Multiple paternity in the socially monogamous of

Diplomarbeit

Zur Erlangung des akademischen Grades Magistra der Naturwissenschaften (Mag. a rer. nat.)

an der Naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz Institut für Biologie

vorgelegt von Helgit Natmessnig

Unter der Betreuung von Univ.-Prof. Dipl.- Ing. Dr.in Kristina Sefc

Graz, 2018

Acknowledgements

First of all, I want to thank my supervisor Dr.in Kristina Sefc, for giving me the opportunity to work in this exciting field of evolutionary biology. Additionally, I would like to thank Kristina for so much more than just the support of my Diploma thesis.

Many thanks also to Holger Zimmermann for all the time he spent in explaining evolutionary relations and giving me an introduction in the lab work and the exciting life of cichlid species. Furthermore I will thank you for the wonderful drawing I got for better understanding how female removal experiments were done in the field. I am forever thankful to Angelika, Aneesh, Florian and again Holger for the funny stories about the field trips to Africa, the help and support during my work on the Institute of Biology and the nice coffee breaks and talks. Thank you all for having such a lovely and unforgettable time together. It was great to be a part of such a wonderful working group!

I want to thank my parents and parents in law for all the support and help during the last years. Thank you for being such lovely and outstanding grandmothers and grandfathers! Further, I want to thank Hedy for believing in me and helping me in order that I was able to finish my courses! Thank you for being such a lovely surrogate grandmother for Erik in Graz. Many thanks also to my siblings, siblings in law and my nephews and nieces Hergund (Goti), Mario (Gete), Adrian (our godchild), Annika, Helke, Bernd, Liam, Lisi, Mathias, Linda, Theo (our godchild), Harald, Oli, Valentin, Franziska, Gerhild, Konstantin, Karli, Herbert, Wolfgang, Eva, Marie and Carmen for your accompaniment the last years!

Moreover, I will thank Karoline (my godchild and the very best witness ever!), Kathi and Elli (the very best future bridesmaids ever!) and Daniel (the man with the sledgehammer) for being more than just friends!

I want to express my greatest gratitude to Markus, Erik and Skyla, my lovely family and the center of my life, for solving technical problems, for making me laugh, for your patience, for your support and for all your love – I love you!

I

Statutory Declaration

I declare that I have authored this thesis independently, that I have not used other than the declared sources/resources, and that I have explicitly indicated all material which has been quoted either literally or by content from the sources used.

Eidesstattliche Erklärung

Ich erkläre an Eides statt, dass ich die vorliegende Arbeit selbstständig verfasst, andere als die angegebenen Quellen/Hilfsmittel nicht benutzt, und die den benutzen Quellen wörtlich und inhaltlich entnommenen Stellen als solche kenntlich gemacht habe.

______(Place, Date) (Signature)

II

Contents

Acknowledgements ...... I

Statutory Declaration ...... II

Eidesstattliche Erklärung ...... II

Contents ...... III

Abstract ...... V

Zusammenfassung ...... VI

1 Introduction ...... 1 1.1 Cichlid species and their significance in ...... 1 1.2 Mating systems and parental care ...... 1 1.3 Differences between fish and birds: Why investigate monogamy and bi-parental brood care in fish? ...... 2 1.3.1 Brood size ...... 2 1.3.2 Bi-parental care incidence and brood care investment/cost ...... 2 1.3.3 Fertilization control ...... 3 1.4 Extra pair paternity (EPP) ...... 4 1.5 Alternative reproductive tactics (ARTs) ...... 5 1.5.1 Plasticity of ARTs ...... 6 1.5.2 ARTs in fish ...... 7 1.6 The target species Variabilichromis moorii ...... 8 1.7 Aim of the study ...... 9

2 Materials and Methods ...... 10 2.1 Sample collection ...... 10 2.2 Female removal experiment ...... 11 2.3 DNA extraction ...... 11 2.4 Polymerase chain reaction (PCR) and genotyping ...... 11 2.5 Fragment length - and data analyses ...... 12 2.6 Statistical analyses ...... 13

3 Results ...... 14 3.1 Sample metric, November 2017 ...... 14 3.1.1 Genetic paternity analysis, November 2017 ...... 14 III

3.2 Effects on extra pair paternity share ...... 15 3.2.1 Season and nest location depth, years 2015-2017 ...... 15 3.2.2 Fry mortality, samples collected across the seasons 2015-2017 ...... 16 3.2.3 Cannibalism of foreign fry, samples collected across seasons 2015-2017 ...... 16 3.3 Female removal, November 2017 ...... 17

4 Discussion ...... 18 4.1 EPP and influencing factors ...... 18 4.2 Genetic relatedness and cannibalism ...... 19 4.3 Did brood size influence the fry size? ...... 19 4.4 Did bi-parental brood care entail monogamy in V. moorii? ...... 20

5 Conclusion ...... 23

6 References ...... 24

7 List of Figures ...... 30

8 List of Tables ...... 30

IV

Abstract

Cichlids feature an astonishing diversity in morphology, ecology and breeding strategies and provide tremendous opportunities for the research of mating systems and brood care. Monogamy and bi-parental brood care are frequent. Exclusively observational approaches may underestimate the gap between social and genetic monogamy. Hence, genetic parentage analyses have become standard in studies of mating systems. Here, I used Variabilichromis moorii, a socially, but not genetically, monogamous and bi-parental Lake Tanganyika cichlid, to estimate reproductive success of paired males and further to investigate the reasons for males to keep up brood care even when facing high rates of cuckoldry. Therefore, I conducted genetic parentage analyses of 14 broods, including seven female removal experiments, in which males were forced to provide brood care without their female partner’s participation. I also used an extended dataset collected over several seasons to find factors affecting variations in paternity shares of paired males. My results showed that most analyzed broods were sired by more than one male. Interestingly, the mean paternity shares of paired males were significantly lower in the rainy season than in the dry season. Further, I found a trend that pair bonded males had a higher paternity share in nests with larger (i.e. older) fry. Finally, when deprived of their social mate, males with larger fry continued caring more often than males with smaller fry, independent of their paternity share. Seasonal differences in paternity share might exist because males might fend off competitors more easily in clear water. Increasing paternity shares when fry get older might indicate the possibility that males cannibalize on foreign fry. Finally, the results of the female removal experiment suggest that males might estimate reciprocal investment in the current brood and benefits of owning a territory rather than care for fry at all costs.

