# Conservation Genetics of the Reintroduced Giant Tortoises (Aldabrachelys gigantea) in Madagascar

Guillaume Besnard, Alexandre Barutel, Rianja Rakotoarivony, Natacha Nikolic, Jonia Rasolofoniaina, F. Gözde Çilingir, Xenia Wietlisbach, Annabelle Constance, Jean-baptiste Ferdy, Miguel Pedrono

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Guillaume Besnard, Alexandre Barutel, Rianja Rakotoarivony, Natacha Nikolic, Jonia Rasolofoniaina, et al.. Conservation Genetics of the Reintroduced Giant Tortoises (Aldabrachelys gigantea) in Madagascar. Ecology and Evolution, 2025, 15 (9), pp.e72005. 10.1002/ece3.72005. hal-05325749

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RESEARCH ARTICLE

OPEN ACCESS

# Conservation Genetics of the Reintroduced Giant Tortoises
*(Aldabrachelys gigantea)* in Madagascar

Guillaume Besnard<fnref n="1" /> | Alexandre Barutel<fnref n="1" /> | Rianja Rakotoarivony² | Natacha Nikolic<fnref n="1" />,³

Jonia Rasolofoniaina⁴ | F. Gözde Çilingir⁵,⁶ | Xenia Wietlisbach⁷ | Annabelle Constance⁸ | Jean-Baptiste Ferdy<fnref n="1" /> |

Miguel Pedrono²,⁴,⁹

**Correspondence:** Guillaume Besnard (guillaume.besnard@utoulouse.fr) | Miguel Pedrono (miguel.pedrono@mnhn.fr)

**Received:** 14 May 2025 | **Revised:** 24 July 2025 | **Accepted:** 5 August 2025

**Funding:** This work was supported by Agence Nationale de la Recherche, ANR-10-LABX-0041, ANR-10-LABX-25-01.

**Keywords:** *Aldabrachelys gigantea* | demographic inference | genetic admixing | major histocompatibility complex (MHC) | microsatellites | paternity tests

## ABSTRACT

The Aldabra giant tortoise is the only surviving lineage of the Malagasy megafauna, which went extinct due to human activities over the last millennium. In 2018, the first rewilding project was launched at the Anjajavy Reserve (NW Madagascar), using 12 founders of unknown origin. They started to mate in 2019, but genetic proximity between individuals and their reproductive output remains to be determined for optimal management of the newly established population. Here, we used 29 microsatellite loci to analyze the genetic diversity of native populations from Aldabra, a coral atoll 420 km from Madagascar, to assign Anjajavy founders to specific genetic groups and assess how well they represent the overall genetic diversity in the native range. Additionally, five of these loci allowed us to investigate the diversity of the genomic region containing the Major Histocompatibility Complex (MHC) genes. The non-MHC microsatellite data were also used to investigate the past demography of the species using a step-wise mutation model (VarEff). Finally, we evaluated the contribution of Anjajavy founders to the offspring. First, the clustering analysis identified two main genetic groups in Aldabra, and demographic inferences then support that population size fluctuations were strongly shaped by climatic shifts and sea-level changes on the atoll. Both genetic groups are represented among the Anjajavy founders, who most likely originated from the southern island of Aldabra. However, the founders contributed unequally to the offspring, as most juveniles analyzed were the result of mating between individuals from the two groups, leading to a significant global increase in heterozygosity in the offspring. We propose measures to minimize inbreeding and maintain genetic diversity within the Anjajavy population, thereby enhancing its potential for long-term viability.

## 1 | Introduction

An extremely diverse megafauna once inhabited Madagascar until human colonization of the island. This fauna included three species of hippopotamus (*Hippopotamus* spp.), 17 species of giant lemurs from nine genera (*Palaeopropithecus*,

*Archaeoindris*, *Babakotia*, *Mesopropithecus*, *Megaladapis*, *Archaeolemur*, *Hadropithecus*, *Pachylemur*, and *Daubentonia*), a giant fossa (*Cryptoprocta spelea*), elephant birds (ratites of the genera *Aepyornis* and *Mullerornis*), a horned crocodile (*Voay robustus*), and two species of giant tortoise (*Aldabrachelys grandidieri* and *Aldabrachelys abrupta*) (Burney et al. 2004).

<footnote n="1">¹Centre de Recherche sur la Biodiversité et l'Environnement (CRBE), UMR 5300 CNRS-IRD-TINP-UT3, Université Toulouse III - Paul Sabatier, Toulouse cedex 9, France | ²UMR Astre, CIRAD, Campus International de Baillarguet, Montpellier cedex 5, France | ³INRAE, Aqua, ECOBIOP, France | ⁴Anjajavy Reserve, Madagascar | ⁵The Swiss Federal Institute for Forest Snow and Landscape Research WSL, Birmensdorf, Switzerland | ⁶Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland | ⁷Department of Biology, University of Fribourg, Fribourg, Switzerland | ⁸Seychelles Islands Foundation, Victoria, Republic of Seychelles | ⁹UAR PatriNat, Muséum National d'Histoire Naturelle, Paris, France</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). *Ecology and Evolution* published by British Ecological Society and John Wiley & Sons Ltd.</footnote>
Overhunting and trophic cascades are thought to have signifi-
cantly contributed to their extinction around 500–1200 years
ago (Crowley 2010; Sandom et al. 2014; Burns et al. 2016; Hixon
et al. 2021). Subfossil records from Holocene deposits provide
further insights into how these various megafauna species
coexisted with other wildlife in Madagascar, suggesting that
large herbivores have shaped the island's ecosystems through-
out much of their evolutionary history. These species played a
crucial role in the dynamics of Madagascar's ecosystems (e.g.,
seed dispersal, bioturbation, fire regimes), and their extinction
continues to impact Malagasy ecosystems, complicating forest
regeneration and species conservation (Pedrono et al. 2013).

While all vertebrates over 20kg in Madagascar were wiped
out, a lineage of giant tortoise (Aldabrachelys gigantea), closely
related to A. abrupta (Kehlmaier et al. 2023), survived and re-
peatedly colonized remote Seychelles islands from Madagascar,
likely aided by ocean currents (Pedrono et al. 2013; Cheke
et al. 2017; Kehlmaier et al. 2023). Today, a large wild population
of A. gigantea survives on Aldabra Atoll, located in the extreme
southwest of the Seychelles, approximately 420km northwest
of Madagascar. The atoll consists of three main islands, sepa-
rated by channels and a shallow lagoon. The tortoises are un-
evenly distributed across its three largest islands: Grande Terre,
Malabar, and Picard (Bourn and Coe 1978). Overexploitation by
Europeans in the 19th century is believed to have caused the
extinction of tortoise populations on the granitic Seychelles
(Cheke and Bour 2014) and nearly led to the extinction of those
on Aldabra. In the 1890s, the tortoise population on the atoll
was estimated to have dwindled to just a few hundred individ-
uals (Bourn et al. 1999). Concerns over the species' potential
extinction prompted the implementation of protective measures
in 1891 (Stoddart and Peake 1979), including the control of inva-
sive species such as goats and the reintroduction of tortoises to
Picard Island through successive translocations from Malabar
and Grande Terre between 1900 and 1980 (Bourn et al. 1999).
These conservation efforts were highly successful, with the pop-
ulation rebounding from just a few thousand individuals in 1960
to over 100,000 today (Grubb 1971; Turnbull et al. 2015).

As the largest herbivore and frugivore on Aldabra, *A. gigantea* is considered an ecosystem engineer or functional species, playing a critical role in shaping and maintaining biological communities typical of reptile-dominated ecosystems (Griffiths et al. 2011; Pedrono et al. 2013; Hansen 2015). As a result, this species has been used in ecological restoration programs to replace extinct giant tortoise species from the Mascarene Islands (*Cylindraspis* spp.), following ecological replacement schemes on Île aux Aigrettes, Round Island, and Rodrigues in Mauritius (e.g., Hansen et al. 2010; Griffiths et al. 2011; Moorhouse-Gann et al. 2022).

By applying a radical conservation approach successfully devel-
oped elsewhere to restore some lost ecological interactions and
processes (i.e., Foreman 2004; Zimov 2005; Donlan et al. 2006),
the genus *Aldabrachelys* was reintroduced to Madagascar after
an absence of more than six centuries (Pedrono et al. 2020).
While the introduction of this genus to the Mascarenes was
novel, since it had never been native there, the reintroduction
in Madagascar represents a return to its historical evolutionary
range. Moreover, *A. gigantea* is genetically and morphologically

similar to the extinct Malagasy taxon *A. abrupta* (Kehlmaier et al. 2023; Bour 1994). This first rewilding project in Madagascar thus aims to enhance the resilience and sustain the biodiversity of the relatively well-preserved ecosystems in the Anjajavy Reserve, in northwest Madagascar (Pedrono et al. 2013; Joseph et al. 2024).

In the 5years since the initial release of 12 founder individ-
uals, over 150 juveniles have been reported, marking the first
giant tortoise births in Madagascar in ~600years (Pedrono
et al. 2020). However, the origin of these founders remains un-
known, and their genetic relatedness is unclear. For effective
population management, it is crucial to promote genetic mixing
to prevent inbreeding in future generations, as this can lead to
genetic erosion, inbreeding depression, and reduced adaptive
potential (Frankham et al. 2002). Therefore, assessing the rel-
ative reproductive contributions of adult breeding males within
the population is essential.

Over the past 30years, the development of molecular tools,
particularly microsatellite markers, has provided crucial infor-
mation for conservation project managers (e.g., Ivy et al. 2009;
Witzenberger and Hochkirch 2011; de Cara et al. 2013; Font
et al. 2024). Several tortoise genomes, including that of A. gi-
gantea (Çilingir, A'Bear, et al. 2022), have recently been as-
sembled, offering new opportunities to develop specific genetic
markers. These molecular tools enable the assessment of genetic
diversity within a taxon, the tracing of individual origins, and
the reconstruction of parentage relationships, thereby support-
ing more effective conservation management, particularly for
tortoise species (Aggarwal et al. 2004; Milinkovitch et al. 2004;
Edwards et al. 2014; Wietlisbach 2017). When combined with
behavioral observations, molecular analyses also provide valu-
able insights into the reproductive strategies of the species (e.g.,
Pearse and Avise 2001; Moon et al. 2006; Cutuli et al. 2013,
2014). This approach has revealed that females of many reptiles
are often fertilized by multiple males (Uller and Olsson 2008),
with some cases of male competition occurring, potentially
within the spermatheca (Moon et al. 2006). Additionally, mo-
lecular analyses have documented the erosion of genetic diver-
sity in populations of endangered tortoise species, particularly
on genes with potential adaptive value (e.g., immune responses)
such as those of the Major Histocompatibility Complex (MHC)
(Elbers et al. 2017).

