I Ben-Gurion University of the Negev The Jacob Blaustein Institutes for Desert Research The Albert Katz International School for Desert Studies

Dispersal in the Colonial citricola: The Role of Sexual Selection and Sexual Cannibalism

Thesis submitted in partial fulfillment of the requirements for the degree of "Master of Science"

By: Na'ama Berner-Aharon

October 2013 II Ben-Gurion University of the Negev The Jacob Blaustein Institutes for Desert Research The Albert Katz International School for Desert Studies

Dispersal in the Colonial Spider Cyrtophora citricola: The Role of Sexual Selection and Sexual Cannibalism

Thesis submitted in partial fulfillment of the requirements for the degree of "Master of Science"

By Na'ama Berner-Aharon

Under the Supervision of Prof. Yael Lubin

Department of Desert Ecology

Author's Signature …………….……………………… Date 31.10.2013

Approved by the Supervisor… ……… Date 29.10.2013

Approved by the Director of the School …………… Date ………….… III

Dispersal in the Colonial Spider Cyrtophora citricola: The Role of Sexual Selection and Sexual Cannibalism

Na'ama Berner-Aharon

Thesis submitted in partial fulfillment of the requirements for the degree of "Master of Science"

Ben-Gurion University of the Negev The Jacob Blaustein Institutes for Desert Research The Albert Katz International School for Desert Studies

2013

Abstract

In this study, I investigated the role of sexual cannibalism in male mate choice and dispersal in the colonial spider Cyrtophora citricola. In group living organisms, dispersal occurs when the benefits and fitness of an individual remaining in the group are reduced, and the costs of staying increase. Breeding dispersal in search for mates can be the result of increasing competition over resources or mates and inbreeding avoidance that limits mating opportunities. Sexual cannibalism and its high reproductive costs potentially could influence dispersal decisions, since sexual selection is predicted to favor male mate choice and male-biased dispersal when male mating opportunities are limited by high costs. Sexual cannibalism in group living organisms is known so far to occur only in C. citricola. The occurrence of sexual cannibalism in a colonial spider is counter-intuitive. As opposed to other sexually cannibalistic species that experience low mate encounter rate, in colonial species, females should be readily available for males. However, if inbreeding is a risk, females might be expected to be selective and prefer to mate with more than one male and lower the risk of inbreeding. Cannibalism IV may prevent a male from copulating with both , while leaving the female with the possibility of re-mating. This conflict of interests between the male and the female may be solved for the male through male mate choice. Many , among them sexually cannibalistic spiders, are known to use sex pheromones in mate search and mate choice. These pheromones, which are found on the body or on the silk of females, may include information on the receptivity, the quality, the relatedness and the reproductive history of the female. I hypothesized that in C. citricola, sexual cannibalism selects for male choosiness, where males are able to assess a female’s reproductive state and react accordingly in terms of dispersal decisions. In order to test this hypothesis I examined the sexual behavior of C. citricola in relation to its mating behavior, mate choice and dispersal decisions under both laboratory and semi-natural condition. I found that sexual cannibalism is a fixed trait and occurs in all matings. This behavior selects for male mate-choice based on web-borne pheromones. Males are able to reach females via aerial dispersal, which may assist in mate choice, as they are able to arrive to remote but perhaps favored females. Males preferred to stay on webs of virgin females and dispersed from webs of sub-adult or mated females. However, males can mate with a mated female and females can re-mate at least once. These finding imply that although males probably have maximum 50% paternity, this is the maximal reproductive success possible under these conditions. This however reinforces the need for careful mate choice. Further research is required for fully understanding the evolution of sexual cannibalism in a colonial spider. V

Acknowledgments

I wish to thank Yael Lubin, my supervisor, for being a real inspiration. Thank you for opening the wonderful world of spiders to me, and for untangling the web around their fascinating behavior.

I wish to thank Iris Musli and Ishai Hoffman, our technicians, for all the technical help and good advice.

I wish to thank my lab members for all their useful, wise comments: Efrat

Gavish-Regev, Eric Yip, Huda Al Beiruti, Iara Sandomirsky, Itai Opatovsky,

Idan Kahnonitch and Vardit Makover.

I wish to thank my family and friends who encouraged and supported me throughout the way.

And to Shlomi, who believed in me and inspired me to follow my dreams. For the endless love I receive from you and from our little spider lover, Tal Devora VI

Table of Contents

1. General Introduction………………………………………………………...1

1.1 Dispersal…………………………………………………………..…2

1.2 Sex-biased Dispersal………………………………………………....3

1.3 Sexual Conflict and Sexual Cannibalism………………………….....5

1.4 Male Mate Choice …………………………………………………...6

2. General Methods…………………………………………………………..…9

2.1 Study Species………………………………………………………....9

2.2 Collecting and Rearing………………………………………………9

2.2.1 Sampling Sites……………………………………………...9 2.2.2 Maintenance of Spiders…………………………………...10 2.3 Statistical Analysis………………………………………………….11

3. Aerial Dispersal in Cyrtophora citricola……………………………………13

3.1 Introduction…………………………………………………………13

3.2 Methods……………………………………………………………..17

3.2.1 Dispersal Trials………………………………………...…17 3.2.2 Statistical Analysis………………………………………..17

3.3 Results………………………………………………………………18

3.4 Discussion…………………………………………………………..20

4. Sexual Cannibalism and Male Mate Choice in Cyrtophora citricola…….24

4.1 Introduction…………………………………………………………24

4.2 Methods……………………………………………………………..30

4.2.1 Laboratory Experiment…………………………………………...30

4.2.1.1 Preliminary Mating Observations……………...30 VII

4.2.1.2 Female Receptivity Experiment…………………30 4.2.2 Field Experiment………………………………………………….31 4.2.2.1 Net-house Dispersal Experiment………………..31 4.3 Results……………………………………………………………….32

4.3.1 Preliminary Mating Observations………………………...32

4.3.2 Female Receptivity Experiment…………………………...35

4.3.3 Net-house Dispersal Experiment……………………….…36 4.4 Discussion…………………………………………………………...37

5. General Discussion………………………………………………………….41

6. References…………………………………………………………………...44 VIII

List of Tables and Figures

Table 1- Description of male and female behaviors during courtship………...52

Table 2- Summery of the female receptivity experiment……………………...52

Figure 1- Fitness as a function of group size …………………………………..5

Figure 2- Map of collecting sites in southern Israel…………………………...10

Figure 3- The proportion of juveniles, males and females showing

dispersal behaviors……………………………………………………..18

Figure 4- Dispersal as a function of body length……………………………...19

Figure 5- Male tiptoeing in the laboratory apparatus …………………………20

Figure 6- The male approaches the female……………………………………54

Figure 7- Copulation…………………………………………………………..54

Figure 8- Sexual cannibalism……………………………………………….....55

Figure 9 - The proportion of males remaining-on or dispersing-from

webs of females………………………………………………………...57 1

1. General Introduction

Group living is a widespread phenomenon that evolved many times in the kingdom.

