# Long-term genetic mark and recapture in Astyanax mexicanus cave populations: demographic inferences and conservation issues

Laurent Legendre, Julie Rode, Isabelle Germon, Marie Pavie, Stéphane Père, Julien Fumey, Maxime Policarpo, Luis Espinasa, Didier Casane, Sylvie Rétaux

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Laurent Legendre, Julie Rode, Isabelle Germon, Marie Pavie, Stéphane Père, et al.. Long-term genetic mark and recapture in Astyanax mexicanus cave populations: demographic inferences and conservation issues. Conservation Genetics, 2026, 27 (1), pp.24. 10.1007/s10592-025-01753-8. pasteur-05477724

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# Long-term genetic mark and recapture in *Astyanax mexicanus* cave populations: demographic inferences and conservation issues

Laurent Legendre<fnref n="1" /> · Julie Rode<fnref n="2" /> · Isabelle Germon<fnref n="1" /> · Marie Pavie<fnref n="3" /> · Stéphane Père<fnref n="3" /> · Julien Fumey<fnref n="4" />,<fnref n="5" /> · Maxime Policarpo<fnref n="6" /> · Luis Espinasa<fnref n="7" /> · Didier Casane<fnref n="1" />,<fnref n="8" /> · Sylvie Rétaux<fnref n="3" />

Received: 15 October 2025 / Accepted: 24 December 2025

© The Author(s) 2026

## Abstract

The cave populations of *Astyanax mexicanus* in northeastern Mexico are the subject of intensive study as a model for investigating evolutionary processes in subterranean environments. Investigating key evolutionary questions alongside conservation challenges requires a well-defined demographic framework. However, few estimates of population sizes have been obtained. In this study, we used genetic identification of cavefish individuals captured at various time intervals, ranging from a few days to several years, and in multiple caves, to gain insights into population size dynamics, population structure, and connectivity. We used 18 to 24 microsatellite markers to genotype and identify unambiguously 702 fish from six caves and several surface locations. The genetic distance between sampled fish, as well as short-term and long-term recaptures of cavefish, demonstrate that the most studied and sampled population, La Cueva de El Pachón, is small (comprising a few hundred individuals) and isolated. In contrast, populations in La Cueva de Los Sabinos and in El Sótano de Las Piedras, which we demonstrate belong to a well-connected cave cluster, could be larger, numbering in the thousands. At a larger geographical scale, our data suggest that the distribution of *A. mexicanus* cave populations in groundwater can be best described as a poorly connected network of oases within a karst desert.

**Keywords** Cavefish · *Astyanax mexicanus* · Population size · Conservation · Genetic identification · Capture-Mark-Recapture

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

## Introduction

The Mexican tetra, *Astyanax mexicanus*, is the leading model for studying evolution in subterranean aquatic environments (Malard et al. 2023). This species exists under two ecomorphotypes, cave-adapted and surface-dwelling populations, that can interbreed despite striking differences in morphology, physiology, and behaviour (Keene et al. 2016). Today, there are 33 caves identified as hosting *A. mexicanus* cavefish (Elliott 2018; Espinasa et al. 2018, 2020; Miranda-Gamboa et al. 2023). The age of these cave populations and the number of independent origins are still a matter of debate. However, the most recent studies converged on the hypothesis that only two or three lineages colonized independently the underground water environment during the Late Pleistocene, in a range of 200,000 to 10,000 years ago (Fumey et al. 2018; Herman et al. 2018; Policarpo et al. 2021, 2024; Moran et al. 2023). The genetic determinism of phenotypic differences between ecotypes has been the subject of intense research; however, knowledge of the ecological background

<footnote>Didier Casane
didier.casane@universite-paris-saclay.fr</footnote>

<footnote>Sylvie Rétaux
sylvie.retaux@cnrs.fr</footnote>

<footnote n="1">¹ UMR Évolution, Génomes, Comportement et Écologie, Université Paris-Saclay, CNRS, IRD, 91190 Gif-sur-Yvette, France</footnote>

<footnote n="2">² Objectif Sciences International NGO, OSI-Panthera program, Geneva, Switzerland</footnote>

<footnote n="3">³ Institut de Neuroscience Paris-Saclay, Université Paris-Saclay and CNRS, 91400 Saclay, France</footnote>

<footnote n="4">⁴ Institut Pasteur, Université Paris-Cité, Microbial Paleogenomic Unit, 75015 Paris, France</footnote>

<footnote n="5">⁵ Institut Pasteur, Université Paris-Cité, Bioinformatics and Biostatistics Hub, 75015 Paris, France</footnote>

<footnote n="6">⁶ Max Planck Institute for Biological Intelligence, Martinsried, Germany</footnote>

<footnote n="7">⁷ School of Science, Marist University, NYPoughkeepsie, USA</footnote>

<footnote n="8">⁸ Université Paris-Cité, UFR Sciences du Vivant, 75013 Paris, France</footnote>
and population demography lags behind (Elliott 2018; Wilson et al. 2021; Legendre et al. 2023, 2025; Espinasa et al. 2024). The ecological and demographic context is essential for understanding the evolutionary mechanisms involved in the fixation of mutations in caves, particularly the relative roles of hybridization, selection, and genetic drift (Rétaux and Casane 2013).

