Behavioral Ecology Advance Access published March 20, 2016

Behavioral
Ecology

The official journal of the

ISBE

International Society for Behavioral Ecology

Behavioral Ecology (2016), 00(00), 110. doi:10.1093/beheco/arw023

Original Article

A prison effect in a wild population: a scarcity

of females induces homosexual behaviors
inmales

Received 9 October 2015; revised 29 January 2016; accepted 2 February 2016.

The high frequency of same-sex sexual behaviors (SSB) in free-ranging animals is an evolutionary puzzle because fitness benefits are
often unclear in an evolutionary context. Moreover, the physiological and genetic underpinnings of SSB remain unclear. We exploited
an extraordinary natural experiment to examine the impact of environmental factors (local sex ratio [SR]) and testosterone (T) levels on
SSB in a dense population of Hermanns tortoises monitored for 7years. Under the combination of high density and extremely skewed
SR (~50 females, >1000 males), males courted and mounted other males more frequently than females. They even exhibited extravagant sexual behaviors, attempting to copulate with dead conspecifics, empty shells, and stones. T levels remained within the species
normal range of variation. SSB was not observed in other populations where SR is not, or less skewed, and where density is lower.
This study reports the first natural example of a prison effect, whereby a high population density combined with female deprivation
triggered SSB as a mere outlet of sexual stimulation. More generally, it supports the hypothesis that SSB can be a nonadaptive consequence of unusual proximate factors rather than reflecting physiological disorders.
Key words: ecophysiology, reptiles, sex ratio, testosterone, tortoise.

INTRODUCTION
Same-sex sexual behaviors (SSB) have been recorded in numerous free-ranging animal species from a wide array of lineages, and
in many cases are common (Sommer and Vasey 2006; Bailey and
Zuk 2009; Poiani 2010; Scharf and Martin 2013). Because sexual
behaviors are tightly linked to procreation and often entail major
survival and energy costs, the high frequency of these apparently
unproductive behaviors is a biological paradox. Both adaptive and
nonadaptive hypotheses have been proposed to explain the origin
and maintenance of SSB. Possibly due to their greater explanatory
power, adaptive hypotheses (e.g., social glue, intrasexual conflict)
have attracted most of the research (Camperio-Ciani et al. 2004;
MacFarlane etal. 2007; Bailey and Zuk 2009; Bierbach etal. 2013;
VanderLaan etal. 2014). Moreover, SSB is considered not only as
the outcome of selection but also as a selective agent per se (Bailey

Address correspondence to X.Bonnet. E-mail: xavier.bonnet@cebc.cnrs.fr.

The Author 2016. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved. For
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and Zuk 2009). Technical difficulties may also explain why investigations of SSB tend to focus on ultimate rather than on proximate
causations. Indeed, sexually active individuals often express a wide
spectrum of behaviors, and SSB can be intermingled with heterosexual behaviors (HSB), posing obstacles to isolate physiological
processes that specifically trigger SSB. Consequently, physiological mechanisms underlying adaptive SSB have rarely been experimentally elucidated in the field (Shine et al. 2000), and there are
only limited data on this topic for nonadaptive SSB (Scharf and
Martin2013).
In captive animals, genetic, anatomical, neurophysiological, or
hormonal investigations have failed to identify clear distinctions
between the mechanisms that promote SSB versus HSB (Poiani
2010; Hoskins etal. 2015). Even in humans, one of the most intensively studied vertebrate species, the abundant efforts to link sexual
orientation with anatomy, physiology, and genetic mechanisms
have revealed complex and equivocal patterns (Banks and Gartrell
1995; Rice et al. 1999; Zitzmann and Nieschlag 2001; Mustanski
et al. 2002; Jannini et al. 2010). Although recent epidemiologic

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XavierBonnet,a AnaGolubovi,b DraganArsovski,a,b Sonjaorevi,b Jean-MarieBallouard,c

BogoljubSterijovski,d RastkoAjti,e ChristopheBarbraud,a and LiljanaTomovib
aCEBC, UMR-7372, CNRS, 79360 Villiers en Bois, France, bFaculty of Biology, Institute of Zoology,
University of Belgrade, 11000 Belgrade, Serbia, cCRCC, SOPTOM, 83590 Gonfaron, France,
dMacedonian Ecological Society, Herpetology Group, 1000 Skopje, Former Yugoslav Republic of
Macedonia, and eInstitute for Nature Conservation of Serbia, 11070 Belgrade, Serbia

Behavioral Ecology

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MATERIALS AND METHODS

Studied species
The Hermanns tortoise (Testudo hermanni) is a medium-sized
Mediterranean species that comprises 3 subspecies, one (e.g.,
Testudo hermanni boettgeri) is locally abundant in the eastern part of
its distribution range (Bertolero et al. 2011). This species exhibits typical tortoise sexual behaviors: Males display a repertoire
of behaviors (e.g., head bobbing, biting) to court and (often forcibly) mount females (Hailey 1990; Sacchi et al. 2013). During
copulation, they produce characteristic high pitch vocalizations (Galeotti et al. 2007). Females usually try to escape before
accepting copulation, sometimes flipping males onto their backs.
Male rivals often engage in vigorous combats characterized by
shell ramming, severe bites, and attempts to flip back opponents,
but without mounting or vocalization. Because adult chelonians
are sexually dimorphic (Bonnet etal. 2010), including the studied
subspecies (Djordjevi et al. 2011, 2013), sex can be accurately

determined and thus SSB can be unambiguously distinguished

fromHSB.
SSB were never observed in the thousands of free-ranging individuals of other Hermanns tortoise populations (T. h. boettgeri and
Testudo hermanni hermanni) or in other Testudo species studied over
more than 20 years (Testudo graeca; Testudo horsfieldi; Bonnet et al.
2001; Lagarde et al. 2003, 2008). Additionally, despite the very
abundant literature, we are not aware of published SSB in freeranging tortoises.