The definition of a group includes social structures of related or unrelated individuals, forming various kinds of groups such as herds, harems, schools, colonies and nests, which remain together through time and space by philopatry of juveniles and adults of both or only one of the sexes (Wilson 1975, Lee 1994). Individuals living in a group interact with members of their group more than with other conspecifics. The evolution of group living is through different selective forces, all favoring the formation of a group under certain environmental and ecological conditions.

Individuals in a group may benefit from this way of living by obtaining more or higher quality resources, increasing the chances of finding a mate and increasing the efficiency of defense against predators (Krause & Ruxton 2002). Additionally, relatedness is expected to be high in group living organisms due to philopatry (Hamilton 1964). Thus, benefiting from indirect fitness through the success of kin can also influence the choice to live in groups. Nevertheless, the cost of group living may be high and includes an increase in disease and parasite transmission, competition over resources and mates, kin competition and conspicuousness to predators (Rubenstein 1978, Krause & Ruxton 2002). As a result, natural selection will favor group living if the benefits from the presence of other individuals are greater than the costs (Pitcher et al. 1983); if the costs exceed the benefits, dispersal will be favored. 2

1.1 Dispersal

Dispersal is the movement of individuals or propagules with potential consequences for gene flow across space. It includes both natal dispersal, where individuals leave their birthplace, and breeding dispersal where potential mates move from one reproduction site to another (Ronce 2007). Howard (1960) defines natal dispersal as “the permanent movement an individual makes from its birth site to the place where it reproduces or would have reproduced had it survived and found a mate”. Breeding dispersal, according to

Howard is “the movement of an adult individual from one home range to another between attempts at reproduction, disregarding whether reproduction was successful”. Therefore, since dispersal influences the genetic and spatial structure of the population, its determinants are under strong selection.

Dispersal is comprised of three stages: emigration, transfer and immigration. Each of these stages is risky since the available patches might be scarce and of poor quality, the probability of finding a mate decreases with time and space (Koenig & Pitelka, 1992;

Emlen, 1994), and dispersing individuals might experience increased predation risk (Yoder et al. 2004).

The conditions in which dispersal is favored can vary. Competition over food and space together with kin-competition are usually the cause of juvenile (natal) dispersal, which is a common phenomenon in many organisms. Competition over mates, which causes high intra-sexual competition [referred to as Local Mate Competition (Hamilton

1967)], together with inbreeding avoidance, are the main causes of breeding dispersal, which is often sex-biased. 3

The competition over resources or mates is intensified in group living organisms due to increasing population density in the group. As a result, the pressure on an individual to disperse may be greater than in solitary species. As density increases, the benefits and fitness of an individual remaining in the group are reduced, and the costs of staying increase. As a result, as population size increases, dispersal away from the colony is expected. The point of dispersal is predicted to be when the fitness of an individual in a group is lower than the fitness of being alone (Figure 1).

Point of dispersal Fitness of being alone

Figure 1- Fitness as a function of group size (adapted from Krause & Ruxton 2002)

1.2 Sex-biased Dispersal

The dispersal of only one of the sexes is common in many organisms, and many studies have looked for the factors that determine which sex disperses. Dobson & Jones (1986) proposed three major hypotheses, all related to sexual selection, explaining sex-biased 4

dispersal: (a) inbreeding avoidance, (b) competition for mates, and (c) competition for resources. These hypotheses are not mutually exclusive and are often offered together.

Sometimes, the conditions favoring the dispersal of either sex are similar for a group of species (taxon or clade), and an inherent phylogenetic bias for the dispersal of one of the sexes evolves [for example, male-biased dispersal in most spiders (Foelix 2011 ) and many mammals and female biased dispersal in many birds (Greenwood 1980, Mabry et al. 2013)]

The evolution of dispersal as a mechanism for the avoidance of inbreeding has been suggested by a number of authors (Greenwood 1980, Johnson & Gains 1990). This mechanism may be particularly relevant for group-living species, where potential mates are likely to be close relatives. In the past two decades, both natural and captive populations

(Lacy 1993, Crnokrak & Roff 1999, Woodworth et al. 2002) have provided empirical demonstrations of inbreeding depression. Others, however, have questioned the assumption that inbreeding in natural populations is always deleterious (Smith 1979, Templeton &

Read 1984). The inbreeding avoidance hypothesis for dispersal predicts that either sex can disperse. Therefore, in order to predict which sex disperses, other factors, namely ones that relate to the mating system, must be considered.

The mating system (polyandry, polygyny or monogamy) and the sex ratio influence the intensity of the intrasexual competition and the resulting sex-biased dispersal (Leturque

& Rousset 2004). For example, if the sex ratio is biased towards females, female mate choice will cause high intra-sexual competition between males. This in turn, will result in a polygynous mating system and usually in male biased dispersal. The opposite mating system, polyandry, will evolve if only a few females can defend territories around a limited resource. This will favor the dispersal of unmated females in search for unoccupied 5

territories. Male mate choice, together with male-biased dispersal, is predicted when mating opportunities for males are limited by high reproductive costs (Bonduriansky 2001). Male reproductive costs include searching costs (Bonduriansky 2001) the production of ejaculates (Dewsbury 1982, Simmons 1993, Wedell et al. 2002), nuptial gifts (Wedell

1993, Vahed 1998, Stalhandske 2001), expensive courtship displays (Kotiaho et al. 1998), and the notorious case of injury or death through sexual cannibalism (Elgar 1992).

1.3 Sexual Conflict and Sexual Cannibalism

Sexual cannibalism, in which a female kills and feeds upon her mate during or after copulation, is a rather uncommon phenomenon found mostly in and in some insects (Elgar 1992). It is an extreme result of the sexual conflict between males and females over reproductive success. This conflict evolved due to the distinct difference between the investment of males and females in their gametes. While females have few large gametes, males have many small ones; and while females benefit from carefully choosing their few mates, males usually maximize their reproductive success by mating multiply (Schneider & Lubin 1998). Being cannibalized prevents the male from mating multiply, and restricts him to one or two mating events only. This unusual situation, in which females have control over mating and males experience forced monogamy, seems to be a case in which the male have 'lost' the battle of sexes. However, although this phenomenon causes high reproductive costs for males, it may also select for male mate choice, which may lower the costs of monogamy and may even include benefits for the male (Gaskett et al. 2004). 6

1.4 Male Mate Choice

Mate choice is based on the ability of an individual to sense cues in the environment indicating the quality or quantity of potential mates. These cues are then translated into actions of either acceptance or rejection of the mate (Fisher 1915, Maynard-Smith &

Harper, 2003). Courtship and potential mating follow the acceptance of a mate. Dispersal in search of another mate follows either the rejection of a mate or a successful mating.

The cues used by mates are various and include visual, olfactory, tactile and chemical cues. Sex pheromones are chemical cues secreted or excreted by various insects, spiders and some vertebrates, with a purpose to attract individuals of the opposite sex.

