THE MAINTENANCE OF MALE COLOUR

POLYMORPHISM IN TELMA THERINA SARASINORUM,

AN ENDEMIC FISH FROM LAKE MATANO, SULAWESI

Suzanne Marie Gray MSc. University of Guelph, 2002 B.Sc. University of Guelph, 1999

THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

In the Department of Biological Sciences

O Suzanne Marie Gray 2007

SIMON FRASER UNIVERSITY

Spring 2007

All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author APPROVAL

Name: Suzanne Marie Gray

Degree: Doctor of Philosophy

Title of Thesis:

The maintenance of male colour polymorphism in Telmatherina sarasinorum, an endemic fish from Lake Matano, Sulawesi

Examining Committee:

Chair: Dr. J.M. Webster, Professor Emeritus

-- Dr. L.M. Dill, Professor, Senior Supervisor Department of Biological Sciences, S.F.U.

Dr. J.S. McKinnon, Professor Department of Biological Sciences. University of Wisconsin-Whitewater

Dr. F. Breden, Associate Professor Department of Biological Sciences, S.F.U.

Dr. B.J. Crespi, Professor Department of Biological Sciences, S.F.U.

- - Dr. J.D. ~T~nolds,Professor Department of Biological Sciences, S.F.U. Public Examiner

Dr. F.H. Rodd, Associate Professor Department of Zoology, University of Toronto External Examiner

22007 -- Date Approved .. I1 1%: SIMON FRASER &<$*Q ",,,,Idi brary

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Simon Fraser University Library Burnaby, BC, Canada ABSTRACT

Colour polymorphism has historically been used to understand the mechanisms

that help to generate within- and between-species diversity. In a broad review of the literature I explored selective processes that contribute to the maintenance of colour polymorphism and may also lead to speciation. Frequency-dependent selection within, and divergent selection between populations, coupled with environmental heterogeneity appear to be important in promoting colour polymorphism. Using Telmatlwrinrr sar*asirior.zrrw,a small fish endemic to Lake Matano, Sulawesi, Indonesia, I tested the hypothesis that selection varies in a spatially heterogeneous light environment, resulting in the maintenance of variation in male colour. A comparative description of the mating behaviour of seven colour polymorphic telmatherinids from the Malili Lakes (including

Matano), for which no behavioural ecology was known, suggests both conservation of reproductive behaviours and adaptations to different environments. The focal species, T. sar.asinorzrm, has five male colour morphs found in varying frequencies in two spawning habitats: shallow beach sites and deeper sites with overhanging roots. Measurements of the light environment and morph colour in each habitat indicate that blue males are more conspicuous in shallow habitats whereas yellow males are more conspicuous in root sites.

I used a model to test if the most conspicuous morph in each habitat had the highest reproductive fitness based on a set of variables derived from extensive behavioural observations in the field. Males that contrast more with the background are expected to have higher pairing and spawning success with females, but may also be cuckolded or have their eggs cannibalized more often (there is a positive relationship between the number of cuckolders at a spawning event and egg cannibalism). In a comparison of the two most abundant morphs, blue males are more frequent and have higher fitness than yellow males in beach sites, and yellow males are predominant and have higher fitness in root sites. This suggests a role for environment-contingent sexual selection in promoting male colour polymoi-phism in this species. This research will help us to understand the processes that maintain diversity and provide insight into how protection of the heterogeneous visual environment should be directed.

Keywords: sexual selection, Telmatherinidae, colour polymorphism, mating behaviour, cannibalism, cuckoldry, vision, environmental heterogeneity ACKNOWLEDGEMENTS

The process that culminates in a finished thesis is an arduous, albeit rewarding,

road that one does not travel alone. There are many people to whom 1 owe great thanks

for their continuing support through the adventurous path that I like to call my thesis.

Family, friends, mentors, perfect strangers, wild animals and a few inanimate objects added to the experience. To all of you, thank you.

First, 1 would like to sincerely thank my senior supervisor, Larry Dill. Larry, you have been a true mentor, your door was always open and for that 1 thank you. Your advice on subjects scientific and academic have been honestly given and much appreciated. I hope to take your advice and the enthusiasm you share about behavioural ecology, and life in general, throughout my career.

I would also like to thank my co-supervisor, Jeff McKinnon, for introducing me to the beautiful fishes of Lake Matano. Although almost all of our contact was via disembodied phone conversations and emails, I thank you for your continuous commitment to my work. The remainder of my advisory committee, Felix Breden, and

Bernie Crespi, were instrumental in developing my ability to do research and to think through difficult research problems. Although not officially on my committee, Arne

~Mooersand Elizabeth Elle have always had open doors and made time to discuss the finer points of evolutionary theory, graduate school, and academia. I would also like to sincerely thank Helen Rodd and John Reynolds, who were on my examining committee, and who provided insightful comments on my thesis. During the course of almost every graduate degree there are those people acting behind the scenes that seem to prevent the world from collapsing. I would therefore like to thank Marlene Nguyen, for keeping me on track and registered, even when 1 was half a world away; Ian Gordon, for helping me actually get to the field with equipment and sanity intact; and, Dave Carmean, for keeping my computer from exploding.

Doing fieldwork in a developing country adds a touch of adventure to any degree, and working in Indonesia was no exception. The Indonesian Science Foundation (LIPI) kindly provided permits to work in the Malili Lakes and Fadly Tantu provided assistance in the field. Fadly taught me a few things about life, not the least of which was patience.

One of the first Indonesians I met upon arrival in Jakarta was Peter Hehanussa. His assistance in understanding the Indonesian system has been invaluable, and his kind- hearted nature was a wonderful welcome to Indonesia.

Even though my work was conducted in a remote part of the world, Pt. Inco made my time there relatively luxurious. I would particularly like to thank Jim Gowans, Berno

Wenzl, Sulvi Suardi, and Lili for facilitating my stays in Salonsa. I would also like to thank our boat drivers Wahab, Nus Allum, and Pak Dahlan. I would like to especially thank Dahlan for trying his best to teach me Indonesian with a smile on his face, even when I came up with such phrases as "Careful, there's a duck on your coconut!", instead of, "Careful, there's a bee on your head!".

There were a number of people who made my visits to Sulawesi memorable, and in some cases life-changing. For her generosity, honesty, perpetual supply of Heineken, and for welcoming me into her home, I thank Tracie Fenato; for her excellent meals that always consisted of meat, I thank Kelly LeFroy; for reminding me that there is life beyond work, I thank the Soroako Hash House Harriers and a particular frog. There is also a swam of bees that I should thank for reiterating the latter point.

None of my fieldwork could have been completed without the dedication and hard work of my field assistants, Amanda Crawford, Daniel Holme, Alex Robertson, and

Sierra van der Meer. Ibu Mandi and Mr. Alex, thank you not only for your perseverance in the field, but for the incredible friendship that you provided during our adventures in the tropical jungles, lakes and seas of Indonesia. Sierra, thank you for your indomitable spirit at a time when being in the field was difficult; you reminded me why I love

Indonesia. Thanks to all of you for laughing hysterically with me when lighting was crashing around our metal boat, we got the fifth tlat tire in as many days, the boat driver dropped us off on an island and forgot about us, and they served tripe at the TAB.

The Dillery has been a place of growth and learning for me. I would sincerely like to thank the Dill Lab for their camaraderie over the past few years, especially Megan

Kerford, Aaron Wirsing, Jeremy Mitchell and Sandra Webster. Sandra, I owe you a special thank you for being such a patient office-mate and friend. I also want to specially thank Andrea Pomeroy for reading en~barrassingdrafts of manuscripts, listening to my rants, and for being such a close friend. Go FAB*Lab; what more do I need to say?

To the girl who bothered to talk to me in that long line-up my first week at SFU, and invited me into her life after only an hour of conversation, thank you, Adrienne. By bringing me into your life, you also introduced me to the "Girls". I hope all of you know how much "Girls Night" meant to me. To all of n1y wonderful friends, both in school and out, your support has been incredible. Thank you! Who would have thought that the girl I fought with over a window more than ten

years ago in a dorm room would be such a constant figure in my life throughout three

science degrees? My Jules, thank you for everything; your support, your understanding,

your honesty; your friendship.

Although I was physically far away from them for most of this degree, I would not be at this point without the loving support of my family. Heather and Erin, you have both been so understanding and supportive, thank you. Mum and Dad, you always said I could be whatever I wanted to be; whether it was an astronaut or a farmer, you were always there, supporting, encouraging, and loving. Thank you for everything.

Finally, the telmatherinids of the Malili Lakes are incredibly beautiful fish; they are not, however, very cooperative when it comes to being handled. Despite this, they revealed themselves to me in surprising and interesting ways, without which this thesis would be considerable shorter. Terima kasih untuk segalanya! TABLE OF CONTENTS

Approval ...... ii ... Abstract ...... 111 Acknowledgements ...... v Table of Contents ...... ix List of Tables ...... xi .. List of Figures ...... xi1 Synthesis ...... 1 Literature Cited ...... 13 Chapter 1 Linking colour polymorphism maintenance and speciation...... 16 1 . 1 Abstract ...... 17 1.2 Introduction ...... 18 1.3 Between-population processes ...... 18 Genetic drift and gene flow ...... 19 Divergent selection and gene flow ...... 20 Sensory bias ...... 23 1.4 Wi thin-population processes ...... 24 Disruptive selection ...... 25 Frequency-dependent selection ...... 26 1.5 Conclusions ...... 30 1.6 Acknowledgements ...... 31 1.7 Literature Cited ...... 32 I .8 Glossary ...... 38 1.9 Boxes ...... 39 Disruptive correlational selection and CP evolution (Box 1.1)...... 39 Outstanding Questions (Box 1.2) ...... 41 1.10 Tables ...... 43 1.1 1 Figures ...... 45 Chapter 2 A comparative description of mating behaviour in the endemic telmatherinid fishes of Sulawesi's Malili Lakes ...... 49 2.1 Abstract ...... 50 2.2 Introduction ...... 51 2.3 Materials and Methods ...... 52 . . Species recogn~t~on ...... 53 Sampling Procedure ...... 54 2.4 Results and Discussion ...... 55 Generalized mating behaviour ...... 55 Interspecific comparison of mating behaviours (See Table 2.2) ...... 58 2.5 Conclusion ...... 64 2.6 Acknowledgements ...... 65 2.7 Literature Cited ...... 65 2.8 Tables ...... 67 2.9 Figures ...... 70 Chapter 3 The role of environment-contingent sexual selection in the maintenance of male colour polymorphism in Tehtatherinn sarasinorrrm ...... 76 3.1 Abstract ...... 77 3.2 Introduction ...... 78 3.3 Methods ...... 83 Colour analyses ...... 84 Between-habitat analysis of reproductive success ...... 87 Within-habitat analysis of reproductive fitness ...... 90 3.4 Results ...... 92 Colour analyses ...... 92 Between-habitat analysis of reproductive success ...... 93 Within-habitat analysis of reproductive fitness ...... 95 3.5 Discussion ...... 96 3.6 Acknowledgements ...... 104 3.7 Literature Cited ...... 105 3.8 Tables ...... 110 3.9 Figures ...... 112 Chapter 4 Cuckoldry incites cannibalism: Male fish turn to cannibalism when perceived certainty of paternity decreases ...... 117 4.1 Abstract ...... 118 4.2 Introduction ...... 119 4.3 Methods ...... 120 Study organism and mating system ...... 120 Observational study and analyses ...... 122 4.4 Results ...... 124 4.5 Discussion ...... 126 4.6 Acknowledgements ...... 129 4.7 Literature Cited ...... 129 4.8 Tables ...... 131 4.9 Figures ...... 132 Appendix 1 Colour photographs and movies on CD-ROM ...... 134 Figure Legends ...... 134 Appendix 2 Microspectrophotometry of cones from Telmatlterina bonti and Marosntlterina Iadigesi ...... 136 Literature Cited ...... I38 Appendix 2 Tables ...... 139 Appendix 2 Figures ...... 140 LIST OF TABLES

Table I .I : Representative examples of model colour polyn~orphicsystems and processes promoting colour polymorphism...... 43 Table 2.1 : Terms used to describe general behaviours performed by males and/or females during mating, using existing terminology where applicable (Mortez & Rogers 2004). Males accompanying a female are called "paired males"; those without a female are called "single males"...... 67 Table 2.2: A comparison of mating behaviours between seven species of telmatherinid fishes from Lakes Matano ...... 68 Table 3.1 : Location of sites (GPS coordinates) and sample sizes for transect counts, and male and female focal follows for each of eight permanent sampling sites (12 transects) around Lake Matano...... 1 10 Table 3.2: Mean difference between blue and yellow males for individual variables (defined in text) influencing reproductive success...... 1 1 1 Table 4.1 : The proportion of spawning events for which females and males of each behavioural category were present and the proportion of spawning events where they were present that each attempted to . . cannibalize eggs...... 13 1 Table 4.2: Proportion of filial cannibalism by focal courting males in the presencelabsence of conspecific males, either sneakers or non- mating males (i.e., cannibals that did not participate in the spawning event)...... 1 3 1 LIST OF FIGURES

Figure 1.1 : Exan~plesof model colour polymorphic species...... 45 Figure 1.2: Visual ecology and a heterogeneous environment ...... 47 Figure 2. I: A map of the Indonesian archipelago with island details...... 70 Figure 2.2: Graphical description of some aspects of generalized telmatherinid mating behaviour ...... 72 Figure 2.3: A graphical representation of Trlmuther-ina sar-asinor-tun sneaking and egg cannibalism behaviour at a root spawning site...... 74 Figure 3. I: Map of Lake Matano, Sulawesi ...... 1 12 Figure 3.2: Mean background radiance spectra (normalized to photon flux of 1) for beach (0;n = 6 sites) and root (+;n = 6 sites) mating habitats...... 1 13

Figure 3.3: Mean morph frequency (nbcach= 6 sites, n,,,, = 6 sites) (+ 1 SE) of all five male colour morphs at beach and root mating habitats. Pair- wise Tukey HSD tests within a habitat show significant differences (a > b > c > d) between morphs at a = 0.05...... 1 14 Figure 3.4: Mean fitness (-+ 1 SE) differs significantly between blue and yellow male colour morphs in the two main spawning habitats (one-tailed t- tests: beach t = 1.938, d.f. = 10, P = 0.04; root t = -2.606, d.f. = 10, P - 0.013)...... 115 Figure 3.5: Sensitivity analyses for the fitness values used in the fitness model ...... 116 Figure 4.1 : Courting T. saruainoi-mz...... 132 Figure 4.2: Probability of filial cannibalism by courting male T. sai-usinontin in the absence (0 sneakers, black bar, n = 41 5) or presence (1 sneaker, white bar, n = 35; 2 or more sneakers, hatched bar, n = 20) of sneakers (plus one binomial standard error (Zar 1984))...... 133

xii SYNTHESIS

Charles Darwin devoted a considerable amount of space in "The Origin of

Species'' to describing the large amount of variation that exists in nature. Particularly

<( perplexing" to Darwin were cases in which distinct varieties occur within a species:

"There is one point connected with individual differences, which is extremely perplexing: I refer to those genera which have been called "protean" or "polymorphic," in which the species present an inordinate amount of variation;" (Darwin 1869, p. 5 1)

Darwin goes on to note several examples of discrete variation such as the shell colour patterns of brachiopods, the plumage variation in male Ruffs (Machetes pupax), and wing colour pattern differences between some Malaysian female butterflies. The latter examples have since become classics in the study of colour polymorphism, especially with advances in genetic knowledge during the mid-1900's brought about by Huxley

(1 955), Ford (1965) and others. Huxley (1 955) formally defined colour polymorphism as the occurrence of two or more distinct colour morphs within an interbreeding population, the rarest of which is too frequent to be due solely to recurrent mutation. Researchers have since spent an enormous amount of time and effort in trying to understand how such variation is maintained in nature. This thesis represents my exploration of questions about the maintenance of intraspecific diversity in nature, using a relatively unknown system of fishes endemic to the island of Sulawesi, Indonesia; and what better place to do so than

on an island so eloquently described by Alfred Wallace in "The Malay Archipelago":

"Situated in the very midst of an Archipelago, and closely hemmed in on every side by islands teeming with varied forms of life, its productions have yet a surprising amount of individuality. While it is poor in the actual number of its species, it is yet wonderfully rich in peculiar forms, many of which are singular or beautiful, and are in some cases absolutely unique upon the globe." (Wallace 1869, p.2 18)

My thesis is structured such that I begin with an exploration of the processes believed responsible for the maintenance of colour polymorphism and which may also

lead to the generation of new species, through a synthesis of a burgeoning literature. I then introduce the telmatherinid fishes of Sulawesi's Malili Lakes; a system likely to be an exceptional model for studying questions about adaptive radiation and animal colouration. I next focus on a single species, Telrnafherina sarasinorum, to further investigate hypotheses about the role of sexual selection and complex mating behaviours in maintaining male colour polymorphism in a spatially heterogeneous environment.

The aim of my review, entitled "Linking colour polymorphism maintenance and speciation" (Gray and McKinnon 2007; Chapter l), was to synthesize a large body of research concerning the processes involved in maintaining colour polymorphism and that may in some cases lead to speciation. Several underlying themes emerged from this synthesis. First, there are two literatures, one concerning questions about how selection can maintain multiple morphs within a species over time, and the other concerned with answering questions about the evolution of reproductive isolation between different colour forms. What is not necessarily appreciated in the literature is that the processes involved may be the same in both cases, but that speciation is not necessarily the end point, rather one extreme of a continuous distribution: at one extreme multiple morphs are maintained, at the other morphs become species, and in between are systems that range from unstable morph frequencies to only weakly or asymmetrically reproductively isolated forms. Second, it is necessary to consider how selection might influence colour variation both between- and within-populations. For example, selective pressures dependent upon the visual environment may be fine-scaled such that individuals within a population experience different environments, or coarse-scaled such that populations experience different environments. Finally, in reviewing the colour polymorphism literature it became apparent that the maintenance of such diversity is rarely due to only one mechanism; often several selective processes work simultaneously to produce the observed variation in colour patterns. Furthermore, it is important to realize that colour may not always be the target of selection, but that colour can be correlated with other traits and maintained by correlational selection. Future investigations should therefore consider multiple processes over multiple scales to really understand when and how colour polyn~orphismevolves, persists, or leads to speciation.

I wanted to directly address questioiis about the relative role of selection in maintaining intraspecific diversity and so chose a system, recently hailed as "Wallace's

Dreamponds" (Herder et al. 2006), that will probably be recognized as a good model for studying adaptive radiation in the near future. However, almost nothing was known about the behavioural ecology of this group of fishes when I started this thesis in 2002 (other than taxonomic descriptions (Kottelat 1990, 199 1)). Consequently, I next introduce the Tel~natherinidae(Atheriniformes), a family of fishes found almost exclusively on

Sulawesi, Indonesia, in a paper entitled, "A comparative description of the mating

behaviour of the endemic telmatherinid fishes of Sulawesi's Malili Lakes" (Gray and

McKinnon 2006; Chapter 2). Given the recent interest in these fishes, especially with

respect to the phylogenetic relationships among them (Roy et al. 2004; Herder et al.

2006), it is essential that some thought be given to the ecology of the group. The

telmatherinids, called 'opudi' in the local dialect, or sailfin silversides by researchers, are small, often brightly-coloured fishes found throughout the five lakes and numerous streams of the Malili Lakes drainage (see Appendix 1, Figure A I. 1 for map). All of the telmatherinids found in Lake Matano, the most isolated lake, are endemic, while several species found in the largest lake, Towuti, are also found in neighbouring Lakes Mahalona and Wawantoa but are not found elsewhere. Several "stream" forms are found throughout the system, collectively called Tdtnufhet.ina honti (although this will likely change as more thorough genetic analyses are conducted). Current research suggests there has been introgressive hybridization between stream and lake forms, which may have contributed to the radiation of the fishes in the system (Herder et al. 2006). Most of the telmatherinids are male colour polymorphic with monomorphic, cryptically coloured females. There are no observed sister-species distinguished by male colouration as is often seen in the

African Great Lakes cichlids (Kocher 2004). They do vary in trophic morphology and appear to have diverged to fill many available niches, often in parallel with other freshwater fishes. For example, Telt~lafherina "whitelips" (Lake Matano) has large fleshy lips and is very similar in appearance to fleshy-lipped cichlids (eg, Cheilochmn~is euchilza) of Lake Malawi, Africa (Arnegard and Snoeks 200 1). My coniparative description of the reproductive behaviours of seven male colour polymorphic telmatherinids, four from Matano and three from Towuti, showed that the general mating behaviours of the group are conserved. However, several potentially adaptive exceptions to the generalized reproductive ethogram were discovered. For example, males of all seven species (from three different genera) perform a circling behaviour near females while trying to entice them to spawn on the substrate, but the size of thc circle differs substantially among species and is severely restricted in a species found in fast-flowing water. One of the most interesting observations was that the two species with the greatest within population colour variation, one from each major lake, utilize at least two distinct spawning habitats: shallow beach habitat and deeper habitats that drop-off steeply from shore, each habitat providing a different visual background

(see Appendix 1, Movies A I. 1, A 1.2). Such spatial variation in the visual environment could influence the direction of selection in each habitat and therefore contribute to the maintenance of male colour polymorphism.

