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\begin{document}

\title{Investigating the reproductive behaviour of the green sea turtle
(Chelonia mydas) using parentage analysis}



\author[1]{Violaine Dolfo}%
\author[2]{Cécile Gaspar}%
\author[3]{Miri Tatarata}%
\author[1]{Emilie Boissin}%
\author[4]{Serge Planes}%
\affil[1]{EPHE PSL}%
\affil[2]{Te mana o te moana Fundation}%
\affil[3]{Direction de l'Environnement de la Polynésie Française}%
\affil[4]{University of perpignan}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The reproductive systems of natural populations can greatly impact their
genetic diversity by preventing or encouraging inbreeding. It is
therefore crucial to have a comprehensive understanding of the mating
system to evaluate a population's ability to maintain genetic diversity
over time. In this study, we examine the mating system of an endangered
population of green sea turtles in Tetiaroa, French Polynesia. We
determine if different mating behaviours serve as strategies to avoid
inbreeding. We genotyped 107 nesting females and 1483 hatchlings from
549 nests and used 23 microsatellite markers to reconstruct the
genotypes of the fathers. We assessed the level of inbreeding and
relatedness of the parent pairs and explored the correlation between
relatedness and fitness parameters in the offspring. We determined the
mating behaviours of both males and females and investigated if specific
behaviours were linked to different levels of relatedness. Our results
showed that 27 fathers and 31 mothers were responsible for the genotypes
of 445 hatchlings from 105 nests. Global Fis was significant, and levels
of relatedness were higher than expected through random mating,
indicating inbreeding and non-random partner selection. However, we did
not find any mating behaviours that were associated with lower
relatedness levels than the general population, suggesting that they are
not part of an inbreeding avoidance strategy. Ultimately, this study
illuminates the reproductive system of green turtles and shows that this
population is susceptible to inbreeding. Additionally, our research
demonstrates the effectiveness of parentage analysis in understanding
the reproductive behaviour of elusive species.%
\end{abstract}%



\sloppy


Investigating the reproductive behaviour of the green sea turtle
(\emph{Chelonia mydas} ) using parentage analysis

\textbf{Running title} : Green Turtle Reproductive Behaviour

Violaine Dolfo \textsuperscript{1,*}, C\selectlanguage{ngerman}écile Gaspar\textsuperscript{2},
Miri Tatarata \textsuperscript{3}, Emilie Boissin \selectlanguage{english}\textsuperscript{1,4,
+}, Serge Planes \selectlanguage{english}\textsuperscript{1,4, +}

\textsuperscript{1} PSL Research University: EPHE - UPVD - CNRS, UAR
3278 CRIOBE, 66860~Perpignan, France

\textsuperscript{2}Te Mana O Te Moana Foundation, Papetoai, Moorea,
French Polynesia

\textsuperscript{3} Direction de l'Environnement de la Polynesie
Francaise, Papeete, Tahiti, French Polynesia

\textsuperscript{4} Laboratoire d'Excellence << CORAIL >>,
66860~Perpignan, France

\selectlanguage{english}+ These authors contributed equally to this work and share senior
authorship

* Corresponding author: violaine.dolfo@lilo.org

Cecile Gaspar: cecile.gaspar@temanaotemoana.org ; Miri Tatarata:
miri.tatarata@environnement.gov.pf. ; Emilie Boissin:
emilie.boissin@univ-perp.fr ; Serge Planes: planes@univ-perp.fr

\textbf{Conflict of interest disclosure}

The authors declare no conflict of interest.

\textbf{Ethics Approval}

\emph{Chelonia mydas} sample collection was authorized and coordinated
by the Direction of the Environment of French Polynesia. Non-lethal skin
and muscle biopsies were performed, which do not require any other
specific permits. Samples were exported to France for processing with
CITES permits ndeg FR1298700118-E and ndeg FR2098700187-E. All samples
remain the DIREN's property.

Abstract

The reproductive systems of natural populations can greatly impact their
genetic diversity by preventing or encouraging inbreeding. It is
therefore crucial to have a comprehensive understanding of the mating
system to evaluate a population's ability to maintain genetic diversity
over time. In this study, we examine the mating system of an endangered
population of green sea turtles in Tetiaroa, French Polynesia. We
determine if different mating behaviours serve as strategies to avoid
inbreeding. We genotyped 107 nesting females and 1483 hatchlings from
549 nests and used 23 microsatellite markers to reconstruct the
genotypes of the fathers. We assessed the level of inbreeding and
relatedness of the parent pairs and explored the correlation between
relatedness and fitness parameters in the offspring. We determined the
mating behaviours of both males and females and investigated if specific
behaviours were linked to different levels of relatedness. Our results
showed that 27 fathers and 31 mothers were responsible for the genotypes
of 445 hatchlings from 105 nests. Global \emph{F\textsubscript{is}} was
significant, and levels of relatedness were higher than expected through
random mating, indicating inbreeding and non-random partner selection.
However, we did not find any mating behaviours that were associated with
lower relatedness levels than the general population, suggesting that
they are not part of an inbreeding avoidance strategy. Ultimately, this
study illuminates the reproductive system of green turtles and shows
that this population is susceptible to inbreeding. Additionally, our
research demonstrates the effectiveness of parentage analysis in
understanding the reproductive behaviour of elusive species.

Keywords: genotype reconstruction, mating behaviour, fitness,
relatedness, multipaternity, inbreeding

Introduction

Reproductive systems strongly influence genetic diversity in natural
populations (Clegg et al., 1992). Loss of genetic diversity may lead to
inbreeding depression, loss of adaptive potential, and accumulation of
deleterious alleles, and ultimately to extinction (Charlesworth, 2009).
The number of breeders, their reproductive success, and the mating
strategies they use are key elements present in the mating system of a
population (Sugg and Chesser, 1994). A good understanding of these
factors is essential to assess the intrinsic capacity of natural
populations to maintain themselves through generations and to maintain
their genetic diversity (Anthony and Blumstein, 2000). Several
components of the mating system may either mitigate or favour
inbreeding. They may be precopulatory such as the operational sex-ratio
(OSR, i.e. relative number of breeders from both sexes, (Emlen and
Oring, 1977)), reproduction frequency, and partner choice and number
(Blouin and Blouin, 1988; Taylor et al., 2014). Postcopulatory factors
include sperm storage and sperm competition (Michalczyk et al., 2011).