Given the small size of the newly established Anjajavy popula-
tion, managing its future dynamics will be crucial. To this end,
we conducted an analysis of the genetic diversity of *A. gigantea*
populations from Aldabra Atoll and Anjajavy. Forty-seven indi-
viduals were characterized with 29 multiallelic genetic markers
(microsatellites) of which 17 were developed in this study (in-
cluding five from the genomic region containing MHC genes).
We compared the genetic diversity of these populations and de-
termined which Aldabra population(s) the individuals released
in Madagascar most likely originated from. Based on these data,
we also investigated the demographic history of the species with
an approximate likelihood approach (i.e., stepwise mutation
model). Paternity tests on 50 juveniles, born between 2019 and
2022, then allowed us to identify the adult individuals contribut-
ing to reproduction in Anjajavy and assess whether gene assort-
ment occurs randomly. Finally, we provide recommendations on
<figure><img src="image_2.png" /></figure>

**FIGURE 1** | Geographic origin of *Aldabrachelys gigantea* individuals analyzed in our study. A zoom on the Aldabra atoll allows visualizing the distribution of analyzed individuals in their native range. Putative barriers to dispersal within islands [i.e., low vegetation cover with limited shade in Grande Terre (Takamaka) and very dense scrub in Malabar] are indicated by black lines. For each location, the number of individuals is indicated in brackets. Our sampling includes 15 individuals from Madagascar [12 from the reserve of Anjajavy, and three from the Parc Botanique & Zoologique de Tzimbazaza (PBZT)] and 32 from Aldabra, to which we added 112 Aldabra individuals genotyped by Balmer et al. (2011). At each location, we also give the number of individuals assigned to each genetic cluster at *K*=3 for *p*>0.8 (see Figure 3). When *p* was inferior to 0.8, individuals were considered as « admixed ».

how to best maintain genetic diversity and minimize inbreeding
within the population.

2 | Material and Methods

2.1 | Reintroduction and Monitoring of the A. gigantea Population in Madagascar

The site for the reintroduction of *A. gigantea* in Madagascar is
located in the Anjajavy Reserve, in the northwest of the island
(Figure 1). This reserve lies within the historical distribution
range of *A. abrupta* (Pedrono 2008) and features an optimal cli-
mate and ecosystem (e.g., shrubby savannah and dry deciduous
forest), which would have supported a large population of giant
tortoises in the past. Moreover, its remoteness reduces the risk
of poaching of released individuals. Finally, the Anjajavy Lodge,
responsible for managing the reserve, ensures the long-term
funding of the project.

The reintroduction began in June 2018, when the Malagasy
government seized 12 sub-adult giant tortoises—five males and
seven females—from a private collection of an animal dealer in
the capital city, Antananarivo, and entrusted their management
to the Anjajavy Reserve. After a quarantine period in a pre-
release paddock, the tortoises were transferred to a two-hectare
natural savannah, surrounded by karst formations (Tsingy).
Local Sakalava rangers monitor and protect the tortoises, but do
not provide supplementary food, encouraging the emergence of
natural behaviors. The first two hatchlings were discovered in
November 2019 (Pedrono et al. 2020), and as of 2020, the nests
were located by monitoring the nocturnal activity of the females
during the laying season (April to July). The nests were left to in-
cubate naturally where the female laid them, but were protected
by a wooden enclosure covered with wire mesh, which prevents

another female from destroying the nest by laying in exactly
the same place. This also allows access to the juveniles as they
emerge. After hatching, the nest is excavated to see how many
unhatched eggs remain and to check whether they have been
fertilized or not. In early 2024, 151 juveniles, resulting from
natural incubation in the wild, were being raised in a nursery
established near the founding population (Table 1). This head-
starting facility allows the juveniles to grow to a larger size in
a protected ex situ environment, increasing their survivorship
compared to wild conditions before they are released back into
the wild once they reach a critical carapace size, offering pro-
tection from predators. Data on nest numbers, clutch size, and
reproductive output have been collected (Table 1), as well as ob-
servations on mating attempts (Table S1). Three males (#4, 6, 8)
were found to regularly mate with five females (#1, 2, 3, 9, 11)
and are considered sexually mature.

2.2 | Genetic Analyses

2.2.1 | Sampling

Blood samples were collected for the 12 founding individu-
als and 50 juveniles, including all those born in 2019, 2020,
2021, and most of those born in 2022 (at least one individual
per nest). In addition, three very old male individuals of the
Tsimbazaza Botanical and Zoological Park in Antananarivo
(PBZT) were also sampled for comparison. They belong to
the main nonreproductive captive group of *A. gigantea* in
Madagascar (three males and one female) and were gifted by
Queen Victoria to the last Queen of Madagascar at the end of
the 19th century. In addition, we analyzed tortoises from the
current native range to assign founder individuals of Anjajavy
and PBZT to genetic groups and assess their degree of repre-
sentativeness of the global species genetic diversity. Today, all
<table><caption>TABLE 1 | Female reproductive output over four years (from 2020 to 2023). For each oviposition, the number of clutches (N<sub>Ciu</sub>), eggs laid (‘Eggs laid’), and births (‘Births’), plus the hatching rate (% hatch.) are given for each female and on the total (per female and/or per year).</caption><thead><tr><th rowspan="2"></th><th colspan="3">Oviposition 2020</th><th colspan="3">Oviposition 2021</th><th colspan="3">Oviposition 2022</th><th colspan="3">Oviposition 2023</th><th colspan="3">Total</th></tr><tr><th>N<sub>Ciu</sub></th><th>Eggs laid</th><th>Births<sup>a</sup></th><th>% hatch.</th><th>N<sub>Ciu</sub></th><th>Eggs laid</th><th>Births<sup>a</sup></th><th>% hatch.</th><th>N<sub>Ciu</sub></th><th>Eggs laid</th><th>Births<sup>a</sup></th><th>% hatch.</th><th>N<sub>Ciu</sub></th><th>Eggs laid</th><th>Births<sup>a,b</sup></th><th>% hatch.</th></tr></thead><tbody><tr><td>Female #1</td><td>0</td><td>—</td><td>—</td><td>—</td><td>4</td><td>56</td><td>2 (1)</td><td>3.6</td><td>4</td><td>48</td><td>33 (19)</td><td>68.8</td><td>1</td><td>11</td><td>7 (7)</td><td>63.6</td><td>9</td><td>115</td><td>42 (27 + 1)</td><td>36.5</td></tr><tr><td>Female #2</td><td>3</td><td>40</td><td>28 (5)</td><td>70.0</td><td>3</td><td>26</td><td>18 (12)</td><td>69.2</td><td>3</td><td>25</td><td>21 (12)</td><td>84.0</td><td>1</td><td>12</td><td>10 (10)</td><td>83.3</td><td>10</td><td>103</td><td>77 (39 + 2)</td><td>74.8</td></tr><tr><td>Female #3</td><td>1</td><td>1</td><td>0</td><td>0.0</td><td>1</td><td>12</td><td>1 (0)</td><td>8.3</td><td>1</td><td>12</td><td>8 (7)</td><td>66.7</td><td>0</td><td>—</td><td>—</td><td>—</td><td>3</td><td>25</td><td>9 (7)</td><td>36.0</td></tr><tr><td>Female #5</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Female #9</td><td>3</td><td>40</td><td>6 (2)</td><td>15.0</td><td>3</td><td>33</td><td>10 (1)</td><td>30.3</td><td>4</td><td>40</td><td>20 (19)</td><td>50.0</td><td>3</td><td>26</td><td>15 (14)</td><td>57.7</td><td>13</td><td>139</td><td>51 (36)</td><td>36.7</td></tr><tr><td>Female #11</td><td>3</td><td>30</td><td>13 (8)</td><td>43.3</td><td>3</td><td>20</td><td>18 (2)</td><td>90.0</td><td>2</td><td>18</td><td>12 (12)</td><td>66.7</td><td>3</td><td>27</td><td>18 (18)</td><td>66.7</td><td>11</td><td>95</td><td>61 (40 + 1)</td><td>64.2</td></tr><tr><td>Female #12</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td><td>0</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Total</td><td>10</td><td>111</td><td>47 (15)</td><td>42.3</td><td>14</td><td>147</td><td>49 (16)</td><td>33.3</td><td>14</td><td>143</td><td>94 (69)</td><td>65.7</td><td>8</td><td>76</td><td>50 (49)</td><td>65.8</td><td>46</td><td>477</td><td>240 (149 + 5)</td><td>50.3</td></tr><tr><td>Mean clutch size</td><td>11.8</td><td></td><td></td><td></td><td>9.8</td><td></td><td></td><td></td><td>18.8</td><td></td><td></td><td></td><td>12.5</td><td></td><td></td><td></td><td>10.4</td><td></td><td></td><td></td><td></td></tr><tr><td>Average incubation duration (in days)</td><td>128.1</td><td></td><td></td><td></td><td>137.3</td><td></td><td></td><td></td><td>141.4</td><td></td><td></td><td></td><td>130.4</td><td></td><td></td><td></td><td>134.3</td><td></td><td></td><td></td><td></td></tr></tbody></table>

<footnote><sup>a</sup>The number of surviving individuals in the nursery in 2024 is given in brackets.</footnote>

<footnote><sup>b</sup>For the total, five additional siblings have been recorded: For female #2, two living siblings were obtained in 2019, but there was no record of clutches and hatchings on this first year of the project; then, from 2020, although we tried to record all clutches every year, three additional progenies were found in the reserve: C12M1_21 in 2021 (assigned to mother #1), C15M1_22 in 2022 (assigned to mother #11), and C8M1_23 in 2023 (from unknown mother; not yet genotyped).</footnote>
<figure><img src="image_3.png" /><figcaption>**FIGURE 2** | Schematic representation of chromosome 12 of *Aldabrachelys gigantea* (Çilingir, A'Bear, et al. 2022) with the location of MHC gene homologs and the five studied SSR loci (MHC_I to V). A zoom allows visualization of the position of the first four loci (MHC_I to IV) on the 520-kb genomic block carrying nine regions identified with blastn of strong homology with genes of MHC class I (in blue; seven copies) and II (in yellow; two copies). The region MHC_X, which combine information from loci MHC_I to IV, is indicated. This genomic block may recombine very rarely according to the very strong linkage disequilibrium revealed between polymorphisms at loci MHC_I to IV (Figure S1). Based on the lack of segregation at these four polymorphic loci, we revealed eight MHC haplotypes, shown below. At each locus, alleles are distinguished by a colored box (with their size in bp inside). Haplotype frequency in Aldabra (on 32 individuals) and Madagascar (15) is indicated on the right.</figcaption></figure>

living *A. gigantea* individuals come from the Aldabra populations (Austin et al. 2003). We thus sampled 32 tortoises from the three islands of the Aldabra Atoll inhabited by *A. gigantea* (11 for Grande Terre and Malabar, 10 for Picard; Çilingir, Hansen, et al. 2022). We also reused the data set of Balmer et al. (2011) that consists of SSR genotypes at eight microsatellite loci of 112 individuals from Grande Terre and Malabar. The list of analyzed individuals is given in Table S2.