In order to test the hypothesis that spatial heterogeneity in the visual environment contributes to the niaintenance of male colour polymorphism, I focused on one species,

Telmcrthei-inu surtrsinorwm, endemic to Lake Matano ("The role of environment- contingent sexual selection in the niaintenance of male colour polymorphism in

Telmtrtherinu sc~r.usinonrml'jChapter 3). Males of this species can be one of five colour morphs while females are cryptically coloured (see Appendix 1, Figures A I .2-A I .7). All morphs are found at varying frequencies in the beach and root mating habitats described above. Under sexual selection theory we expect females to prefer the most conspicuously coloured males (i.e., the ones that contrast most with the background) (Andersson 1994). However, the perception of male conspicuousness is expected to depend upon the visual

background against which he is viewed; thus the direction of sexual selection may change depending upon the environnient in which mating takes place (Endler 1990; Endler

1992), a process known as en\rironment-contingent sexual selection. The beach habitat provides a more yellow background than the root habitat and the root habitat is bluer than the beach habitat. Blue inales are expected to contrast more in the beach habitat and therefore be favoured by sexual selection while yellow niales should be favoured in the root habitat. This prediction was met. The fact that we find all morphs in both habitats suggests that some form of opposing selection may favour less conspicuous morphs or that there is movement of the morphs between mating habitats. Conspicuous morphs might be selected against via increased male-male competition and cuckoldry since they are also more conspicuous to other males. A second form of opposing selection could be predation against the more conspicuous morphs. Evidence supporting a balance between sexual selection for, and natural selection against, conspicuousness is found i11 other systems where niales have conspicuous secondary sexual characteristics (Andersson

1994). However, there are no known large piscivores in Lake Matano (personal observation). A survey of the largest fish in the lake, Glossogobita sp. did not find telinatherinids in their diet, although it was a limited sample. Some pisciverous birds, such as sea eagles (Hulj~~e1~1.~sp.) and cormorants (Phal~~cr-ocorurxsp.), are sometimes seen on the lake but seldoni near the shores where mating takes place. The most likely source of predation is conspecific egg cannibalism, which still fits the general prediction that more conspicuous males, while favoured by females, may be more susceptible to egg cannibalism after spawning because they are more visible. Finally, while the direction of selection may vary between habitats and thereby allow all morphs to persist within the

lake, it is possible that males move between habitats, resulting in the observed frequencies of morphs within habitats. Despite several valiant attempts, I was unable to collect mark-recapture data because these fish are particulasly sensitive to being handled and unfortunately did not survive the initial procedures. 1 have observed T. snrnsinor~~m moving from the mating habitat to deeper waters late in the afternoon and returning in early morning, suggesting that movement between habitats, especially when they are adjacent, is likely. I do not know if fish found in particular habitats represent distinct populations; however, even if individual males experience both environments, the direction of selection should remain the same within a habitat.

Using a model parameterized with data collected from extensive field observations of mating behaviour over three field seasons, I tested for differences in iltness between colour morphs in two visual environments. By comparing the fitness values derived from the model for the two most conspicuous and common n~orphs,blue and yellow, I found that blue males have higher fitness in the beach habitat where they contrast more against the yellowish background than yellow males and are more frequent, and that the opposite is true in the root habitat where yellow males dominate. Despite knowing very little about the movement of fish between habitats, the sesults suggest that spatial variation in the visual environment influences the maintenance of male colour polymorphism in T. sctr.nsinor.um because the direction of selection depends on the habitat.

While observing the complex mating behaviours of T. surtrsinorz~rnI discovered a relationship between the presence of cuckolders and egg cannibalism, which I examine in detail in a paper entitled " Cuckoldry incites cannibalism: Male fish turn to cannibalism

when perceived certainty of paternity decreases" (Gray et al. 2007; Chapter 4). I found

that the probability that a courting male attempts to cannibalize eggs (that he has possibly

just fertilized) increases significantly in the presence of cuckolders. Furthermore, I found

that this relationship is positively associated with the number of sneakers present: as the

number of sneakers present at a spawning event increases, so does the probability that the

courting male will attempt to cannibalize the eggs. This suggests that as a male's

perceived certainty of paternity decreases, he is more likely to trade-off current

reproductive success with future reproductive effort by consuming eggs that were less

likely to be fertilized by his sperm. The probability of a blue male being cuckolded in the

beach habitat is higher than for yellow males, and vice versa in the root habitat,

supporting the prediction that conspicuousness can be disadvantageous. By extrapolation,

males experiencing increased cuckoldry should also have their eggs eaten more often.

What is particularly interesting about this finding is that the theory pertaining to

the evolution of filial cannibalism (the eating of one's own offspring) as an adaptive

behaviour has focused entirely on systems in which males provide some form of parental

care (Rohwer 1978; Dominey and Blumer 1984: Lindstrom 2000; Manica 2002). Neither

male nor female T. sui.usinorzm provide care, rather they abandon the eggs immediately

after spawning. I believe that this finding will help advance the fields of cannibalism

evolution and parental care evolution. For example, by including this case in a general model, it would be possible to examine filial carmibalism over a range of mating systenls from no parental care with decisions to eat or not to eat, through to full parental care and decisions about how much care to give, including whether to eat some or all of the offspring being cared for.

Ultimately it would be useful to have more detailed information on the role of female mate choice and male-male competition in T. sninsit~ot-z~m.It may not be possible to test for female preferences until captive populations can be maintained so that it can be determined how many eggs are being laid in different circumstances (e.g., with one male present versus two) and if females actually prefer more conspicuous males (i.e., using experiments that manipulate lighting conditions). On two occasions I observed an extremely interesting female behaviour. In the middle of a swarm of courting male fish I noticed a female that was not moving, but rather floating on her side and slowly drifting away from the group of males. I assumed she was dead, but at some distance from the males (who were vigorously fighting with each other) she quickly flipped upright and swam away. This 'playing dead' behaviour could be a tactic for females to avoid mating in situations where there would likely be several males present as sneakers and thus very likely that any eggs laid would be eaten.

With respect to male-male competition, future investigations should focus on determining how much sperm males expend when they are alone with a female versus when sneakers are present, or when they are sneaking (e.g., see Engqvist and Reinhold

(2006)). Also, knowing something about fertilization success of males when there is competition in the form of sneaker ejaculates is cnicial. Such information could be added to the fitness model as the probability of fertilization, and hence provide an even more direct measure of male reproductive fitness. The influence of male fighting on female behaviour could also be very interesting to examine, but again experimental work will be necessary.

On a larger scale there is an interesting relationship between T. sa~nsino~urnand a closely related species, T. antoniae, also endemic to Lake Matano. Male T. antoniae are either yellow or blue, or (less frequently) blue with yellow fins. This species spawns predominantly in shallow beach zones with mating occurring mostly during mid- morning. Male T, .sar.asinort/nz follow courting T. antoniae pairs and will often attempt to eat their eggs (I have found T. antoniae-sized eggs in the stomachs of T. sar-asinor-trm males). Male T. snmsinorurn appear to guard a single T. antoniue pair by fighting off all other approaching fish, including male T. antorline males that might otherwise have challenged the paired T. untonine male to a fight. In this way it is conceivable that the paired T. antonine males tolerate the presence of T. sarasinomm during spawning because they do the fighting for them, at a cost of occasionally losing eggs to cannibalism. One of the most unusual behaviours I have observed in T. sarasinorrrm males is what I tern1 'sneaky eating'. The male T, snrt~sinorvmthat is guarding a pair of mating T. antonicre will chase off the paired T. antonicre male and begin courting the T. trntoninr female. When the mis-matched pair goes to the substrate to spawn, the female

T. antoniue quivers as per usual but the T. surasino~-ummale does not; instead he turns around immediately and attempts to eat the eggs that the heterospecific female has just laid. The relationship between guarding heterospecific pairs and egg-eating is bizarre and deserves further attention, especially with respect to the colour of both the T. surusinorurn male and the T. antonicre male whose eggs are being cannibalized, and the effect that this might have on female T, trntoniue mate choice. If egg predation pressure is higher on more conspicuous morphs, females may prefer to mate with less conspicuous morphs. It is possible that ten~poralvariation in the lighting environment in beach habitats may play a role in maintaining male colour polymorphism in T. untonitre, however egg predation pressure might be much stronger for this species than in T. sar~lsinorzrrnwhere it is generally associated with cuckoldry, which only happens during

12% of spawning events. Further investigation and modelling of this relationship are necessary. The even more bizarre 'sneaky eating' behaviour would also certainly be worth investigation, although this may be difficult as the behaviour was only observed four times over the course of three extensive field seasons.

Relationships between telmatherinids of the Malili Lakes are only beginning to be explored, as we know so little about the system as a whole. There are many opportunities for parallel studies between colour polymorphic species within lakes and between lakes.

For example, the difference in spatial versus temporal variation in the visual environment for T. .sur-~lsitior.2rn-1and T, antoniue, respectively; both T. sur-tlsinot-urn and T. celebensis

(Lake Towuti) are egg eaters; T. satminorzrm and Toniincrnya species (Lake Towuti) both utilize beach and root habitats for spawning and have the most colour morphs. In the future it should be possible to advance our understanding of this system and put it into a more general theoretical context of adaptive colouration and adaptive radiation, by examining the system at several different scales: within species, between species within a lake, between similar species in different lakes, and at the whole system scale. Further to this, examination of the genetic relationships between groups and the visual physiology of the telmatherinids, combined with more detailed assessments of the natural visual environments, may shed some light on the evolution of colour polymorphism in this

unique family of fishes.

One of the crucial findings of my thesis is that spatial heterogeneity in the visual

environment contributes to the maintenance of colour polymorphism in at least one, and I

suspect several, of the telmatherinids. I would be remiss to neglect to report, at least

briefly, on the significance of this finding with respect to the conservation of such unique

animals. If variation in the visuaI environment influences the maintenance of colour

diversity, then it is safe to predict that the homogenization of the environment will

directly affect the maintenance of this diversity. Unfortunately, this prediction has been

tested in Lake Victoria, Africa through human-induced increases in water-turbidity. A

significant decrease in cichlid diversity has been reported in areas where eutrophication

has decreased visibility and changed the colour of the water (Seehausen 2006; Seehausen

et al. 1997). It is thought that a break-down in reproductive isolation based on colour-

specific female mate choice has led to hybridization between insipient species-pairs.

Increases in turbidity are known to affect both the survival (Abrahams and Kattenfeld

1997) and reproduction (Jarvenpaa and Lindstrom 2004; Seehausen 2006) of numerous

other fish species around the world. Lake Matano is still considered a pristine tropical

lake, yet it in the four years that I have studied this system, areas of the lake are already experiencing at least short-term bursts of turbidity due to lake-side deforestation, road construction, pollution and other mining activities. The effects of such changes to the visual environn~entare not known in this lake but this study, combined with evidence from other sirnilas systems, suggests that pro-active measures should be taken to protect the natural environmental heterogeneity in the Malili Lakes before it is irrevocably damaged.

Finally, my exploration of the question, how is intraspecific colour diversity maintained in nature, has led to several conclusions. Given the inherent link between colour signal perception and the environment in which signals are viewed, it is extremely important to consider variation in the visual environment with respect to understanding how selection might act on such signals. In a broader context, the scale of environmental variation is also important, especially with respect to population structure and the scale over which selection occurs and therefore its effect on the maintenance of colour polymorphism.

Literature Cited

Abrahams, M., and M. Kattenfeld. 1997. The role of turbidity as a constraint on predator- prey interactions in aquatic environments. Behavioral Ecology and Sociobiology 40: 169- 174. Andersson, M. 1994, Sexual Selection. New Jersey, Princeton University Press. Arnegard, M. E., and J. Snoeks. 2001. New three-spotted cichlid species with hypertrophied lips (Teleostei: Cichlidae) from the deep waters of Lake MalawiINyasa, Africa. Copeia 200 1 :705-7 17. Danvin, C. 1869, The Origin of Species. New York, The Modern Library. Dominey, W. J., and L. S. Blunier. 1984. Cannibalism of early life stages in fishes, Pages 43-64 in G. Hausfater, and S. B. Hrdy, eds. Infanticide: Comparative and Evolutionary Perspectives. New York, Aldine Publishing Co. Endler, J. A. 1990. On the measurement and classification of colours in studies of animal colour patterns. Biological Journal of the Linnean Society 41 :3 15-352. -. 1992. Signals, signal conditions and the direction of evolution. American Naturalist 139:s 125 -s 153. Engqvist, L., and K. Reinhold. 2006. Sperm competition games: optimal sperm allocation in response to the size of competing ejaculates. Proceedings of the Royal Society of London B Online: doi: 10.1098/rspb.2006.3722. Ford, E. B. 1965, Genetic Polymorphism. Cambridge, The MIT Press. Gray, S. M., and J. S. McKinnon. 2006. A conlparative description of mating behaviour in the endemic telmatherinid fishes of Sulawesi's Malili Lakes. Environmental Biology of Fishes 75:469-480. -. 2007. Linking color polymorphism maintenance and speciation. Trends in Ecology and Evolution 22:7 1-79. Gray, S. M., L. M. Dill, and J. S. McKinnon. 2007. Cuckoldry incites cannibalism: male fish turn to cannibalism when perceived certainty of paternity decreases. American Naturalist 169:258-263. Herder, F., A. W. Nolte, J. Pfaender, J. Schwarzer, R. K. Hadiaty, and U. K. Schliewen. 2006. Adaptive radiation and hybridization in Wallace's Dreamponds: evidence from sailfin silversides in the Malili Lakes of Sulawesi. Proceedings of the Royal Society of London B 273:2209-22 17. Huxley, J. 1955. Morphism and evolution. Heredity 9: 1-52. Jarvenpaa, M., and K. Lindstrom. 2004. Water turbidity by algal blooms causes mating system breakdown in a shallow-water fish, the sand goby Pomaloschislus min~l~u.Proceedings of the Royal Society of London B 27 1 :2361-2365. Kocher, T. D. 2004. Adaptive evolution and explosive speciation: the cichlid fish model. Nature Genetics 5:288-298. Kottelat, M. 1990. Sailfin silversides (Pisces: Telmatherinidae) of Lakes Towuti, Mahalona and Wawontoa (Sulawesi, Indonesia) with descriptions of two new genera and two new species. lclithyological Exploration of Freshwaters 1 :35-54. -. 199 1. Sailfin silversides (Pisces: Telmatherinidae) of Lake Matano, Sulawesi, Indonesia, with descriptions of six new species. Ichthyological Exploration of Freshwaters 1 :32 1-344. Lindstrom, K. 2000. The evolution of filial cannibalism and female mate choice strategies as resolutions to sexual conflict in fishes. Evolution 54:617-627. Manica, A. 2002. Filial cannibalism in teleost fish. Biological Reviews 77:261-277. Rohwer, S. 1978. Parent cannibalism of offspring and egg raiding as a courtship strategy. The American Naturalist 12:429-440. Roy, D., M. F. Docker, P. Hehanussa, D. D. Heath, and G. D. Haffner. 2004. Genetic and morpl~ologicaldata supporting the hypothesis of adaptive radiation in the endemic fish of Lake Matano. Journal of Evolutionary Biology 17: 1268- 1276. Seehausen, 0. 2006. Conservation: Losing biodiversity by reverse speciation. Current Biology 16:334-337. Seehausen, O., J. J. M. van Alphen, and F. Witte. 1997. Cichlid fish diversity threatened by eutrophication that curbs sexual selection. Science 277: 1808- 18 1 1. Wallace, A. R. 1869, The mal lay Archipelago. Singapore, Periplus Press. CHAPTER 1

LINKING COLOUR POLYMORPHISM MAINTENANCE AND SPECIATION

Gray, S. M., and J. S. McKinnon. 2007. Linking color polymorphism maintenance and speciation. Trends in Ecology and Evolution 22: 7 1-79. 1.1 Abstract

Here, we review the lately burgeoning literature on colour polymorphisms, seeking to integrate studies of the maintenance of genetic variation and the evolution of reproductive isolation. Our survey reveals that several mechanisms, some operating between and others within populations, can contribute to both colour polymorphism persistence and speciation. As expected, divergent selection clearly can couple with gene flow to maintain colour polymorphism and mediate speciation. More surprisingly, recent evidence suggests that diverse forms of within-population sexual selection can generate negative frequency-dependence and initiate reproductive isolation. These deserve additional study, particularly concerning the roles of heterogeneous visual environments and correlational selection. Finally, comparative studies and more comprehensive approaches are required to elucidate when colour polymorphism evolves, persists, or leads to speciation. 1.2 Introduction

Ever since Huxley (Huxley 1955), colour polymorphism (CP) has been defined as the presence of two or more distinct, genetically determined colour morphs within a single interbreeding population, the rarest of which is too frequent to be due solely to recurrent mutation. A recent resurgence in CP research has focused on two areas fundamental to evolutionary biology: the study of the processes maintaining genetic variation in nature and the study of speciation. Here we review the literahlres of these two fields, seeking in particular to identify insights arising from joint consideration of these topics and to highlight areas ripe for further research. We begin by considering processes operating between populations, including differences in the visual environment that may influence both natural and sexual selection. We then consider those processes that may act entirely within populations to maintain CP; these are of particular interest because they could contribute to sympatric speciation by sexual selection, a controversial process

(van Doom et al. 2004; Gavrilets and Hayashi 2005). We focus our attention on those systems that have been studied the most thoroughly, and especially those in which both

CP persistence and the possible contribution of CP to the evolution of reproductive isolation have been investigated (e.g., Table 1.1, Figure 1.1). Although plants provide many examples of CP, we limit our survey to animals.

1.3 Between-population processes

Regardless of the evolutionary forces acting within a population (e.g., random genetic drift or directional selection), we expect that gene flow (see 1.8 Glossary) between divergent populations will play a nearly ubiquitous role in determining the degree to which populations diverge. Here we address two issues: 1) the contribution of non-adaptive processes, genetic drift and gene flow, to CP maintenance, and 2) the

selective pressures involved in divergence in colour morph frequencies between

populations, particularly the scale and consistency of the local visual environment and

how these interact with gene flow to mediate CP persistence and speciation.

Genetic drift and gene flow

In a few well-studied cases, there appears to be a role for random genetic drift in

maintaining CP (Hoffman et al. 2006), despite the fact that within populations, random

genetic drift should lead to the fixation of one colour morph. We only expect drift to

maintain CP in con-junction with other evolutionary forces (e.g., frequency-dependent

selection, temporal variation in selection, or gene flow) acting on the population;

consequently, drift might be difficult to detect (e.g., see Svensson and Abbott (2005)).

The 'locus comparison approach' compares genetic variation between genes involved in

CP and putatively neutral genetic markers (Hoffman et a]. 2006). In one case, the

northern leopard frog, Rar~upipiens, the use of this approach suggests that drift rather than selection maintains CP (Hoffman et a]. 2006). Perhaps most interesting, however, is the finding that genetic drift can intermittently influence morph frequencies if the strength of selection varies temporally (O'Hai-a 2005; Oxford 2005; Hoffman et al. 2006).

Oxford (2005) proposes that when the frequency of one morph (mdimita) of the candy- stripe spider (Enoplognathn ova6~1)is low (between -0.05 and 0.3), weak selection operates and drift appears to mediate morph frequencies across generally small populations. By contrast, strong selection and possibly gene flow are thought to protect

CP when a perturbation changes morph frequencies, although the agent of selection is not known. More long-term studies that test for a genetic signature of selection, both between and within populations, are needed to help determine the relative contribution of drift to

the maintenance of CP in a larger sample of CP systems (O'Hara 2005; Hoffman et al.

2006).

Divergent selection and gene flow

The terms divergent and disruptive selection are often used interchangeably in the

CP literature; however, we distinguish them to clarify how each might contribute to CP evolution. Divergent selection occurs between two environments, each experienced by different populations (Schluter 2000), whereas disruptive selection in the generic sense describes selection for extreme phenotypes over intermediate forms within a single population (Rueffler et al. 2006). The scale of environmental variation is important, in that we expect divergent selection when individuals generally experience only one environment (broad-scaled variation), whereas we expect disruptive selection if individuals experience multiple environments or "n~icrohabitats"(fine-scaled variation).

Unfortunately the associated line in nature is seldom so easy to draw, especially along environmental gradients.

Divergent selection in different visual environments could favour one colour type while gene flow might simultaneously enable alternative colour types to persist within the same populations (Kapan 200 1; Hoekstra et al. 2004; Crispo et al. 2006). Environment- contingent natural and sexual selection, the processes whereby morphs experience differential fitness dependent upon the environment (see Figure. 1.2) (Boughman 200 1 ; Fuller et al. 2005b) should favour alternate morphs in alternate light environments.

Distinct visual environments should play a role in promoting CP, depending on the consistency (or predictability) of environmental variation (Endler 1992; Endler et al.

2005; Roulin 2004; Fuller et al. 2005b). In general, consistent variation and selection are more likely to lead to divergence.

One of the best emerging examples of CP maintained by divergent natural selection and gene flow between alternate habitats is the convergent evolution of blanched and dark dorsaI coloration in the lizards of White Sands Ecotone (Rosenblum

2006). Distinct light and dark habitats select for crypsis via predation. Three lizard species have each evolved two genetic colour inorphs (i.e., polymorphism in the melanocortin-1 receptor (Mcli.) gene (Rosenblum et al. 2004)). In a set of elegant tests,

Rosenblum (2006) showed that the degree of CP (i.e., amount of phenotypic divergence) is directly related to the level of gene flow between the habitats based on species-specific population structure: Holhrookiu mactrlutu is patchily distributed and shows the greatest amount of phenotypic and genetic divergence relative to Scelopotus trnu'2rlatz4s and

A.~pic/osc.eli.~inornuta, which range continuously among the habitats. Striking similarities in light and dark CPs (e.g., in the beach mouse (Peroi?i~~sctr.spolionotz~s)(Hoekstra et al.

2006); as well as other mammals and birds (Mundy 2005; Nachman 2005)), coupled with variations at the gene encoding ~llclr(Hoekstra 2006), suggest that this pattern is common and that a balance between divergent selection and gene flow may frequently act to maintain CP.

In a less intuitive situation, multiple Miillerian mimic morphs can be maintained if multiple co-models are spatially segregated as separate mimicry rings, creating a mosaic of alternate selective environments (Kapan 2001; Symula et al. 2001; Joron and

Iwasa 2005; Kronforst et al. 2006). Helico~izabutter'rlies that face positive frequency-

dependent selection in a given locality, but that can migrate between mimicry rings,

provide the best example of this form of between-population morph persistence ( Kapan

200 1 ; Joron and Iwasa 2005). Where mimicry rings are separated on a larger geographic

scale (e.g., in the sister species H. cycr'r.70 yalan!h~rsand H. pachinus, which are separated

by a mountain range), there is evidence for reproductive isolation in the form of male

mating preference for wing colour (Kronforst et al. 2006). This finding supports the idea

that the scale of environmental heterogeneity is a key factor determining when CP is

maintained by divergent selection and gene flow, and when it may lead to speciation between populations.

