Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

José F Meléndez-Cal-y-Mayor; Jasmin Winkler; Ramon Müller; Beatrice Lüscher; Janine Bolliger; Arpat Ozgul; Benedikt R Schmidt

doi:10.1371/journal.pone.0340580

. 2026 Jan 8;21(1):e0340580. doi: 10.1371/journal.pone.0340580

Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

José F Meléndez-Cal-y-Mayor ^1,^*, Jasmin Winkler ¹, Ramon Müller ¹, Beatrice Lüscher ², Janine Bolliger ³, Arpat Ozgul ¹, Benedikt R Schmidt ^1,⁴

Editor: Sven Winter⁵

PMCID: PMC12782421 PMID: 41505422

Abstract

Genetic diversity and structure are rarely assessed in populations established through conservation translocation. Here, we analysed the genetic structure and diversity of populations of an endangered pond-breeding amphibian, the common midwife toad, Alytes obstetricans, by comparing translocated populations against two types of populations: (i) populations which have recently colonized newly created ponds and (ii) natural populations which have been known to be present for a long time. Bottleneck events and dispersal patterns were analysed to describe the outcome of the translocations. In addition, we simulated trajectories of genetic diversity (H_e) of populations over time. The genetic diversity of natural colonized and translocated populations was similar to that of natural populations. However, there were signatures of genetic bottleneck events in three colonized populations and in a natural population. Simulations of genetic diversity over time showed that number and frequency of dispersers and population size are important parameters determining genetic diversity in the populations in the future. We conclude that, translocated, natural and colonized populations are genetically comparable, indicating that translocations can serve as an effective tool in conservation efforts. However, constructing ponds and waiting for natural colonization is also known to work well.

Introduction

Conservation biology has two main goals. The first goal is to understand how anthropogenic stressors lead to the loss of biodiversity. The second equally important goal is to work on solutions that mitigate the negative effects of anthropogenic stressors on populations [1]. To bend the curve of biodiversity loss, solutions are needed to restore habitats and populations or to slow or halt the decline of populations to allow the recovery of declining species [2–4]. Once solutions are available and implemented by conservation practitioners, it is necessary to assess their effectiveness [5–7]. Solutions such as habitat creation or restoration allow target species to colonize those habitats [6,8,9]. However, in some cases, natural colonizations may not be possible or take a long time [10]. In addition, colonized populations may have low genetic diversity due to the founder effect (i.e., bottleneck), caused by the small number of founder individuals and the small number of founder populations [11,12]. Conservation translocations may therefore have a role in wildlife conservation because one may select a translocation strategy that maximizes genetic diversity [13].

Translocations are commonly used in conservation practice [14–16]. A conservation translocation is the intentional release of organisms at a site with the goal to establish, reestablish, or augment a population [14,17]. The conservation benefit is achieved, and a translocation is viewed as successful, when it has led to a viable population [18–20]. However, the success of a viable translocated population may be jeopardized if translocated individuals have little genetic diversity [21,22]. Evidence suggests that some translocated populations have reduced levels of genetic diversity when compared to long established populations [23–26]. There are many reasons for reduced genetic diversity in translocated populations. For instance, the number of translocated founders, admixture, genetic drift, and bottlenecks may determine the genetic diversity of the translocated population [27,28]. A high number of translocated individuals and multiple source populations reduce the likelihood of bottlenecks, inbreeding and genetic drift [29], and should therefore lead to translocated populations with levels of genetic diversity similar to natural population. Inbreeding and reduced levels of genetic diversity can affect population viability [30–32]. Because genetic diversity can affect long-term population viability [33–35], it is important to compare the genetic diversity of translocated populations to long-established natural populations and to populations which have recently colonized previously empty patches. This is also important because genetic diversity is often neglected in conservation practice [36].

Amphibians are declining globally [37–40]. To aid the recovery of amphibians, translocations are commonly used [4,29,41,42]. In this study, we evaluated translocations using microsatellites to document the genetic diversity and structure of populations of the common midwife toad, Alytes obstetricans. We used data from two conservation programs which had the aim to increase the number of populations in the Swiss Emmental and in the Lucerne regions (genetic rescue was not an aim; [6,43–46]) to compare three population types in two regions within Switzerland: natural populations, translocated populations and populations which resulted from natural colonizations (the local amphibian conservation officer Beatrice Lüscher made sure that no translocations took place in these naturally colonised populations) after the creation of new ponds. Ponds in the amphibian populations were surveyed decades ago; therefore, we can be confident that natural populations were present already decades ago and persisted (for reviews of the surveys, see [43] (Lucerne) and [44] (Emmental). This allows us to compare natural versus translocated populations and natural versus colonized populations. We do not expect the effect of translocations and natural colonizations on the genetic structure of the new populations to be necessarily different. While conservationists worry about the genetic consequences of translocations, this is usually not the case for natural colonizations. We believe that a comparison of the two types of new population may be useful for conservation science and practice. The lessons learned from this study can improve future conservation actions because the genetic consequences of translocations are better understood and compared to demographic processes such as colonization within a metapopulation.

Materials and methods

Research permits and ethical considerations

Permission to capture specimens of Alytes obstetricans were granted by the nature conservation authorities of the Swiss cantons Lucerne and Bern (to Benedikt Schmidt). Animal welfare permits to collect tissue samples were granted by the animal welfare agencies of the Swiss cantons Zurich and Bern (ZH66/2014(an intercantonal permit), BE17/15). All procedures have been carried out in accordance with relevant guidelines and regulations and, where applicable, reported in accordance with ARRIVE guidelines. To minimise suffering, the tadpoles were anaesthetised using ethyl 3-aminobenzoate methanesulfonate (Sigma-Aldrich, Buchs, Switzerland, code MS-222) before tissue sampling. Tadpoles were subsequently released. No animals were sacrificed.

Study species and sampling sites

The midwife toad (Alytes obstetricans) is classified as “least concern” on the global IUCN Red List. However, as in many other European countries [47], it is endangered in Switzerland due to ongoing population declines [48–50].

The sampling sites are located in central Switzerland around Lucerne and Emmental (Fig 1). Genetic samples from all known populations of A. obstetricans in Emmental and Lucerne (n = 33) were collected between April and July 2015. Twenty-one sites were in Emmental of which twelve were natural populations and nine were naturally colonized (Table 1). In Lucerne, there were twelve populations. Six were natural populations (four out of six EHRL, CHAL, SSSL and STAL were used as donor populations) and six were translocated populations (Table 1). In Emmental we sampled 199 and 145 individuals from natural and colonized populations, respectively. In Lucerne we sampled 170 and 160 individuals from natural and translocated populations, respectively. More information on the number of individuals sampled per population is provided in Table 1. We disinfected field equipment and boots with Virkon S each time we collected samples from different sites to prevent the spread of disease during field work [51].

Table 1. Gene diversity indicators for natural, colonized and translocated populations at the study sites. The table lists the area, the population type, the population, sample size and the estimated genetic metrics. The confidence intervals (95%; values in brackets) for effective population size (N_e) were obtained using the jackknife resampling method.