V

Zusammenfassung

Cichliden (Buntbarsche) zeigen eine außergewöhnliche Vielfalt in ihrer Morphologie, Ökologie und Fortpflanzung und stellen damit ein gewaltiges Repertoire an Möglichkeiten zur Erforschung von Fortpflanzungs- und Brutpflegesystemen zur Verfügung. Monogame Fortpflanzung und bi-parentale Brutpflege sind weit verbreitet und ausschließlich empirische Forschung ist nicht im Stande eine genaue Aussage über die tatsächlichen Vaterschaften zu treffen. Daher sind genetische Vaterschaftsanalysen zur Erforschung von Paarungssystemen erforderlich. In dieser Studie wurden 14 Bruten der Buntbarschart Variabilichromis moorii, einer sozial monogamen Fischart mit bi-parentaler Brutpflege, einer Vaterschaftsanalyse unterzogen, um den Reproduktionserfolg der territorialen Männchen zu untersuchen. Des Weiteren wurde nach Gründen gesucht, die eine weiterführende Brutpflege trotz hohen Fremdvateranteils rechtfertigen würden. Die Resultate zeigten, dass im Großteil der Bruten mehr als ein Männchen an der Vaterschaft beteiligt war. Interessant war, dass in der Regenzeit der durchschnittliche Anteil der Fremdväter höher ausfiel als in der Trockenzeit. Ein Grund dafür könnte die erleichterte Verteidigung ihrer Fertilisationen im klaren Wasser sein. Zusätzlich konnten höhere Vaterschaften von territorialen Männchen in Nestern mit größeren (älteren) Jungfischen gezeigt werden. Das könnte auf kannibalistisches Verhalten des territorialen Männchens hinweisen. Abschließend konnte festgestellt werden, dass territoriale Männchen mit größeren (älteren) Jungfischen eher dazu tendieren die Brutpflege bei Entfernen des territorialen Weibchens beizubehalten. Man würde annehmen, dass territoriale Männchen dazu in der Lage sind, den noch zu leistenden Aufwand an bevorstehender Brutpflege und den Vorteil eines Territoriums abschätzen zu können. Die vorliegende Arbeit stellt eine fundierte Grundlage für weitere Analysen zu den Fortpflanzung- und Brutpflegeverhalten der Buntbarschart V. moorii dar.

VI

1 Introduction

1.1 Cichlid species and their significance in speciation

Lake Tanganyika (LT: 9-12 million years old) is one of the most diverse freshwater ecosystems in the world (Sturmbauer, 1998) and comprises the most species-rich fish family Cichlidae (Salzburger & Meyer, 2004). feature an astonishing diversity in morphology, ecology and breeding strategies (Koblmüller et al., 2008) and provide tremendous opportunities for the research of evolutionary mechanism (Salzburger, 2009). More than 250 endemic cichlid species are described for LT (Koblmüller et al., 2008) and studies suggest that this species richness was founded by only a few ancient lineages making it one of the best studied examples of adaptive radiations (Salzburger & Meyer, 2004).

One important factor presumably associated with the success of cichlid fish and their high diversity in the east African crater lakes is their breeding behavior. Almost all cichlids engage in more or less sophisticated brood care (Salzburger, 2009) and there is extraordinarily large diversity in behaviors related to mating and parental care (Salzburger & Meyer, 2004; Koblmüller et al., 2008; Salzburger, 2009). Additionally, there are several other factors which might be important for their evolutionary diversification like the development of a pharyngeal jaw, adaptations of their visual system, or divergence triggered by abiotic factors (e.g. fluctuation of water level; see Salzburger (2009) for more examples).

1.2 Mating systems and parental care

The Lake Tanganyika cichlid flocks encompasses the entirety of mating and parental care behaviors found in cichlids. Mating systems can be either monogamous or polygamous but there are also cases where both strategies can be found either facultatively or obligatorily within and among species or even populations (Sefc, 2011). Brood care includes various forms of mouthbrooding and substrate breeding comprising cooperative breeding, whereby the latter exhibits the most social form of substrate breeding (Sefc, 2011). Socially monogamous cichlid species are characterized by bi-parental brood care (care by both parents) or consistent spawning with the same partner. Adult males and females form exclusive pair bonds with specific individuals of the opposite sex over an extensive time- period (Whiteman & Côté, 2004; Liebgold et al., 2006; Sefc, 2011). Between the partners of a pair, a division of labor is common with males often building and maintaining nests and / or guarding and defending mates and territories while females are often more engaged in direct 1 brood care (Sturmbauer et al., 2008; Coleman & Jones, 2011). For species which protect and defend eggs and offspring like socially monogamous fish with bi-parental brood care, it seems that the breeding area (i.e. territory) is an important resource (Sturmbauer et al., 2008). In polygamous mating systems, individuals of one sex mate frequently with more than one partner of the opposite sex during one reproduction bout.

1.3 Differences between fish and birds: Why investigate monogamy and bi-parental brood care in fish?

1.3.1 Brood size

There are differences in brood size, costs for parental care and fertilization mechanisms between fish and birds. Birds are the most studied group of concerning monogamous mating, bi-parental brood care and extra pair paternity (Griffith et al., 2002; Wan et al. 2013). One important difference is that brood sizes of fish species are larger than brood sizes of birds. The clutch sizes of substrate breeding cichlid fishes range from 10-20 up to hundreds or even thousands of eggs (Sefc, 2011). Birds in comparison to fish have small clutch sizes (Coleman & Jones, 2011). For example, in the family of passerines, one of the most studied socially monogamous birds with high rates of extra pair paternity (EPP), breeding pairs usually have a clutch size of less than ten eggs per clutch (Bauer et al., 2012). Therefore, clutch size might be a limiting factor on the maximum number of mates per brood (Coleman & Jones, 2011) and hence may have an effect on extra pair paternity rates and mating respectively brood care behavior.

1.3.2 Bi-parental care incidence and brood care investment/cost

The incidence of bi-parental brood care is another factor that differs between fish and birds. Generally, in birds bi-parental care is accomplished in the majority of species (90-95%; Balshine, 2012). In fishes, most of the species provide no parental care and only few fish families provide parental brood care (approx. 31%, Mank et al., 2005). Most of these fish provide exclusive uniparental care. Bi-parental brood care is the least prevalent form of brood care among fishes, although it is relatively common in cichlids. Only about 10% of fish families that contain species caring for their offspring also contain species that care for their offspring conjointly as a pair (Mank et al., 2005; Balshine, 2012).

Further, the investment in parental care comprises remarkable differences among fish and birds. Birds provide care by building nests, incubating their eggs and defending and

2 feeding the young. In comparison, fish typically provide parental care by hiding and fanning their eggs and guarding their offspring (Balshine, 2012).

1.3.3 Fertilization control

Sperm competition in birds is high because of the capability of females to store sperm for extended periods and ejaculates from different males may coexist in the female reproductive tract simultaneously (Wan et al. 2013). Wan et al. (2013) suggest that sperm competition is the main driver of sexual selection, leads to the disparity in reproductive success of individuals and makes extra pair paternity (EPP) possible in socially monogamous birds. The opportunity for females to control male choice entails that female birds became the key players in the interaction between females and their mates, between females and extra- pair males and between pair bonded and extra pair males. Hence, females might have control over the existence of EPP (Wan et al., 2013). Contrary, fertilization in most fishes happens externally. The eggs and sperm of the vast majority of fish species are released in the water column or are attached on the ground on / in the substrate, where fertilization occurs (Taborsky, 2012). Therefore, in socially monogamous cichlids with bi-parental brood care and external fertilization the females have no, respectively only little, control over fertilization. One example for female control over reproduction might be the European bitterling, a of the family Cyprinidae. Females of this fish species lay their eggs in the gill chambers of freshwater mussels with the help of their long ovipositors. During the spawning seasons, males defend territories around mussels against other territorial males, non-territorial males and females which are not ready for spawning. Additionally, sexually mature males develop remarkable nuptial coloration. If a female, ready for spawning (i.e. with extended ovipositor) enters the territory of a territorial male, the male begins to court the female immediately. If courtship was successfully (i.e. female pursued him to his mussel), the territorial male releases sperm in the close vicinity of the siphon of his mussel (external fertilization). After the pre-oviposition ejaculation, territory owners defend the mussel while the female inspect the siphon of the mussel (Smith et al., 2004). Smith et al. (2004) mentioned the importance of the mussel quality (i.e. embryos already present in the mussel) to determine the oviposition choice of a female. The interesting issue regarding spawning is that female bitterlings adjust their egg numbers per spawning and display a remarkable behavior close to sneaker males during spawning to get the attention of these additional males (Smith & Reichard, 2005). The authors suggest that females might benefit from an increase of sperm volume. Furthermore, sneaker males might be genetically more compatible to the females

3 than the territorial males (Smith & Reichard, 2005). The breeding behavior of the female bitterlings implies an influence on breeding male decision and fertilization control despite external fertilization.