These pheromones may include information on the receptivity, the quality and the reproductive history of an individual and may assist mate search and mate choice

(Maynard-Smith & Harper, 2003). For example, males may differentiate between mated and unmated females using evidence left by previous males (e.g. chemical traces or `mating plugs'), or signals emitted by the females themselves. Males may be selected to reject recently mated females and accept virgin females if first-male sperm precedence predominates (Simmons et al. 1994). However, if there is last male sperm precedence, males may be selected to perform mate-guarding (Parker, 1974) and to reject females likely to mate again before ovipositing (e.g. females with immature eggs).

Many spiders are known to use sex pheromones in mate search and mate choice

(Gaskett 2007). These pheromones are usually found on the body or on the silk of females, and are received by the male via air or contact. Airborne pheromones mainly promote male searching behavior and attract males towards the female. Contact pheromones, received by 7

the chemoreceptors on male pedipalps, stimulate male courtship behavior and provide specific information about the female. Male spiders are known to be choosy and are often most attracted to adult virgin females or to juvenile females prior to their final molt. This preference suggests that the first male to mate with a female has considerable advantages, possibly due to sperm priority patterns, or lower receptivity of mated females (Gaskett

2007).

Several sexually cannibalistic spiders were found to use sex pheromones

(Kasumovic et al. 2007, Gaskett et al. 2004, Segoli et al. 2006, Stoltz et al. 2007). Some of these males were found to differentiate sub-adult females from adult females, and virgin females from mated ones showing a distinct preference for virgin females (Kasumovic et al.

2004, Gaskett et al. 2004, Stoltz_et al. 2007). These results are predicted by the high reproductive costs the male experiences and the obligation to channel all his reproductive effort into one mating.

Although sexual cannibalism has been recorded in at least 30 groups of

(Polis 1981), the few studies of sexual cannibalism have focused on mate choice in laboratory experiments. Even less is known about the dynamics of mate choice and male dispersal under natural conditions. Sexual cannibalism in group living is an extremely rare phenomenon and is known so far to occur in only one colonial spider species, Cyrtophora citricola Forskål 1775 (Araneidae). The first and only mention of this behavior is found in a comprehensive study done by Blanke (1972). This study includes a description of the courtship and the mating, but lacks information on the females' reproductive status and does not use the term "sexual cannibalism" but rather describes the end of the mating by saying that the female "spinnt es ein" e.g. wraps the male. 8

The occurrence of sexual cannibalism in a colonial spider raises interesting questions regarding its evolution in this unique social structure: how and why did sexual cannibalism evolve in a group living species with supposedly low mate searching costs and high mate encounter rate? And, why is aggressiveness absent between females and juveniles but is directed towards males?

In this study, I asked how does sexual cannibalism influence male breeding dispersal in the colonial spider C. citricola. In order to answer this question I examined the sexual behavior of C. citricola in relation to its mating behavior, mate choice and dispersal decisions under both laboratory and semi-natural condition. I hypothesized that sexual cannibalism selects for male choosiness, where males are able to assess a female’s reproductive state and react accordingly in terms of dispersal decisions.

In order to test this hypothesis, I first tested the dispersal propensity of juveniles, adult males and adult females. Then, I examined the mating behavior, recording the frequency of occurrence of sexual cannibalism and the willingness of a female to re-mate.

Last, I combined the sexual behavior and the dispersal abilities of males and tested the ability of males to discriminate between females with different reproductive status and to respond accordingly by remaining or dispersing. In this research, I aimed at studying the unusual occurrence of sexual cannibalism in a group living spider, and exploring the mating behavior and mate choice of males and females C. citricola. 9

2. General Methods

2.1 Study Species

Cyrtophora citricola (Araneidae) is a predominantly group-living colonial spider found worldwide, mainly in tropical and subtropical regions. Its geographical distribution is undergoing a range expansion in the past 15 years towards Central and South America and the Caribbean islands (Blanke 1972; Leborgne et al. 1998, Levi 1997; Alayón 2001, 2003).

In Israel, C. citricola is found in the Jordan and Arava valleys and the western Negev. The spiders can either build their webs interconnected with each other to form a colony of spiders or they can live a solitary life. The spiders build a unique tent-like three dimensional non-sticky web, consisting of a horizontal orb web and silk threads above and below it connecting the web to other webs and to thorny plants such as Acacia trees, cacti, and shrubs. The spider rests in the center (hub) of the horizontal orb-web waiting for prey to strike the web. When an insect strikes the upper barrier threads, it is knocked down to the orb-web where it is restrained until the spider attacks it.

2.2 Collecting and Rearing

2.2.1 Sampling Sites

Cyrtophora citricola spiders were collected from six sites in southern Israel: Beer Sheva

(BS, 31°25′87″N 34°74′62″E) in the northern Negev, Urim-Re’im (UR, 31°34′45″N

34°50′35″E), Daya garbage disposal site (DY, 31°22′44″N 34°49′67″E), Retamim-Revivim

(RR, 31°05′45″N 34°71′67″E ) and Shuva orchard (SO, 31°46′17″N 34°53′20″) in the north-western Negev and from Tzofar (TZ, 30°33′38″N 35°10′53″E) in the Arava valley 10

(Figure 2). Spiders were found on individual webs or in colonies of 10-100 individuals each on Acacia raddiana (Mimosoideae) trees at TZ site, on Cereus repandus (Cactaceae) cacti at BS, UR and DY sites, on dry Bassia indica (Chenopodiaceae) and dry Alhagi graecorum

(Papilionaceae) at RR site and on sp. at SO site.

SO

UR BS DY

RR

TZ

Figure 2- Map of collecting sites in southern Israel: BS, UR, DY, RR, SO and TZ

2.2.2 Maintenance of Spiders

All spiders were brought to the laboratory and kept in constant conditions of 250C and 45% humidity, with 12 hours of light a day. Large females were placed in terrariums while males and juveniles were placed in mesh covered plastic cups. The spiders were fed twice a week according to their size with Drosophila flies or early instar grasshoppers (Locusta sp.). Before each experiment, spider body length (cephalothorax and abdomen) was measured using a digital caliper (±0.02mm accuracy). 11

For the experiments, four groupings by age and sex were recognized: juveniles (less than 2 mm, before sex is distinct), adult males (with distinct, mature pedipalps), sub-adult females (with distinct, pre-adult genitalia) and adult females (with distinct, mature genitalia).

2.3 Statistical Analysis

The data was analyzed using Statistica 11.0 (StatSoft 2012). 12

"Then came a quiet morning. A warm draft of rising air blew softly through the barn cellar. The air smelled of the sweet springtime. The baby spiders felt the warm updraft. One spider climbed to the top of the fence.

The spider stood on its head, pointed its in the air, and let loose a cloud of fine silk. The silk formed a balloon.

