Article

Eunuch supremacy: evolution of post-mating spider emasculation

Abstract and Figures

Emasculation—males becoming effectively sterile by self-removing their genitals—has long been considered a peculiar evolutionary phenomenon with unknown function, taxonomically restricted to few spiders and flies. In spiders, emasculation results in half or full eunuchs when males sever one or both sperm transferring organs, palps. Three types of emasculation, pre-maturation, mating, and post-mating are known in spiders, all having evolved multiple times. Males practicing pre-maturation emasculation sever one of their palps while still immature, then engage in strict monogyny via genital plugging and spontaneous death. Emasculation during mating also results in genital plugs, but half eunuchs have another chance to mate. So far, the behavior of those males that become eunuchs post-mating by self-removing disfigured palps has not been investigated empirically. We test the mechanism and adaptive significance of post-mating emasculation in coin spiders (Herennia multipuncta) and use phylogenetic reconstruction to understand its evolutionary history. Our laboratory assays corroborate three hypotheses related to mate monopolization: (1) The plugging hypothe-sis—predicting genital plugs to prevent female remating; (2) The better-fighter hypothesis—predicting enhanced eunuch aggressiveness toward rivals; and (3) The gloves-off hypoth-esis—predicting increased eunuch endurance. The support for these hypotheses in spiders practicing emasculation during and after mating reinforces recent phylogenetic interpretations of these two emasculation types being evolutionarily linked in the family Nephilidae. We weigh the evidence in support of three different, but equally parsimonious scenarios of nephilid emasculation evolution. We conclude that emasculation is an adaptive, sexually selected trait that calls for further compar-ative and experimental research.
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ORIGINAL PAPER
Eunuch supremacy: evolution of post-mating spider emasculation
MatjažKuntner &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
#Springer-Verlag Berlin Heidelberg 2014
Abstract Emasculationmales becoming effectively sterile
by self-removing their genitalshaslongbeenconsidereda
peculiar evolutionary phenomenon with unknown function,
taxonomically restricted to few spiders and flies. In spiders,
emasculation results in half or full eunuchs when males sever
one or both sperm transferring organs, palps. Three types of
emasculation, pre-maturation,mating, and post-mating are
known in spiders, all having evolved multiple times. Males
practicing pre-maturation emasculation sever one of their
palps while still immature, then engage in strict monogyny
via genital plugging and spontaneous death. Emasculation
during mating also results in genital plugs, but half eunuchs
have another chance to mate. So far, the behavior of those
males that become eunuchs post-mating by self-removing
disfigured palps has not been investigated empirically. We test
the mechanism and adaptive significance of post-mating
emasculation in coin spiders (Herennia multipuncta) and use
phylogenetic reconstruction to understand its evolutionary
history. Our laboratory assays corroborate three hypotheses
related to mate monopolization: (1) The plugging hypothe-
sispredicting genital plugs to prevent female remating; (2)
The better-fighter hypothesispredicting enhanced eunuch
aggressiveness toward rivals; and (3) The gloves-off hypoth-
esispredicting increased eunuch endurance. The support for
these hypotheses in spiders practicing emasculation during
and after mating reinforces recent phylogenetic interpretations
of these two emasculation types being evolutionarily linked in
the family Nephilidae. We weigh the evidence in support of
three different, but equally parsimonious scenarios of nephilid
emasculation evolution. We conclude that emasculation is an
adaptive, sexually selected trait that calls for further compar-
ative and experimental research.
Keywords Terminal investment .Sperm competition .
Genital plugs .Sexual conflict .Monogyny .Polyandry
Introduction
Emasculationmale genital self-removal in spiders
(Robinson and Robinson 1980) and dipterans (Downes
1978)has long been considered an obscure evolutionary
phenomenon. Eunuch spiders have been almost completely
neglected, being known only in a few nephilids (genera
Herennia,Nephilengys,Nephilingis), araneids (Caerostris,
Argiope), and in a tiny fraction of theridiids (genera Tidarren
Communicated by N. Wedell
MatjažKuntner, Urška Pristovšek, and Simona Kralj-Fišer contributed
equally to this work.
