Behav Ecol Sociobiol (2015) 69:117–126 DOI 10.1007/s00265-014-1824-6

ORIGINAL PAPER

Eunuch supremacy: evolution of post-mating emasculation

Matjaž Kuntner & Urška Pristovšek & Ren-Chung Cheng & Daiqin Li & Shichang Zhang & I-Min Tso & Chen-Pan Liao & Jeremy A. Miller & Simona Kralj-Fišer

Received: 11 April 2014 /Revised: 30 September 2014 /Accepted: 1 October 2014 /Published online: 15 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Emasculation—males becoming effectively sterile via genital plugging and spontaneous death. Emasculation by self-removing their genitals—haslongbeenconsidereda during mating also results in genital plugs, but half eunuchs peculiar evolutionary phenomenon with unknown function, have another chance to mate. So far, the behavior of those taxonomically restricted to few and flies. In spiders, males that become eunuchs post-mating by self-removing emasculation results in half or full eunuchs when males sever disfigured palps has not been investigated empirically. We test one or both sperm transferring organs, palps. Three types of the mechanism and adaptive significance of post-mating emasculation, pre-maturation, mating, and post-mating are emasculation in coin spiders ( multipuncta) and use known in spiders, all having evolved multiple times. Males phylogenetic reconstruction to understand its evolutionary practicing pre-maturation emasculation sever one of their history. Our laboratory assays corroborate three hypotheses palps while still immature, then engage in strict monogyny related to mate monopolization: (1) The plugging hypothe- sis—predicting genital plugs to prevent female remating; (2) The better-fighter hypothesis—predicting enhanced eunuch Communicated by N. Wedell aggressiveness toward rivals; and (3) The gloves-off hypoth- Matjaž Kuntner, Urška Pristovšek, and Simona Kralj-Fišer contributed esis—predicting increased eunuch endurance. The support for equally to this work. these hypotheses in spiders practicing emasculation during M. Kuntner (*) : U. Pristovšek : R.