OSR is a key feature of the mating system, especially for species with a
temperature-dependent sex determination (TSD) like the marine turtles
(Standora and Spotila, 1985). The primary sex-ratio of TSD species is
solely dependent on environmental conditions and can be heavily biased
under climate warming situations (Janzen, 1994). The primary sex-ratio
of several marine turtle populations is female-biased (Casale et al.,
2000; Jensen et al., 2018; Santidrian Tomillo et al., 2014; Zbinden et
al., 2007), but the OSR is usually much more balanced (Hays et al.,
2022), mitigating concerns about the vulnerability of a population.
However, as highlighted by Wright et al. (2012a), if the OSR reflects a
small number of males that breed more frequently than females, it will
still lead to a loss of genetic diversity and inbreeding. Due to the
high energetic cost of reproduction, which may include long migrations
between foraging and nesting grounds, female marine turtles typically
reproduce at intervals of several years (Hays et al., 2014). For the
green turtle, \emph{Chelonia mydas} , reproduction frequency in females
is estimated between 2 and 5 years (Seminoff et al., 2015). In contrast,
males can mate more frequently, and annual migration to breeding grounds
has been observed in loggerhead (\emph{Caretta caretta} ), leatherback
(\emph{Dermochelys coriacea} ) and green turtles (James et al., 2005;
Limpus, 1993; Wibbels et al., 1990). Knowing the reproduction frequency
of both sexes is thus important to correctly interpret the OSR
concerning inbreeding risk.

Both male and female marine turtles can reproduce with several partners
(i.e. polygynandry), which tends to equilibrate the OSR (Jensen et al.,
2013). Polyandry is observed through multiple paternity in clutches,
which has been found at various degrees in all marine turtle species.
For green turtles, it ranges from 15\% to 92\% of the clutches depending
on the populations (reviewed in Lee et al., 2018). Multiple paternity is
a direct consequence of seasonal sperm storage in females, a capacity
well established in Testudines (Pearse and Avise, 2001) and observed in
six of the marine turtle species (Crim et al., 2002; FitzSimmons, 1998;
Kichler et al., 1999; Phillips et al., 2013; Sakaoka et al., 2011;
Theissinger et al., 2009). Satellite tracking and direct observations
showed that breeding usually occurs before the beginning of the nesting
season and all the clutches laid within a season are usually sired by
the same fathers (Hays et al., 2022). However, sperm storage across
multiple seasons has not been formally demonstrated in marine turtles,
as opposed to terrestrial and freshwater turtles (Owens, 1980; Whitaker,
2006), but several studies have suggested that it is likely (Howe et
al., 2017; Theissinger et al., 2009; Wright et al., 2013).

It has been proposed that females would benefit from polyandry and sperm
storage. The `good genes' hypothesis relies on the assumption that by
mating with different partners, better quality sperm would outcompete
lower quality sperm, which would lead to an increased fitness of embryos
sired by the dominant father (Kokko et al., 2002). However, while many
studies have attempted to prove this hypothesis, no correlation has yet
been found between multipaternity and fitness parameters in the clutches
(Jensen et al., 2006; Lee et al., 2018; Lee and Hays, 2004; Wright et
al., 2013, but see Howe et al., 2017). On the other hand, the `genetic
compatibility' hypothesis predicts that paternity would be biased
towards genetically dissimilar males to avoid inbreeding (Bretman et
al., 2009; Zeh and Zeh, 1997). In an attempt to test this latter
hypothesis in a hawksbill turtle population in the Republic of
Seychelles, Philips et al. (2013) did not find any correlation between
relatedness and paternity contribution in the clutches. Ultimately, all
of these studies tend to conclude that polyandry is likely to occur as
an energy cost trade-off between mating several times and avoiding
mating harassment (i.e., convenience polyandry, Lee and Hays, 2004).
Male sea turtles are known to actively and aggressively attempt mating,
and females may try to avoid it (Booth and Peters, 1972). Thus, levels
of multipaternity may simply reflect the density of breeders on
reproductive grounds and the number of encounters between the two sexes
(Lee et al., 2018).

Understanding the mating system of sea turtles is challenging, notably
due to the lack of observations of males and breeding grounds. For
species as elusive as these, molecular analyses can provide important
insights. The use of microsatellite markers allowed for the genotypes of
unsampled fathers to be reconstructed and to thus assess the mating
system parameters in several marine turtle populations (e.g. Bernatchez
and Duchesne, 2000; Figgener et al., 2016; Horne et al., 2022; Phillips
et al., 2013; Wright et al., 2012). Together, these studies reveal that
regional variations in these parameters are the rule rather than the
exception, and many regions are still lacking this key information for
their populations.

French Polynesia, in particular, is an archipelago composed of 118
islands distributed over an exclusive economic zone of 5 million
km\textsuperscript{2}, a surface as wide as Europe. The country is
thought to host approximately 1,000 female green turtle breeders,
although no recent assessment exists (Groombridge and Luxmoore, 1989;
Seminoff et al., 2015). In the centre of French Polynesia, Tetiaroa
Atoll (Society archipelago) is one of the major nesting grounds. It is
estimated that around 100 females nest annually on the atoll (Seminoff
et al., 2015; Touron et al., 2018), and mating behaviours are observed
around the island (Gaspar, pers. comm.). This location provides a unique
opportunity to closely investigate the mating system of green turtles
with molecular marker analyses. Here, we genotyped nesting females and
hatchlings from Tetiaroa with 23 microsatellite markers, reconstructed
male genotypes, and conducted parentage analysis to study the mating
behaviours of both sexes.

The aim of this study is threefold. First, we characterize the level of
inbreeding and relatedness in the population of green turtles nesting in
Tetiaroa and explore their effect on fitness, by measuring three fitness
parameters in the clutches: the number of eggs, successful hatchlings,
and dead embryos. Second, using reconstructed pedigrees, we determined
the features of the mating system and nesting parameters of this
population. These parameters, which are key to inbreeding depression
dynamics, include the OSR, the reproductive frequency, the level of
multipaternity, the female's nesting intervals, and the number of
partners for the males. Third, we test whether the mating strategies
deployed reduce the overall relatedness and contribute to avoiding
inbreeding by comparing the relatedness of couples involved in these
strategies with the overall relatedness of the population. To our
knowledge, this work is the first to explore the relatedness bias of
mating behaviours in green turtles. It improves our general
understanding of the drivers of mating strategies in sea turtles.