Study sites and study population

A large population was monitored for 7 years (20082014) on
Golem Grad Island (Former Yugoslav Republic of Macedonia, ~18
ha; N4052; E2059) by 212 people (6 on average) during 13
field sessions (total 137 searching days). The location and behavior
of each tortoise were first recorded in order to limit disturbance by
the observer; individuals were then captured, sexed, permanently
marked using a notche-code on the marginal-scutes, and rapidly
released (Djordjevi et al. 2011; Golubovi et al. 2013). During
resightings, behavior was also recorded first; individuals were then
recaptured to check identity. Importantly, sexual activity, although
influenced by climatic conditions (Tomovi L, Bonnet X, Sterijovski
B, orevi S, unpublished data), was observed during the entire
activity period, from April to September. We marked 1737 tortoises: 1208 adults (and 529 juveniles, discarded from analyses) and
collected abundant recaptures (N=7829).
On Golem Grad Island, a very particular landscape (Figure1) splits
the population into 2 almost-distinct subpopulations. Approximately
20% of the adults live on the shoreline (~1 ha), which is separated by
vertical cliffs (10- to 25-m high) from the plateau subpopulation (~17 ha;
71% of individuals). Afew tortoises (9%, mostly males [79%]) have been
recorded to commute between subpopulations through 2 narrow steep
paths (Tomovi L, Bonnet X, Sterijovski B, orevi S, unpublished
data). For unknown reasons (perhaps reflecting temperature-dependent
sex determination coupled with a scarcity of potential nest sites that provide daughter-producing temperatures; Pieau 1971), the SR is highly
male biased on the shoreline subpopulation (23% adult females) and
even more strongly male biased on the plateau (5% adult females).
As in other Testudo populations, males patrol their habitat, search
for females, and harass them to copulate, whereas females never
chase conspecifics (Hailey and Willemsen 2000). The very high population density (~67 adults per hectare) means that many substratedeposited pheromonal trails intersect in the field whereas body size
and coloration overlap between sexes, reducing males ability to
locate females using olfactory or visual clues. Consequently, for a
male tortoise locating a female is like looking for a needle in a haystack of rivals, notably for males living on the plateau where adult
females are very scarce. In contrast, shoreline tortoises are confined
within a narrow 1-ha zone where female density is 10 times greater
than on the plateau. Thus, shoreline males can find females more
easily, but they cannot avoid many malemale encounters. We
observed a high frequency of male SSB in the 2 subpopulations of
Golem Grad (see Results for details).
A neighboring population situated on the mainland (Konjsko, 4
km away from the island, 407 individuals marked; 322 adults; ~20
ha) was monitored between 2010 and 2014 using similar method.
Although not the main focus of this study, it provided comparative
data regarding possible effects of SR and T levels on sexual behaviors. In this neighboring mainland population, the SR is close to 1:1
(53% adult females) and density (~20 individuals per hectare) lower
compared with Golem Grad. We never observed any male SSB in

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studies revealed that genetic factors influence human sexuality

(Jannini et al. 2015), underlying genetic and physiological mechanisms of sexual orientation are not yet understood such that adaptive hypotheses remain controversial (Rahman2005).
Nevertheless, there is no doubt that physiological factors (e.g.,
hormones) influence behaviors and thus SSB (Nelson 2005;
Goldey and van Anders 2015). In vertebrates, investigations have
concentrated on mechanisms that involve sex steroids (Poiani
2010; Goldey and van Anders 2015). Such studies have explored
several experimentally induced disorders (e.g., using knockout
mice; Bakker et al. 2002, 2006). Experimental masculinization
can induce SSB but also perturbs fertility and reproductive behaviors, with hyperaggressiveness or behavioral asexuality as possible
outcomes (Balthazart, et al. 1997). Consequently, such disorders
represent reproductive impasses and are unlikely to be selected
in free-ranging animals. Moreover, empirical results suggest that
male vertebrates that engage in SSB do not exhibit behavioral
anomalies, are able to discriminate between the sexes, reproduce
normally, and do not display unusual circulating testosterone
levels (T) (Poiani 2010). Overall, available results regarding hormones and sexual behaviors provide little insights into the prevalence of SSB in natural populations. However, the difficulty of
identifying proximate physiological mechanisms does not mean
that sexual hormones are not key factors. Examining these issues
remains important to test adaptive versus nonadaptive causes, to
understand why individuals engage in SSB in natural populations,
and thus to frame physiological investigations in a natural nonpathological context.
We benefited from a quasi-experimental natural situation to
investigate one of the main nonadaptive hypotheses proposed by
Bailey and Zuk (2009): The prison effect where depriving individuals of members of the opposite sex causes them to engage in
sexual interactions with members of the same sex. This notion
received support in a recent review on insects studied in captivity:
The paucity of sexual partner (i.e., one sex kept in isolation) combined with high population density and exposure to sexual chemical stimulation is assumed to trigger SSB (Scharf and Martin 2013).
However, comparable data are lacking in vertebrates or in natural
populations. In the current study, a strong spatial and population
contrast allowed us to explore the effect of environmental (female
scarcity) and physiological (T levels) factors on the high SSB frequency exhibited by free-ranging males.