D. Li : S. Zhang Department of Biological Sciences, National University of Singapore, Singapore, Singapore Introduction

< : < I. M. Tso C. P. Liao Emasculation—male genital self-removal in spiders Department of Life Science, Tunghai University, Taichung, Taiwan (Robinson and Robinson 1980) and dipterans (Downes J. A. Miller 1978)—has long been considered an obscure evolutionary Naturalis Center, Leiden, The Netherlands phenomenon. Eunuch spiders have been almost completely neglected, being known only in a few nephilids (genera S. Kralj-Fišer University of Primorska, Faculty of Mathematics, Natural Sciences Herennia, , ), araneids (Caerostris, and Information Technologies, Koper, Slovenia Argiope), and in a tiny fraction of theridiids (genera Tidarren 118 Behav Ecol Sociobiol (2015) 69:117–126 and Echinotheridion) (Robinson and Robinson 1980;Kuntner 2012), and Latrodectus (Snow et al. 2006) and of emascula- et al. 2014). Emasculation rendering males sterile imposes tion in Tidarren (Knoflach and van Harten 2001; Ramos et al. high costs on their further mating opportunities and thus future 2004) and in two nephilid species, the SE Asian Nephilengys reproductive output. Relatively recently, however, studies in- malabarensis and the Malagasy Nephilingis livida (Kralj- vestigating reproductive strategies such as sperm competition, Fišer et al. 2011; Kralj-Fišer and Kuntner 2012; Lee et al. genital damage and plugging, male terminal investment, sex- 2012;Lietal.2012). However, mating plugs consisting of ual cannibalism, female polyandry, male monogyny, and sex- male spider genital parts are not always fully effective in ual conflict triggered a shift in focus among researchers of preventing female remating (Schneider and Elgar 2001; spider biology (Andrade 1996; Elgar and Fahey 1996; Schneider and Elgar 2002; Kralj-Fišer et al. 2011;Fromhage Schneider and Lubin 1998; Schneider et al. 2000;Elgar and Schneider 2012) and thus the plugging spider male may et al. 2003a, b; Andrade and Kasumovic 2005; Fromhage increase his monopolization of the female through post- and Schneider 2006; Miller 2007; Fromhage et al. 2008; copulatory mate guarding (Robinson and Robinson 1980; Kuntner et al. 2009b, c; Uhl et al. 2010;Fromhageand Fromhage and Schneider 2006; Kralj-Fišer et al. 2011). In Schneider 2012; Herberstein et al. 2012). These studies solid- accordance with the “asset protection principle” (Clark 1994), ified the view that most sexual strategies, including those that predicting that a male should adjust his behavior to its future relate to genital and body sacrifice pose certain fitness advan- residual reproductive potential, the better-fighter hypothesis tages such as securing paternity and monopolizing partners predicts escalated eunuch aggressiveness toward rival males (Austad 1984; Andrade 1996; Kuntner et al. 2009c; Uhl and with intact genitals (Kralj-Fišer et al. 2011). A male with Busch 2009; Welke and Schneider 2010). damaged genitalia and thus low future residual reproductive Male insects and spiders sometimes damage their genitals potential is expected to be more aggressive and to take more during copulation (Monnin and Peeters 1998; Fromhage and risk in male–male contests than a male with intact genitalia Schneider 2006;Snowetal.2006; Nessler et al. 2007, 2009; and high future residual reproductive potential (Fromhage and Uhl et al. 2010; Ghione and Costa 2011; Herberstein et al. Schneider 2005). 2012). In spiders, however, genital mutilation ranges from The remaining hypotheses explaining the adaptiveness simple embolic breakage (i.e., breakage of the genital tip) to of emasculation are specific to eunuchs (those spiders practic- emasculation, where males break off the entire genital or- ing emasculation) and do not apply to other spiders practicing gan(s). Kuntner et al. (2014) established a distinction between lesser genital damage. In this case, the possession of the “lesser genital damage,” a widespread practice where male disproportionally heavy palp should be more costly than its spiders break only a distal part of the embolus (i.e., the removal. In fact, the proximate cause of eunuch superior terminal portion of the male palp—the intrommitent organ in fighting abilities has been explained by the gloves-off spiders) (Uhl et al. 2010), and “(full) emasculation” (hence- hypothesis, which predicts the male endurance and stamina forth emasculation) resulting in palpless eunuchs (Robinson to increase with each severance of the heavy palp (Ramos and Robinson 1980; Kuntner 2005, 2007). Beyond the obvi- et al. 2004; Kuntner et al. 2009c; Lee et al. 2012). ous anatomical