### 2.2.2 | DNA Extraction, Microsatellite Development and Genotyping of Individuals

DNA from each Anjajavy blood sample was extracted using a QIAGEN kit (DNeasy Blood & Tissue Kit) following the supplier's protocol and stored at 4°C (then at -20°C after PCR). Twenty-nine microsatellites were used to characterize the individuals (Tables S3 and S4). The location of the loci in the genome was considered in order to study linkage disequilibrium (LD) between markers physically linked on a chromosome. Nine loci were developed by Ciofi et al. (2002) on *Chelonoidis nigra* and have shown good transferability to *Aldabrachelys* in previous studies (Palkovacs et al. 2003; Balmer et al. 2011); three others were developed on *Astrochelys radiata* by Paquette et al. (2005), and finally, 17 were newly developed from the recently published genome of *A. gigantea* (Çilingir, A'Bear, et al. 2022). The markers developed previously have di-nucleotide motifs. For the loci developed here, di-nucleotide (4 markers), tri-nucleotide (11) or tetra-nucleotide (2) microsatellite motifs (> eight repeats) were targeted. Five of the newly developed loci are located close to MHC genes and are potentially affected by selective processes via hitchhiking (see below).

MHC genes code for transmembrane glycoproteins, whose main function is to present a peptide code of self and nonself antigens to lymphocytes (i.e., CD8 and CD4, coded for by genes of class I and II, respectively). The MHC is involved in activating immune responses in the presence of a pathogen. MHC gene diversity can be affected by genetic drift in small populations (e.g., Elbers et al. 2017); however, it has been shown that MHC haplotypes are not randomly assorted (by disassortative mating) in many animal species, which would contribute to maintaining a high diversity of MHC genes (e.g., Tregenza and Wedell 2000; Milinski 2006; Dandine-Roulland et al. 2019; Nishita et al. 2024). In the reference genome of *A. gigantea* (Çilingir, A'Bear, et al. 2022), the MHC genes are located at the beginning of chromosome 12 (20Mb). Seven regions of high homology with MHC class II genes (six arranged in tandem and the last in antisense) followed by two for MHC class I genes are present on a chromosomal segment of ~520kb, between positions 392,000 and 913,000 (Figure 2). In order to study the diversity of this genomic region and the assortment of its haplotypes in the descendants of the Anjajavy population, we targeted five microsatellite loci distributed over a genomic segment of 1.5Mb (Figure 2; Table S4). MHC_III is located in a noncoding part (intron) of the first copy of class II, while MHC_IV is located between the two class I copies. The three remaining loci are in the flanking parts in 5' (MHC_I and II) or 3' (MHC_V), a few 100s of kb apart of the genomic region carrying the MHC genes (Figure 2).

PCR amplifications were performed in 25µL according to the conditions reported by Besnard et al. (2011), using the method of Schuelke (2000) to integrate fluorescence using a labeled M13 primer on the generated fragments. For each locus, one of the following four fluorochromes was used: FAM, YAK,
ATTO550, or ATTO565. The PCR program used was as follows: denaturation of the DNA at 95°C for 2 min, followed by 25 cycles with 30 s at 95°C (denaturation), 30 s at 55°C (for 24 markers) or 58°C (for MHC markers) (primer annealing), and 90 s at 72°C (elongation), followed by ten additional cycles in which the hybridization temperature was lowered to 51.5°C to allow integration of the labeled M13 primer into amplified fragments, before a final elongation for 20 min at 72°C. The markers were then multiplexed in groups of four to six (using different fluorochromes, or a range of non-overlapping allele sizes for loci sharing the same fluorochrome). The PCR products were diluted 1:100 in formamide with LIZ-500 (Applied Biosystems) before being sent to GenoScreen in Lille. The chromatograms obtained on an ABI 3730 capillary sequencer were read on GENEIOUS v.9.1.8 (Kearse et al. 2012). To limit reading errors, all loci were read twice independently by G.B. and A.B.

### 2.2.3 | Genetic Diversity Parameters

Firstly, we studied the linkage disequilibrium between markers, especially those that are physically linked (Tables S3 and S4). LD is expected to be higher the closer the loci are on a chromosome, as the probability of a recombination event between them decreases with their physical proximity. LD is created by genetic drift and founder effects. However, it will decrease over time (due to recombination and new mutations). Comparing LD between populations can therefore reveal contrasting demographic histories, for example for old native populations and those recently expanding in an introduced area (e.g., Balboa et al. 2024). Given that the *A. gigantea* population underwent a major size reduction at the end of the 19th century (Bourn et al. 1999), we can expect significant LD between markers physically linked over several Mb. In our data set, we have 27 pairs of markers less than 50 Mb apart on the genome. The LD between these marker pairs was measured using the R package *Poppr* (Kamvar et al. 2014) on mature individuals from Anjajavy, PBZT, and Aldabra (47 individuals) with the association index $r_D$ (Agapow and Burt 2001), which was directly compared to the physical distance (in Mb) between markers on the chromosomes.

We then estimated genetic diversity parameters for mature individuals from Aldabra (across the atoll and by island) and Anjajavy, as well as for their 50 progenies. Observed ($H_O$) and expected ($H_E$) heterozygosities under the Hardy-Weinberg hypothesis (i.e., population of infinite size, panmictic reproduction and non-overlapping generations), and the number of alleles per locus were calculated using *GENETIX* v.4.05 (Belkhir et al. 2004). Allelic richness ($R_a$) was estimated for a minimum of ten individuals (i.e., population sample size at Picard) using the R package *HIERFSTAT* (Goudet 2005) in order to compare the number of alleles between groups (populations or subpopulations). The presence of null alleles was tested using the Brookfield (1996) method with a 95% confidence interval using *MICROCHECKER* v.2.2.3 (van Oosterhout et al. 2004). The inbreeding coefficient ($F_{IS}$; Weir and Cockerham 1984) was estimated with *HIERFSTAT*, and its significance was assessed with permutations. In the case of juveniles, the significance of $F_{IS}$ departure from 0 was also assessed at each locus using random sampling of parental alleles to produce the null distribution (see below; last paragraph of the section on the paternity analyses).

For the following analyses on the population genetic structure, we removed the genetically linked loci, keeping only the most informative locus for each series of loci in linkage disequilibrium. A very high LD was revealed for the five MHC markers (see results), especially from MHC_I to IV, for which a strict association between alleles was observed on all samples we analyzed (i.e., 47 mature individuals and 50 progenies). Combining polymorphism from these four markers allowed us to define eight haplotypes of this genomic block (MHC_X; Table S2; Figure 2). Diversity of the MHC region (MHC_X) was thus compared with the genome as a whole between mature individuals and juveniles at Anjajavy.

### 2.2.4 | Genetic Structure of Populations and Origins of Founding Individuals in Anjajavy

The measure of genetic differentiation between populations or sub-populations was first calculated with the fixation index ($F_{ST}$; Weir and Cockerham 1984) using *GENETIX* with 10,000 permutations. To measure the genetic similarities between the individuals analyzed, without any preconceived ideas about their origin, a Principal Component Analysis (PCA) was performed using *HIERFSTAT*. We considered two data sets: the one completed in our study on 22 independent loci for 47 mature individuals, and another combining our data and that of Balmer et al. (2011) on eight independent loci for 159 individuals.

To determine the number of genetic clusters present in our sample, we used *STRUCTURE* v.2.3.4 (Pritchard et al. 2000), which uses a Bayesian approach to assign individuals to genetic clusters on the basis of their genotype. All the analyses were carried out using the admixture model with 200,000 generations after a burn-in period of 50,000 generations. We carried out two analyses for a maximum number of clusters ($K$) of six: (i) the first with 12 founder individuals from Anjajavy, three individuals from PBZT, and the 32 individuals from Aldabra; then (ii) a second analysis including the data from Balmer et al. (2011) completed for eight loci on 112 additional individuals from Aldabra (with missing data on 14 loci). For the latter, the probability of having missing data at a particular locus is expected to be independent of what allele the individual has there, and this should not strongly impact the results (Pritchard et al. 2000). For each of these analyses, ten iterations were performed for each $K$ value to estimate the standard deviation error for each membership ancestry component. The most likely number of clusters was determined using the $\Delta K$ and $Ln(K) \pm s.d.$ criteria defined by Evanno et al. (2005), which were estimated using *STRUCTURESELECTOR* (Li and Liu 2018).

In order to determine the possible origin of the 12 founder individuals from Anjajavy and three males of the PBZT, we assigned them to the five Aldabra subpopulations (i.e., 'Grande Terre East', 'Grande Terre South', 'Malabar East', 'Malabar West', and 'Picard') using *GENECLASS* v.2 (Piry et al. 2004). The standard criterion described by Rannala and Mountain (1997) was used to calculate, using Bayesian statistics, the probabilities of their genotype in the source populations. The population assignment simulation algorithm of Paetkau et al. (2004) was used to simulate 10,000 genotypes for each sub-population. An arbitrary
threshold probability value of 0.05 or more was then applied to
identify the possible origin of each Malagasy individual.