"Good-bye!" it said, as it sailed through the door-way. Then another spider crawled on top of the fence, made a balloon, and sailed away. Then another. Then another. The air was soon filled with tiny balloons, each balloon carrying a spider.

"Good-bye!" they called. "We are aeronauts and we are going into the world to make webs for ourselves, wherever the wind takes us high, low.

Near, far. East, west. North, south. We take the breeze, we go as we please."

Charlotte's Web, E.B. White 13

3. Up, Up and Away, In My Beautiful Balloon:

Aerial Dispersal in Cyrtophora citricola

3.1 Introduction

Both juvenile-natal and adult-breeding dispersal occur in spiders. Natal dispersal is common in many spider families (Greenstone et al. 1987), where juveniles after their 2nd-

3rd molt leave their birthplace to look for a new place to build a web or to claim a terrestrial territory. Breeding dispersal, where an adult individual searches for a mate is an essential part of any adult life, and occurs in all spiders, usually in males (but see the case of social spiders in Lubin et al. 2009).

Spiders can disperse both by walking between patches, and by moving aerially.

Spiders do not have flight organs, but they can disperse aerially using silk threads, which facilitates the movement between patches (Szymkowiak et al. 2007).

Silk- based aerial dispersal of spiders occurs in enormous numbers, many times seasonally, and contributes to long distance movement in both web-building and non web- building spiders (Greenstone et al. 1987). There are two types of aerial dispersal in spiders: long-distance ballooning and short-distance bridging, and both can occur in different ages and under different circumstances. A spider’s attempt to disperse begins when it first climbs to the top of any substrate available. Then, it stands on the tips of the tarsi of all 4 pairs of legs (i.e. tiptoeing) and extrudes silk, which is lifted by airstreams. The silk either sticks to a close-by substrate and the spider uses it as a bridge (i.e. bridging), or it is carried up-high, the spider takes off and moves long distances (i.e. ballooning) (Suter 1999, Lubin 14

and Suter 2013). These two different mechanisms are responsible for the gene flow within and between populations and maintain their heterogeneous genetic structure.

Body size and weight are important determinants of the capability to disperse aerially. Both theoretical and empirical findings show that primarily there is a bias towards small size aeronauts, because in order to aerially disperse, large individuals must spend more energy [e.g. emitting large amount of silk (Suter 1999, Schneider et al. 2001)] and overcome unfavorable conditions such as sub-optimal turbulent conditions for large body mass and greater conspicuousness to predators (Suter 1999). Research done by Greenstone et al. (1987) on aerial dispersal in spiders found that almost all ballooners were small and light (weight < 1.0 mg) immature individuals undergoing natal dispersal, except for the

Linyphiidae, where juveniles and adults are both small sized and were found to disperse in similar proportions. Larger, heavier adult spiders of some species were found to balloon long distances (Schneider et al. 2001) using a somewhat different mechanism. It is unclear what proportion of adults undergo aerial dispersal is and what mechanisms they use.

In web building spiders, adult males frequently have smaller body size than adult females, and while females are sedentary, males leave their web and disperse in search for mates (Foelix 2011). The distance between mates and the spatial distribution of females, whether clustered or scattered, affects the nature of the dispersal - whether done by walking very short distances or by bridging or ballooning longer distances.

Group living spiders, by definition, have reduced or limited dispersal, as they need to remain philopatric to create a group. Spiders are not generally known to be social as they are predatory and cannibalistic, yet around 100 species are group living (Lubin and Bilde, 15

2007). Permanent group living in spiders occurs in two fundamentally different forms: cooperative breeding (sociality) and coloniality. Colonial spiders do not cooperate, as opposed to social spiders, but they benefit from this way of living by sharing silk and capturing larger prey [the ricochet effect (Uetz 1992)]. While social spiders are highly inbred as a result of philopatry and post-mating dispersal, colonial spiders are not necessarily closely related as they experience both natal and pre-mating dispersal (Blanke

1972, Leborgne et al.. 1998, Rao & Lubin 2010, Bilde & Lubin 2011).

Clustered populations such as group living spiders form permanent colonies consisting of females and males in different developmental stages. These colonies may exhibit various mating opportunities for males. Males maturing in a colony may remain to mate within it, and may disperse either short or long distances in search for foreign colonies. As colonies are often established by a cohort of related young (Lubin 1980), mating within the natal colony harbors a risk of inbreeding and dispersal away from the colony should be favored. In addition, colony size and the intensity of the intra-specific competition between males over females may also influence dispersal decisions. The genetic structure of populations of group living spiders was not well studied (Johannesen et al. 2012) and it is unclear to what extent spiders within and between colonies are related to each other and how exactly a group is formed. It is assumed that philopatry of some individuals combined with natal dispersal of others maintains on the one hand existing colonies and on the other hand creates new ones. As a result, it remains unknown to what distance a male must disperse in order to avoid inbreeding, and whether it uses aerial dispersal as its main mechanism to reach females. 16

The colonial spider, Cyrtophora citricola (Araneidae), lives in Israel in arid and hyper-arid habitats. Juveniles are known to disperse and establish new colonies (Blanke

1972). Juvenile dispersal was found to be influenced by the presence of prey, by both attracting juveniles to webs with food remains and by reducing dispersal in prey rich environments (Mestre and Lubin 2011). These two factors contribute to the establishment and maintenance of colonies around prey-rich resources such as water sources and agricultural areas as the ones found in the Negev Desert and the Arava Valley (N. Berner-

Aharon, personal observations). Using genetic analysis it has been found by Johannesen et al. (2012) that alongside with natal dispersal there are some evidence of male-biased breeding dispersal, while females are philopatric. Still, the mechanisms of both natal and breeding dispersal in colonial spiders in general and in C.citricola in particular, where never studied.

I hypothesize that C. citricola spiders have a low dispersal tendency, yet juveniles will have a higher tendency than adults of either sex. The hypothesis is based on the assumption that costs of dispersal are lower for juveniles than for adults. In addition, the hypothesis is based on the assumption that dispersal in web-building spiders is silk-based dispersal (i.e. ballooning or bridging) and not by means of walking.

In order to study to what extent C.citricola spiders use aerial dispersal, I examined both juveniles and adults using a testing apparatus, imitating natural conditions favoring aerial dispersal. I predicted that both juveniles and males will display typical dispersal behaviors such as tiptoe behavior and the release of silk threads, while females will not demonstrate such behaviors. 17

3.2 Methods

3.2.1 Dispersal Trials

Dispersal trials were done between March-July 2012 within 48 hours after spiders were collected from BS and UR colonies. Juveniles, adult males and adult females were used in these trials. Spiders were placed on a platform with two vertically positioned 24 cm high and 1 cm diameter wide wooden sticks. The platform was placed inside a shallow 5 cm high plastic box. A ventilator was placed 1m from the platform and was angled 450 towards the platform. The wind speed varied between 0.5-1.1 m/s. Each spider was placed individually on the platform and observed for 5 minutes during which its behavior was recorded. At the end of the 5 minutes, the spider was removed from the platform. Behaviors such as climbing on one of the wooden sticks, silk release, tiptoeing and bridging were recorded. Ballooning was not observed in the trials. We interpreted the demonstration of tiptoeing while releasing silk and of bridging as a tendency to disperse (Bonte 2012)

(Figure 5).