However, in fishes, the external fertilization of eggs is thought to promote the development of alternative reproductive tactics (ART, e.g. cuckoldry). Usually, this results in remarkable extra pair paternity rates (Coleman & Jones, 2011). Consequently, the frequent occurrence of ARTs respectively EPP implies severe uncertainties of parentage in socially monogamous pairs as can be seen in birds (Wan et al., 2013).

1.4 Extra pair paternity (EPP)

The extra pair paternity rate is defined as the proportion of fertilizations within a brood resulting from foreign males other than the pair bonded male (PBM) (Griffith et. al., 2002). Evidently, field observations alone may not be sufficient to evaluate mating behavior and reproduction strategies comprehensively. Thus, genetic methods are a good supplement to provide accurate data and detect the success of extra-pair sires in cichlid fishes and other animals too (Westneat, 1987; Avise et al., 2002; Griffith et al., 2002; Sefc et al., 2009). Genetic analyses have demonstrated that monogamous mating and bi-parental brood care behavior do not obviate multiple paternity (Sefc et al., 2008). For example, in the salamander Plethodon cinereus, high rates of multiple paternity were determined although they are socially monogamous. Likewise, discrepancies between true and observed paternity are also known in socially monogamous mammals (e.g. the island fox; Roemer et al., 2001), reptiles (e.g. the Australian lizard; Bull et al., 1998) and birds (Liebgold et al., 2006). Contrary to the high prevalence of social monogamy and bi-parental brood care in birds (90 – 95%, see above), EPP is frequent and occurs in most of the studied species. Currently, more than 150 studies concerning EPP in birds have been published (Griffith et. al., 2002; Westneat & Stewart, 2003). For example, only 14% of analyzed passerine bird species were indeed genetically monogamous (Griffith et al., 2002). However, EPP levels of passerines are higher and more variable than EPP rates in non-passerine birds and therefore, EPP in other orders might differ in frequency (Westneat & Stewart, 2003; Crouch & Mason-Gamer, 2018). Hence, authors proposed various hypotheses to explain the factors that shape the variation in EPP and how extra pair copulations (EPC) may increase the fitness of individuals (Yasui & Yoshimura, 2017). Generally, the reasons why males pursue various mating strategies seem perspicuous because males are able to increase their fitness by additionally fertilizing eggs outside the social pair bond (Coleman & Jones, 2011; Wan et al., 2013) when the costs for 4 extra pair copulations are not higher than the benefits (Wan et al., 2013). Extra pair mating by females includes direct and indirect benefits. Direct are non-genetic or environmental benefits as for example the “fertility assurance hypothesis”. Following this hypothesis, females pursue EPC to obviate infertility of male mates. Indirect gains are genetic benefits as for example proposed the “good genes hypothesis”, the “genetic compatibility hypothesis” and the “genetic diversity hypothesis” (Wan et al., 2013; Yasui & Yoshimura, 2017). The “Bet- hedging hypotheses” is a further theory, which may explain ubiquitous cuckoldry in birds as well as in various other taxa. Bet-hedging is a risk-spreading strategy where goods (e.g. different phenotypes to increase the fitness of the individual) which are exposed to danger (e.g. rapid changing of environmental conditions or high number of predators) will be portioned to minimize the risk of losing well adapted traits at once. For example, female polyandrous mating behavior may lead to new genetic acquisitions of direct or genetic benefits. Through this bet-hedging derived benefit (i.e. multiple mating) the risk of failed reproduction (e.g. male infertility, low genetic quality and / or genetic compatibility of the pair bonded male) would be minimized (Philippi & Seger, 1989; Garcia-Gonzales, 2015). Furthermore, a wide range of additional factors could play a role in the variation of EPP rates as for example the breeding density (i.e. higher density of males may increase EPP rates; Brouwer et al., 2017), paternal care (i.e. reduced or absent care may increase EPP rates; Emlen & Oring, 1977; Wan et al., 2013), adult mortality (species with a short reproductive life span may tolerate higher EPP rates; Mauck et. al., 1999; Wan et al., 2013). Considering these various factors and although EPP is well studied in avian mating systems and various other animal taxa, the factors that explain variation in EPP rates and the variability in mating systems are still poorly understood and strongly depend on the species studied (Westneat & Stewart, 2003; Grunst et al., 2017; Yasui & Yoshimura, 2017; Brouwer et al., 2017; Crouch & Mason-Gamer, 2018).

However, the consequences of EPP for a socially monogamous fish with bi-parental brood care are the loss of fitness (lifetime reproduction success of future offspring; Gross, 2005) due to stolen fertilizations.

1.5 Alternative reproductive tactics (ARTs)

Alternative reproductive tactics (ARTs) are alternative ways to increase the reproduction success of individuals. Usually, alternative mating tactics refer to traits chosen to maximize fitness in more than one alternative way among conspecific same-sex competitors. Social and ecological competition between individuals within populations and the resulting strategies 5 may lead in to remarkable phenotype polymorphism, which are characterized by a discontinuous distribution of behavioral, physiological and morphological traits (e.g. males using attracting traits such as plumage variation in birds or color and size variation in fish) (Sinervo & Zamudio, 2001; Taborsky et al., 2008; Kuerthy et.al., 2015). Typically, ARTs are expected to evolve more often in males because reproduction is less costly for them than for females (assume e.g. the costs of egg production for females even when parasitic, Taborsky et al., 2008). Further, the oviposition behavior of females is costlier and takes longer contrary to fertilization of males. Taborsky (1997) recommended two functional terms to describe ARTs. First, the term suggested for the tactic where males pursuing to maximize their reproductive success by stealing fertilizations from pair bonded conspecifics is called “parasitic”. Second if males invest effort in developing special features to obtain access to females (monopolize females), defend a spawning site or nest they are called “bourgeois” (Taborsky, 1997; Taborsky et al., 2008, Neff & Clare, 2008; Engqvist & Taborsky, 2016). In this study, we will use the terms “pair bonded male” (PB males) instead of “bourgeois male” and “cuckolder” instead of the term “parasitic male”. Usually, PB males invest more into growth, and defending and obtaining resources. Cuckolders potentially benefit from a smaller unobtrusive body shape and invest more into sperm production and testis size (Fagundes et al., 2012; Kuerthy et. al., 2015). Resource allocation and investment patterns can diverge significantly between PB males- or cuckoldry males. This may generate various adjustments (Kuerthy et. al., 2015). For example, in a shell-brooding cichlid fish pair bonded males accumulate considerable evisceral fat stores (i.e. included all the fat in the liver and muscles) during development and stored hardly any visceral fat (i.e. all fat stored within the peritoneum). Contrary, fat reserves of immature males rises with increasing body size (i.e. age) and cuckolders exhibits visceral and evisceral fat stores which did not relate to body size (i.e. age) (Kuerthy et. al., 2015).