2.2.5 | Inference on the Past Demography of the Aldabra Giant Tortoise

To estimate historic fluctuations in the effective population size ($N_e$) of the Aldabra giant tortoise, we utilized the *VarEff* model (Chevalet and Nikolic 2010; Nikolic and Chevalet 2014), implemented as an R package. This model is specifically designed for microsatellite markers and is based on a stepwise mutation model (SMM). It employs an approximate likelihood approach derived from theoretical results and uses a Markov Chain Monte Carlo (MCMC) framework to simulate past demography through sampling step functions. The analysis was performed with a mutation rate ($\mu$) of 0.00015, based on estimates for microsatellite markers of the herpetofauna and long-lived vertebrates (Zhang and Hewitt 2003), and a generation time (g) of 60 years, estimated as the average of the maturity age (20–25 years) and the mean longevity (ca. 100 years; Swingland and Lessells 1979). For the MCMC simulations, we followed the recommendations of Nikolic and Chevalet (2014) by using 10,000 dememorization steps and a total length of 1,000,000 steps, and by extracting 10,000 uncorrelated states for parameter estimation. Output parameters were provided in normalized scales ($\Theta=4N_e\mu$) for effective population size and generation time (g), then converted to natural scales such as $N_e$, assuming a constant mutation rate ($\mu$) and a time scale in years. Analyses were conducted using a data set of 24 microsatellites (excluding loci of the MCH region, which is potentially under strong selection) on the 47 individuals of Aldabra and Madagascar for which there was no missing data. Analyses were also repeated on the full data set for Aldabra (145 individuals) and on individuals of ‘Grande Terre South’ only (46 individuals). All individuals from Aldabra were either considered as a single population or by separating them based on the known population structure of the atoll, but this did not impact the analysis (so that we finally considered a single population).

2.2.6 | Paternity Tests and Gene Assortment in Juveniles From Anjajavy

To determine the contribution to reproduction of each found-
ing individual at Anjajavy, we used paternity tests based on the
microsatellite profiles of the 50 juveniles analyzed. A likelihood
method implemented in CERVUS v.3.0.7 (Marshall et al. 1998)
enabled us to confirm the maternal identity and to assign the
most likely paternity to each juvenile. Paternity was assigned ac-
cording to the Δ criterion, which corresponds to the difference
between the LOD scores of the two most probable male genitors.
The critical value of Δ was estimated by running 100,000 simu-
lations with an input error rate (or mutation) of 0.01 and five in-
dividuals as potential male genitors (i.e., number of males in the
population). The Δ values were determined for attribution confi-
dence thresholds of 95% (‘strict’) and 85% (‘extended’). Paternity
is attributed to the individual with the highest LOD score only if
Δ exceeds its critical value.

Finally, as we observed an excess of heterozygosity in progenies
compared to founder individuals, we tested the significance of

departure from panmixia for each locus. For that purpose, we
randomly sampled alleles from the known parental population
and simulated 500 sets of 50 progenies. We used HIERFSTAT to
compute the FIS distribution for each simulated set. From these
simulations, we obtained the distribution of FIS expected under
the null hypothesis of panmixia. In order to test whether the
excess of heterozygosity could stem from selection, we also ran
simulations where both females and males were sampled ac-
cording to fitness proxies. For each female, we used either their
total number of progenies (Table 1) or their number of proge-
nies in the 50 genotyped juveniles. For males, we used either the
number of mating attempts observed in 2022 (Table S1) or the
number of progenies in the 50 genotyped juveniles.

3 | Results

3.1 | Development of Markers, Linkage Disequilibrium Between Loci, and Estimation of Genetic Diversity Indices

The 29 loci tested were amplified on the 47 individuals analyzed, and most revealed polymorphism within populations (Tables 2 and S2). Most of the alleles detected in Madagascar (Anjajavy and PBZT) are also observed in Aldabra (except one MHC haplotype, AGI_2B-172, AGI_4-160, and AGI_5-144), while 15 alleles observed in Aldabra (all with frequency <20%, and including one MHC haplotype and five alleles of GAL_136) were not introduced into Anjajavy and PBZT (Table S2). Analyses with MICROCHECKER suggest a possible presence of null alleles on RAD_573 and AGI_5, which show a significant excess of homozygotes in our data set (Table 2). This could, however, also be due to a structure of diversity within populations (see below). Furthermore, no null alleles were detected on the descendants of the five main breeding individuals in Anjajavy (#1, 2, 6, 9, 11; with at least seven descendants/parent; see below for paternity analyses). Consequently, most of our loci should have a low frequency of null alleles and be therefore suitable for measuring population genetic parameters.