Material and Methods

\textbf{Study site and sample collection}

Sampling was conducted on Tetiaroa atoll (16deg59' S, 149deg34' E), French
Polynesia, during eleven nesting seasons from 2010/11 to 2020/21. Sample
collection was authorized and coordinated by the Direction of the
Environment of French Polynesia. Tetiaroa atoll has a total surface of 6
sq. km, about 585 hectares of sand, and is divided into 12 islets
(Figure 1). Between 2010/11 and 2017/18, 53 to 1316 nesting events per
season (July - April) were recorded by the local NGO \emph{Te mana o te
moana} (Touron et al., 2018). Biopsies of approximately
0.5cm\textsuperscript{3} of skin and muscle tissues were collected from
the posterior fin of all the observed females and on all dead hatchlings
and embryos found. For a total of 6 nests, more than 10 hatchlings were
sampled. The laying date was either recorded when laying was directly
observed, or estimated when the nest was discovered. In this case, a
confidence interval of +- 3 days was applied to all of the estimations.
Since 2010/11, monitoring has gradually increased and by 2016/17, almost
all nests were sampled each season. However, nesting females were not
always observed. (Touron et al., 2018). Nest parameters such as clutch
size, number of hatchlings (estimated from empty eggshells), and number
of dead embryos were recorded for each nest. Samples were stored in 90\%
ethanol and kept at 4degC or -20degC until processing.

\textbf{Molecular analyses}

Total genomic DNA was extracted using the QIAamp 96 DNA QIAcube HT Kit
and the QIAcube DNA extraction robot (QIAGEN, Hilden, Germany) following
the manufacturer's protocol with modification as described in Dolfo et
al. (2023). Samples were genotyped at 23 microsatellite loci using five
multiplex reactions as per Dolfo et al. (2023) (GenBank accession
number: OQ162049-OQ162073). Allele sizes were visually assessed using
GENEMAPPER software v.5 (Applied Biosystems). All ambiguous peak
profiles were considered as missing data and only individuals with
missing data which occurred at 3 loci or less were kept for the
analyses. MICROCHECKER v.2.2.3 (Van Oosterhout et al., 2004) was used to
identify null alleles, stuttering errors, and large allele dropout. In
total, 1592 hatchling and female genotypes were kept for further
analyses.

\textbf{Conceptual workflow}

To answer the three goals defined above, analyses were carried out with
the following conceptual workflow (Figure 2). First, the population
state was described through the number of breeders, the OSR, and the
level of inbreeding and relatedness between reconstructed couples. The
effect of the relatedness on the fitness parameters in the clutches was
investigated. Then, the reproductive behaviours of the population were
described for both sexes, i.e. the reproduction frequency, nesting
and/or reproductive parameters within a season, and levels of
multipaternity. Finally, we tested whether these behaviours mitigate or
favour inbreeding by comparing the relatedness of couples involved with
the relatedness of all reconstructed couples.

\textbf{Identification of recaptured individuals}

The R package \emph{RClone} (Bailleul et al., 2016) was used to identify
duplicate genotypes in the dataset and to detect recaptured individuals.
Using the allelic frequencies of the dataset, \emph{RClone} simulates a
reproduction event and compares the genetic distances between genotypes
in the simulated and the observed populations. This allows for a
threshold to be determined, below which genotypes in the observed
population may be too similar to occur as a result of reproduction.
Thus, the genotypes of all individual pairs that had less than 5
differences at any locus were double-checked with GENEMAPPER, and the
data were crosschecked with field information to identify recaptured
individuals.

\textbf{Calibration of the set of loci}

To test the accuracy of the motherhood assignment provided by the
software COLONY v.2.0.6.6 (Jones and Wang, 2010), we calibrated the
assignment probability based on known mother-offspring relationships.
The probability of assignment of a mother was plotted against its true
motherhood, based on the known mother-offspring relationships from the
field, which involved 58 mothers and 275 hatchlings from 105 nests.
COLONY uses a full likelihood model to assign sibship and parentage
relationships using multilocus genotype data. Its algorithm allows for
missing data and genotyping errors. Three long runs were performed with
high likelihood precision, allowing polygamy for both sexes under an
inbreeding model, and assuming an error rate of 0.01 for both allelic
dropout and genotyping error. It is assumed that hatchlings sampled in
2010/11 did not reach sexual maturity by 2020/21 based on the mean age
at maturity of 25 years (Chaloupka et al., 2004), therefore, we did not
consider the occurrence of overlapping generations. All the hatchlings
were considered as potential offspring and all the females were
candidate mothers.

To determine the effect of genotyping errors on the accuracy of
motherhood assignment, the above calibration was run with three
different subsets of markers to determine which one would best perform
in assigning true mother-offspring relationships: subset 1 comprised all
23 loci described in Dolfo et al. (2023); in subset 2 the loci which
were deviant from Hardy-Weinberg equilibrium, as emphasized by the
authors, were removed, leaving 14 loci (list in Supplementary Data 1);
in subset 3 the loci which were given the highest error rate by COLONY
were removed (CMY19, CMY22, and CMY35), leaving 20 loci. Based on this
calibration (Supplementary Data 1) all 23 loci were retained for the
analyses.

\textbf{Parentage analyses}

COLONY was run with 23 loci in the same configuration as for marker set
calibration, to assign candidate mother-offspring relationships and to
infer the genotype of unknown parents. For genotyped mothers (n=107), an
offspring assignment probability threshold of 0.9 was used based on the
results of the above calibration. For genotypes that were reconstructed,
COLONY only provides a probability per locus. A probability threshold of
0.9 is usually a good choice for conservative results, however, in our
case, this threshold would discard 93\% of the genotypes. Thus,
probability thresholds from 0.4 to 0.9 were examined to determine the
best trade-off between conservative and robust results (i.e. number of
samples retained) (Supplementary Data 2). Finally, only reconstructed
loci with a probability superior to 0.8 were retained for the analyses,
while the others were considered as missing data. Like genotyped
individuals, individuals with missing data at more than 3 loci were
discarded.

Finally, the dataset was checked for inconsistencies (for example, when
more than one mother was found for a single nest) and the least likely
relationship (i.e. with the lowest probability) was removed. Ultimately,
a parent-offspring relationship was only retained when both parents
(assigned or reconstructed) passed the different thresholds: 0.9 for
assigned mothers, 0.8 for reconstructed loci, and less than 3 missing
loci for a given genotype.