The effect of gene flow on the evolution of reproductive isolation remains controversial, but theoretical analyses increasingly support the plausibility of reinforcement, in which matings between incipient species promote the evolution of reproductive isolation (Servedio and Noor 2003). These issues have been studied in exceptional detail in the walking stick insect Tin7cma cristinae (Figure I. lgh) (Nosil

2004; Nosil and Crespi 2006). Divergent selection on different host plants against less cryptic migrants, hybrids and migrant-native mating pairs makes a major contribution to reproductive isolation in this species (Nosil and Crespi 2006; Nosil et al. 2006).

Assortative mating is strongest at intermediate frequencies of migrants, when encounter rates are sufficient to promote reinforcement but not so great as to swamp divergence.

Although colour pattern is the most conspicuous phenotypic difference between populations of this species, it is not the basis of pre-mating isolation; instead the best candidates are presently pheromones (Nosil et al. 2006). This finding suggests an important caution: even when colour differences between incipient species are obvious, their role in reproductive isolation cannot be assumed automatically.

Sensory bias

Heterogeneous environments provide alternative visual habitats in which the visual systems of colour-signal receivers are expected to be under natural selection

(Boughman 2001 ; Fuller and Travis 2004; Roulin 2004; Carleton et al. 2005; Endler et al.

2005; Fuller et al. 2005a; Maan et al. 2006) (Figure 1.2). We expect natural selection to often favour. visual systems that match the local environment by shifting visual sensitivity to increase contrast (e.g., by shifting toward the predominant colour of the environment,

Figure 1.2b) (Endler 1992). The evolution of visual systems in this way can influence a female's perception of male colour traits and hence her preference for those traits in a given environment, a process known as sensory bias (Fuller et al. 2005b). For example, female visual sensitivity in threespine sticklebacks Gasteroste~~sacl~leut~ls varies depending upon the degree of red-shift in the local light environment, with a corresponding shift in male nuptial coloration (Boughman 200 1). Thus, divergent sexual selection favouring different morphs in alternative light environments could be driven by sensory bias.

Theory predicts that the scale and consistency of environmental variation will influence the evolution of female visual systems (and consequently of mate preferences) and whether speciation or a stable CP should ultimately result. Consistent variation is more likely to lead to consistent selection and divergence, whereas unpredictable changes in the visual environn~entcould favour flexibility in mate choice and the maintenance of CP (Jennions and Petrie 1997). The attenuation of light with depth underwater provides an example of stable environmental variation for which some evidence shows divergent sensory biases and mate preferences in the incipient cichlid species P~ri~c~urnilia piri~cl~~rniliaand P. i~yeiwei(Maan et al. 2006). Variation in cone opsin expression in bluefin killifish (and perhaps many cichlids (Spady et al. 2005)) is determined both genetically and environmentally, suggesting that female preferences could vary as plastic responses to spatial and temporal fluctuations in the visual environment but also be subject to selective pressures (Fuller and Travis 2004).

1.4 Within-population processes

The non-overlapping phenotypic distributions that define CPs can result from genetic or developmental constraints, but are typically expected to be shaped by disruptive selection, which will often be accompanied by the negative frequency- dependent selection important to CP persistence within populations (Rueffler et al. 2006).

Both forms of selection may result from the same mechanisms if, for example, polymorphic female preferences lead simultaneously to poor mating success for intermediate males and greater success for the rarer morph. We consider these two fonns of selection in turn. Genetic drift provides a non-adaptive alternative to these processes, but available evidence (see above) suggests that it typically plays a secondary role to selection and is not considered further here. Disruptive selection

Evidence for the role of disruptive selection in CP evolution has mainly been indirect. In a comparative study of birds, for example, CP was associated with use of both open and closed habitats and with daily rhythms extending across day and night (Galeotti et al. 2003). Disruptive selection was thought to arise because different colour patterns are cryptic in different lighting conditions. Of the few examples providing direct evidence of disruptive selection on colour pattern within a species, one of the best is a study of sexual selection on yearling male lazuli buntings Pnsserina nmoenn (Greene et al. 2000). Very dull and very conspicuous male buntings achieve greater mating success than intermediates, but colour pattern variation is not discretely distributed. The paucity of direct evidence for disruptive selection on CPs might arise from a lack of appropriate studies or from weakened selection owing to previous evolutionary responses (Rueffler et al. 2006). Another plausible explanation, however, is that non-linear selection frequently acts not on individual traits but on suites of characters through 'correlational selection'

(Blows and Brooks 2003), with potentially important implications for speciation (Box

1.1).

Speciation might sometimes be driven by disruptive sexual selection on colouration, with cichlids providing possible examples (Table 1.1). Here, females of different incipient species often prefer males with different colour patterns and assortative mating is reduced under narrowed ambient light spectra ( Seehausen and van

Alphen 1998; Seehausen 2006). Recently, females have been shown to possess directional colour preferences within a morph, thereby selecting for at least one extreme

(Pauers et al. 2004; Maan et al. 2006); disruptive selection, however, has not yet been demonstrated directly. Studies of the passion-vine butterflies H. cydno and H. rnelpomene

(Naisbit et al. 200 1) and of sticklebacks (McKinnon and Rundle 2002) provide evidence

of disruptive sexual selection against hybrid males, although not necessarily because of

their intermediate colour patterns.

Heterosis represents the opposite of disruptive selection because (genetically)

intermediate forms are expected to be favoured. It was historically considered to be a

major process maintaining allelic diversity, including CP (Fuller and Travis 2004; Roulin

2004); however, little recent evidence supports this possibility for CP systems (but see

Tuttle (2003)).

Frequency-dependent selection

Frequency-dependent natural selection, whether relative or absolute (i.e., density

(Sinervo and Calsbeek 2006)) has long been hypothesized to lead to CP maintenance

within populations, particularly through predation (Ford 1965; Punzalan et al. 2005). A

recent study of guppies provides the best experimental evidence yet obtained for a natural

system in support of this hypothesis. Olendorf et al. (2006) manipulated male morph

frequencies in the field and found that rare morphs experienced elevated survival rates.

The mechanism responsible for this apostatic selection is not definitively known, and

recent work suggests that complexities of predator behaviour may have important

implications for CP persistence (Shigemiya 2004; Punzalan et al. 2005). Nevertheless,

laboratory studies using artificial prey provide considerable support for the straightforward hypothesis that predators form a search image for the most common prey types, making them less likely to eat rare inorphs (Punzalan et al. 2005; Bond and Kamil

2006). Such apostatic selection may not contribute directly to speciation, but it might

contribute indirectly by favouring the persistence of multiple colour morphs. This could,

in turn, create conditions under which disruptive frequency-dependent selection on

females, provided males are in some respect limited, leads to a preference polymorphism

and assortative mating (van Doorn et al. 2004).

Negative frequency-dependence can also arise through sexual selection. For

males, several mechanisn~sthat favour lower frequency morphs can lead to a 'rare male

effect'. In probably the best-known scenario, females consistently shift their preference

toward whichever morph is uncommon or novel (eg, as in guppies (Hughes et al. 1999;

Eakley and Houde 2004)), although this effect has been demonstrated only infrequently.

Alternatively, female preferences can be polymorphic, with some females preferring one

male morph and other individuals preferring a different morph. For example, individual

females of the swordtail fish Xiphophorzis cur-tmi strongly prefer either males with or without bar patterns (Morris et al. 2003). Although heritable preference polymorphisms have rarely been confirmed directly in species with CP, extensive documentation of CP- based assortative mating in birds raises the possibility that such polymorphisms are widespread (Roulin 2004).

These two intersexual mechanisms of negative frequency-dependence have very different implications for speciation. Genetically-based female preference polymorphisms are key elements of sympatric speciation models and should facilitate the evolution of reproductive isolation (van Doorn et al. 2004). By contrast, homogenous but shifting preferences for rare morphs would be expected to facilitate gene flow and prevent divergence, thereby slowing speciation. Phenotypically plastic preferences, possibly

evolving as an adaptation to unpredictable environments, (e.g., Grether et al. (2005)),

could have a similar effect.

Studies of divergent selection suggest additional possible mechanisms of

frequency-dependent sexual selection. Microhabitat (fine-scaled) or temporal variation in

visual backgrounds could result in distinct visual 'niches' experienced by all morphs, but

with each morph favoured, for example as a result of high contrast, in one microhabitat

(Figure 1.2). Overall, it appears that disruptive sexual selection and negative frequency-

dependence could arise as a result, but a formal model is needed. Sensory bias could

similarly have a within-population analogue, namely the evolution of polymorphic

female visual sensitivity leading to polymorphic mate preference. Few studies have

shown within-population polymorphic vision (e.g., as in spider monkeys Ateles geo@oyi

(Riba-Hernindez et al. 2004) and guppies (Archer and Lythgoe 1990)), but advances in

studying opsin expression and visual sensitivity should facilitate such research (Carleton

et al. 2005; Endler and Mielke 2005). In future work, it will be important to look at causes of variation in sexual selection within populations and at the scale of variation in the light regime (Endler et al. 2005).

Recent studies have highlighted the role of intrasexual selection in maintaining male CPs. In the side-blotched lizard Uta stunsburiana, a cyclical, 'rock-paper-scissors' game is played between three male colour morphs, each with their correlated mating strategy (Figure 1. l a,b,c) (Sinervo et al. 200 1). Remarkably, the different morphs also exhibit different patterns of settling and altruism. Some blue males with high genetic similarity across diverse elements of the genome establish adjacent territories disproportionately often and behave mutualistically and/or altruistically toward each

other. Thus, colour pattern may be one aspect of a 'greenbeard' genotype in this species

(Sinervo and Calsbeek 2006; Sinervo et al. 2006). One model suggests that such

mechanisn~scould contribute to the evolution of reproductive isolation (Hochberg et al.

2003).

A different intrasexual mechanism may promote male CP in members of Lake

Victoria's Pzrndumiliu complex of cichlids, in which males range from blue to red both

within and between species (e.g., Figure 1.1 ij). Patterns of territory distribution and

community assemblage suggest that, between distinct species with either blue- or red-

coloured males, higher aggression between males that share the same colour pattern

might mediate species coexistence (Dijkstra et al. 2006). Seehausen and Schluter (2004)

build on these and other results to develop a sympatric speciation model in which the

territorial advantage enjoyed by a rare morph causes an increase in its frequency and a

correlated increase in the frequency of rare females preferring such males, thereby

initiating reproductive isolation. Male-male social interactions, which take place initially

within species, ultimately become interspecific and, as long as such interactions persist, it

should remain difficult for the alternate morph to reinvade either incipient species.

Further support for this scenario comes from evidence of higher aggression by blue

males, fi-om relatively 'pure' populations and from reproductively isolated blue and red sister species, toward males of the same colour. However, for partially isolated incipient species with different colour patterns, stronger aggression toward same colour males is not observed (Dijkstra et al. In press). Further study of colour and patterns in male-male interactions is clearly warranted in cichlids as well as other taxa, particularly those that are unequivocally polymorphic within a population.

Sexual conflict, in which the evolutionary interests of males and females differ

(see Parker (2006)), has recently received much attention as both a generator of within- species diversity and of speciation (Tregenza et al. 2006). In the damselfly Ischt~ura elegans (Svensson and Abbott 2005; Svensson et al. 2005), rare female colour morphs suffer less male harassment and have higher fecundity relative to common morphs, thereby contributing to CP maintenance. A comparison of inter-locus sexual conflict models has shown that CP maintenance is more probable than sympatric speciation in such cases because speciation would also require the male population to divide (Gavrilets and Hayashi 2005). In cichlids, intra-locus conflict over offspring sex-ratios, which are determined by a colour pattern-linked locus, might have contributed to male mating preferences and assortative mating (Seehausen et al. 1999b). The details of this scenario are complex, however, and empirical evidence for some elements of it is limited (Lande et al. 2001 ; Kocher 2004).

1.5 Conclusions

Our review reveals that several different mechanisms can contribute to CP maintenai~ceand influence the evolution of reproductive isolation. In the most straightforward scenario, considerable evidence suggests that selection and, to a lesser extent, drift can cause divergence between populations, with the balance between selection and gene flow influencing the likelihood of speciation versus CP persistence. Environment-contingent sexual selection and sensory bias increasingly appear to be important in driving divergence.

More (and better) evidence is also accumulating in support of a diversity of within-population mechanisms of CP maintenance; however, we continue to have only a very limited understanding of the relative importance of each mechanism. In guppies for example, apostatic selection by predators, disruptive correlational selection, preferences for rare male morphs and sensory biases have all been implicated in CP maintenance, as well as divergent selection coupled with gene flow. However, we know little about which mechanism is most important or how they might interact. Several of these mechanisms, together with intrasexual frequency-dependence, have also been implicated in the evolution of reproductive isolation and could contribute to sympatric speciation, thereby raising the plausibility of that controversial process.

A variety of studies are called for in order to better elucidate the processes that maintain CP and when these processes should contribute to speciation versus CP persistence (Box 1.2). Although considerable progress has been made since Huxley coined his definition, our understanding of CP maintenance and the role of CP in speciation remains fragmented. Even so, some of these fragments hold tremendous promise and rapid progress may be possible with a more integrated approach.

1.6 Acknowledgements

We sincerely thank F. Breden, B. Crespi, L. Dill, P. Nosil and the FAB*Lab at Simon

Fraser University for insightful discussions and their comments on early versions of the ms. We also thank J. Dale, A. Lindholm, D. Kapan, C.L. Peichel, M. Pauers, M.

Servedio and K. Summers for their comments. The following provided articles in press or in prep: J.K. Abbott, J. Dale, A. Hendry, H. Hoekstra, D. Kapan, M.R. Kronforst, A.

Meyer, 0. Seehausen, B. Sinervo and E.I. Svensson. Financial support was provided to

JSM by a U.S. National Science Foundation REU grant; and to SMG by a Simon Fraser

University President's Stipend and National Science and Engineering Research Council of Canada graduate scholarship.

1.7 Literature Cited

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Journal of Evolutionary Biology 18: 1503- 15 14. Svensson, E. I., J. Abbott, and R. Hardling. 2005. Female polymorphism, frequency dependence, and rapid evolutionary dynamics in natural populations. American Naturalist 165:567-576. Symula, R., R. Schulte, and K. Summers. 200 1. Molecular phylogenetic evidence for a mimetic radiation in Peruvian poison frogs supports a Miillerian mimicry hypothesis. Proceedings of the Royal Society of London B 265:2415-242 I. Tregenza, T., N. Wedell, and T. Chapman. 2006. Sexual conflict: a new paradigm? Philosophical Transactions of the Royal Society of London B 361 :229-234. Tuttle, E. M. 2003. Alternative reproductive strategies in the white-throated sparrow: behavioural and genetic evidence. Behavioral Ecology 14:425-432. van Doom, G. S., U. Dieckmann, and F. J. Weissing. 2004. Sympatric speciation by sexual selection: A critical re-evaluation. American Naturalist 163:709-725. 1.8 Glossary

Anti-apostatic selection: positive frequency-dependent selection by predators such that rare prey suffer higher predation; often found in aposematic mimicry systems.

Aposematism: warning colouration that advertises unpalatability of prey to predators.

Apostatic selection: negative frequency-dependent selection by predators such that rare prey suffer less predation than common prey.

Correlational selection: non-linear selection in which multiple traits have an interactive effect on fitness; specific suites of tsaits are favoured over other others.

Density-dependent selection: the case where the fitness of an allele (or trait) in the population depends upon its density.

Disruptive selection: selection where extremes of a phenotypic distribution are favoured over intermediate phenotypes within a population.

Divergent selection: the case where different phenotypes are favoured, over intermediates in different environments and populations.

Frequency-dependent selection: selection where the fitness of an allele depends upon its frequency in the population; selection favouring the rare allele is negative frequency- dependent, selection favouring the common allele is positive frequency-dependent.

Gene tlow: the movement of alleles between populations via migration and subsequent survival and successful reproduction between immigrants and non-immigrants.

Greenbeard: a gene or linkage group that can produce a signal, detect the signal in others and direct benefit to others possessing that signal. Heterosis: the case where individuals heterozygous for an allele have a fitness advantage compared to homozygotes (also known as heterozygote advantage or over-dominance); it can be associated with disassortative mating between homozygotes.

Intra-locus sexual conflict: antagonism between genes at the same locus in the two sexes.

Inter-locus sexual conflict: antagonism between male and female phenotypes encoded by alleles at different loci in each sex.

Mimicry ring: a group of sympatric species with a common mimetic pattern.

Miillerian mimicry: two or more unpalatable species converge on the same warning colour pattern.

Rare male effect: negative frequency-dependent sexual selection favouring rare male morphs over common ones (often via female mate choice).

Sexual conflict: the situation where characteristics that enhance the reproductive success of one sex reduce the fitness of the other.

1.9 Boxes

Disruptive correlational selection and CP evolution (Box 1.1)

A recent analysis suggests that disruptive selection (and non-linear selection generally) might often be correlational, involving suites of traits that do not appear to be under disruptive selection individually (Blows and Brooks 2003). Documentation of disruptive correlational selection involving CPs raises the possibility that such selection may also be important in CP maintenance (Sinervo and Calsbeek 2006), particularly since CPs are often correlated with other characters (e.g., behaviour, immune function, and other colour traits: Table I. I). In guppies, for example, three favoured trait- combinations occupy adaptive peaks and experience disruptive selection (Blows et al.

2003), whereas such selection was not detected in analyses of the individual traits (Blows and Brooks 2003).

The genetic mechanisms underlying correlations between CPs and other traits are now being elucidated, for example in Heliconizis sp. Miillerian mimics. Here, linkage of son-~emajor colour pattern genes (Naisbit et al. 2003; Joron et al. 2006) leads to a genetic architecture minimizing production of individuals with mixed trait sets that mimic co- models poorly. In a more surprising result, Heliconizw forewing colour and male mate- preference genes both map to the regulatory gene 'wingless' (Kronforst et al. 2006).

Consequently, natural selection for different colour patterns in the young sister species H. cydno and H. pachinus would also cause evolution of corresponding mate preferences, thereby facilitating speciation. Consistent with this hypothesis, male preferences in a polymorphic population of H. cydno (Figure l.ld,e) (whose morphs experience divergent natural selection for Miillerian mimicry on different models (Kapan 2001)) are also assortative, at least for one morph; the second mates at random, possibly because most individuals are heterozygous at the mimicry locus (Kronforst et al. 2006). The use of a single pigment in both colour patterns and the eye is a potential mechanism for pattem- preference pleiotropy (Kronforst et al. 2006).

Correlations between CPs and other traits could increase the potential for CP to contribute to speciation generally, even when selection results mainly from social interactions as in the side-blotched lizard Urn srrmsbzlriana (Sinervo et al. 2006). As frequency-dependent, disruptive coirelational selection builds up co-adapted trait complexes in alternative sympatric colour morphs, assortative mating should be favoured to reduce the production of 'hybrids' with poorly adapted con~binationsof characters

(Sinervo and Svensson 2002). Moreover, as additional characters are added to a multi- trait CP, the probability that one will initiate positive assortative mating pleiotropically might increase (although other preferences might also appear).

Outstanding Questions (Box 1.2)

What is the relative importunce of between- versus within-population processes for CP maintenance?

When morph frequencies differ between adjacent populations, CP can be maintained primarily by gene flow or by within-population processes. Rarely do we know which is more important.

Do CPs evolve mainly as cr result of selection on the CPs themselves, on trrrits correlated with the CPs, or on suites of traits that include CPs?

Because the few studies that impinge on this question (e.g., Blows et al. (2003)) have suggested that combinations of traits are sometin~esthe target of selection, it is essential that we increase our understanding of the correlation(s) between colouration and other traits. Genomic analyses will be central to elucidating the underlying genetics of colour and associated phenotypic traits. Analyses of correlational selection and innovative experimental n~anipulationswill be ciucial to understanding the evolutionary significance of the genomic results. Such work could have important implications for speciation. I.Vltut is the role of (micro) lteterogeneous visual envirnnrrtents in maintaining CP and initiating speciution?

Differential colour pattern conspicuousness and different directionslpatterns of selection in alternate visual environments have now been demonstrated, but we have limited evidence on how important such mechanisms are within populations. We also know little about whether sensory polymorphisms might be maintained by within-population processes and thereby contribute to CP persistence or initiate speciation. Both theoretical and experimental work are called for.

Airtong populations and species, what are the correlates of CP persistence, absence of

CP and CP as a transient stage in speciation?

Ultimately, comparative studies will be needed to test hypotheses of CP maintenance and speciation in the broader context of Ford's (1965) questions about how genetic diversity is maintained in nature. Several analyses do this to some extent by examining relationships between CP, ecology and behaviour in birds (Roulin 2004) and mating system in fish (Seehausen et al. 1999a). Future work on CP should strive to match and broaden this approach, at least in part through use of the comparative method.

, ,. . . . -, - ,t-- OD=ca- 725.ggz -3,,.,,< ,_zs zc=,z - ?a 'f, 3- sGs- L- 54'25-T-TIJ>5.2 y,:_, - 7 3 GC' 1;s 3 3 z;=- --zS= - - --;c~,$~z:=~m~ PF, .;c% 1.1 1 Figures

Figure 1.1: Examples of model colour polymorphic species. Three male colour morphs of the side-blotched lizard Utcl stansbrrriuna play a cyclical 'rock-paper-scissors' game with correlated mating strategies: (a) aggressive orange males usurp the smaller territories of blue mate-guarders; (b) blue morphs defeat yellow sneakers by guarding mates; and (c) yellow sneakers defeat orange territory holders by cuckolding fertilizations (Sinervo et al. 200 1) (photos by B. Sinervo). This frequency- dependent male game is linked with the two-year cycle of two alternative female colour morphs (and life-history strategies) whose frequency fluctuates with population density (Sinervo and Svensson 2002). In Miillerian mimic butterflies, Heliconizrs cydno, (d) white and (e) yellow morphs are found in sympatry, probably as the result of multiple, sympatric mimicry rings acting as a heterogeneous environment in which anti-apostatic selection favours different morphs (Kapan 2001) (Photos by D. Kapan). (f)The Cuman5 guppy (geographical variant of Poecilicl reticulatcl, commonly known as Endler's Livebearer) displays extreme variation in individual male colouration, as do most guppy populations (Alexander and Breden 2004) (Photo by H. AlexanderIF. Breden with permission ti-om Blackwell Publishers). In walking stick insects Timema cristinuc, (g) striped and (h) unstriped morphs are cryptic on alternative host plants and have a suite of reproductive isolating mechanisms, although some gene flow still occurs (Nosil 2004) (Photos by P. Nosil). The sympatric sibling species of African cichlid (i) Pzrndamilia i?yererei red males and (j)P. p~rndrmiliclblue males (Photos by 0.Seehausen) are one of the best examples of sensory bias promoting colour-assortative mating, which, when coupled with frequency-dependent male-male interactions, possibly leads to speciation (Maan et al. 2006).