Region	Population type	Population	Sample size (n)	Allelic richness (A_R)	Observed Heterozygosity (H_o)	Expected heterozygosity (H_e)	Fixation index (F_ST)	Private alleles (P_a)	Inbreeding coefficient (F_is)/Median values	Effective population size (N_e)
Emmental	Colonized	ARM	15	1.45	0.184226	0.163407	0.364	2	−0.133/-0.167	Infinite (10.9-Infinite)
		CHNM	8	1.72	0.265625	0.258333	0.305	0	−0.03/0.000	Infinite (3.4-Infinite)
		DBM	10	1.25	0.150000	0.111184	0.439	0	−0.376/-0.378	11.8 (0.5-Infinite)
		EGSM	20	1.93	0.298191	0.303590	0.291	3	0.018/0.059	86.8 (18.7-Infinite)
		FEM	17	1.94	0.330882	0.326822	0.231	6	−0.013/-0.016	12.4 (2.1-Infinite)
		HNM	20	1.97	0.302303	0.326531	0.247	1	0.076/0.036	Infinite (31.1-Infinite)
		HOM	20	1.85	0.275822	0.274193	0.263	4	−0.006/0.028	2.7 (1.5-9.1)
		SBM	20	1.38	0.121875	0.112420	0.446	1	−0.087/-0.042	8.1 (1.7-Infinite)
		TFM	15	1.76	0.236310	0.243183	0.278	0	0.029/0.029	1.8 (0.6-17.0)
	Natural	SO2M	6	2.00	0.229167	0.223485	0.298	1	−0.028/0.000	Infinite (3.5-Infinite)
		S13M.	20	2.21	0.308224	0.312276	0.225	6	0.013/-0.042	Infinite (23.9-Infinite)
		S15M.	20	2.03	0.290625	0.288301	0.258	0	−0.008/-0.006	50.3 (12.9-Infinite)
		S18M.	12	1.74	0.234375	0.229846	0.269	0	−0.021/0.000	20.4 (4.3-Infinite)
		S21M.	20	1.74	0.275000	0.239423	0.283	0	−0.153/-0.148	47.1 (5.9-Infinite)
		S22M.	7	1.95	0.339286	0.287088	0.246	0	−0.2/-0.163	Infinite (4.7-Infinite)
		SO3M	20	1.55	0.218750	0.189984	0.327	3	−0.156/-0.137	49.5 (6.1-Infinite)
		SO7M	20	1.60	0.225000	0.198157	0.376	0	−0.14/-0.101	8.4 (2.1-73.3)
		S11M.	14	1.63	0.252232	0.237747	0.307	0	−0.064/-0.030	5.5 (1.0-Infinite)
		S12M.	20	1.76	0.270703	0.257939	0.282	0	−0.051/-0.027	7.4 (1.7-214.0)
		S5BM.	20	1.74	0.233717	0.223537	0.306	1	−0.047/-0.071	6.1 (1.7-41.7)
		S6CM.	20	2.03	0.322606	0.291544	0.239	0	−0.11/-0.065	8.7 (2.6-36.8)
Lucerne	Translocated	KAP	30	1.87	0.245833	0.232839	0.312	10	−0.057/-0.084	171.4 (20.7-Infinite)
		GEI	21	1.79	0.254315	0.257341	0.367	2	0.012/-0.060	16.2 (6.2-87.4)
		OTT	19	1.65	0.240439	0.240995	0.378	1	0.002/0.016	2.3 (1.3-8.0)
		CHR	30	2.13	0.295833	0.303884	0.256	1	0.027/0.008	25.9 (12.9-82.2)
		ERS	30	2.19	0.358333	0.352295	0.267	10	−0.017/-0.039	14.8 (6.7-40.3)
		SON	30	2.48	0.400431	0.378446	0.206	15	−0.059/-0.073	4.9 (2.6-8.9)
	Natural	EHR	30	2.10	0.304167	0.292373	0.270	0	−0.041/-0.057	Infinite (34.4-Infinite)
		CHA	30	1.81	0.261422	0.232035	0.346	0	−0.129/-0.122	64.4 (14.1-Infinite)
		HER	30	1.86	0.249138	0.238425	0.303	1	−0.046/-0.047	1063.4 (29.5-Infinite)
		LAT	20	1.93	0.337500	0.306170	0.366	28	−0.105/-0.082	3.2 (1.7-13.7)
		SSS	30	1.97	0.308333	0.310134	0.257	5	0.006/-0.012	11.5 (6.1-22.2)
		STA	30	2.06	0.281250	0.278143	0.275	16	−0.011/-0.031	Infinite (37.9-Infinite)

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Genetic data

A. obstetricans tadpoles were collected by dip-netting. For every population a maximum of 30 tadpoles were caught. Three mm of the tail tip were cut off with a scalpel blade. This is a method approved by the Swiss federal conservation and animal welfare agencies [52]. Tissue samples were put in 94% ethanol and stored in a freezer (−20.5 °C ± 0.8 °C) prior to laboratory analysis. We extracted DNA from the tail of a tadpole.

For DNA extraction, we used the BioSprint 96 DNA Blood Kit (Qiagen, Hombrechtikon, Switzerland, code 940054) following its protocols. The genetic data was obtained by microsatellite markers. The markers were previously used to analyse the genetic structure of Swiss populations of Alytes obstetricans [46]. Polymerase chain reaction (PCR) was performed with fluorescent labelled primers. Twelve markers were developed by Tobler et al. (2013) and four new markers were developed by ecogenics GmbH (Zurich, Switzerland; S1 Table). Linkage disequilibrium (LD) and deviation from Hardy-Weinberg equilibrium (HWE) were calculated with the R package adegenet (v2.1.7; [53,54]). Probable presence of null alleles was checked with FreeNA program [55]. The percentage of missing alleles in both datasets, Emmental and Lucerne, was calculated with the R package poppr (v2.9.5; [56,57]) using the functions read.genalex () and info_table (). On average, Emmental has 0.8% of missing alleles (S1 Fig) and Lucerne has 0.78% (S2 Fig).

Genetic diversity and bottleneck

Inference of the genetic parameters rarefied allelic richness (A_r), observed and expected heterozygosity (H_o and H_e, respectively), and fixation index (F_st; between populations within each region) was calculated using FSTAT (v2.9.4; [58]) and GDA (v1.1; [59]). The average of all the pairwise F_st values per population was also used for analysis. The number of private alleles (P_a) per population was calculated using the private_alleles function in the package poppr (v2.9.5; [56,57]). The inbreeding coefficient (F_is) was estimated using FSTAT (v2.9.4; [58]). We used a one-sample t-test to assess the alternative hypothesis that the median F_is values are not equal to zero with the function t.test () in R (v4.3.1; [60]). Effective population size (N_e) was estimated using the Linkage Disequilibrium (LD) method [61–63], as implemented in N_eEstimator (v2.1; [64]). The N_e values for the populations in Emmental (ARM, CHNM, HNM, SO2M, S13M, S22M) and in Lucerne (EHR and STA) were excluded from further analysis because they were infinite. As explained by [63], infinite estimates of N_e can arise due to sampling variation when the number of individuals in the sample is small. We believe that this interpretation of infinite N_e values is more likely than genuinely high N_e (which we believe are unlikely). Although the LD method yielded infinite N_e estimates, the lower limits of the 95% confidence intervals were small, thus suggesting that N_e are small rather than large, or noise caused by kinship or rare alleles [63,65]. Other explanations include strong gene flow (which masks genetic drift, resulting in low linkage disequilibrium and therefore high N_e values) [66]. Additionally, violations of the assumptions of the linkage disequilibrium method, for instance closed populations, non-recent bottleneck, or population structure, could also contribute to these results [66]. The genetic parameters A_r, H_o, H_e, F_st, P_a, F_is and N_e from translocated and colonized populations were compared against natural populations using R [60] and the packages lme4 (v1.1.26; [67]), lmerTest (v3.1.3; [68]), lmtest (v0.9.38; [69]), and fBasics (v3042.89.1; [70]). The comparisons were done with linear models (genetic parameter ~ region + population type) using the lm function. The factor “region” had two levels (Emmental and Lucerne) and the factor “populations type” had three levels (natural, translocated, and colonized). The model assumptions of normality and independence of residuals were tested for each model using the function jarqueberaTest () and dwtest (), respectively, from the packages fBasics (v3042.89.1; [70]) and lmtest (v0.9.38; [69]). The tests showed that the assumptions of the linear models were met.