Demographic parameters of interest are the census size (Nc) of the subterranean populations, their connectiv-ity, and the migration rate from the surface. It is expected that these parameters vary across the distribution area and have changed over time. Nc can be measured directly, or can be inferred indirectly using population genetics meth-ods estimating effective population size (Ne). For *Astyanax* cavefish, most population genetic studies have suggested a small Ne, in the order of a few hundred or less (Panaram and Borowsky 2005; Bradic et al. 2012; Fumey et al. 2018), while others have proposed a larger Ne, higher than 10³ and up to 10⁵ (Herman et al. 2018). For such a prolific fish, with up to a few hundred eggs per spawn (Simon et al. 2019), Nc may be several orders of magnitude larger than Ne (Charles-worth 2009). Thus, a small Ne is somewhat expected, in line with recent in situ cavefish number estimates and counts (Legendre et al. 2023; Espinasa et al. 2024). A Ne larger than 10⁵ would suggest that the fish accessible to human cave exploration represent a tiny fraction of much larger, well-mixed and hidden subterranean populations.

To further examine these hypotheses, which are par-ticularly important for conservation purposes, estimates of current Nc are also necessary. For cavefish, which are mobile animals and cannot easily be counted directly over their whole distribution area, census population size can be estimated using Capture-Mark-Recapture (CMR) methods. However, data are still scarce. More than 50 years ago, in 1971, CMR studies were performed to estimate the size of *Astyanax mexicanus* populations in two caves, La Cueva de El Pachón (hereafter Pachón Cave) and El Sótano de Yer-baniz (hereafter Yerbaniz Cave), using caudal fin clippings of more than 200 individuals at one to three days inter-vals. After tentative corrections for death rates induced by fin clipping, the population size was estimated to be 9,781 (95% confidence interval (CI) 1,179–18,283) in Pachón Cave and 8,671 (95% CI 1,810–15,534) in Yerbaniz Cave (Elliott 2018). In 2009, Reynoso et al. captured and marked 50 individuals in Pachón Cave and 36 individuals in La Cueva Chica (hereafter referred to as Chica Cave) by caudal fin clip. Unfortunately, they were unable to estimate popu-lation sizes because no marked fish were found among the 54 and 14 fish, respectively, captured two days later (Elliott 2018), which may result from small sample sizes and/or high mortality rates of marked fish.

Caves undergo cycles where, during the dry season, the water level decreases and isolated water units, known as pools, form, allowing cavefish to be segregated and isolated into different pools. During the rainy season, the water level rises, streams of moving current can form, and the different pools can be joined into a single body of water, allowing the mixing of resident fish (Legendre et al. 2025). Short-term (a few days interval) CMRs are appropriate to estimate popu-lation sizes at the pool level, but long term CMRs are neces-sary to estimate at cave or cave cluster levels. Between 2019 and 2022, we re-examined the population size in Pachón Cave using a novel, minimally invasive CMR method based on swabbing and genetic identification of fish captured at different time intervals, ranging from a few days to several years (Legendre et al. 2023). The estimated population size for the main pool in Pachón Cave during the 2022 dry sea-son was 218 cavefish (95% CI 40–395). Moreover, compar-ing individuals captured in 2019 and 2022, we found three pairs of identical genotypes, suggesting that these fish had been recaptured three years after their first capture. Alto-gether, these results indicated that the population in Pachón Cave is currently small, of an order of magnitude of a few hundred individuals. Furthermore, the comparison of the 1971 and 2022 population size estimates revealed a decline in the Pachón cavefish population, raising serious conserva-tion concerns and questions within the *Astyanax* cavefish community (Legendre et al. 2023). However, the confidence intervals of these Nc estimates are large, and they must be reassessed several times to be reliable.

Here, we aimed to fill this gap and to examine whether small population sizes applies to all *Astyanax* caves. We present novel genetic CMR data collected in 2023 and 2024 from the Pachón Cave, which now includes 24 microsatel lite markers for statistical robustness. These data confirm the alarmingly small size of this cavefish population. To compare the situation with other *Astyanax* caves, we also performed CMR in La Cueva de Los Sabinos (hereafter Sabinos Cave) and in El Sótano de Las Piedras (hereaf- ter Piedras Cave), which suggest slightly larger or similar population sizes, respectively. We interpret and discuss our CMR results within the frame of the global genetic differen-tiation pattern of *Astyanax* cavefish and surface fish popula-tions in the geographical area.

# Materials and methods

## Fish sampling

Since 2011, we have assembled a library of cavefish DNA samples. Adult fish were gently caught with telescopic han-dle fishing nets during fishing sessions that could last up to
several hours. Fresh fin tissue or skin cells were collected
through fin clipping (2011–2019) and swabbing (since

then), respectively. Geographic positions of sampling loca-
tions are shown in Fig. 1, and sampling statistics are shown
in Table 1.

In four caves, fish were sampled and released several
times. In Pachón Cave, we sampled 20 fish in 2016, 35 fish
in 2019, 30 and 29 fish at three-day interval in 2022 (22nd
and 25th February 2022), 31 and 30 individuals at five-
day interval in 2023 (4th and 9th February 2023), and 30
fish in 2024 (18th February 2024). All samples were taken
from the main pool. In Sabinos Cave, we sampled 2 fish
in 2011 (pool1), 16 fish in 2013 (pool1), 15 fish in 2017
(10 in pool1 and 5 in pool2), 52 fish in 2019 (36 in pool1
and 16 in pool2), 30 and 27 fish in 2022 (pool2, 19th and
20th February 2022), 30 and 25 fish in 2023 (pool2, 6th and
11th February 2023), 30 and 15 fish in 2023 (pool2, 20th
and 23rd February 2023), plus 30 fish also in 2023 (pool1,
23rd February 2023). In Piedras Cave, we sampled 15 fish in
2022 (pool5, the first large transverse pool of the cave), 15
fish in 2023 (pool5), and 24 fish in 2024 (16 in pool5; 4 in
pool9; 4 in pool10, which is the final sump). In La Cueva del
Río Subterráneo (hereafter Subterráneo Cave), we sampled
16 fish in 2013 (pool2), 10 fish in 2016 (pool2), 24 fish in
2019 (pool2), and 6 fish in 2022 (small deeper pools3 and
4) (Table 1).