1.5.1 Plasticity of ARTs

Alternative reproductive tactics may be fixed as a life-long individual specialization or adjustable to temporary conditions. In the latter case, individuals are able to adapt their reproductive tactics to specific environmental conditions. These tactics are called plastic or flexible tactics. Inflexible or fixed tactics refer to all cases where individuals retain one specific phenotype the whole lifespan (Oliveira et al., 2008; Taborsky & Brockmann, 2010). The inflexible tactic is preferred if the costs of switching from one form in the other outweighs the benefits of doing so (e.g. environmental restrictions; Brockmann & Taborsky,

6

2008). Contrary, flexible tactics were chosen if optimal mating requirements vary permanently over a period (Taborsky et al., 2008). However, ARTs may be influenced by genetic (i.e. different genotypes) and environmental factors simultaneously (Taborsky, 1998; Taborsky & Brockmann, 2010).

1.5.2 ARTs in fish

Fish provide valuable insights in the most exceptional variability of alternative reproductive tactics among vertebrates. Sometimes, three or more alternative reproductive tactics may occur within one species (Taborsky, 2008). Cuckolders respectively parasitic males exhibit ARTs in form of “sneaker males” by spending time in the vicinity of the nest and stealing fertilizations by secret attendance during spawning (Mank & Avise, 2006). Additionally, there are cuckolder males which are able to mimic the parental female by expressing female behavior and color (Neff & Gross, 2001) and cooperative males which gain fertilizations through cooperative behavior with the pair bonded male (Mank & Avise, 2006; Taborsky, 2008). For example, among others (Taborsky, 2008), “external fertilization” and “indeterminate growth” might be important factors for the frequency and variability in fish. Thus, to monopolize fertilizations seems to be difficult for pair bonded males because conspecific competitors may easily get access to eggs during spawning. Additionally, to secure fertilizations externally a vast amount of sperm is needed. Thus, males should produce large amounts of sperm and this, consequently, favors their competitive abilities in sperm competition (Taborsky, 2008). Furthermore, the indeterminate growth (i.e. individuals do not stop growing after maturation; Taborsky, 2016) of the vast majority of fish may be important for the high diversity of ARTs in fish because smaller (i.e. younger) fish could not be competitive against bigger (i.e. older) conspecifics (Taborsky, 2001; Taborsky, 2008; Taborsky & Brockmann, 2010). Therefore, the smaller individuals have to find other strategies for successful reproduction. For example, in many fish species males display parasitic tactic (cuckoldry) when small and turn into pair bonded males when large (Taborsky & Brockmann, 2010). Consequently, ARTs could result in sperm competition (usually, pair bonded males have smaller testes sizes than cuckolders; Taborsky, 2008). Moreover, high rates of extra pair paternity, which are a consequence of ARTs, could lead in to cannibalism of foreign offspring to minimize misdirected parental care of the pair bonded male (i.e. caring for non-related offspring; e.g. bluegill sunfish; Neff & Gross, 2001; Neff, 2003; Alonzo & Klug, 2012).

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1.6 The target species Variabilichromis moorii

Fig. 1 V. moorii in their natural Fig. 2 Adult V. moorii in habitat in LT, left side, lower the laboratory of the corner: offspring (picture© University of Graz Aneesh Bose)

Variabilichromis moorii (formerly moorii) belongs to the tribe Fig. 2 Adult V. moorii in , the most species rich cichlid lineage inthe Lake laboratory Tanganyika. of the This cichlid species is endemic to this African Lake (Sturmbauer et al., University2008; Sefc of et Graz al., 2008). Adults of this species reach a total length of 7 – 10 cm and occupy shallow, rocky areas in the littoral zone of the southern seaside of Lake Tanganyika (Rossiter, 1991; Sefc et al., 2008). Each individual of a breeding pair holds a sub-territory which slightly overlaps with the sub- territory of the pair bonded mate. The territory border is defended aggressively in cooperation of both parents and territory size ranged from <1 to almost 4 m2 (Sturmbauer et al., 2008; Sefc et al., 2008). The territory size increases with increasing water depth and serves as both breeding sites and feeding territories. Consequently, population density decreased with increasing territory size (Karino, 1997; Ota et. al., 2012). V. moorii is a socially monogamous, substrate breeding cichlid fish which provides bi-parental brood care (Sefc et. al., 2008). Brood sizes may reach >100 fry and brood care continues up to 100 days. Whether pair bonds persist after the brood care period has not yet been resolved (Sefc et al., 2008). Based on a former study, high rates of extra pair paternity were already determined (Sefc et al., 2008) and this usually leads to caring for non-descendent young (i.e. alloparental care; Wisenden, 1999).

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1.7 Aim of the study

Males of Variabilichromis moorii face high rates of extra pair paternity in their nests. This was already found in a former study, which, however, did not include genotyping of parental fish (Sefc et al. 2008). Moreover, there is a large body of literature that suggests the importance of paternity when studying brood care behavior (e.g. Westneat & Sherman, 1993; Queller, 1997). Fromhage & Jennions (2016) predicted that broods with non-descendent young create an asymmetry in parental brood care investment and may lead to reduced care by one parent (i.e. the male in this study) over evolutionary time scales. Therefore, I focus my work on the examination of the reproductive success of pair bonded males with respect to parental care. Further, I want to know if the paternity share of males was rather consistent or variable between different seasons and different nest depths. Therefore, I used an extended dataset collected in three field seasons for further analyses. Nest depth might be interesting because in birds, extra pair paternity rates are high at high population densities (Westneat & Sherman, 1997). In V. moorii the differences between population densities a manly based on algae abundance and hence water depth (Karino, 1997; Ota et al., 2012). Seasonal differences might occur because the water turbidity might interfere with defense against potential cuckolders. Alin et al. (1999) suggest that the water turbidity and sediment discharge reach their maximum at the rainy season. High rates of extra pair paternity rates may cause misdirected care for unrelated offspring (i.e. alloparental care). Thus, cannibalism could counteract misdirected paternal effort when sacrificing fry which were non-descendent to the pair bonded male. To test this, a possible association of fry size (i.e. age) with extra pair paternity rates was examined. If parental males progressively cannibalize foreign offspring, their paternity share should increase with brood age. Although socially monogamous, former studies (unpublished data) exhibited that females of V. moorii are able to care for their brood without the pair bonded male for some time. This raises the question whether bi-parental care is accountable for maintain monogamy in this species or if there are other factors (e.g. fry size or paternity) affecting the brood care behavior of pair bonded males. To test this, I examined fry loss suffered by seven males after removal of their mates and compared these changes in fry numbers with their paternity shares. Further I looked at a possible correlation between fry number decrease with the fry size because it might be possible that males anticipate the costs of care until the fry reach independency and decide upon this cost whether to continue brood care or not.