M. Kuntner (*):U. Pristovšek :R.<C. Cheng:S. Kralj-Fišer
Institute of Biology, Scientific Research Centre, Slovenian Academy
of Sciences and Arts, Novi trg 2, 1000, Ljubljana, Slovenia
e-mail: kuntner@gmail.com
M. Kuntner :D. Li
Centre for Behavioural Ecology and Evolution, College of Life
Sciences, Hubei University, Wuhan, Hubei, China
M. Kuntner
National Museum of Natural History, Smithsonian Institution,
Washington, DC, USA
D. Li :S. Zhang
Department of Biological Sciences, National University of
Singapore, Singapore, Singapore
I.<M. Tso :C.<P. Liao
Department of Life Science, Tunghai University, Taichung, Taiwan
J. A. Miller
Naturalis Biodiversity Center, Leiden, The Netherlands
S. Kralj-Fišer
University of Primorska, Faculty of Mathematics, Natural Sciences
and Information Technologies, Koper, Slovenia
Behav Ecol Sociobiol
DOI 10.1007/s00265-014-1824-6
and Echinotheridion) (Robinson and Robinson 1980;Kuntner
et al. 2014). Emasculation rendering males sterile imposes
high costs on their further matingopportunities and thus future
reproductive output. Relatively recently, however, studies in-
vestigating reproductive strategies such as sperm competition,
genital damage and plugging, male terminal investment, sex-
ual cannibalism, female polyandry, male monogyny, and sex-
ual conflict triggered a shift in focus among researchers of
spider biology (Andrade 1996; Elgar and Fahey 1996;
Schneider and Lubin 1998; Schneider et al. 2000;Elgar
et al. 2003a,b; Andrade and Kasumovic 2005; Fromhage
and Schneider 2006; Miller 2007; Fromhage et al. 2008;
Kuntner et al. 2009b,c; Uhl et al. 2010;Fromhageand
Schneider 2012; Herberstein et al. 2012). These studies solid-
ified the view that most sexual strategies, including those that
relate to genital and body sacrifice pose certain fitness advan-
tages such as securing paternity and monopolizing partners
(Austad 1984; Andrade 1996; Kuntner et al. 2009c; Uhl and
Busch 2009; Welke and Schneider 2010).
Male insects and spiders sometimes damage their genitals
during copulation (Monnin and Peeters 1998; Fromhage and
Schneider 2006;Snowetal.2006; Nessler et al. 2007,2009;
Uhl et al. 2010; Ghione and Costa 2011; Herberstein et al.
2012). In spiders, however, genital mutilation ranges from
simple embolic breakage (i.e., breakage of the genital tip) to
emasculation, where males break off the entire genital or-
gan(s). Kuntner et al. (2014) established a distinction between
lesser genital damage,a widespread practice where male
spiders break only a distal part of the embolus (i.e., the
terminal portion of the male palpthe intrommitent organ in
spiders) (Uhl et al. 2010), and (full) emasculation(hence-
forth emasculation) resulting in palpless eunuchs (Robinson
and Robinson 1980; Kuntner 2005,2007). Beyond the obvi-
ous anatomical distinction (Kuntner et al. 2014), the former
does not necessarily result in functional sterility (Snow et al.
2006), while the latter does (Kuntner et al. 2014). In this paper,
we follow thisdistinction and only discuss genital damage if it
relates to emasculation.
Various hypotheses have been proposed to explain male
emasculating behaviors (reviewed in Kuntner et al. 2014). The
plugging hypothesis predicts that the brokenmale genital parts
lodged in female copulatory organs must effectively prevent
female remating, thereby reducing sperm competition and
increasing paternity share (Kuntner 2005; Kuntner et al.
2009c; Uhl et al. 2010). However, as genital plugs are a
widespread phenomenon in spiders, this hypothesis may ap-
ply to any genital damage and is not strictly restricted to
emasculating eunuchs. In general support of the plugging
hypothesis, numerous studies demonstrate paternity benefits
of plugs made of male genital parts. For example, empirical
research has shown adaptive function of lesser genital damage
in Nephila (Fromhage and Schneider 2006), Argiope (Nessler
et al. 2007,2009;GhioneandCosta2011; Herberstein et al.
2012), and Latrodectus (Snow et al. 2006) and of emascula-
tion in Tidarre n (Knoflach and van Harten 2001; Ramos et al.
2004) and in two nephilid species, the SE Asian Nephilengys
malabarensis and the Malagasy Nephilingis livida (Kralj-
Fišer et al. 2011; Kralj-Fišer and Kuntner 2012; Lee et al.