distinction (Kuntner et al. 2014), the former Accordingly, Tidarren males remove one of their palps before does not necessarily result in functional sterility (Snow et al. mating rendering them more agile and thereby likely allowing 2006), while the latter does (Kuntner et al. 2014). In this paper, them to win contests against rival males (Ramos et al. 2004). we follow this distinction and only discuss genital damage if it In an untested hypothesis, Kuntner (2005) suggested that relates to emasculation. males chew off their damaged palps in order to avoid Various hypotheses have been proposed to explain male haemolymph leakage. Finally, it has been reported that the emasculating behaviors (reviewed in Kuntner et al. 2014). The entire male genital severance resulting in whole palp plug plugging hypothesis predicts that the broken male genital parts allows for continuous sperm transfer into female spermathecae lodged in female copulatory organs must effectively prevent after the eunuch male has been “detached” from copula (Li female remating, thereby reducing sperm competition and et al. 2012). This remote-copulation hypothesis predicts con- increasing paternity share (Kuntner 2005; Kuntner et al. tinuous sperm transfer into female spermatheca after palpal 2009c; Uhl et al. 2010). However, as genital plugs are a removal (Kralj-Fišer et al. 2011;Lietal.2012). Recent empir- widespread phenomenon in spiders, this hypothesis may ap- ical research has demonstrated adaptive function of emascula- ply to any genital damage and is not strictly restricted to tion in the South-East Asian N. malabarensis, with support for emasculating eunuchs. In general support of the plugging the above hypotheses (haemolymph leakage not tested), and in hypothesis, numerous studies demonstrate paternity benefits part in the Malagasy N. livida, with support for the plugging of plugs made of male genital parts. For example, empirical hypothesis, but not for better-fighter hypothesis (Kralj-Fišer research has shown adaptive function of lesser genital damage et al. 2011;Kralj-Fišer and Kuntner 2012; Lee et al. 2012;Li in (Fromhage and Schneider 2006), Argiope (Nessler et al. 2012). Another emasculating nephilid spider, Herennia, et al. 2007, 2009;GhioneandCosta2011; Herberstein et al. remains untested as do the araneids Caerostris and Argiope, Behav Ecol Sociobiol (2015) 69:117–126 119 and with the exception of the gloves-off hypothesis, so do the self-emasculate the disfigured palp post-mating (Kralj-Fišer theridiids Tidarren and Echinotheridion (Kuntner et al. 2014). et al. 2011). Such voluntary, post-mating palp removal has Emasculation in spiders can occur during pre-maturation, also been anecdotally reported in coin spiders, genus at mating,andpost-mating and has evolved multiple times Herennia (Kuntner 2005; Kuntner et al. 2009c). However, it (Kuntner et al. 2014). In the theridiids, Tidarren and remains unknown whether post-mating emasculation is the Echinotheridion emasculation occurs both during pre- sole mechanism leading to eunuchs in Herennia,andno maturation and at mating (Knoflach and van Harten 2001; empirical study has investigated the biology of post-mating Agnarsson 2006; Knoflach and Van Harten 2006). While emasculation and its function in detail. immature, these spiders first self-remove one of the paired To understand the evolutionary significance of differ- palps, then use the second one to plug the female during ent types of emasculation, a phylogenetic understanding mating. Mating emasculation—convergent in the nephilids is necessary that would inform on the number of evo- Nephilengys and Nephilingis—also results in whole palpal lutionary origins and modifications of the behavior. plugs, but since adult intact males have two functional palps However, the currently understood evolution of emascu- filled with sperm, the first emasculation only results in half lation in nephilids is equivocal (Fig. 1). The behavior is eunuchs; these have another chance to mate with the same or clearly convergent, but parsimony reconstructions am- another female, and thus be monogynous or bygynous biguously resolve the ancestral emasculation type as (Kuntner et al. 2014). While the males of N. livida always either mating or post-mating (Kuntner et al. 2014). break their palps during mating (Kralj-Fišer and Kuntner Here, we test the mechanism and function of post- 2012), those of N. malabarensis predominantly practice mat- mating emasculation in Herennia multipuncta, the South ing emasculation (in 87.5 % of cases) but sometimes (12.5 %) and South-East Asian coin spider (Kuntner 2005), and use