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2 Materials and Methods

2.1 Sample collection

The samples I used in my study had been collected by my colleagues during three field trips to Lake Tanganyika. The field trips took place in October 2015 and November 2017 (dry season) and in April 2016 (rainy season). The study site (~ 1000m2) was located on Mutondwe Island near Mpulungu, Zambia. Nests were located and marked in depths between 3.0m and 6.4m (mean ± s.d. = 4.1 ± 1.0m, n = 22) during the rainy season and between 1.7m and 5.5m (mean ± s.d. = 3.4 ± 1.0m, n = 45) during the dry season. For each nest, the nest holding male and female were marked individually by fin clipping to distinguish between the sexes (males: dorsal part of the caudal fin, females: ventral part of the caudal fin). Fin clips were stored in 99.9% ethanol for further genetic parentage analysis (see below). In total, 77 adult and 545 offspring individuals of Variabilichromis moorii were used for genetic analyses to determine the extra pair paternity (November 2017 season, see Table 1; authors part).

Table 1 Summary of nest numbers and individuals, November 2017, n=14

Nest N WP O EP O1 EP O2 EP O3 A O FR B7 67 46 21 yes E2 29 24 5 4 34 9 16 4 1 4 yes 5 35 13 4 6 12 6 24 17 4 2 1 yes 7 39 27 3 3 6 yes 8 47 22 9 15 1 9 12 5 3 2 1 1 19 55 12 33 9 1 28 74 2 58 6 7 29 32 29 3 yes 36 34 27 1 6 yes 39 32 17 13 2 yes 42 31 29 2 Adults 77 FemaleTotal removal experiment622 Table 1 N, individuals genotyped; WP O, within pair offspring, EP O1, extra pair offspring of cuckolder 1; EP ForO2, sevenextra pair nests offspring (Table of 1,cuckolder FR), female 2; EP O3, removal extra pair experiments offspring of wercuckoldere done 3; in A theO, adopted field. First, offspring; FR, female removal;

Table 1 Summary of nest numbers and individuals, November 2017, n=14

Nest N WP O EP O1 EP O2 EP O3 A O FR B7 67 46 21 yes E2 29 24 5 4 34 9 16 4 1 4 yes 5 35 13 4 6 12 6 24 17 4 2 1 yes 7 39 27 3 3 6 yes 8 47 22 9 15 1 9 12 5 3 2 1 1 10 19 55 12 33 9 1 28 74 2 58 6 7 29 32 29 3 yes 36 34 27 1 6 yes 39 32 17 13 2 yes 2.2 Female removal experiment

For seven nests (Table 1, FR), female removal experiments were done in the field. First, approximately half of the brood of each nest was removed for genetic parentage analysis using SSR (Simple Sequence Repeats). The rest of the brood was caught with transparent plastic bags and pictures of trapped offspring were taken to count brood sizes. To record all fry, the bottom of the area was gently scanned with both hands and the remaining fry was herded together at the nest center and counted by the observers. Afterwards, fry were carefully released back into the nest. The nest was observed and all incoming intruders were expelled until at least one parent was back defending the remaining fry. Next, the female of each nest was removed and stored in a net-cage approx. 100 m distant to the nest. After the pair bonded male returned to the nest, it remained for 24h without further observation. On the next five consecutive days remaining fry was counted as describe above. After the last count, females were released in close vicinity to their nests.

2.3 DNA extraction

Total length of fry was measured to the nearest mm. DNA from fin clips was extracted from tissues with 5 % Chelex and Proteinase K digestion following a protocol of Gariepy et al. (2012). 2µl proteinase K and 100µl 5% Chelex resin solution was used for each extraction. Subsequently, the samples were incubated at 55°C and 550rpm on a thermomixer over night. Finally, the samples were heated up to 95°C to inactivate the Proteinase K and centrifuged for 10 minutes at 4°C.

2.4 Polymerase chain reaction (PCR) and genotyping

PCR was done by using 2.5µl Qiagen Type-it Multiplex PCR Master Mix, 0.5µl Primer-Mix 17a and 3µl respectively 2 µl DNA-extract. Program parameters were as follows: 5min at 95°C Hot-Start, followed by 28 cycles of 30sec at 95°C, 90sec at 54°C and 30sec at 72°C, followed by a final extension step at 60°C for 30min, after which the PCR-products were cooled down to 8°C. Qiagen Type-it Multiplex PCR Master Mix contains HotStarTaq Plus

DNA Polymerase, Type-it Microsatellite PCR Buffer with 6mM MgCl2, and dNTPs. 9 highly polymorphic microsatellite loci were genotyped in one multiplex arrangement (see Table 2 & 3 for loci details). The Multiplex stock solution (named 17a, total volume: 400 μl) was mixed as follows: 8 μl of microsatellite loci TmoM11, UNH2075, Hchi94 and Ppun 20; 4 μl of microsatellite loci Pmv17, Ppun21 and Hchi59 and 32 μl of microsatellite loci Pzeb3 and

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Ppun9. The mixed primer stock solutions (in total: 152μl  forward and reverse) were mixed with 248 μl TE-Buffer (Tris-EDTA Buffer system).

Table 2 Summary of used microsatellite loci. Forward primer was labeled with the indicated fluorescent dye. Locus Dye RA SR Source Autor

TmoM11 FAM (CA)n 150-230 moorii Zardoya et al., 1996 UNH2075 HEX (GT)n 150-250 Labeotropheus fuelleborni X Albertson, Streelman and Metriaclima zebra Kocher, 2003 Hchi94 NED (CA)n 150-200 Maeda et al., 2008 chilotes Ppun20 NED (GACA)n (GATA)n 300-720 Pundamilia pundamilia+ Taylor et al., 2002 P. nyererei F1 Pmv17 FAM (TAGA)n 100-230 Pseudocrenilabrus multicolor victoriae Crispo et al., 2007 Ppun21 HEX (GATA)n (GACA)n 250-400 Pundamilia pundamilia+ Taylor et al., 2002 P. nyererei F1 hybrid Ppun9 HEX (GATA)n 350-500 Pundamilia pundamilia+ Taylor et al., 2002 P. nyererei F1 hybrid Hchi59 NED (CA)n 100-150 Haplochromis Maeda et al., 2008 chilotes Pzeb3 FAM (GT)n 300-400 Pseudotropheus zebra Oppen et al., 1997 Table3 2 RA, repeat array of the cloned allele; SR, size range (bp); Source, species from which microsatellite was isolated;

2.5 Fragment length - and data analyses

DNA fragments were sized against an internal size standard (GeneScan-500 LIZ, Applied Table 2 Summary of used microsatellite loci. Forward primer were labeled with the indicated fluorescent dye. Biosystems).Locus ForDye each sampleRA we used 1SR µl of PCR product Sourceand 9.9 µl Sequencer MasterAutor Mix