2012;Lietal.2012). However, mating plugs consisting of
male spider genital parts are not always fully effective in
preventing female remating (Schneider and Elgar 2001;
Schneider and Elgar 2002; Kralj-Fišer et al. 2011;Fromhage
and Schneider 2012) and thus the plugging spider male may
increase his monopolization of the female through post-
copulatory mate guarding (Robinson and Robinson 1980;
Fromhage and Schneider 2006; Kralj-Fišer et al. 2011). In
accordance with the asset protection principle(Clark 1994),
predicting that a male should adjust his behavior to its future
residual reproductive potential, the better-fighter hypothesis
predicts escalated eunuch aggressiveness toward rival males
with intact genitals (Kralj-Fišer et al. 2011). A male with
damaged genitalia and thus low future residual reproductive
potential is expected to be more aggressive and to take more
risk in malemale contests than a male with intact genitalia
and high future residual reproductive potential (Fromhage and
Schneider 2005).
The remaining hypotheses explaining the adaptiveness
of emasculation are specific to eunuchs (those spiders practic-
ing emasculation) and do not apply to other spiders practicing
lesser genital damage. In this case, the possession of the
disproportionally heavy palp should be more costly than its
removal. In fact, the proximate cause of eunuch superior
fighting abilities has been explained by the gloves-off
hypothesis, which predicts the male endurance and stamina
to increase with each severance of the heavy palp (Ramos
et al. 2004; Kuntner et al. 2009c; Lee et al. 2012).
Accordingly, Tid arren males remove one of their palps before
mating rendering them more agile and thereby likely allowing
them to win contests against rival males (Ramos et al. 2004).
In an untested hypothesis, Kuntner (2005) suggested that
males chew off their damaged palps in order to avoid
haemolymph leakage. Finally, it has been reported that the
entire male genital severance resulting in whole palp plug
allows for continuous sperm transfer into female spermathecae
after the eunuch male has been detachedfrom copula (Li
et al. 2012). This remote-copulation hypothesis predicts con-
tinuous sperm transfer into female spermatheca after palpal
removal (Kralj-Fišer et al. 2011;Lietal.2012). Recent empir-
ical research has demonstrated adaptive function of emascula-
tion in the South-East Asian N. malabarensis, with support for
the above hypotheses (haemolymph leakage not tested), and in
part in the Malagasy N. livida, with support for the plugging
hypothesis, but not for better-fighter hypothesis (Kralj-Fišer
et al. 2011;Kralj-Fišer and Kuntner 2012; Lee et al. 2012;Li
et al. 2012). Another emasculating nephilid spider, Herennia,
remains untested as do the araneids Caerostris and Argiope,
Behav Ecol Sociobiol
and with the exception of the gloves-off hypothesis, so do the
theridiids Tidarren and Echinotheridion (Kuntner et al. 2014).
Emasculation in spiders can occur during pre-maturation,
at mating,andpost-mating and has evolved multiple times
(Kuntner et al. 2014). In the theridiids, Tidarren and
Echinotheridion emasculation occurs both during pre-
maturation and at mating (Knoflach and van Harten 2001;
Agnarsson 2006; Knoflach and Van Harten 2006). While
immature, these spiders first self-remove one of the paired
palps, then use the second one to plug the female during
mating. Mating emasculationconvergent in the nephilids
Nephilengys and Nephilingisalso results in whole palpal
plugs, but since adult intact males have two functional palps
filled with sperm, the first emasculation only results in half
eunuchs; these have another chance to mate with the same or
another female, and thus be monogynous or bygynous
(Kuntner et al. 2014). While the males of N. livida always
break their palps during mating (Kralj-Fišer and Kuntner
2012), those of N. malabarensis predominantly practice mat-
ing emasculation (in 87.5 % of cases) but sometimes (12.5 %)
self-emasculate the disfigured palp post-mating (Kralj-Fišer
et al. 2011). Such voluntary, post-mating palp removal has
also been anecdotally reported in coin spiders, genus
Herennia (Kuntner 2005; Kuntner et al. 2009c). However, it
remains unknown whether post-mating emasculation is the
sole mechanism leading to eunuchs in Herennia,andno
empirical study has investigated the biology of post-mating
emasculation and its function in detail.
To understand the evolutionary significance of differ-
ent types of emasculation, a phylogenetic understanding
is necessary that would inform on the number of evo-
lutionary origins and modifications of the behavior.