Fig. 1 The evolution of emasculation in nephilid spiders and their predominant type of emasculation was scored, but note that both types immediate outgroups and the study species. The evolutionary reconstruc- may be present in Nephilengys. This study focused on post-mating tion is partially ambiguous: while emasculation in nephilids is convergent emasculation in Herennia.PhotographshowsH. multipuncta female in with an independent origin in Nephilingis, parsimony cannot unequivo- nature being guarded by a partially emasculated male having severed one cally establish which type of emasculation was ancestral. Only the of the two pedipalps (arrow) 120 Behav Ecol Sociobiol (2015) 69:117–126 the new data to infer the currently ambiguous evolution- examined them for male genital parts under a Leica MZ16 ary history of mating and post-mating emasculation in stereomicroscope following established protocols (Kuntner nephilids. Testing the remote copulation hypothesis (Li et al. 2009c). et al. 2012) in a spider known only for post-mating emasculation would be illogical. We therefore focused Test of the better-fighter hypothesis on the remaining hypotheses. By staging laboratory assays, we tested the plugging effectiveness in preventing Staged male–male contests followed established methodology female remating (Kuntner 2005), the better-fighter (Kralj- (Kralj-Fišer et al. 2011). A subset of eunuchs survived (N=11) Fišer et al. 2011), and the gloves-off (Lee et al. 2012) and were used repeatedly in the experimental treatments (N= hypothesis validity. Finding support for these hypotheses 23). We introduced a eunuch on a web of the female he would render them more general for all spider eunuchs previously mated with together with another virgin male irrespective of the mechanism leading to emasculation or (N=18). In one trial, the female consumed the eunuch early the phylogenetic placement of their bearers. in the experiment, and this trial was ignored. Due to the low number of surviving males, eunuchs and virgin males were reused in 2.2±0.8 and 1.3±0.6 experimental trials, respective- Materials and methods ly. In the control treatment (N=22; N of males=23), we introduced two virgin males on a web of a virgin female and Individuals of H. multipuncta were collected in 2011–2012 in observed their behavior. Some virgin males were reused in the China (Hainan), Vietnam (Cuc Phuong), Singapore (Bukit control treatment; each male was tested in 1.9±1 control Timah, Pulau Ubin, Kent Ridge), and Taiwan (Nantou), trans- experiments. In both treatments, we observed male agonistic ferred to the laboratory, placed in individual plastic cups, and behaviors; males were assigned the number of points depend- reared under constant temperature (27 °C) and photoperiod ing on intensity of aggressiveness during a 30-min trial (walk- (12DL/12NL). They were watered three to five times and fed ing toward a rival, 1 point; shaking web, 2 points; chasing a fruit flies and mealworms twice a week. Days prior to testing, rival, 3 points; attacking a rival, 4 points; biting a rival, 5 adult females were relocated into glass frames (50×50× points). “Shaking the web” referred to the tested male shaking 10 cm) to gain space for web building. his body when a rival male was approaching him or the female (Christenson and Goist 1979), not when the male shook to the Type of emasculation female. Total male aggressiveness was assessed as the sum of points (Kralj-Fišer et al. 2011). To observe whether males became (half) eunuchs (amputating During the trials, we also observed male locomotory ac- one or both palps) during or after copulation, thus establishing tivity on the web (number of walking initiatives) and behav- the type of emasculation (Kuntner et al. 2014), mating trials iors indicating mate guarding or courtship in eunuch and (N=60) were staged with a virgin female and a virgin male. virgin males, respectively (average distance to the female: We fed the female a day prior to mating trial to reduce recorded each minute; number of touching female; signaling). cannibalism due to hunger. Onto her web we gently introduced The number of touching female was counted as a number of a male using a paintbrush and observed the spiders for 2 h but initial male touches. The male “signaling” refers to the num- left the pair together until the male was found emasculated or ber of initial web tapping using legs (Kuntner et al. 2009c). for up to 7 days. In the 60 trials, 28 males remained virgin, nine males severed one palp (half eunuchs) and 23 males severed Test of the gloves-off hypothesis both palps (eunuchs). The courtship and mating sequence is described in our prior study (Kuntner et al. 2009c). To test the gloves-off hypothesis, we measured the time until exhaustion, i.e., endurance of males in laboratory. As in the Test of the plugging hypothesis original study (Lee et al. 2012), a tested male was placed in a 25×20×10 cm box, and time was recorded since his first The genital plug efficiency in