Bonnet etal. Same-sex sexual behaviors in wild tortoises

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this population, whereas classical HSB (e.g., courting, mating) were

frequent (e.g., N=45).

Adapting behavioral data to a capturemark

recapture framework
Capturemarkrecapture (CMR) analyses have been developed to
assess demographic parameters (e.g., population size, annual survival) of populations when the detectability of individuals is imperfect (Jolly 1965; Seber 1965). The encounter history of each animal
is implemented as a basic input in the analyses, thus, estimates of
model parameters can be performed using numerical maximum
likelihood techniques (Lebreton et al. 1992). Various factors and
their interactions can be modeled, individuals can be assigned into
groups and covariates can be used. The robustness and flexibility
of this approach explain the exponentially increasing popularity
of CMR analyses in the ecological literature (Kendall and Nichols
2002; Choquet, Lebreton, et al. 2009). Because the probability to
observe a given behavior in the field is subjected to survival, sighting, and observation biases, we adapted the CMR approach to our
behavioral observations. In other words, we evaluated the annual
probabilities of male tortoises to engage in SSB using the individual CMR data from 2008 to 2014. Indeed, many individuals
were observed repeatedly during the study (resighted 5.53.9 times
on average, range 127), enabling us to build individual capture/
behavioral histories (Supplementary Annex 1) and to use matrix
analyses where transition probabilities among successive behaviors
within the histories can be estimated.

In the current study, one crucial aspect of the construction of

the CMR dataset was the assignment of each individuals behavioral capture history to a biologically relevant group allowing us
to take into account the effects of sex and site: males on plateau,
females on plateau, males on shoreline, and females on shoreline
(Supplementary Annex 2). Site assignment was based on the place
of capture of each individual during sampling sessions. Data for
the few individuals captured within the 2 narrow paths, commuting
between the plateau and shoreline, were discarded. The assignment
of each individual to a site allowed us to include SR and density
into modeling. Although possible interaction between SR and density could not be disentangled, the principal difference between the
2 subpopulations was due to skewed SR (5.4 times more skewed on
the plateau than on the beach; this difference was only 2.1 for density). Consequently, our null hypothesis was a lack of difference in
the probability to exhibit SSB between plateau and shoreline males.
Conversely, a significant site effect would suggest an influence of
SR and density.
The dataset consisted of 1208 adult capture histories (we
retained only 1 observation per year and per individual). In case of
multiple resighting of individuals within a given year, we retained
the most informative behavior. For example, SSB was preferred
over heterosexuality because this study focuses on SSB. In practice,
this selection was applied on 13 occasions only, and possible associated bias was negligible considering the large number of sexual
behaviors observed (removing these 13 cases from the analyses did
not change the results).

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Figure1
Golem Grad island is an 18-ha natural prison where tortoise population density is high, ~60 individuals per hectare. (a) Vertical cliffs separate a plateau
subpopulation (highly skewed SR: 5% of females) from a narrow shoreline subpopulation (skewed SR: 23% of females) of Hermanns tortoises (photographed
by X.B.). (b) Amale mounting (+ vocalization) another male, which himself is mounting a juvenile male (left) (photographed by A.G.). (c) Amale mounting a
stone (center) (photographed by A.G.). (d) Amale mounting a female (right) (photographed by X.B.).

Behavioral Ecology

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Designing the multistate markrecapturemodel

Building biological scenarios

Survival (S) was constrained to be constant over time and state but
was allowed to vary with sex and site. Transitions between sexual
behaviors () were held constant over time but they were sex, site,
and state dependent. Detection probability (P) was time, sex, site,
and state dependent to allow us to take into account variations in
detectability. Our initial model was thus S(sex site) (sex site state) P(sex
site state t). We then built alternative models to test specific hypotheses by using a backwards stepwise procedure while removing
effects from each parameter in order to obtain the best linear combination at each step. Model selection was first performed on detection probability by testing time, sex, site, and state dependencies.
We then tested for sex and site dependencies for survival. Finally,
we tested for site dependency on transition probabilities between
behavioral states, thus testing for an effect of SR and density.
We tested the goodness-of-fit of the time-dependent MSMR model
using U-CARE (Lebreton and Pradel 2002; Choquet Lebreton, etal.
2009). Model selection was based on the quasi-likelihood counterpart
of Akaikes information criterion (QAIC) and all models were run
under program E-SURGE 1.9.0 (Lebreton and Pradel 2002; Choquet,
Rouan, etal. 2009). Resighting probability decreased during the study
period for all states (Figure 2a) due to decreasing search effort over
time and was influenced by the type of behavior exhibited (Table 2,
Figure2a), but did not vary between subpopulations orsexes.