From parentage assignments, OSR, female and male reproduction
frequencies were determined. Additionally, within-season parameters such
as multipaternity, female nesting interval, and the number of partners
for males, were reported. The effect of multipaternity on fitness
parameters (clutch size, number of hatchlings, number of dead embryos)
was investigated by performing a two-tailed Student's t-test on the
average.

\textbf{Relatedness and inbreeding}

The inbreeding coefficient (\emph{F\textsubscript{is}} ) was calculated
with GENETIX v4.05.2 (Belkhir et al., 2004) on all genotyped females and
one offspring per nest (n=665). Pairwise relatedness coefficients
(\emph{r} ) were calculated with the R package \emph{related} v1.0. (Pew
et al., 2015) for all parent pairs found using COLONY, using sample
allele frequency. Simulations were performed based on the observed
allele frequency to determine which of the four relatedness estimators
implemented in the package performs best on our data (i.e. is the
closest to expected relatedness values). A hundred simulated genotypes
for each of the four relationships were generated and their
\emph{r}coefficient was calculated (parent-offspring, expected \emph{r}
= 0.5; full-sibling, expected \emph{r} = 0.5, half-sibling, expected
\emph{r} = 0.25, unrelated, expected \emph{r} = 0). The Wang estimator,
which yielded the best fit to expected relatedness values, was then used
for relatedness calculation of reconstructed parent pairs (Supplementary
Data 3). Relatedness of reconstructed vs potential parent pairs was
calculated, and a two-tailed Student's t-test on the mean was performed
to test for significant differences between the two relatedness
distributions. To explore the effect of relatedness on fitness, clutch
size, total number of hatchlings, and number of dead embryos were
plotted against the relatedness of parent pairs. Additionally, the
relatedness of pairs involving i) a male that mated with multiple
partners (within a season or across seasons), ii) a female that mated
with multiple partners (within a season or across seasons), iii) the
female and dominant male from clutches with multipaternity, and iv) a
female that that nested over several years, were compared with the
relatedness of all the reconstructed parent pairs to determine whether
any of these behaviours led to a deviation of the observed relatedness,
and significant differences on the mean relatedness were tested with a
two-tailed Student's t-test.

Results

\textbf{Recapture identification}

Genotypes were obtained from 1483 hatchlings representing 549 nests and
107 females. Sample size per nest ranged from 1 to 83, with 15 nests
having more than 10 samples. The 23 loci had a
mean\emph{H\textsubscript{e}} of 0.66 +- 0.04 and a
mean\emph{H\textsubscript{o}} of 0.62 +- 0.04. Using the R
package\emph{RClone} , two genotype clones were identified (Table 1).
They correspond to two females, both of which were sampled for the first
time in 2016-2017 and resampled after 4 years in 2020-2021. One of these
females was correctly identified in the field by its flipper tag, while
the other was thought to be an unknown female and was given a different
name at the second encounter.

\textbf{Parentage analysis}

After COLONY's parentage reassignment, genotype reconstruction, and
filtering with the different thresholds, 105 nests were assigned to 27
genotyped mothers, and 13 were assigned to 4 reconstructed females
between seasons 2014/15 and 2020/21. The total of 118 nests accounted
for 448 offspring. One hatchling that was not assigned to any nest was
linked with a genotyped mother. Out of these 118 nests, 70 counted more
than one genotyped hatchling. Once the father's genotypes were
reconstructed, a total of 27 fathers explained the genotypes of the 448
hatchlings. The operational sex ratio (OSR) ranged from 0.8 to 1.3 males
per female depending on the season, averaging at 0.87, but the sample
size was small with fewer than 10 males and females per season. (Table
2). Thus, temporal variations between the nesting seasons were not
investigated.

\textbf{Inbreeding and relatedness in the population}

Global \emph{F} \textsubscript{is} was significant (\emph{F}
\textsubscript{is} = 0.06, p\_value \textless{} 0.001) showing
inbreeding in the population. Pairwise relatedness was calculated for
all possible male-female dyads and reconstructed couples. Mean
relatedness among reconstructed couples (mean +- sd = 0.097 +- 0.21) was
higher than the mean relatedness of all possible dyads (mean +- sd =
-0.003 +- 0.15) (Figure 3). This difference was found to be significant
when a t-test was performed on the mean (p = 0.008). The percentage of
pairs with a relatedness greater than 0.25 and 0.5 was also higher for
the reconstructed couples than for the potential pairs
(r\textsubscript{0.25}: 17\% vs 4\%, r\textsubscript{0.5}: 3\% vs
0.17\%, Table 3). When looking at the effect of the relatedness of the
parents on the fitness parameters in the clutches, a slightly positive
correlation was found with the number of successful hatchlings, and no
correlation with the clutch size and number of dead embryos was
observed. (Supplementary Data 4).

\textbf{Frequency of reproduction and within-season nesting parameters}

Out of the 31 females, 26 of them nested for only one season. The
remaining five females were observed nesting across consecutive seasons.
Among these, four had offspring in two seasons and one had offspring for
three seasons, indicating an annual reproduction cycle for these females
(Table 1). Each of them laid between 3 and 9 clutches sired by a unique
male over the period. The interval between the first and last clutch was
359 to 673 days. For these females, we did not find any other clutch
sired by other males within this period.

In a single season, females laid between 1 and 10 clutches
(Supplementary Data 5), with an average of 3.2 nests per female. The
most common nesting interval was 11 to 14 days, with variations ranging
from 0 to 94 days (Figure 4). Six clutches followed a first egg-laying
event by only 0 to 3 days (+- 3 days), indicating that a single female
can lay several clutches in a very short time interval, possibly on the
same day. The size of these clutches varied between 39 and 103 eggs
(mean 79 eggs), which was not different from the mean clutch size of the
total sample (84 eggs, t-test p-value= 0.43) (Supplementary Data 6).

Of the 27 reconstructed males, 18 were assigned offspring in only one
season. Eight males were assigned offspring in 2 seasons, with 7 of them
doing so consecutively and one with a gap period of 3 years. One male
was assigned offspring in three consecutive seasons. Among these 9
males, 6 sired different females in the consecutive seasons. Within one
season, 24 of the 27 males sired a single female. Two males sired 2
different females, and 1 male sired 3 females in the same season.

\textbf{Multiple paternity}

Multipaternity was confirmed from genotype analysis in 5 of the 70
clutches in which we identified more than one genotyped hatchling (7\%).
Two fathers per female were responsible for all of the offspring sampled
in each clutch. The occurrence of multipaternity was not correlated with
the number of genotyped hatchlings per clutch, as multipaternity was
found in clutches with counts of 2 to 20 genotyped hatchlings
(Supplementary Data 7). One male was dominant in each clutch and sired
between 60\% and 66\% of the genotyped hatchlings (Figure 5) (except in
the clutch with only 2 genotyped hatchlings).