3.2.2 Statistical Analysis

The tendency to disperse was tested with the χ2 statistic on the full contingency table and against a 50:50 distribution for juveniles, males and females separately. The tendency to disperse as a function of body length was tested using a logistic regression. 18

3.3 Results

Overall there was a significant tendency to disperse (χ2=16.098, p<0.001). Juveniles did not show a significant tendency to disperse (n=28/45, χ2=2.688, p=0.1). Males did not show a significant tendency to disperse (n=15/24, χ2=1.5, p=0.22). Females showed a significant tendency not to disperse (n=4/22, χ2=8.9, p=0.002). Juveniles had a greater dispersal tendency than females, (χ2=15.227, p<0.001). Males and juveniles did not differ in their dispersal tendency (χ2=0.287, p=0.591). Males tended to disperse more than females

(χ2=9.299, p=0.002) (Figure 3).

***

n.s ***

Figure 3- The proportion of juveniles, males and females showing dispersal behaviors

Body length negatively affected the overall tendency to disperse (all data combined, y=1.133-0.305*body length, p<0.001) (figure 4). The body size of individuals within each group, however, did not explain the probability of dispersal (juveniles: y=-6.782+7.124* 19

body length, p=0.087; males: y=-5.842+2.582* body length, p=0.069 ;females: y=-

10.455+1.103* body length, p=0.321).

Figure 4- Dispersal (0= no dispersal behavior, 1=dispersal behavior observed) as a function of body length (mm) of juveniles (empty circles), males (dashed circles) and females (filled circles) 20

Figure 5- Male tiptoeing in the laboratory apparatus

3.4 Discussion

Both male and juvenile C. citricola were found to demonstrate dispersal behaviors such as tiptoeing and bridging in more than 50% of the trials. The vast majority of females on the other hand, did not demonstrate these behaviors at all.

These results demonstrate that aerial dispersal takes place in C. citricola juveniles as found in other spiders (Greenstone et al. 1987), but a percent of non-dispersing individuals exists. The presence of dispersal in most juveniles and absence in some may be explained by the dynamics of group living. Juveniles may remain in the colony after their

3rd molt and maintain it, since group living can be beneficial. When the colony size reaches a threshold, and the benefits of group living decrease, dispersal occurs, and juveniles leave in order to establish a new colony. Juvenile aerial dispersal is present, and is responsible for the foundation of new colonies as found in another two colonial spiders Cyrtophora moluccensis and Philoponella republicana (Lubin 1980). However, this dispersal is to a 21

lower extent compared to solitary species as a result of juvenile philopatry in colonial spiders, which is responsible for colony maintenance. If all juveniles disperse, the colony dies out and a new colony is founded in a new site.

The results also suggest that aerial dispersal takes place in males to a moderate extent, occurring in some individuals and absent in others. Several factors might influence male dispersal, such as relatedness, population density and availability of females. If the relatedness within a colony is high, then male dispersal may occur in high numbers in order to avoid inbreeding. However, if the relatedness is not high, then male dispersal occurs, but to lower extent. In addition, if the competition over mates is intense, males will disperse in greater numbers than if the competition is relaxed. The results may imply a moderate level of relatedness in some colonies and higher levels in others. These results may also be explained by variation between colonies in the competition between males. Some colonies might exhibit higher competition levels due to limited numbers of receptive females per male, either because they are still sub-adults or because they have already mated. A combination of inbreeding avoidance and competition could result in variation in male behavior, where some males demonstrate dispersal behavior and some do not. Since I did not record the colony size, density and sex ratio and I did not test for relatedness within the original colonies, I do not know the levels of competition or inbreeding these individuals experienced. Nevertheless, Johannesen et al. (2012) found that the genetic differentiation among large colonies in natural populations was heterogeneous, with colonies being either little or highly differentiated. These findings support the results of this research regarding the inbreeding avoidance hypothesis, suggesting that inbreeding avoidance may play a role in male dispersal. 22

In C. citricola, adult female dispersal via bridging generally does not occur nor does ballooning, in agreement with other observations of many web-building female spiders in general (Foelix 2011) and group-living female spiders in particular. Both are absent in this species probably because of body size limitation and as a result of high cost of leaving a colony vs. high benefits of philopatry in colonial spiders.

Body length influenced the overall tendency to disperse. Smaller individuals were more likely to demonstrate dispersal behavior. These results are in agreement with other spiders (Greenstone et al.1987). Body length did not influence the tendency to disperse within each group separately although there was a non-significant tendency for both males and juveniles.

In conclusion, I found that juvenile C. citricola have a high tendency to disperse, males have a moderate tendency to disperse, and females do not have a tendency to disperse. These results suggest that partial juvenile dispersal and female philopatry can explain the dynamics of C. citricola colonies. 23

Mr. Blackwall has sometimes seen two or more males on the same web with a single female, but their courtship is too tedious and prolonged an affair to be easily observed. The male is extremely cautious in making his advances, as the female carries her coyness to a dangerous pitch. De Geer saw a male that “in the midst of his preparatory caresses was seized by her in a web and then devoured”, a sight as he adds "filled him with horror and indignation”.

The Descent of Man, and Selection in Relation to Sex

C. Darwin (1871) 24

4. Here She Comes, She's A Man Eater:

Sexual Cannibalism and Male Mate Choice in Cyrtophora citricola

4.1 Introduction

Sexual cannibalism is an extreme result of the sexual conflict between males and females over reproductive success (Parker 1979). This phenomenon occurs in certain insect species, some scorpions but mostly in spiders. The frequent occurrence of sexual cannibalism in spiders, and especially in web-building species, suggests it has an important role in their courtship and mating behavior (Elgar 1992). According to the adaptive male sacrifice hypothesis, sexual cannibalism evolves when evolutionary and ecological factors create conditions that enable the female to prey on the male, while the male has relatively low costs, and possibly even benefits from the female’s actions (Buskirk 1984). Alternatively, sexual cannibalism can be maladaptive for males, and occur simply because females are predatory and males are unable to escape them. In this case, male traits that enable the female's actions, such as small body size, were selected in other contexts, and now prevent the males from having an adaptive mating behavior (Gould 1984).

Both hypotheses include a basic requirement of a female's ability to control the mating and prey on the male. Sexual size dimorphism (SSD) where females are larger than males may fulfill this requirement, as it gives females a physical advantage over males.

Although previous comparative study failed to correlate SSD with sexual cannibalism

(Elgar 1992), a recent study by Wilder & Rypstra (2008) found that SSD predicts the frequency of sexual cannibalism within and among species of spiders.

