Ethics Approval
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 n° FR1298700118-E and n° 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 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.
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; Santidrián 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, 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 (Caretta caretta ), leatherback
(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
km2, 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