In three other caves, fish were sampled and released only once: 8 fish in La Cueva de El Refugio (hereafter Refugio Cave) in 2024, 16 fish in El Sótano de La Tinaja (hereafter Tinaja Cave) in 2016, and 29 fish in El Sótano del Toro (hereafter Toro Cave) in 2019. Additionally, 85 surface fish were sampled at various locations (Fig. 1).

Fish genotyping

From 2011 to 2019, fish were sampled by cutting a small-
sized fin-clip at the caudal fin, and DNA was extracted from
the fin fragments conserved in ethanol. Since 2022, we have
used a less invasive procedure. Gentle swabs were per-
formed on each flank of the fish to obtain two independent
samples per individual (Legendre et al. 2023). That proce-
dure minimizes physical damage to the fish and probably
reduces stress significantly compared to fin clipping. We
have never observed any consequences or mortality assos-
ciated with this sampling method, either in fish from our
laboratory facility or in the field with wild fish. In all cases,
individuals were rapidly released back into their natural
pond after sampling, whether fin-clipped or swabbed.

The procedure for sample storage and DNA extraction
was optimized to account for the fieldwork conditions, as
follows. DNA was extracted from swabs (FLOQSwabs<sup>R</sup>,
COPAN Diagnostics Inc.) stored in tubes with silica gel
beads at room temperature. Lysis with a mix of buffer
(Tris HCl pH 8 100 mM, EDTA 2 mM, triton 0.2%) and

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Map with sampling localities. The 33 caves with *A. mexicanus* cavefish (CF caves) are indicated (orange and red circles). The caves studied in this paper are indicated by a large red circle and their name is written on the map. The names of the caves where CMR was performed are in bold. Surface fish sampling localities (river stations) are indicated with letters: (A) Pozo de Pachón Praxedis Guerrero, (B) Arroyo Tampemole El Barranco, (C) Arroyo La Pagua Otates, (D) Nacimiento del Río Choy, (E) Nacimiento del Arroyo El Nacimiento, (F) Río Gallinas Puente Sur Rascón, (G) Estanque del Cocodrilo Tani-nul, (H) Río Tampaon Puente Tanchachin</figcaption></figure>
<table><caption>**Table 1** Sampling schedule and summary statistics</caption><thead><tr><th></th><th>2013</th><th>2016</th><th>2017</th><th>2019</th><th>2022</th><th>2023</th><th>2024</th><th>TOTAL=702</th></tr></thead><tbody><tr><td>Pachón</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>N</td><td>19</td><td></td><td></td><td>35</td><td>(1)</td><td>(1)</td><td>(2)</td><td>202</td></tr><tr><td>He</td><td>0.19</td><td></td><td></td><td>0.20</td><td>29</td><td>31</td><td>29</td><td></td></tr><tr><td>P<sub>uni</sub>(x 10<sup>-4</sup>)</td><td>2.1</td><td></td><td></td><td>2.1</td><td>2.8</td><td>2.2</td><td>3.5</td><td>0.18</td></tr><tr><td>Refugio</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>N</td><td></td><td></td><td></td><td></td><td></td><td></td><td>8</td><td>8</td></tr><tr><td>He</td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.6</td><td></td></tr><tr><td>P<sub>uni</sub></td><td></td><td></td><td></td><td></td><td></td><td></td><td>&lt; 10<sup>-13</sup></td><td></td></tr><tr><td>Sabinos</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>N</td><td>2 + 16</td><td></td><td></td><td>15</td><td>(1)</td><td>(1)</td><td>(1)</td><td>264</td></tr><tr><td>He</td><td>0.23</td><td></td><td></td><td>0.21</td><td>29</td><td>27</td><td>30</td><td>15</td></tr><tr><td>P<sub>uni</sub>(x 10<sup>-4</sup>)</td><td>0.7</td><td></td><td></td><td>1.9</td><td>0.24</td><td>0.23</td><td>0.22</td><td>0.21</td></tr><tr><td>Tinaja</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>N</td><td>16</td><td></td><td></td><td></td><td></td><td></td><td></td><td>16</td></tr><tr><td>He</td><td>0.33</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P<sub>uni</sub></td><td>&lt; 10<sup>-6</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Piedras</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>N</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>54</td></tr><tr><td>He</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P<sub>uni</sub>(x 10<sup>-4</sup>)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Toro</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>29</td></tr><tr><td>N</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>He</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P<sub>uni</sub></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Subterráneo</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>N</td><td>15</td><td>7</td><td></td><td></td><td></td><td></td><td></td><td>50</td></tr><tr><td>He</td><td>0.54</td><td>0.38</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P<sub>uni</sub></td><td>&lt; 10<sup>-11</sup></td><td>&lt; 10<sup>-6</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Surface</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>N</td><td>79</td><td></td><td></td><td></td><td></td><td></td><td></td><td>79</td></tr><tr><td>He</td><td>0.84</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P<sub>uni</sub></td><td>&lt; 10<sup>-26</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Samples from Sabinos obtained in 2011 and 2013 were pooled because only two fish were sampled in 2011. Sampling rank in a given year is shown in parentheses. N: number of samples genotyped at all 18 microsatellite loci and used in population analyses. He: mean expected heterozygosity. P<sub>uni</sub>: probability that two unrelated fish have the same genotype. Surface: surface fish samples at different localities, see Fig. 1 for sampling locations

Global P<sub>uni</sub> where several samples are available: Pachón: 2.2 x 10<sup>-4</sup>, Sabinos: 5.5 x 10<sup>-5</sup>, Piedras: 4.5 x 10<sup>-5</sup>, Subterráneo: 3.5 x 10<sup>-9</sup>

<figure><img src="image_5.png" /></figure>
proteinase K (250 µg/mL) overnight at 55 °C was followed by inactivation of proteinase K for 10 min at 98 °C. Aliquots of DNA were transferred to new tubes and stored at -20 °C.