Out of these five clutches, two were laid by the same female, CMY0188
(1415\_ONE8 and 1415\_ONE9) (Figure 5). She laid these two clutches
within 13 days in season 2014/15. They were sired by the same fathers
*129 and *133, with the contribution of father *133 slightly decreasing
in the second clutch (66\% to 60\%). In this season, eight other
clutches laid by the same female were sired only by father *133, showing
no multipaternity (Table 4). Three of them were laid before the clutches
with multipaternity and counted 3, 17, and 6 genotyped hatchlings,
respectively. The remaining 5 were laid after 1415\_ONE9 and counted 8,
9, 7, 1, and 6 hatchlings (Table 4). Hatchlings from father *129 thus
occurred only after several egg-laying events, possibly indicating
mating in the inter-nesting interval.

Two other clutches laid one year apart were also sired by the same
fathers (*62, *53) but these were from two different females, CMY1384
and CMY2419 respectively (Figure 5). For the female CMY1384, we found
three nests in 2017/18 counting 1, 3, and 2 genotyped hatchlings, and
multipaternity was found only in the second clutch, with father *53
absent from the other clutches. For CMY2419, 4 nests were identified in
season 2018/19 (2, 2, 1, and 2 genotyped hatchlings) and father *62 was
found only in the second clutch, the only one to show multipaternity
(Table 4). Finally, the last clutch with multipaternity, 2021\_ONE17,
was laid by female CMY3468 in season 2020/21 and sired by fathers *97
and *337. Another clutch was laid by the same female after 36 days, from
which two hatchlings were sampled. Only father *97 was found responsible
for the genotype of these hatchlings (Table 4). Thus, multipaternity was
never found in all the clutches laid by a female within a season.

We then investigated the effect of multipaternity on fitness. When
comparing the fitness parameters in clutches with and without
multipaternity, no correlation was found between the number of fathers
and any of the three parameters (t.test p\_values: 0.91, 0.95, and 0.83
for the clutch size, the total number of hatchlings, and the number of
dead embryos respectively. Figures in supplementary data 4).

\textbf{Mating behaviour and relatedness}

Finally, none of the deduced mating behaviour, i.e mating with multiple
partners, breeding more frequently, or having a higher share of
paternity in multipaternity clutches was correlated with a change in the
relatedness compared to the relatedness of all reconstructed couples
(Figure 6). T-test p-values on the mean relatedness ranged from 0.16 to
0.5 (Table 3). However, fathers that were dominant in multipaternity
clutches were consistently more related to the female than to the other
father, although that comparison involved only 8 pairs and the
difference was not significant with the t-test (p-value: 0.180, Table
3). Relatedness of couples with the dominant father ranged between 0.044
and 0.391 (mean r= 0.252), while relatedness with the non-dominant
father ranged between -0.180 and 0.212 (mean r= 0.130).

Discussion

The present parentage analysis on the green turtle nesting population of
Tetiaroa allowed for the genotype of 31 females and 27 males to be
rebuilt from the genotype of 443 hatchlings from 105 nests, leading to
an OSR of 0.87. The highly conservative thresholds we adopted in the
analysis process accounted for the small number of males and females
retained, which should be interpreted as a minimal number of breeders
rather than the real number. As a principle, we favoured type II errors,
assuming that reconstructed males and females are real and provide
certainty in the following analysis. Nonetheless, patterns related to
the mating system of this green turtle population emerged. First, the
relatedness of couples was higher than expected by random mating,
coupled with significant \emph{F\textsubscript{is}} , regardless of the
mating behaviour considered. Neither the multipaternity nor the global
relatedness was correlated with the fitness parameters in the clutches.
Second, a low level of multipaternity was observed, and individuals
showed some plasticity in their mating behaviours. Noticeably, both
males and females appeared able to reproduce annually. Within a single
season, we discover that females can lay several clutches over short
time intervals, and males can sire several females, both before and
during the nesting season. All of these outcomes are quite new in the
depiction of green turtle reproductive behaviour and features.

\textbf{Level of inbreeding and strategies to avoid relatedness in the
population}

Inbreeding was significant in this population
(\emph{F\textsubscript{is}} =0.06, p\_value \textless{} 0.001) and
coupled with a higher relatedness than expected by chance. This
indicates that these turtles breed non-randomly and overall do not avoid
inbreeding. A similar situation was found by Horne et al. (2022) in a
Hawaiian hawksbill turtle population, and was correlated with strong
philopatry to nesting complexes less than 100 km apart. Here, the atoll
of Tetiaroa has a total area of only 6 sq. km, but each female was found
nesting on only one or two of the 11 islets surveyed (Supplementary Data
6). Such a level of male philopatry (\textless{} 1-2km) would be
surprising given the scale, the absence of a swimming barrier around the
island, and the capacity of males to connect rookeries (Bradshaw et al.,
2018; Roberts et al., 2004).

The levels of relatedness observed could not be correlated with a loss
of fitness when measuring clutch parameters. Philips et al. (2017)
suggested that relatedness levels and parental multilocus heterozygosity
can interact in both negative and positive ways in turtle populations
and that relatedness alone does not explain levels of fitness, but
investigating these interactions is beyond the scope of this study. None
of the mating behaviour deduced from the parentage analysis (mating with
multiple partners, breeding more frequently, or having a higher share of
paternity in multipaternity clutches) seemed to efficiently mitigate
inbreeding, as the relatedness of pairs involved in these behaviours was
similar to the overall relatedness. This is in discordance with the
inbreeding avoidance theory that predicts that populations would tend to
avoid inbreeding with pre and postcopulatory mechanisms (Blouin and
Blouin, 1988; Cornell and Tregenza, 2007; but see Szulkin et al., 2013).
Inbreeding avoidance is suspected in a small population of leatherback
turtles in the South West Atlantic (Vargas et al., 2022). On the
contrary, mating choices in this population seem to be driven by other
factors. At the scale of French Polynesia, three populations were
identified regardless of their nesting islands and mitochondrial lineage
(Dolfo et al., submitted). When including reconstructed male genotypes
from Tetiaroa in this analysis, most of them belonged to the same
``genetic population'' as their female mates (Supplementary Data 8).
However, in Tetiaroa, three distinct genetic populations coexist,
indicating a deliberate selection of partners rather than random
pairing. As proposed by the authors, these populations may be linked
with phenotypical or ecotypical preferences in partner choice (e.g.
carapace colour and shape) or may be conditioned to breeding grounds
that occur irrespective of nesting islands. However, in Tetiaroa,
breeding behaviours are observed in the vicinity of the island (Gaspar,
pers. comm.), suggesting that breeders then nest on this island.
Courtship is usually observed as a result of repetitive attempts by
males, as females can also refuse mating (Booth and Peters, 1972). Green
turtle courting behaviour should thus be investigated to further
determine what may drive such a non-random mating choice.