Figure 1.2: Visual ecology and a heterogeneous environment. Many organisms live in visually heterogeneous environments in which the visual background can change spatially and temporally. A colour morph can therefore shift from being conspicuous to relatively cryptic depending upon the environment. Environments I (El; a: green line) and 2 (E2; a: light blue line) differ in visual properties (either spatially or temporally distinct habitats). The blue (B) and yellow (Y) morphs contrast differently with the background in each environment such that B (a: dark blue line) is more conspicuous in El and Y (a: yellow line) in E2. The measurement of radiance (a) reveals a greater departure of the most conspicuous morphs from the background in each environment, whereas the measured spectrum for the more cryptic morph more closely matches the background spectrum. If the receiver (here, a female choosing a mate, although it could be a predator choosing prey) experiences both El and E2, her choice of mate may be environment-contingent. If the receiver only experiences one of the environments (e.g., there are two female populations, A and B, or females are polymorphic within a population), the visual sensitivities of receivers might differ between environments because of divergent natural selection on visual systems. Even a small shift (e.g., a few nm, exaggerated here for illustrative purposes) in the long wavelength-sensitive opsins (b) can result in differences in the perception of a receiver that could affect her mating preferences substantially (i.e. sensory bias (Fuller et al. 2005b)). The arrows show the direction of the predicted shift in peak sensitivity in each environment, assuming selection favours sensitivity to the predominant background spectrum, with dotted spectra representing the derived state. Environment 1 Environment 2

a

Wavelength (nm) Wavelength (nm) L -- L .VvT 349~

350 Wavelength (nm) 750 CHAPTER 2

A COMPARATIVE DESCRIPTION OF MATING BEHAVIOUR IN THE ENDEMIC TELMATHERINID FISHES OF SULAWESI'S MALILI LAKES

Gray, S. M., and J. S. McKinnon. 2006. A comparative description of mating behaviour in the endemic telmatherinid fishes of Sulawesi's Malili Lakes. Environmental Biology of Fishes 75:469-480. O With kind permission from SpringerIKluwer Academic Publishers 2.1 Abstract

The telmatherinid fishes of the Malili Lakes, Sulawesi, Indonesia provide a new and promising system for studying the processes maintaining diversity in nature, and especially for testing the generality of the influential findings emerging from studies of other fish systems. Here we develop the telmatherinid system by providing the first detailed descriptions of mating behaviour for seven species representing both of the major Malili lakes and all three genera. The mating behaviour of all seven species can be generalized, suggesting that particular behaviours are conserved within the group. For example, male-male competition in the form of lateral fin displays and physical fighting is evident in all seven species. Males also perform a circling behaviour alongside females that they are paired with, although the size of the circle varies across species. In some species egg cannibalism and/or sneaking behaviour are also prevalent. Interspecific comparisons of mating behaviour show that habitat may play an important role in driving bel~aviouraldifferences between species. Parallel intraspecific variation in use of habitat and mating behaviours is also noted for two species. This study will facilitate future behavioural and evolutionary ecology research with this system.

Keywords: Telmatherinidae, Si~lawesi,endemic freshwaler fish, maling syslem 2.2 Introduction

Fish radiations have been intensively studied by evolutionary ecologists seeking

to understand the forces maintaining diversity in nature (Schluter 2000). To date, these

studies have focused on a small number of taxa but it is important to test the generality of

current findings with a wider range of lineages. The study of new groups may also lead to

unanticipated insights and novel avenues of research. The Telmatherinidae are a

relatively unknown family of fishes, the majority of which are restricted to the Malili

Lakes of South-Central Sulawesi, Indonesia (Figure 2.1). Taxonomic descriptions of

several of the species were first published by Boulenger (1 897) and have most recently

been revised by Kottelat ( 1990, 199 1) and Aam et al. (1 998). However, these papers

provide only very preliminary ecological and behavioural descriptions. Our goal is to

provide a more detailed assessment of mating behaviour in this group of fishes as a step

toward developing a promising new study system.

Two large lakes, Matano and Towuti, dominate the Malili Lakes system (Figure

2. Ic). Each harbours at least seven distinct telmatherinid species according to Kottelat

(1990, 1991) (but see Roy et al. (2004) for an argument on species descriptions in

Matano). The system is currently the subject of intense study by several groups interested

in the radiations of the telmatherinids and many other endemic organisms (Roy et al.

2004; von Rintelen et al. 2004; von Rintelen and Glaubrecht 2005; Herder et al. 2006).

Lake Matano is separated from the other lakes in the system by 90 m elevation and was

formed approximately 1.7 million years ago (Haffner et al. 200 1). Phylogenetic analysis suggests an ancient (Martens 1997; McKinnon 2002) monophyletic origin of the Malili

Lakes telmatherinids (Aam et al. 1998; Roy et al. 2004), although more refined molecular techniques are now being used to test this (F. Herder & U. Schliewen and D. Roy, D.

Heath, D. Haffner, P. Hehenussa, personal communications) and some species designations may change.

The telmatherinids are notable for the male colour polymorphism displayed by at least half of the described species (Kottelat 1990, 199 1). Males in these species tend to have blue, grey or yellow colouration, although some Lake Towuti species have additional red and purple elements. All species appear to be sexually dimorphic with the females tending to be dull grey or sandy coloured and the males having much larger and elongated dorsal and anal fins as well as being much more colourful. The male colour polymorphisn~(described in Chapter 3) is similar to that seen in several African Rift Lake cichlids (Seehausen et al. 1999) but is generally less complex than in that system. In order to develop the telmatherinid system for evolutionary ecology investigations it is necessary to first learn more about the ecology and behaviour of these fish. Here we describe the mating behaviour of seven telnlatherinid species as observed in nature.

2.3 Materials and Methods

Mating behaviours (see Table 2.1 for terminology used to describe behaviours) of the family Telmatherinidae were observed in the natural habitat over three time periods:

January to March 2003, January to May 2004, and October to December 2004. The following seven species were observed mating: Tefmutherinu untoniae, T. sur.usinorurr.1,

T. wd~jzli,and T. "whitelips" (undescribed species) from Lake Matano; and, T. c.efehen.sis, Tominurlgu sp., and Parutherinu sp. from Lake Towuti. Species recognition

Four of the seven observed species are found only in Lake Matano: T. un/oni~rr,

T. sarasinotzm, T. kv~rhj~li(see Kottelat (1 991) for morphological species descriptions)

and T. "~vhi/elips". Reproductive adult T. ~rntoni~rt!(and possibly T. sorasinorum) varied

greatly in size and shape and furtlier molecular analyses may show species-level

distinctions between them (F. Herder, personal communication). We categorized T.

anlonioe males into two major size classes, the smaller fish that have relatively shallow

bodies and range from approximately 39-56 mm standard length and the larger

individuals, which have deeper bodies and can reach over 80 mm in standard length (see

also Kottelat (1991)). Here we do not distinguish between them since their mating

behaviour is largely conserved and the great ma-jority of our observations are of the

smaller size class. T. "whitelips" is an undescribed species, or one that is misdiagnosed

among another species from Lake Matano.

The remaining three observed species, T, celehensis, Totnincrngcr sp. and

Pot-nthwincr sp. are found i11 Lake Towuti (see Kottelat (1990) for morphological species descriptions). T. celebensis is also reportedly found in Lakes Mahalona and Wawontoa

(Kottelat 1990). Species descriptions for the genus Tornin~~nycrare based on only 24

specimens from two lakes (Lakes Towuti and Mahalona) and possibly the adjoining

Toniinanga River (Kottelat 1990). We hesitate to name the species observed here because our observations in Lake Towuti do not entirely match those described by Kottelat

(1 990), although we are sure the fish observed belong to that genus. Tornincrngu are niorphologically distinct from Tc.ltno/he~in~r(Aarn et al. 1998); however, mating behaviour appears to be mostly conserved between the two genera. Only two observations of courting pairs of Put*~itllet-inawere made on a single visit to Lake Towuti

in February 2003. We were unable to assign the observed specimens to the species

described by Kottelat (1 990).

Sampling Procedure

Transects, 80 m long and 1 to 2 m deep, were set at ten sites in Lake Matano and

two sites in Lake Towuti. Male fish density was determined by counting the number of

male fish 2 m in front and 1 m to each side of the transect by an observer snorkelling

along the transect. All observations of mating were recorded on or near the transect lines

by snorkelling. Male and female focal follows were conducted for T. antonitre, T.

sar.usinorutn, T. crlebet7sis and Torninanga sp. but only male follows for the remaining

species. Only two mating events were observed in Parather-ina sp. and both males and

females were followed simultaneously. The obvious sexual dimorphism in all species

(males are colourful and have larger and elongated anal and dorsal fins) allows us to

easily distinguish between males and females in the water (Figure 2.2a).

~Mulefhculfhllo~vs:A male focal follow was performed by haphazardly selecting

a male paired with a female along or near a transect and following that male for 4 to 10

minutes with an underwater video camera (Sony miniDV camcorder, OceanImages

unde~watervideo housing). The male was followed regardless of whether or not he lost

the original female. Mating behaviour was later determined from video analyses, noting position of the male relative to the female, activity of the male and interactions with other males, and putative spawning events. Fent(rle focal follo~vs: Information from all female follows was recorded directly by the observer on underwater paper because the cryptically coloured females are too difficult to follow in the LCD screen of the camera. A female paired with a male was again haphazardly chosen along or near a transect and observed for 4 to I0 minutes. The coloui- morph of the original male was recorded, as were male-male interactions and spawning events as above. Having both male and female follow data allow us to ask questions about the mating behaviour of each sex.

Mating events, such as spawning and male fighting, take place continuously throughout the day and an individual fish can experience several spawning bouts, exchange of partners, and fights within only a couple of minutes. We set a minimum and maximum time for observation in order to standardize results and to make sure we captured an individuals' behavioural pattern.

2.4 Results and Discussion

Generalized mating behaviour

There are several generalities in the mating behaviours in all seven telmatherinid species we observed. First, there is 110 parental care; both males and females leave the spawning site iinmediately after apparent spawning, often together. All species are substrate spawners. Neither males nor feinales exhibit territoriality, although male fidelity to a particular spawning area may exist in some species (individually identifiable males were observed spawning along the same transect over a three month period; personal observation). Both males and females appear to be promiscuous in all species observed, switching partners frequently within a 10 min period and often spawning with different partners within that time frame.

The following is a generalized description of mating behaviour in the seven observed telmatherinids (Figure 2.2); species-specific differences are noted in Table 2.2 and further highlighted below. A male finds a female 011 the spawning habitat and either swims slightly behind or in front of the female, who appears to be searching for a place to spawn (Figure 2.2a). If the female slows, the male circles fonvard and away from the female, ending parallel and very close to her (Figure 2.2b). Circling continues until the female presses her abdomen to the substrate, followed immediately by the male. The pair then quiver, apparently releasing sperm and egg (Figure 2.2~).The pair immediately leaves the spawning site and generally repeats the searching and circling behaviours unless interrupted by an approaching male. Sometimes the female will instead leave the male and the spawning habitat for deeper water.

All seven species experience inale-male competition over females. When a single male approaches a pair, the paired male displays laterally, forcing himself between the female and the approaching male (Figure 2.2d) while simultaneously nudging the female in the direction opposite to the approaching male. Males display vigorously to approaching males but rarely (and in a less vigorous manner) to females when other males are absent. The female may swim away or attempt to swim back toward the approaching male. If the approaching male does not retreat in response to the paired male's display, the altercation may escalate into a fight (Figure 2.2e), although occasionally a paired male will give up a female without a fight. The winner of the fight takes on the role of paired male while the loser may attempt to sneak, depending upon the species (Figure 2.3a). A female may switch partners often depending on the outcome of

fights, however she does not spawn with every male she is paired with and pairs

spawning infrequently or not at all occasionally split up without a tight. This suggests

that despite strong male-male competition, the female may be exercising some mate

choice. In the species where male colour polymorphism exists, we have also observed all

colour morphs fighting over and mating with the same females, suggesting that they are

indeed morphs of the same species, not separate species.

Sneaking is a male mating tactic in this group, meaning that a male can either

court a female or be a sneaker, and chooses which tactic to en~ploydepending upon his

current situation (e.g., a paired male that loses a fight may immediately choose to sneak

on another pair). In telmatherinids that employ sneaking as a mating tactic, all colour

morphs and adult males of various sizes have been observed sneaking indicating that

sneaking is not an alternative reproductive strategy as seen in other fish such as the

bluegill sunfish, Lepmis mcrchr.ocl?ir-z~s(Gross and Charnov 1980). We have often

observed aborted spawning events if a female and paired male detect sneakers before

commencement of quivering (i.e. apparent spawning). The pair usually then continues

repeating the general courting and spawning behaviour as described above.

Conspecific egg predation (i.e. cannibalism) is also prevalent in several species.

This behaviour takes place inimediately after spawning and often involves several males

(Figure 2.3b): the paired male, sneakers, and single males in the vicinity but not close enough to sneak. Paired males (and sneakers if present) are therefore practising filial cannibalism (defined as the eating of ones' own offspring (Rohwer 1978)). The male

turns around and attempts to eat the eggs immediately after he has (potentially) just fertilized then]. Thus, both filial and conspecific egg cannibalism (by males not involved

in the mating) can take place simultaneously (Figure 2.3b).

Interspecific comparison of mating behaviours (See Table 2.2)

Mating Habitat

One of the most striking differences in mating behaviour that we see among these

seven species is the use of alternative mating habitats by T. sarasinonrm and Tonzinarga

sp. (Table 2.2a). The two habitats are: shallow beaches (1 to 2 m deep) composed of

small cobble and sand typical of most other species; and deeper sites that drop-off

quickly from the shoreline where overhanging, algae-covered branches, deadfall and

roots compose the spawning habitat. Mating behaviour in beach sites follows the general

telmatherinid model although there are several notable exceptions. T. sarasinor-um males

fight intensely, can switch mating tactics between courting a female and sneaking

copulations, and frequently practise egg cannibalism, especially when sneakers are

present (Gray et a1. 2007; Chapter 4). Tomir~cmgamating behaviour in beach sites also deviates slightly from the general pattern in that males often form small courting groups

(average group size = 4 males to 1 female, range = 3 - 7 males to 1 - 2 females, n = 16 observed groups). One male appears to be paired with the female and male-male interactions take place within the group. If another male approaches, the paired male stays close to and parallel with the female while displaying to the approaching male.

Instead of escalating to fighting, paired males vigorously nudge females while performing a zigzag manoeuvre that alternately displays each side of his body to the approaching male. This is performed while swimming quickly away from the approaching male in a flee rather than a fight. The female increases swimming speed away from the approaching males. Sneaking, but not cannibalism, has also been observed in this species.

In root sites males and females form swarms around the branches: groups of 5 to

400 fish for T, su~u.sit~ot.z/mand 20 to approximately I000 fish for Torninungu in an arca approximately 1 m2 (n = 50 and 4 swarms, respectively). Pairing between males and females in roots swarms is short-lived as males are constantly fighting and switching partners. Circling does not occur. Spawning in the swarms often involves sneaking

(Figure 2.3a), sometimes involving up to 10 sneaker males. This is frequently followed by egg cannibalism in T. .sur-usi~lor.zm~(Figure 2.3b; Gray et al. 2007) but not Totnitlangu.

It is interesting to note that T. sut-asinotwm, with five distinct male colour morphs, and

Totnitzut~gu.with at least 14 observed colour morphs (Table 2.2j), have the greatest morph diversity in Lake Matano and Lake Towuti, respectively, and are the only species known to use two spatially distinct mating habitats.

Another distinction in mating habitat that appears to influence mating behaviour is the flowing water in which T. wuhjui lives and spawns (Table 2.2~1).T. ~vu/zj~/iis found only at the outlet of Lake Matano where the water flows quickly into the Petea River.

Courting pairs generally swim against the current. When another male approaches, the paired male displays laterally while nudging the female downstream, using the flow of the current to increase speed away from the approaching male. Fights also tend to go with the current rather than against it. The space between the paired male and female is reduced (less than 10 cm) and the circling diameter of the male is much tighter relative to the other species (Table 2.2d,e). The availability of spawning habitat may have played a role in the evolution of

mating behaviour diversity in this group. All species are substrate spawners and therefore

require suitable substrate upon which to lay their eggs. Differences in spawning habitat

may be lake-specific, although the within-lake variation in mating habitat (e.g. beach

versus roots; Table 2.2a) may be more important given that behaviour appears conserved

across the two lakes. However, Lake Matano has steeper sides and is less productive than

Lake Towuti (Haffner et al. 2001), which may make beach habitat more limiting in

Matano. Shallow beach habitats in Lake Towuti are also more varied, providing level areas of sand or mud with small cobble, as is typically found in Lake Matano, and also

beaches coniposed of large algae-covered rocks. We may then expect to see mating at greater depths in Matano (Table 2.2b) as shallow substrate is relatively limited.

Circling behaviour

Circling behaviour seems to be the most variable courting element of courtship, and alternatives may be contingent on the mating habitat (Table 2.2e). For instance, in T. cvul?jz/i, which mates in fast-flowing water, the paired male remains closer to the female

(5 to 1 Ocm) than in any other species, and has a circling diameter of approximately 5 cm, staying veiy close to the female at all times. In beach-spawning Torninanga only a half circle is performed: the male swims from behind the female, turns away from her and makes a half circle back toward the female such that his head aligns with her abdomen, and their bodies are perpendicular. The male then straightens out and resumes position slightly behind the female. Circling behaviour is completely absent in root spawning T. sumsinorurn and Tomincmngu where male fish density is much higher relative to beach sites (Table 2.2). Male-male competition

Male-male competition in the form of displays and fighting is obvious in this family (Table 2.2g; Figure 2.2d,e). Fighting intensity differs substantially between species with some notable behavioural differences and in some cases may be influenced by fish density (Table 2.2). Fights among T. cmfoniae males are often fierce, with the males forming a tight head to tail circle, both displaying and biting the other male

(medium intensity). As the circling of the fight progresses the two move vertically in the water colunin toward the surface, sometimes even breaking the surface of the water.

Males often lose the female while fighting, either to another male or because the female leaves the area. T. celebemis males appear to be the most aggressive as approaching males make darting movements toward the female, escalating a display to a full fight earlier than in most other species. 7'. "whilelips" fight least frequently and less aggressively relative to the other species (low intensity), which may be due to the low density of T. "whilelips" at the spawning habitat (Table 2.2: mean density = 4.7 fish per transect). Fighting behaviour among Pwafhet-inumales was the least intense in terms of the speed at which displaying and fighting took place; but it was continuous. In the two independently observed courting events in this species, courtship was similar to the other telrnatherinids with the exception that in both cases, two males appeared to accompany the female as she searched. The males alternated in their role of paired or single male.

The paired male would display toward the approaching male, escalating immediately to a fight. The two males swam in a circle, head to tail, slowly rising to the surface while the female apparently continued to search. Both males returned to the female, the winner taking on the role of paired male with the loser trailing behind. Among males of T, snrasii~oiwmand Tominangu fighting intensity differs between spawning habitats and may reflect significant differences in density between habitats (Table 2.2: t-test = 1 1.87, df = 1. p < 0.000 1 ; t-test = 12.57, df = I, p < 0.000 1, respectively). In root sites, where density is higher, pairing between males and females is short-lived as males are constantly fighting aggressively and switching partners (high intensity). In beach sites, where density is much lower, T. su~.asinorurnfight in the typical telmatherinid way, whereas Tornii~~~ngc-rfighting behaviour differs in the small courting groups (see above).

Alternative male tactics

In several species males employ two mating tactics: courting and sneaking.

Sneaking behaviour was observed in four of the seven species: T. celebensis, T. surwsinoi-urn, T. wul?jtii, and Tomin~~ng~~(Table 2.2h). We hypothesize that increased density may influence sneaking behaviour as sneaking appears more frequent in the high- density roots sites used by T. sui-~~siim-ziinand Tomiimngcr. 111 high density sites it may be more difficult for paired females and males to detect sneakers. For example Reichard et al. (2004) experimentally showed a significant increase in sneaking with male density in

European bitterling (Rhoc/eza ser.icezi.s). However, further quantification of sneaking and observations of the remaining telmatherinid species are needed to confirm this. Sneaking in T, sulwsir~nrzimis also closely linked with egg cannibalisn~(Table 2.2i, Figure 2.3b): egg cannibalism in paired males increases substantially in the presence versus the absence of sneaker males (Gray et 211. 2007; Chapter 4). Corresponding data for T. cp1ebc.nsi.s, the only known egg predator in Lake Towuti, are lacking. This interaction between mating, sneaking and cannibalism in some species but not others is intriguing and further investigation of these phenomena will be enlightening with respect to the

evolution of mating behaviour in telmatherinids (Gray et al. 2007). Fuhire studies will

address such questions as, how often do males eat in the presence versus the absence of

sneakers? It will also be important to map the evolution of egg predation, sneaking and

cannibalism when a robust phylogeny becomes available.