Previously (Legendre et al. 2023), we selected eighteen microsatellite loci for genotyping on the basis that: (1) they were highly polymorphic (Bradic et al. 2012), (2) they could be easily amplified by PCR, and (3) the amplicon sizes allowed the amplification of these eighteen loci through only three multiplexed PCRs. In the present study, we selected six additional loci that were genotyped only when a pair of 18-loci genotypes were identical, corresponding to a putative recapture, to confirm the recapture and reduce the probability of a false recapture. To estimate the probability that two unrelated fish have an identical genotype at these six loci, we genotyped a random sample of 30 fish from Pachón Cave and 30 fish from Sabinos Cave and estimated allele frequencies in each.

Before setting up the PCR reactions, we prepared a 10X primer mix containing 2 µM of each primer (Multiplex1, Multiplex2, and Multiplex3). A fourth Mix (Multiplex4) was used to get the additional 6-loci genotype when necessary. The PCR reactions were carried out in 10 µL of final volume with: 1 µL of template DNA, 5 µL 2X Platinum™ Multiplex PCR Master Mix (ThermoFisher Scientific), 1 µL 10X primer mix, and 3 µl H₂O. The program used was: 2 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 90 s at 60 °C, and 60 s at 72 °C, and a final extension for 30 min at 60 °C.

Genotypes were scored using an ABI 3130 XL Genetic Analyzer, with GeneScan™ 500 LIZ™ size standard (ThermoFisher Scientific) and GeneMapper™ software v4.1 (Applied Biosystems™). For each locus, several alleles with different sizes were sequenced using homozygous specimens. These sequences enabled us to translate the relative allele sizes obtained with GeneMapper into actual allele sizes (Supplementary Data S1).

For each specimen captured in 2022, 2023, and 2024, the two independent samples (swabs, left and right side) from each fish were genotyped. It could be seen as a control of the method's efficacy. With most DNA samples, we could determine the genotype at each of the eighteen or twenty-four loci. If a genotype was missing or the two genotypes differed at a locus for a given DNA sample, another round of genotyping for that locus was performed. However, for a few fish, the genotype at one or several loci was not unambiguously identified (24 out of 726 fish genotyped). For statistical analyses, only complete genotypes were used (Table 1, Supplementary Data S1).

The probability that two unrelated fish have the same genotype ($P_{uni}$) was computed as previously described in Legendre et al. 2023. The probability that two full siblings

have the same genotype was calculated as described by Woods et al. 1999.

In addition, we performed extensive simulations to evaluate the effect of undetected genotyping errors, considering allelic dropout (technical failure for some alleles to amplify) only, false heterozygosity (addition of a false allele taken at random among existing alleles) only, or both types of genotyping errors. For each model, we considered a percentage of genotyping errors per locus ranging from 1% to 10%. Under the different models of errors, independent 18-Loci genotypes were generated, taking into account allele frequencies at each locus in each cave. In no case did genotyping errors increase the number of identical genotypes among unrelated individuals above the number expected by chance without genotyping errors. That is because allelic dropout and false alleles tend to produce ghost genotypes (false new individuals), and not false identical genotypes. Only an unrealistic error rate of 10% led to a dramatically reduced number of identical genotypes. In conclusion, genotyping errors tend to lead to an underestimate rather than an overestimate of the number of recaptures based on genetic identification, further ensuring that the results reported below are reliable.

## Population size estimation by genetic CMR

We conducted CMR studies to estimate the population size by sampling fish in the main accessible pools of different caves during the dry season (in February and March). That could represent only a fraction of the actual population that can exchange individuals, not only from pools accessible to humans, but also from any unexplored pools. To estimate the population size, CMR studies were conducted within and between years from 2013 to 2024, allowing for several flooding events of the caves between captures, particularly during rainy seasons. That accounts for the subpopulations of Astyanax that can readily interchange individuals during “normal” years. It may be that perched or distant pools are only accessible during intense hurricanes or once-in-a-hundred-year flooding events, but these infrequent events are not accounted for in our experimental design.

The principle of a CMR is as follows: we capture, “mark”, and release a fish out of a total population of size $x$. After the “marked” fish have freely mingled with the unmarked, we re-catch a random sample of size $n$, of which $r$ are found to be “marked”. That allows for the estimation of the maximum likelihood estimate of $x$ and its variance (Bailey 1951). To take into account a bias when the expectation of $r$ is small, Bailey proposed an adjusted estimate of $x$ and of its variance (Bailey 1951, 1952):

$$ \hat{x} = a(n + 1)/(r + 1) \quad (1) $$
$$ \text{var } \bar{x} = a^2(n+1)(n-r)/(r+1)^2(r+2) \quad (2) $$

These estimators have been used in previous CMR studies of *A. mexicanus* population sizes, and, for comparison purposes, we also used them in the present study. The validity of the estimates depends on several assumptions that should be verified: (1) the population is closed, that is the total number of individuals is not changing through births, deaths, immigration or emigration over the sampling time period, (2) all animals are equally likely to be captured and recaptured, (3) capture and marking do not affect catchability, (4) marks are not lost. The last two assumptions are a matter of the methodology. Researchers should use permanent marks, and marking should be as little invasive as possible to limit the effect on catchability. Whether the studied population is closed and restricted to a well-defined distribution area, with neither immigration nor emigration, is often an unsettled issue. This last assumption may not be satisfied if we assume that caves are only a small part of the subterranean world in which cavefish thrive. As marked and unmarked animals must have freely mingled before recapture, the animals must be mobile enough, and the time interval between samplings must be long enough to allow mixture. Thus, the time interval between samplings also depends on the size and connectivity of the distribution areas.