The linkage disequilibrium between markers was then analyzed (Figure S1). A significant LD is detected on all pairs of physically linked loci less than ~3 Mb apart. A similar pattern is observed in Anjajavy and Aldabra (data not shown). To keep only independent markers and avoid redundancy of information, a few loci were then excluded for certain analyses (22 independent loci being retained). For each series of non-MHC loci in LD, we kept the most informative locus (i.e., GAL_263 and AGI_1B, after exclusion of AGI_2B and AGI_9/AGI_12) for genetic diversity and STRUCTURE analyses. Furthermore, a very strong LD on the five MHC markers was detected on chromosome 12. In the 50 Anjajavy juveniles studied, no recombination event was observed (in particular between the most distant loci MHC_I and MHC_V) both for the breeding male (50 observations; see below for paternity analyses) and the breeding females (49 observations; heterozygous at the MHC locus), meaning that the targeted 1.5 Mb region was systematically transmitted as a block. The same strict association between alleles of MHC loci was also observed in the 35 Aldabra and PBZT individuals, but only from MHC_I to MHC_IV. These four loci are distributed over a
<table><caption>TABLE 2 | Diversity genetic indices for each group of individuals (min. 10) from Anjajavy (Madagascar) and Aldabra (Seychelles).</caption><thead><tr><th rowspan="3">Locus</th><th colspan="10">Anjajavy</th><th colspan="10">Aldabra</th></tr><tr><th colspan="3">Founders (12)</th><th colspan="3">Juveniles (50)</th><th colspan="3">Malabar (11)</th><th colspan="3">Grande Terre (11)</th><th colspan="3">Picard (10)</th><th colspan="3">Total (32)</th></tr><tr><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th></tr></thead><tbody><tr><td>RAD313</td><td>3</td><td>2.9</td><td>0.35</td><td>0.33</td><td>0.09</td><td>3</td><td>2.5</td><td>0.23</td><td>0.26</td><td>-0.10</td><td>2</td><td>2.0</td><td>0.40</td><td>0.55</td><td>-0.33</td><td>3</td><td>2.9</td><td>0.38</td><td>0.27</td><td>0.32</td><td>2</td><td>0.10</td><td>0.10</td><td>0.00</td><td>3</td><td>2.7</td><td>0.32</td><td>0.31</td><td>0.03</td></tr><tr><td>RAD573</td><td>1</td><td>1.0</td><td>—</td><td>—</td><td>—</td><td>1</td><td>1.0</td><td>—</td><td>—</td><td>—</td><td>2</td><td>2.0</td><td>0.24</td><td>0.09</td><td>0.64</td><td>2</td><td>1.9</td><td>0.09</td><td>0.09</td><td>0.00</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>2</td><td>1.8</td><td>0.12</td><td>0.06</td><td>0.48</td></tr><tr><td>RAD932</td><td>2</td><td>2.0</td><td>0.38</td><td>0.50</td><td>-0.29</td><td>2</td><td>1.9</td><td>0.48</td><td>0.36</td><td>0.26</td><td>2</td><td>2.0</td><td>0.40</td><td>0.36</td><td>0.13</td><td>2</td><td>2.0</td><td>0.30</td><td>0.18</td><td>0.43</td><td>2</td><td>0.18</td><td>0.20</td><td>-0.06</td><td>2</td><td>2.0</td><td>0.30</td><td>0.25</td><td>0.20</td></tr><tr><td>AGI_1B</td><td>5</td><td>4.7</td><td>0.68</td><td>0.50</td><td>0.31</td><td>4</td><td>3.8</td><td>0.66</td><td>0.72</td><td>-0.09</td><td>5</td><td>4.9</td><td>0.74</td><td>0.82</td><td>-0.06</td><td>7</td><td>6.8</td><td>0.75</td><td>0.73</td><td>0.08</td><td>6</td><td>0.66</td><td>0.80</td><td>-0.17</td><td>8</td><td>5.8</td><td>0.74</td><td>0.78</td><td>-0.05</td></tr><tr><td>AGI_2B</td><td>5</td><td>4.6</td><td>0.64</td><td>0.83</td><td>-0.26</td><td>4</td><td>3.4</td><td>0.60</td><td>0.66</td><td>-0.09</td><td>5</td><td>4.9</td><td>0.68</td><td>0.73</td><td>-0.02</td><td>5</td><td>4.8</td><td>0.71</td><td>0.82</td><td>-0.10</td><td>5</td><td>0.70</td><td>0.80</td><td>-0.09</td><td>8</td><td>5.7</td><td>0.78</td><td>0.78</td><td>0.02</td></tr><tr><td>AGI_3</td><td>3</td><td>2.9</td><td>0.40</td><td>0.42</td><td>0.01</td><td>2</td><td>1.9</td><td>0.16</td><td>0.18</td><td>-0.09*</td><td>3</td><td>2.9</td><td>0.38</td><td>0.45</td><td>-0.16</td><td>3</td><td>2.9</td><td>0.24</td><td>0.27</td><td>-0.07</td><td>2</td><td>0.10</td><td>0.10</td><td>0.00</td><td>4</td><td>2.6</td><td>0.25</td><td>0.28</td><td>-0.10</td></tr><tr><td>AGI_4</td><td>4</td><td>3.9</td><td>0.52</td><td>0.42</td><td>0.24</td><td>3</td><td>2.9</td><td>0.52</td><td>0.66</td><td>-0.26***</td><td>2</td><td>2.0</td><td>0.30</td><td>0.36</td><td>-0.18</td><td>4</td><td>3.8</td><td>0.58</td><td>0.73</td><td>-0.20</td><td>2</td><td>0.46</td><td>0.50</td><td>-0.05</td><td>5</td><td>3.2</td><td>0.51</td><td>0.53</td><td>-0.02</td></tr><tr><td>AGI_5</td><td>3</td><td>2.8</td><td>0.23</td><td>0.25</td><td>-0.07</td><td>1</td><td>1.0</td><td>—</td><td>—</td><td>—</td><td>2</td><td>2.0</td><td>0.40</td><td>0.36</td><td>0.13</td><td>2</td><td>2.0</td><td>0.40</td><td>0.18</td><td>0.57</td><td>2</td><td>0.50</td><td>0.70</td><td>-0.37</td><td>3</td><td>2.0</td><td>0.44</td><td>0.41</td><td>0.09</td></tr><tr><td>AGI_6</td><td>4</td><td>3.9</td><td>0.70</td><td>0.83</td><td>-0.14</td><td>4</td><td>3.9</td><td>0.70</td><td>0.72</td><td>-0.02</td><td>4</td><td>3.8</td><td>0.55</td><td>0.64</td><td>-0.12</td><td>4</td><td>3.9</td><td>0.69</td><td>0.55</td><td>0.26</td><td>3</td><td>0.67</td><td>0.80</td><td>-0.15</td><td>5</td><td>3.5</td><td>0.67</td><td>0.66</td><td>0.03</td></tr><tr><td>AGI_7</td><td>3</td><td>2.9</td><td>0.61</td><td>0.50</td><td>0.22</td><td>3</td><td>2.9</td><td>0.37</td><td>0.44</td><td>-0.19</td><td>3</td><td>2.9</td><td>0.54</td><td>0.82</td><td>-0.48</td><td>3</td><td>2.9</td><td>0.51</td><td>0.36</td><td>0.33</td><td>3</td><td>0.66</td><td>0.80</td><td>-0.16</td><td>4</td><td>3.0</td><td>0.62</td><td>0.66</td><td>-0.05</td></tr><tr><td>AGI_8</td><td>2</td><td>1.9</td><td>0.15</td><td>0.00</td><td>1.00</td><td>2</td><td>1.9</td><td>0.23</td><td>0.26</td><td>-0.14</td><td>2</td><td>1.9</td><td>0.17</td><td>0.18</td><td>-0.05</td><td>2</td><td>2.0</td><td>0.43</td><td>0.27</td><td>0.41</td><td>2</td><td>0.46</td><td>0.50</td><td>-0.05</td><td>3</td><td>2.0</td><td>0.38</td><td>0.31</td><td>0.18</td></tr><tr><td>AGI_9</td><td>2</td><td>2.0</td><td>0.47</td><td>0.42</td><td>0.15</td><td>2</td><td>1.9</td><td>0.49</td><td>0.52</td><td>-0.06</td><td>2</td><td>2.0</td><td>0.50</td><td>0.55</td><td>-0.05</td><td>2</td><td>2.0</td><td>0.48</td><td>0.27</td><td>0.47</td><td>2</td><td>0.50</td><td>0.60</td><td>-0.15</td><td>3</td><td>2.0</td><td>0.50</td><td>0.47</td><td>0.08</td></tr><tr><td>AGI_10</td><td>4</td><td>3.8</td><td>0.64</td><td>0.58</td><td>0.13</td><td>3</td><td>2.8</td><td>0.53</td><td>0.82</td><td>-0.54***</td><td>5</td><td>4.9</td><td>0.72</td><td>0.82</td><td>-0.08</td><td>5</td><td>4.9</td><td>0.68</td><td>0.73</td><td>-0.02</td><td>4</td><td>0.67</td><td>0.70</td><td>-0.00</td><td>6</td><td>4.8</td><td>0.74</td><td>0.75</td><td>-0.00</td></tr><tr><td>AGI_11</td><td>3</td><td>2.8</td><td>0.53</td><td>0.58</td><td>-0.06</td><td>3</td><td>2.6</td><td>0.54</td><td>0.48</td><td>0.12</td><td>4</td><td>3.8</td><td>0.48</td><td>0.45</td><td>0.10</td><td>2</td><td>2.0</td><td>0.30</td><td>0.36</td><td>-0.18</td><td>4</td><td>0.70</td><td>1.00</td><td>-0.39</td><td>5</td><td>3.8</td><td>0.57</td><td>0.59</td><td>-0.03</td></tr><tr><td>AGI_12</td><td>4</td><td>3.7</td><td>0.57</td><td>0.33</td><td>0.45</td><td>3</td><td>2.9</td><td>0.51</td><td>0.70</td><td>-0.35***</td><td>3</td><td>2.9</td><td>0.37</td><td>0.45</td><td>-0.19</td><td>4</td><td>3.9</td><td>0.53</td><td>0.55</td><td>0.02</td><td>4</td><td>0.48</td><td>0.50</td><td>0.01</td><td>5</td><td>3.6</td><td>0.47</td><td>0.50</td><td>-0.05</td></tr><tr><td>GAL50</td><td>3</td><td>2.9</td><td>0.59</td><td>0.75</td><td>-0.22</td><td>3</td><td>2.9</td><td>0.65</td><td>0.90</td><td>-0.37**</td><td>3</td><td>2.9</td><td>0.38</td><td>0.36</td><td>0.08</td><td>3</td><td>3.0</td><td>0.48</td><td>0.36</td><td>0.29</td><td>2</td><td>0.10</td><td>0.10</td><td>0.00</td><td>3</td><td>2.8</td><td>0.34</td><td>0.28</td><td>0.20</td></tr><tr><td>GAL85</td><td>3</td><td>2.8</td><td>0.23</td><td>0.25</td><td>-0.07</td><td>2</td><td>1.8</td><td>0.11</td><td>0.12</td><td>-0.05</td><td>3</td><td>2.9</td><td>0.38</td><td>0.45</td><td>-0.16</td><td>3</td><td>3.0</td><td>0.48</td><td>0.27</td><td>0.47</td><td>3</td><td>0.47</td><td>0.30</td><td>0.40</td><td>3</td><td>2.9</td><td>0.44</td><td>0.34</td><td>0.24</td></tr><tr><td>GAL94</td><td>5</td><td>4.9</td><td>0.75</td><td>0.92</td><td>-0.19</td><td>5</td><td>4.4</td><td>0.70</td><td>0.82</td><td>-0.17</td><td>5</td><td>4.9</td><td>0.71</td><td>0.55</td><td>0.28</td><td>5</td><td>4.9</td><td>0.77</td><td>0.73</td><td>0.10</td><td>4</td><td>0.73</td><td>0.60</td><td>0.22</td><td>5</td><td>4.8</td><td>0.77</td><td>0.63</td><td>0.20</td></tr><tr><td>GAL100</td><td>3</td><td>2.8</td><td>0.29</td><td>0.25</td><td>0.18</td><td>3</td><td>2.5</td><td>0.35</td><td>0.44</td><td>-0.23</td><td>3</td><td>3.0</td><td>0.43</td><td>0.55</td><td>-0.21</td><td>3</td><td>3.0</td><td>0.60</td><td>0.64</td><td>-0.02</td><td>2</td><td>0.10</td><td>0.10</td><td>0.00</td><td>3</td><td>2.9</td><td>0.42</td><td>0.44</td><td>-0.02</td></tr><tr><td>GAL127</td><td>6</td><td>5.7</td><td>0.75</td><td>0.83</td><td>-0.06</td><td>7</td><td>5.5</td><td>0.79</td><td>0.98</td><td>-0.23***</td><td>4</td><td>4.0</td><td>0.73</td><td>0.64</td><td>0.18</td><td>6</td><td>5.9</td><td>0.78</td><td>0.64</td><td>0.23</td><td>5</td><td>0.73</td><td>0.80</td><td>-0.05</td><td>7</td><td>5.8</td><td>0.79</td><td>0.69</td><td>0.15</td></tr><tr><td>GAL136</td><td>4</td><td>3.8</td><td>0.46</td><td>0.42</td><td>0.14</td><td>4</td><td>2.3</td><td>0.15</td><td>0.16</td><td>-0.05</td><td>4</td><td>3.7</td><td>0.25</td><td>0.27</td><td>-0.05</td><td>7</td><td>6.7</td><td>0.69</td><td>0.64</td><td>0.13</td><td>6</td><td>0.55</td><td>0.70</td><td>-0.22</td><td>9</td><td>5.5</td><td>0.53</td><td>0.53</td><td>0.01</td></tr><tr><td>GAL159</td><td>1</td><td>1.0</td><td>—</td><td>—</td><td>—</td><td>1</td><td>1.0</td><td>—</td><td>—</td><td>—</td><td>1</td><td>1.0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>2</td><td>1.9</td><td>0.17</td><td>0.18</td><td>-0.05</td><td>2</td><td>0.38</td><td>0.50</td><td>-0.29</td><td>2</td><td>1.9</td><td>0.19</td><td>0.22</td><td>-0.11</td></tr><tr><td>GAL247</td><td>2</td><td>1.9</td><td>0.28</td><td>0.17</td><td>0.44</td><td>2</td><td>1.9</td><td>0.38</td><td>0.52</td><td>-0.34**</td><td>2</td><td>2.0</td><td>0.43</td><td>0.45</td><td>-0.00</td><td>2</td><td>2.0</td><td>0.48</td><td>0.27</td><td>0.47</td><td>2</td><td>0.26</td><td>0.30</td><td>-0.13</td><td>2</td><td>2.0</td><td>0.42</td><td>0.34</td><td>0.19</td></tr><tr><td>GAL263</td><td>6</td><td>5.8</td><td>0.72</td><td>0.83</td><td>-0.11</td><td>6</td><td>4.6</td><td>0.64</td><td>0.72</td><td>-0.12</td><td>5</td><td>4.7</td><td>0.62</td><td>0.64</td><td>0.02</td><td>5</td><td>5.0</td><td>0.71</td><td>0.82</td><td>-0.10</td><td>5</td><td>0.76</td><td>1.00</td><td>-0.27</td><td>7</td><td>6.0</td><td>0.77</td><td>0.81</td><td>-0.03</td></tr><tr><td>Global<sup>a</sup></td><td>3.3</td><td>3.2</td><td>0.44</td><td>0.46</td><td>0.03<sup>ts</sup></td><td>3.1</td><td>2.7</td><td>0.39</td><td>0.46</td><td>-0.16*</td><td>3.1</td><td>3.1</td><td>0.44</td><td>0.47</td><td>-0.02<sup>ts</sup></td><td>3.6</td><td>3.5</td><td>0.50</td><td>0.44</td><td>0.16*</td><td>3.1</td><td>0.44</td><td>0.50</td><td>-0.10*</td><td>4.3</td><td>3.4</td><td>0.49</td><td>0.47</td><td>0.06<sup>ts</sup></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 2 | (Continued)</caption><thead><tr><th rowspan="3">Locus</th><th colspan="10">Anjajavy</th><th colspan="10">Aldabra</th></tr><tr><th colspan="3">Founders (12)</th><th colspan="3">Juveniles (50)</th><th colspan="3">Malabar (11)</th><th colspan="3">Grande Terre (11)</th><th colspan="3">Picard (10)</th><th colspan="3">Total (32)</th></tr><tr><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th><th>Na</th><th>Ra</th><th>H<sub>E</sub></th><th>H<sub>O</sub></th><th>F<sub>IS</sub></th></tr></thead><tbody><tr><td>MHC_I</td><td>4</td><td>3.7</td><td>0.57</td><td>0.50</td><td>0.17</td><td>4</td><td>3.8</td><td>0.63</td><td>0.66</td><td>-0.04</td><td>4</td><td>3.9</td><td>0.57</td><td>0.64</td><td>-0.08</td><td>4</td><td>3.9</td><td>0.61</td><td>0.55</td><td>0.15</td><td>4</td></tr><tr><td>MHC_II</td><td>4</td><td>3.8</td><td>0.60</td><td>0.50</td><td>0.21</td><td>4</td><td>3.5</td><td>0.62</td><td>0.70</td><td>-0.13</td><td>4</td><td>3.9</td><td>0.57</td><td>0.64</td><td>-0.08</td><td>4</td><td>3.9</td><td>0.61</td><td>0.55</td><td>0.15</td><td>5</td></tr><tr><td>MHC_III</td><td>5</td><td>4.6</td><td>0.64</td><td>0.67</td><td>0.01</td><td>5</td><td>4.4</td><td>0.67</td><td>0.68</td><td>-0.01</td><td>4</td><td>3.9</td><td>0.61</td><td>0.64</td><td>-0.00</td><td>4</td><td>3.9</td><td>0.68</td><td>0.73</td><td>-0.03</td><td>5</td></tr><tr><td>MHC_IV</td><td>3</td><td>2.9</td><td>0.35</td><td>0.42</td><td>-0.15</td><td>3</td><td>2.9</td><td>0.43</td><td>0.54</td><td>-0.26</td><td>2</td><td>2.0</td><td>0.30</td><td>0.18</td><td>0.43</td><td>2</td><td>2.0</td><td>0.40</td><td>0.55</td><td>-0.33</td><td>4</td></tr><tr><td>MHC_V</td><td>4</td><td>3.8</td><td>0.64</td><td>0.75</td><td>-0.14</td><td>4</td><td>3.8</td><td>0.68</td><td>0.82</td><td>-0.20</td><td>4</td><td>3.9</td><td>0.68</td><td>0.73</td><td>-0.03</td><td>4</td><td>3.9</td><td>0.63</td><td>0.55</td><td>0.18</td><td>4</td></tr><tr><td>MHC_X<sup>b</sup></td><td>6</td><td>5.5</td><td>0.67</td><td>0.67</td><td>0.05<sup>ts</sup></td><td>6</td><td>4.7</td><td>0.68</td><td>0.72</td><td>-0.06<sup>ts</sup></td><td>5</td><td>4.8</td><td>0.62</td><td>0.64</td><td>0.01<sup>ts</sup></td><td>5</td><td>4.9</td><td>0.69</td><td>0.73</td><td>-0.01<sup>ts</sup></td><td>5</td></tr></tbody></table>