We tested for evidence of genetic bottlenecks using BOTTLENECK (v1.2.02; [71]). We used three models, the stepwise mutation model (SMM) and the two-phase model (TPM) are the most appropriate for testing bottleneck events using microsatellites, and the allele frequency distribution which can differentiate between a bottleneck event and a stable population [72]. The SMM assumes that a mutation happens in a change in one repeat unit. The TPM assumes that a microsatellites mutation changes between the SMM and that a mutation involves any number of tandem repeats (IAM: Infinite alleles model) [73]. The allele frequency distribution shows the lack of rarest alleles rapidly lost after a bottleneck event [74]. The TPM was run using a recommended parameter set to 95% SMM and 5% IAM, and variance between infinite allele mutations of 12% [72]. Significance was evaluated using a Wilcoxon signed-rank test because of its power and robustness when used with few polymorphic loci [72]. We considered populations bottleneck when two tests, SMM and TPM, were significant because of the uncertainty regarding which mutation model is the most appropriate for microsatellite loci.

Genetic population structure and migration rates

To evaluate the genetic effects of translocated populations for the Lucerne sites, the analyses (isolation by distance, genetic pattern as assessed by STRUCTURE and principal component analysis) was conducted for natural and translocated populations together and for natural populations. This was to observe any effect because of the translocations.

The genetic structure of populations from Emmental and Lucerne was analysed using STRUCTURE (v2.3.4; [75–78]). The most probable number of genetic clusters was determined using the highest ΔK values (in STRUCTURE), the observation of the assignment probabilities of each individual for each analysed cluster, and supported by principal component analysis (PCA). The PCA analysis was carried out in the R package adegenet (v2.1.3; [53,54]) using the allele frequencies, centered and scaled, and missing allelic information was substituted with mean values. The results from STRUCTURE were plotted using DISTRUCT (v1.1; [79]).

Markov chain Monte Carlo (MCMC) method using BayesAss (v3.0.4; [80]) were used to estimate the migration rates between populations. The parameters used in the BayesAss analysis for the Emmental populations were the following: random seed = 100, Markov Chain Monte Carlo (MCMC) iterations = 10000000, burn-in = 1000000, sampling interval = 100 and mixing parameters: migration rate (dM) = 0.9, allele frequencies (dA) = 0.9, inbreeding coefficients (dF) = 0.9. For the Lucerne populations the parameter were: random seed = 100, MCMC iterations = 10000000, burn-in = 1000000, sampling interval = 100 and mixing parameters: dM = 0.15, dA = 0.6, dF = 0.6). With this parameterization, the MCMC converged for both populations, Emmental and Lucerne (S3 and S4 Figs, respectively). The obtained migration rates were used in the program quantiNemo ([v2.0.0; [81]) to support the projection of genetic diversity and to assess the future status of the studied populations.

Projecting genetic diversity

Microsatellite analysis provides information on the current status of the populations. To learn about the future status, we simulated genetic diversity over time to model long-term trends in genetic diversity. To do so, we used the program quantiNemo (v2.0.0; [81]). We used the estimated values of expected heterozygosity (H_e) to develop the projections of genetic diversity. H_e is a sensitive genetic parameter to detect reductions in genetic diversity in populations inhabiting disturbed areas [82], to model genetic diversity under different scenarios for 30 generations. We considered a metapopulation selection level, a mean fecundity of 50 with zero fluctuations, a random mating proportion of 0.95 and a promiscuous mating system. Eight different scenarios were simulated for the study areas Emmental and Lucerne. Scenarios accounted for variation in dispersal and population size [83,84]. We assumed different dispersal types, population sizes and dispersal frequencies (for details, see S2 Table). 100 replicates were simulated for each scenario. The results obtained with quantiNemo (v2.0.0; [81]) were analysed with the generalised linear model function glm in R [63]: H_e Final ~ H_e Start + Population type (natural vs colonization; natural vs translocated) + Dispersal type + Population size + Dispersal frequency + Population type * Dispersal type + Population type * Population size + Population type * Dispersal frequency. H_e Final and H_e Start refers to the last and first value in the projection of 30 generations, respectively. Separate models were fit to the Emmental and Lucerne data.

Results

Genetic diversity and bottlenecks

The genotype data for all 21 and 12 (Emmental and Lucerne, respectively) populations showed negligible frequencies of null alleles (S3 Table). No locus showed consistent deviations from Hardy-Weinberg equilibrium (S3 Table). The few detected deviations from HWE were probably due to population substructure which caused and excess of homozygotes (a Wahlund effect; [85]). Furthermore, there was no strong evidence of linked loci because a low percentage of the variation in one microsatellite marker is shared with the other microsatellite marker (Standardized Index of Association over all loci (rbarD) = 0.019 and 0.062; for Emmental and Lucerne, respectively).

The metrics used to describe genetic diversity in the populations are shown in Fig 2 and Table 1.

The results of the regression models (Table 2) showed no significant effect of region (Emmental and Lucerne) and population type (natural, colonized, and translocated) on the genetic parameters A_r, H_o, H_e, F_st and F_is. This suggests that there were no differences (Fig 2). The genetic parameter P_a and N_e showed a significant effect of region (lower values in Emmental than in Lucerne). F_is values in the Emmental colonized populations and in the Lucerne translocated populations were not significantly different from zero (t(8) = −1.075, p = 0.314 and t(5) = −2.251, p = 0.074, respectively), but F_is values in the Emmental natural populations and Lucerne natural populations were (t(11) = −3.874, p = 0.003 and t(5) = −3.666 p = 0.015, respectively).

Table 2.

Linear model results for the gene diversity indicators. Allelic richness (A_R), observed and expected heterozygosity (H_o and H_e, respectively), fixation index (F_st), private alleles (P_a), inbreeding coefficient (F_is) and effective population size (N_e). A significant value is shown by *(P < 0.05). Values in square brackets are 95% confidence intervals. This analysis shows the absence of significant differences in the genetic diversity indicator between population types (natural, translocated, and colonized). Only P_a and N_e showed significant differences between regions (Emmental and Lucerne).

	A_r	H_o	H_e	F_st	P_a	F_is	N_e
Intercept	1.83	0.27	0.25	0.28	0.92	−0.08	20.60
Intercept	[1.69 – 1.97]	[0.23 – 0.30]	[0.21 – 0.28]	[0.25 – 0.32]	[−2.37–4.20	[-0.13 – -0.03]	[-149.07 – 190.27]
Region: Lucerne	0.12	0.02	0.03	0.02	7.42	0.03	263.03
	[-0.11 – 0.36]	[-0.03 – 0.08]	[-0.03 – 0.09]	[-0.04 – 0.08]	[1.72 – 13.11]	[-0.06 – 0.11]	[13.27 – 512.78]
	p = 0.298	p = 0.402	p = 0.353	p = 0.547	p = 0.012*	p = 0.551	p = 0.04*
Population type: Translocated	0.06	0.01	0.02	−0.01	−1.83	0.04	−244.38
	[-0.21 – 0.34]	[-0.06 – 0.07]	[-0.05 – 0.09]	[-0.08 – 0.07]	[-8.41 – 4.74]	[-0.06 – 0.14]	[-590.72 – 101.97]
	p = 0.641	p = 0.784	p = 0.597	p = 0.882	p = 0.573	p = 0.441	p = 0.15
Population type: Colonized	−0.14	−0.03	−0.01	0.03	0.97	0.02	2
	[-0.35 – 0.07]	[-0.08 – 0.02]	[-0.07 – 0.04]	[-0.02 – 0.09]	[-4.05 – 5.99]	[-0.06 – 0.10]	[−217.05–221.05
	p = 0.192	p = 0.293	p = 0.625	p = 0.212	p = 0.695	p = 0.561	p = 0.985
R²	0.225	0.148	0.134	0.054	0.249	0.073	0.213

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The bottleneck analysis found evidence for recent bottlenecks in three populations resulting from colonizations from Emmental (DBM, FEM, and TFM; S4 Table). This revealed significant heterozygosity excess (Wilcoxon test, p < 0.05) and the shifted mode test showed a distortion in the allele frequency distribution. The loss of rare alleles and excess of heterozygosity are consistent with a bottleneck occurring within the las few generations. In one of the natural populations in Lucerne (SSS) there was too heterozygosity excess (Wilcoxon test, p < 0.05).