Cannibalism

Congeneric egg predation and cannibalism of conspecific eggs in T. .surusinor.un~

and T. cekehensis may also influence the evolution of mating behaviour with respect to

predator avoidance (Table 2.29. Telmatherinids as a whole appear to lay very few eggs at

a time and spawn multiply in different locations throughout the day and year, likely

decreasing the impact of egg predation. For example, T. crnloniae defend the female they

are paired with from all approaching conspecific males. They do, however, appear to

tolerate the presence of one T. snrusinor.irnl egg predator, which guards the pair from

other would-be suitors and predators (Gray; personal observation). T. antoniae may then

be trading off the loss of a few eggs to predation if the presence of a single T. samsinortrm lowers the intensity of male-male competition over the female. Further

theoretical and observational work on this phenomenon is ongoing and will hopefully provide insight into the relationship between egg cannibalism and mating behaviour.

Furthermore, many other vertebrates, as well as some larger invertebrates in Lake Matano

(and possibly Lake Towuti) are equipped to avoid egg predation. For example, endemic

Gkossogohita sp. form pairs and guard nests of eggs; endemic halfbeaks

(Henniramphidae) are livebearers; endemic rice fish (Oryzius) carry egg sacks under their pelvic fins and, endemic snails (Qkornekuni~~)inte~nally brood young. This observation leads us to believe that egg predation may be a strong selective force shaping the species assemblage in the system and the mating behaviour of telmatherinids. Finally, we have found it very difficult to collect telmatherinid eggs after apparent spawnings, suggesting that eggs are highly cryptic and raising the possibility that some apparent spawnings are deceptive and eggs are not always deposited (Kraak and van den Berghe 1992; Kume et al. 2002).

2.5 Conclusion

The mating behaviours of the telmatherinids observed here can be generalized, suggesting conservation of mating behaviour in this radiation. Promiscuity and intense male-male interactions are common throughout the family, as is a lack of parental care.

Putative adaptations to different habitats, such as the modifications to circling distance in the flowing water-dwelling T. wnhjzii, and increased intensity of fighting in T. sntersinotwn-1 and Tott?it?nngnin root site swarms, also exist. In addition, there is a potential correlation between high fish density in root sites and mating behaviours, especially with respect to sneaking and cannibalism.

The mating behaviour of the seven telmatherinid species described here gives only a tantalizing initial view of the diversity within this almost unstudied group of fishes. The fields of behavioural and evolutionary ecology will certainly benefit from further work that integrates the phylogenetic history, mating behaviour and colour patterns within in this radiation. 2.6 Acknowledgements

We thank L. Dill and the Dill-lab for their critical review and comments on the ms and F.

Herder and U. Schliewen for insightful discussions. The following people helped with the fieldwork: A. Crawford, D. Holm, A. Robertson, S. van der Meer and F.Y. Tantu. We also thank Tantu for the important support he provided as our Indonesian collaborator. Pt.

Inco provided logistical support, especially R.A. Lolo, S. Suardi, F. Iskandar, B. Wenzl, and J. Gowans. LIP1 (Indonesian Science Foundation) provided permits to conduct research in Indonesia. This pro-ject was funded by an NSF Grant to J.S.M, CIDA -

EIUDP funding to L.M.D., and NSERC, PAD1 Project AWARE and Sigma-Xi Grants-

In-Aid-Of-Research to S.M.G and equipment donations from Aqualung Canada.

2.7 Literature Cited

Aarn, W. Ivanstoff, and M. KotteIat. 1998. Phylogenetic analysis of Telmatherinidae (Teleostei: Atherinomorpha) with description of hfarosatl~er-ina,a new genus from Sulawesi. Ichthyol. Explor. Freshwaters 9.3 1 1-323. Boulenger, G. A. 1897. An account of the freshwater fishes collected in Celebes by Drs. P. and F. Sarasin. Proceedings of the Zoological Society of London l897:426- 429. Gray, S. M., L. M. Dill, and J. S. McKinnon. 2007. Cuckoldry incites cannibalism: male fish turn to cannibalism when perceived certainty of paternity decreases. American Naturalist 169:258-263. Gross, M. R., and E. L. Charnov. 1980. Alternative inale life histories in bluegill sunfish. Proceedings of the National Academy of Sciences 77:6937-6940. Haffner, G. D., P. E. Hehanussa, and D. Hartoto. 2001. The biology and physical processes of large lakes of Indonesia: Lakes Matano and Towuti, Pages 183-192 in M. Munawar, and R. E. Hecky, eds. The Great Lakes of the World: Food-web, health and integrity. Ecovision World Monograph Series. Leiden, Backhuys Publishers. Herder, F., A. W. Nolte, J. Pfaender, J. Schwarzer, R. K. Hadiaty, and U. K. Schliewen. 2006. Adaptive radiation and hybridization in Wallace's Dreainponds: evidence from sailfin silversides in the Malili Lakes of Sulawesi. Proceedings of the Royal Society of London B 273:2209-22 17. Kottelat, M. 1990. Sailfin silversides (Pisces: Telmatherinidae) of Lakes Towuti, Mahalona and Wawontoa (Sulawesi, Indonesia) with descriptions of two new genera and two new species. Ichthyological Exploration of Freshwaters 1 :35-54. -. 199 1. Sailfin silversides (Pisces: Telmatherinidae) of Lake Matano, Sulawesi, Indonesia, with descriptions of six new species. Ichthyological Exploration of Freshwaters 1 :32 1-344. Kraak, S. B. M., and E. P. van den Berghe. 1992. Do female fish assess paternal quality by means of test eggs? Animal Behaviour 43:865-867. Kume, G., A. Yamaguchi, and I. Aoki. 2002. Dummy egg production by female cardinalfish to deceive cannibalistic males: oogenesis without vitellogenesis. Environmental Biology of Fishes 65:469-472. Martens, K. 1997. Speciation on ancient lakes. Trends in Ecology & Evolution 12: 177- 182. McKinnon, J. S. 2002. Aquatic hotspots: speciation in ancient lakes 111. Trends in Ecology Sr. Evolution 17:542-543. Mortez, J. A., and W. Rogers. 2004. An ethological analysis of the breeding behavior of the fantail darter, Efl?eosfoma,fi~be//u~~e.American Midland Naturalist 152: 140- 144. Reichard, M., C. Smith, and W. C. Jordan. 2004. Genetic evidence reveals density- dependent mediated success of alternative mating behaviours in the European bitterling (Rhoderw sericeus). Molecular Ecology 13: 1569-1578. Rohwer, S. 1978. Parent cannibalism of offspring and egg raiding as a courtship strategy. The American Naturalist 12:429-440. Roy, D., M. F. Docker, P. Hehanussa, D. D. Heath, and G. D. Haffner. 2004. Genetic and morphological data supporting the hypothesis of adaptive radiation in the endemic fish of Lake Matano. Journal of Evolutionary Biology 1 7: 1268- 1 276. Schluter, D. 2000, The Ecology of Adaptive Radiation. Oxford, Oxford University Press. Seehausen, O., P. J. Mayhew, and J. J. M. van Alphen. 1999. Evolution of colour patterns in East African cichlid fish. Journal of Evolutionary Biology l2:5 14-534. von Rintelen, T., and M. Glaubrecht. 2005. Anatomy of an adaptive radiation: a unique reproductive strategy in the endemic freshwater gastropod Tylomelnnia (Cerithioidea: Pachychilidae) on Sulawesi, Indonesia and its biogeographical implications. Biological Journal of the Linnean Society 85:513-542. von Rintelen, T., A. B. Wilson, A. Meyer, and M. Glaubrecht. 2004. Escalation and trophic specialization drive adaptive radiation of freshwater gastropods in ancient lakes on Sulawesi, Indonesia. Proceedings of the Royal Society of London B 27 1 :254 1-2549. 2.8 Tables

Table 2.1: Terms used to describe general behaviours performed by males andlor females during mating, using existing tel-minology where applicable (Mortez & Rogers 2004). Males accompanying a female are called "paired males"; those without a female are called "single males". Behaviour Term Description Peijbimed by both sexes: Putative spawning Male and female place abdomens together on the substrate and quiver. Egg and sperm are difficult to see by humans during these events; hence we use "putative" Quiver Performed by both sexes, usually simultaneously, while pressing abdomens to the substrate in a putative spawning ~Mcllebehaviozrrs: Approach A single male swims toward a paired male Circle A paired male circles next to a female while the female searches Display Erection of the first and second dorsal fins and the anal fin by the paired male upon approach of a single male. If approach continues, the paired male moves his body laterally toward the other male, maintaining erect fin display Eat After a putative spawning a fish attempts to eat the freshly spawned egg(s) Fight Physical interaction between two males, usually between a paired male and an approaching single male. The two males circle each other head to tail, often biting each other on the flanks and tail Nudge Upon approach of a single male the paired male touches his head to the side of the female, pushing her away from the approaching male Sneak A single male rushes alongside the female when the paired male and female go to the substrate to spawn, and begins to quiver with the pair in an attempt to fertilize eggs Femule Belzcrviolri-s: Search Searching for a place to spawn; swimming is slow and erratic Leave The female swims quickly away from the male, usually into deeper water '- 'I. = E-d 2 >, - .; .-C.5" - -=;-m39--=o <=C2z-z 5 5 -0gqgzgz Id - Ga 5ZC /.=> Es Zzr? - cm , , - : m- - 0 - --= z - %I, -, C;i 5 xi, - 7 - :P .g -. 5 - c; Ts L - = ."' = .- -"C "2 - 3.5C) - * 2oCv5 2,;' Fs,g -, 4 n/2 z0.- ,: 6 ZJ? .... y %? ,--, - -.- &E ...... 2.T z&2&+2z: uxC.5 2.9 Fi,oures

Figure 2.1: A map of the Indonesian archipelago with island details. a) the Island of Sulawesi boxed; b) The Island of Sulawesi with the Malili Lakes system boxed; and, c) The Malili Lakes system which drains east into the ocean, Bay of Bone (maps were modified from Pt. lnco Ltd documents). Shaded areas represent land. All observations were recorded in Lakes Matano and Towuti, the two major lakes in the system. L. Wawantoa

OfBone Figure 2.2: Graphical description of some aspects of generalized telmatherinid mating behaviour (see Table 2.1 for definitions of behavioural terminology). a) Male and female pair up, where (i) is the distance the paired male swims behind and to the side from the female and (ii) is the distance the pair swims from the substrate; b) the male circles alongside the female as she searches where (iii) is the diameter of the males7 circle; c) after searching a feinale chooses a place to spawn and the male and female press their abdomens to the substrate, and quiver in a putative spawning. Male-male interactions include: d) the approach of a single male (black) causing the paired male to display; and, e) if the approaching male persists, a fight between the two males ensues. The winner of the fight pairs with the female and spawns (c) or spawning can occur in the presence of a sneaker. often the loser of the fight (see Figure 2.3). Note that colour differences are for illustrative purposes only as all male morphs have been observed fighting each other. (Illustration by S.M. Gray)

Figure 2.3: A graphical representation of Telil~ufherinasarnsinorrinr sneaking and egg cannibalism behaviour at a root spawning site. a) The female (F) and paired male (M) spawn as in Figure 2.2~but here are joined by a sneaker male (S) attempting to fertilize the eggs being spawned by the pair. b) After spawning the female leaves the spawning site while the paired male and sneaker male turn around and attempt to eat the eggs and are joined by a non-sneaking cannibal (C). All colour morplx have been observed performing all behaviours and differences in shading here are for illustrative purposes. (Illustration by S.M. Gray)

CHAPTER 3

THE ROLE OF ENVIRONMENT-CONTINGENT SEXUAL SELECTION IN THE MAINTENANCE OF MALE COLOUR POLYMORPHISM IN TELMATHERINA SARASINORUM 3.1 Abstract

The role of sexual selection in maintaining intraspecific diversity is gaining

recognition, especially in systems that display male colour polymorphism. Variation in the visual environment is expected to influence the direction of selection on colour signals and may therefore be important in promoting colour polymorphism. Here, we use

Tclmatherii7tr sarasii~oruin,a small flsh endemic to Lake Matano, Sulawesi, Indonesia, to test the hypothesis that selection varies in a spatially heterogeneous light environment, resulting in the maintenance of variation in male colour. The flve male colour morphs are found in varying frequencies in two spawning habitats: shallow beach sites and deeper sites with overhanging roots. Measurements of the light environment and morph colour in each habitat indicate that blue males are more conspicuous than other morphs in beach sites whereas yellow males are more conspicuous than other colour types in root sites. A model based on a set of variables derived from extensive behavioural observations in the field was used to test if the most conspicuous morph in each habitat had the highest reproductive fitness. Males that contrast more with the background are expected to havc higher pairing and spawning success with females, but may also be cuckolded or have their eggs cannibalized more often. In a con~parisonof the two most abundant morphs, blue males were more frequent and had higher fitness than yellow males in beach sites, and yellow males were predominant and had higher f'itness in root sites. This suggests a role for environment-contingent sexual selection in promoting rnale colour polymorphism in this species and perhaps others in the Malili Lakes telmatherinid radiation.

Keywords: sexual selection, reproductive fitness. helerogeneous visual environment, Telmatherinidae. mating behaviour, fish 3.2 Introduction

Understanding how intraspecific diversity is maintained in nature is a

fundamental problen~in biology. For decades researchers have used easily observable

colour polymorphisms, defined as the occurrence of two or more interbreeding colour

morphs in the same population (Huxley 1955), to investigate the mechanisms promoting

the maintenance of within-species diversity. More recently, male colour polymorphisms

have been used to elucidate the relative roles of sexual and natural selection in

maintaining polyn~orphisms(Endler 1983; Endler 1992; Anderson 1994; Boughman

200 1 ; Sinervo et al. 200 1 ; Sinervo and Calsbeek 2006) and in generating new species

(Seehausen and van Alphen 1999; Seehausen et al. 1999; Carleton et al. 2005). Several

mechanisms, such as negative frequency-dependent selection and correlational selection, are thought to promote both the maintenance of intraspecific colour diversity and, in some cases, speciation (reviewed in Gray and McKinnon 2007; Chapter 1). Due to the inextricable link between colour signals and the light environment (Lythgoe 1979; Endler

1990; Endler and Mielke 2005), the visual environment should be important in shaping the direction of selection and has lately received more attention in analyses on colour variation.

Environment-contingent selection is the process whereby a particular phenotype is favoured depending on the environment in which selection takes place, such that different forms are favoured in different places or times (Gray and McKinnon 2007). The role of environment-contingent selection in maintaining colour polymorphism is only beginning to be appreciated and both theoretical and empirical treatments are lacking.

Numerous studies quantify the relationship between the visual background and colour morphs, for example the lizards of Whitesands Ecotone (Rosenblum 2006), pocket

(CI?~~etoclipusinlet-medius) and beach (Pcrtmysc~apolionotus) mice (Hoekstra et al.

2004; Hoekstra et al. 2006), Cepaea snails (Jones et al. 1977), sticklebacks (Ga.~te~.osletr.s

~~c~rkeat~rs)( Reimchen 1989; Boughman 200 I), and some birds (Roulin 2004). Fewer

studies, however, empirically examine the actual processes responsible for maintaining

colour polymorphism within and between habitats in a heterogeneous visual environment

(but see Gamble et al. (2003); Carleton et al. (2005); Maan et al. (2006)); fewer still

examine the behaviour of the animals in natural settings.

The scale at which animals experience environmental heterogeneity is important

in understanding how selection acts. The visual environment can vary spatially (e.g.,

between open and closed canopy forest patches) or temporally (e.g., between dawddusk

and mid-day) (Lythgoe 1979; Endler 1990) and can be either coarse- or fine-scaled (Gray

and McKinrion 2007). For example, populations may experience different visual

backgrounds, leading to monomorphic populations and spatial differences in colour,

unless gene flow between populations is strong or other processes are acting within

populations. Within populations, colour polymorphism may be maintained by multiple

processes acting together, or in opposition. For example, if sexual selection favours conspicuous male colouration, directional sexual selection could be opposed by some other form of selection, such as predation. Other possible processes leading to the maintenance of within-population polymorphism include negative frequency-dependent selection (Gray and McKinnon 2007; Olendorf et al. 2006), directional selection with gene flow (Crispo et al. 2006; Gray and McKinnon 2007; Nosil et al. 2006), and disruptive selection (Blows et al. 2003; Rueffler et al. 2006). Some of the best-studied colour polyn~orphicsystems are fishes, possibly because many teleosts are colourful (Braasch et al. 2006) and the aquatic environment tends to provide more intense differences in background lighting and colouration than terrestrial environments (Lythgoe 1979; Boughman 2002). Guppies (Poecilia t-eticzrlata) represent one of the niost extreme cases of male colour polymorphism in nature. Colour pattern varies between male guppies within populations and also among populations. Differences between populations are often the result of a balance between sexual selection favouring conspicuousness and natural selection by predators favouring crypsis (Endler 1980, 1983;

Millar et al. 2006). In high predation populations, males that are more cryptic against the gravel substrate are favoured, whereas males in low predation populations tend toward more conspicuous patterns preferred by feinalcs during mate-choice (Endler 1980, 1983).

Within guppy populations individual variation is influenced by a variety of factors including negative frequency-dependent survival (Olendorf et al. 2006) and mating success (Eakley and Houde 2004). The complexity of colour patterns in this system has made it difficult to elucidate how different forms of selection (e.g., disruptive correlational selection (Brooks and Couldridge 1999; Blows et al. 2003)) collectively contribute to the maintenance of male colour polymorphism within and among populations. In the bluefin killifish (Lucuniu goodei), variation in the lighting environment predicts the relative abundance of five male colour morphs within populations (Fuller 2002). Males with blue anal fins are relatively more abundant and contrast more against tea-stained streams, whereas red- and yellow-finned males doniinate in clear springs where they are more conspicuous. Some form of sexual selection favouring the most conspicuous morph is thought to maintain colour poly~norphism(Fuller 2002), although no empirical work has tested this. Here, we

describe the role of environment-contingent selection in maintaining male colour

polymorphism in a sailfin silverside fish from the Malili Lakes, Indonesia, by examining

not just the relationship between morph frequency and the environment, but also the direction of selection operating in different habitats. This study is quite novel compared

to other studies of colour polynlorphisn~as we were able to quantify colour and environmental light variation in the natural state and link to this direct measures of reproductive success.

The Malili Lakes (Sulawesi, Indonesia; see Appendix 1, Figure A 1.1 for detailed maps) are rapidly becoming known as a valuable model for studying adaptive radiation due to the extreme levels of endemism in such a small system, especially relative to the more con~plexAfrican Rift Valley cichlid models (Sturrnbauer 1998; Kocher 2004;

Seehausen 2006). Two large (Matano and Towuti) and three small lakes formed approximately 1-2 million years ago (W. Ahmed; Pt. INCO, personal communication), and their flora and fauna has likely diversified since then (Roy et al. 2004; von Rintelen et al. 2004; von Rintelen and Glaubrecht 2005; Herder et al. 2006). The small, brightly coloured sailfin silverside fishes (Telmatherinidae) are found throughout the lakes and drainages, with several species endemic to single lakes (Kottelat 1990, 199 1). Recent molecular evidence suggests hybridization events between some lake and riverine species, which may have influenced radiation of the fishes into novel habitats (Herder et al. 2006). Until very recently almost nothing was known about the behavioural ecology

(see Gray and McKinnon (2006) (Chapter 2) and Gray et al. (2007) (Chapter 4)) or the adaptive role of colour variation among the telmatherinids of the Malili Lakes. We focus on one of the sharpfin species, Teltn~rtlzerit~asarasitmtwm (Kottelat

1991), endemic to Lake Matano. This species is found throughout the lake and can easily be observed performing complex mating behaviours throughout the day and year (Gray and McKinnon 2006; Gray et al. 2007). Female T. sorasinot-urn appear to be cryptic

(sandy-grey colour), while males display five colour morphs (Appendix 1, Figures A 1.2-

A 1.7: blue body with blue or white fins; yellow body and fins; blue body with yellow f'ins and head patch; grey body with grey or black fins; and, grey body with yellow head patch). The mating system of T. scrrasit~otwnis described in detail in Gray and

McKinnon (2006) and Gray et al. (2007). In brief, T. sarasinorun? are promiscuous substrate spawners that provide no parental care. Individual males employ two mating tactics interchangeably. They can court and pair with females, during which time males will fight off other males and guard the female. Alternatively, when unpaired, males often try to sneak fertilizations and cuckold other paired males, while also initiating fights with paired males in an attempt to win a female. Both paired males and those acting as sneakers often appear to try to eat the eggs that have just been laid (Gray et al. 2007). All five colour morphs are found in varying frequencies in each of two studied mating habitats: beach sites are shallow sand and rock beaches; root sites drop off steeply from the shore and spawning takes place mainly on over-hanging algae-covered roots. All morphs have been observed mating with the same females and so are assun~edto be a single species. The fact that all morphs use both mating habitats, and that some appear to be more frequent in one or the other, allows a test of the hypothesis that spatial variation in the visual environment influences the direction of selection in each mating habitat and may therefore influence the maintenance of multiple colour morphs. We test three predictions of this hypothesis. First, if the two mating habitats

provide visually different backgrounds against which the fish are viewed, then the male

colour morphs are expected to be perceived differently (i.e., have differential contrast) in

each habitat. We therefore evaluate the difference in contrast between each morph and

the background lighting environment in each habitat. Differential contrast of morphs

between habitats is expected to influence the direction of selection in each mating habitat;

therefore we next examine the direction of selection between habitats, expecting that one

niorph is favoured over others in each habitat (i-e., environment-contingent selection).

Under sexual selection theoly, the morph that contrasts the most with the background

should be favoured by some forms of selection (e.g., female mate-choice) but be at a

disadvantage by others (e.g., egg cannibalism) because they are more visible. Finally, we

derive an equation to test the prediction that the morph with the highest colour contrast in

each habitat has the highest rclative reproductive fitness in that habitat.

3.3 Methods

We visited Lake Matano during two wet seasons (Janua~yto March 2003 and

January to May 2004) and one dry season (October to December 2004). In order to facilitate consistent observations in each of the two predominant mating ha b' I tats over

time, we established eight permanent sampling sites around the lake (Table 3.1, Figure

3.1). At four of these sites we placed two, 80 m long by 1-2 m deep transects, one each in a root habitat and a beach habitat, separated by a minimum distance of 100 m and a maximum distance of 500 m. The remaining four sites had a single transect placed along the shore in the centre of either root habitat (two sites) or beach habitat (two sites) and not in proximity to the alternate habitat, making a total of six root transects and six beach transects.

