboring populations through sexual isolation. 
These patterns indicate that population divergence in song and preference may initiate 
processes that lead to reproductive isolation through assortative mating, and that the 
initial barriers to reproduction are likely behavioral.  
 
 
 
 85 
 
TABLES 
Table 1. Average song values (and standard errors) for males used in phonotaxis 
trials. Pulse rate in pulses per second (pps), pulse duration in milliseconds (ms), and 
carrier frequency in Hertz (Hz) are averaged across all female trials for each male 
population. The ?c? denotes L. cerasina populations and the ?e? denotes L. eukolea. 
For each male population, there was no difference between the songs given to the 
homotypic vs. heterotypic female population of each pair (p>0.05 all comparisons).  
 
Male 
population/sp 
Pulse Rate 
(pps) 
Pulse Duration 
(ms) 
Carrier Frequency 
(Hz) 
Kalopa Park/c 2.391 (0.014) 41.6 (0.70) 4811 (19.9) 
Laupahoehoe/c 2.620 (0.010) 40.9 (0.79) 4764 (16.0) 
        
Naulu/c 2.429 (0.011) 38.2 (0.78) 4856 (23.1) 
Glenwood/c 2.612 (0.013) 40.3 (0.95) 4816 (24.6) 
        
Muliwai/c 2.597 (0.011) 40.3 (0.93) 4809 (27.2) 
Ginger Camp/e 3.931 (0.021) 39.6 (0.72) 4707 (29.4) 
        
Waimea/c 2.460 (0.013) 40.8 (0.78) 4744 (23.5) 
Ginger Camp/e 3.955 (0.019) 39.9 (0.75) 4715 (28.9) 
 
 
 
 86 
 
Table 2. Comparison of mean male pulse rate (PR), pulse duration (PD), and carrier 
frequency (CF) between contrasted populations/species. Populations designated ?c? 
are members of L. cerasina, while those designated ?e? are L. eukolea. All contrasted 
populations were significantly differentiated in pulse rate, but the other song 
characters rarely showed significant differences between populations. 
 
Female pop/sp 
 
Comparison 
PR  
difference 
PD  
difference 
CF  
difference 
Kalopa Park/c KP vs LP *** NS NS 
Laupahoehoe/c KP vs LP *** NS NS 
         
Naulu Trail/c NT vs GR *** NS NS 
Glenwood Road/c NT vs GR *** NS NS 
         
Muliwai Plateau/c MP vs GC *** NS NS 
Ginger Camp/e MP vs GC *** NS * 
         
Waimea Reservoir/c WM vs GC *** NS NS 
Ginger Camp/e WM vs GC *** NS NS 
    *p<0.01 
    **p<0.001 
    ***p<0.0001 
    NS: p>0.05 
 
 
 
 
 
 
 
 87 
 
FIGURE LEGENDS 
 
Figure 1. Map of Hawaii, showing the populations used for intraspecific and 
interspecific mate choice trials. All Big Island populations are L. cerasina, and the 
Maui population (Ginger Camp) is L. eukolea. 
 
Figure 2. Phonotaxis arena. The mesh tubes on either end of the linear arena were 
removable, and could be detached from the rest of the setup. Once a female entered 
one of the tubes, she proceeded to the mating phase of the trial.  
 
Figure 3. Female phonotaxis preference. Error bars indicate the 95% confidence 
intervals for the proportion based on the binomial distribution. The light gray bars 
indicate preference for the homotypic male, and the dark gray indicates preference for 
the heterotypic male. a) Intraspecific trials of L. cerasina, with KP-LP comparison on 
the left and GR-NT comparison on the right. b) Interspecific trials between L. 
cerasina and L. eukolea, with MP-GC comparison on the left and WM-GC 
comparison on the right. 
 
Figure 4. Mating success. The proportion of trials ending in a macrospermatophore 
transfer is indicated for each population. The light gray bar indicates homotypic 
pairings, while the dark gray bar shows heterotypic pairings. a) Intraspecific trials of 
L. cerasina. The proportion of successful matings are contrasted between KP and LP 
populations and between GR and NT populations, plus a final column provides a 
 
 88 
 
summary across all populations b) Interspecific trials between L. cerasina and L. 
eukolea. The proportion of successful matings from MP and WM populations of L. 
cerasina are each contrasted with the GC population of L. eukolea. 
 
 89 
 
FIGURES 
Figure 1. 
 
 
 
 
  
 
 
 
 90 
 
Figure 2. 
 
 
 
 
 
 
 91 
 
Figure 3. 
 
a) 
 
 
 
 
 
b) 
 
 
 
 
 
 
 92 
 
Figure 4. 
 
 
a) 
 
 
 
 
b) 
 
 
 
 
 
 93 
 
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