(consistsTmoM11 of: FAM 10 µl HiDi(CA) andn 0.2 µl150 Gene-230 Scan-500 Tropheus LIZ). Aftermoorii that, the samplesZardoya et were al., 1996 UNH2075 HEX (GT)n 150-250 Labeotropheus fuelleborni X Albertson, Streelman and denaturated at 95°C for 5 min and transferred to a cold plateMetriaclima before zebra being analyzed onKocher, an ABI 2003 Hchi94 NED (CA)n 150-200 Haplochromis Maeda et al., 2008 3130xl automatic sequencer (Applied Biosystems). Scoringchilotes of fry and adult genotypes was Ppun20 NED (GACA)n (GATA)n 300-720 Pundamilia pundamilia+ Taylor et al., 2002 done with GeneMapper 3.7 (Applied Biosystems). TheP. number nyererei F1 of hybrid alleles (NA), the expected Pmv17 FAM (TAGA)n 100-230 Pseudocrenilabrus multicolor victoriae Crispo et al., 2007 (HE)Ppun21 and observedHEX (HO)(GATA) heterozygosityn (GACA)n 250 -per400 locus andPundamilia population pundamilia (Table+ 3) were Taylorcalculated et al., 2002 P. nyererei F1 hybrid withPpun9 the computerHEX software(GATA) Identityn (Sefc350-500 & Wagner,Pundamilia 1999). The pundamilia nine very+ high polymorphicTaylor et al., 2002 P. nyererei F1 hybrid microsatelliteHchi59 NEDmarkers had(CA) a n low probability100-150 of null allelesHaplochromis (ƒ (0); Table 3). ParentageMaeda et wasal., 2008 chilotes reconstructedPzeb3 FAM by using COLONY(GT)n (v 300 2.0.6.1,-400 Jones &Pseudotropheus Wang 2010) zebra, a maximumOppen-likelihood et al., 1997 Table3 2 RA, repeat array of the cloned allele; SR, size range (bp); Source, species from which microsatellite was isolated; method to group fry in full-sib and half-sib families with the help of genetic microsatellite marker data (Wang, 2004). We identified extra pair sires in each brood and in seven broods low numbers of foreign offspring with extra pair sires and extra pair mothers. To determine the minimum number of contributing sires per brood and to identify genotyping errors we double-checked the output of COLONY carefully because of the occasionally overestimation of the minimum value of sires by COLONY (Sefc & Koblmüller, 2009). In broods where two extra pair sires were represented just one allele of each offspring, we combined these alleles add up to only one genotype (i.e. one extra pair sire).

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Table 3 Summary statistics for nine polymorphic microsatellite markers in the population sample. Locus NA HE HO ƒ (0)

TmoM11 17 0,835 0,807 0,015 UNH2075 24 0,938 0,961 -0,011 Hchi94 17 0,903 0,883 0,010 Ppun20 26 0,920 0,883 0,019

Pmv17 17 0,917 0,896 0,011 Ppun21 22 0,933 0,910 0,012 Ppun9 24 0,923 0,909 0,007

Hchi59 12 0,738 0,727 0,006 Pzeb3 13 0,827 0,857 -0,016

Table 3 NA, number of alleles; HE / HO, expected and observed heterozygosity; ƒ (0), estimated frequency of null alleles;

2.6 Statistical analyses

Computer software R v. 3.4.4 (R Development Core Team) was used for all statistical analyses. To determine the difference of PBM paternity share between dry and rainy season we calculated the mean of EPP in November 2017 and across the seasons 2015-2017. We fit a generalized linear mixed-effect model (GLMM, R package glmmADMB; Fournier et al., 2012) with a negative binomial error distribution (nbinom) to test for effects of nest depth and season on the average PBM paternity share.

To detect any aberration from continuous fry mortality over time, a Spearman rank correlation test was used to find a possible correlation between brood size and fry size (which is used as a proxy of fry age). Moreover, the same test was used to examine the relationship between PBM paternity share and fry size in order to find an evidence for filial cannibalism. All samples for Spearman`s rank correlation tests were collected across field seasons October 2015, April 2016 and November 2017.

Finally, a generalized linear mixed-effect model (GLMM, R package glmmADMB; Fournier et al., 2012) with a negative binomial error distribution (nbinom) was used to test for effects of nest depth and season on the average PBM paternity.

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3 Results

3.1 Sample metric, November 2017

Total brood size ranged from 12 to 119 fry (mean ± s.d. = 56.57 ± 27.73 fry, n=14) and the total length of the fry ranged from 6 to 22 mm (mean ± s.d. = 14.36 ± 5.83 mm). In seven nests, foreign (adopted) offspring were found (1 to 12, mean ± s.d. = 3.43 ± 3.95 fry; Fig. 3).

3.1.1 Genetic paternity analysis, November 2017

Fig. 3 Paternity of PB males, extra-pair sires and adopted offspring for each of the 14 broods sampled during dry season, November 2017. Blue parts of the bars indicate the number of within-pair offspring. Alternating green shaded segments represent the offspring per extra pair sire in the broods. Red segments of the bars represent adopted

offspring per nest.

Fig. 4 Paternity shares (in %) of PB males and extra-pair sires for each of the 14Fig. sampled 3 Paternity broods of PB during males, dry extra season,-pair sires and adopted offspring for each of November 2017. Blue parts of the bars indicatethe 14 sampled the proportion broods during of withindry season, pair offspring.November 2017. Alternating Blue parts green of the shaded bars

segmentsindicate the represent proportion the shares of within of extra pair pairoffspring. sires in Alternating the broods. Note: green paternity shaded sharessegments were represent calculated the shares using maternal of extra broodpair sires size in (i.e. the numberbroods. Redof fry segments shared byof the same bars representmother). adopted offspring per nest.

Paternity share of the pair bonded male

(PBM paternity) ranged from 3% (in 1 Fig. 4 Paternity of PB males and extra- nest) to 100% (in 2 nests), with a mean of 59.2 ± 28.3% (n = 14 nests; Fig. 4). The number of pair sires for each of the 14 sampled extra-pair sires per brood ranged from 0-3 (mean 1.9broods ± 1.1 sires during; Fig. dry 4). Paternity season, Novsharesember of 2017. Blue parts of the bars indicate the PB males decreased with increasing extra pair sire numbers (Spearman`s rank correlation, r = proportion of within pair offspring.s -0.63, p = 0.016, n = 14). Alternating green shaded segments represent the shares of extra pair sires in the broods. Note: paternity shares were calculated using maternal brood size (i.e. number of fry shared by the same mother). 14

3.2 Effects on extra pair paternity share

3.2.1 Season and nest location depth, years 2015-2017

Multiple paternity occurred in most of the broods and was significantly more frequent in the rainy season compared to the dry season (Fig. 5, Table 4). Nest depth exhibits no influence on PBM paternity share (Fig. 6, Table 4).

Fig. 5 Seasonal differences in

PBM paternity share. Fig. 6 PB male paternity share is not related to nest depth. Nests were

pooled across seasons 2015-2017, n=68. Line represents linear Fig. 5 Seasonal differences in regression. PBM paternity share.

Fig. 6 PB male paternity share is not Table 4 GLMM output of model on the related to nest depth. Nests were factors affecting the paternity share of PB pooled across seasons 2015-2017. Line males. Significant values are in bold, n=68 represents linear regression. nests; seasons 2015-2017; Estimate Std. Error z value Pr(>|t|)

(Intercept) -0.40 0.31 -1.27 0.203 Season -0.36 0.18 -2.03 0.042 (spring) depth in m -0.01 0.10 -0.06 0.948

Table 4 GLMM output of model on the factors affecting the paternity share of PB males. Significant values are in bold, n=68 nests; seasons 2015-2017; Estimate Std. Error z value Pr(>|t|) 15

(Intercept) -0.40 0.31 -1.27 0.203 Season -0.36 0.18 -2.03 0.042 (spring) 3.2.2 Fry mortality, samples collected across the seasons 2015-2017

No correlation between brood size and fry size was detected (Spearman`s rank correlation, rs= -0.10, p= 0.420, n= 68, Fig. 7). This contradicts the assumption that offspring mortality occurs steadily during the whole brood care period.