We report fitness and contrast values for only the blue and yellow colour morphs as they are the most frequent morphs in the beach and root habitats, 43% and 35% respectively (Figure 3.2), and because together they represent 60% of the entire population. They are also expected to show the greatest extremes of colour contrast.

Colour analyses

To determine if each mating habitat provides a different visual background and if the relative contrast of each colour morph differs in each habitat, we combined measures of radiance of the fish in their natural habitat, radiances from the background against which they are viewed, and the spectral sensitivity of photoreceptors in a closely related species, T, bonli (a riverine species with blue and yellow male colour morphs, used because T. sat-nsinorzm was unavailable). This approximates how a male fish may be perceived by a conspecific in a particular habitat.

We used a quantum spectroradiometer (Oceanoptics 2000) with a radiance probe

(Hobi Labs) in the field to measure the colour reflected from the fish and the background against which it is viewed under ambient conditions. All measurements were taken on transects between 10:OO and 12:00, the time interval when mating behaviour peaks.

Individual fish were captured and immediately euthanized in an overdose of clove oil (5.0 ppt). The fish was placed on a mechanical frame (Appendix 1, Figure A 1.8) that holds the fish suspended in the water (at 1.0 m depth) and maintains a constant distance (1 0.0 cm) between the fish and the radiance probe. The probe could then be moved across the surface of the fish to take radiance measures of different patches (patch = 0.4 cnl

diameter circle). Three patches on each fish were measured: on the head above and

slightly behind the eye; mid-body below the anterior insertion of the second dorsal fin;

and on the anterior basal portion of the second dorsal fin itself (Appendix 1, Figure

A 1.9). After the patches were measured the fish was moved out of the field of the probe and a measurement of the ambient background light was taken. The entire procedure for one fish was completed in less than 10 minutes to mini~nizepost-mortem loss of colour

intensity.

Radiance values were standardized following Endler ( 1990). Radiance spectra were smoothed between 350 and 700 nm with 5 nm intervals, then standardized to an integration time of 200 msec and by a calibration correction factor and converted to watts/m2. To generate photon flux values (the number of photons, rather than the energy, reaching a surface in a unit of time) we n~ultipliedby wavelength and 0.00835 19 (Endlcr

1990). Photon flux values were corrected by the immersion correction factor (R.

Maffione, personal communication) and the lens transmittance obtained for a related telmatherinid species, ~blmmtr!herinalcrdigtzsi (Marshall and McKinnon; unpublished data).

To determine if the visual background provided by beach and root habitats differed with respect to light intensity (i.e., peak (corrected) photon flux) and colour (i.e., h,,,,, of peak photon flux), we calculated the average background spectnm for each beach and root site. We then normalized (i.e., forced the peak photon flux to unity) the mean background spectra to examine the relative difference in colour (rather than brightness) between the two backgrounds. To incorporate a measure of photoreccptor spectral sensitivity of the fish in our

calculation of contrast, we used n~icrospectrophotometry(MSP) (Loew 1994; Fuller et al.

2003; see Appendix 2 for MSP methods) to determine the major classes of cone pigments

found in the retina of four T. honti (four lab-bred individuals from a population

originating in the River Petea near the outlet of Lake Matano). We used template fitting

to determine the maximum absorbance (A,,,, +/- standard deviation) of each scanned

cone, but only used those spectra with a standard deviation less than 10 nm (Fuller et al.

2003). All cones appear to be fitted best with an A 1 (rhodopsin chromophore) template.

From the mean I.,,,,, for each cone class we generated nomograms that were used in

combination with the corrected photon flux spectra for each measurement to determine

Euclidean contrast values (as per Endler ( 1990) and Pauers et al. (2004)). Photon flux

spectra for the patch and for the background were each multiplied by the nomogram

spectrum (standardized for cone sensitivity) for a cone class and summed over all

wavelengths. The difference between the summed patch and background values for each

cone class was then squared. Squared values were summed among cone types and the

square root taken of the sum to give the contrast index (CI) for that patch. This procedure was repeated for all fish (each colour patch separately) in both the beach and root habitats. We performed one-tailed t-tests between the difference in body CI for blue and yellow males in each habitat (i.e., blue CI - yellow CI beach compared to blue CI - yellow CI root) to test if blue males have higher contrast relative to yellow males

(positive values) against the beach background and yellows have higher contrast relative

to blues (negative values) against the root background. Between-habitat analysis of reproductive success

We predicted differential reproductive success for male colour morphs between the two studied mating habitats. To test this, we collected data for a set of variables representing components of reproductive success using behavioural observations in the wild. The following directional predictions were made based on the expectation that, under sexual selection theoly (Andersson 1994), the morph with the highest contrast is the most successful in each habitat. First, the frequency of the most conspicuous morph should be higher relative to other morphs and this should differ between habitats. Second, the morph with the highest contrast should be paired more often and spawn more frequently when paired; however, because he is more conspicuous to females, he should also be more visible to other males and so should be cuckolded relatively more often and subsequently suffer more egg cannibalism. The more conspicuous morph should also be attacked relatively more often. Lastly, less visible morphs should sneak more when unpaired than higher contrast males.

Collection of observational data

Each site was visited between two and six times per field season, at least one week apart with the order of site visits chosen randomly. On each visit to a site, the day was divided into five, two-hour time periods between 6:30 and 16:30. During each period, we collected the following data: water visibility (measured with a horizontal secchi disk in m), estimated cloud cover (096, 25%, 50%, 75% or 100% cloud cover), the presence or absence of shade covering the transects, transect count data of males, and mating behaviour data using focal follows of females and males. All observations were made on or near the transect by two or three observers using snorkel gear. A single observer snorkelled at a slow, constant pace along the transect (marked with flagging tape every 5.0 m) and counted the number of male fish 2.0 m in front and

1.0 n~on either side of the transect line. Male colour inorphs were identified and whether or not they were paired with a female was recorded. The density and frequency of each colour morph and the proportion of paired to unpaired males (of each morph) on transects were calculated for each time period. We estimated the operational sex ratio on the transect (OSR; females:males) using the number of males paired with a female relative to the total number of males counted per observation.

A focal follow was performed by haphazardly selecting either a female or a paired male along or near a transect and following that individual for 4 to 10 minutes. Female follows were recorded directly on underwater paper by an observer, during which apparent spawnings, the colour of the paired male and (if present) the colour and number of cuckolders and apparent cannibals were recorded. Male follows were recorded with an underwater video camera (Sony miniDV camcorder, OceanImages underwater video housing). Males were followed regardless of whether they lost the original female, providing data on male behaviour both while paired and unpaired. When the male was paired with a female the observer recorded the number of apparent spawnings, the colour and number of cuckolders and cannibals (if present), attacks by other males and the winner of those fights (defined as the male that, after a fight, pairs with the contested female), and the number of females that left him without spawning. When the male was solitary, we recorded the number of females he approached, paired with, or was rejected by. and the number of attempted sneak fertilizations. Estimation of reproductive success

Using transect data we estimated the frequency of each morph at each time period and place (transect, site) they were counted (e.g. no. blue n~alesltotalno. males) (see

Table 3.1 for sample sizes per site). We estin~atedthe probability that a male of a given morph was paired (P,,) or unpaired (P,,) with a female at any given time and place (e.g. no. blue males pairedltotal no. blue males counted). From the female follow data (N =

570), we calculated the total number of spawnings a morph had within a habitat and site and determined the probability that a male spawns in the absence of cuckolders and cannibals (P,); in the presence of cannibals (including himself and non-mating males) but the absence of cuckolders (P,); in the presence of one cuckolder (no cannibals) (PkI);in the presence of two or more cuckolders (no cannibals) (Pk3);and, in the presence of both cuckolders and cannibals (including himself, sneakers, and non-mating males) (Pb). From individual male follows (N = 285) we determined the mean spawning rate (no. spawns per minute per female) when paired and the mean sneaking rate (no. sneaks per minute) when unpaired for each site and transect. While a male is unpaired, he also spends time attempting to pair with females, so we estimated several variables that could influence a male's ability to pair with and retain a female: the rate at which he approaches single fen~ales(approach rate: no. females approached per minute); the probability of successfully pairing with approached females (P,: no, females a male pairs withltotal no. females a male approaches); the mean attack rate (no. of attacks by other males per minute while paired); the probability that the paired male wins a fight (P,,); and, the probability that a fe~naleleaves a male without spawning with him (no. females that leave a male/ total no. females a male pairs with). All probabilities were arcsine square root transformed and were calculated within a single morph, so that differences in morph frequency are removed from our estimations. Sample sizes (e.g., male follows and

spawnings within female follows) were too small to calculate probabilities per time

period per day so were pooled for a site and habitat (see Table 3.1).

Each of these predictions was tested individually (one-tailed t-tests) by asking if

the difference in the mean value between blue and yellow males differed between

habitats. For example, if we expect blue males to have a higher probability of being

paired than yellow males in the beach site, and yellow males to have higher pairing

success in root sites, then the difference between blue and yellow for the beach and root

sites should be positive and negative, respectively.

Within-habitat analysis of reproductive fitness

An increased ability to pair and successfully spawn with a female within a habitat should increase a male's reproductive fitness. However, since all morphs are present in each habitat, fltness is likely to be offset by opposing forces such as cuckoldry and cannibalism. Using the components of fitness described above we estimated the relative fitness of morphs within each habitat (beach and root) by building a simple fitness equation that sums the estimated reproductive success gained by a male when paired with a female through spawning (I a), and that gained while unpaired though sneaking behaviour (I b). Although we did not measure survival, we assume that, all else being equal, a male's fitness will depend upon his reproductive success; hereafter, we refer a male's reproductive success as fitness. The fitness values (a)for each possible means of gaining fitness were assigned according to a set of basic assumptions. We assume that in the absence of cuckolders and cannibals a spawning event is successful so the male gains a full unit of fitness (01, = I). Any spawning event that involved cannibalism yields no fitness because it is assumed that all of the egg(s) are eaten, therefore fitness gained in the presence of cannibals (o,) and cannibals and cuckolders (wb) is zero. The presence of one cuckolder at a spawning event reduces the paired male's chance of fertilizing the egg(s) by half (akl= 0.5), as two males are competing for fertilization. Likewise, the presence of two or more cuckolders would reduce the male's chance of a successful spawning event by two thirds (ak2= 0.33).

Although we have observed up to 10 males cuckolding a single spawning event, this happens rarely coinpared to cuckoldry by one or two sneakers at a time (median no. cuckolders = 1.0). When unpaired and acting as a sneaker (cuckolding paired males) we assume a male has only half a chance of fertilizing the egg(s) (to, = 0.5). Without further work on the determinants of the actual fertilization success of sneakers (e.g., the distance and position of the cuckolder to the female may contribute to the chance of fertilizing an egg when sneaking), this is our best approximation of the possibility of gaining fitness from this behaviour. Sensitivity analyses were performed on each of the fitness values separately to test the robustness of our assumptions. Each fitness value was increased andlor decreased up to 50% (at 5% intervals) and we then determined the change in the p- values (u significance 0.05) generated from the one-tailed t-tests of the difference in

relative fitness between blue and yellow males in each habitat.

We used separate one-tailed t-tests to test for a difference in relative fitness

between blue and yellow morphs within each habitat because our npi-iori prediction was

that the most conspic~ious(and frequent) n~orphin a habitat would have the highest

fitness. Mean values per sitelhabitat were used as the sampling unit (nbCaCh= 6, n,,,, = 6).

3.4 Results

Colour analyses

The mean background radiance spectra of each habitat were different in that the beach habitats had higher peak photon flux (mean beach = 0.45 w/n12 (+I- 0.09 SE); mean root = 0.1 1 w/m2 (+/-0.04 SE); t-test = 3.62, d.f. = 8, P = 0.007) indicating higher levels of light intensity in beach compared to root sites. The maximum wavelength at which photon flux peaked (I,,,,) also differed between beach (A,,, = 579 nm, +/- 1.87

SE) and root (h,,,,, = 565 nm, +I- 0 SE) sites (t-test = 7.483, d.f. = 10, P < 0.0001). We compared the normalized spectra (Figure 3.2; relative photon flux) of the two backgrounds in two areas of the spectrum where they deviate from each other because that difference is what we are interested in and showed that the beach site reflected more yellow and red than did the root site (between 600 and 680 nm; mean beach = 0.66 (+/-

0.03 SE) cf. mean root = 0.44 (+I- 0.05 SE), t = 3.53, d.f. = lo, P = 0.008), while the root site reflected more in the blue end of the spectrum (betn~een435 and 5 15 nm; mean beach

= 0.43 (+I-0.02 SE) cf. mean root=0.59 (+I- 0.03 SE), t=4.15, d.f. = 10, P=0.003) than did the beach site. Male colour morphs contrast differently in each of the two mating habitats. We

estimated the contrast indices for 8 blue and 11 yellow males in the beach habitat and 9

blue and 8 yellow males in the root habitat. Blue males had significantly higher contrast

in the beach site than did yellow niales (mean blue CI = 8.6, mean yellow CI = 4.9, one-

tail t-test = 1.96, d.f. = 17, P = 0.03). The opposite was true in the root site where yellow

males had higher contrast values than did blue males (mean blue CI = 0.80, mean yellow

CI = 1.8, one-tail t-test = -1 35, d.f. = 15, P = 0.04). The relative difference in CI between blue and yellow morphs differed significantly between beach (mean = 3.7) and root

(mean = -1 .O) habitats (t-test = 3.629, d.f. = 6, P = 0.01), indicating higher relative contrast of blue males against the beach background and yellow males against the root background, as predicted.

Between-habitat analysis of reproductive success

The frequency of male colour morphs differs significantly within (separate

ANOVAs and posthoc Tukey's HSD tests (Figure 3.3): Beach F = 29.6, d.f. = 29, p <

0.000 1 ; Root F = 68. I, d.f. = 29, p < 0.000 1) and between (two-way ANOVA with habitat and ~norplias independent variables: F = 37.0, d.f. = 59, p < 0.0001) habitats

(Figure 3.3). There were no within habitat differences in morph frequencies between paired (have adjacent beach and root habitats) and unpaired sites (single transect in one habitat) (Beach: morph*site pairing F = 4.48, d.f. = 1, P = 0.42; Root: morph*site pairing

F = 1.29, d.f. = I, P = 0.29), therefore all further analyses treat sites singularly. Male density also differed significantly between habitats (mean density beach = 22.1 males/transect (+I- 4.7 SE); mean density root = 72.2 malesltransect (+I- 2 1.5 SE), t-tesl = 2.28, d.f. = 10, p = 0.04), but OSR did not (mean beach = 0.23 (+I-0.02 SE), mean root

= 0.28 (+I- 0.02 SE); t = 1.70, d.f. = 10, P = 0.12). A univariate ANCOVA with morph

frequency as the dependent variable, male colour morph as the covariate and four

independent variables representing spatial and temporal observational units (habitat, site,

time period. and season) showed that only habitat (morph*habitat: P < 0.001) and site

(morph*site P < 0.001) influenced differences in frequency between morphs. Time period

(morph*period P = 0.07) and season (morph*season P = 0.66) had no effect; therefore, all

subsequent analyses use pooled values for transect (habitat) and site (where appropriate)

as the sampling unit. A similar analysis showed no influence of shade (morph*shade P =

0.67), cloud cover (morph*cloud cover P = 0.30), or water visibility (morph*visibility P

= 0.67) on differences in frequency between morphs.

We evaluated the relative difference between blue and yellow males with respect to several variables that could influence their reproductive success (Table 3.2). As predicted, blue males had a higher probability of being paired (P,) than yellow males in the beach site (positive value) compared to the root habitat, as indicated by a negative value. Similarly, while paired, the blue morph had a higher spawning rate in the beach habitat, while yellows did better in the root environment. The morph being cuckolded more often by one sneaker (Pkl)differed significantly and as predicted between habitats, although no difference was found between the probability of two or more cuckolders

(PQ) being present at a spawning event, possibly because this happens so infrequently

(less than 4 '% of all spawnings). There was also no difference in the probability of both cuckoldry and cannibalism (PI,) occurring during a spawn, again likely because this happens relatively rarely. When unpaired (P,,), sneaking rate differed significantly between blue and yellow males in each habitat as predicted; the less frequent, lower

contrast morph had a higher rate of sneaking than did the more common, higher contrast

morph. There was no difference in the number of females that males approached while

unpaired (approach rate), however blue males were on average more successful at pairing

with females they approached (Pa) in the beach habitat, yellows in the root habitat as

predicted. There was no difference in the rate at which morphs were attacked by other

males (although values lie in the predicted direction). Yellow males were more likely to

win fights (P,.) in either habitat although significantly more so in the root habitat. There

was no difference between morphs or habitats in the probability that a female leaves a

male without spawning with him.

Within-habitat analysis of reproductive fitness

The results of our fitness model suggest that overall blue males have higher

reproductive success than yellow males in the beach site (relative fitness beach: blue = 1,

yellow = 0.42, one-tailed t-test P = 0.04), where they are more frequent, and that yellow

males have higher fitness relative to blue males in the root habitat (relative fitness root: blue = 0.09, yellow = 1, one-tailed t-test P = 0.005), where they are more frequent (Figure

3.4). The difference in fitness between blue and yellow males across habitats was also significant (two-tail t-test = 3.75, d.f. = 10, P = 0.004). The relative difference in fitness between blue and yellow males in the beach habitat is only 58% compared to 91% in the root habitat. There was no difference in fitness between paired and unpaired sites within a habitat (Beach: morph* site pairing F = 0.003, d.f. = 1, P = 0.96; Root: morph*site pairing F = 0.335. d.f. = 1, P = 0.57). Sensitivity analyses (Figure 3.5) reveal that assumptions about fitness parameters for only two scenarios, spawning in the absence of cuckolders or cannibals (Figure 3.5~1)and sneaking while unpaired (Figure 3.5f) affect the final outcomes, and only for the beach site.

3.5 Discussion

Our results suggest that environmental heterogeneity, coupled with environment- contingent selection favouring the male colour morph that contrasts most strongly with the background, helps to maintain male colour polymorphism in T. sntwsinor~~m.

Significant differential morph success between habitats was found in seven of the 14 individual variables that we examined, while within-habitat differential morph fitness was also found, indicating the importance of environmental heterogeneity in maintaining this male colour polymorphism. In the beach habitat, where blue males are more common than yellow males, they are also more conspicuous against a more yellow background and have 58% higher relative reproductive fitness. The opposite is true in the root habitat, which provides a relatively more blue background compared to the beach habitat, and against which yellow males contrast more strongly than blue males, are more comnion and have 9 1% higher relative reproductive fitness.

The genetic mechanism controlling colour expression in T. sar~~sinot-ummales is not currently known. Several attempts to raise split-clutch crosses, which would help identify the pattern of colour inheritance, were unsuccessful. There is reason to expect a large genetic component moderating colour expression in this species, as has been shown in many other inale colour polymorphic taxa (e.g., Sinervo and Lively 1996; Seehausen et al. 1999b; Brooks and Endler 2001; Fuller and Travis 2004). For example, the five male morphs of Poecilinpcrrar are determined by a relatively simple, one locus, five allele, Y-

linked genetic system (Lindholm et al. 2004). Much evidence in other colourful taxa also

suggests that environmental conditions will influence colour expression (e.g. carotenoid

availability (Olsen and Owens 1998; Grether et al. 2001)). In order to understand the

relative influence of the environment on colour expression we need to assess both the

genetic basis of colour, and also the physical nature of colour expression (i.e., structural

colours versus pigment-based colours). Instead of being determined by different alleles,

male colour could alternativcly be a product of a genetic switch mechanism (i.e.,

individuals are genetically similar) and variation in the environment (West-Eberhard

2003). In some locusts, for example, colour pattern is determined in the developmental

stage by rearing temperature and conspecific density (Song 2004). Many examples of

plolyphenisms (phenotypically plastic morphs) exist and it is possible that the "switch"

mechanism is maintained by selection if there is an advantage for multiple forms in the

population (West-Eberhard 2003). Until we can successfully raise progeny from test

csosses we will not be able to determine the genetic basis of colour polymorphism or the

influence of the environment on colour expression in this species.

Understanding the relationship between animal colour patterns and the environment relies on how light is measured and subsequently interpreted. Methods for measuring colour pattems and backgrounds have improved considerably (Endler 1990;

Endler and Mielke 2005; Endler et al. 2005; Fleishman et al. 2006), however, choosing an appropriate method remains difficult and is often logistically constrained.

Incorporating information about the visual system of the animal substantially improves the interpretation as it allows an approximation of how a colour pattern is perceived by conspecifics (Endler and Mielke 2005; Endler et al. 2005). Here, we use a method that directly measures colour and background in the natural environment and incorporates some basic information about the spectral sensitivity of photoreceptors. Other methods are much more data intensive and allow for more detailed statistical analyses (see Endler and Mielke (2005)), however we were not able to acquire such data. Very few studies include field measurements of colour and background and even fewer incorporate any visual properties of the organisms receiving colour signals. Therefore, our use of

Euclidian contrasts (as per Endler (1990) and Pauers et al. (2004)) that incorporate cone spectral sensitivity is a good first approximation of the contrast differences between morphs in different environments.

Radiance measurements of fish colour were taken at approximately I m depth, where spawning typically takes place, with the probe aimed horizontally from I0 cm away, a distance we estimate to be behaviourally appropriate for females to examine males and for other males to examine each other before combat. Females may sometimes be slightly below approaching males and so would be looking at them at a slight upward angle rather than to the side. This may influence the background against which males are viewed by females, making temporally dynamic elements of the background (e.g., cloud cover, shade) relatively more important. However, while paired, males are often to the side and slightly ahead of females when tlying to entice them to spawn (Gray and

McKinnon 2006; Chapter 2), and so our horizontal measurement should still reflect the contrast a female sees before choosing to spawn with a particular male in a given environment. Males approach each other directly and so this is an appropriate angle for intrasexual viewing. The outcome of our model combines the probability of gaining and losing fjtness

by opposing forms of selection and so represents a conservative estimate of relative

fitness. Our estimates of fitness values were based on several sets of assumptions. The

first was that if a male spawned in the absence of cuckolders and/or cannibals then he

received a full unit of fitness for the spawn. We have no direct observations of how many

eggs are laid at each spawning event; however, we assume the number to be quite low as

females have on average 20 ripe eggs in their ovaries at a time (Gray; unpublished data)

and they spawn multiple times throughout the day. A change of 50% in the fitness value

used for the probability of a successful spawning did not change the difference in fitness

between blue and yellow males in the root habitat, although a decrease of 25% did

equalize blue and yellow fitness in the beach habitat. This is reflected in the fact that

there is a smaller difference in fitness between blue and yellow males in beach compared

to root sites, and may indicate that selection is weaker in the beach habitat.