Testosterone and behaviors

To assign a precise behavior with T level, we rapidly took a blood
sample (less than 3min on average, range 0.27min) on a random

Table1
Tortoise behaviors observed in the field
Behavior

Definition

Category

Burrowed
In the shade
Basking
Foraging
Hiding
Walking
Chased
Chasing
Courted
Courting
Fighting
Sex group
Mounting
Necrophilia
Skeletophilia
Sex train
Not classified

Partly burrowed, head and legs retracted

Motionless in the shade
Fully exposed to sunrays, legs deployed
Eating plants, fungi, or animals
Moving toward a shelter
Walking alone
Trying to escapea from the sexual assaults of a male
Male pursuing a conspecific
Courted by a male
Male courting a conspecific, mounting attempts
Male to male combat, violent shell shocks, and bites
Sexually active group, e.g., 25 males courting a female
Male mounting any conspecific, vocalizing, ejaculation
Male mounting a dead tortoise, vocalizing
Male mounting an empty shell (bones), vocalizing
Several males forming a line while pursuing a female
Information missing, ambiguous behavior (e.g., partly shaded), dead individual

Inactive
Inactive
Active
Active
Active
Active
Sexual (P)
Sexual (A)
Sexual (P)
Sexual (A)
Sexual (A)
Sexual
Sexual (A)
Sexual (A)
Sexual (A)
Sexual

677
2513
1729
195
718
755
30
31
22
22
30
101
667
5
5
3
2075

SSB (%)

25 (83)
25 (81)
10 (45)
10 (45)
0 (0)
36 (36)
506 (76)
5 (100)
5 (100)
0 (0)

Behaviors (resightings of 1737 individuals, 20082014) were classified into 3 main categories: inactive, active, or sexual; (A) and (P) mean, respectively, sexually
active versus sexually passive. Sample size (N) indicates the total number of observations; for example, 15 pairs of fighting males involve 30 individuals (some
information was missing). SSB (%) indicates the actual number and proportion of SSBs observed; these involved only males (except for one female mounting
another female).
aPossible confusion with a male breaking the fight. Two males courting stones were included in the not-classified category.

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Over time, individuals exhibit different behaviors, shifting from sexual activity to inactivity, etc. Therefore, we used a multistate mark
recapture (MSMR) model to estimate transition probabilities among
behaviors at each sighting occasion (e.g., shifts from SSB to HSB).
MSMR can distinguish between the behavioral states within a CMR
probabilistic framework in order to take into account imperfect
detectability of tortoises during capture sessions in the field (Burnham
and Anderson 2002). This CMR approach allowed us to analyze
the annual probability of detecting different behaviors, while taking
into account possible differences in detectability and survival between
years, sites, sexes, and states. It also allowed us to take into account
the previous behavioral state of individuals (e.g., successive behaviors might not be independent), which might be important to obtain
unbiased estimates of demographic parameters (Lebreton and Pradel
2002; Choquet, Lebreton, etal. 2009; Choquet, Rouan, etal.2009).
In practice, from an initial departure state of initial state, individuals could be categorized in 4 distinct states (see Table1 for details):
sexually inactive or sexually passive (SP, individual exhibiting no
sexual behavior or passive during sexual behavior), sexually active
(SA, individual displaying active HSB or involved in male-to-male
combat), SA with the same sex (SSB, individual observed in an
active homosexual behavior, essentially a male mounting another
male, thus excluding combats), and dead (). Three state transitions
were modeled to account for survival probability between capture
occasions (matrix S; Supplementary Annex 2): SSP, SSA, and SSBB
were the respective survival probabilities of SP (line 1), SA (line 2),
and SSB (line 3) tortoises. Six state transitions were implemented
to model behavioral shifts between capture occasions (matrix ;
Supplementary Annex 2): SPSA and SPSSB were the respective
probabilities that a SP tortoise would become SA or display SSB
(line 1); SASP and SASSB were, respectively, the probabilities that
a SA tortoise would become SP or SSB (line 2); SSBSP and SSB-SA
were the respective probabilities that a tortoise displaying same sex
behavior would become SP or SA (line 3). Detection probability
was modeled as follow (matrix P; Supplementary Annex 2): P SP,

P SA, and P SSB were the probabilities that a tortoise was recaptured
at a capture occasion and observed as SP, SA, or displaying SSB,
respectively. Note that the state dead was not observable. This
is an absorbing state representing death (there was no emigration
from the island), used to ensure that model parameters would be
estimable (Lebreton and Pradel 2002; Pradel 2005; Choquet,
Lebreton, etal. 2009; Choquet, Rouan, etal.2009).

