University of Groningen Better together Groenewoud, Frank

Better together

Groenewoud, Frank

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Groenewoud, F. (2018). Better together: Cooperative breeding under environmental heterogeneity.

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Cooperative breeding under environmental heterogeneity

(GELIFES) at the University of Groningen and the Behavioural Ecology group at the Univer-sity of Bern, Institute of Ecology and Evolution.

This thesis is financially supported by the Netherlands Organisation for Scientific Rese-arch (NWO-TOP-854.11.003 to JK/DSR; NWO-ALW-823.01.014 to JK), the European Commu-nity’s Sixth Framework Programme (028696 to JK), the Swiss National Science Foundation (310030B_138660 and 31003A_156152 to MT; 31003A_144191 and 31003A_166470 to JGF), the Rektorenkonferenz der Schweizer Universitäten (CRUS) for their contribution within the framework of the “Cotutelles de these” program. Printing was supported by the University of Groningen and the Faculty of Science and Engineering..

ISBN (printed book): 978-94-034-1156-9

ISBN (e-book PDF without DRM): 978-94-034-1155-2

Cover: Tim Holland

Illustration: Jacqueline van Rhijn

Layout: Ilse Modder, www.ilsemodder.nl

Printing: Gildeprint - Enschede, www.gildeprint.nl

© Frank Groenewoud, 2018

For all articles published, the copyright has been transferred to the respective publisher. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without written permission from the author or, when appropriate, from the publisher.

Cooperative breeding under environmental heterogeneity

PhD thesis

to obtain the degree of PhD of the University of Groningen

on the authority of the Rector Magnificus Prof. dr. E. Sterken

and in accordance with the decision by the College of Deans.

and

to obtain the degree of PhD of Science in Ecology and Evolution of the University of Bern

on the authority of the Rector Prof. dr. C. Leumann and

the Dean of the Faculty of Science Prof. dr. Z. Balogh.

Double PhD degree

This thesis will be defended in public on Friday 16 November 2018 at 12.45 hours

by

Frank Groenewoud

born on 25 October 1984

Prof. dr. M. Taborsky

Co-supervisors

Dr. S. A. Kingma

Assessment Committee

Prof. dr. I.R. Pen Prof. dr. H. Kokko Prof. dr. S. Alonzo Prof. dr. J.M. Gaillard

Chapter 1 General introduction

Chapter 2 Predation risk drives social complexity in cooperative breeders

Chapter 3 Spatio-temporal resource variation, group formation and the

benefits of cooperative breeding in the Seychelles warbler

Chapter 4 Subordinate females in the cooperatively breeding Seychelles

warbler obtain direct benefits by joining unrelated groups

Chapter 5 Predation risk mediates the benefits of sociality and

suppresses within-group conflict in a cooperatively breeding

cichlid fish

Chapter 6 Experimentally induced anti-predator responses are sex

specific and mediated by social and environmental factors in a cooperatively breeding passerine

Box A Box A: Anti-predator benefits drive communal breeding in the

Seychelles warbler Chapter 7 Synthesis References Nederlandse samenvatting Dankwoord/Acknowledgements 9 23 43 67 87 107 125 131 141 159 169

Chapter 1

INTRODUCTION

How I have enjoyed watching the simple life of this happy and affectionate family. Perfect harmony reigns between all; I am beginning to believe these birds incapable of a show of anger toward each other. Better such a life in the open fields, on a diet of cockroaches and grasshoppers, than life in a palace where the board groans under the cream and honey of the land, with the constant disagreements and bickerings which so often disfigure the con-duct of the wealthy. From: “Groove-billed Ani – some reflections on their family rela-tions,” Journal, Vol. 5, September 22, 1930 – Alexander F. Skutch

The passage above was written in 1930 by the then twenty six year old Alexander Skutch (who died in 2004 – just eight days short of his one hundredth birthday), where he describes the pleasantly simple life of a group of groove-billed anis (Crotophaga sulcirostris). A few years later, Skutch would formalize these and other observations in an article where he coined the term “helpers at the nest”, which is still in use today to refer to particular types of coop-erative breeding (Skutch 1935). In the broadest sense, coopcoop-erative breeding is an umbrella term for any system where more than two individuals are engaged in raising offspring. Even without the aid of modern molecular techniques to confirm relatedness, and the use of banding to distinguish between individuals, Skutch appreciated by careful observations that different categories of “helpers at the nest” existed, and made the distinction between (i) juvenile helpers, which are retained offspring that provided alloparental care to their younger siblings, (ii) unmated sexually mature helpers, which cannot breed themselves due to a lack of mates or breeding vacancies, and decide to help others, and (iii), mutual helpers, which are breeding birds that assist each other in rearing their own respective fam-ilies. While many of the cooperatively breeding birds, fishes, mammals and insects known today could be assigned to one (or a combination) of these categories, our understanding of the environmental, social and genetic factors that drive these behaviours within- and be-tween species, on both proximate and ultimate levels, has improved considerably (Koenig & Dickinson 2016). However, we are still far from a general “theory of cooperative breeding”, as it has proven difficult to find the right balance between simple, generalizable hypotheses and predictions, and the number of systems to which these would apply. Many different reasons have contributed to this conundrum. First, within-species variation (i.e. between individuals and/or between populations) is the rule rather than the norm. Different sub-ordinates in the same species could be providing care for different reasons depending on potential future fitness benefits that may depend on sex, age, relatedness or body condition. Between-population variation poses a particular problem for studies that try to develop insights into the evolution of cooperative breeding using comparative methods. These ap-proaches usually resort to taking some kind of average trait value, or using data only from

a single population, leading to a loss of valuable information on the ecological drivers of cooperative breeding. Second, while cooperative breeding was initially studied in birds, it also occurs in mammals, insects and fishes, further complicating generalization due to vastly different modes of reproduction (i.e. oviparity vs viviparity), and life histories (e.g. growth patterns, number of offspring; Wilson 1971; Cockburn 1998; Clutton‐Brock 2006; Ta-borsky 2016). Last, cooperative breeding encompasses a whole range of different breeding and social systems (Cockburn 1998; Hatchwell 1999; Cockburn 2006; Riehl 2013), such as groups that consist of unrelated coalitions (e.g. dunnocks) and those that form (primarily) through the delayed dispersal of offspring (e.g. Seychelles warblers), and these systems like-ly had very different evolutionary origins (Ligon & Burt 2004; Clutton-Brock 2009; Wong et al. 2012; Riehl 2013). Despite all these reservations, the field of cooperative breeding, and the study of its genetic, social and ecological drivers has come a long way since its first descrip-tions by early observers.

Providing a comprehensive overview of the cooperative breeding literature over the past decades is beyond the scope of this introduction. Instead I would like to highlight some important contributions that focus on the ecological drivers of cooperative breeding, spe-cifically the environmental conditions that affect the costs and benefits of delayed dispersal and helping behaviours (i.e. alloparental care). After this more general background, I will briefly introduce my two study species: the Seychelles warbler (Acrocephalus sechellensis) and the cooperatively breeding cichlid Neolamprologus pulcher. Lastly, I will summarize the questions that will be the focus of the remaining chapters of my thesis.