However, the currently understood evolution of emascu-
lation in nephilids is equivocal (Fig. 1). The behavior is
clearly convergent, but parsimony reconstructions am-
biguously resolve the ancestral emasculation type as
either mating or post-mating (Kuntner et al. 2014).
Here, we test the mechanism and function of post-
mating emasculation in Herennia multipuncta, the South
and South-East Asian coin spider (Kuntner 2005), and use
Fig. 1 The evolution of emasculation in nephilid spiders and their
immediate outgroups and the study species. The evolutionary reconstruc-
tion is partially ambiguous: while emasculation in nephilids is convergent
with an independent origin in Nephilingis, parsimony cannot unequivo-
cally establish which type of emasculation was ancestral. Only the
predominant type of emasculation was scored, but note that both types
may be present in Nephilengys. This study focused on post-mating
emasculation in Herennia.PhotographshowsH. multipuncta female in
nature being guarded by a partially emasculated male having severed one
of the two pedipalps (arrow)
Behav Ecol Sociobiol
the new data to infer the currently ambiguous evolution-
ary history of mating and post-mating emasculation in
nephilids. Testing the remote copulation hypothesis (Li
et al. 2012) in a spider known only for post-mating
emasculation would be illogical. We therefore focused
on the remaining hypotheses. By staging laboratory
assays, we tested the plugging effectiveness in preventing
female remating (Kuntner 2005), the better-fighter (Kralj-
Fišer et al. 2011), and the gloves-off (Lee et al. 2012)
hypothesis validity. Finding support for these hypotheses
would render them more general for all spider eunuchs
irrespective of the mechanism leading to emasculation or
the phylogenetic placement of their bearers.
Materials and methods
Individuals of H. multipuncta were collected in 20112012 in
China (Hainan), Vietnam (Cuc Phuong), Singapore (Bukit
Timah, Pulau Ubin, Kent Ridge), and Taiwan (Nantou), trans-
ferred to the laboratory, placed in individual plastic cups, and
reared under constant temperature (27 °C) and photoperiod
(12DL/12NL). They were watered three to five times and fed
fruit flies and mealworms twice a week. Days prior to testing,
adult females were relocated into glass frames (50×50×
10 cm) to gain space for web building.
Type of emasculation
To observe whether males became (half) eunuchs (amputating
one or both palps) during or after copulation, thus establishing
the type of emasculation (Kuntner et al. 2014), mating trials
(N=60) were staged with a virgin female and a virgin male.
We fed the female a day prior to mating trial to reduce
cannibalism due to hunger. Onto her web we gently introduced
a male using a paintbrush and observed the spiders for 2 h but
left the pair together until the male was found emasculated or
for up to 7 days. In the 60 trials, 28males remained virgin, nine
males severed one palp (half eunuchs) and 23 males severed
both palps (eunuchs). The courtship and mating sequence is
described in our prior study (Kuntner et al. 2009c).
Test of the plugging hypothesis
The genital plug efficiency in a female mated in both genital
openings (N=23) was estimated by staging two subsequent
mating trials within 2 weeks, each with a virgin male. Plugs
were deemed effective if both virgin males aimed but were
unable to insert their palps into the plugged copulatory
openings.
At the end of experiments, we euthanized the females
mated in both genital openings (see above, N=23) and pre-
served them in 70 % ethanol. We excised their epigyna and
examined them for male genital parts under a Leica MZ16
stereomicroscope following established protocols (Kuntner
et al. 2009c).
Test of the better-fighter hypothesis
Staged malemale contests followed established methodology
(Kralj-Fišer et al. 2011). A subset of eunuchs survived (N=11)
and were used repeatedly in the experimental treatments (N=
23). We introduced a eunuch on a web of the female he
previously mated with together with another virgin male
(N=18). In one trial, the female consumed the eunuch early
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-
ly. In the control treatment (N=22; Nof males= 23), we
introduced two virgin males on a web of a virgin female and
observed their behavior. Some virgin males were reused in the
control treatment; each male was tested in 1.9 ± 1 control
experiments. In both treatments, we observed male agonistic
behaviors; males were assigned the number of points depend-
ing on intensity of aggressiveness during a 30-min trial (walk-
ing toward a rival, 1 point; shaking web, 2 points; chasing a
rival, 3 points; attacking a rival, 4 points; biting a rival, 5
points). Shaking the webreferred to the tested male shaking
his body when a rival male was approaching him orthe female
(Christenson and Goist 1979), not when the male shook to the
female. Total male aggressiveness was assessed as the sum of
points (Kralj-Fišer et al. 2011).