Bonnet etal. Same-sex sexual behaviors in wild tortoises

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subsample of adults monitored for the CMR study on Golem

Grad. We notably ensured that the individuals did not detect the
observers (i.e., no sign of disturbance) at capture. This fast procedure limited the possible rapid negative effect of stress on steroid
levels (Michel and Bonnet 2014), thus enabling us to consider that
T levels and behaviors were recorded simultaneously. We also collected blood samples in adult tortoises from the nearby mainland
Konjsko population using the same method. In total, we collected
283 blood samples (N=216 on Golem Grad [31 females and 185
males] and N=67 on Konjsko [46 females and 21 males]).
Blood samples (100300L, adjusted to tortoise body size) were
taken during activity time (morning and afternoon) from the jugular vein or cervical sinus using very small needles (2730 G) and
slightly heparinized 1-mL syringes. Blood was centrifuged in the
field, plasma samples were immediately stored in liquid nitrogen,
and then at 25C in the laboratory until assays. Plasma T levels
were measured by radioimmunoassay at the CEBC (Lagarde etal.
2003)on 50L of plasma after diethyl ether extraction (efficiency
0.930.1, standard deviation [SD]). Cross-reactivity of the specific antibody (Sigma Laboratory) with other steroids was low (B/

B0: 5-dihydrotestosterone: 17.8%; 5-androstene-3, 17-diol:

1.4%; 5-androstene-3, 17-diol: 1.2%; androstenedione: 1.4%;
epitestosterone: 0.7%; progesterone: 0.07%). The assays sensitivity
was 7.8 pg by tube, that is, 156 pg/mL. Intra-assay and interassay
variations were 6% and 12%, respectively.

Ethicalnote
All procedures were performed in accordance with Macedonian,
Serbian, and French guidelines and regulations. No individual was
mistreated or injured. Permits no. 03-246, 353-01-46/2012-03 and
no.11-4093/5 to perform population monitoring were issued by Galiica
National Park authorities (FYROM) and the Ministry of Environment,
Mining and Spatial Planning (Serbia). Ethical procedures were approved
by the ethical committee COMETHEA (permit no. CE2013-6).

Between 2008 and 2014, we collected 7503 behavioral observations

on Golem Grad (Table 1). Adult females were more frequently
(b)

1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

Active SSB
Active heterosexual
Sexually inactive

2009

2010

2011

2012

2013

2014

Probability to shift from

sexually inactive to SSB
from t to t+1

Sighting probability

(a)

0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00

Plateau

Shore

Figure2
Annual resighting probabilities (mean standard error [SE]) of adult male tortoises as a function of their behaviors. (a) Active heterosexual refers to
males courting females or involved in male-to-male combats; active SSB refers to males courting other males (e.g., mounting); and sexually inactive includes
individuals observed: 1)inactive (e.g., resting), 2)active but not involved in sexual activity (e.g., feeding), and 3)SP (e.g., mounted) (see Methods for details).
(b) Annual probabilities for a male in year t (sexually inactive) to engage in SSB in year t + 1 in each of the 2 subpopulations of Golem Grad (mean SE).
Table2
MSMR analyses of male tortoise behaviors
np

Deviance

QAIC

QAIC

1
2
3
4
5

90
34
58
40
28

10220.4
11092.4
10257.3
10277.2
10391.5

10400.4
11160.4
10373.3
10357.2
10447.5

43.2
803.2
16.1
0
90.3

6
7

38
37

10218.3
10305.2

10357.3
10379.2

0.1
22

8
9

34
37

10290.1
10284.9

10358.1
10358.9

0.9
1.7

Model
Modeling resighting probability
S(sex site) (sex site state) P(sex site state t)
S(sex site) (sex site state) P(sex site state)
S(sex site) (sex site state) P(sex state t)
S(sex site) (sex site state) P(state t)
S(sex site) (sex site state) P(t)
Modeling survival probability
S(sex) (sex site state) P(state t)
S(.) (sex site state) P(state t)
Modeling transition probability
S(sex site) (sex state) P(state t)
S(sex site) (site state) P(state t)

Modeling probabilities of survival (S), resighting (P), and transition between behavioral
states () of Hermanns tortoises on Golem Grad, 20082014. The
2
selected model is indicated in bold. The initial model (model 1)fitted to the data ( 105 =110.2, P=0.345). np, number of parameters; sex, sex dependence;
site, site dependence; state, state dependence; t, time dependence; , interaction; . constant parameter.

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RESULTS
Sexual behaviors

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We estimated the annual number of heterosexually active individuals versus individuals exhibiting SSB using raw numbers of
individuals captured each year along with their capture probabilities from the best-supported model (Table 2). In order to estimate
confidence intervals, we used parametric bootstrapping. Each
year on average, the total estimated number of adult tortoises was
108040 (standard error); 9727 displaying HSB versus 13934
exhibitingSSB.
Following the same approach, we also estimated the total population size and the numbers of females and males. Mean annual
population size was 1038228 adult tortoises, 5615 females, and
979228 males. The mean estimated SR was 17.5.