Fig. 7 Brood size is not related to mean offspring size. Line represents linear

regression.

Fig. 7 Brood size is not related to mean offspring size. Line represents linear regression.

3.2.3 Cannibalism of foreign fry, samples collected across seasons 2015-2017

There was a trend that PB males had a higher paternity share in nests with larger (i.e. older) fry (Spearman`s rank correlation, rs= 0.23, p= 0.060, n= 68, Fig. 8).

Fig. 8 The relationship between mean offspring size and PBM paternity share. Line represents linear regression.

Fig. 8 The relationship between mean offspring size and PBM paternity share. Line represents linear regression.

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3.3 Female removal, November 2017

Total length of offspring in the female removal experiments ranged from 6 to 22 mm (mean ± s.d. = 14.36 ± 6.76 mm, n=7). Total offspring numbers on observation day 1 ranged from 17 to 54 fry (mean ± s.d. = 32.14 ± 11.80 fry, n=7) and dropped to 0 to 49 fry after removal of the female (mean ± s.d. = 22.30 ± 19 fry, n=7). PBM paternity share did not influence the loss of offspring (GLMM, beta= -0.15, sd= 3.00, z= -0.05, p= 0.961, n= 7, Fig. 9). Our data suggests that pair bonded males with smaller offspring lost more of their brood than pair bonded males with bigger offspring (GLMM, beta= -0.26, sd= 0.10, z= -2.80, p= 0.006, n= 7, Fig. 10).

Fig. 9 Relationship between the Fig. 10 The relationship between PBM paternity share and the relative mean offspring length and the loss of offspring, observed in decline in offspring numbers November 2017. n= 7; (share of total brood size) during female removal, observed in

November 2017. n= 7; Fig. 9 Relationship between the

PBM paternity share and the relative loss of offspring, observed in Fig. 10 The relationship between November 2017. n= 7; mean offspring length and the decline in offspring numbers (share of total brood size) during female removal, observed in November 2017. n= 7; 17

4 Discussion

4.1 EPP and influencing factors

EPP is very common in fish species and microsatellite-based studies are paramount instruments to determine EPP rates in mating systems and to gain an insight in the staggering diversity of alternative reproduction tactics and reproduction in fishes. Therefore, studying the factors that affect mating systems as well as EPP is essential to understand the evolution of behaviours related to parental care (Coleman & Jones, 2011).

My data show that the majority of the analysed broods were sired by more than one male (i.e. 12 of 14 broods, Table 1, Fig. 4 & 5). The maximum number of extra pair sires in the broods was three. In seven nests a small number of adopted offspring (different mother and father) was found but the vast majority of the offspring in a nest shared the same mother (Fig. 4 & 5). The mean extra pair paternity share is significantly higher in the rainy season than in the dry season. One possible explanation for the difference in mean PB male paternity share could be water turbidity. The turbidity and sediment discharge reach their maxima during the rainy season (Alin et al., 1999). If PB males depend on a good visibility to fend off potential cuckolders during spawning, a change in water clarity might cause varying paternity shares.

Further, the results of my study showed no correlation between nest depth and PB male paternity share. My initial expectation was that paternity share might increase with increasing nest depth due to lowered population density and thus relaxed competition over fertilizations during spawning events. Unfortunately, the study quadrat provided no variation in water clarity, light penetration and food abundance between the uppermost border and the deeper part down to six meters. In one study, it has been documented that V. moorii is much more frequent, has a better sperm production and is in a better physiological condition in shallow habitats around 4-6 meters than in deeper habitats (Ota et al., 2012). Thus, further examinations of a possible effect of nest depth on PB male paternity share should also include deeper areas where changing environmental conditions might constrain population densities.

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4.2 Genetic relatedness and cannibalism

V. moorii is characterized by alternative reproductive tactics with “breeding mates (parents)” and “cuckolders (parasitic males)” sensu Neff (2003). This competition over fertilizations leads to allopaternal care (Farmer & Alonzo, 2008) as a consequence of high extra pair paternity rates. Theoretical studies predict that brood-rearing and minimizing misdirected parental effort (i.e. caring for non-related offspring) is beneficial for males to maximize their fitness (Neff, 2003; Klug & Bonsall, 2007; Bose et al., 2016). One possibility to avoid misdirected paternal care might be to favour own offspring and / or to cannibalize foreign offspring. Consequently, brood caring males must be able to distinguish between their own offspring and foreign offspring. Hence, kin-recognition is important in species where cuckoldry is present. For example, in the bluegill sunfish the cannibalism increases if care- providing males were cuckolded (Neff & Gross, 2001; Neff, 2003; Alonzo & Klug, 2012). This fish species uses olfactory cues to distinguish the foreign fry from the own offspring and brood-caring males adapt their parental investment in reaction to the changing paternity during development of offspring (Neff & Gross, 2001; Alonzo & Klug, 2012). Furthermore, bluegills may use the visible intrusion rates of sneaker males as a cue to estimate the risk of being cuckolded and adjust their paternal care accordingly (Neff & Gross, 2001). Here I show that in V. moorii the paternity share of nest holding male declined with increasing extra pair sire numbers. Further, the results suggested that, even though just as a trend, a higher paternity share of PB males in broods with larger (i.e. older) offspring could be found (Fig. 7). This suggests that V. moorii might be able to detect related young or expected parentage based on the presence of potential competitors during spawning and / or based on chemical cues. Hence, PB males might be able to adjust parental care decisions according to their own paternity shares and reduce their current parental effort in the face of a low paternity share or cannibalize foreign offspring to reduce misdirected parental effort.

4.3 Did brood size influence the fry size?

Brood size showed no correlation with fry size. My expectation was that brood size decreases with increasing fry size because of continuous offspring mortality and/or offspring losses due to . Territories of V. moorii provide both, a save refuge for offspring and a sustainable source of nourishment. Consequently, pairs defend their territories fiercely. The offspring of a breeding pair is always located between the parents whereby the female is more associated to the brood and the male is more engaged in territorial duties (Zimmermann et al., in preparation; Smith & Wootton, 2016). However, temporal and spatial traits may lead to 19 non-continuous ambushes. Sturmbauer et al. (2008) described more hetero-specific attacks in the afternoon than in the morning and found that this might be for example due to a higher mobility of some species during the afternoon or due to short-term environmental changes like for example water turbidity, water current changes or waves that can be used by predators to overcome brood defence. Randomly distributed predation may lead to a scattered distribution of offspring mortality and hence, the predicted negative correlation between fry size and brood size may disappear.