## Genetic distances and multidimensional scaling

The genetic distance between each pair of individuals was computed as the number of allelic differences at the 18 loci. To represent the matrix of genetic distances as a two-dimensional scatter plot, we performed a metric multidimensional scaling (MDS) using the cmdscale function from the stats package in R (R Core Team 2021).

## Results

### Fish genotyping and genetic CMR accuracy

Each fish was identified by genotyping at eighteen microsatellite loci, which are known to be highly polymorphic in surface populations but less polymorphic in caves (Legentre et al. 2023). Since 2022, we have taken two independent samples per fish, and most specimens could be genotyped independently at all 18 loci. At the end of the process, 99.75% of the genotypes were determined unambiguously. Those loci that we could not genotype (0.25%) were often loci that could not be amplified and found in a few specimens, suggesting a problem with the quality of the DNA. Because only 24 of the 726 samples could not be genotyped at all 18 loci, we removed these samples with incomplete

genotypes from the analyses. Summary statistics of the sampling are shown in Table 1.

In surface fish locations and in caves where surface and hybrid fish are found (Refugio, Toro, and Subterráneo caves), the probability that two unrelated fish have the same genotype was very low (~10⁻⁹ to 10⁻²⁶) (Table 1). On the other hand, in caves where no surface fish or hybrids have been observed (Pachón, Sabinos, Piedras, and Tinaja caves), the probability that two unrelated fish have the same genotype was higher (2.2 × 10⁻⁴, 5.5 × 10⁻⁵, 4.5 × 10⁻⁵, and 4.0 × 10⁻⁷, respectively). Because the number of genotype comparisons was high in Pachón and Sabinos caves (20301 and 34716, respectively), we expected 4.5 (2.2 × 10⁻⁴ × 20301) and 1.9 (5.5 × 10⁻⁵ × 34716) identical genotypes among unrelated fish in these caves, respectively. In Piedras Cave, the number of comparisons was lower (1431) and the number of expected false recapture was lower than one (4.5 × 10⁻⁵ × 1431 = 0.06).

To reduce the number of potential false recapture counts in Pachón and Sabinos caves, the polymorphism at six additional loci was assessed. We genotyped 30 randomly selected DNA samples from Pachón Cave, Sabinos Cave and surface fish (Supplementary Data S1). Using twenty-four loci, the probability of a false recapture fell significantly below one in Pachón and Sabinos caves (0.20 and 0.05, respectively). Moreover, the probability that two full sibs have the same genotype was low in Pachón and Sabinos caves (0.0042 and 0.0018), which excludes the possibility that strong kinship could explain identical genotypes, and ensures that the genetically identified recaptures reported below are reliable.

### Genetic differentiation of cave populations

To estimate the level of connectivity between caves and the relationship with surface populations, we plotted the genetic distances between all 702 fish with complete 18-Loci genotypes (Table 1) on a MDS representation. The resulting graph highlighted several clusters that are fully consistent with the geographic distribution of the caves and their level of isolation relative to one another and to surface streams (Fig. 2).

First, surface fish collected in several locations (Fig. 1) all belong to a large yet homogeneous cluster (grey crosses in Fig. 2A, see Supplementary Figure S1 for a detailed representation of each surface locality), in accordance with data suggesting virtually no differentiation of surface populations at this geographic scale (Avise and Selander 1972). Second, cavefish populations form more compact clusters, in line with the restricted genetic diversity found in smaller populations (See He in Table 1), and with varying degrees of connection to other caves and surface populations.
## A - Multidimensional scaling (MDS) of genotypes

<figure><img src="image_6.png" /><figcaption>**Fig. 2 A**, MDS (Multidimensional Scaling) based on genetic distances between all fish. The color code for cave and surface populations is indicated. Each dot or cross is an individual fish. Dots labelled S indicate a cavefish with a surface phenotype; Dots with an H indicate a cavefish with a hybrid phenotype. Dots without a letter indicate cavefish with a cavefish phenotype. Individuals with a black asterisk are shown in B, along with their ID numbers for reference in the pictures. **B**, Selected examples of genotype-to-phenotype relationships in the Refugio Cave (blue) and in the Toro Cave (green). The individuals</figcaption></figure>

## B - Genotype to phenotype relationship

<figure><img src="image_7.png" /></figure>

shown are those with a black asterisk in panel A. Complete IDs are indicated; only the last two digits are reported on panel A. The first column displays cavefish with typical troglomorphic phenotypes, characterized by eyelessness and depigmentation. The middle column shows cavefish with a hybrid phenotype, i.e., with small eyes and/or pigmentation loss (labelled H in panel A). The third column shows surface fish with large eyes and strong pigmentation (labelled S in panel A). Photos are not at the same scale

In agreement with our previous suggestion that the Pachón Cave population is small and isolated (Legendre et al. 2023), we observed a small and compact cluster, well separated from other caves and surface fish (yellow dots in Fig. 2A). The fish closest to Pachón Cave were in the Refugio Cave (light blue dots in Fig. 2A), which is only a few kilometers from the Pachón Cave. Yet there was no evidence of mixture between these two caves. On the other hand, among the eight individuals sampled in Refugio Cave, two had a surface fish phenotype and one had a hybrid phenotype (Fig. 2AB). They consistently clustered with surface fish and/or were placed between Refugio cavefish and surface fish on the scatter plot, respectively.