Sexual size dimorphism may have evolved through selection for male dwarfism or female gigantism. Vollrath and Parker (1992) proposed a selection mechanism for male 25

dwarfism in spiders based on differences in male and female adult life style. While females are usually sedentary, adult males search for mating opportunities. These males might experience high mortality rates that result in a female-biased sex ratio. This in turn reduces male-male competition and decreases the selection for large adult male size. Not only is there reduced selection for larger body, but selection favors smaller males that have better dispersal abilities, lower energetic costs and lower predation risk. There is some debate over this hypothesis because it is not fully supported by phylogenetic analysis or empirical studies (Coddington et al. 1997, Prenter et al. 1998, Walker & Rypstra 2003, but see De

Mas et al. 2009). Instead, the hypothesis of female gigantism due to fecundity selection was offered as an alternative mechanism for sexual size dimorphism. Whether caused by male dwarfism or female gigantism, the result of the SSD in predatory organisms such as spiders, is large females that are able to cannibalize their small mates.

SSD is not the only driving force behind sexual cannibalism, as not all sexually dimorphic spiders are sexually cannibalistic. Other factors such as low mate encounter rate, risky mate search or low residual reproductive value after mating (i.e. as a consequence of genital mutilation and functional sterility) were found to correlate with sexual cannibalism.

Risky mate search and low female encounter rate occurs in several sexually cannibalistic spiders. In Nephila plumipes (Araneidae) less that 40% of males survive mate search

(Kasumovic et al. 2007). In Latrodectus hasselti (Theridiidae) only 20% of the male survive mate search and succeed to mate (Andrade 2003). A similar low encounter rate exists in Latrodectus pallidus (Theridiidae) where less than 20% of males reach a female

(Segoli et al. 2006). Lowered future reproductive value is often found in spiders and insects that exhibit male sacrifice behavior. These males have genitalia that break off or are 26

mutilated after a single copulation (Miller 2007) and consequently the male cannot mate multiply. These factors lower the male's chances of a successful mating, and may select for male sacrifice, if the adaptive value from being cannibalized is higher than the adaptive value of escaping the female after mating and continuing mate search.

The timing of the sexual cannibalism, whether done before, during or after sperm transfer is crucial in understanding the evolutionary pathways leading to the female's and the male's behaviors. Cannibalizing the male before sperm transfer can be mere predation for the female, but it can also be a form of female choice where the female aggressively rejects the male (Prenter et al. 2006, Elgar 1992). Cannibalizing the male during sperm transfer can also be a form of female choice, where the female controls the copulation duration, and hence the amount of sperm transferred by the male.

From the male’s perspective, sexual cannibalism before sperm transfer should always be avoided as it lacks any adaptive value. However, sexual cannibalism after sperm transfer can be evolutionary advantageous for males, if selection acts on mechanisms that maximize the reproductive success such as paternal investment and male mate choice

(Bonduriansky 2001; Andrade and Kasumovic 2005).

Mechanisms of paternal investment or ensured paternity may increase the number or quality of the offspring compared to offspring of non-cannibalized males (Buslirk et al.

1984). Such mechanisms, some very complex, are found in several post-mating sexually cannibalistic spiders. For example, Latrodectus revivensis (Theridiidae) males can break the tip of their embolus inside the female genitalia before dying, creating a physical barrier against sperm of subsequent males (i.e. mating plug) (Berendonck & Greven 2000). 27

Another case of self-mutilation occurs in male Nephilengys malabarensis (Nephilidae) who detach the whole during mating. The detached genitals can continue pumping sperm into the female while she is consuming the male (Li et al. 2012). In Latrodectus hasselti, males shift the abdomen within reach of the of their mates during copulation and self-sacrifice themselves. In this species, cannibalized males (65% of matings) increase their paternity relative to males that are not cannibalized by having longer copulation durations and by reducing the female's receptivity for re-mating (Andrade

1996). Another mechanism that may increase the male benefits is female monogamy.

Female monogamy can be either natural via female choice or forced via male intervention.

Males can lower the female chances of re-mating using mating plugs as described above.

Males can also remove the female's web silk that includes male attracting pheromones, and by this reduce her advertisement for other males (Ross & Smith, 1979, Breene & Sweet,

1985, Anava & Lubin, 1993).

The high reproductive costs males experience because of sexual cannibalism risks also selects for male mate choice. Males that are able to discriminate between females of different reproductive value will maximize their reproductive success and have higher fitness. The few studies done on mate discrimination in sexually cannibalistic spiders, support the prediction that males will favor virgin females over mated females. Male

Latrodectus hasselti can discriminate female maturity and mating status based on web- borne chemicals. The study showed that male activity on extracts from webs of virgin females was higher than on extracts of webs of juveniles, mated females or controls (Stoltz el al 2007). In the spider Argiope keyserlingi (Araneidae) males strongly preferred 28

penultimate and virgin females to mated females based on both airborne and web borne pheromones (Gaskett et al. 2004).

In most cases, sexual cannibalism is not an obligatory part of copulation, and some males are not cannibalized. However, if sexual cannibalism is a fixed trait and all males are cannibalized post-mating, an even stronger selection is predicted to act on both paternity insuring mechanisms and male mate choice.

Cyrtophora citricola is a colonial spider with pronounced sexual size dimorphism, where females are three times larger than males. Males mature earlier than females and reach adulthood within five molts whereas females require at least nine. Males are sedentary until their maturation molt, after which they leave their web in search for females

(Blanke 1972). Mature females were found to attract males by olfactory cues, but it is not clear whether the scent is emitted onto the web or on the female's body, and the reproductive history of the females in this study was not known. The courtship and mating are described by Blanke (1972). The mating ends when the female "wraps the male". This description is not quite clear, and the term "sexual cannibalism" is not used. In addition, the frequency of this behavior is not mentioned.

The occurrence of sexual cannibalism in a colonial spider is counter-intuitive. As opposed to other sexually cannibalistic species that experience low mate encounter rate, in colonial species, females should be readily available for males. Therefore, males are expected to be less choosy and less likely to allow themselves to be cannibalized. However, if inbreeding is a risk, females might be expected to be selective and prefer to mate with more than one male and lower the risk of inbreeding. Cannibalism may prevent a male from copulating with both pedipalps, leaving the female with the possibility of re-mating. This 29

conflict of interests between the male and the female may be solved for the male through male mate choice or through paternity insuring mechanisms.

The early maturation of males may lead to increased competition over mates in the beginning of the season, when mature females are scarce. If cannibalism occurs with high frequency, it might be a male's only chance of mating. Consequently, the male should choose its mating options carefully and try to maximize its reproductive success. If a male mates with a non-virgin female, he will fertilize only a proportion of her eggs and consequently will have lower paternity than if he mates with a virgin female. Therefore, males should avoid mated females while virgin females should be favored.

In this part of my study, I examined the mating behavior of both males and females and the likelihood of female re-mating in the laboratory. I also examined male mate choice under semi-natural conditions.