Note: These parameters were estimated for each locus, and then globally for 21 independent loci and the genomic region carrying the MHC genes (MHC_X). For Anjajavy, estimates were computed both on founding individuals and their 50 progenies. For Aldabra, estimates are given for each island and on all 32 analyzed individuals. For each considered group, the number of individuals is given in brackets. For juveniles, the significance of F<sub>IS</sub> departure from panmixia was assessed at each locus using random sampling of parental alleles to produce a null distribution. Na, number of alleles; Ra, Allelic richness estimated for 10 individuals (Na = Ra for Picard); H<sub>E</sub>, expected heterozygosity under Hardy-Weinberg; H<sub>O</sub>, observed heterozygosity; F<sub>IS</sub>, inbreeding coefficient (Weir and Cockerham 1984). ***/*#/*F<sub>IS</sub> significantly different from 0 at p<0.001, 0.01, 0.05, respectively; ns, F<sub>IS</sub> non-significantly different from 0.

<sup>a</sup>Global estimates were based on 21 independent loci (not including those of the MHC region, and excluding AGI 2B, AGI 9 and AGI 12).

<sup>b</sup>MHC_X corresponds to the region carrying MHC genes for which we have defined eight haplotypes using the four loci MHC_I, II, III, and IV.

region of ~645 kb carrying all MHC homologs of Class II and the first copy of Class I, while the second copy of Class I is only ~20 kb apart in 3' of MHC_IV (Figure 2). We therefore defined eight haplotypes from this region (MHC_X) by combining the information from these four markers (Figure 2). In comparison, the LD is less marked in the few observations we have for pairs of physically linked loci on chromosome 8 (Figure S1). Furthermore, we observed some recombination events in the progenies on these genomic regions [i.e., AGI_1B vs. AGI_9 (1.84 Mb apart): four recombinants in the breeding male for 50 observations, and zero in the breeding females for 13 observations, i.e., average genetic distance of 6.3 cM; and AGI_2B vs. GAL_263 (3.04 Mb apart): two recombined in the male for 50 observations and three in females for 50 observations (5 cM)].

On the 21 independent loci excluding MHC_X, an overall allelic richness (Ra) of 3.4 was measured at Aldabra (varying between islands from 3.1 to 3.5; Table 2). In comparison, the founding Anjajavy population presents an allelic richness slightly lower than that of the atoll (3.2). Likewise, expected heterozygosity (H<sub>E</sub>) is significantly higher in Aldabra (0.49) than in Anjajavy (0.44). For MHC_X, Ra and H<sub>E</sub> of the founding Anjajavy population are generally similar to those of the atoll. In Anjajavy, although Ra and H<sub>E</sub> are slightly reduced in juveniles compared to their founding population at the 21 loci, the observed heterozygosity (H<sub>O</sub>) is higher than expected. A slight H<sub>O</sub> increase on MHC_X is also observed in juveniles. Likewise, H<sub>O</sub> is higher than expected in the recently founded Picard population.

## 3.2 | Genetic Diversity Structure and Origin of Malagasy Individuals

PCAs of individuals based on our microsatellite data (explaining 18% and 24.6% of the genetic variance depending on the data set considered; Figure S2) show that the different populations studied are not clearly differentiated. However, we note that Malagasy individuals from Anjajavy and PBZT mainly cluster with individuals from 'Grande Terre East'. Similarly, individuals from Picard are mainly found with individuals from Malabar. F<sub>ST</sub> analyses indicate a weak but significant differentiation between most pairs of populations considered, ranging from approximately 3%–9% when each island is considered as a distinct population, and from 0% to 9% when an intra-island subdivision is considered on Grande Terre and Malabar (Table S5). As observed in the PCA analysis, the population of Anjajavy is not differentiated from that of 'Grande Terre East'; on the other hand, it is quite clearly differentiated from Picard (~9%).

STRUCTURE analyses (without a priori on the origin of individuals) indicate the presence of a genetic structure in our tortoise sampling (47 individuals) with a most likely number of clusters at K=2 according to the ΔK and Ln(K)±SD criteria (Figure 2 and Figure S3). However, these criteria also suggest a structure at K=3, and on the complete data set (159 individuals) at K=5 (Figure S3). At K=5, the pattern is not easy to interpret as more individuals appeared as admixed compared to K=2 and 3. In addition, in contrast to K=2 and 3, individuals from the two main islands analyzed in our study and in Balmer et al. (2011) are not similarly assigned to genetic clusters (Figure S3). Actually, spurious substructure in the complete data set could be due to two
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>FIGURE 3 | Legend on next page.</figcaption></figure>

factors: (i) different level of missing data between both studies and (ii) the slight difference in the allele scoring (for instance on GAL_136, for which long alleles [>120bp] are missing in Balmer et al. 2011). Consequently, results at K=5 have to be interpreted with caution, and we will thus only consider K=2

and 3 (Figures 1 and 3). According to the latter, individuals from ‘Grande Terre East’ and ‘Grande Terre South’ tend to be assigned to two distinct clusters, namely, GTE and GTS, while individuals from Picard are almost all assigned to the PIC cluster at K=3 (or GTS at K=2). Individuals from Malabar appear more
**FIGURE 3** | Inferences of the structure of populations in the native range (Aldabra, Seychelles) and Madagascar (Anjajavy, PBZT) based on 22 independent nuclear loci and using Bayesian simulations with STRUCTURE v2.3.4 (Pritchard et al. 2000). The inferences were performed (A) on the 47 individuals sampled in our study, and then (B) on 159 genotypes after including the 112 individuals analyzed by Balmer et al. (2011) from Aldabra (see Table S2 for the data set). Barplots represent the percentage of assignment (p) of each individual to the clusters averaged over ten iterations at K=2 and 3. Each vertical bar represents an individual. The six breeding individuals from Anjajavy are indicated with their identification number at the top of panel A (females in black, and the male in bold gray). For the analysis based on the whole sample (B), the genotypes produced by Balmer et al. (2011) are distinguished by the black horizontal lines under the barplots. More details of these analyses, including the Ln(K) and ΔK criteria of Evanno et al. (2005), are given in Figure S4.

mixed overall. The Anjajavy individuals were not all assigned to the same genetic cluster. Females #2, 9 and 11 show a higher affinity with GTS individuals, while the rest were assigned to the GTE cluster. The three PBZT individuals are also assigned to GTE. GENECLASS analysis largely confirms STRUCTURE results with three individuals of Anjajavy (#2, 9, and 11) strongly assigned to 'Grande Terre South', seven individuals of Anjajavy (#1, 5, 6, 7, 8, 10, and 12) and two of PBZT mainly assigned to 'Grande Terre East,' and finally two individuals of Anjajavy (#3 and 4) and PBZT1 respectively more assigned to 'Malabar East' and 'Malabar West' (Table S6). However, for the latter, the assignment of 'Anjajavy 3' and 'PBZT 1' is equivocal, possibly the consequence of admixture on Malabar as revealed by the STRUCTURE analysis (Figure 3).