Population structure

STRUCTURE inferred that the Emmental populations were divided into K = 2 (∆K = 129.24; S5 Fig). However, based on the hierarchical comparison Ks, and the PC analysis (S5 and S6 Figs; respectively), we concluded that the Emmental population is more likely divided into K = 4 clusters (Fig 3). Additionally, the natural and colonized populations in the Emmental are admixed and no population is isolated.

Fig 3 — The STRUCTURE plot for Emmental represents K = 4 (top) and for Lucerne K = 3 (bottom). Each vertical bar in the STRUCTURE plot represents an individual, and the colours within each bar indicate the proportion of that individual’s genome assigned to each genetic cluster **(K)**. The number of colours corresponds to the number of clusters (K) inferred in the analysis. Individuals with a single colour are strongly assigned to one cluster, whereas individuals with multiple colours show admixture among clusters. The black lines separate different populations.

STRUCTURE divided the Lucerne populations (natural and translocated) into K = 2 (∆K = 384.42; S7 Fig). However, the hierarchical comparison of Ks and the PC analysis (S7 and S8 Figs; respectively) led us to conclude that the most probable number of clusters was K = 3 (Fig 3). STRUCTURE divided the Lucerne natural populations into K = 4 (∆K = 5.94; S9 Fig). However, the hierarchical comparison of Ks and the PC analysis (S9 and S10 Figs; respectively) led us to conclude that the Lucerne natural populations are divided into K = 3. Both separated analysis (natural vs translocated and natural populations) showed us that the number of clusters in the Lucerne sampled area was three. We showed that natural and translocated populations were admixed. However, the natural population LATL remains as a single cluster. This population was not used as donor population in the translocation plan (S11 Fig).

Projecting genetic diversity

The results on the simulation of genetic diversity (H_e) over time for the Emmental population are presented in Table 3 and S12 Fig.

Table 3. Generalized linear model results of the genetic diversity (H_e) obtained from the simulations in quantiNemo for the Emmental populations. This analysis considered a metapopulation selection level, with a mean fecundity value of 50 with zero fluctuations, 0.95 of random mating proportion and promiscuity mating system. R² = 0.471. The values in bold are statistically significant (P < 0.05).

	H_e Final
Predictors	Estimates	95% CI	p
(Intercept)	0.25	0.22–0.28	<0.001
H_e Start	0.35	0.23–0.46	<0.001
Population type (Natural)	−0.12	−0.15 – −0.09	0.001
Dispersal type (BAYESSAss)	−0.00	−0.02–0.02	0.866
Population size (100)	−0.01	−0.03–0.01	0.392
Dispersal frequency (First generation)	−0.06	−0.09 – −0.04	<0.001
population type (Natural) * Dispersal type (BAYESSAss)	0.11	0.09–0.14	<0.001
Population type (Natural) * Population size (100)	−0.05	−0.08 – −0.02	<0.001
Population type (Natural) * Dispersal frequency (First generation)	0.06	0.04–0.09	<0.001

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The projection of genetic diversity for the Emmental study sites showed that there were significant differences in H_e between the genetic diversity of natural and colonized populations at the end of the simulated 30 generations. Average final H_e was 0.236 for natural populations and 0.290 for colonized populations. Plots which visualize the interactions between the eight different scenarios are shown in S13 Fig. The number of dispersers had no effect on H_e in colonized populations. In contrast, natural populations had higher H_e with the BayesAss dispersal rates than with five dispersers. Dispersal frequency affected H_e in colonized populations but had no effect in natural populations. In colonized populations, constant dispersal led to higher H_e than dispersal only in the first generation. Population size had no effect on H_e in colonized populations. In contrast, larger natural populations (500) had higher H_e than the smaller natural populations (100).

In the projected genetic diversity for the Lucerne populations, natural and translocated, we found that there were no statistically significant differences in the final genetic diversity values between natural (0.317) and translocated populations (0.337) (Table 4; S14 Fig). Population size and dispersal frequency affected final H_e, i.e., smaller population showed lower H_e and regular dispersal increased H_e. There were no significant interactions.

Table 4. Generalized linear model results of the genetic diversity obtained from the projections of genetic diversity done in quantiNemo for the natural and translocated populations in Lucerne. This analysis considered a metapopulation selection level, with a mean fecundity value of 50 with zero fluctuations, 0.95 of random mating proportion and promiscuity mating system. R² = 0.366. The values in bold are statistically significant (P < 0.05).

	H_e Final
Predictors	Estimates	95% CI	p
(Intercept)	0.30	0.23–0.37	<0.001
H_e Start	0.26	0.02–0.50	0.034
Population type (Translocated)	0.01	−0.03–0.05	0.606
Dispersal type (BAYESSAss)	0.00	−0.03–0.03	0.849
Population size (100)	−0.03	−0.06 – −0.00	0.030
Dispersal frequency (First generation)	−0.07	−0.10 – −0.04	<0.001
Population type (Translocated) * Dispersal type (BAYESSAss)	−0.02	−0.07–0.02	0.255
Population type (Translocated) * Population size (100)	0.02	−0.02–0.07	0.261
Population type (translocated) * Dispersal frequency (First generation)	0.01	−0.03–0.05	0.700

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Discussion

Conservation practice should focus more on genetic diversity [36]. Translocations are a good example where this can be done. Translocations involve the intentional movement of individuals to a new place with the aim to establish a new population. When a new population is founded, it may be genetically less diverse than the population from which the translocated individuals originated. Reduced levels of genetic diversity in translocated populations have been reported in a few cases [23–25] and may compromise the long-term viability of the new population. Reassuringly, our results show that natural, translocated, and populations resulting from natural colonizations had a similar genetic make-up. This is an important result, because if translocated or colonized populations had less genetic diversity than natural populations, then they could be more susceptible to external stressors such as climate change, pathogens or pollution [35,86].

The populations in Lucerne and Emmental showed a genetic structure of multiple spatial clusters. This inference is based on the STRUCTURE analysis and the PCA. The clustering suggests that gene flow is limited, probably because of IBD and landscape heterogeneity [87–89]. Limited gene flow might increase the effects of genetic drift. That is, deleterious alleles may rise in frequency and populations may have a reduced ability to respond to environmental conditions that individuals may encounter in the new habitat [32], especially in the colonized and translocated populations. An increase in population connectivity through the construction of new ponds [6,9] might help to reduce spatial genetic structure and may increase population viability because there would be a greater exchange of individuals among populations.

The genetic diversity of colonized and translocated populations was similar to that of natural populations. Even though, the median F_is values of the Emmental and Lucerne natural populations are significantly different from zero, they do not suggest the presence of inbreeding but of heterozygous (i.e., outbred) individuals due to the mating of distantly related individuals [90]. This heterogeneity may be a characteristic of colonized and translocated populations without the negative costs of outbreeding [91].