a female mated in both genital move. During stops, the spider movement was encouraged openings (N=23) was estimated by staging two subsequent by gentle paintbrush touches. Trials stopped when the male mating trials within 2 weeks, each with a virgin male. Plugs became exhausted judging from a succession of five touches were deemed effective if both virgin males aimed but were with no male response. The spider endurance was measured as unable to insert their palps into the plugged copulatory the total time between the first and the last spider move. openings. We were unable to produce functional “artificial eunuchs” At the end of experiments, we euthanized the females (Lee et al. 2012) because most males died following experi- mated in both genital openings (see above, N=23) and pre- mental palp removal due to haemolymph leakage. In order to served them in 70 % ethanol. We excised their epigyna and control for individual differences and age, we thus conducted Behav Ecol Sociobiol (2015) 69:117–126 121 a repeated design, where a male was tested twice when virgin the males performed voluntary emasculation by chewing off (with two functional palps) and once as a full eunuch (without their used and disfigured palp(s), always within 24 h after palps; N=9). Because many males were cannibalized during copulation. This suggests that in H. multipuncta post-mating mating, 24 males were tested twice as virgin but never as emasculation is obligate. eunuchs. Plugging hypothesis Character evolution All copulating males initially damaged their palps via embolic We traced the evolution of emasculation types via parsimony breakage; i.e., the breakage of the male embolus (the terminal ancestral reconstruction in the package Mesquite (Maddison portion of the male palpal organ; termed lesser genital damage and Maddison 2012) on the optimal nephilid phylogeny in Kuntner et al. 2014). These embolic parts were left lodged (Kuntner et al. 2013). in female genital openings (N=23). Subsequent to trials, we found parts of broken male emboli in all examined genitalia of the mated females (N=23), each used genital tract possessing Statistical analyses a single plug. In the remating trials (N=46), virgin males attempted copulation but were unable to insert their palps into We tested the better-fighter hypothesis using six general linear previously plugged female copulatory openings (N=23 fe- mixed models (GLMM; each for one dependent variable); the males). Unsuccessful short insertion attempts and no dependent variables were body weight aggression score pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi hematodochal expansion thus implied a 100 % genital plug ðÞþ 1 ðÞþ 1 ( x ), distance to female ( x ), touch female, effectiveness. signaling, and walking on female’s web; the fixed variable was mating status (odd spider ID in one virgin vs another Better-fighter hypothesis virgin male; even spider ID in one virgin vs another virgin male; one virgin vs another eunuch; one eunuch vs another Inthestagedmale–male contests on the female web, eunuchs virgin male). The random factors include experimental ID and were in total more aggressive and remained closer to the spider ID. Experimental ID coped with comparison of depen- female compared with virgin males (Table 1; Figs. 2 and 3). dent variables within each treatment, and spider ID coped with However, eunuchs and virgin males did not significantly differ correlated results from the same spider ID and in signaling, walking on the web, and in touching the females pseudoreplications. We used the following tools and processes (Table 1). The two virgin males in the control group did not for statistical modeling and tests: GNU R (ver. 3.0.2.), differ in any observed behaviors, except in total walking on Package gmodels (ver. 2.15.4.1) for creating dummy variables the web (Table 1). in accordance to specific contrasts, and Package lme4 (ver. – 1.0 5) for GLMM. All estimates were chosen to optimize the Gloves-off hypothesis restricted maximum likelihood (REML) criterion. We used – Package ImerTest (ver. 2.0 3) for coefficient testing. Type The measured endurance of the retested virgin males did not III ANOVA (to test marginal effect) was always applied. change over time (F32, 1=0.387; P=0.538; Fig. 4), suggesting Degrees of freedom for T distribution were all calculated that age and prior experience do not affect an individual’s ’ based on Satterthwaite sapproximation. endurance level. After becoming full eunuchs, however, the The endurances of virgin and eunuch individuals were measured endurance tended to increase, albeit not significant- compared by repeated measure design of the general linear ly (F8, 1=3.027; P=0.12). This was due to an outlier in the model. We used PASW 18 statistics. Statistically significant eunuch data belonging to a male that was tested as late as difference was set at P<0.05. 52 days after becoming a eunuch. After omitting this datum, we found the predicted outcomes with high statistical signif-