4.4 Did bi-parental brood care entail monogamy in V. moorii?

In bi-parental care, females and males cooperate in providing care for their offspring but with varying responsibilities as described above (Zimmermann et. al., in preparation; Smith & Wootton, 2016). Gross (2005) argued that bi-parental care will evolve when pair bonded individuals gain their maximum lifetime reproductive success through cooperative care. Furthermore, bi-parental care is necessary if the care of one parent is not sufficient to ensure the survival of some offspring and might be the response to high predation rates (Smith & Wootton, 1995). Bi-parental brood care systems are particularly related with socially monogamous mating systems (Smith & Wootton, 2016). However, social monogamy in V. moorii seems to be incongruent with genetic parentage (Sefc et al., 2008). Brood-caring males achieved a paternity share of 61.2 ± 32.4% (mean ± sd) across three field seasons (October 2015, April 2016 and November 2017 (my study)). Thus, the very low paternity share, compared to other fish (DeWoody, 2003), may reduce paternal effort over evolutionary time (Westneat & Sherman, 1993; Formhage & Jennions, 2016; Griffin et al., 2013) and finally lead to maternal-only care (Formhage & Jennions, 2016; Griffin et al., 2013). This raises the question, if bi-parental brood care and monogamous mating is needed for offspring survival in V. moorii. Therefore, female and male removal experiments were done in the field (female removal: n=27, male removal: n = 19).

The male removal study showed that for the observed period, females were capable to provide successful care without PB males. Therefore, monogamous mating combined with bi- parental brood care may not a requirement for offspring survival in V. moorii.

The female removal experiments were done to determine first, if males are able and willing to take care of their offspring and second, which factors affect the brood care of single males. The present study demonstrated that in female removal experiments with sample size n=7, the majority of males are able to care for their brood alone for some time (i.e. 5 males

20 kept their brood and 2 males lost the brood). Further, the male paternity share was not correlated to the relative loss of offspring. The GLMM I calculated that larger (older) offspring might have a better chance of survival than smaller offspring under exclusive paternal care. One possible explanation for this could be that parental males are able to estimate the residual investment needed to raise the fry until independency. This may allow brood-caring males to balance out their current reproductive success and costs on future reproductive prospects. However, fry size might not only affect male decisions about further brood care but it might also be that larger fry are already more capable of predation avoidance by themselves. A sample size extension of the female removal experiment (n = 27) demonstrated a high rate of offspring losses for the caring males (unpublished data). This result indicates that some males might be less able to provide exclusive care for their offspring than others. Therefore, they might 1) not be able to sustain the territory required for offspring development and/or 2) might not be sufficiently able to fend off brood predators. Further, possible life-history trade-offs might explain the mixed results in the female removal treatment. Older males might already be at the end of their reproductive phase and future reproductive prospects might be scarce for them. Therefore, it might be beneficial for them to care for their current brood, as competition for a new spawning bout might not pay off. Contrary to that, younger males with prospering reproductive prospects may sacrifice their current brood to save energy for future reproduction. In the latter case, it might also be beneficial for males to cannibalize their brood to replenish past energy expenditures (energy- based hypothesis; Manica, 2002; Vallon & Heubel, 2016). A study on dietary contents of V. moorii showed that 7.1% of food intake consisted of fry (Hori, unpublished). Unfortunately, the sex of the sampled individuals was not documented in this data set. In addition, the fry found in the intestines were not determined to any specific species. Therefore, it was not possible to estimate the possibility of male filial cannibalism. Interestingly, an examination of the intestinal contents of twenty-five PB males (examinations were done after finalization of female removal experiments in the field in November 2017) revealed no fry intake, so filial cannibalism of caring males seems rather unlikely (H. Zimmermann & A. Bose, personal conversation). These two studies combined with a video sequence (done by Aneesh Bose) of a female feeding on fry undisturbed by the resident male after the female removal allow speculations that juveniles might be a “nuptial gift” for a potential future partner. This may enable the male to retain the territory and may increase the chance to reproduce again within a short time, comparable to the food gifts given by males to females at mating in insects (Sakaluk, 2000). Whiteman and Côté, (2004) proposed that monogamous mating may likely

21 occur in species where pair bonded males cannot comprise and secure the territory of more than one female and additionally, most monogamous pair bonding adults defend separate territories which overlap. The results of the male removal experiment suggest that bi-parental brood care is not a prerequisite for successful offspring survival in V. moorii. Females are capable of caring for their offspring without the help of the pair bonded males (mentioned above) for some time. In contrast to that, not all males take care of their offspring without their mate. What factors may affect male decision whether to stay or not is not totally examined yet. First, pair bonded males benefit strongly from holding a territory because of the higher reproductive success contrary to cuckolders (Bose et al., 2018). Second, territoriality might entail monogamy in V. moorii because the population density is high and suitable rocks are limited. This may lead into territory scarcity (i.e. suitable territories are rare or occupied, Whiteman & Côté, 2004). Consequently, if pair bonds outlive more than one breeding bout, pair bonded males may be able to increase their future reproductive success when they retain their territory and their mate compared to males that desert their females (Morley & Balshine, 2002; Sefc et al., 2008). Therefore, to occupy and maintain a territory might be indispensable for males and it might be that territoriality has a greater influence on the maintenance of monogamy in V. moorii than bi-parental brood care.

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5 Conclusion

In my study, I found a high proportion of extra pair paternity within broods of V. moorii. Additionally I could show that EPP varies with season. One possible reason for this might be that males, although generally facing high rates of cuckoldry, try to fend off potential competitors during spawning which might be more difficult in turbid water during the rainy season. Furthermore, my investigations raised the possibility that pair bonded males might have a higher paternity share in nests with larger (i.e. older) fry. I suggest that both a preference for the fry he sired himself and cannibalization of foreign fry could lead to an increase of paternity over the care period. Further studies including sampling at the beginning and the end of the care period as well as an observational examination of possible filial cannibalism will be needed to test these predictions. Finally, in female removal experiments, most males were able to care for their offspring alone and further, uniparental males were more successful with older broods than with younger ones. This result raises the possibility that PB males might be able to estimate their remaining investment in further brood care until the offspring is ready to gain independency. While the increased readiness of widowed males to care for large offspring may reflect fine-tuned care decisions when they are left alone, the possibility for males and females to successfully provide brood care alone raises the question if bi-parental brood care is necessary for offspring survival in V. moorii and further, if the bi- parental care can be seen as a prerequisite for the maintenance of monogamy in this cichlid. This study provides several open questions about the brood care system in V. moorii and the findings of this study could be a contribution for further research on brood care allocation and mating behaviour in this species.

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7 List of Figures

Fig. 1 V. moorii in their natural habitat in LT ...... 8

Fig. 2 Adult V. moorii in the laboratory of the University of Graz ...... 8

Fig. 3 Share of total nest offspring ...... 14

Fig. 4 Paternity shares ...... 14

Fig. 5 Seasonal differences in PBM paternity share ...... 15

Fig. 6 Nest depth related to PBM paternity share ...... 15

Fig. 7 Fry mortality ...... 16

Fig. 8 Relationship between mean offspring size and PBM paternity share...... 16

Fig. 9 Relationship between the PBM paternity share and the relative loss of offspring ...... 17

Fig. 10 Relationship between mean offspring length and the decline in offspring numbers ...... 17

8 List of Tables

Table 1 Summary of nest numbers and individuals ...... 10

Table 2 Summary of used microsatellite loci ...... 12

Table 3 Summary statistics for nine polymorphic microsatellite markers ...... 13

Table 4 Significance of factors affecting the paternity share of PB males ...... 15

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