Cooperative breeding: a two-step process

While various evolutionary routes to cooperative breeding have been proposed, most forms of cooperation occur in family groups (Cockburn 2006; Riehl 2013). Two likely explanations exist for why this is so. First, delayed or limited dispersal of offspring is one of the primary modes by which cooperative breeding groups form (Emlen 1982; Koenig et al. 1992; Griesser et al. 2017). Second, kin selection – selection on genes through its effects on others carry-ing the same gene (Hamilton 1963) – is an important evolutionary driver of cooperation (West-Eberhard 1975; Foster, Wenseleers & Ratnieks 2006; Bourke 2014). It should be noted that these two statements relate to different processes: group formation in the former, and the benefits of cooperation, or helping, in the latter. This distinction is why it is generally accepted that the evolution of cooperative breeding is best approached as a two-step pro-cess where delayed dispersal is a nepro-cessary, but not a sufficient condition for the evolution of cooperative breeding. While it is difficult to overstate the importance of kin selection theory in the last half century of research in the field of cooperation (West-Eberhard 1975; Bourke 2014), its importance for the evolution of cooperative breeding has been put into

question (Clutton-Brock 2002, 2009). This has generally not been because of flaws in the theory itself, but rather because most studies that invoked kin selection were correlation-al, and other (direct) benefits could therefore be in place (e.g. Wright 2007; Taborsky 2013; Kingma et al. 2014). Despite these criticisms, support for kin selection theory is strong, both within (e.g. Emlen & Wrege 1989; Komdeur 1994b; Richardson, Komdeur & Burke 2003b; Wright et al. 2010), and between species (Hughes et al. 2008; Cornwallis et al. 2010; Lukas & Clutton-Brock 2012; Dillard & Westneat 2016). Furthermore, empirical tests of the benefits of cooperative breeding in any particular species only apply to the selective forces that are currently maintaining cooperative breeding, but not necessarily to its evolutionary origins. Kin selection might therefore have been a necessary initial driver of cooperation, but once these conditions were set, other direct benefits may have evolved that were not necessarily dependent on genetic relatedness (Cockburn 2013). This thesis does not focus on the indi-rect (kin selected) or diindi-rect benefits of cooperative breeding per se, but rather on the eco-logical and environmental factors that shape the costs and benefits of dispersal and group formation, cooperative breeding and the stability of groups.

Environmental heterogeneity and the evolution of delayed dispersal

Delayed dispersal is often seen as a necessary first step for the evolution of cooperative breeding, at least in so called “helpers at the nest” type systems, where the majority of help-ers are the offspring of previous breeding attempts (Emlen 1982; Koenig et al. 1992; Riehl 2013). Such delayed dispersal does not necessarily lead to cooperative breeding, if there are no further benefits of helping. Consequently, many family living species exist, where subor-dinates do not show alloparental care (Griesser et al. 2017). Two main hypotheses have been proposed that make predictions regarding the environmental conditions under which de-layed dispersal should be the preferred strategy by offspring. The “ecological constraints” hypothesis suggests that constraints on dispersal and independent breeding reduce the fitness benefits of leaving the natal territory, so that offspring achieve higher fitness by postponing breeding, and potentially staying at home (Selander 1964; Brown 1974; Emlen 1982). This hypothesis predicts that constraints on independent reproduction could arise through stable environments leading to habitat saturation and thus a lack of available suit-able breeding opportunities, or alternatively, fluctuating environmental conditions lead-ing to high costs of dispersal or rearlead-ing young in bad years (Emlen 1982). Habitat saturation has been criticized as being an insufficient explanation for the evolution of cooperative breeding, because of the observation that many, if not most species, experience severe com-petition for suitable breeding positions, yet do not show delayed dispersal (Stacey & Ligon 1991; Koenig et al. 1992). The “benefits of philopatry” hypothesis argues that subordinate individuals should forego dispersal if the benefits in their resident territory (e.g. food, pro-tection) exceed the benefits of dispersal (Stacey & Ligon 1991). This hypothesis assumes that

variation in quality between territories is required to offset the cost of delayed reproduc-tion by increased fecundity or survival when a high-quality territory is acquired. Although it has generally been agreed that these two hypotheses are effectively two sides of the same coin, and differ only in their focus on the benefits of staying versus the costs of leaving (Emlen 1994), there are notable differences. First, if the ecological constraints hypothesis only includes the costs and benefits of independent reproduction, but not dispersal, off-spring should not necessarily remain philopatric. In fact, unless there are some additional benefits of philopatry, offspring could disperse and roam through the population without association to any group or territory, a strategy generally referred to as “floating” (Koenig et al. 1992; Ridley, Raihani & Nelson-Flower 2008; Kingma et al. 2016a). In contrast, the benefits of philopatry hypothesis explicitly emphasizes the benefits that can be gained on the natal territory, thereby excluding the possibility of floating as a viable option. However, most individuals will experience some combination of ecological constraints and benefits in the natal territory, potentially leading to selection for natal philopatry, and delayed reproduc-tion (Koenig et al. 1992; Emlen 1994; Komdeur 1992).

The ecological constraints and benefits of philopatry hypotheses also differ in whether they emphasize temporal or spatial variation in environmental conditions as the primary driver of delayed dispersal. The ecological constraints” hypothesis derives its predictions mainly from environmental fluctuations (or lack thereof) through time that determine the costs and benefits for offspring to disperse or breed independently. For example, the number of breeding vacancies might be higher following a year with high breeder mortality, and years with high food availability might lead to lower costs of dispersal or independent breeding. In both examples, temporal variation in environmental conditions lead to decreased costs, or increased benefits, of dispersal. In contrast, when there is no variation in quality between territories, offspring have no prospect of obtaining a higher quality territory by initially delaying dispersal. Thus, the benefits of philopatry hypothesis requires consistent spatial variation in order for delayed dispersal to evolve.

Both the ecological constraints and the benefits of philopatry hypothesis emphasize the rel-ative costs and benefits of dispersal vs natal philopatry for subordinates, but do not include the costs and benefits of these subordinates for the rest of the group. These can be especial-ly important when (i) individuals have an effect on the fitness of other group members, and (ii) when a few group members (e.g. dominants) can control group membership (e.g. through eviction). While not further explored here, recent studies have taken such effects into account and have consequently provided important insights into the conditions un-der which groups form (Shen et al. 2017).

Environmental heterogeneity and the benefits of helping

In order for natal subordinates to be able to provide alloparental care, they have to be (i) present during a breeding attempt and (ii) capable of providing alloparental care. Obvi-ously, there are large differences in rates of maturation, and inter-reproductive intervals between different species and taxa that could affect these two conditions. For instance in the facultative eusocial hover wasp Liostenogaster flavolineata, nesting is year-round due to a lack of seasonality, and newly emerged females are almost immediately capable of provid-ing alloparental care. Conversely, white-wprovid-inged choughs (Corcorax melanorhamphos) only breed during the breeding season, which is a yearly occurrence. This necessitates offspring to remain with their parents for at least a year to be able to provide help. Subordinates can provide a wide range of alloparental care behaviours that are mostly an extension of the types of parental care provided by breeding pair. Help can consist of providing food to de-pendent young, egg-cleaning, incubation, nest-building, territory defense and territory maintenance (Brown 1987; Heinsohn & Legge 1999; Taborsky 2016).