Although the power to detect a bottleneck event is small because little time has elapsed since the translocations and colonizations [92], there was evidence for genetic bottleneck events in some colonized and natural populations. Bottlenecks were found in three populations in the Emmental (populations DBM, TFM and FEM) and in a natural (i.e., old) population in Lucerne (populations SSS). The populations in the Emmental are all populations which colonized newly created ponds. We assume that a small number of dispersers may have led to genetic bottlenecks (founder effect [11,12]). In other words, many alleles from the population from where the immigrants originate are not passed on, rare alleles are lost immediately, and genetic drift is strongest in the first generations; even if the population grows rapidly, this does not restore the loss of alleles [93,94]. It is more challenging to explain the bottleneck in the natural population SSS in the Lucerne area. This population inhabits a well and other bodies of water in the grounds of an eighth-century castle. It is conceivable that the maintenance of the well led to the mortality of larvae and thus to bottlenecks. No translocated populations went through a detectable bottleneck event. This suggests that the translocation program might have found a good number of donor populations, distance between natural and translocated populations, quality of the release habitat and the number of translocated individuals [17,95,96]. Despite the bottleneck events that were detected, the genetic structure of the three population types did not differ much (Fig 2, Table 2). Nevertheless, bottleneck reduces genetic diversity, and such a reduction may affect population viability [97]. It could therefore be interesting to compare the demography and viability of the bottlenecked populations with those that show no signs of bottlenecks [26] and to explore whether some form of genetic rescue would be feasible and necessary.

We used simulations to predict future levels of genetic diversity (He) in the populations. Despite the simplifying assumptions that had to be made in the simulations, we found that populations resulting from the colonization of new ponds had higher average final H_e values than natural populations. A possible explanation for the higher mean final H_e values is that the naturally colonized populations may have been colonized by dispersers from multiple populations [98]. This seems likely because, we found that the Emmental populations are admixed and not isolated. However, interactions between dispersal type, dispersal frequency and population size used in the simulations may also have contributed to these results. This suggests that to positively influence dispersal, conservation management should provide high quality habitat for the species by increasing the number of ponds in the study area and by improving the terrestrial habitat (e.g., dry-stone walls and hangslide), and ponds should be allowed to function for long periods [6,9]. To maintain high dispersal rates, one should focus on the three stages of dispersal: emigration, transience and settlement [99]. Emigration could be improved if ponds are constructed and managed in such a way that they produce large numbers of offspring which may emigrate (e.g., through the removal of non-native fish; [100]). During the transient phase, dispersal can be enhanced through the removal of potential barriers to dispersal or the creation of microhabitats which may serve as daytime shelters on potential dispersal corridors [101,102]. Similarly, settlement can be improved by providing suitable habitat. In the case of the midwife toads, microhabitats such as dry-stone walls are important [6]. By doing so, dispersers from source populations would reach other sites in higher numbers and more frequently. All this would prevent genetic diversity from decreasing.

We found that translocations and natural colonizations resulted in populations that were genetically similar to natural populations. Thus, the conservation programmes achieved their primary goal of increasing the number of populations in the study area. Although maintaining genetic diversity was not an explicit aim of the translocations and pond construction, both programmes achieved this main goal through different approaches. This suggests that both natural colonizations following pond construction and translocations can be used to achieve conservation goals, as long as the risks of translocations are considered (e.g., outbreeding depression, pathogen introduction and adaptive potential; [21,103,104]). However, the evidence of genetic bottlenecks suggests that the translocations can be improved. Because A. obstetricans does not disperse over long distances [6,105], the creation of additional ponds could help to increase the number of dispersers and the effective populations size (N_e) by expanding breeding opportunities, reducing density-dependent larval mortality, lowering variance in reproductive success, enhancing connectivity among populations, and reducing the impact of environmental stochasticity on recruitment [106,107], and thereby further improve the viability of the population network, both genetically and demographically. Further assessments of the conservation actions should include monitoring of adaptive genetic variation and life history traits related to population viability to detect outbreeding depression and other processes that may reduce population viability in the future [103,104,108,109].

Supporting information

S1 Fig. Percentage of missing data for Emmental populations by Locus (on x-axis) and population (on y-axis).

The lowercase letters at the end of the population names indicates the population type (n = natural and c = colonized). Emmental region shows 0.8% missing alleles.

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pone.0340580.s001.eps^{(64KB, eps)}

S2 Fig. Percentage of missing data for Lucerne populations by Locus (on x-axis) and population (on y-axis).

The lowercase letters at the end of the population names indicates the population type (n = natural and t = translocated). Lucerne region shows 0.78% missing alleles.

(EPS)

pone.0340580.s002.eps^{(46.5KB, eps)}

S3 Fig. Trace plot of Markov Chain Monte Carlo (MCMC) for inference of migration rate estimates in BayesAss for the Emmental populations.

The grey part of the chain indicates the samples discarded before the migration rate estimates were obtained. The MCMC converged (the trace fluctuates around a stable mean), meaning that the chain ran long enough for the obtained migration rates to be valid and stable.

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pone.0340580.s003.eps^{(1.9MB, eps)}

S4 Fig. Trace plot of Markov Chain Monte Carlo (MCMC) for inference of migration rate estimates in BayesAss for the Lucerne populations.

(EPS)

pone.0340580.s004.eps^{(1.8MB, eps)}

S5 Fig. Bayesian clustering results of the STRUCTURE and delta K plot (in the corner) analysis for microsatellite data of populations sampled in Emmental (natural and colonized populations) of A. obstetricans (344 individuals).

The STRUCTURE results show the distribution of clusters from K = 2 to K = 8 using no a priori information on geographic or population. The most suitable K based on the higher delta K (∆K) is K = 2 (observe ∆K plot too). However, considering the ∆K value, the hierarchical observation of the different clusters and the PCA, K = 4 is best supported. Each vertical bar represents an individual, and the colours within each bar indicate the proportion of that individual’s genome assigned to each genetic cluster. The number of colours corresponds to the number of clusters (K) inferred in the analysis. Individuals with a single colour are strongly assigned to one cluster, whereas individuals with multiple colours show admixture among clusters. The black lines separate different populations.

(EPS)

pone.0340580.s005.eps^{(22.9MB, eps)}

S6 Fig. Principal components analysis (PCA) for the Emmental, natural and colonized, populations based on 344 individuals and 16 microsatellite loci.

The n and the c at the end of the population’s labels mean natural and colonized, respectively. Each point represents an individual, with colours indicating populations. The PCA reveals clustering into 4 groups. The bold bars in the inset graph (eigenvalues) indicate the first two principal components shown in the PCA figure.

(EPS)

pone.0340580.s006.eps^{(622.6KB, eps)}

S7 Fig. Bayesian clustering results of the STRUCTURE and delta K plot (in the corner) analysis for microsatellite data of populations sampled in Lucerne (natural and translocated populations) of A. obstetricans (330 individuals).

The STRUCTURE results show the distribution of clusters from K = 2 to K = 10 using no a priori information on geographic or population. The most suitable K based on the higher delta K (∆K) is K = 2 (observe ∆K plot too). However, considering the ∆K value, the hierarchical observation of the different clusters and the PCA, K = 3 is best supported. Each vertical bar represents an individual, and the colours within each bar indicate the proportion of that individual’s genome assigned to each genetic cluster. The number of colours corresponds to the number of clusters (K) inferred in the analysis. Individuals with a single colour are strongly assigned to one cluster, whereas individuals with multiple colours show admixture among clusters. The black lines separate different populations.