## 3.3 | Past Demography of the Aldabra Tortoise

Using *VarEff*, very similar results were obtained whatever the sampling considered. We thus only present here results on the 47 Aldabra tortoises for which there is no missing data (see Figure S4 for more details). The time to the most recent common ancestor (TMRCA) was estimated at ~75,000 generations ago (~4.5 Mya; Figure S4A, green peak), which aligns with the estimated divergence of *A. gigantea* from its last common ancestor with the now-extinct Malagasy relative *A. abrupta* (Austin et al. 2003; Kehlmaier et al. 2023). Another major shift in population size was observed after ~2000 generations (~120,000 years ago; Figure S4B), and it may represent a signal of colonization at the end of Aldabra's submersion (Thomson and Walton 1972; Taylor et al. 1979; Plummer 1995; Braithwaite 2016). Finally, the *VarEff* analysis revealed a continuous decline in *N*<sub>e</sub> over the past several thousand generations (Figure S4C), rather than an expected sudden bottleneck in recent history. The most pronounced demographic event detected includes a marked population contraction around 20,000 years ago (~333 generations ago; Figure S4A, orange peak), likely corresponding to the end of the Last Glacial Maximum (LGM) and subsequent sea level rise, which likely fragmented tortoise populations and reduced genetic diversity. Over the last few generations, *N*<sub>e</sub> appears to be decreasing gradually, but without a distinct signal of a recent severe bottleneck (Figure S4C,D).

## 3.4 | Contribution of Founders in the Reproduction at Anjajavy and Maintenance of Genetic Diversity in the Offspring

The maternity test first confirmed the expected maternal identity of each juvenile, allowing us to perform the paternity test, considering that the maternal identity was known. Forty-nine out of the 50 juveniles could be assigned to a single male with more than

95% confidence, and they all share the male #6 (with a positive LOD-score only for him). However, for C2M4_2022 (from female #2), it is uncertain whether the paternal contributor is #7 or #6 (LOD-Scores: #7=9.01, and #6=8.99). Given that male #7 seems to mate only occasionally (Table S1) and that male #6 is overrepresented as the father of the juveniles, it seems highly likely that all 50 juveniles originated from this same male genitor. The majority of juveniles (43 out of 50) thus come from the cross between a male assigned to GTE (#6) and females assigned to GTS (#2, 9 and 11; Figure 3; Table S6). This should explain the excess heterozygosity in juveniles (Table 2). These crosses between pairs of parents from different genetic clusters produced a significant number of clutches (> 10 between 2020 and 2023) and generally had a higher hatching rate (in particular for #2 and 11; with 75% and 64% hatching success, respectively) compared to crosses between individuals from the same cluster (#1 and 3; with nine and three clutches respectively, and a hatching of 36%; Table 1). Among the six breeding individuals, female #3 is the only one homozygous at the MHC_X locus and has contributed the least to the offspring (currently representing 4.6% of the juveniles in the nursery). The largest male (#4) and female (#5) of the Anjajavy population (Table S1) are both among the nonreproducing individuals, and also homozygous on MHC_X. At PBZT, two of the three males are homozygous at MHC_X, and none have reproduced in the past.

We found significantly negative *F*<sub>IS</sub> values in the juvenile population: for seven loci, heterozygotes were more frequent than expected under the null hypothesis of random mating in the parental population (Table 2). To test whether this excess of heterozygosity is due to selection in parents, we ran simulations in which the probability of a parent contributing to the juvenile population was proportional to a fitness proxy. When only female fitness was considered in the simulations, GAL247 and AGI_3 do not deviate anymore from expected heterozygosity. When male fitness was considered and estimated from the number of copulation attempts, only AGI_10 became nonsignificant. When male fitness was instead estimated from the paternity analysis, AGI_12, AGI_10, AGI_4, and GAL127 became nonsignificant. Finally, when both male and female fitness are considered, only GAL50 remained significant (albeit marginally: *p*=0.024). In summary, these results suggest that the observed heterozygosity excess in juveniles reflects fitness differences in the parental population.

## 4 | Discussion

In this study, we developed new genetic markers to characterize, at a relatively low cost, the genetic diversity of Aldabra tortoises, particularly in genomic regions that are expected to play important roles in immunity (MHC). These findings have potential applications in reintroduction and rewilding programs, such as defining
the genetic profile of each individual, conducting paternity analyses, and managing translocations between populations to optimize genetic mixing and limit inbreeding. We observed strong LD between pairs of physically linked markers, which persists over at least 3 Mb. This has important implications for population genetic studies that require the use of independent loci. Moreover, the high levels of LD observed may reflect past demographic instability (e.g., Bourn et al. 1999), as bottlenecks and fluctuations in effective population size can extend LD over larger genomic regions. When studying LD decay profiles between populations, we recommend considering a sliding window of at least 5-10 Mb; although the LD intensity can vary considerably across genomic regions (Stapley et al. 2017).

## 4.1 | On the Low Recombination Rate in the Genomic Region Carrying MHC Genes

While a recombination rate of 1.64 and 4.29cM/Mb was preliminarily estimated from progenies in two regions of chromosome 8, which is consistent with other observations in vertebrates (avg. = 2.52cM/Mb; Stapley et al. 2017), no recombination was observed in the 50 juveniles within the genomic block carrying the MHC genes (i.e., a 1.5 Mb segment on chromosome 12). Combined with the fact that the same MHC haplotypes (defined by loci MHC_I to MHC_IV; segment of 645 kb) were detected in mature individuals of both Anjajavy and Aldabra, these observations suggest that the recombination rate in this genomic region is very low compared to the rest of the genome. Yet, in other vertebrate species, the MHC region has been reported as a recombination hotspot (~50cM/ Mb in the domestic horse; Beeson et al. 2019). However, there is a large variation between related species, such as in primates, where it has sometimes been reported as a 'low-spot' of recombination (e.g., Malfroy et al. 1997; de Groot et al. 2014). For any species, one can expect that the demographic history and the selection pressures linked to pathogens strongly impact the evolution of this genomic region. A recurrent fluctuation in the population size of the Aldabra giant tortoise (Quesada et al. 2019) is expected to lead, at least transiently, to a strong erosion of genetic diversity, including that of MHC genes, as previously observed in the gopher tortoise (Elbers et al. 2017). When gene assortment is limited, for instance during colonization by very few individuals, it would be essential that the maintained MHC haplotypes retain functional and complementary genes to optimize immune responses. If assortative mating (i.e., mating choice) contributes to maintaining diversity at the MHC locus (see below), the block transmission of such haplotypes could be favored in the long term. A low recombination rate could be linked to a particular genomic architecture, such as a centromeric position or a high divergence between haplotypes that restrict pairing during meiosis (Stapley et al. 2017). In addition, the recombination rate could also be influenced by epigenetic factors (e.g., DNA methylation; Mirouze et al. 2012) and may explain the relatively rapid evolution of this trait among related species (e.g., de Groot et al. 2014).

## 4.2 | Population Structure and Demographic History of the Aldabra Tortoise

Our population genetic analyses confirm the presence of a structure within the Aldabra Atoll, with two main clusters, as observed

in Balmer et al. (2011) and Çilingir, Hansen, et al. (2022). The first, GTE, groups together the majority of individuals from 'Grande Terre East' as well as some individuals from Malabar, while the second, GTS, mainly includes individuals from 'Grande Terre South' and Picard. $F_{ST}$ analyses between subpopulations of Aldabra showed low genetic differentiation (from 0% to 9%), compared to those reported, for example, in the Galápagos giant tortoise, which can reach up to 50% (Ciofi et al. 2002). Nevertheless, we note a genetic differentiation between 'Grande Terre East' and 'Grande Terre South' suggesting that connectivity along the south-eastern axis of the island may be limited (see below). Furthermore, we did not observe genetic differentiation between 'Malabar East' and 'Malabar West', contrary to the results obtained by Çilingir, Hansen, et al. (2022), but this may be due to an insufficient sample size for 'Malabar West' ($n=6$). As reported by Wietlisbach (2017), genetic diversity (measured with $R_a$) is relatively low in Picard, which is expected, given that this island was recolonized by a few individuals from Grande Terre and Malabar in the 20th century (Bourn et al. 1999). $F_{ST}$ values between Picard and other islands remain high, likely due to the small effective size of its founding population that led to genetic drift (Wietlisbach 2017).

The genetic structure observed on Aldabra may result from past demographic fluctuations. In particular, the re-expansion during the 20th century may have involved only a few genitors (Bourn et al. 1999), potentially with strong biases in their contribution, across a few recolonization foci located on Grande Terre and Malabar, leading to the formation of subpopulations (Balmer et al. 2011; Çilingir, Hansen, et al. 2022). Despite the remarkable longevity of this species (> 100 years), the maintenance of differentiation between subpopulations likely results from strong limitations in gene flow between and within islands, caused by physical and natural barriers to dispersal [e.g., deep channels between islands, low vegetation cover in Grande Terre (Takamaka) and very dense scrub in Malabar (Grubb 1971; Balmer et al. 2011; Çilingir, Hansen, et al. 2022)]. Additionally, the fragmentation of suitable habitats, representing only 19% of the atoll's surface area (Walton et al. 2019), could contribute to this pattern.