Helping is not a necessary consequence of delayed dispersal: offspring could delay dis-persal and remain in the natal group, but not provide help (Drobniak et al. 2015; Griesser et al. 2017). Thus, there need to be additional benefits that select for helping behaviour by subordinates, and these benefits – which can be broadly categorized into indirect benefits (i.e. benefits accrued through kin selection), and direct benefits – have been extensively dis-cussed and reviewed (e.g. Cockburn 1998; Clutton-Brock 2002; Bergmüller et al. 2007; King-ma et al. 2014). The costs and benefits of subordinate help can be affected by abiotic and biotic conditions. For instance, when nestling starvation is the main cause of reproductive failure, subordinates in cooperatively breeding birds are expected to have a larger impact on reproductive success when food conditions are poor than when these are high (i.e. the “hard life” hypothesis; Koenig, Walters & Haydock 2011). The main reasoning behind this is that when conditions are good and food availability is high, the additional food provisioned by helpers is less valuable than when food conditions are poor and additional food might mean the difference between reproductive failure and success. A similar argument could be made with regards to any other ecological pressure that affects the reproductive success or survival or groups that can be modulated through subordinate help, such as reducing the risk of predation. One additional benefit of improving reproductive success under harsh environmental conditions is that offspring produced under such conditions are more valu-able, because fewer total offspring are produced in such years, and relative contribution to population growth (i.e. reproductive value; Fisher 1930; Taylor 1990) is therefore higher. In addition, there are other benefits of reducing variance in reproductive success that is in-duced by temporal variation in environmental conditions. Such strategies (i.e. rein-duced fe-cundity variance at the expense of mean fefe-cundity) are generally referred as “bet-hedging”

strategies (Gillespie 1977; Lehmann & Balloux 2007; Starrfelt & Kokko 2012). Several studies have recently suggested that cooperative breeding might similarly be a bet-hedging strat-egy that buffers against fecundity variance induced by temporal fluctuations in environ-mental conditions (Rubenstein 2011; Koenig & Walters 2015). Further support for coopera-tive breeding as a bet-hedging strategy comes from comparacoopera-tive studies that show that the occurrence of cooperative breeding is positively associated with climatic uncertainty and variability (Jetz & Rubenstein 2011; Cornwallis et al. 2017; Lukas & Clutton-Brock 2017). Thus, while it has been suggested that the benefits of cooperative breeding can depend on (fluc-tuations in) the environment, how and under what conditions subordinates in cooperative groups can improve reproductive success has received little attention.

The suppression of within-group conflict

Living in social groups provides many benefits for group members, but also comes with costs (Alexander 1974). One of the major costs of group living concerns conflict over the dis-tribution of limited resources (i.e. food, reproduction) between members of these groups. Such conflict leads to behaviours that aim to secure a larger share of resources, thereby negatively affecting the stability of groups and potentially negating the benefits of social-ity (Shen, Akçay & Rubenstein 2014). Investigating the factors that reduce conflict is thus important for understanding the evolutionary stability of groups (West et al. 2015). One important way by which conflict could be reduced between members of a social group, is when environmental conditions improve the fitness benefits of group living relative to leaving the group. These conditions should select for individuals refraining from using ag-gression to obtain a larger share of resources, because in doing so, they are reducing the fitness benefits they accrue through other group members (Alexander 1974; Brown 1982; Shen et al. 2014). While predation risk is often invoked as an important factor selecting for group-living (Inman & Krebs 1987; Krause & Ruxton 2002), its role in the evolution of more complex social organisation, such as that of cooperative breeders, has been largely neglect-ed. This is surprising, because predation risk can have substantial consequences for (i) off-spring survival, which might require cooperation between individuals and (ii) the costs of dispersal and independent reproduction, selecting for limited dispersal. Predation risk can therefore influence both the benefits of group-living and cooperation, as well as the costs of leaving the group and breeding independently. Consequently, increased levels of preda-tion risk should minimize the willingness of individuals to obtain a larger share of group resources at the expense of others, and reduce conflict between group members.

Ecological factors such as predation risk and food availability can have important conse-quences for group formation through delayed dispersal, the benefits of cooperation and levels of within-group conflict, all important aspects in the evolution of complex social

sys-tems and cooperative breeding. Investigating how these ecological factors shape the social systems and behaviours of cooperative breeders can therefore provide insights into the pro-cesses that underlie transitions from simple to complex social organization. In the coming paragraphs, I will introduce the species that I will use throughout the rest of my thesis to address these questions, the cooperatively breeding Seychelles warbler Acrocephalus sechel-lensis and the social cichlid Neolamprologus pulcher.

Island life

The Seychelles warbler is a small (13-19 g) facultative cooperatively breeding passerine en-demic to several islands in the Seychelles archipelago (Fig. 1.1A; Komdeur et al. 2016, 2017). By the 1960’s, the last decimated population of Seychelles warblers (26-50 individuals) was confined to the island of Cousin (ca 29 ha; 04º20’S, 55º40’E). Habitat restorations and sev-eral translocations to other islands have restored the population to viable numbers and have provided a unique opportunity to study the cooperative breeding system of this once critically endangered species. Most Seychelles warblers on the island of Cousin (Fig. 1.1B), which is our main study population, live in pairs (ca 60% of all territories), but a proportion lives in groups consisting a dominant breeding pair and 1-5 subordinates (mean ± SE = 0.59 ± 0.02; 1996-2016; see chapter 3) of either sex. These helpers are usually, but not always the offspring of the dominant breeding pair (Kingma et al. 2016a; see chapter 3). Territories are defended year-round and breeding pairs often remain pair-bonded on the same territory throughout their lives. Seychelles warblers on Cousin Island typically produce single egg clutches, but around 13% of clutches contain 2-3 eggs (Richardson et al. 2001). Offspring can remain nutritionally dependent on their parents and other group members for a period of up to three months, which is extremely long for a passerine species.

The population of Seychelles warblers on Cousin Island is contained, with virtually no mi-gration on or off the island (Komdeur et al. 2004a). Additionally, annual resighting prob-abilities of birds on the island are extremely high: up to 0.98 for adult birds, and 0.92 for younger individuals (Brouwer et al. 2010). This combination makes the Seychelles warbler an excellent system to study the factors associated with natal dispersal, because these are not confounded by individuals dispersing from the study site. Seychelles warblers are strictly insectivorous, taking most of their arthropod prey from the underside of leaves (Komdeur 1991). They are thus highly dependent on the relatively short peaks in abundance of these prey following monsoon rains, which occur twice per year (Komdeur & Daan 2005). Reproduction therefore mostly occurs following such rains, during June-September (major breeding season), or January-March (minor breeding season).

FIGURE 1.1 An adult Seychelles warbler (A) and a bird’s eye view of our main study site – Cousin Island – with our research

sta-tion in the foreground and the neighbouring island of Cousine in the background (B). Photo (A) by Sjouke A. Kingma and (B) by Martin Harvey, courtesy of Nature Seychelles.

A

Detailed measurements of the abundance of prey have been collected since the start of the study on Cousin Island, and earlier studies have indicated that between-territory variation plays an important role in group formation, with offspring being more likely to delay dis-persal on high quality than on low quality territories (Komdeur 1992). However, there is substantial between-year variation in arthropod abundance, which could have consequenc-es for delayed dispersal and other aspects of cooperative breeding, which were until recent-ly unknown (see chapter 3).