(EPS)

pone.0340580.s007.eps^{(24.7MB, eps)}

S8 Fig. Principal components analysis (PCA) for the Lucerne, natural and translocated, populations based on 330 individuals and 16 microsatellite loci.

The n and the t at the end of the population’s labels mean natural and translocated, respectively. Each point represents an individual, with colours indicating populations. The PCA reveals clustering into 3 groups. The bold bars in the inset graph (eigenvalues) indicate the first two principal components shown in the PCA figure.

(EPS)

pone.0340580.s008.eps^{(470.7KB, eps)}

S9 Fig. Bayesian clustering results of the STRUCTURE and delta K plot (in the corner) analysis for microsatellite data of populations sampled in Lucerne (natural populations) of A. obstetricans (170 individuals).

The STRUCTURE results show the distribution of clusters from K = 2 to K = 6 using no a priori information on geographic or population. The most suitable K based on the higher delta K (∆K) is K = 4 (observe ∆K plot too). However, considering the ∆K value, the hierarchical observation of the different clusters and the PCA, K = 3 is best supported. Each vertical bar represents an individual, and the colours within each bar indicate the proportion of that individual’s genome assigned to each genetic cluster. The number of colours corresponds to the number of clusters (K) inferred in the analysis. Individuals with a single colour are strongly assigned to one cluster, whereas individuals with multiple colours show admixture among clusters. The black lines separate different populations.

(EPS)

pone.0340580.s009.eps^{(16.9MB, eps)}

S10 Fig. Principal components analysis (PCA) for the Lucerne natural populations based on 170 individuals and 16 microsatellite loci.

The n at the end of the population’s labels means natural. Each point represents an individual, with colours indicating populations. The PCA reveals clustering into 3 groups. The bold bars in the inset graph (eigenvalues) indicate the first two principal components shown in the PCA figure.

(EPS)

pone.0340580.s010.eps^{(292.8KB, eps)}

S11 Fig. Circos plot showing the translocations.

Number of individual translocated from donor populations are represented by the arrows from left (donor/natural) to right (translocated). Each arrow represents a translocation event. The size of the axis shows the total of individuals translocated. The letters n and t at the end of the names represent natural and translocated populations, respectively.

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S12 Fig. Projecting 30 generations of genetic diversity in the Emmental populations under eight scenarios considering a metapopulation selection level, with a mean fecundity value of 50 with zero fluctuations, 0.95 of random mating proportion and promiscuity mating system.

1 = ARME colonized, 2 = CHNM colonized, 3 = DBME colonized, 4 = EGSME colonized, 5 = FEME colonized, 6 = HNME colonized, 7 = HOME colonized, 8 = SBME colonized, 9 = TFME colonized, 10 = SO2ME natural, 11 = SO3ME natural, 12 = SO7ME natural, 13 = S11ME natural, 14 = S12ME natural, 15 = S13ME natural, 16 = S15ME natural, 17 = S18ME natural, 18 = S21ME natural, 19 = S22ME natural, 20 = S5BME natural and 21 = S6CME natural. Lines correspond to the heterozygosity respond to the migration rate estimates in BayesAss and the eight simulated scenarios explained in Table S2. The variation over time in H_e values among Emmental population types (natural and colonized) is more sensitive to changes in dispersal type, population size, and dispersal frequency (see Table 3 for details).

(EPS)

pone.0340580.s012.eps^{(124.9KB, eps)}

S13 Fig. Final expected heterozygosity (H_e) for colonized and natural Emmental populations under various simulated scenarios (dispersal type, population size, and dispersal frequency).

Points represent mean H_e (obtained over 30 generations), with error bars showing standard error. Colonized populations consistently exhibit higher H_e across scenarios compared to natural populations. Thus, demographic assumptions strongly influence the magnitude of H_e in natural populations.

(EPS)

pone.0340580.s013.eps^{(31.2KB, eps)}

S14 Fig. Projecting 30 generations of genetic diversity in the Lucerne populations (translocated and natural) under eight scenarios considering a metapopulation selection level, with a mean fecundity value of 50 with zero fluctuations, 0.95 of random mating proportion and promiscuity mating system.

1 = KAPL translocated, 2 = GEIL translocated, 3 = OTTL translocated, 4 = CHRL translocated, 5 = ERSL translocated, 6 = SONL translocated, 7 = EHRL natural, 8 = CHAL natural, 9 = HERL natural, 10 = LATL natural, 11 = SSSL natural and 12 = STAL natural. Lines correspond to the heterozygosity respond to the migration rate estimates in BayesAss and the eight simulated scenarios explained in Table S2. In general, smaller populations showed lower heterozygosity (H_e) and regular dispersal increased H_e. The variation over time in H_e values does not show variation between Lucerne, translocated and natural populations (see Table 4 for details).

(EPS)

pone.0340580.s014.eps^{(81.9KB, eps)}

S1 Table. New microsatellite DNA markers of Alytes obstetricans developed by ecogenics GmbH.

Repeat type is based on genomic DNA sequence analyzed on an Illumina MiSeq platform. Size bp (base pairs) is based on the fragment analysis of 15 individuals on an ABI373.

(DOCX)

pone.0340580.s015.docx^{(22.3KB, docx)}

S2 Table. Details for the simulations made in quantiNemo (v2.0.0) for Emmental and Lucerne populations.

This table shows eight simulated scenarios for dispersal frequency, population size, and dispersal type.

(DOCX)

pone.0340580.s016.docx^{(22.5KB, docx)}

S3 Table. Estimated deviation from Hardy-Weinberg equilibrium (HWE) and null allele frequencies per population and for the 16 microsatellite loci.

A) Shows the p-values of the HWE exact test, Monte Carlo permutations of alleles. Significant deviation from HWE is shown by *(P < 0.05). B) Shows the estimates of null allele frequencies. None of the loci showed consistent deviation from Hardy-Weinberg equilibrium, and null allele frequencies were negligible.

(DOCX)

pone.0340580.s017.docx^{(48.7KB, docx)}

S4 Table. Results on the Bottleneck analysis on natural, colonized and translocated populations from canton Lucerne and Emmental using three different calculation models.

The * represents statistically significant results (p < 0.05). L-shaped distributions indicate a stable population with numerous low-frequency alleles, whereas mode-shifted distributions reflect a recent bottleneck, characterized by the loss of rare alleles and a shift toward intermediate allele frequencies.

(DOCX)

pone.0340580.s018.docx^{(24.6KB, docx)}

Acknowledgments

We thank all pond owners and conservation authorities in Emmental and Lucerne for granting us access, allowing us sampling the endangered amphibians, and sharing information on the populations. For field assistance we thank Sam Cruickshank and Ursina Tobler, and Chris Funk for comments on the manuscript. Our appreciation to the group of Prof. Lukas Keller, especially to Glauco Camenisch and Martina Schenkel for their help during the lab work. Last but not least, we would like to thank all the people whose conservation efforts improved the conservation status of the midwife toad and thereby made this study possible.

Data Availability

The Zenodo DOI is already available: https://doi.org/10.5281/zenodo.15052694.

Funding Statement

The author José F. Meléndez-Cal-y-Mayor received a scholarship from the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT; Award Number: 438370). The author Benedikt R. Schmidt received an award from the University of Zurich. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0340580.r001

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

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Reviewer #1: I Don't Know

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The PLOS Data policy

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: This paper provides a valuable contribution to understanding the genomic patterns of naturally and artificially colonized ponds, and compares them to original source populations. I appreciate the authors' use of existing opportunities to assess the genomic status of an organism of conservation concern, a commendable approach to monitoring these populations. The manuscript employs a range of population genetic tools and statistical approaches to assess and project genetic diversity across population types. Overall, the methods appear thoughtfully selected and generally appropriate for the study's objectives. I offer the following comments and suggestions to help improve clarity and robustness:

Introduction:

The statement, “Beatrice Lüscher made sure that no translocations took place in these naturally colonised populations,” is mentioned. However, without additional context regarding who Beatrice Lüscher is outside the author section and how they ensured no translocations, it is difficult to fully understand the significance of this statement.