The demographic history of *A. gigantea*, as reconstructed using the *VarEff* model, provides crucial insights into the evolutionary and environmental processes that have influenced it. The consistency across analyses, regardless of whether the population structure was considered, suggests that the historical demographic signals are robust and likely reflect large-scale, species-wide events rather than localized or population-specific phenomena. It implies that the origin of *A. gigantea* and its key demographic events, such as the colonization of Aldabra and responses to climatic shifts, were influential enough to leave detectable genetic signatures across the entire species. Our findings thus highlight key events in the species' history, including its divergence from common ancestors (ca. 4.5 Mya; Figure S4), population expansions, bottleneck events (ca. 120,000 and 20,000 years ago), and recent declines. These events seem to have been strongly influenced by geological and climatic changes impacting sea levels (Thomson and Walton 1972; Taylor et al. 1979; Plummer 1995; Braithwaite 2016), underscoring the interconnectedness of biological and environmental factors. These findings are also consistent with genetic
studies suggesting a long and distinct evolutionary history for *Aldabrachelys* (Austin et al. 2003; Kehlmaier et al. 2023). Our demographic inference, however, suggests a long post-LGM decline rather than a recent collapse (Bourn et al. 1999). Because effective population size estimations are influenced by coalescent timescales, recent demographic contractions may not yet be fully reflected in the genetic data, especially in a long-lived species such as *A. gigantea*. The long generation time (~60 years) of Aldabra tortoises may slow the genetic response to recent population changes, making short-term declines more difficult to detect with coalescent-based approaches. *VarEff* can theoretically detect recent demographic declines, but in this case, the signal is likely masked by the stronger effect of older fluctuations. Furthermore, historical gene flow between subpopulations may have buffered the genetic signal of a recent bottleneck, maintaining higher diversity.

## 4.3 | South Aldabra Origin of Anjajavy Individuals and Strong Bias in Their Reproductive Output: Implications for Population Management

Most of the alleles detected in the 15 Malagasy founder individuals were also found in Aldabra and the genetic differentiation between Anjajavy and Grande Terre is particularly low ($F_{ST} < 3\%$). These results suggest that most Malagasy individuals likely originated from Grande Terre. This hypothesis is strongly supported by clustering and assignment analyses, with most individuals assigned to 'Grande Terre East' and 'Grande Terre South'. Our analyses also indicate that representatives of the two main genetic clusters of Aldabra (GTE and GTS) were introduced to Anjajavy (in contrast to PBZT; Figures 1 and 3).

Although three males regularly mate with adult females, only one (#6) successfully contributes to reproduction, while five females (#1, 2, 3, 9, and 11) participate unevenly. The main reproductive females (#2, 9, and 11) are assigned to a different genetic cluster than male #6, and their mating thereby maintains high genetic diversity (notably a higher $H_O$) within the population, as observed in the first 50 juveniles. A similar pattern (increased $H_O$ with significantly negative $F_{IS}$) has been observed in the populations of Picard (this study) and Rodrigues (Wietlisbach 2017), which were recently founded, probably by individuals belonging to both genetic clusters. Such fitness differences in the parental population could result from preferential mating between genetically distant individuals. Furthermore, the largest male (#4; Table S1) has no descendants despite numerous mating attempts. Body size could not be a good proxy of reproductive success in *A. gigantea*, as reported in the gopher tortoise (White et al. 2018), but we note here that male #4 is homozygous for the MHC region. In the Chinese alligator, homozygous males for the MHC region also exhibit very low reproductive success (Han et al. 2019). Given these observations, it is possible that mate choice in *A. gigantea* is partially mediated by MHC compatibility. Such genetic control of mating choice is expected to reduce the availability of compatible mates, potentially leading to a decrease in reproductive success in small populations, as already observed in Aldabra giant tortoises by Lavigne et al. (2025). In the literature, three main hypotheses have been proposed to explain the role of the MHC in mate

choice: (1) The 'good gene' hypothesis proposes that females prefer to mate with males possessing specific alleles or genotypes that enhance offspring fitness against pathogens (Mays and Hill 2004; Cutrera et al. 2012); (2) the 'heterozygosity advantage' hypothesis suggests that males with higher overall heterozygosity at MHC genes are more resistant to a broader range of pathogens and are therefore preferentially selected by females (Landry et al. 2001; Mays and Hill 2004); and (3) the 'compatible gene' hypothesis states that females select males with MHC genotypes different from their own, thus providing a large MHC diversity in the offspring (Olsson et al. 2003; Tregenza and Wedell 2000). Furthermore, it is important to note that our observations need to be validated over the longer term, once all founder individuals have fully reached sexual maturity. It would also be highly relevant to study MHC gene assortment in other wild or captive populations.

Finally, having only one breeding male represented in the offspring may have long-term consequences for the viability of this population, although the very high generation interval in this species will delay the erosion of genetic diversity. This bias in breeding individuals could lead to the emergence of high rates of inbreeding in future generations, as crosses between full and half-sibs could potentially result in inbreeding depression and a reduction in the population's adaptive potential. To address this issue, it is necessary to implement measures to isolate male #6, allowing other adult males (#4, 7, 8, and 10) to contribute to reproduction.

## 5 | Conclusion and Perspectives

This study provides the first genetic assessment of Madagascar's only population of giant tortoises, shedding light on their origins, genetic diversity, and reproductive patterns. Our data suggest that the founding individuals are representatives of the two main gene pools found in Aldabra and likely originated from Grande Terre. These individuals contribute to reproduction in a heterogenous manner, with a single male fertilizing five reproductive females. Most of the juveniles analyzed result from crosses between individuals from the two genepools, which leads to increased heterozygosity in progenies. Further studies are needed to determine if this could be due to disassortative mating and at which stages mate choice or reproductive failure may occur (Lavigne et al. 2025).

The Anjajavy rewilding project aims to achieve an effective population size sufficient for long-term viability, reaching a density necessary to support the rewilding of the reserve's dry forest ecosystem (approximately 500 individuals expected by 2030, and approximately 2000 in 2040). It would be beneficial to organize exchanges of juveniles with reintroduction and rewilding projects on several islands in the western Indian Ocean (Hansen et al. 2010; Griffiths et al. 2011) to create an artificial metapopulation (i.e., translocation of individuals between sites; see Pedrono et al. 2004). These exchanges would maximize genetic mixing between populations, thereby reducing inbreeding. As all living *A. gigantea* individuals either come directly from Aldabra or are descendants of individuals taken from Aldabra (Austin et al. 2003), they can be considered to be closely related. Consequently, outbreeding depression,
as observed in the gopher tortoise (Loope et al. 2025), is very unlikely. The selection of translocated individuals could be guided by their genetic cluster membership, genotype, and heterozygosity at the MHC locus. Furthermore, because this large species does not reach sexual maturity until around 20 years of age, there is a trade-off in selecting translocated individuals. Although it is preferable to use adults or subadults so they can contribute to reproduction more quickly, transporting small juveniles by air is simpler and much less expensive. An adult male weighs about 80 kg, which significantly increases the cost of transporting several individuals by air between countries. In practice, translocations would preferentially involve juveniles around 10 years of age or subadults (min 15 years), offering a reasonable compromise between these two constraints. The older PBZT captive individuals, although already present in Central Madagascar, would not be suitable for strengthening the Anjajavy population, as they belong to the GTE group, which is already well represented at Anjajavy.

In the long term, this first rewilding project in Madagascar will also enhance our understanding of the ecological roles and interactions that giant tortoises once played within Malagasy ecosystems. For the first time, it is possible to study the ecology and genetics of one of the species from the Malagasy megafauna in real time; rather than indirectly through their subfossil remains (Pedrono et al. 2013, 2020).

## Author Contributions

**Guillaume Besnard:** conceptualization (lead), data curation (lead), formal analysis (equal), funding acquisition (equal), supervision (equal), writing – original draft (lead). **Alexandre Barutel:** data curation (equal), formal analysis (equal), writing – original draft (equal). **Rianja Rakotoarivony:** resources (equal), writing – review and editing (equal). **Natacha Nikolic:** formal analysis (supporting), writing – review and editing (equal). **Jonia Rasolofoniaina:** resources (equal), writing – review and editing (equal). **F. Güzde Çilingir:** resources (equal), writing – review and editing (equal). **Xenia Wietlisbach:** resources (equal), writing – review and editing (equal). **Annabelle Constance:** resources (equal), writing – review and editing (equal). **Jean-Baptiste Ferdy:** formal analysis (supporting), writing – review and editing (equal). **Miguel Pedrono:** conceptualization (lead), funding acquisition (equal), project administration (lead), resources (equal), supervision (equal), writing – original draft (lead).

## Acknowledgments

Anjajavy le Lodge is the founder of the giant tortoise rewilding project. G.B., A.B., N.N., and J-B. F. are members of the CRBE laboratory, which is supported by the Laboratoire d'Excellence (LabEx) CEBA (grant ANR-10-LABX-25-01) and LabEx TULIP (grant ANR-10-LABX-0041), both managed by the Agence Nationale de la Recherche in France. The local *Sakalava* rangers play a vital role in this rewilding project and assisted us with the collection of data on each individual. We would also like to thank Nancy Bunbury, Claudio Ciofi, Glauco Camenisch, Christine Grossen, Hervé Gryta, Emilie Lecompte, Célia Paris, and Dennis Hansen for their constructive comments and/or help with obtaining Aldabra tortoise samples from the native range (including previous published data). We thank the Parc Botanique & Zoologique de Tsimbazaza (PBZT) for samples of captive individuals held in Antananarivo.

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

Genotype data generated in this study are provided in the Table S2.

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**Supporting Information**

Additional supporting information can be found online in the Supporting Information section. **Figure S1:** Test of the linkage disequilibrium between pairs of loci. **Figure S2:** Projection of *Aldabrachelys gigantea* individuals on the first two axes of PCAs performed on microsatellite data. **Figure S3:** Detailed STRUCTURE analyses of native and introduced populations. **Figure S4:** *VarEff* analysis of 47 giant tortoises based on 24 microsatellites markers. **Table S1:** Observed attempts of reproduction between individuals of the Anjajvy Reserve during the breeding season 2022. **Table S2:** Tortoise sampling and genotyping data set. **Table S3:** Characteristics of the 24 microsatellite loci used to analyze Aldabra giant tortoises. **Table S4:** Characteristics of the five SSR loci designed in the genomic region containing the MHC class I and II genes in *Aldabrachelys gigantea*. **Table S5:** Genetic differentiation between each pair of sampled (sub)populations. **Table S6:** Results of the assignment test comparing the genotypes of the 12 founding individuals of Anjajavy and the three individuals of PBZT to genotypes of five sub-populations of Aldabra.