Seychelles warblers on Cousin suffer virtually no adult predation, but predation of eggs by the endemic Seychelles fody Foudia sechellarum is an important cause of nest failure (Kom-deur & Kats 1999). However, several aspects of the Seychelles warbler breeding biology re-duce the risk of nest predation. First, (dominant) males perform nest guarding behaviour (i.e. remaining vigilant close to the nest during female off-bouts). Such behaviour reduces the likelihood of egg predation because eggs are only taken from unprotected nests. Nest guarding is probably costly for males since male investment into nest guarding behaviour increases with territory quality (see chapter 6; Komdeur & Kats 1999). Second, incubation by subordinate females reduces the time that the nest is unprotected, and should therefore also reduce the risk of nest predation by fodies. Third, Seychelles warblers will attack and chase off any fodies that come to close to their nest. However, several aspects of Seychelles warbler behaviour with regards to nest predation and the benefits of cooperative breeding remain elusive. For instance, the extent to which subordinate dispersal, dominant toler-ance of subordinates and co-breeding by subordinate females is associated with the risk of nest predation was hitherto unknown. Additionally, we knew very little about the tradeoffs that determine anti-predator behaviours for Seychelles warbler parents. These have now been addressed in box 1 and chapter 6, respectively.

On the southern shores of Lake Tanganyika

Neolamprologus pulcher is a highly social cooperatively breeding cichlid fish (Fig. 1.2A), en-demic to Lake Tanganyika (Fig. 1.2B), where it inhabits the sublittoral zone between 2-40 meters depth (Konings 1998). It lives in groups that consist of a dominant pair and up to 25 subordinates of either sex (Taborsky & Limberger 1981; Groenewoud et al. 2016). Territories are centred on some kind of substrate, usually one or several small rocks, but fish have also been seen to use shells or crevices, which they use for both shelter and breeding (Josi et al. in prep.). Subordinates engage in various types of helping behaviours, including territory maintenance (i.e. removing sand and debris to create shelters), egg cleaning and fanning, defense against con- and heterospecifics space competitors and predators (Taborsky 1984).

FIGURE 1.2 A group of Neolamprologus pulcher in their territory (A) and the sunset over Lake Tanganyika as seen from the field

station at Kasakalawe bay (B). Photo (A) by Dario Josi and (B) by Arne Jungwirth.

A

Subordinates are of different sizes and show age-related task specialization: smaller, sexu-ally immature subordinates ususexu-ally engage in maintenance and defence against non-dan-gerous predators and space competitors, and large sexually mature subordinates defend against larger predators (Bruintjes & Taborsky 2011; chapter 5). Territories are aggregated into colonies, which are made up of several dozen, to several hundreds of territories (Heg et al. 2008).

The evolution of cooperative breeding in N. pulcher – and other cooperative breeding fish – is likely distinct from the evolution of cooperative breeding in most other vertebrate systems, in that it is not based on habitat saturation or increased levels of within-group relatedness. N. pulcher does not suffer from habitat saturation in the strict sense, since breeding sub-strate appears to be plentiful. However, most of this free habitat occurs outside of the boundaries of the colony, and individuals seem unwilling to disperse to such habitat. When similar habitat was experimentally made available inside the colony, fish usually dispersed there within a few days (Heg et al. 2008). One of the main obstacles to dispersal, and the main drivers of delayed dispersal, is the risk of predation by large predatory cichlids such as Lepidiolamprologus elongatus and L. attenuatus. These fish are highly mobile predators, swimming through N. pulcher colonies in small groups (often together with Mastacembelid eels). Individuals that do not belong to a group and are devoid of protection, are easy prey, and several studies have shown that increased predation risk leads to delayed dispersal by larger, sexually mature subordinates (Heg et al. 2004a; see chapter 2). As a result of frequent dominant turn-over, subordinate-dominant relatedness decreases with increasing subor-dinate size (Dierkes et al. 2005). Larger, sexually mature helpers are therefore mostly not related to the dominants in whose territory they reside and to whom they provide help. As group membership is a prerequisite for survival and large subordinates impose costs on dominant breeders, large helpers have to compensate for these costs by providing help (i.e. “pay-to-stay”; Gaston 1978; Fischer et al. 2014).

The role of predation risk in the evolution of cooperative breeding, and other forms of so-cial complexity, have generally been understudied. The cooperative breeding system of N. pulcher offers a unique opportunity to study such effects because fish can be easily observed in the wild and there is high natural variation in predation risk (see chapter 2). The broader implications of predation risk on the structural complexity of social groups and the extent to which individuals show behavioural adjustments to increase the benefits of cooperative breeding under high predation risk were previously unexplored – but have now been ad-dressed in chapter 2 and 5, respectively.

Environmental heterogeneity and the evolution of cooperative breeding

Developing a hypothesis based on well-supported assumptions is the beginning of any in-vestigation. I hope I have been able to provide a strong argument for the hypothesis that the ecology of cooperative breeders is an important factor determining the costs and bene-fits of (delayed) dispersal, helping behaviour and behavioural strategies that maximize the benefits of cooperation. In the coming chapters, I will further explore these questions. In the second chapter, I will explore the consequences of predation risk for dispersal and the so-cial organization of N. pulcher, and discuss the general implications of our results for other cooperative breeding systems. In the third chapter, I will show that spatio temporal variation in food availability has important consequences for group formation in the Seychelles war-bler, but that the benefits that are obtained through cooperative breeding are not affected by such variation. In the fourth chapter, I will presents the results of an investigation into al-ternative dispersal strategies in the Seychelles warbler, and show that subordinate females can disperse into unrelated group to obtain reproductive benefits. In the fifth chapter, I will return to N. pulcher to show how groups reduce within-groups conflict to maximize the ben-efits of cooperative breeding under high predation risk, with wider implications for evo-lutionary transitions to higher social complexity. In my sixth chapter, I will present results of a field experimental study showing the social and environmental factors that mediate tradeoffs in anti-predator defenses in the Seychelles warbler. Box A contains an investiga-tion into the extent to which the risk of nest predainvestiga-tion drives the benefits of communal breeding for females in the Seychelles warbler. In the final chapter, I will present a synthesis the results of this thesis and their implications, and offer some perspectives for future stud-ies investigating the ecological and environmental drivers of cooperative breeding.

Chapter 2

Predation risk drives social

complexity in cooperative breeders

Frank Groenewoud*, Joachim Gerhard Frommen*, Dario Josi, Hirokazu Tanaka, Arne Jungwirth and Michael Taborsky.

Published in Proceedings of the National Academy of Sciences of the United States of America, 113, 4104–4109 * Equally contributing authors

ABSTRACT

Predation risk is a major ecological factor selecting for group living. It is largely ignored, however, as an evolutionary driver of social complexity and cooperative breeding, which is attributed mainly to a combination of habitat saturation and enhanced relatedness levels. Social cichlids neither suffer from habitat saturation, nor are their groups composed pri-marily of relatives. This demands alternative ecological explanations for the evolution of advanced social organization. To address this question, we compared the ecology of eight populations of Neolamprologus pulcher, a cichlid fish arguably representing the pinnacle of social evolution in poikilothermic vertebrates. Results show that variation in social orga-nization and behavior of these fish is primarily explained by predation risk and related eco-logical factors. Remarkably, ecology affects group structure more strongly than group size, with predation inversely affecting small and large group members. High predation and shelter limitation leads to groups containing few small but many large members, which is an effect enhanced at low population densities. Apparently, enhanced safety from predators by cooperative defense and shelter construction are the primary benefits of sociality. This finding suggests that predation risk can be fundamental for the transition toward complex social organization, which is generally undervalued.