Sampling:

The study uses a good narrow sampling timeframe, which is especially relevant for a species that reproduces annually. This strengthens the validity of their results.

One point of clarification: I am unsure if the tadpoles sampled survived the sampling process. If the organisms do not survive, it would be helpful to specify this to avoid any potential confusion.

Methods:

The laboratory methods used are sound, and bioinformatics techniques for assessing linkage disequilibrium (LD), Hardy-Weinberg equilibrium (HWE), null alleles, and percent missing data appear appropriate and well-executed. For genetic diversity and bottleneck detection, AR and private alleles are strongly associated with sample size. Larger sample sizes typically contain more alleles compared to smaller samples. It seems in your results that this pattern doesn't obscure your results, which I think makes them even more meaningful. The authors use mean Fis to assess whether it significantly differs from zero. However, Fis values are often not normally distributed across microsatellites. Given this, I suggest using the median Fis to summarize the data, as it may offer a more robust estimate, or demonstrate normality.

Statistical Analyses:

The use of linear models to compare genetic diversity indicators across regions and population types is valid for continuous response variables. However, I recommend clarifying whether the model assumptions (e.g., normality) were checked, particularly for metrics like Fst and Fis. These metrics may require transformations before fitting a linear model, and this should be addressed in the methods section.

On line 160, the authors suggest that the Ne values reaching infinity could be due to sampling error. I encourage the authors to review the literature for other possible explanations of this phenomenon, as it may not be solely a result of sampling error.

The use of BOTTLENECK is appropriate for detecting recent bottlenecks, and the authors selected models suited to microsatellite data. However, I recommend reporting the specific significant p-values, rather than just using the asterisk notation, to allow for clearer interpretation. Additionally, given the weight placed on bottlenecks in the discussion, the results section could benefit from more detailed reporting of the bottleneck results, including data from the supplemental section.

The use of quantiNemo to simulate genetic diversity loss under different demographic and dispersal scenarios is a strong approach for understanding future genetic diversity trajectories. However, I recommend clarifying whether multiple replicates were simulated for each scenario, as stochasticity plays a significant role in forward-time simulations.

Figures:

I was unable to view Figure 1 properly, and I expect the version in the PDF is distorted compared to what was submitted. However, even considering that, the figure is tricky to interpret. I only see orange dots on the main map, and I wonder if the purple dots are covered. If the final version of the figure does not contain purple dots, this should be addressed. In addition, the inset map could benefit from using the same color-coding as the main map. To make the figure more accessible at a glance, I recommend changing the shape of the dots for different site types, in addition to using lowercase letters for differentiation.

Supplementary Information:

I believe Supplemental Table 1 provides important information that would benefit from being moved to the main paper rather than remaining in the supplements. Its content appears central to the study and would improve the overall readability of the paper if included in the main manuscript.

Supplemental Table 4 is difficult to read due to formatting issues. Despite attempts to adjust the table in my Word document, I was unable to view all the columns at once. I recommend improving the formatting for better accessibility.

Overall, this paper presents a compelling study on genetic diversity patterns in a conservation context, and the methodology is generally sound. A few clarifications and additions would strengthen the manuscript, particularly in terms of statistical analysis and presentation of results. The authors use of current genetic diversity data and forward simulations is a notable strength, and I believe these findings will make a valuable contribution to the field.

**********

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Reviewer #1: No

**********

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PLoS One. 2026 Jan 8;21(1):e0340580. doi: 10.1371/journal.pone.0340580.r002

Author response to Decision Letter 1

5 Sep 2025

Dear Editor,

In this document we explain how we have revised the manuscript in response to the comments made by the editor and the reviewers. Our responses start with RESPONSE >. Line numbers correspond to the files with track changes.

The reviews were very constructive and helped to improve the manuscript.

Best wishes,

on behalf of all authors

PONE-D-25-16547

Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

PLOS ONE

Dear Dr. Meléndez,

RESPONSE > Thank you very much for your comments. We are happy to continue improving this paper. Below, we describe all of the changes we have made in response to your comments and those of the reviewer.

RESPONSE > Thank you for the comments. We explain below how we revised the manuscript in response to the comments. Here we would just like to point out that we reran the statistical analysis with Fis as suggested and that we fixed the problem with the figures.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

RESPONSE > We have made the required changes following the suggested templates to fulfil the journal requirements.

RESPONSE > We added the request information to the manuscript (from line 97 to 99 and from 132 to 137). Please note that we had an animal welfare permit to conduct the study. In this permit, all animal welfare issues are described and specified.

RESPONSE > The data used for this study is already available on Zenodo with DOI: https://doi.org/10.5281/zenodo.15052694

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

RESPONSE > We have removed the funding information from the Acknowledgements Section and from the manuscript.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

RESPONSE > The Funding Statement we have provided is correct. We don’t wish to do any amendments.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

RESPONSE > We made a new map using data which is all free to use. Specifically, we used map data which only uses publicly available data. The data is available in the R package “rnaturalearth” and “rnaturalearthdata”.

RESPONSE > We made a new map for which we do not need a permit.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

RESPONSE > Thank you for the suggested resources. However, we used a different package in R for which we do not need a permit.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

RESPONSE > Thank you for your positive assessment of our manuscript.

Introduction:

RESPONSE > We added the information to the manuscript that Beatrice Lüscher is the local amphibian conservation officer.

Sampling:

The study uses a good narrow sampling timeframe, which is especially relevant for a species that reproduces annually. This strengthens the validity of their results.

One point of clarification: I am unsure if the tadpoles sampled survived the sampling process. If the organisms do not survive, it would be helpful to specify this to avoid any potential confusion.

RESPONSE > It is a threatened species. Therefore, there was no lethal sampling. This is now explained in the section “Research permits and ethical consideration”.

Methods:

RESPONSE > We used the median Fis values as suggested by the reviewer to assess whether it significantly differs from zero. Only the result for Lucerne natural populations changed from no significant to significantly different from zero (line 157; from line 259 to 264; line 362; Table S1).

Statistical Analyses:

RESPONSE > The model assumption of normality of residuals was checked for each model. For all models, the null hypothesis of normality was not rejected (From line 172 to 175).

RESPONSE > We have added two possible more explanations in accord to our findings (from line 162 to 166).

RESPONSE > We have added the p-values in Table S4 for the models SMM and TPM. For the allele frequency distribution there is not p-values but frequencies across loci and tends to follow an L-shaped distribution a shifted mode. After a recent bottleneck rare alleles are lost more rapidly than intermediate-frequency alleles. This shifts the allele frequency distribution. This is known as shifted mode. So, we can only report if the frequencies of alleles across loci have a L-shape distribution or a shifted mode (presence of bottleneck). This all is explained in the references we cited in the manuscript

Attachment

Submitted filename: ResponseToReviewers.docx

pone.0340580.s020.docx^{(26.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0340580.r003

Decision Letter 1

Sven Winter

22 Oct 2025

Dear Dr. Meléndez-Cal-y-Mayor,

Please submit your revised manuscript by Dec 06 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Sven Winter

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

**********

Reviewer #2: I have carefully examined the authors’ point-by-point responses to the previous review as well as the revised version of the manuscript. Overall, it is clear that the authors have made a substantial effort to address the earlier concerns, and many of the revisions have improved the clarity, methodological transparency, and overall quality of the paper. In particular, the handling of the statistical comments appears sound: the use of median Fis values was implemented as suggested, model assumptions were tested and reported, and the treatment of bottleneck results has been improved. Ethical aspects related to animal handling have been clarified with commendable precision, and the data availability now meets the journal’s requirements. These are all meaningful improvements.