During the trials, we also observed male locomotory ac-
tivity on the web (number of walking initiatives) and behav-
iors indicating mate guarding or courtship in eunuch and
virgin males, respectively (average distance to the female:
recorded each minute; number of touching female; signaling).
The number of touching female was counted as a number of
initial male touches. The male signalingrefers to the num-
ber of initial web tapping using legs (Kuntner et al. 2009c).
Test of the gloves-off hypothesis
To test the gloves-off hypothesis, we measured the time until
exhaustion, i.e., endurance of males in laboratory. As in the
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
move. During stops, the spider movement was encouraged
by gentle paintbrush touches. Trials stopped when the male
became exhausted judging from a succession of five touches
with no male response. The spider endurance was measured as
the total time between the first and the last spider move.
We were unable to produce functional artificial eunuchs
(Lee et al. 2012) because most males died following experi-
mental palp removal due to haemolymph leakage. In order to
control for individual differences and age, we thus conducted
Behav Ecol Sociobiol
a repeated design, where a male was tested twice when virgin
(with two functional palps) and once as a full eunuch (without
palps; N=9). Because many males were cannibalized during
mating, 24 males were tested twice as virgin but never as
eunuchs.
Character evolution
We traced the evolution of emasculation types via parsimony
ancestral reconstruction in the package Mesquite (Maddison
and Maddison 2012) on the optimal nephilid phylogeny
(Kuntner et al. 2013).
Statistical analyses
We tested the better-fighter hypothesis using six general linear
mixed models (GLMM; each for one dependent variable); the
dependent variables were body weight aggression score
(ffiffiffiffiffiffiffiffiffiffiffiffiffiffi
xþ1ðÞ
p), distance to female ( ffiffiffiffiffiffiffiffiffiffiffiffiffiffi
xþ1ðÞ
p), touch female,
signaling, and walking on females web; the fixed variable
was mating status (odd spider ID in one virgin vs another
virgin male; even spider ID in one virgin vs another virgin
male; one virgin vs another eunuch; one eunuch vs another
virgin male). The random factors include experimental ID and
spider ID. Experimental ID coped with comparison of depen-
dent variables within each treatment, and spider ID coped with
correlated results from the same spider ID and
pseudoreplications. We used the following tools and processes
for statistical modeling and tests: GNU R (ver. 3.0.2.),
Package gmodels (ver. 2.15.4.1) for creating dummy variables
in accordance to specific contrasts, and Package lme4 (ver.
1.05) for GLMM. All estimates were chosen to optimize the
restricted maximum likelihood (REML) criterion. We used
Package ImerTest (ver. 2.03) for coefficient testing. Type
III ANOVA (to test marginal effect) was always applied.
Degrees of freedom for Tdistribution were all calculated
based on Satterthwaitesapproximation.
The endurances of virgin and eunuch individuals were
compared by repeated measure design of the general linear
model. We used PASW 18 statistics. Statistically significant
difference was set at P<0.05.
Results
Type of emasculation
In the 60 mating experiments, 28 males remained virgin with
undamaged palps, and of the males that mated, nine severed a
single palp to become half eunuchs and 23 severed both of
their palps to become (full) eunuchs. Subsequent to mating,
the males performed voluntary emasculation by chewing off
their used and disfigured palp(s), always within 24 h after
copulation. This suggests that in H. multipuncta post-mating
emasculation is obligate.
Plugging hypothesis
All copulating males initially damaged their palps via embolic
breakage; i.e., the breakage of the male embolus (the terminal
portion of the male palpal organ; termed lesser genital damage
in Kuntner et al. 2014). These embolic parts were left lodged
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
a single plug. In the remating trials (N=46), virgin males
attempted copulation but were unable to insert their palps into
previously plugged female copulatory openings (N=23 fe-
males). Unsuccessful short insertion attempts and no
hematodochal expansion thus implied a 100 % genital plug
effectiveness.
Better-fighter hypothesis
Inthestagedmalemale contests on the female web, eunuchs
were in total more aggressive and remained closer to the
female compared with virgin males (Table 1; Figs. 2and 3).
However, eunuchs and virgin males did not significantly differ
in signaling, walking on the web, and in touching the females
(Table 1). The two virgin males in the control group did not
differ in any observed behaviors, except in total walking on
the web (Table 1).