inactive (i.e., partly sheltered and motionless) compared with adult

males (2=30.6, P<0.001, Table1).
We observed many classical HSB and male-to-male combats,
but most of the sexual behaviors recorded on this small island were
SSB (Table1): For instance, on 295 occasions, a male was observed
mounting another male (with vocalizations and/or ejaculation).
SSB (i.e., mounting + courting and so on, Table 1) largely dominated among plateau-males (74% SSB, N = 786), whereas HSB
were more frequent among shoreline-males (33% SSB, N = 102).
SSB was exhibited by different individuals (N=363) and thus was
attributable to males at large (not to few individuals).
CMR modeling results suggest that resighting probability was lowest for SA individuals involved in HSB, highest for sexually inactive
individuals, and intermediate for individuals displaying SSB (Table2,
Figure 2a). Transition probabilities between SSB versus HSB were
sex and site dependent (Table2, model 4). However, QAIC between
models 4, 6, and 8 were low. We selected model 4 (lowest absolute
QAIC) for biological reasons (Burnham and Anderson 2002). Indeed,
this model takes into account differential survival between sites, and
this conforms, for example, to the fact that females face greater environmental difficulties on the plateau compared with the shoreline
(e.g., water unavailability during summer). Furthermore, by selecting
the second best model (6), the transition probabilities between sexual
behaviors were still site and sex dependent.
Individual males were not strictly homosexual or heterosexual,
shifting between SSB and HSB. However, the annual probabilities
for a sexually inactive male in year t to engage in SSB in year t +
1 were significantly higher on the plateau than on the shoreline
(Figure2b).

TESTOSTERONE PLASMA
CONCENTRATION (ng/ml)

GOLEM GRAD

We first compared T levels between sexes and mainland/island

populations (using generalized linear model [GLM; Statistica 12.0,
StatSoft 2013, www.statsoft.fr] [log Poisson model] with sex [male/
female] and population [mainland/island] as fixed effects and
testosterone level as the dependent variable). Mean T levels differed between sexes (Wald test=215.82, P<0.001) and between
mainland and island populations (Wald test = 24.81, P < 0.001).
Females displayed markedly lower T levels compared with males;
the difference between mainland and island populations appeared
weak (Figure3). However, removing the population factor from the
model decreased adjustment quality (AIC increasing from 1015.81
to 1036.47, AIC > 2), suggesting a significant effect. This population effect was caused by a difference between mainland and
island males: T levels were slightly albeit significantly lower on
Golem Grad (4.63.5 [SD] ng/ml), compared with Konjsko males

KONJSKO

c
3

10
bc
13

bc
8
6

26
a
123

ab
13

abc
4

17

4
2

21

31
0

E
E SSB SSB NG
T
I
BA
AL
ON
AL SIVE TIVE MAT COM FEM
S AC
RO
PA
TE
HE

E
E NG
I
AL
ON
AL MAT FEM
RO
TE

HE

BEHAVIOUR AT BLOOD SAMPLING

Figure3
Mean plasma levels of testosterone ( standard error [SE]) and sexual behaviors in randomly sampled free-ranging adult tortoises from Golem Grad Island
(circles, left panel) and from a neighboring mainland population (Konjsko, diamonds, right panel). The vertical dashed line facilitates the distinction between
island and mainland populations. Numbers above symbols stand for sample sizes. Males found alone (ALONE) or mounted by another male (PASSIVE SSB)
were considered as SP (gray circles and gray diamond). Males mounting another male (ACTIVE SSB), mounting a female (HETERO MATING), or during
male-to-male combat (COMBAT) were considered as SA (black circles and black diamond). Females (open circle and open diamond) were not observed
SA. Note that SSB were observed only on Golem Grad. Strong sex differences and population effects were observed (see text for details). Letters denote
significance differences among groups of males.

Downloaded from http://beheco.oxfordjournals.org/ at CNRS-CEBC on March 22, 2016

12

Testosterone

Bonnet etal. Same-sex sexual behaviors in wild tortoises

Page 7 of 10

in the most active males) with sufficient statistical power, we used

broad behavioral categories: SA (N = 46) versus sexually inactive
males (N = 157). Mean T levels differed between these groups
(GLM, Wald test=19.86, P<0.001) with a mainland/island effect
(GLM, Wald test=20.77, P<0.001). Overall, our results suggest
a relationship between T level and sexual activity in males without
specific effect of SSB.

DISCUSSION
The expression of any phenotypic trait, including sexual behaviors, can be examined at proximate and ultimate levels. Laboratory
studies of SSB in vertebrates suggest that intrinsic drivers, prenatal
factors notably, influence sexual orientation in adults (Balthazart
2011). In natural populations, mostly adaptive hypotheses have
been proposed for the high frequency of SSB observed in different taxa (Bailey and Zuk 2009). Yet, a recent review on arthropods
revealed that SSB are principally displayed by sexually stimulated
males and result essentially from nonadaptive mistaken identifications, especially under high density and strongly skewed SR (Scharf
and Martin 2013). Our data collected in wild tortoises monitored
during 7 years support this option where SSB is triggered by specific environmental factors and where adaptive explanations play a
limitedrole.
In free-ranging male Hermanns tortoises, high population densities combined with strongly male-biased SRs possibly generated a
prison effect (Bailey and Zuk 2009) and SSB became more common than HSB in this situation only (Table3). Hormonal investigations suggested that the high expression of SSB correlated with high
T levels. These high T values remained within the range of natural

Table3
Population density (number of individuals per hectare [D]), numbers of adults sampled (N), and SR (adult males/adult females)
among 16 populations/subpopulations of Testudo hermanni boettgeri monitored in Greece (data from the Tables 2 and 3 in Hailey
and Willemsen 2000)and in Macedonia (Golem Grad, Konjsko) and in 3 other species of Testudo (Testudo hermanni hermanni,
Ballouard JM, unpublished data; Testudo horsfieldii, Bonnet etal. 2001; Lagarde etal. 2002; and Testudo graeca, Lagarde etal. 2008;
Bonnet X, unpublished data)
Species