icance: eunuch endurance was superior to virgin males (F7, 1= 15.613; P=0.006; Fig. 4). The remaining eunuchs aged 8.3± Results 6.9 days.

Type of emasculation Emasculation evolution

In the 60 mating experiments, 28 males remained virgin with A parsimony reconstruction of emasculation types on the undamaged palps, and of the males that mated, nine severed a phylogeny suggests three mutually exclusive interpretations single palp to become half eunuchs and 23 severed both of as equally parsimonious (Fig. 5a–c). In the first scenario their palps to become (full) eunuchs. Subsequent to mating, (Fig. 5a), mating and post-mating emasculation evolved in 122 Behav Ecol Sociobiol (2015) 69:117–126

Table 1 Comparison of behav- iors in control male–male contests Dependent parameter Experimental contest Estimate Std. Error df t P (twovirginmalesinthewebofa virgin female; N=19) and in ex- Body weight Virgin vs eunuch 0.001 0.002 34.44 0.519 0.607 perimental male–male contests Virgin vs virgin 0.001 0.002 34.54 0.500 0.620 (eunuch and a virgin male in the Aggression score Virgin vs eunuch 0.8612 0.2396 35.93 3.595 0.001 web of a eunuch’smate;N=21) − − using GLMM Virgin vs virgin ( )0.4387 0.2322 33.92 ( )1.889 0.067 Distance to female Virgin vs eunuch (−)0.3339 0.1599 24.04 (−)2.088 0.048 Virgin vs virgin 0.0371 0.1621 28.57 0.229 0.821 Touch female Virgin vs eunuch 0.6667 0.4535 75.99 1.470 0.146 Virgin vs virgin (−)0.8421 0.4768 75.99 (−)1.766 0.0811 Signaling Virgin vs eunuch (−)0.002 1.3384 32.95 (−)0.001 0.999 Virgin vs virgin (−)2.1703 1.3050 27.50 (−)1.663 0.108 Walking Virgin vs eunuch 0.2524 1.7497 37.78 0.144 0.886 Virgin vs virgin (−)6.6589 1.7643 39.64 (−)3.774 <0.001 parallel in Nephilengys and Herennia. In the second scenario the better-fighter hypothesis, and the gloves-off hypothesis. (Fig. 5b), mating emasculation evolved in the common ances- Although emasculation during mating has been shown to be tor to Nephilengys and Herennia with one to several modifi- adaptive in Nephilengys (Kralj-Fišer et al. 2011; Lee et al. cations of the strategy into obligate or conditional post-mating 2012; Li et al. 2012) and at least partially in Nephilingis emasculation. In the third scenario (Fig. 5c), an ancestral post- (Kralj-Fišer and Kuntner 2012), we so far lacked any exper- mating emasculation was retained in Herennia but was re- imental evidence for adaptiveness of post-mating emascula- placed with mating emasculation in Nephilengys.As tion (Kuntner et al. 2014). Therefore, both mating and post- discussed below, we deem the third scenario postulating mating emasculation types share adaptive values: eunuchs post-mating emasculation as the ancestral trait as the most effectively plug female epigyna, then mate-guard and engage likely. in aggressive fights with intact rival males. Furthermore, our finding that palp self-removal increases Herennia eunuch endurance and stamina provides new support for the gloves- off hypothesis and draws a parallel with pre-maturation emas- Discussion culation type as known in Tidarren and Echinotheridion (Knoflach and van Harten 2001; Ramos et al. 2004; This study confirmed anecdotal reports that the obligate mech- Agnarsson 2006;KnoflachandVanHarten2006). An addi- anism of emasculation in H. multipuncta was indeed post- tional adaptive trait, remote copulation, is only possible in mating (Kuntner et al. 2009c) and provided support for three those spiders that practice mating emasculation, since post- adaptive hypotheses explaining it: the plugging hypothesis,

Fig. 3 In H. multipuncta, eunuchs remain closer to the defended female. Fig. 2 In H. multipuncta, eunuchs are more aggressive. Total aggression Average distances (cm) to the female by virgin males (left) and eunuchs scores of virgin males (left) and eunuchs during male–male contests (right) during male–male contests Behav Ecol Sociobiol (2015) 69:117–126 123

full eunuchs are effectively sterile with no chances for further mating. Their strategy is to fight off the rival males thereby preventing them from mating with their mate; aggressive eunuchs monopolize their females to reduce sperm competi- tion and secure their paternity (e.g., Kralj-Fišer et al. 2011). Similar strategies have been observed in other spiders known for lesser genital damage that also renders them sterile, e.g., Nephila fenestrata (Fromhage and Schneider 2005). It should be noted that in Herennia, genital damage during mating is obligate, and thus in this system there is a possibility of confounding aggressiveness due to emasculation with aggres- sion due to past mating. As to the possible proximal cause of eunuch’sbetterfight- ing, we showed that eunuch’s endurance is increased through palp self-removal, reinforcing the conclusions from the stud- Fig. 4 In H. multipuncta, eunuchs endure more. Endurance/stamina (seconds to exhaustion) of virgin males and of eunuchs. Control group ies on Tidarren (Ramos et al. 2004)andNephilengys (Lee (virgin 1 vs virgin 2) showed no difference: F32, 1=0.387,P=0.538.Inthe et al. 2012). In the latter, intact virgin males exhibited lower experimental group (virgin 1 vs eunuch), eunuchs showed an increased endurance due to disproportionally large and heavy palps endurance: F7, 1=15.613, P=0.006 amounting to 9 % of total body weight. Similarly, Tidarren male palps amount to 10 % of total body weight, and their mating emasculation always takes place subsequent to palp removal results in 44 and 63 % increase in maximum speed detachment from copula. and endurance, respectively (Ramos et al. 2004). In the similar vein, we suggest that the costs of bearing a dysfunctional Adaptiveness of post-mating emasculation genital organ in Herennia males after copulation (e.g., heavy mass; haemolymph leakage) may be higher than the benefits We showed that H. multipuncta eunuchs exhibit higher ag- of its removal; i.e., higher endurance and thus likely higher gressiveness levels during male–male contests and remain chances to fight off a rival male and secure paternity. The closer to their mates compared with their virgin rivals, resem- gloves-off hypothesis receives strong support also in bling N. malabarensis (Kralj-Fišer et al. 2011). This mirrors Herennia and seems to be a good explanation of all three the predictions from the theory: A male with high future types of spider emasculation. reproductive potential should not escalate fights to avoid In Tidarren and Herennia, males voluntary self-remove injury, but when his residual reproductive potential is insig- their palps, whereas Nephilengys males commonly break off nificant, he should fight forcefully (Enquist et al. 1990;Clark the entire palp during copulation and only rarely employ self- 1994; Fromhage and Schneider 2005;Kralj-Fišer and Kuntner removal of disfigured palps after copulation. Our attempts of 2012). Although half eunuchs have another chance to mate, experimental palp removal in Herennia males prior to mating