INTRODUCTION

Predation risk is a key ecological factor selecting for adaptive responses in morphology, behavior, and life history decisions in animals (Barbosa & Castellanos 2005; Caro 2005). In particular, it constitutes a fundamental selective force for group living (Alexander 1974; Krause & Ruxton 2002). Group members benefit from predator dilution or confusion (Wro-na & Dixon 1991; Krause & Ruxton 2002) and from joint effort in antipredator behavior, such as mobbing and vigilance (Caro 2005). However, living in groups also entails costs; thus, group living should only evolve when, on average, the benefits of group living exceed its costs (Alexander 1974).

Long-term, stable groups mainly form in the context of reproduction. The most highly structured and complex groups occur when offspring are raised cooperatively, which often involves division of labor between group members (Wilson 1971; Clutton-Brock, Russell & Sharpe 2004). In such cooperative groups, sexually mature individuals typically refrain from reproduction to raise the offspring of others (Taborsky & Limberger 1981; Cockburn 1998; Jennions & Macdonald 2007), which may involve lifetime reproductive sacrifice (eusocial-ity). The evolution of cooperative breeding is generally understood as a two-step process, where delayed dispersal is accompanied by the decision to provide alloparental brood care to dependent young (Koenig & Dickinson 2016). Limited dispersal resulting from habitat saturation may facilitate the evolution of cooperation by kin selection through the creation of kin neighborhoods (Frank 1998). Empirical evidence is provided by correlations between relatedness and helping effort (Emlen & Wrege 1989; Komdeur 1994b; Wright et al. 2010), and by interspecific correlations between monogamous mating and the incidence of co-operative breeding and eusociality (Hughes et al. 2008; Briga, Pen & Wright 2012). However, this paradigm has limited explanatory power where habitats are not saturated and where cooperation occurs between unrelated individuals, demanding alternative explanations to account for the evolution of complex social organization (Clutton-Brock 2009). Despite its central role in group formation, predation risk has rarely been recognized as an evolution-ary force in the transition from simple to complex social organization, where subordinate nonbreeders provide alloparental care. This represents an important gap in our under-standing of the evolution of complex social organization. It should be noted that the term “social complexity” has different connotations, especially when used in connection with different taxa, but there seems to be consensus that social complexity is not merely synon-ymous with group size. Instead, this term typically refers to social systems incorporating different types (or roles) of individuals within groups, accounting also for the nature and diversity of interactions among these individuals (Blumstein & Armitage 1997, 1998; Free-berg, Dunbar & Ord 2012). Here, we demonstrate that variation in predation risk between

populations can explain social organization and complexity in cooperatively breeding fish. Predation risk may affect delayed dispersal, and hence group formation, in two possi-ble ways. First, it increases the costs of dispersal by causing mortality when individuals disperse from their natal area to unfamiliar territory (Yoder 2004). This effect can be exac-erbated when individuals need to sample their environment for suitable dispersal options (Clobert et al. 2009; Bocedi, Heinonen & Travis 2012), as is the case in cooperative breeders keeping their territories all year round (Bergmüller et al. 2005a). Second, predation may render independent breeding unprofitable or impossible if the joint effort of group mem-bers is required for successful reproduction (Balshine et al. 2001; Caro 2005; Kingma et al. 2014). Hence, predation risk is an important ecological constraint selecting for reduced or delayed dispersal, fulfilling two of three conditions (see conditions ii and iii below) pro-posed by the ecological constraints hypothesis (Emlen 1982). This hypothesis predicts de-layed dispersal when there is: (i) a shortage of vacant breeding territories or mates, (ii) high mortality risk during dispersal, and (iii) a low chance of independent reproduction when a breeding territory has been established. These effects may be additive and can cause strong selection for delayed dispersal under high levels of predation.

The cooperatively breeding cichlid fish Neolamprologus pulcher is a highly suited model sys-tem to unveil the role of predation risk as ecological constraint, as this species suffers from high predation (Balshine et al. 2001), but usually not from a shortage of breeding sites (Heg et al. 2008). N. pulcher breeds colonially in sandy to rocky patches along the sublittoral zone (2- to 45-m depth) of Lake Tanganyika. Individual groups consist of a dominant breeding pair and up to 30 subordinates (Taborsky 1984; Balshine et al. 2001; Bergmüller et al. 2005a). Groups are frequently attacked by predatory fish (Balshine et al. 2001), which significantly affects the survival probability of group members, especially small subordinates and fish devoid of protection through large conspecifics (Heg et al. 2004a). Hence, membership in a group is a precondition for survival. Within groups, subordinates have to pay by helping in brood care, territory maintenance (i.e., removing sand and debris from underneath rocks), and defense to compensate for the costs they impose on dominant breeders (Taborsky 1985; Balshine-Earn et al. 1998; Bergmüller, Heg & Taborsky 2005b; Fischer et al. 2014), to which they are often unrelated (Dierkes et al. 2005). The lack of relatedness between larger sub-ordinates and dominants reduces the opportunity for selective benefits of assisting kin, which differs sharply from the situation proposed for many other cooperative breeders (c.f. Cockburn 1998; Solomon & French 2007; Hatchwell 2009). This finding suggests that other mechanisms are in place to explain helping behavior by larger subordinates (Bergmüller et al. 2005b; Taborsky 2016). We investigated the effects of predation on the social organization of N. pulcher, using natural variation in predation risk and ecological factors shaping mor-tality, group composition, and behavioral decisions among eight distinct populations in

Lake Tanganyika. Specifically, we focused our attention on shelter availability and sand cov-er as ecological factors. Sheltcov-er availability has been shown to affect group size in this spe-cies (Balshine et al. 2001), but not all group members may be equally dependent on access to shelters for their survival. In addition, territory maintenance has been shown to be one of the most costly activities in terms of time and energy expenditure (Grantner & Taborsky 1998; Taborsky & Grantner 1998), and the extent to which helpers engage in these behaviors is likely dependent on the presence of sand in their territories.

MATERIALS AND METHODS

Sampling sites, predation risk, and ecological factors

We collected data on eight different populations of N. pulcher between September and De-cember 2012 and 2013 by SCUBA-diving at the southern end of Lake Tanganyika. Populations were on average 1,796 m apart (range = 150–22,450 m), with seven populations all being with-in 9 km of each other and one population located about 20 km away from the rest.

We estimated predation risk in each population along four transects of 10 × 1 m2 _{by counting}

the number of fish predators (Lepidiolamprologus elongatus, Lepidiolamprologus attenuatus, and Lamprologus lemairii). We repeated these scans between 6 and 10 times per population on different days to capture the variation in fish activity. For each population we estimated pre-dation risk on adult N. pulcher by calculating the mean number of large (>10 cm) L. elongatus and L. attenuatus per transect (Heg et al. 2004a). L. elongatus and L. attenuatus are the most common predators of N. pulcher in our study area (Balshine et al. 2001; Heg et al. 2004a, 2008) and they were also the two most abundant species in the surveyed populations. These are highly mobile predators, usually observed in small groups moving through our populations at 20-30 cm above ground looking for prey, which in the case of N. pulcher consists mainly of smaller fish or fish devoid of protection by a group (Taborsky 1984; Heg et al. 2004a).