Sabinos and Tinaja caves belong to an interconnected group of caves (Mitchell et al. 1977). Accordingly, fish sampled in these two caves (orange and red dots in Fig. 2A) belonged to a homogeneous cluster on the scatter plot. More surprisingly, fish from Piedras Cave (dark blue dots) were entirely embedded within the cluster of Sabinos and Tinaja fish - even though they are located five kilometers further south - indicating some level of population mixture between these three caves (see Supplementary Figure S1 for a detailed representation of each cave).

The Toro cavefish (green dots in Fig. 2A) constituted a cluster separated from other cavefish, but very close to the Sabinos/Tinaja/Piedras cluster, in accordance with their geographic proximity in south El Abra (Fig. 1). In the Toro Cave, a mixture of fish with cave, surface, and hybrid phenotypes was observed (Policarpo et al. 2024). Indeed, like Refugio fish, the Toro fish with a surface-like phenotype clustered with surface fish from surface stations, and hybrids were found between surface fish and cavefish from the Sabinos/Tinaja/Piedras/Toro cluster (Fig. 2AB).

Finally, fish captured in the Subterráneo Cave (purple dots in Fig. 2A) were the closest to surface fish among all caves analyzed, in agreement with the description of surface fish being regularly washed into this cave every year during the rainy season (Bibliowicz et al. 2013). Most of them had a troglomorphic phenotype, but two were surface-like fish, and one had a hybrid phenotype, which was reflected in their corresponding locations on the plot.

Altogether, our MDS analysis suggests that the Pachón cavefish population in the north El Abra is isolated, whereas other cavefish populations in the south El Abra show greater connectivity (Sabinos, Tinaja, Piedras). Notably, the three caves where we have performed CMR (Pachón,
Sabinos, Piedras) seem devoid of significant surface fish introgression.

## Population size estimation

With 18-loci genotypes, we observed twenty-three pairs of identical genotypes (hence putative recaptures) in Pachón Cave, three in Sabinos Cave, and one in Piedras Cave. Using the 24-loci genotypes, we identified four “false” recaptures in Pachón Cave and two “false” recaptures in Sabinos Cave. Notably, these numbers corresponded to the expected false positive rates computed above (4.5 and 1.9, respectively), confirming the necessity and efficacy of increasing genotyping power for accurate CMR results.

The patterns of recaptures in each cave, within and between years, are shown in Fig. 3 and Supplementary Data S1.

Strikingly, most recaptures occurred in Pachón Cave, where 17 fish were captured at least twice, of which two were captured 3 times (twice the same year and once during another year) (Fig. 3AA'). With similar short-term CMR sample sizes in 2022 (29 and 29) and 2023 (31 and 29), we identified three fish recaptured in both cases. We

obtained similar estimates of the population size in the main pool, specifically 218 cavefish in 2022 (95% CI: 40–395) and 233 cavefish in 2023 (95% CI: 43–422), respectively. Moreover, between 2022 (45 fish genotyped) and 2023 (47 fish genotyped), four fish were recaptured. The (over-)estimated population size from this long-term, one-year CMR would be 432 (an overestimate because the assumption that no fish would die between samplings is likely not valid). We observed numerous other recaptures between years. In 2019, 35 fish were genotyped, three were recaptured in 2022 among 55 fish (the (over-)estimated population size from this long-term 3-year CMR would be 490). Two other fish were captured in 2023 among 57 fish, and two other fish in 2024 among 30 fish. None of the 19 fish captured in 2016 was recaptured later. Together, these data show that, despite relatively small samples, short-term and long-term recaptures were repeatedly observed, and the number of recaptures was similar within and between years over a five-year window. This suggests that Pachón cavefish were not significantly washed out of the cave nor mixed with cavefish from other caves over several years, demonstrating that the population is closed, in agreement with our genetic distance analyses (Fig. 2A).

<figure><img src="image_8.png" /><figcaption>**Fig. 3** ABC, CMR plan, and recapture pattern. The number of fish captured and genotyped each year is given in parentheses (see also Table 1). The number of recaptures within and between years is indicated under the lines linking samples. Two fish captured twice a given year during short-term CMR and recaptured another year are indicated with an asterisk. For Pachón, the data from 2016, 2019, and 2022 were presented in Legendre et al. (2023). In Sabinos, the two pools were sampled (pool1 and pool2, indicated). Samples from Sabinos obtained in 2011 and 2013 were pooled because only two fish were sampled in 2011. In Piedras, CMR was mainly designed in pool5. A'B'C', Sche-</figcaption></figure>

matic maps of caves (detailed topographic maps can be found in Supplementary Figure S2). Note that each cave is represented at a different scale (see bars). The water pools are represented here at the dry season, i.e., as observed when humans can safely enter the caves; however, the different pools within a cave may be connected during the rainy season (see Discussion). The sampling sites are indicated by a pink dot in their respective pools. Empty grey arrows indicate final sumps that humans cannot access, and which can connect to the underground aquifer (and other caves)
In striking contrast to Pachón Cave, in Sabinos Cave, only one fish captured in 2013 was recaptured in 2024 (Fig. 3BB') – and this was despite a similar sampling effort (264 fish genotyped). In particular, the three rounds of short-term CMR performed successively in 2022, 2023, and 2024 in Sabinos pool2 led to zero identified recaptures. These results suggest that the population in Sabinos Cave is too large to be reliably estimated with small samples. Notably, the single Sabinos cavefish we recaptured was more than 11 years old, since it was large enough in 2013 to be sampled by fin clipping.