Site

T.h.hermanni
T.h. hermanni
T.h. hermanni
T.h. boettgeri
T.h. hermanni
T.h. boettgeri
T.h.hermanni
T.h.boettgeri
T.horsfieldii
T.h.boettgeri
T.graeca
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri
T.h.boettgeri

Callas
Saint Daumas
Lambert
Deskati
Redon
Mikra Volvi
Riaux
Litochoron
Bukhara
Langadia
Marrakech
Kilkis
Parga
Olympia
Kalamata
Agios Dimitrios
Konjsko
Meteora
Kastoria
Sparta
Igoumenitsa
Golem Grad Plateau
Golem Grad Beach

1.2
1.2
1.6
3.7
4.2
4.6
5.2
5.3
5.4
5.9
5.9
11.3
12
15.4
18.2
20
20
20.2
20.5
20.6
39.6
59.7
130

258
123
64
201
105
338
78
102
354
71
192
99
187
1378
262
230
322
4855
265
814
237
1075
131

SR

SSB %

HSB

0.93
0.84
1.21
4.02
1.55
3.69
1.69
2.09
1.21
0.55
1.21
2.81
0.83
1.61
1.65
0.84
1
1.9
1.85
2.31
6.41
18.19
3.36

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
74
33

27
71
4

29

27

314

192

45

204
68

SSB were observed in males only (expressed as % relative to all sexual behaviors recorded). The last column indicates the number of HSB observed. Populations
were ranked according to increasing density.

Downloaded from http://beheco.oxfordjournals.org/ at CNRS-CEBC on March 22, 2016

(6.93.3ng/ml; Tukey post hoc test P < 0.01); no difference was

observed in females (P=0.916).
SSB and other SA behaviors (e.g., courting, mounting, fighting),
and high T levels were exclusively observed in males. Therefore, we
focused on this sex. Breaking up the males behaviors into categories was required to gain a more fine-scale view of how T related to
different behaviors. Using the main behaviors exhibited by males,
we distinguished 5 types of behaviors: males found alone, mounted
by another male, mounting another male, mounting a female, or
during male-to-male combats (Figure 3). A GLM revealed a significant effect of behavior types on T levels (Wald test = 38.41,
P < 0.001) and a significant difference between mainland and
island populations (Wald test = 16.87, P < 0.001; as above AIC
was lower in the full model, AIC=13.7). Further analyses (comparing all parameters estimated or using post hoc tests) suggested
that SP males (alone or mounted) exhibited lower T levels compared with males involved in the most vigorous sexual behaviors
(combat) whereas other active males (mounting) displayed intermediate T levels (Figure3). Because SSB was observed exclusively
in Golem Grad males, we restricted some analyses to these individuals: Results were unchanged (GLM analysis revealed a strong
effect of behavior type, Wald test = 37.37, P < 0.001). Thus, we
observed a trend of higher T levels in those males sampled during most demanding sexual behaviors, without an influence of SSB.
Furthermore, the significant effect of mainland/island populations
and examination of Figure3 suggest that this trend was specific to
GolemGrad.
Sample sizes were small in males from the continent and in
fighting island males, shedding uncertainty on some of the results
above. Therefore, to examine the main effect (i.e., higher T level

Behavioral Ecology

Page 8 of 10

Overall, SSB may emerge and persist in natural populations because

the costs for total SSB inhibition outweigh the costs of nonadaptive
SSB (Thornhill and Alcock 1983; Burgevin etal. 2013; Scharf and
Martin 2013; Engel et al. 2015). Mechanisms that eliminate SSB
may negatively impact on HSB, for example, through a decrease of
general sexual motivation if common neuroendocrine pathways are
involved. Precisely, in vertebrates, T stimulates hypothalamic areas
that promote both heterosexual and homosexual activity (Bakker
etal. 2002, 2006). We speculate that the development, maintenance,
and functioning of specific neuroendocrine structures that selectively
filter out SSB might be too expensive (and useless) in male tortoises.
Other unusual sexual behaviors observed on Golem Grad Island,
especially on the plateau, reinforce the notion that the most SA adult
males deprived of females were highly stimulated (Figure 1). Males
(N=12) were observed mounting juveniles, dead males, and tortoise
skeletons (empty shells of males). Two were seen attempting to copulate with stones that vaguely resembled a tortoise (Figure 1). In captivity, male SSB plus strange sex-toy behaviors have been reported in
tortoises (Internet search using keywords tortoise and shoe). These
cases of juvenile mounting, homosexual Davian behaviors, skeletophilia, or petrophilia illustrate that the strong sexual motivation of
males can overrule accurate discrimination of appropriate sexual partners. In the nearby mainland Konjsko population, in other Hermanns
tortoise populations, and in other Testudo species, uneven SR (generally
unbiased or male biased) and varying population densities have been
documented (Table3). However, the combination of extremely skewed
SR with very high population density and SSB was observed only on
Golem Grad. We thus suggest that frequent SSB are expressed above a
threshold that combines high density and skewed SR in physiologically
predisposed individuals (e.g., male tortoises with high T levels).
Considering other adaptive or nonadaptive explanations does
not change our main conclusions. For example, mate selection by
females would reinforce the scarcity of partners for the males; the
effect of practice (Immature individuals learn through SSB;
Bailey and Zuk 2009) is unlikely in tortoises because sexual behaviors were never observed in juveniles or in other contexts than Golem
Grad. The tortoises of Golem Grad provide the first documented
example of a prison effect in free-ranging animals and thus offer
a strong support to the hypothesis of nonadaptive causation for SSB.
This finding suggests that proximate factors underlying SSB are not
necessarily restricted to physiological disorders or require an adaptive
basis (although indirect links between nonadaptive SSB and mating
success exist; Logue etal. 2009). Under conditions with strong social
stimulation and a scarcity of partner, SSB may be common.