Gloves-off hypothesis
The measured endurance of the retested virgin males did not
change over time (F
32, 1
=0.387; P=0.538; Fig. 4), suggesting
that age and prior experience do not affect an individuals
endurance level. After becoming full eunuchs, however, the
measured endurance tended to increase, albeit not significant-
ly (F
8, 1
=3.027; P=0.12). This was due to an outlier in the
eunuch data belonging to a male that was tested as late as
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 (F
7, 1
=
15.613; P=0.006; Fig. 4). The remaining eunuchs aged 8.3±
6.9 days.
Emasculation evolution
A parsimony reconstruction of emasculation types on the
phylogeny suggests three mutually exclusive interpretations
as equally parsimonious (Fig. 5ac). In the first scenario
(Fig. 5a), mating and post-mating emasculation evolved in
Behav Ecol Sociobiol
parallel in Nephilengys and Herennia. In the second scenario
(Fig. 5b), mating emasculation evolved in the common ances-
tor to Nephilengys and Herennia with one to several modifi-
cations of the strategy into obligate or conditional post-mating
emasculation. In the third scenario (Fig. 5c), an ancestral post-
mating emasculation was retained in Herennia but was re-
placed with mating emasculation in Nephilengys.As
discussed below, we deem the third scenario postulating
post-mating emasculation as the ancestral trait as the most
likely.
Discussion
This study confirmed anecdotal reports that the obligate mech-
anism of emasculation in H. multipuncta was indeed post-
mating (Kuntner et al. 2009c) and provided support for three
adaptive hypotheses explaining it: the plugging hypothesis,
the better-fighter hypothesis, and the gloves-off hypothesis.
Although emasculation during mating has been shown to be
adaptive in Nephilengys (Kralj-Fišer et al. 2011; Lee et al.
2012; Li et al. 2012) and at least partially in Nephilingis
(Kralj-Fišer and Kuntner 2012), we so far lacked any exper-
imental evidence for adaptiveness of post-mating emascula-
tion (Kuntner et al. 2014). Therefore, both mating and post-
mating emasculation types share adaptive values: eunuchs
effectively plug female epigyna, then mate-guard and engage
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-
culation type as known in Tidarren and Echinotheridion
(Knoflach and van Harten 2001; Ramos et al. 2004;
Agnarsson 2006;KnoflachandVanHarten2006). An addi-
tional adaptive trait, remote copulation, is only possible in
those spiders that practice mating emasculation, since post-
Fig. 2 In H. multipuncta, eunuchs are more aggressive. Total aggression
scores of virgin males (left) and eunuchs during malemale contests
Fig. 3 In H. multipuncta, eunuchs remain closer to the defended female.
Average distances (cm) to the female by virgin males (left) and eunuchs
(right) during malemale contests
Tabl e 1 Comparison of behav-
iors in control malemale contests
(twovirginmalesinthewebofa
virgin female; N=19) and in ex-
perimental malemale contests
(eunuch and a virgin male in the
web of a eunuchsmate;N=21)
using GLMM
Dependent parameter Experimental contest Estimate Std. Error df t P
Body weight Virgin vs eunuch 0.001 0.002 34.44 0.519 0.607
Virgin vs virgin 0.001 0.002 34.54 0.500 0.620
Aggression score Virgin vs eunuch 0.8612 0.2396 35.93 3.595 0.001
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
Behav Ecol Sociobiol
mating emasculation always takes place subsequent to palp
detachment from copula.
Adaptiveness of post-mating emasculation
We showed that H. multipuncta eunuchs exhibit higher ag-
gressiveness levels during malemale contests and remain
closer to their mates compared with their virgin rivals, resem-
bling N. malabarensis (Kralj-Fišer et al. 2011). This mirrors
the predictions from the theory: A male with high future
reproductive potential should not escalate fights to avoid
injury, but when his residual reproductive potential is insig-
nificant, he should fight forcefully (Enquist et al. 1990;Clark
1994; Fromhage and Schneider 2005;Kralj-Fišer and Kuntner
2012). Although half eunuchs have another chance to mate,
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 eunuchsbetterfight-
ing, we showed that eunuchs endurance is increased through
palp self-removal, reinforcing the conclusions from the stud-
ies on Tidarre n (Ramos et al. 2004)andNephilengys (Lee
et al. 2012). In the latter, intact virgin males exhibited lower
endurance due to disproportionally large and heavy palps
amounting to 9 % of total body weight. Similarly, Tidarren
male palps amount to 10 % of total body weight, and their
removal results in 44 and 63 % increase in maximum speed
and endurance, respectively (Ramos et al. 2004). In the similar
vein, we suggest that the costs of bearing a dysfunctional
genital organ in Herennia males after copulation (e.g., heavy
mass; haemolymph leakage) may be higher than the benefits
of its removal; i.e., higher endurance and thus likely higher
chances to fight off a rival male and secure paternity. The
gloves-off hypothesis receives strong support also in
Herennia and seems to be a good explanation of all three
types of spider emasculation.