Additionally, our results on multipaternity clutches contradict the
`gene compatibility' theory, as the dominant father in multipaternity
clutches was more related to the female than the other male. Dominance
in shares of multipaternity is thought to be linked with first male
sperm precedence (Fitzsimmons, 1998). This, for example, is confirmed in
nests 1415\_ONE8 and 1415\_ONE9. In these nests, the dominant male was
found in all of the other nests earlier in the season (Table 4), while
the non-dominant male was not found in any of the nests. Although the
latter might have been missed in the early nests, this pattern may also
reflect a second mating occurring later in the nesting season. In this
case, multipaternity might mitigate inbreeding by mating with other
partners when the first one has a high level of relatedness. This
mechanism is observed in the Whites' skink (\emph{Egernia whitii} ),
where females sometimes seek a second and less related partner outside
their social group, which increases the offspring heterozygosity (While
et al., 2014). In Chinese alligators (\emph{Alligator sinensis} ),
polyandry females mate with males that are less related to them than
monogamy females, mitigating inbreeding in the population (Wang et al.,
2017). To date, very few studies have explored relatedness in multiple
paternity clutches of marine turtles (Phillips et al., 2013). This
hypothesis therefore needs to be confirmed with a higher number of
multipaternity clutches and deserves further investigations in other
populations.

\textbf{Number of breeders and population density}

The operational sex ratio (OSR) was slightly biased toward females
(0.87:1 male: female), indicating that more females than males breed in
this population. OSR in sea turtles has typically been reported as
skewed towards males or even between both sexes (Hays et al., 2022). For
example, Prakash et al. (2022) found an OSR of 1:1 in a population of
hawksbill turtles in Fiji, and Wright et al. (2012b) found an OSR of
1.4:1 in a green turtle population in Cyprus. The primary sex-ratio of
this population in Tetiaroa does not seem to suffer heavy female bias as
observed elsewhere (Laloe et al., 2020), and so the discrepancy between
the number of male and female breeders is likely small. However, a low
level of multipaternity was observed (7\% of the clutches, 16\% of the
females) and green turtles demonstrated multipaternity in as much as
92\% of their clutches (Alfaro-Nunez et al., 2015). Our finding
constitutes the lowest estimation of multipaternity level in this
population, considering that 70 clutches were analysed over a total of
549 observed. Plus, the number of hatchlings genetically sampled was
uneven in the clutches (most often \textless{} 10), thus the
contribution of some fathers may have been missed. With no apparent
fitness benefits, a low level of multipaternity may indicate low chances
of encountering multiple males, directly linked to the density and
number of male breeders (Lee et al., 2018). However, a high density of
breeders is observed near the island (Gaspar, pers. comm.), and 11\% of
the males mated with multiple females within a season, indicating that
males may encounter several available females. Coupled with the low
level of multipaternity, it suggests that not all males are equally
capable of mating with multiple females. In contrast, Phillips et al.
(2013) found no male hawksbills that mated multiple times within a
season and concluded that a low density and high turnover of males
prevented this from occurring.

Another deduced behaviour of interest arose from the two reconstructed
males *53 and *62. They both sired female CMY1384 in 2018/19 and a
different female CMY2419 in the next season. This finding is surprising
given the low number of breeders retained and the low level of
multipaternity observed, though the probability of such an occurrence
increases when the number of male breeders is low and the density in the
mating area is high. This may indicate that some males are dominant on
the breeding ground. Dominance hierarchies in males have been described
in other species of freshwater turtles like the common snapping turtle
(\emph{Chelydra serpentina} ) and the wood turtle (\emph{Clemmys
insculpta} ), and in the gopher tortoises (\emph{Gopherus agassizii} )
(Pearse and Avise, 2001). Such dominance could occur regularly on the
breeding ground, or be year-specific in the case where CMY2419 was sired
in 2018/19 and able to store sperm until the next season. Alternatively,
non-random mating choices driven by a restrictive criterium may limit
the number of suitable partners for a given female, increasing the
probability of mating with the same pair of males. Nonetheless, these
contrasting results are the first to look into the number of male
breeders in French Polynesia and show that differences between the two
sexes are likely to be small and that this number seems sufficient to
maintain fitness in the population despite high levels of relatedness.

\textbf{Plasticity in mating behaviours}

\textbf{Reproduction frequency and long-term sperm storage}

Parentage assignment revealed that at least 5 females (16\%) returned to
lay once or twice during the 11-year study period. These returning
events were not observed in field surveys, as recapture identification
found that only 2 females were resampled during a returning event. One
of these females was among the 5 females identified from their genotype,
matching the genetic approach (CMY1113, Table 1). Interestingly, these
females were found nesting in consecutive seasons. According to data
available so far, annual reproduction frequency has never been observed
in female marine turtles. For example, Hays et al. (2014) revealed with
satellite tracking that while no female loggerhead turtles migrated
annually to breeding grounds in the Mediterranean Sea, 76\% of the males
did. Studies focusing on female reproduction frequency are based on
field observations, and thus it is possible that in some cases, annual
reproduction has been overlooked due to the absence of direct
observation in the field.