We measured the number of shelters and the percentage of sand cover per square meter

(hereafter “sand cover”) for each population by surveying four transects of 10 × 1 m2 _of

bot-tom substrate, starting from the center of a colony and moving outwards in four directions at 90° angles. We also determined how many of these shelters were used by N. pulcher or other species, whether the respective square meter contained an N. pulcher territory and if so, the proportion of area covered by N. pulcher territories. To check for within- population correlations between group and habitat characteristics, we recorded the group composi-tions according to different size classes for all territories located within these transects, de-scribed in detail below.

Group compositions, group sizes, and densities

For between-population comparisons, we used data from 20 territories sampled at random from each population. We searched for N. pulcher territories in each of the eight popula-tions until we were confident that all territories had been detected. The boundaries of N. pulcher colonies were established where no other territories were found within 5 m of the outermost territories of the colony, except for two very large populations where, because of practical considerations, artificial boundaries were established despite other territories being close by. All territories were individually marked with small numbered stones (~5 cm in diameter). In each population we determined the group composition for each randomly selected territory. We estimated the standard length (SL; from tip of the snout to the pos-terior end of the last vertebrae) of individuals, and assigned them to different size classes: fry (<0.5 cm), nonhelpers (0.5-1.5 cm), small helpers (1.6-2.5 cm), medium helpers (2.6-3.5 cm), large helpers (>3.5 cm) following Heg et al. (2004a). Our analyses focus mainly on the differences between small and large helpers, because these represent nonoverlapping size classes and previous studies have shown clear differences in behavior and mortality risk as a result of predation (Heg et al. 2004a; Bruintjes & Taborsky 2011). For example, small helpers typically do not disperse, whereas large helpers do disperse if conditions allow (Stiver et al. 2004; Jungwirth, Walker & Taborsky 2015b). In addition, predation risk differs markedly between small and large helpers also with regard to group size effects (Heg et al. 2004a). Medium-sized helpers, in contrast, form a transitional state that is intermediate in both life history decisions and behavior, and their predation risk is also intermediary (Heg et al. 2004a). Therefore, there are no clear hypotheses regarding the variation of their numbers according to ecological factors. However, we provide information about these relationships in Fig. S2.1. We also recorded the presence or absence of dominant breeding females and males. Dominants can easily be distinguished from subordinates based on size and be-havior. Because not all subordinates in a group were always visible (e.g., as a result of time spent hiding or feeding in the water column) we estimated group composition repeatedly for each territory (median = 3 times, range = 1–4). For one population, only a single measure of group composition per territory could be obtained. From these group compositions we also calculated total group size (i.e., the total number of helpers). For each focal territory, we measured the distance to the nearest neighboring territory from the center of each ter-ritory to the nearest 5 cm and counted the total number of territories present within a 2-m radius, as a measure of territory density.

Behavioral observations

In each focal group we recorded the behavior of both dominant breeders and of one haphaz-ardly selected individual from each subordinate size class. All behaviors were recorded con-tinuously for 7 minutes using a handheld computer (Psion Teklogix Workabout Pro-7525)

in a waterproof plastic bag, running Noldus Pocket Observer (v3.0). Recorded behaviors included aggression against predators, group members, and other conspecifics, which was either overt (chasing, ramming, biting, mouth fighting, or other forms of elevated aggres-sion) or restrained (spreading of fins or opercula, head down display, s-bend swimming, or fast approach); submissive behaviors toward dominants and other group members (tail quiver, hook swimming, and bumping); and maintenance behaviors (removal of sand and debris from the territory; Taborsky & Limberger 1981; Taborsky 1984). We also observed the spacing behavior of focal individuals continuously during the observation and recorded whether they were inside their home territory (±30-cm semispherical dome around breed-ing shelter; Taborsky & Limberger 1981), visitbreed-ing another territory, outside of any territory, or in a shelter. In total, we collected 77 h of behavioral data of 660 individual fish in 154 different territories.

Statistical analyses

We analyzed between-population effects on the total number of subordinates and the number of large and small subordinates by fitting these as separate response variables in a generalized linear mixed model (GLMM) assuming a Poisson error distribution. To see how the number of small helpers related to territory density and the number of large helpers in the territory, we fitted a Poisson GLMM. Territory defense was analyzed as a binary trait in a GLMM where we included individual class, the number of small and large helpers in the territory, nearest neigh-bor distance, and predation risk as predictor variables. To assess whether shelter maintenance was affected by sand cover and predation risk, and whether this relationship varied between different individuals, we fitted a Poisson GLMM with interactions between both sand cover and predation risk, and individual class and predation risk. We analyzed the time spent hiding in shelters by fitting a linear mixed model with predation risk, nearest neighbor distance, in-dividual class, and the interaction between predation risk and inin-dividual class as predictors. For all models, we fitted varying intercepts for Territory ID, and where necessary Population ID, to account for the nonindependence of repeated measurements within these groups. We used an information theoretic model selection approach to find the most parsimonious mod-el. Variables were removed from the model if dropping that variable resulted in a model with a minimum difference of two Akaike Information Criterion (AIC)c values (Burnham & Anderson

2002). We calculated conditional R2 _{based on Nakagawa and Schielzeth (Nakagawa &}

Schiel-zeth 2013) as an estimator of the explained variance. Parameter significance was inferred based

on likelihood ratio tests of deviances assuming a χ2_{-distribution. All models were inspected for}

violations of model assumptions, such as overdispersion, deviations from normality, and het-eroscedasticity. All data were analyzed in R v3.1.2 (R Development Core Team 2008) using the packages “lme4” (Bates et al. 2014), “nlme” (Pinheiro et al. 2012), and “AICcmodavg” (Mazerolle 2013) for parameter inference and model selection.

RESULTS

Between-population comparisons

Habitat characteristics varied substantially between different populations (Fig. 2.1). The to-tal number of helpers was not associated with predation risk (mean ± SE = 0.026 ± 0.020, P = 0.19). However, predation risk showed an inverse relationship with small and large helpers: the number of large helpers increased with higher levels of predation (mean ± SE = 0.093 ± 0.021, P < 0.001), whereas the number of small helpers decreased (mean ± SE = −0.07 ± 0.031, P = 0.019; Fig. 2.1A and B). The total number of helpers increased with rising numbers of shelters (mean ± SE = 0.052 ± 0.016, P = 0.001), which was mostly because of the relationship between shelter availability and the number of small helpers. The number of small helpers increased significantly with a rising number of shelters (mean ± SE = 0.063 ± 0.024, P = 0.008), and this relationship was more than twice as strong as for large helpers, where it was not significant (mean ± SE = 0.025 ± 0.017, P = 0.132; Fig. 2.1C and D). Sand cover was positively correlated to total helper number (mean ± SE = 0.011 ± 0.004, P = 0.003), again mainly because of the relationship between sand cover and the number of small helpers (mean ± SE = 0.015 ± 0.005, P = 0.004), while there was no significant correlation with the number of large helpers (mean ± SE = 0.004 ± 0.004, P = 0.293; Fig. 2.1E and F). Results of the relationship between these ecological variables and the number of medium-sized helpers are presented in Fig. S2.1 and Table S2.1.