In Piedras Cave, where a less intensive sampling effort was realized almost exclusively in pool5 (54 fish genotyped), only one fish captured in 2022 was recaptured in 2023 (Fig. 3CC'). The (over-)estimated population size from this long-term, one-year CMR in pool5 would be 120. The population in Piedras pool5 is probably small because we could recapture one fish with very small sample sizes. However, a more intensive sampling effort would be necessary to obtain reliable estimates in pool5, as well as to extend the study to deeper pools of this cave.

## Discussion

In the present study, we have sought to better understand the current demography of *A. mexicanus* cavefish in different areas of their main geographic distribution, the Sierra de El Abra. Reiterated fish sampling and genotyping over several years allowed us to perform short- and long-term CMR analyses and estimate census population sizes. We also had the ability to detect fish that might have migrated from one cave to another one, or from the surface to a cave. We obtained contrasting results in the north and south of El Abra region which are discussed below.

### North El Abra: Pachón cave demography

We have significantly increased the dataset, first obtained in a previous study of the demography in the Pachón Cave (Legendre et al. 2023), with three additional samplings in 2023 and 2024. The new census population sizes are congruent with previous estimates, and we observed several recaptures across the years from 2019 to 2024. Moreover, and importantly, previous putative recaptures have been confirmed, beyond reasonable doubt, by adding six new microsatellite loci in the genotyping scheme (from eighteen loci to twenty-four loci).

Our previous study suggested that the population in Pachón Cave is small and isolated, which raised emotions and critiques within the *Astyanax* cavefish community. We agree that this type of analysis must be repeated several

times, because the confidence intervals of the population size estimates are large. It can be argued that, in our previous study, we have recaptured several fish in Pachón by chance, even though the cave is part of an extensive cave network with a large but hidden population in inaccessible groundwater.

However, we obtained several independent estimates, and we found similar values with short-term (218/233 fish) and long-term (432/490 fish) recaptures. Notably, estimates of population size based on long-term recaptures are less accurate and tend to be overestimates, as the assumption that no fish would die between sampling is likely not valid. However, long-term recaptures were expected in a small, isolated population because the adult life expectancy of *A. mexicanus* cavefish in the wild is several years, approaching and perhaps exceeding ten years (Simon et al. 2017) – they can live up to 15 years in the lab (personal observations and Cobham et al. 2025).

Repeated recaptures over the years also suggest that Pachón cavefish are not intermixed with cavefish from other caves during the rainy season, when the water regime fluctuates (Legendre et al. 2025). This conclusion from CMR is in line with our results on genetic differentiation in caves, where Pachón individuals form a compact cluster, distant from other cave and surface fish. Thus, multiple CMRs and cavefish genetic proximity converge to support the idea that the Pachón cavefish population is small and isolated.

### South El Abra: Sabinos area demography

In Sabinos Cave, despite a sampling effort from 2011 to 2023 similar to the one in Pachón Cave, we obtained only one recapture: a fish in the 2024 sampling that was first captured in 2013, in the same pool. This pattern suggests that the sampling effort must be increased to recapture fish in this cave and calculate the proper population size, and that the Sabinos population may be slightly larger than in Pachón – but not much higher, otherwise we would not have recaptured the single 11-years old individual in pool1. Rather, the Sabinos population may be in the order of a few thousands, as suggested by the results of a contemporary study using a transect sampling method in the 310-meter-long Sabinos pool1 (Espinasa et al. 2024). Notably, the discovery of an 11-year-old *Astyanax* cavefish in Sabinos is not unexpected, as it falls below the expected lifespan of these fish in captivity. Additionally, sclerochronology studies in the wild have previously reported cavefish reaching up to 8 years old (in Tinaja Cave), albeit with a small sample size (Simon et al. 2019).

A recent study of the water regime and fluctuations in Sabinos Cave has demonstrated that during the rainy season, when the cave is flooded, the two pools of Sabinos
Cave can merge (Legendre et al. 2025), suggesting that the resident fish of pool1 and pool2 can mix on these occasions. This result predicts that genetic differentiation cannot occur between the two pools of this cave. This prediction was confirmed here with the finding of a single compact cluster of Sabinos individuals in the MDS analysis, regardless of their pool of origin. Moreover, the possible mixing of pool1 and pool2 fish, which occurs during intense flooding in some years, suggests that in future CMRs, we may recapture in one pool a fish that was initially captured in the other pool.

Finally, in pool5 of Piedras Cave, we recaptured one fish in 2023 that was first captured in 2022, a result indicating that the population in this relatively small pool, located in the middle of the cave, could be small. More recaptures are necessary to consolidate this hypothesis for this particular pool and for other pools of the cave. Studies of the water regime in Piedras showed that floods induce a “cascading effect”, with water from pool5 being washed deeper in the cave into pool9 and then pool10 (Legendre et al. 2025). Probably, fish are also entrained in the process, depleting pool5 and increasing the number of fish in the deeper pools, which aligns with our personal observations of a larger fish population in pool10. Therefore, it is expected that in the following years, we will recapture the fish previously captured in pool5 in 2022-2023-2024 in Piedras’ deep pools.