In Tidarren and Herennia, males voluntary self-remove
their palps, whereas Nephilengys males commonly break off
the entire palp during copulation and only rarely employ self-
removal of disfigured palps after copulation. Our attempts of
experimental palp removal in Herennia males prior to mating
Fig. 4 In H. multipuncta, eunuchs endure more. Endurance/stamina
(seconds to exhaustion) of virgin males and of eunuchs. Control group
(virgin 1 vs virgin 2) showed no difference: F
32, 1
=0.387,P=0.538.Inthe
experimental group (virgin 1 vs eunuch), eunuchs showed an increased
endurance: F
7, 1
=15.613, P=0.006
Fig. 5 Three equally parsimonious scenarios of emasculation evolution in nephilid spiders. Phylogenetic and behavioral evidence supports the third (c)
as the most likely
Behav Ecol Sociobiol
were unsuccessful resulting in loss of adult males due to
haemolymph leakage. In contrast to Herennia,emasculation
in Nephilengys is also experimentally possible; since in
Nephilengys it naturally occurs during mating, we presume
there must be additional anatomical weak points in their palps.
Our results, showing that virgin males were never
able to mate with a previously plugged female, also
fully support the plugging hypothesis (e.g., Kralj-Fišer
et al. 2011; Kralj-Fišer and Kuntner 2012). We also
showed that H. multipuncta eunuchs remain closer to
their mates compared with virgin males (e.g., Kralj-Fišer
et al. 2011). Mate guarding is thus an additional eunuch
strategy to protect paternity. But, while mate guarding is
logical as an additional monopolizing strategy in
Nephilengys, where plugging was only 75 % effective,
why do Herennia eunuchs additionally mate-guard de-
spite fully effective mating plugs? In addition to our
laboratory results, morphological and experimental evi-
dence is solid that mated females remain plugged with
elaborate male embolic parts (Kuntner 2005; Kuntner
et al. 2009b,2009c). Therefore, we find unlikely the
possibility that in their natural environment, males
should be able to remove a previously lodged plug.
But what if intact males are able to inseminate plugged
females without removing prior plugs, e.g., bypassing
them or resorting to traumatic insemination? Both pos-
sibilities seem unlikely. In our remating trials, no en-
larged hematodocha were observed which would indi-
cate sperm transfer. Although traumatic insemination has
been reported in spiders (Rezac 2009), no known cases
exist in nephilids or their orb web relatives. However,
high levels of sexual size dimorphism, sperm competi-
tion, and sexual conflict may suggest that it is prema-
ture to discard the possibility. As the last resort, perhaps
mate guarding in Herennia is a phylogenetic leftover
from ancestors, where the strategy served a similar
function to that in Nephilenygs.
Evolution of emasculation types
A recent review found a complex pattern of spider emascula-
tion evolution (Kuntner et al. 2014). In araneoid spiders,
emasculation evolved multiply (between five and eleven
times) and became repeatedly lost or modified. That study
also found that emasculation was phylogenetically significant-
ly correlated with lesser genital damage, sexual cannibalism,
and extreme sexual size dimorphism (Kuntner et al. 2014).
Furthermore, within the clade Nephilidae, mating and post-
mating emasculation, while technically clearly different, were
nevertheless linked phylogenetically in evolutionary time, but
it remained ambiguous whether emasculation in Nephilengys
and Herennia eunuchs evolved ones (then became modified)
or the two cases evolved in parallel (Fig. 1). Although our
study cannot in itself provide a definitive answer to this
phylogenetic problem, we are now able to interpret the key
differences between emasculation in the two sister genera
according to three equally parsimonious evolutionary
scenarios.