Satellite tracking on adult females in French Polynesia revealed that
several individuals stay within the territory, while others migrate to
Fijian neritic feeding grounds between the nesting seasons (Craig et
al., 2004; Piovano et al., 2019; DIREN personal comm.). Whether those
breeding annually are those that remain in French Polynesia throughout
the year needs to be determined, but it is difficult to imagine long
annual migrations to Fiji (\textgreater{} 2000km), given the time and
energy that it requires, adding to the energy needed for the
reproduction itself. Interestingly, the same fathers consistently sired
the clutches of annually breeding females over the given period (from
359 to 673 days). This may indicate that females are capable of storing
sperm across seasons or exhibiting narrow male choice for reproduction,
implying fidelity to what are currently unknown mating criteria.
Alternatively, cross-seasonal sperm storage has neither been
demonstrated nor quantified in marine turtles, although it is suspected
to occur. For example, Wright (2013) found that multipaternity was more
common in returning females than in primary nesters, and proposed
cross-seasonal sperm storage as one of the possible causes.
Cross-seasonal sperm storage over several years has been observed in
other taxa, including birds (Feldschuh et al., 2005), insects (Baer et
al., 2003), and reptiles (Ewing, 1943; Levine et al., 2021). Although
the possibility of multiple mating among the same individuals cannot be
ruled out (Sakaoka et al., 2013), cross-seasonal sperm storage coupled
with relatively sedentary life traits may help females to minimize
energy costs linked with migration and mating. To date, however, adult
green turtles are rarely observed outside the nesting season in French
Polynesia, and no feeding ground has been identified throughout French
Polynesia or nearby. Hatase et al. (2006) showed, using satellite
tracking and stable isotope analysis, that 31\% (n=89) of the females
nesting on Ogasawara Islands, Japan, were oceanic planktivores rather
than neritic herbivores. Further investigation using these complementary
techniques should reveal whether females in French Polynesia also feed
in oceanic habitats close to nesting grounds.

For males, 6 (22\%) sired different females in consecutive seasons,
indicating a possible annual reproduction frequency. However, caution
should be taken as this might also reflect long-term sperm storage in
the case when the females could not be identified in one of the
consecutive seasons. Male reproduction interval is thought to be shorter
than for females due to a smaller energy cost of reproduction that
allows them to reproduce more often (Hays et al., 2022). For example,
the male green turtle remigration interval was 2.1 years in the southern
Great Barrier Reef (Limpus, 1993), and 1 year in Hawaii (Balazs, 1983),
based on tagged recapture. Wright et al. (2012a) determined the
reproduction frequency of male green turtles in northern Cyprus with
parentage analysis and found that 3\% bred more than once within the 3
years. Overall, annual male reproduction frequency linked to a
residential strategy must be considered as a strong possibility in
French Polynesia, and in this population, a discrepancy between male and
female reproduction frequency is not observed.

\textbf{Within-season nesting parameters}

Within a single season, females laid, on average, 3.2 clutches. Field
estimations based on the length of the nesting season and the observed
inter-nesting interval proposed that each female laid on average 6 nests
in 2017/18 (Touron et al., 2018). The true number is likely to lie
between these two estimations and is coherent with clutch frequencies
for other green turtle populations in the Pacific Ocean (reviewed in
Pilcher 2021). They vary from 1.8 in French Frigate Shoal to 7 +- 1.3
clutches per female per season in the Northern Mariana Islands (Balazs,
1980; Summers et al., 2018). Additionally, through parentage analysis,
74 nests without observed females in the field were associated with a
female, demonstrating the efficiency of the analysis to supplement field
observations.

Inter-nesting interval was highly variable (between 0 and 94 days) and
an interval of 11 to 14 days was the most common interval in this
population. This is coherent with field observations (Touron et al.,
2018), and aligns with findings in other populations (reviewed in
Robinson et al., 2022). As not all nests could be analysed, the longest
inter-nesting interval could not be determined. However, and more
surprisingly, our study reveals the ability of females to lay
regular-sized clutches within a very short interval (0 to 3 days). This
represents a new finding in the reproductive strategy of green turtles.
This shows a capacity for plasticity in nesting behaviour, a potential
advantage in allowing a population to adapt to changing environmental
and weather conditions on nesting beaches if one supposes that females
might search for optimal nesting conditions. Plasticity in nesting
behaviour has been observed in Tetiaroa, with an increase in the nesting
season length over the years (Touron et al., 2018), possibly indicating
that females may look to nest during cooler months to produce more male
offspring in the ongoing warmer conditions (Laloe et al., 2020).

For almost all females, clutches were sired by the same father within a
season, coherent with the fact that breeding occurs before nesting (Hays
et al., 2022). However, one male (*129) sired 2 clutches in the middle
of the nesting season, which induced multipaternity in these clutches.
The genes of this male were not found in the first three clutches nor
the last five clutches laid by the same female (CMY0188) during the
season, although the number of sampled hatchlings (1 to 17) was enough
to detect multipaternity in some of these clutches. The first clutch
sired by this male was laid 40 days after the first recorded clutch for
this female. Female marine turtles are thought to be available for
reproduction only for a short interval of approximately one week, before
nesting (Comuzzie and Owens, 1990). Here we suggest for the first time
in a natural sea turtle population that mating occurs during the
inter-nesting interval (but see Sakaoka et al., 2011, on a captive
population of loggerhead turtles), and provide another indication that
green turtles are capable of unexpected plasticity in their mating
behaviour.

\textbf{Implication for the conservation}

The green turtle population of Tetiaroa shows a significant level of
relatedness and inbreeding, with no clear inbreeding avoidance strategy
and an apparent preference for related partners that rather favour it.
This indicates that the reproductive system of green turtles makes them
intrinsically vulnerable to inbreeding and its potential negative
consequences on the capacity of a population to maintain itself through
generations. Although it does not seem to affect the fitness of
Tetiaroa's population at this stage, it could have negative effects if
the number of breeders is not maintained, which highlights the
importance of turtle conservation programs. On Tetiaroa, a conservation
and monitoring program has been in place since 2007, and an increasing
number of breeders has been recorded on the island, indicating that it
is likely efficient and can preserve the genetic diversity of this
population (Touron et al., 2018). Finally, unexpected plasticity in
green turtle mating behaviours and nesting parameters shows some
adaptive capacity which may help maintain the population's resilience
under changing environmental conditions.

\textbf{Acknowledgements}

This research was funded by the Direction of the Environment of French
Polynesia. We want to thank the volunteer network coordinated by the
Direction of the Environment in French Polynesia for providing all of
the samples used in this work, including the NGO Te mana o te moana for
the samples from Tetiaroa and Moorea and the Bora Bora Turtle Center for
the samples from the Leeward Islands. Thank you to Jeanine Almany for
the English corrections.

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\textbf{Data Accessibility and Benefit-Sharing}

\textbf{Data availability}

Microsatellite sequences are available on GenBank under accession number
OQ162049-OQ162073. All other data generated or analyzed during this
study are included in this published article and its supplementary
information files.