Fig. 5 Three equally parsimonious scenarios of emasculation evolution in nephilid spiders. Phylogenetic and behavioral evidence supports the third (c) as the most likely 124 Behav Ecol Sociobiol (2015) 69:117–126 were unsuccessful resulting in loss of adult males due to study cannot in itself provide a definitive answer to this haemolymph leakage. In contrast to Herennia,emasculation phylogenetic problem, we are now able to interpret the key in Nephilengys is also experimentally possible; since in differences between emasculation in the two sister genera Nephilengys it naturally occurs during mating, we presume according to three equally parsimonious evolutionary there must be additional anatomical weak points in their palps. scenarios. Our results, showing that virgin males were never The first possibility is that mating and post-mating emas- able to mate with a previously plugged female, also culation evolved in parallel in Nephilengys and Herennia fully support the plugging hypothesis (e.g., Kralj-Fišer (Fig. 5a). Hypothetically, independent origins would mirror et al. 2011; Kralj-Fišer and Kuntner 2012). We also the key biological differences between those eunuchs who showed that H. multipuncta eunuchs remain closer to perform direct palp severance during mating or escalated their mates compared with virgin males (e.g., Kralj-Fišer fighting (Kuntner et al. 2009a). et al. 2011). Mate guarding is thus an additional eunuch The second scenario would evolve direct, mating emascu- strategy to protect paternity. But, while mate guarding is lation in the common ancestor to Nephilengys and Herennia logical as an additional monopolizing strategy in and would imply one to several modifications of the strategy Nephilengys, where plugging was only 75 % effective, into obligate or conditional post-mating emasculation why do Herennia eunuchs additionally mate-guard de- (Fig. 5b). Such modification(s) would perhaps be a response spite fully effective mating plugs? In addition to our to a relaxed cannibalism pressure from the females or a relaxed laboratory results, morphological and experimental evi- sperm competition among males. A conditional post-mating dence is solid that mated females remain plugged with emasculation strategy would be retained in Nephilengys but elaborate male embolic parts (Kuntner 2005; Kuntner would evolve into obligate post-mating emasculation in et al. 2009b, 2009c). Therefore, we find unlikely the Herennia. Insofar as emasculation evolution is linked with possibility that in their natural environment, males sexual cannibalism, sexual size dimorphism, and genital plug- should be able to remove a previously lodged plug. ging (Kuntner et al. 2014), this scenario would predict lower But what if intact males are able to inseminate plugged levels of these traits in Herennia compared with Nephilengys. females without removing prior plugs, e.g., bypassing There is some support for this, as sexual cannibalism is highly them or resorting to traumatic insemination? Both pos- frequent at 75 % in Nephilengys (Kralj-Fišer et al. 2011)andat sibilities seem unlikely. In our remating trials, no en- 83 % in Nephilingis (Kralj-Fišer and Kuntner 2012), and much larged hematodocha were observed which would indi- less so in Herennia at50%(Kuntneretal.2009c). However, cate sperm transfer. Although traumatic insemination has sexual size dimorphism in Herennia is on average more female been reported in spiders (Rezac 2009), no known cases biased than in Nephilengys, while it is more extreme in exist in nephilids or their orb web relatives. However, Nephilingis (ratio females to males in H. multipuncta=4.1; high levels of sexual size dimorphism, sperm competi- N. malabarensis=2.7; Nephilingis cruentata=6.5) (Kuntner tion, and sexual conflict may suggest that it is prema- and Coddington 2009). ture to discard the possibility. As the last resort, perhaps In the third scenario, an ancestral post-mating emasculation mate guarding in Herennia is a phylogenetic leftover (Fig. 5c) would be conditionally replaced with mating emas- from ancestors, where the strategy served a similar culation in Nephilengys (in response to high incidences of function to that in Nephilenygs. sexual cannibalism), while Herennia would retain the ances- tral behavior. This scenario seems to be best supported by the Evolution of emasculation types phylogenetic optimizations because the ancestors to the two alternative nephilid outgroups, araneids and zygiellids, are A recent review found a complex pattern of spider emascula- believed to exhibit post-mating emasculation (Kuntner et al. tion evolution (Kuntner et al. 2014). In araneoid spiders, 2014). This scenario would require a modification of the emasculation evolved multiply (between five and eleven ancestral state into a conditional mating emasculation in times) and became repeatedly lost or modified. That study Nephilengys and an independent origin of obligate emascula- also found that emasculation was phylogenetically significant- tion in Nephilingis. At least in Nephilengys, this strategy ly correlated with lesser genital damage, sexual cannibalism, enables remote copulation, a continuation of sperm transfer and extreme sexual size dimorphism (Kuntner et al. 2014). after males are detached from copula, which is an additional Furthermore, within the clade Nephilidae, mating and post- mechanism to secure eunuch paternity (Li et al. 2012). mating emasculation, while technically clearly different, were Although Nephilengys papuana eunuchs have not been sub- nevertheless linked phylogenetically in evolutionary time, but ject to experimental testing, early research reported incidents it remained ambiguous whether emasculation in Nephilengys of post-mating emasculation in this species (Robinson and and Herennia eunuchs evolved ones (then became modified) Robinson 1980) and thus its behavior is likely to closely or the two cases evolved in parallel (Fig. 1). Although our resemble that of its sister species. Behav Ecol Sociobiol (2015) 69:117–126 125