I hypothesized that C. citricola males are able to assess the female’s reproductive state based on web-borne pheromone cues, and react accordingly in terms of dispersal decisions. I predicted that males would show preference by remaining or dispersing from webs of females in different reproductive status. Specifically, I predicted that males would prefer to leave the webs of mated females or sub-adult females but would stay on the webs of virgin adult females. In addition, I suggest that male self-sacrifice should be favored if their paternity is secured by reducing the chance of a female mating with another male.

Therefore, I predict males to either plug the female’s genitalia by breaking the tip of their embolus or by reducing the female's ability to re-mate using a different mechanism. 30

4.2 Methods

4.2.1 Laboratory Experiment

4.2.1.1 Preliminary Mating Observations

The mating began when an adult male was carefully added into a terrarium with a virgin adult female. He was placed at the edge of her web to prevent female’s aggressiveness towards the male. In addition, females were fed a day prior to mating to ensure they were satiated. Both male and female behaviors were recorded (leg tapping, web shaking, silk pulling, etc.). In addition, mating success, time to copulation, copulation length and male survival were recorded. The mating trials were done at mid-day and each couple was observed for two hours. If they failed to mate within two hours, the male was removed. If the mating succeeded, the female was left to produce an egg sac. Some mated females were taken to be examined for post-mating condition and if broken pedipalp tips had been deposited inside the insemination duct or in the spermatheca, using external and internal examination under dissecting microscope. Female were dissected and placed in KOH solution for 10 minutes. Then, they were placed in 75% ethanol for examination under the dissecting microscope. The mating trails took place between April-

June 2011, using spiders from TZ, BS, UR, RR and SO populations. In each mating trial, I used males and females from the same population.

4.2.1.2 Female Receptivity Experiment

Three groups of females (n=20) from the SO population were mated in the laboratory under the same conditions as the previous experiment. One group of females was mated again three days after the first mating and the second group was mated again ten days after the 31

first mating. The third group was not re-mated as a control for natural mating propensity.

Mating success, time to copulation, copulation length, male survival and male and female behaviors were recorded. The experiment took place between August and September 2013.

4.2.2 Field Experiment

4.2.2.1 Net-House Dispersal Experiment

In the laboratory, large sub adult, virgin adults and mated adult females were placed in terrariums containing Acacia tree branches. The females were given ten days to construct their typical three dimensional non-sticky webs. By the end of the ten days, the terrariums were taken to an outdoor net house (10mX40m) where 1-1.5m high potted Acacia trees were placed in two rows. The rows of trees were East-West and trees were three meters apart. Each cluster of branches holding a web was carefully taken out of the terrarium, and placed on the south-facing side of a tree, selected haphazardly. The female was removed from the web and replaced by a male. The trees with the webs and males were visited over the next four days to check whether males stay or leave the female’s web. The experiment took place between September and December 2012. Twenty-three mated adult females, 27 virgin adult females, 23 sub-adult females and 73 males from DY and RR sites were used in the experiment. Males were always placed on webs of females from their own site of origin. 32

4.3 Results

4.3.1 Preliminary Mating Observations

I conducted 38 mating trails, 20 of which ended with a successful mating and 18 failed. All successful matings ended with the female cannibalizing the male, 18 after the first pedipalp insertion, and two after the second pedipalp insertion. A successful mating was one in which at least one insertion took place and a fertile egg sac had hatched. The copulation took place between 20-90 minutes after the male was introduced to the female.

During my observations, I could identify typical courtship and mating behaviors (Table 1):

Table 1- Description of male and female behaviors during courtship

Behavior Description

Laying silk thread At the beginning of the courtship, the male produces a thread

starting from the irregular part of the female's web, and

strengthens it by walking up and down.

Plucking the thread & The male plucks his thread and the web with his third legs.

web The female plucks the web with her third legs.

Cleaning the The male cleans his pedipalps with his second and third legs

pedipalps

Shaking the web The female shakes the orb web, sometimes causing the male

to drop on a drag-line.

Female approaching The female walks beneath the orb web towards the male.

the male Sometimes the male drops on a dragline. 33

Male resumes A male that drops can return to the web and resume courting courtship

Female mating The female approaches the male and hangs by the fourth posture legs, while plucking the web with the third legs.

Male approach The male approaches the hanging female (Figure 6)

Male approach- The male approaches the hanging female but withdraws withdrawal before inserting the pedipalps. He can cease courting or

continue until the female returns to the mating posture.

Mating with one The male inserts one pedipalp (Figure 7) and after pedipalp and sexual approximately 5-10 seconds, the female inserts her cannibalism chelicerae into the abdomen of the male. After a few more

seconds, the female pulls the male and his pedipalp is

detached from the epyginum (Figure 8). The female then

wraps the male with silk and returns to her hub with the male

either in her chelicerae or held on a thread by the fourth leg.

Mating with two The male inserts the first pedipalp and after a few seconds pedipalps withdraws from the female. He draws the pedipalps through

the mouthparts and then returns to the hanging female. Then

the mating continues with the second pedipalp as described

above.

Male or female lack of The male or the female do not respond to the presence of the interest other, and freeze in their places for more than two hours. 34

The epigynes of ten mated females were examined under the microscope. The females' genitalia, both external and internal did not have any sign of a male mating plug. It was not possible to discern on the cannibalized males if they had lost any part of the embolus during mating because of the damage the females had made to their bodies.

Figure 6- The male approaches the female

Figure 7- The male inserts his pedipalp into the female epygine 35

Figure 8- The female inserts her chelicerae into the abdomen of the male while the pedipalp is still attached. Then the female pulls the male towards her mouthparts and the pedipalp is detached

4.3.2 Female Receptivity Experiment

The three groups did not differ in the females' propensity to mate on day 0 (χ2=0.022 p=0.988) where an average of 10.3  1.52 females mated from each group (n=20). There was no difference between the propensity of females to mate in day 0 and to re-mate three days later (χ2=0.01 p=0.917). Similarly, the same proportion of females mated in day 0 and ten days later (χ2=0.006 p=0.937). There was no difference between the propensity of females to mate three days after the first mating and ten days after the first mating

(χ2=0.038 p=0.843) (Table 2). 36

Table 2- Summery of the female receptivity experiment, presenting the number of successful matings within each group

Mating success Successful Successful re-mating, Successful re-mating,

mating, day 0 day 3 day 10 Group

Control- 9 ------mate once (n=20)

Mate twice-

day 0 & day 3 12 5 ---

(n=20)

Mate twice-

day 0 & day 10 10 --- 6

(n=20)

4.3.3 Net-house Dispersal Experiment

The reproductive status of the females influenced the dispersal decision of males (χ2=8.382 p= 0.015). Males remained on webs of virgin females and dispersed from webs of mated females and sub-adult females (χ2=7.935 p=0.004, χ2=3.687 p=0.05). Males reacted similarly to webs of mated females and sub-adult females and dispersed from both in a similar proportion (χ2=0.839 p=0.359) (Figure 9). The location of the tree, whether in the northern row or the southern row did not influence the males' dispersal decision (χ2= 0.23 p=0.891). 37

n.s

* ***

Figure 9 - The proportion of males remaining on (dark bars) or dispersing from (grey bars) webs of sub adult, virgin and mated females

4.4 Discussion

Sexual cannibalism is a fixed trait in C. citricola and all males are cannibalized post- mating, usually after inserting one pedipalp. The mating is extremely rapid and lasts only a few seconds. Both the female and the male do not mate readily, and a successful mating occurs only around 50% of the times. In both the preliminary observations and the re- mating experiment, I could observe the male or the female lack interest in their mate, as they did not respond to the presence of the other on the web. It was difficult to distinguish 38

whether it was the male or the female who rejected the other, probably because of a cryptic mate choice, based on cues I could not detect.