The only other value that, when changed, caused a decrease in the difference

between blue and yellow fitness was that assigned to the probability of gaining fitness when unpaired and attempting to sneak fertilizations. Again, the effect was only seen in the beach habitat, where a 20% increase in the fitness value of sneaking negated the fitness advantage of blue males. There is a difference in the rate of sneaking between habitats, with yellow males sneaking at a greater rate than blues in the beach habitat and blues sneaking more often in the root habitat. This suggests that in the beach habitat, less coi~spicuousyellow males are better able to sneak fertilizations while blues are better in the root sites where they are more cryptic. Sneaking may be the only viable tactic for less conspicuous morphs if they are less likely to attract females. The actual difference between sneaking rate among blue and yellow males in the beach habitat is larger than

the difference between the two in the root habitat by 52%, which may explain why

increasing the amount of fitness obtained by sneaking negates the relative fitness

difference between blue and yellow males only in the beach habitat. However, cuckoldry

only happens during 12% of spawning events (Gray et al. 2007) and may not have a large

effect on overall reproductive success. There is a theoretical possibility that, when

sneaking, males ejaculate more sperm per spawn than paired males thereby giving

sneakers an advantage in sperm competition (Parker 1998; Engqvist and Reinhold 2006),

although we have no data to test this in T. .sni.crsino,-urn.

Disentangling the effects of intersexual selection (i.e., female mate choice) from

intrasexual selection (i.e., male-male competition) has proven difficult in this system. The

role of female choice in driving the difference in the probability of a particular morph

being paired at any given time and place is uncertain. Females have been observed

leaving a male without spawning. They have also been observed 'aborting' a spawning

event: at the point where the male and female place their abdomens on the substrate, but

before 'quivering' (i.e., putative spawning) takes place, the female swims rapidly away

from the male. This generally happens when other males try to cuckold, which may

indicate that the female is making a choice about who she wants to fertilize her eggs or is attempting to avoid potential egg cannibalism. Further investigation of the female's role

in maintaining male colour polymorphism is warranted, but may only be possible once

lab populations have been established. Ideally one would want to test whether females actively choose to pair with differently coloured males under different visual situations as has been shown in several other systems (e.g., guppies (Gamble et al. 2003); cichlids (van

Doorn et al. 1998)): or if they accept whichever male wins fights against competitors.

The importance of male-male competition in sexual selection and the generation

of colour variation has recently been treated theoretically (see Seehausen and Schluter

(2004)) and empirically in the Pundumillia cichlid complex of Lake Victoria, Africa

(Dijkstra et al. 2005; Dijkstra et al. 2006). Male aggression is biased towards like-

coloured males, giving rare colour morphs an advantage in securing good territories.

Thus, negative frequency-dependent selection appears to be important in the generation

of colour polymorphism and possibly speciation in the cichlid system. It appears that the

yellow T. s~rrusiiiorzrmmorph has a higher probability of winning fights, although the

most conspicuous morph is attacked proportionately more often by other males (although

not significantly so). There could be more subtle interactions between males of different

or same colouration. There is a link in many aniinals between carotenoid-based

secondary sexual traits, increased aggression and testosterone levels that could influence

interactions between males (reviewed in Folstad and Karter (1992)) and so we plan

further investigation to determine which colour morphs are attacking most in each habitat

and whether the outcon~eof fights is dependent upon the colour of the attacking male in

each habitat. It is possible that intrasexual male interactions drive environment- contingent selection and therefore influence the maintenance of colour polymorphism in this species.

If male colour polymorphism in T. sorusinorzmi has a largely genetic basis, then several processes could maintain the polymorphism, including heterozygote advantage, divergent selection between populations with gene flow, or negative frequency-dependent selection (Ford 1965). Heterozygote advantage, or Iieterosis (heterozygous individuals

have a fitness advantage over homozygotes), could only maintain the polymorphism if T.

sat-asiimwnfemales carrying specific colour alleles are able to recognize males

homozygous for the alternate allele, thereby facilitating dis-assortative mating. Given that

individual females mate with multiple colour morphs and that sneaking often negates

female choice, this scenario does not seem plausible. Although we show here that the

direction of selection differs between the two mating habitats, we do not know if mating

habitats contain isolated populations, mostly isolated populations with some gene flow,

or if the there is constant movement of morphs between habitats meaning that the lake is

effectively a single population. It is very unlikely that each mating habitat represents an

isolated population as there are no obvious boundaries to fish movement and the habitats are often adjacent. Therefore, divergent selection between mating habitats with gene flow

is probably not driving the maintenance of polymorphism in this species. Negative frequency dependent selection is expected to favour rare morphs. This could be facilitated by females preferring rare colour morphs (e.g., Hughes et al. 1999) or if competition between like-coloured males is more intense (e.g., Dijikstra et al. 2005).

Whether negative frequency dependent selection occurs at a flne scale (i.e., within a mating habitat) or at a larger scale (i.e., across habitats within the lake) will depend on whether mating habitats represent isolated populations. If they do, then perhaps the advantage that rare, less conspicuous niales enjoy via higher sneaking rates and lower egg cannibalism allows them to persist within the population. However, if movement between habitats is common, then negative frequency dependent selection is more difficult to imagine. One possible scenario is that fish density and resource limitation (in terms of both energy and mating habitat availability) drive the system: when a morph becomes

abundant within a habitat, a lack of resources may force males to move to the alternate

mating habitat where they are less successful. All of this depends on movement of fish between habitats, something for which we currently have no data.

Given the intensity of fighting and pressures of sneaking and cannibalism, one might expect males to move between habitats such that they maximize their fitness based on their colour contrast. The two mating habitats are often adjacent and fish can move easily between them. There are, however, large tracts where few T. scrrwsinor-tun are ever observed, and these may pose barriers to movement. Both male and female T. strrusinorzrni move out of the spawning habitat to deeper water each afternoon and return starting early in the morning (Gray; personal observation). We have only anecdotal evidence of some site fidelity (i.e., the same recognizable individual was repeatedly observed in the same general area, but on a larger scale than one transect). Tagging and individually marking fish by clipping tins proved unsuccessf~~l.We do know that within a focal follow, neither males nor females tend to exceed a total distance travelled of -1 0 m

(Gray, unpublished data). It is possible that some males are moving to the habitat where they can gain the highest reproductive success while trading-off the cost of time spent moving through potentially unsuitable habitat.

Parallel processes of environnient-contitigeiit selection may be involved in the maintenance of colour polymorphism in other telmatherinids in the Malili Lakes. For example, a related species of fish, Tornintrrlg~~sp., found in neighbouring Lake Towuti, uses two similarly distinct mating habitats, beaches of algae-covered boulders and very deep shore sites with large fallen trees covered in algae (Gray and McKinnon 2006). We have observed up to 14 colour morphs incorporating red elements, in this species, and similar mating behaviour including sneaking but not cannibalism. Temporal variation in the visual background (i.e., dawn versus mid-day) may play a role in the persistence of blue and yellow Telmnthrri~mmtnnitre males that spawn mainly in beach habitats in

Lake Matano (J. McKinnon, unpublished data). There is a need for further exploration of the role of environment-contingent processes involved in the telmatherinid radiation.

Furthermore, understanding the link between behavioural mechanisms and proximate processes, such as tuning of the visual system in alternate environments, would improve our knowledge of how male colour polymorphism is maintained in these and other species.

3.6 Acknowledgements

We are grateful for the assistance of our Indonesian collaborator, F. Tantu (Tadulako

University, Sulawesi). We also thank F. Breden, B. J. Crespi, F. Herder, P. Nosil, S.

Pavey, A. Pomeroy, J. Thomson, S.J. Webster and the Dill, FAB*, and Cdte Labs at SFU for their insightful discussions and critical review of the ms. Field assistants A. Crawford,

D. Holm, A. Robertson, and S. van der Meer made the fieldwork possible. J. Ross provided a template for preliminary colour analyses. F. Herder graciously donated 7'. honti for MSP analyses and E. Loew (Cornell University) provided training and facilities for all MSP work. The Indonesia Science Foundation (LIPI) provided research permits.

Logistical support in Sulawesi was facilitated by Pt. Inco. This work was funded by NSF to JSM, EIUDP-CIDA to LMD, Sigma-Xi GIAR, PAD1 Project AWARE and Aqualung

Canada donations to SMG. 3.7 Literature Cited

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Table 3.1: Location of sites (GPS coordinates) and sample sizes for transect counts, and male and female focal follows for each of eight permanent sampling sites (1 2 transects) around Lake Matano. Site Latitude Longitude Habitat Sample Transect Male Female Code" (s) (W) days Counts Follows Follows (B,Y)~ 1B 2" 18' 12.1" 121" 11'42.3" Beach 7 24 30(11, 10) 4 5 Root 121" 12' 12.4" Beach 121" 14' 18.0" Beach 121"15'14.3" Root 121' 15' 50.2" Beach Root 121" 15' 53.5" Beach Root 12 1" 16' 39.4" Root 12 1" 15' 24.7" Beach Root a) The number denotes the site and the letter shows whether the transect was in beach (B) or root (R) habitat, with four sites having transects in both habitats. b) Values in brackets represent the subset of follows for blue and yellow males, respectively. Table 3.2: Mean difference between blue and yellow males for individual variables (defined in text) influencing reproductive success. Fitness Variables" Beach (+I- SE) Root (+I- SE) P" P pi, 0.17 (0.04) -0.30 (0.04) <0.0001 Spawning rate P"

PC pk l Pk2 Pb P" Sneaking rate Approach rate pa Attack rate put PI a) The differential for each variable was calculated by subtracting the mean yello\v value from the mean blue value, with the mean values estimated from six values, one from each site for each habitat. b) P-values reported are for one-tailed t-tests (d.f. = 10) between beach and root habitats, in bold where significant at u = 0.05. Figure 3.1: Map of Lake Matano, Sulawesi with permanent sampling site numbers marked. Site letters indicate (a) sites with two transects, one in both beach and root habitat, (b) unpaired sites with only one transect in beach habitat, and (c) unpaired sites with only in transect in root habitat. 400 450 500 550 600 Wavelength (nm)

Figure 3.2: Mean background radiance spectra (normalized to photon flux of 1) for beach (0:n = 6 sites) and root (e;n = 6 sites) mating habitats. Blue IYellow Blue Yellow IGrey Grey-Yellow

Beach Root Mating Habitat

Figure 3.3: Mean morph frequency (nb,,,,, = 6 sites, n,,,, = 6 sites) (+ 1 SE) of all five male colour morphs at beach and root mating habitats. Pair-wise Tukey HSD tests within a habitat show significant differences (a > b > c > d) between morphs at a = 0.05. Beach Root

Figure 3.4: Mean fitness (+ 1 SE) differs significantly between blue and yellow male colour morphs in the two main spawning habitats (one-tailed t-tests: beach t = 1.938, d.f. = 10, P = 0.04; root t = -2.606, d.f. = 10, P = 0.013). a b C.08 - 0.05 - 0.0; - * 0.04 " 4 z *+*, *** -d ;0.02 - ****** ;0.cc - *440 m a 2 0.02 - 0.02 - C,OII1 ' = = m ...,..... =O . . . C 1 0 1 0.4 0.8 0.8 1 0 C.; C .: C.E f(neither) f(can11ibalism) C d 3.X --- 3.05 - t 4 +**0***.+ 3 32 - 0.05 - *****o***** al al a 3.33 - -a 3.32 - 3Jw~==-mmD 0: ..... &3 3." - ... .om m., 331 - 0.01 - .

3 -I 3 7 02 0.6 o C' 0.8 c 0.2 c .L 0.6 f(l cuckolded f(2+ cuckolders) e f 0 35 , 308 334<>4444* t t t t i 3w- P) I QI ma 333- I3 332- 44O 2 l 3 32 3 31 Fm~w.mm.mm. z034: mmmmmO.~mmm

3, 3 3 31 3: 3? 34 3-E 3E' 33 33 3s 3F 3e 37 38 f(cuckoldry and cannibalism) f(sneaks)

Figure 3.5: Sensitivity analyses for the fitness values used in the fitness model (open symbols represent the values used in the model to generate fitness values for blue and yellow males in beach (e)and root (B) habitats). Each fitness value was increased and/or decreased up to 50% in increments of 5% while all other values were held constant in the model. Each point represents the p-value for a test between blue and yellow male fitness at each fitness level for the beach and root sites separately. CHAPTER 4

CUCKOLDRY INCITES CANNIBALISM: MALE FISH TURN TO CANNIBALISM WHEN PERCEIVED CERTAINTY OF PATERNITY DECREASES

Gray, S. M., L. M. Dill, and J. S. McKinnon. 2007. Cuckoldry incites cannibalism: malc fish turn to cannibalism when perceived certainty of paternity decreases. American Naturalist 169:258-263. 4.1 Abstract

Perceived certainty of paternity is expected to influence a male's behaviour

toward his offspring: if he is uncertain of his reproductive success with a current brood due to the presence of cuckolders, it may benefit him to invest instead in future reproduction. A decrease in perceived certainty of paternity incites filial cannibalism (the eating of one's own offspring) in some teleost fishes that provide parental care; however, no work has yet demonstrated that cannibalism increases proportionately with increased

levels of cuckoldry. Here we show for the first time in a fish with no parental care that as the number of cuckolders at a spawning event increases, so does the probability that a male will cannibalize eggs. 111 field observations of Telmatherina sarasinorwn, a small fish endemic to Sulawesi, Indonesia, males increased filial cannibalism behaviour three- fold in the presence of one cuckolder and nearly six-fold in the presence of two or more cuckolders. This suggests that males may use detection of cuckolders as an indication that the paternity of current offspring has been compromised.

Keywords: filial cannibalism, sneaking, Tc~lmcr~lwrir~u.vcrr.uvit~onirn 4.2 Introduction

The evolution of filial carmibalism (the consumption of one's own offspring) is thought to be driven by an energetic trade-off between caring for the current brood and investing in future reproductive success by eating offspring (Rohwer 1975; Manica

2002b). In species with male parental care, a male's assessment of the value of the current brood can incorporate information on his own condition (i.e., energy reserves)

(Neff 2OO3b). the quality and availability of mates (Okuda et al. 2004), prey availability

(Vinyoles et al. 1999), and his perceived certainty of paternity (Neff and Gross 200 1 ;

Neff 2003a: Manica 2004). If a male perceives that the value of his current brood is low, then eating the current brood, either wholly or partially, may improve his future reproductive success (Manica 2002b).

Most work, and all theory, on the evolution of filial cannibalism has focused on understanding the energetic trade-off in teleost fishes that provide parental care, n~ostly by males (Sargent 1992; Manica 2002b). Parental care requires an investment both in time and energy spent rearing or guarding offspring whereas in the absence of parental care there is no energetic cost beyond mating. In both cases, eating offspring is expected to add to future reproductive success through an energy gain (Manica 2002b), however parental care-givers could also benefit by relieving themselves of low value offspring. In species with parental care it is hard to distinguish how the value of current offspring is evaluated because there can be multiple cues about paternity, availability of food and mates, etc. over an extended period of time (Neffand Sherman 2002), but by investigating cannibalism in the absence of parental care we can examine the effect of perceived certainty of paternity in isolation. Perceived certainty of paternity based on visual detection of cuckolders by a male could be an immediate indicator of the value of potential offspring by providing information on expected relatedness (Neff and Sherman 2002). If this is true then a male could use information on sneakers (i.e., their presence and number) to estimate his mating success and adjust his behaviour toward current offspring accordingly. As a male's certainty of paternity decreases, such as when his spawning attempt is cuckolded by

"sneaker" males, so does the value of the brood. Filial cannibalism could be a tactic to recoup energy lost to mating efforts (e.g., through aggressive male-male interactions over mates) and hence increase energy for the next mating bout (Manica 2002b). The cost to a male of mistakenly consuming some of his own eggs may be high with respect to his reproductive fitness. However, this cost may be outweighed by the benefit of securing more energy to find another mating opportunity where his certainty of paternity is higher.

We examined the relationship between filial cannibalism and sneaker presence and abundance using field observations of a small fish that does not provide parental care for young, predicting that the presence of sneaker males and increased numbers of sneakers would both result in increased cannibalism attempts by males.

Study organism and mating system

Telm~~/her.iric~scrr.a.sinor-zim is a small, colourful fish endemic to Lake Matano,

Sulawesi, Indonesia (Kottelat 199 l), that spawns on the substrate and provides no parental care (Gray and McKinnon 2006; Chapter 2) (Figure 4.1). Neither males nor females hold territories (Gray, personal observation). A detailed description of mating behaviour in this species is given by Gray and McKinnon (2006); here we describe only the relevant details. Three behavioural tactics are employed by males: 1) "courting

males" are identified as those males actively paired with and courting a female at the time of observation; 2) "sneaker males" are those males that at the time of spawning between a female and a courting male, rush into a position parallel to the spawning pair and attempt

to fertilize some of the eggs being spawned (indicated by quivering behaviour typical of sperm-release in many fishes, e.g, Rainbow darters, Etheostomn cner~zrlezrrn(Fuller

1998)); and, 3) "non-mating cannibals" are identified as males that dart in toward the spawning pair and attempt to eat the eggs but are not directly involved in the spawning event. An individual male may perform any of these behaviours depending upon his current situation and may switch between tactics in a matter of seconds. For example, a courting male may lose a fight to another male and imnlediately switch to the sneaking tactic if the opportunity presents itself. Males fight intensely over females and females can be paired and spawn with many different males in a short period of time, although females do not spawn with all males they are paired with. Males do not invest any energy in care of their offspring post-mating; however, they do spend a large amount of time

(and thus energy) courting and retaining fen~alespre-spawning.

Lake Matano is relatively resource-poor (Haffner et al. 2001) and T. .strr.asinor.zrm regularly consume congeneric eggs (Kottelat 199 l), as well as the eggs of other fish species (e.g., endemic gobies, Glossogobizrs sp.) (Gray and McKinnon, personal observation). Immediately after spawning, males turn to inspect the area where eggs were apparently laid and have often been observed "picking" at the substrate in that same spot in a similar manner to when they are consuming the eggs of other species. Picking behaviour has been used to identify filial cannibalism in other systems (e.g., bluegill

sunfish Lepornis rtracr-ochirus (Neff 2003b)).

Observational study and analyses

This study took place in Lake Matano, Sulawesi, Indonesia over three field

seasons: Januaiy to March 2003, January to May 2004 and October to December 2004. T.

.sur.n.sir~orun~spawn throughout the year in two distinct habitats: beach sites that are

relatively shallow and flat, and root sites that drop off steeply from shore and where

spawning takes place on overhanging 1,egetation (Gray and McKinnon 2006). We do not

distinguish spawning events based on habitat here, as there was no significant effect of

habitat on sneaking and cannibalism (Wald Chi-square = 0.23, d.f. = I, P = 0.63).

Females are cryptically coloured whereas five male colour rnorphs coexist (Figure 4.1).

The role of male colour will be explored elsewhere (Gray; unpublished data); here data for all morphs are pooled. All male colour morphs employ all three behavioural tactics and do not represent alternative reproductive strategies as seen in other systems (e.g., bluegill sunfish (Gross and Charnov 1980)).

Focal follow observations (n = 576) of female T, snrminorzrrn were made by snorkelling along 13 transects (80 n~ long, 1-2 m deep) at nine sites around Lake Matano.

Each site was visited between five and seven times over three field seasons with each visit separated by at least one week. The number of observations included in our data set per site, per date visited range from one to 28 (mean eight observations per site per visit) and were conducted by at least two (often three) observers, meaning that at one time two or three individual females were being followed. Male density in mating habitats is high (approx. 47 per 80 m by 2m transect (Gray and McKinnon 2006)). The operational sex ratio (females to males) was derived indirectly as the number of males paired with a

female relative to the total number of males counted during a particular observation period and was found to be strongly male-biased (range 0.19 to 0.34, mean 0.26 (+I-SD

= 0.06)). The relatively high density of males in the mating habitat, coupled with the observation that these fish are mobile and often roam across or out of a particular site

(Gray, personal observation) suggests that this is an open system and that the likelihood of re-sanlpling males is very small.

Focal observations were conducted by haphazardly selecting a female paired with a male and observing her for four to 10 minutes. If the original courting male left the female (either by choice or because he lost a fight with another male), we continued to follow the female. We recorded all apparent spawning events, the number of males involved in the spawning event, whether each male was a courting male or a sneaker during that mating event, and any apparent incidence of cannibalism, (i.e., picking behaviour at the site of spawning). We also recorded the presence of non-mating males that attetnpted to cannibalize eggs but were not directly involved in spawning. To confirm oophagy we dissected the stomachs of I3 male fish caught on one of the transects during a time of peak mating (and conspecific cannibalism) and measured any eggs to determine if they were likely 7'. scrr-usitm-zrm eggs. It was impossible, logistically, to catch male fish directly after observing picking behaviour and so our measure is an indirect one.

Spawning events were only observed during 385 of the 576 female follows included in this survey. Females often spawn with more than one male and several times with each male during an observation period. We considered the first spawning event for each different courting niale (i.e., only males distinguishable by colour and/or size, n =

470 courting males) to be independent of previous events and only this subset of data was used for analyses (i.e., only 470 of 1089 spawning events). We used a binomial logistic regression on courting male cannibalism (response variable) to determine the relative importance of the predictor variables sneaker presence (n = 55) and non-mating cannibal presence (n = GO). We used Pearson Chi-square tests to evaluate a) if the incidence of filial cannibalism by courting males (i.e., those paired with the female at the time that spawning took place (Gray and McKinnon 2006)) is associated with the presence of sneaker males, and b) if filial cannibalism by courting males increases with the number of sneakers present at a given spawning event. Relatively few spawning events with multiple sneaker males present remained in the data set after removing non-independent events and so we pooled those into one category representing events where two or more males were present.