Conclusions Elgar MA, Bruce MJ, De Crespigny FEC, Cutler AR, Cutler CL, Gaskett AC, Herberstein ME, Ramamurthy S, Schneider JM (2003a) Male mate choice and patterns of paternity in the polyandrous, sexually Emasculation or genital self-removal is an extreme form of cannibalistic orb-web spider Nephila plumipes. Aust J Zool 51:357– genital damage in spiders and insects (Kuntner et al. 2014). 365 Becoming a spider eunuch is adaptive as recent studies find Elgar MA, De Crespigny FEC, Ramamurthy S (2003b) Male copulation – solid support for the plugging hypothesis, the better-fighter behaviour and the risk of sperm competition. Anim Behav 66:211 216 hypothesis, the gloves-off hypothesis, and the remote copula- Enquist M, Leimar O, Ljungberg T, Mallner Y,Segerdahl N (1990) A test tion hypothesis. This study confirms the validity of the first of the sequential assessment game—fighting in the cichlid fish three hypotheses in explaining post-mating emasculation in Nannacara anomala. Anim Behav 40:1–14 Herennia. Fromhage L, Schneider JM (2005) Virgin doves and mated hawks: contest behaviour in a spider. Anim Behav 70:1099–1104 Emasculation seems to be a male strategy intended to Fromhage L, Schneider JM (2006) Emasculation to plug up females: the reduce or avoid sperm competition with their rival males and significance of pedipalp damage in Nephila fenestrata. Behav Ecol thereby secure their paternity share. This behavior may only 17:353–357 be in the male interest or in the interest of both sexes if the Fromhage L, Schneider JM (2012) A mate to die for? A model of conditional monogyny in cannibalistic spiders. Ecology and eunuch behavior conserves female energy that would other- Evolution 2:2572–2582 wise be wasted on deterring unwanted copulations. A recently Fromhage L, McNamara JM, Houston AI (2008) A model for the evolu- elucidated macroevolutionary pattern of spider emasculation tionary maintenance of monogyny in spiders. 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