2 4 6 8 10 0 1 2 3 4 5 6 7 Predators transect -1 0 1 2 3 4

Number of helpers

Number of shelters m-2 0 _{Sand cover m}10 20 30 40 50-2 _(%)

A

C

E

B

D

F

FIGURE 2.1 The relationship between predation risk (A and B), the number of shelters (C and D), and sand cover (E and F) with

total number of helpers (Top) and group composition (Bottom) in different populations of N. pulcher. Population means and bootstrapped 95% confidence intervals are given for total helper number (filled circles in A, C, and E), small helpers (filled circles in B, D, and F), and large helpers (open squares in B, D, and F). Data points are slightly offset horizontally to avoid overlapping confidence intervals. Solid regression lines represent the model predicted values with bootstrapped predicted 95% confidence

in-Within-population comparisons

We used a subset of the data — all territories located within the habitat transects — to test whether these effects were also present within populations. We found a correlation between the number of shelters and the number of small helpers similar to that of the between-popula-tion comparison (mean ± SE = 0.040 ± 0.021, P = 0.049; Fig. 2.2A). However, within populabetween-popula-tions we found no significant correlation between sand cover and small helper numbers (mean ± SE = 0.006 ± 0.005, P = 0.255). The number of large helpers was not correlated with the number of shelters or sand cover, but similar to the between-population data, both relationships were sig-nificantly greater for small helpers than for large helpers (number of shelters: number of small helpers vs. number of large helpers: mean ± SE = −0.070 ± 0.023, P = 0.002; sand cover: number of small helpers vs. number of large helpers: mean ± SE = −0.022 ± 0.005, P < 0.001).

Territories had fewer small helpers at lower densities (i.e., when neighbors were further away; mean ± SE = −0.007 ± 0.002, P < 0.001), and this effect was stronger under high preda-tion risk (nearest neighbor distance x predapreda-tion risk: mean ± SE = −0.014 ± 0.005, P = 0.003; Fig. 2.3). There was no significant correlation between the number of large and small help-ers within groups (mean ± SE = 0.052 ± 0.032 P = 0.11), and the number of large helphelp-ers did not depend on the distance to the nearest neighbor (mean ± SE = −0.001 ± 0.001, P = 0.413).

< 0.001

BrM

BrF

LH

SH

Time in shelter (s)

A

B

Number of shelters

Number of helpers

FIGURE 2.2 (A) Correlation between the number of small (shaded circles, solid line) and large (open triangles, dashed line)

N. pulcher helpers with the availability of shelters. Data points are slightly offset to provide information on data density, and regression lines are drawn of mean predicted values based on mixed models accounting for between-population variance and the nonindependence of observations within territories. (B) Time spent in shelter for different individual classes of N. pulcher during 7-min observation periods. BrF, breeding female; BrM, breeding male; LH, large helper; SH, small helper. Error bars indicate 95% confidence intervals.

Behaviors

At higher densities, individuals showed fewer aggressive behaviors against predators (mean ± SE = 0.014 ± 0.005, P = 0.008; Fig. 2.4A), suggesting group members benefit from having nearby neighbors. In contrast, groups with more small helpers showed increased per capita predator defense rates (mean ± SE = 0.116 ± 0.051, P = 0.026), whereas there was no effect of the number of large group members on per capita defense (mean ± SE = 0.174 ± 0.127, P = 0.171), even though large helpers attack predators frequently (Fig. 2.4B). Shelter maintenance behavior increased in populations with higher average sand cover (mean ± SE = 0.071 ± 0.020, P < 0.001), and this effect was similar for all individuals (sand cover x individ-ual class: df = 3, χ2 = 3.479, P = 0.323). Individindivid-uals lowered their shelter maintenance behav-ior with increasing predation risk, and small helpers decreased shelter maintenance more strongly than large helpers (slope small helpers vs. large helpers: mean ± SE = 0.301± 0.114, P = 0.008). Predation risk was positively correlated with the time spent in shelters (mean ± SE = 3.384 ± 1.689, P = 0.046), and this effect did not diverge between individuals of different sizes (predation risk x individual class: df = 4, χ2 = 6.040, P = 0.196). However, small helpers in general spent significantly more time hiding in shelters than did other group members (mean ± SE = 116.791 ± 8.839, P < 0.001; Fig. 2.2B and Table S2.2) and also showed the greatest effort in the maintenance of these shelters (Table S2.3).

0 50 100 150 200 0 5 10 15 20

Nearest neighbor distance (cm)

Number of small helpers

Predation risk high

low

FIGURE 2.3 Relationship between the number of small helpers in N. pulcher groups and the distance to the nearest neighboring

group. The original data points are slightly offset to provide information on data density. Conditional regression lines are plotted for high (>median) and low (<median) predation risk based on mixed model mean predicted values accounting for the noninde-pendence of measurements within the same territories and colonies.

0 50 100 150 200 Nearest neighbor distance (cm) 0.0 0.2 0.4 0.6 0.8 1.0 De fense probability < 0.01 0.07 BrM BrF LH SH 0.00 0.10 0.20 De fense probability

A

A

B

FIGURE 2.4 Per capita probability of predator defense of N. pulcher in relation to nearest neighbor distance (A), and class of

group members (B). BrF, breeding female; BrM, breeding male; LH, large helper; SH, small helper. The solid and dashed lines in A indicate the predicted values and boot- strapped 95% confidence intervals, respectively. In B, means and SEs are shown. Model parameter estimates for the significance values are given in Table S4.

DISCUSSION

Habitat saturation and kin selection have been proposed as the primary explanations for the evolution and maintenance of the social complexity characteristic of cooperative breed-ers. Our results highlight that predation risk plays an important role in shaping the social organization and behavior of a cooperatively breeding fish, where habitat saturation and kin-selected benefits are arguably of negligible importance. Furthermore, the influence of predation risk apparently interacts with other important ecological factors. Consistent with our predictions, the social organization relates to the substantial variation between populations in predation risk and habitat characteristics. Predation risk poses a threat es-pecially for small group members, which seem to benefit from enhanced access to shelters and from having close neighbors (Figs. 2.2A, 2.3, and 2.4A). Given that the number of small helpers within groups does not correlate with the number of large helpers, this finding sug-gests that small helpers may benefit more from enhanced security by the presence of close neighbors than from protection by large helpers in their own group (Jungwirth & Taborsky 2015). This pattern is consistent with our behavioral data: per capita predator defense in-creases with nearest neighbor distance, but not with the number of large group members (Fig. 2.4A and Table S2.4). This finding suggests that either predator dilution or benefits of shared defense are the primary contributors to the increased numbers of small helpers. A recent study on N. pulcher indeed revealed that individuals show reduced antipredator defense in response to the presence of close neighbors (Jungwirth et al. 2015a). Addition-ally, experimental data showed that a greater number of large group members increases

survival in the group, except for small helpers (Heg et al. 2004a). Another positive effect of close neighbors might be improved predator detection, enabling small helpers to take refuge from incoming predators in time. Increased predator vigilance is a major benefit of living in dense aggregations and colonies, especially when predators cannot be fended off by mobbing (Krause & Ruxton 2002; Caro 2005). The importance of access to shelters for small helpers is illustrated further by the substantial time and energy expenditure that small helpers invest in creating and maintaining these shelters (Table S2.3), and by the pos-itive relationship between shelter availability and the number of small helpers (Fig. 2.2A). Shelter maintenance has been shown to be the most energetically costly behavior in N. pul-cher, raising standard metabolic rate sixfold (Grantner & Taborsky 1998), which strongly af-fects the helpers’ behavioral energy budget (Taborsky & Grantner 1998). In addition, a field experiment revealed that group size depends on the number of shelters in the territory (Balshine et al. 2001).