Sabinos and Tinaja caves are assumed to belong to a well-connected network of caves, named “El Sistema de Los Sabinos” (Fish 2004; Elliott 2018), which also includes the cave named El Sótano del Arroyo. This hypothesis is well supported in the present study by the MDS analysis. There is no genetic differentiation among these caves, but the migration rate between caves could be low enough to allow recapture within caves but not between caves (especially with our sample sizes). More CMR experiments would be necessary to test this possibility. Sabinos and Tinaja caves are in proximity (1.8 km between the two entrances), and there are likely connections in the underground aquifer that allow water and fish to flow, which humans cannot access. Our MDS results show that cavefish from Piedras, located five kilometers south, display genotypes clustered with those from Sabinos and Tinaja, suggesting that Piedras is part of the same cave cluster or system.

## Underground cavefish distribution models

Population genetics analyses, estimating long-term effective population sizes and migration rates, led to opposed opinions about the demography of *A. mexicanus* cavefish, which we represented as the “Oasis”, the “Lake”, and the “Sea” models (Legendre et al. 2023). The present study allows for further discussion of these models (Fig. 4). Notably, the

demography may be different in different areas and may have changed over time.

In the “Sea” model, it is assumed that we have access to only a few subterranean pools that are well-connected and form a single network over a large distribution area, particularly when the water level is high (Fig. 4, left). In this model, caves serve as small windows into a large and inaccessible groundwater world, and the large and homogeneous cavefish population cannot be accurately estimated using a CMR approach based on small and local samples, particularly when the water level is low. From our results, this type of demography is not expected for the three caves we have studied.

At the other extreme stands the “Oasis” model. There, the fish distribution is very patchy, whatever the water level, with few small isolated populations that are rarely connected (Fig. 4, right). It fits well with the definition of closed populations. For populations that are accessible, population size and its variation over time can be estimated using CMR, and this method does not depend significantly on water level or the time interval between captures. That is because flooding events throughout the years do not give access to other pools where a larger population exists. Thus, estimates of population size within a year or among multiple years with intervening rainy seasons give similar size estimates. CMR and MDS analyses presented here strongly suggest that the Pachón Cave population corresponds to this “Oasis” model.

The intermediate “Lake” model proposes the existence of clusters of caves, with caves well connected within a cluster, but distant clusters poorly interconnected (Fig. 4, middle). A cluster of caves fits the definition of a closed population, but a cave only gives access to a fraction of the population. We propose that the “Sabinos-Tinaja-Piedras” complex (also including Arroyo and other caves, see Fig. 1) would correspond to the “Lake” model. Extensive sampling and genotyping efforts would be necessary to confirm this.

## Conclusion

Contrary to some inferences from population genetics analyses, cave populations are small, in accordance with the “Oasis” or the “Lake” model, rather than the historical “Sea” model. In terms of evolutionary biology, this implies that genetic drift must be significant in these populations. In terms of conservation biology, this means that we cannot expect a cave population to easily recover from extinction through the arrival of new cavefish when a natural disaster or ineffective management occurs. The situation in the Pachón Cave is particularly severe, as we conclusively demonstrate here that its population has decreased by an order of magnitude -from a few thousand to a few hundred- since
**Fig. 4** Models of the distribution of cavefish populations. Dark blue represents water bodies that are accessible to humans and where fish can be observed and sampled (arrows); note that sampling in caves is almost exclusively done at the end of the dry season, when the water level is low, for obvious safety reasons. Light blue represents large or restricted, permanent or temporary water bodies that connect accessible pieces of water. Each model is represented with low (top panels) and high water levels (bottom panels). The “Sea” model corresponds to a large population of cavefishes distributed over a large area in fully connected aquifers. The “Oasis” model corresponds to a patchy distribution of small populations with little connections (dashed lines). The “Lake” model is an intermediate. Modified from (Legendre et al. 2023).

<figure><img src="image_9.png" /></figure>

the 1970s (Elliott 2018). Regardless of the reasons for such a population collapse, the *Astyanax* scientific community, local authorities, and local associations should collaborate to take measures to protect the most emblematic and well-studied cavefish population.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s10592-0 25-01753-8.

**Acknowledgements** This work was supported by a CNRS MITI (Mission pour les Initiatives Transverses et Interdisciplinaires) grant “Instrumentation in situ en conditions extrêmes” to SR and LL, collaborative grants from Agence Nationale de la Recherche (BLINDTEST) and Institut Diversité Écologie et Évolution du Vivant to DC and SR, Équipe FRM grants (DEQ20150331745 and EQU202003010144) to SR, an ECOS-Nord Franco-Mexican exchange program grant (M15A03) to SR and Patricia Ornelas-García. Our field work currently benefits from the label “SEE-Life” from CNRS Écologie & Environnement to LL, SR and DC. Many thanks to Patricia Ornelas-García for sharing field permits over the years, to current and former members of the Rétaux team for help and collaborative spirit during the field expeditions, and to Jean-Louis Lacaille Múzquiz and Alejandro Durán (CONANP) for helping with access to caves located close to La Reserva de la Biosfera Sierra del Abra Tanchipa.

**Author contributions** D.C., L.L. and S.R. conceived and designed the research; L.L., M.P., S.P., J.F., L.E. and S.R. conducted field surveys and collected samples; I.G. and M.P. performed molecular work; J.R. and D.C. analysed data; D.C., L.L., and S.R. wrote the manuscript. All authors read and approved the final version of the manuscript.

**Funding** Open access funding provided by Université Paris-Saclay.

**Data availability** Python and R scripts can be found here: https://github.com/jmorode/genetique-analysis-python.

## Declarations

**Competing interests** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright
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