The first possibility is that mating and post-mating emas-
culation evolved in parallel in Nephilengys and Herennia
(Fig. 5a). Hypothetically, independent origins would mirror
the key biological differences between those eunuchs who
perform direct palp severance during mating or escalated
fighting (Kuntner et al. 2009a).
The second scenario would evolve direct, mating emascu-
lation in the common ancestor to Nephilengys and Herennia
and would imply one to several modifications of the strategy
into obligate or conditional post-mating emasculation
(Fig. 5b). Such modification(s) would perhaps be a response
to a relaxed cannibalism pressure from the females or a relaxed
sperm competition among males. A conditional post-mating
emasculation strategy would be retained in Nephilengys but
would evolve into obligate post-mating emasculation in
Herennia. Insofar as emasculation evolution is linked with
sexual cannibalism, sexual size dimorphism, and genital plug-
ging (Kuntner et al. 2014), this scenario would predict lower
levels of these traits in Herennia compared with Nephilengys.
There is some support for this, as sexual cannibalism is highly
frequent at 75 % in Nephilengys (Kralj-Fišer et al. 2011)andat
83 % in Nephilingis (Kralj-Fišer and Kuntner 2012), and much
less so in Herennia at50%(Kuntneretal.2009c). However,
sexual size dimorphism in Herennia is on average more female
biased than in Nephilengys, while it is more extreme in
Nephilingis (ratio females to males in H. multipuncta=4.1;
N. malabarensis=2.7; Nephilingis cruentata =6.5) (Kuntner
and Coddington 2009).
In the third scenario, an ancestral post-mating emasculation
(Fig. 5c) would be conditionally replaced with mating emas-
culation in Nephilengys (in response to high incidences of
sexual cannibalism), while Herennia would retain the ances-
tral behavior. This scenario seems to be best supported by the
phylogenetic optimizations because the ancestors to the two
alternative nephilid outgroups, araneids and zygiellids, are
believed to exhibit post-mating emasculation (Kuntner et al.
2014). This scenario would require a modification of the
ancestral state into a conditional mating emasculation in
Nephilengys and an independent origin of obligate emascula-
tion in Nephilingis. At least in Nephilengys, this strategy
enables remote copulation, a continuation of sperm transfer
after males are detached from copula, which is an additional
mechanism to secure eunuch paternity (Li et al. 2012).
Although Nephilengys papuana eunuchs have not been sub-
ject to experimental testing, early research reported incidents
of post-mating emasculation in this species (Robinson and
Robinson 1980) and thus its behavior is likely to closely
resemble that of its sister species.
Behav Ecol Sociobiol
Conclusions
Emasculation or genital self-removal is an extreme form of
genital damage in spiders and insects (Kuntner et al. 2014).
Becoming a spider eunuch is adaptive as recent studies find
solid support for the plugging hypothesis, the better-fighter
hypothesis, the gloves-off hypothesis, and the remote copula-
tion hypothesis. This study confirms the validity of the first
three hypotheses in explaining post-mating emasculation in
Herennia.
Emasculation seems to be a male strategy intended to
reduce or avoid sperm competition with their rival males and
thereby secure their paternity share. This behavior may only
be in the male interest or in the interest of both sexes if the
eunuch behavior conserves female energy that would other-
wise be wasted on deterring unwanted copulations. A recently
elucidated macroevolutionary pattern of spider emasculation
suggests that it predictably evolves together with an extreme,
female-biased sexual size dimorphism, sexual cannibalism,
and genital plugging via embolic breakage (Kuntner et al.
2014). This coevolutionary pattern can be tested further via
experimental and comparative research, both in spiders and
perhaps also in insects known for emasculation.
Acknowledgments We thank Ingi Agnarsson for feedback on an early
draft and two anonymous reviewers for valuable suggestions. This re-
search was supported in part by a Raffles Museum for Biodiversity
Research (RMBR) Short-term Fellowship and the grants P10236 and
MU-PROM/12-001 from the Slovenian Research Agency to M.K. and
by the NSFC grant (31272324), Singapore Ministry of Education (MOE)
AcRF grant (R-154-000-476-112), and Ah Meng Conservation Fund
(R-154-518-720) to D.L.
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... Males will sacrifice one or both palps during or after mating, thereby becoming emasculated-effectively eunuchs (74,76,77). These eunuch males are superior fighters and fiercely defend the female from intruders (89). Eunuch male hermit spiders endure longer and fight more aggressively than intact males (71,92). ...
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