Delayed dispersal is regarded as an important first step in the evolution of cooperative breeding, and advanced sociality (Hochberg, Rankin & Taborsky 2008; but see Cockburn 2013; Riehl 2013 for alternative pathways to group formation), and our results suggest that predation can be an important ecological factor selecting for this trait. Predation risk can affect individuals both during and after dispersal, as it also influences the reproductive po-tential of individuals that have obtained dominance in a new group. Subordinate N. pul-cher have been shown to delay dispersal when the risk of predation is increased (Heg et al. 2004a), and group members prefer to disperse to territories in the center of a colony (Heg et al. 2008), apparently to reduce the risk of predation by improved antipredator defense and vigilance. This may both increase survival of small helpers (Fig. 2.3) and decrease workload of group members in general, because of the combined antipredator defense of neighbors (Jungwirth et al. 2015a). One of the most pervasive results of this study is the obvious impor-tance of access to shelters, especially for the survival probability of small individuals. The ability of groups to monopolize and provide access to shelters for small group members seems to be a crucial determinant of their survival and hence, of the reproductive success of breeders.

Our data show that per capita defense rates increase with the number of small helpers in the territory. Three alternative hypotheses can explain this observation. (i) Group members may be more aggressive toward potential predators when there are more juveniles present that are in need of protection. Active defense of helpers in need of protection by dominant group members has been shown experimentally in this system (Taborsky 1984). (ii) Preda-tors may preferentially target territories with a large number of small helpers, and hence they need to be repelled more often. The main predators of this species occur at higher

densities inside of N. pulcher colonies than in adjacent areas (Heg et al. 2008), and it is con-ceivable that within colonies these predators focus on small group members because they are easier prey. Size-dependent choice of prey by piscivore predators has been observed in other species, and optimal foraging theory explains this as resulting from selection on the highest yield per time spent foraging (Holmes & McCormick 2010). In addition, selection of small prey may involve a lower injury risk to predators. (iii) A large number of small helpers present in a group may increase conspicuousness to predators. The relationship between group size, conspicuousness, and predation risk has been well documented in numerous species (e.g. Botham et al. 2005). In accordance with this hypothesis, small subordinates in our study show reduced maintenance behaviors when faced with high predation risk (Table S2.3), which likely reduces conspicuousness. Similarly, in Perisoreus jays (Perisoreus spp.), subordinates seem to show helping behavior primarily under low risk of nest preda-tion, which might reflect the necessity to conceal the nest location if predators of young are abundant (Jing et al. 2009). We should point out that these three hypotheses are not mutually exclusive. They may in fact jointly explain the observed correlation between the number of small group members and individual defense effort.

In addition to these apparent direct effects of predators on group structure and behavior, between populations the number of small helpers per group increased significantly with sand cover. Two mutually nonexclusive explanations could account for this pattern. First, shelter maintenance by digging is the most energetically costly behavior in this species (Ta-borsky & Grantner 1998), which is likely to be partly responsible for the stunted growth of helpers (Taborsky 1984; Taborsky & Grantner 1998). This might result in group compositions being skewed toward small individuals, because investment in maintenance declines with increasing body size (Table S2.3). Second, sand allows for ecological niche construction by digging out shelters, and thereby manipulating the environment (Kylafis & Loreau 2008). Although rocky habitat may provide shelters that are more easily accessible, requiring a smaller initial investment and potentially lower maintenance costs, their number and size cannot be modified. Hence, group composition in N. pulcher may partly depend on the en-vironmental potential for niche construction, which might be an important evolutionary driver of social organization both in N. pulcher and in general.

The effects of predation risk on the transition to complex social organization have been underexplored. Our study demonstrates significant effects and interactions between pre-dation risk and other ecological factors affecting survival, social structure, and behavior of a cooperatively breeding vertebrate in the wild. An important strength of this study is that most of our studied populations were in close vicinity to each other, therefore geographical distance and correlated patterns of gene flow cannot explain the results. In fact, the two

populations that shared the smallest between-population distance showed the highest eco-logical differentiation in almost every aspect. The significance of the results of this study ex-tends far beyond the evolutionary ecology of cooperative breeding. Predation risk has been invoked as a prime ecological driver in the evolution of group living of primates and early hominids, mostly by relating predation risk to group size (Dunbar & Hill 1998; van Schaik & Hörstermann 2014). However, our study shows that predation may affect group compo-sition and behavior more strongly than group size, especially when interactions between predation risk and other ecological factors are considered. Hence, this study highlights the importance of predation risk as a major factor, selecting not only for the formation of groups but for complex social organization.

Acknowledgements

We thank the Department of Fisheries, Ministry of Agriculture and Livestock of Zambia, for the permission to conduct this work; Harris Phiri, Danny Sininza, and the team of the Department of Fisheries at Mpulungu for logistical help; Jan Komdeur and two anonymous referees for comments on the manuscript; and Celestine Mwewa and the staff at the Tang-anyika Science Lodge for their hospitality. This work was supported by Swiss National Sci-ence Foundation Projects 310030B_138660 and 31003A_156152 (to M.T.) and 31003A_144191 (to J.G.F.).

SUPPLEMENTARY INFORMATION

We analyzed between-population effects on the total number of subordinates and the number of large and small subordinates by fitting these as separate response variables in a GLMM assuming a Poisson error distribution. In each model, we fitted: (i) the average number of predators per transect, (ii) the average number of shelters per square meter con-taining a Neolamprologus pulcher territory, and (iii) the average sand cover per square meter containing an N. pulcher territory as predictor variables. We included Territory ID as a ran-dom factor in each model to account for the repeated measures of group compositions for each territory. Furthermore, we fitted the number of small helpers as a response variable in a Poisson GLMM, and added territory density, the number of small helpers, and the num-ber of large helpers as explanatory variables. Nearest neighbor distance and the numnum-ber of large group members were included as predictor variables, and Population ID and Territory ID were included as nested random effects to account for population identity and the repeated measures of territories, respectively.

Territory defense behavior was converted to a binary variable (1/0) to indicate whether in-dividuals had or had not performed aggressive behaviors toward predators during their respective 7-min observation period. We fitted this binary response variable in a logistic GLMM with a logit-link function and included individual class (i.e., dominant male or fe-male or subordinate size class), the number of large and small helpers in the territory, near-est neighbor distance, and predation risk as predictor variables. The interaction between nearest neighbor distance and predation risk was also included. For these purposes, we trans- formed predation risk into a two-level factor indicating high (>overall population median) or low (<overall population median) predation risk. We included Territory ID nest-ed with Population ID to account for population differences and repeatnest-ed measures within the same territory, respectively. We investigated whether shelter maintenance behavior in-creased with rising predation risk or higher sand cover, and whether this differed between individual group members, by fitting the total number of shelter maintenance behaviors in a Poisson GLMM. We included interactions both between sand cover and predation risk, and between predation risk and individual class. Territory ID was included as a random effect to account for the nonindependence of observations within territories. We analyzed the time individuals spent hiding in their shelters as a result of predation risk by fitting the time spent in shelters as a response variable, and predation risk, nearest neighbor distance, and individual class as predictors, in a linear mixed model. We included Territory ID as a ran-dom effect and the interaction between predation risk and individual class.