4.4 Results

We found T. s~lrcrsinorwmeggs in the stoniachs of males suggesting that picking behaviour may result in the consumption of eggs. Of 13 male fish sampled, 1 1 had eggs of a similar size to T. s~~rusinorxmeggs (average egg diameter = 1.34 mm, n = 76 eggs from 3 females; Gray, unpublished data) present in their stomachs, consistent with conspecific cannibalism. Females have never been observed practising filial cannibalism although both sexes appear to eat heterospecific eggs (Kottelat 199 1; Gray and

McKinnon, personal observation). Twelve percent of all spawning events involved one or more sneaker males (range

1 to 4 sneakers, mean = 1.6 (+I- 0.8 SD)). When sneakers were absent (n = 4 15), courting

males attempted to cannibalize eggs after 13% of the spawnings (Figure 4.2). This

behaviour increased significantly in frequency to 62% when one or more sneakers were

present (n = 55), suggesting that when a male's perceived certainty of paternity decreases

lie is more likely to cannibalize (2= 70.6, d.f. = I, P < 0.0001). Certainty of paternity

should be a decreasing function of the number of sneakers present. Accordingly, when

only one sneaker was present (n = 35), filial cannibalism by the courting male was three

times more likely (5 1 %), while there was a nearly six-fold increase (80%) when two or

more sneakers (n = 20) were present (Figure 4.2: X2 = 4.40, d.f. = 1, P = 0.04).

We observed conspecific cannibalism by inales not involved in the spawning

event (Figure 4.1, Appendix 1 : Movie A 1.2). These males are often involved in the fights

that take place prior to spawning and it is unclear whether they were not close enough or

fast enough to attempt a sneak fertilization or chose to have a meal in lieu of mating.

Such non-mating males attempted to cannibalize eggs at 13% of the spawning events

(Table 4.1). There is a 10% increase in the probability of filial cannibalism by courting

males in the presence of these non-mating cannibalistic males (Table 4.2). The binomial

logistic regression used to determine the effects of sneakers and non-mating males on cannibalism by courting males (data presented in Table 4.2) showed that both were significant. However, the effect of sneaker presence was stronger than the effect of the presence of non-mating cannibals (Wald Chi-square value: sneakers = 52.7 cf. non- mating males = 4.44, P sneakers <0.000 1 cf. P lion-mating males = 0.04, d.f. = I), and there was no interaction between these factors (Wald Chi-square interaction = 0.004, P = 0.94, d.f. = 1). Filial cannibalism by the courting male was three times more likely in the presence of sneakers compared to the presence of non-mating cannibal males, suggesting that certainty of paternity is a better predictor of filial cannibalism than the risk of egg loss to non-mating males (Table 4.2).

4.5 Discussion

The probability of cannibalism by T. sarmii~orwmcourting males increases with number of sneakers present (Figure 4.2). This positive relationship between male filial cannibalism and the number of sneaker males is consistent with the hypothesis that males use the presence of sneakers as an indicator of the value of the current brood and adjust their behaviour based on perceived certainty of paternity.

Genctic tests of paternity, which at this time are not possible for T. sur~tainorzirn, could help to determine if males are consuming only non-related eggs, in which case eating eggs after cuckolded spawning events would be of no detriment to a male's fitness.

Only one previous study has attempted to document filial cannibalism in nature in this manner. DeWoody et al. (2001) used microsatellite paternity analyses on embryos found in the stomachs of nest-tending male tessellated darters (Etheostorna olmstedi) and two sunfish species (Leporni.~awitlrs and L, prrncttrtzrs). They determined that none of the males seemed able to discriminate between related and non-related embryos because both were consunied. Given that darter and sunfish males spend a significant amount of time caring for their offspring and still have not evolved the ability to differentiate between related and unrelated embryos when cannibalizing, it is unlikely that T. strr~asinonrr~~ males, who spend no time caring for young, would evolve such an ability, especially since fertilization may not even be complete at the time of consumption. Male bluegill sunfish do change their parental behaviour when cuckoldry is detected and paternity of the brood is mixed, and can even use olfactory cues to detect unrelated juvenile fish in the nest post-hatch and adjust their effort accordiilgly (Neff 200321; Neff and Sherman

2003); however, there is no evidence that they differentiate between kin and non-kin when cannibalizing.

A courting male's information on sneaker presence may not always be perfect

(Neff 2OO3a), which may explain why there is some filial cannibalism even in the absence of sneakers (Figure 4.2,O sneakers). Males also may cannibalize if the chance of the eggs being eaten by others is high; if so, we would expect males to cannibalize to the same extent in the presence of other, non-mating cannibals as in the presence of sneakers, but there is no evidence for this. Females may also be expected to eat the eggs if the chance of them being eaten is high, however the possibility that some eggs are fertilized and not eaten probably outweighs the energetic benefit she would gain by consuming the eggs. We have never observed females performing picking behaviour after spawning

(Table 4. I), although we have observed them picking at the substrate after heterospecitic spawning events.

Sometimes neither courting males nor sneakers attempt to cannibalize (Table 4.1) and it is possible that females do not always release eggs when appearing to spawn, as a tactic to avoid the loss of costly eggs to males. Although we have no intormation on the number of eggs released by females at each spawning event (there is no obvious egg mass laid), that number is likely to be low because they spawn continuously throughout the day. Also, a large proportion of the time that males and females make the effort to perform the typical quivering behaviour on the substrate (see Gray and McKinnon (2006)

for details) we expect that at least one egg is laid. Accordingly, we expect that eggs are

present and being cannibalized at least some proportion of the time that males perform

the picking behaviour because otherwise the cost of the repeated behaviour would be too

high. "Testing" of a male's cannibalistic behaviour by females has been shown in other

fish, such as the Mediterranean blenniid Aidablent~itrssyhyrzx; Kraak and van den Berghe

(1992) found that females only laid small clutches of eggs in empty males' nests. Those

males that did not cannibalize the eggs received more eggs at a later date than did males

that did consume the small clutches. Although it is not known if the same female returns to inspect the same nest, the benefit to the male is obvious. Theory is being developed to

incorporate female choice in studies of filial cannibalism (Lindstrom 2000; Manica

2002b) but little empirical work has yet been done. Further investigation of the female's role should enhance understanding of the evolution of filial cannibalism and mating strategies in T. sarcuit~orzrrnand other systems.

Our results clearly show that the probability of filial cannibalism attempts in T. strrvrsinor-z~nicourting males increases in the presence of sneaker males, suggesting that they can use detection of sneakers as an indicator of lowered certainty of paternity. Since

T. scrrwsinor.trrrr males do not invest in their offspring after egg-laying, canniba 1'~sm cannot be a tactic used to optimize the trade-off between present and future reproduction

(Rohwer 1978; Manica 2002a; Manica 2002b; Manica 2004) except with respect to recouping energy lost through mating efforts. Instead it seems the male may benefit from the energy contained in the eggs, and is increasingly likely to eat the eggs the less likely he is to have fertilized them. 4.6 Acknowledgements

Assistance with fieldwork was provided by our Indonesian collaborator Fadly Y. Tantu,

P. Hehanussa, LIP1 (Indonesian Science Foundation) and our field assistants A.

Crawford, D. Holm, A. Robertson, and S. van der Meer. Logistical support was provided by Pt. Inco. We are grateful to the FAB*Lab, the Dill Lab, F. Breden, 1. C6td and B.

Crespi for their comments on the ms and E. Gray and S. MacDonald for technical assistance. Financial support was provided by an NSF Grant to J.S.M, CIDA - EIUDP funding to L.M.D., and NSERC, PAD1 Project AWARE, and Sigma-Xi Grants-ln-Aid-

Of-Research to S.M.G.

4.7 Literature Cited

DeWoody, J. A., D. E. Fletcher, S. D. Wilkins, and J. C. Avise. 2001. Genetic documentation of filial cannibalism in nature. Proceedings of the National Academy of Sciences 98:5090-5092. Fuller, R. C. 1998. Sperm con~petitionaffects male behaviour and sperm output in the rainbow darter. Proceedings of the Royal Society of London B 265:2365-237 1. Gray, S. M., and J. S. McKinnon. 2006. A comparative description of mating behaviour in the endemic telmatherinid fishes of Sulawesi's Malili Lakes. Environmental Biology of Fishes 75:469-480. Gross, M. R., and E. L. Charnov. 1980. Alternative male life histories in bluegill sunfish. Proceedings of the National Academy of Sciences 77:6937-6940. Haffner, G. D., P. E. Hehanussa, and D. Hartoto. 2001. The biology and physical processes of large lakes of Indonesia: Lakes Matano and Towuti, Pages 183- 192 in M. Munawar, and R. E. Hecky, eds. The Great Lakes of the World: Food-web, health and integrity. Ecovision World Monograph Series. Leiden, Backhuys Publishers. Kottelat, M. 199 1. Sailfin Silversides (Pisces: Telmatherinidae) of Lake Matano, Sulawesi, Indonesia, with descriptions of six new species. Ichthyological Exploration of Freshwaters 1 :32 1-344. Kraak, S. B. M., and E. P. van den Berghe. 1992. Do female fish assess paternal quality by means of test eggs? Animal Behaviour 43:865-867. Lindstrom, K. 2000. The evolution of filial cannibalism and female mate choice strategies as resolutions to sexual conflict in fishes. Evolution 54:617-627. Manica, A. 2002a. Alternative strategies for a father with a small brood: mate, cannibalise or care. Behavioral Ecology and Sociobiology 5 1 :3 19-323. -. 2002b. Filial cannibalism in teleost fish. Biological Reviews 77:261-277. -. 2004. Parental fish change their cannibalistic behaviour in response to the cost-to- benefit ratio of parental care. Animal Behaviour 67: 1015- 102 1. Neff, B. D. 2003a. Decisions about parental care in response to perceived paternity. Nature 422:7 16-7 19. -. 2003b. Paternity and condition affect cannibalistic behavior in nest-tending bluegill sunfish. Behavioral Ecology and Sociobiology 54:377-384. Neff, B. D., and M. Gross. 2001. Dynamic ad-justment of parental care in response to perceived paternity. Proceedings of the Royal Society of London B 268: 1559- 1565. Neff, B. D., and P. W. Sherman. 2002. Decision making and recognition mechanisms. Proceedings of the Royal Society of London B 269: 1435- 144 1. -. 2003. Nestling recognition via direct cues by parental male bluegill sunfish (Lepornis mcrci-ochii-us).Animal Cognition 6:87-92. Okuda, N., S. Ito, and H. Iwao. 2004. Mate availability and somatic condition affect filial cannibalism in a paternal brooding goby. Behaviour 141:279-296. Rohwer, S. 1978. Parent cannibalisn~of offspring and egg raiding as a courtship strategy. The American Naturalist l2:429-440. Sargent, R. C. 1992. Ecology of filial cannibalism in tish: theoretical perspectives, Pages 38-62 in M. A. Elgar, and B. J. Crespi, eds. Cannibalism: Ecology and Evolution among diverse taxa. Oxford, Oxford University Press. Vinyoles, D., I. M. Cote, and A. de Sostoa. 1999. Egg cannibalism in river blennies: the role of natural prey availability. Journal of Fish Biology 55: 1223-1232. Zar, J. H. 1984, Biostatistical Analysis. New Jersey, Prentice-Hall, Inc. 4.8 Tables

Table 4.1: The proportion of spawning events for which females and males of each behavioural category were present and the proportion of spawning events where they were present that each attempted to cannibalize eggs. % Present at spawning event % Cannibalism attempts

Courting male Sneaker male Non-mating cannibal (male)

Table 4.2: Proportion of filial cannibalism by focal courting males in the presencelabsence of conspecific males, either sneakers or non-mating males (i.e., cannibals that did not participate in the spawning event). Sneakers

I present Absent

Present 0.75 (n=4) 0.23 (n=S6) mating males Absent 0.62 (n=51) 0. I3 (n=359) 4.9 Figures

Figure 4.1: Courting T. sarasinorrrm. Five male colour morphs with elaborate secondary sexual characteristics fight intensely over and mate with the same cryptically coloured females. All colour nlorphs can switch mating tactics between courting and sneaking very quickly and all morphs appear to practicc Mia1 cannibalism. 0 sneakers 1 sneaker 2+ sneakers

Figure 4.2: Probability of filial cannibalism by courting male T. sarasinvrum in the absence (0 sneakers, black bar, n = 415) or presence (I sneaker, white bar, n = 35; 2 or more sneakers, hatched bar, n = 20) of sneakers (plus one binomial standard error (Zar 1984)). APPENDIX 1 COLOUR PHOTOGRAPHS AND MOVIES ON CD-ROM

The appended CD-ROM forms part of this thesis.

Figures in JPEG form may be clicked to open. Movies in AVI format may be opened with any movie software (Windows Media Player recommended). The following figure legends correspond with files on the CD-ROM.

Figure Legends

Figure A1.1: a) A colour map of the Indonesian archipelago with the Island of Sulawesi boxed; b) The Island of Sulawesi with the Malili Lakes system boxed; and, c) The Malili Lakes system, which drains east into the ocean, Bay of Bone. Land is green and water is blue. The red dots represent sites that were surveyed over the course of three field seasons (see Figure 3.1 for permanent sampling sites in Lake Matano). (Figure-A1 .jpeg, 304 kb) Figure A1.2: Blue male Tc.lmuther-ina sar-usinorwm, collected from Lake Matano, Sulawesi. (Figure- A2 - blue.jpeg, 109 kb) Figure A1.3: Yellow male Telm~rther-inustrrusirm-zln~, collected from Lake Matano, Sulawesi. (Figure-A3-yellow.jpeg, 126 kb) Figure A1.4: Grey male Telmutherincr sar.u.si~~orwn,collected from Lake Matano, Sulawesi. (Figure-A4-grey.jpeg, 128 kb) Figure A1.5: Grey-yellow male Telrncrther.inu .sarminorum, collected from Lake Matano, Sulawesi. (Figure-AS-grey-yellow.jpeg, 1 16 kb) Figure A1.6: Blue-yellow male Telrnatl~erinusurasinor-urn, collected from Lake Matano, Sulawesi. (Figure-A6-blue-yellow.jpeg, 127 kb) Figure A 1.7: Female Telrnu~her-inasalrr,sinor-~~rn, collected from Lake Matano, Sulawesi. (Figure- A7 - female.jpeg, 98 kb) Figure A1.8: Mechanical frame used in the field to hold a radiance probe and take measurements of the colour of the fish and background against which they are viewed in the (a) root and (b) beach habitats. In the root habitat the frame was suspended at a depth of 1.0 nl while in the beach habitat it was placed on the substrate at a depth of l .O m. (Figure-AS.jpeg, 434 kb) Figure Al.9: Digital image of a fish with white circles denoting the patches of the fish measured with a radiance probe in the field. The field of capture for the probe is 0.4 cm at a distance of 10.0 cm from the face of the probe to the body of the fish. The patches were measured while the fish was suspended on a mechanical frame in the water (see Figure A8). (FigurePA9.jpeg, 195 kb) Movie Al.l: Mating behaviour of Telm~l/herir~usclmsinor~un in a beach habitat (site 2, Yacht Club, Lake Matano, Sulawesi). An apparent spawning takes place between a blue male and female at 28s, with subsequent cannibalism attempts by the paired and other non-mating males. Total video length is 36s. (Movie-A 1 .avi, 126 m b) Movie A1.2: Mating behaviour of Tdrncl/herintr.surminorutn in a root habitat (site 4, South Point, Lake Matano, Sulawesi). An apparent spawning takes place at 1 Is, with subsequent sneaking and cannibalism attempts by a number of males. Total video length is 24 s. (Movie- A2.avi, S5mb) APPENDIX 2 MICROSPECTROPHOTOMETRY OF CONES FROM TELMATHERINA BONTI AND MAROSA THERINA LADIGESI

I used microspectrophoton~etry(MSP) to determine the visual pigment complement of two fish species related to Telnlcr~herinasarasinorzrm, for the purpose of combing this information with the colour nleasurenlents taken of the fish under natural conditions to estimate how a male might be perceived by conspecifics. This technique requires that the fish be alive immediately prior to the extraction of the retina. Since I was unable to obtain live samples of T. sarasinorztrn, I initially tested five specimens of

Mnrosatherina ladigesi in August 2004 in the lab of E.R. Loew, Cornell University,

Ithaca, New York. This species is found in small, clear streams in the southern part of

Sulawesi and has been colleted and used in the aquarium trade for many years. Suppliers now breed the fish in captivity and few, if any, import wild stock. Similar to other telmatherinith of the Malili Lakes, M. ludigesi is colour polymorphic, having both blue and yellow male forms and is often used as the outgroup for the Malili Lakes telmatherinids. However, in October 2005 I received four Telmatherinu bonti individuals

(collected from the Petea River by F. Herder and bred in captivity in Bonn, Gernlany) and had them shipped to Cornell. T. bonti is a close relative of the telmatherinids of the

Malili Lakes and is found throughout the drainage system (Herder et al. 2006). It also exhibits blue and yellow male colour polymorphism, although the colours appear to be more muted than lake forms. I used the photoreceptor sensitivities obtained for T. bonfi in

my calculations of contrast (see Chapter 3) because of the closer relationship of that

species with T. .sur.asinoi.t~m.

I followed precisely the MSP methodology as outlined in (Loew 1994; Fuller et

al. 2003) and only briefly describe it here. Fish were dark-adapted overnight before being

euthanized. All procedures were carried out in a darkroom with miniinal infrared

illumination to prevent bleaching of the photoreceptor cells. The extracted eye was

placed in cold phosphate buffer (Sigma) to prevent the lysis of photoreceptor cells. The eyecup was hemisected and the lens discarded. The retina was then teased from the

pigment epilhelium and placed on a glass slide in a drop of buffer where it was macerated

using two razor blades to expose the outer segments of the photoreceptor cells. A coverslip, the edge lined with silicon grease, was placed on top of the macerated retina.

The MSP instrument is exactly that described in (Loew and Wahl 199 1): a computer- controlled single beam instrument with a 100 W tungsten-halogen lamp, a 50 x mirror objective lens as the condenser and a 100 x Zeiss lens as the objective, allowing accurate absorbance measurement into the UV. Individual cells were scanned from 750 to 350 at

1.0 nm intervals with odd nrn scanned on the downward pass and even nm on the return pass to 750 nm. The actual absorbance maxima (I,,,,, +I- standard deviation) for each scanned cone was detennined using the template fitting method (nomograms). Only those spectra with a standard deviation less than 10 nm were kept. All were fit best with an A1

(rhodopsin chromophore) template.

Teltncrtherina hotlti has five photoreceptor classes (Table A2. I; Figure A2. l), including a cone maximally sensitive to UV light. All classes were found in each of the four specimens sampled. itl~rrosutherinolcru'igesi had five or six different cone classes

(Table A2.2; Figure A2.2), with some variation between individuals, consistent with the findings of (Reckel et al. 2002).

Literature Cited

Fuller, R. C., L. J. Fleishman, M. Leal, J. Travis, and E. Loew. 2003. Intraspecific variation in retinal cone distribution in the bluefin killifish, Lucanin goodei. Journal of Comparative Physiology A 1 89:609-6 16. Herder, F., A. W. Nolte, J. Pfaender, J. Schwarzer, R. K. Hadiaty, and U. K. Schliewen. 2006. Adaptive radiation and hybridization in Wallace's Dreamponds: evidence from sailfin silversides in the Malili Lakes of Sulawesi. Proceedings of the Royal Society of London B 273:2209-22 17. Loew, E. 1994. A third, ultraviolet-sensitive, visual pigment in the Tokay gecko (Gecko geklio). Vision Research 34: 1427- 143 1. Loew, E. R., and C. M. Wahl. 1991. A short wavelength-sensitive cone mechanism in juvenile yellow perch Percn.flavescens. Vision Research 3 1 :353-360. Reckel, F., R. R. Melzer, J. W. L. Parry, and J. K. Bowmaker. 2002. The retina of five Atherinomorph Teleosts: photoreceptors, patterns and spectral sensitivities. Brain, Behavior and Evolution 60:249-264. Appendix 2 Tables

Table A2.1: Maximum wavelengths for five cone classes as determined by MSP for Telnmtl?er.inuAonti.

Cone Class Mean kmax +I- SD N Min. Max. (genomic (nm) (no. cones) kmax Am ax nomenclature) UV (SWS 1) 354.1 3 .O 12 349.0 358.8 Violet (SWS2a) 42 1.7 4.1 14 4 15.2 428.6 Blue (SWS2b) 44 1.7 7.4 2 5 43 1.4 455.7 Green (RH2) 523.4 4.0 42 515.3 533.0 Yellow (LWS) 542.9 3.1 11 539.3 550.0

Table A2.2: Maximum wavelengths for the cone classes as determined by MSP for ~Mcrr.osatherintrladigesi.

Cone Class Mean hmax +I- SD N Min. ~Max. (genomic (nm) (no. cones) kmax kmax nomenclature UV (SWS 1) 352.6 4.4 16 350 3 60 Violet (SWS2a) 412.8 7.7 4 5 400 425 Blue (SWS2b) 45 1.5 10.9 18 43 0 465 Green (RH2) ? 492.8 12.5 7 480 5 10 Green (RH2) ? 53 1.2 7.8 25 520 540 Yellow (LWS) 560.8 12.0 12 545 575 Appendix 2 Fi,oures

Figure A2.1: Frequency distribution of cones and rod cells as measured using microspectrophoton~etryon Teln?~~fl?er.im~honri, a closely related species of Teln~~~ther~ino sut-~~sinot-~~r?~.

Figure A2.2: Frequency distribution of cones as measured using microspectrophoton~etry on M~~rosurher-innladigrsi, a species related to the telmatherinids of the Malili Lakes.