SUPPLEMENTARY MATERIAL
Supplementary material can be found at http://www.beheco.
oxfordjournals.org/

FUNDING
The Ministry of Education, Science and Technological
Development of Republic of Serbia provided fund (grant #173043).
We thank the Galiica National Park authorities for issuing permits. We
thank Mitko Tasevski, colleagues, and students for support in the field.
R.Shine offered valuable help to improve the English. Anonymous reviewers provided very useful comments.
Handling editor: John Fitzpatrick

Downloaded from http://beheco.oxfordjournals.org/ at CNRS-CEBC on March 22, 2016

variations however (e.g., Golem Grad vs. Konjsko), thus SSB did
not require specific adaptive or pathological explanation. In both
the mainland and island populations, T levels were high in adult SA
males, in accordance with the paradigm that androgenic steroids
(e.g., T, 11-keto testosterone) are primary stimulators of sexual activity in male vertebrates. Moreover, the most SA males (i.e., mating
with females, engaged in SSB or combats, Figure3) exhibited higher
T levels, as observed in other reptiles (Aubret etal. 2002; King and
Bowden 2013). Because an individual is not continuously SA, some
of the inactive individuals monitored during random-focal blood
sampling undoubtedly were SA animals that we observed between
bouts of reproductive behavior (especially in mainland tortoises
where lower density decreases encounter rate). Nonetheless, we
recorded higher T in males displaying the most vigorous behaviors:
fighting or mounting other males. Therefore, this suggests that the
combination of high density and skewed SR led to frequent SSB in
males, notably in individuals strongly stimulated by high T levels.
External sexual stimuli (e.g., visual/chemical signals from conspecifics) can activate the brain structures involved in sexual arousal in
adults (Arnow etal. 2002), high density promotes courtship behaviors
in male tortoises (Hailey and Willemsen 2000), and T level correlates
with sexual reactiveness (Amstislavskaya and Popova 2004; Goldey
and van Anders 2015). On Golem Grad, these factors were strongly
amplified in males. Under strong sexual stimulation caused my multiple factors (high density + scarcity of females + high T levels), males
attempted to copulate with the first encountered partner, generally
females on the shoreline but more often with males on the plateau
(video in Supplementary Material). A relatively similar behavioral
libido syndrome where the occurrence of heterosexual courtships
positively correlates with SSB has been described in captive cockroaches (Logue etal. 2009). Interestingly, libido scores also correlated
with mating success, suggesting that selection for high heterosexual
courtship intensity entailed nonadaptive SSB (Logue etal.2009).
The studied population presents several exceptional characteristics and thus suffers from a lack of replication. Experiments where
density and SR are manipulated are needed to gauge the validity
of our conclusions. However, our results are based on a large sample size and long-term monitoring. Furthermore, the quasi-experimental shoreline/plateau situation of Golem Grad, along with the
comparison with other populations belonging to different species
(Table3), offers support to the nonadaptive prison effect hypothesis (Eigenberg 2000; Bailey and Zuk 2009). Plateau-males and to
a lesser extent shoreline-males can rarely find adult females, but
instead encounter many male tortoises that morphologically resemble females while they are under T stimulation. Hyperstimulation
and/or frustration created by a similar situation may promote frequent SSB as documented in feral-domestic cats (Yamane 2006).
Deprivation of member(s) of the opposite sex generates sexual
strain that can be relaxed by different behavioral outlets including
onanism, SSB, or interspecific sexual behaviors (Exton et al. 2001;
Yamane 2006; Sakaguchi et al. 2007). A positive impact of abstinence (a proxy of frustration) on both T levels and sexual motivation
has been documented in humans (Exton etal. 2001; Sakaguchi etal.
2007). Yet, nonadaptive SSB may simply reflect behavioral mistakes,
such as inaccurate sexual partner discrimination (Scharf and Martin
2013). Although these explanations are not exclusive, the hypothesis
that males cannot discriminate sex has been rejected experimentally
(Galeotti et al. 2007, 2011). Many studies demonstrated the strong
ability of tortoises, and of nonavian reptiles in general, to use chemical cues to distinguish sexual partner (Cooper and Prez-Mellado
2002; Shine etal. 2003; Poschadel etal. 2006; Ibez etal. 2012).

Bonnet etal. Same-sex sexual behaviors in wild tortoises

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