\textbf{Benefit-Sharing}

Benefits Generated: A research collaboration was developed with
associations from French Polynesia providing genetic samples, all
collaborators are included as co-authors, the results of the research
have been shared with the provider communities and the broader
scientific community, and the research addresses a priority concern, in
this case, the conservation of organisms being studied. More broadly,
our group is committed to international scientific partnerships, as well
as institutional capacity-building.

\textbf{Author Contributions}

\emph{Chelonia mydas} sample collection was coordinated by Miri Tatarata
and Cécile Gaspar. Miri Tatarata and Serge Planes designed the research.
Sample processing and analysis were performed by Violaine Dolfo,
reviewed by Emilie Boissin, and supervised by Emilie Boissin and Serge
Planes. The first draft of the manuscript was written by Violaine Dolfo,
and all authors commented on previous versions of the manuscript. All
authors read and approved the final manuscript.

Table 1: Females found nesting in multiple seasons using
\emph{Rclone}analysis (marked with °) and parentage analysis (marked
with\textsuperscript{+}).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Ind \# & Ind. ID & 1\textsuperscript{st} sampling season &
2\textsuperscript{nd} encounter & 1\textsuperscript{st} genotype
reassignment & 2\textsuperscript{nd} genotype
reassignment\tabularnewline
\midrule
\endhead
1 & CMY1113\selectlanguage{ngerman}°\textsuperscript{,+} & 2016/17 & 2020/21 & 2017/18 &
-\tabularnewline
2 & CMY1139\selectlanguage{ngerman}° & 2016/17 & 2020/21 + & - & -\tabularnewline
3 & CMY1104\textsuperscript{+} & 2016/17 & - & 2017/18 &
-\tabularnewline
4 & CMY1105\textsuperscript{+} & 2016/17 & - & 2017/18 &
-\tabularnewline
5 & CMY1101\textsuperscript{+} & 2016/17 & - & 2015/16 &
2017/18\tabularnewline
6 & \#75\textsuperscript{+} (reconstructed) & NA & - & 2016/17 &
2017/18\tabularnewline
\bottomrule
\end{longtable}

+ Different ID given on the second encounter

Table 2: Number of females and males deduced from parentage analysis for
each season, and operational sex ratio (OSR, number of males for one
female)\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Season} & \textbf{Nb. females} & \textbf{Nb. males} &
\textbf{OSR}\tabularnewline
2014/15 & 3 & 4 & 1.33\tabularnewline
2015/16 & 2 & 2 & 1.00\tabularnewline
2016/17 & 9 & 8 & 0.90\tabularnewline
2017/18 & 8 & 8 & 1.00\tabularnewline
2018/19 & 3 & 3 & 1.00\tabularnewline
2019/20 & 6 & 5 & 0.83\tabularnewline
2020/21 & 6 & 7 & 1.17\tabularnewline
total & 31 & 27 & 0.87\tabularnewline
\bottomrule
\end{longtable}

Table 3: Mean relatedness between potential and reconstructed couples,
couples involved in different mating behaviours, and Student's t-test
p-values. Significant p-values are indicated in bold.
r\textsubscript{0.25} (r\textsubscript{0.5}): percentage of pairs with a
relatedness greater than 0.25 (0.5).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
mean r\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
t-test p\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\# pairs\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
r\textsubscript{0.25}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
r\textsubscript{0.5}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
All potential couples\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-0.003\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2880\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4\%\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.17\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
All reconstructed couples\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.097\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.008 (vs potential)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
17\%\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Couples with female nesting in consecutive seasons\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.294\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.16 (vs reconstructed)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Couples with multipaternity\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.162\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.39 (vs reconstructed)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Dominant male in clutches Non dominant male in clutches\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.252 0.130\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.13 (vs reconstructed) 0.18 (vs dominant)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
4 4\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Males mating multiple times\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.143\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.50 (vs reconstructed)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Table 4: Father ID and number of offspring in all clutches of the 4
females involved in polyandry. Clutches with multipaternity are
indicated in bold.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Female ID}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY0188}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY0188}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY1384}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY1384}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY2419}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY2419}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY3468}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{CMY3468}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Season\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2014/15\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2014/15\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2017/18\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2017/18\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2018/19\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2018/19\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2020/21\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2020/21\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Nest number\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Father ID\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Offspring nb\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Father ID\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Offspring nb\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Father ID\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Offspring nb\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Father ID\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Offspring nb\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*62\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*53\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{*97/*337}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{20}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
17\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{*62/*53}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{3}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{*53/*62}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{2}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*97\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*62\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*53\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{*133/*129}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{12}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*53\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{*133/*129}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{7}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#7\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#8\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#9\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\#10\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
*133\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure Captions}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/698137/articles/685943-investigating-the-reproductive-behaviour-of-the-green-sea-turtle-chelonia-mydas-using-parentage-analysis}

Figure 1: Sampling locations on Tetiaroa Atoll, French Polynesia.
Sampled nests are represented with black dots around the islets.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/698137/articles/685943-investigating-the-reproductive-behaviour-of-the-green-sea-turtle-chelonia-mydas-using-parentage-analysis}

Figure 2: Conceptual workflow for all the analyses performed for this
study

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/698137/articles/685943-investigating-the-reproductive-behaviour-of-the-green-sea-turtle-chelonia-mydas-using-parentage-analysis}

Figure 3: Density and boxplot of the relatedness of all the potential
(full line) and reconstructed (dash line) parent pairs.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/698137/articles/685943-investigating-the-reproductive-behaviour-of-the-green-sea-turtle-chelonia-mydas-using-parentage-analysis}

Figure 4: Inter-nesting interval between two successive nesting events
for every female between 2014/15 and 2020/21 in Tetiaroa atoll.

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/698137/articles/685943-investigating-the-reproductive-behaviour-of-the-green-sea-turtle-chelonia-mydas-using-parentage-analysis}

Figure 5: Relative fathers' contribution in the five clutches where
multipaternity was found. Each vertical bar represents one clutch and
each shade of grey represents one male. The number of hatchlings in the
clutches is displayed above each bar.

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/698137/articles/685943-investigating-the-reproductive-behaviour-of-the-green-sea-turtle-chelonia-mydas-using-parentage-analysis}

Figure 6: Relatedness of couples involved in different reproduction
behaviours compared with relatedness of all reconstructed couples. Grey
line: all reconstructed parent pairs, black line: multiple mating of
males, two-dash line: annual nesting of females, dot line: dominant
father in multipaternity clutches.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