There are not many examples of such extreme and obligatory sexual cannibalism as found in this species. In C. cicatrosa, a solitary species of the genus Cyrtophora, sexual cannibalism occurs frequently and possibly always (Blanke 1975). A similar case to the

100% cannibalism that was observed in C. citricola is found in Argiope aemula where 90% of the males are cannibalized after the insertion of the second pedipalp (Sasaki & Iwahashi

1995). Elgar (1992) suggested that in these species a successful copulation is enabled only by sexual cannibalism. It is possible that this is also the case in C. citricola.

Due to the pronounced sexual size dimorphism found in C. citricola, the male has a disadvantage in the means of control over copulation. The male is embraced by the female while copulating, and approaches her chelicerae. In contrast to the somersault male of

Araneus pallidus and Latrodectus hasselti undergo (Elgar 1992), here the mating posture is risky to begin with, as the male approaches the hanging female when his abdomen is almost touching her chelicerae. The small size of the male, however, enhances his dispersal abilities, as both my observations (discussed in the previous chapter) and Blanke's observations (Blanke 1972), confirm the male's ability to disperse aerially via ballooning and bridging. Additionally, the smaller body size of males means a shorter development.

The faster the development time, the greater the probability of reaching maturity. It seems likely that the costs and benefits of the SSD balance each other evolutionarily.

No mating plugs were found in the female epyginum. A comparison to non- cannibalized males in terms of paternal investment and quality of offspring is impossible 39

since all males are cannibalized. Therefore, an obvious benefit for the male from sexual cannibalism remains unknown. However, males do show preference to virgin females based on air-borne or silk-borne pheromones and avoid sub-adult females and mated females with egg sacs. These results support the observations of Blanke (1972) on the attraction of males to females over great distances including to foreign colonies. Blanke suggests the pheromones are secreted from the body of the female, and here I show the presence of pheromones on the web itself. Similar preferences were found by Blanke

(1975) in C. cicatrosa, where air-borne pheromones secreted by virgin females only, attracted males from large distances (>1 m). In another Cyrtophora species, the colonial C. moluccensis, males were found to spend more time on webs of adult females that had recently matured than on webs of sub-adult females or mated females with egg sacs (Lubin

1980).

While males are always monogamous, females are not monandrous, as they mate more than once. I observed females re-mate three and ten days after they first mated.

However, I did not test whether females will mate more than twice, or mate after they produced an egg sac. There is a possibility that females mate twice, each time filling a different spermatheca, but do not continue mating more than that. In this manner, unless there is cryptic female choice of sperm from one or the other spermatheca, males may assure their paternity over at least half of the offspring, and the costs of the sexual cannibalism are lowered.

To summarize, I found 100% sexual cannibalism in C. cirtophora, mostly happening after the insertion of the first pedipalp. Male mate choice exists as males prefer virgin females to sub-adult females or mated females with egg sac based on web-borne 40

pheromones. However, males will mate with a previously mated female, and females will re-mate shortly after their first mating. In addition, I found no mating plug in the female's epigyne, and hence no obvious paternity insuring mechanism for the male. 41

5. General Discussion

In this study, I investigated the role of sexual cannibalism in male mate choice and dispersal in the colonial spider Cyrtophora citricola. Sexual cannibalism is a fixed trait and occurs in all matings. This behavior selects for male mate choice, which was found to be based on web-borne pheromones. Males can reach females via aerial dispersal or bridging, which may assist in mate choice, as they are able to arrive to remote and perhaps favorable females. Males prefer to stay on webs of virgin females and disperse from webs of sub- adult or mated females. However, males are able to mate with a mated female and females can re-mate at least once.

Fromhage et al. (2005) have shown that monogamy as a form of paternity protection can evolve when the effective sex ratio is male biased. Under conditions of extreme sexual size dimorphism, males mature faster and survive to maturity more often than females, producing a male-biased effective sex ratio in the population. This can result in the accumulation of multiple males around each female, a phenomenon observed in some extreme sexual size dimorphic spiders (Andrade 1996; Vollrath 1998). This increased competition may be reduced if there is selection for self-sacrificial behavior that confers a paternity advantage to the male and restricts the female from re-mating.

In the beginning of the mating season of C. citricola, there are more mature males available than mature females because of their shorter development time. These males undergo dispersal for short or long distances in search for mates. Field observations

(Blanke 1972, N. Berner-Aharon, personal observations) indicate that there is competition between males, as up to 11 males were observed accumulating on a single female's web. It 42

is possible that in accordance to the Fromhage et al. model (2005), a male-biased sex ratio in the beginning of the season can provide an advantage to sexual cannibalism and male monogamy as a form of paternal investment. However, we still lack knowledge regarding the exact benefits males achieve from sexual cannibalism in this species.

The additional factors that correlate with sexual cannibalism such as low mate encounter rate and high mortality during mate search were not tested in this study. These factors are especially interesting since theoretically, colonial spiders should not experience such constraints during mate search. In the sexually cannibalistic Nephila plumipes

Kasumovic et al. (2007) show that despite the high-density aggregations of this species, male survival during mate searching is extremely low (36%). They suggest that male choosiness causes this high mortality, where males continue mate search, although costly and dangerous, with a goal of reaching a favorable female. High quality females can be virgin females or sub-adult females if mate guarding exists. Additionally, inbreeding avoidance may play an important role in male choosiness and male complicity in sexual cannibalism. A foreign female, having low relatedness may be favored especially in group- living organisms, where the likelihood of encountering a related mate and experiencing the costs of inbreeding are high. Although males are able to disperse aerially, as shown in this study, searching for a foreign female may result in high mortality and low encounter rate, which in turn will select for male self-sacrifice especially if it increases the quality or quantity of his offspring (Buskirk et al. 1984). 43

This study is the first to test for male mate choice and female receptivity in the sexually cannibalistic colonial spider C. citricola. However, in order to further understand the evolution of sexual cannibalism in a colonial spider additional investigation is required.

Studying the dynamics of mate search and mate choice in the field, together with population genetics to determine the degree of inbreeding, will shed more light on this rare and interesting phenomenon. 44

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