The manuscript itself presents a well-defined and relevant study that addresses the important question of genetic similarity of translocated, colonized, and natural populations. The research is well framed and generally methodologically solid. The use of multiple analytical approaches and the integration of empirical data with forward simulations are notable strengths that make this study potentially valuable to the conservation genetics community.

However, some of the responses remain rather superficial. This is especially true for the interpretation of infinite Ne values, which is acknowledged but not convincingly discussed, and for the section on bottleneck events, where additional depth and contextualization would strengthen the argument (please also check all references again, some are missing, e.g., l. 180 "residuals were tested for each model using the function jarqueberaTest () and dwtest ()"). The adjustments to figures and tables still fall short of making the results fully accessible at a glance. For example, the revised map is likely clearer than the original, but the description suggests that the overall visualization strategy remains basic rather than genuinely improved, and the whole map (still?) appears broken to me. Please check your visualization tool. Moreover, all figures and tables need a proper caption, which is self-explanatory; e.g., table 1 lacks a conclusive caption.

A general pattern throughout the responses is that several replies address the comments formally, but without fully exploiting the opportunity to clarify or deepen the presentation of the results. For example, the discussion of the infinite Ne values just lists two additional explanations without critically evaluating their likelihood in the specific study context or providing supporting literature. The response to the bottleneck-related comment adds some details but remains descriptive and lacks an interpretation of how these results inform the study’s main conclusions. Likewise, the comment on improving figure clarity was addressed by creating a new map, but without any clear rationale for how the new visualization strategy better communicates the underlying patterns. While the core analyses seem sound and the technical points were largely addressed, the interpretation and communication of the findings could still be more thorough.

In sum, the authors have clearly engaged with the reviewers’ feedback and improved the manuscript in several important respects. Yet some areas would benefit from more thoughtful revision, particularly regarding interpretation of key genetic results (biological context) and data presentation. I would therefore recommend minor revisions, with a focus on strengthening the clarity and depth of interpretation as well as improving the accessibility of figures and tables.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

**********

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PLoS One. 2026 Jan 8;21(1):e0340580. doi: 10.1371/journal.pone.0340580.r004

Author response to Decision Letter 2

17 Dec 2025

Dear Editor,

Thank you for giving us another opportunity to revise our manuscript. In this document we explain how we have revised the manuscript in response to the comments made by the editor and the reviewers. Our responses start with RESPONSE >. Line numbers correspond to the files with track changes.

We deleted text from the previous decision letter that is not relevant here.

The reviews were very constructive and helped to improve the manuscript.

Best wishes,

on behalf of all authors

Subject: PLOS ONE Decision: Revision required [PONE-D-25-16547R1]

PONE-D-25-16547R1

Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

PLOS ONE

Dear Dr. Meléndez-Cal-y-Mayor,

I agree with the new reviewer that the manuscript did greatly improve and that many of the previous comment have been addressed but there are still a few comment that have not been answered thoroughly enough.

RESPONSE > Thank you. Below we explain how we improved the manuscript. In the last revision, we gave the most consideration to those comments that served to improve the answers to our scientific questions.

Please submit your revised manuscript by Dec 06 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Sven Winter

Academic Editor

PLOS ONE

Journal Requirements:

RESPONSE > We have checked our reference list, and they are complete and correct.

6. Review Comments to the Author

RESPONSE > Thank you for the positive assessment of the manuscript and the revision.

However, some of the responses remain rather superficial. This is especially true for the interpretation of infinite Ne values, which is acknowledged but not convincingly discussed,

RESPONSE > We added more specific explanations of the potential reasons why we found infinite Ne values based on previous works and on our data (from line 160 to 165). We explain that we think that infinite Ne are most likely an artefact.

and for the section on bottleneck events, where additional depth and contextualization would strengthen the argument

RESPONSE > We added more additional information to strengthen our argument (from line 372 to 385). We explain why we believe that there were bottlenecks in the recently colonized populations (simply because colonization caused the bottleneck). In one of the “old” natural populations in Lucerne area, we believe it was due to maintenance of the well.

(please also check all references again, some are missing, e.g., l. 180 "residuals were tested for each model using the function jarqueberaTest () and dwtest ()").

RESPONSE > The citation brackets in PlosOne are []. The brackets that the reviewer mentioned as lacking a citation correspond to the functions jarqueberaTest and dwtest (it is R language). These brackets indicate that both are functions, and inside the brackets, the parameters are specified. The functions jarqueberaTest and dwtest do not require a citation because they are included in the fBasics package, which is already cited (lines 176-177).

The adjustments to figures and tables still fall short of making the results fully accessible at a glance. For example, the revised map is likely clearer than the original, but the description suggests that the overall visualization strategy remains basic rather than genuinely improved, and the whole map (still?) appears broken to me. Please check your visualization tool.

RESPONSE > It is a simple map which shows the location of the adjacent study areas and the populations. We used different colours for the two study areas. We believe that the map serves its purpose. We double-checked all files during the submission process to make sure that the maps are displayed correctly.

Moreover, all figures and tables need a proper caption, which is self-explanatory; e.g., table 1 lacks a conclusive caption.

RESPONSE > We have included more details in the captions of all figures and tables.

RESPONSE > We added some additional explanations for Ne and bottlenecks. We don’t think that a further discussion is useful because it would be speculative. Regarding the infinite Ne values, we added additional explanations and supporting literature in the manuscript (from line 160 to 165). This new information explains the possible reasons why the Ne value was infinite for each population.

The response to the bottleneck-related comment adds some details but remains descriptive and lacks an interpretation of how these results inform the study’s main conclusions.

RESPONSE > We added in the manuscript more interpretation on the presence of bottleneck in 3 colonized populations and in a natural population (from line 374 to 385). This connects with the discussion we do on the presence of bottleneck from line 424 to 430. The main conclusion is that the three population types do not differ much despite the bottlenecks. We added some text in the discussion which clarifies this.

Likewise, the comment on improving figure clarity was addressed by creating a new map, but without any clear rationale for how the new visualization strategy better communicates the underlying patterns.

RESPONSE > We believe that the new maps convey that necessary information. Fig 1 shows the two study areas and all the populations while Fig 3 shows that spatial genetic structure. The maps are simple, but all the necessary information is there. There is no additional information in the maps that may distract (e.g. Hill shade).

While the core analyses seem sound and the technical points were largely addressed, the interpretation and communication of the findings could still be more thorough.

RESPONSE > We added additional explanations in the discussion in response to the previous comments and hope that this made the interpretation and discussion of the results more thorough.

In sum, the authors have clearly engaged with the reviewers’ feedback and improved the manuscript in several important respects.

RESPONSE > Thank you.

Yet some areas would benefit from more thoughtful revision, particularly regarding interpretation of key genetic results (biological context) and data presentation. I would therefore recommend minor revisions, with a focus on strengthening the clarity and depth of interpretation as well as improving the accessibility of figures and tables.

RESPONSE > We explain in the replies to the previous comments how we improved the manuscript.

Attachment

Submitted filename: ResponseToReviewers_auresp_2.docx

pone.0340580.s021.docx^{(23.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0340580.r005

Decision Letter 2

Sven Winter

23 Dec 2025

Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

PONE-D-25-16547R2

Dear Dr. Meléndez-Cal-y-Mayor,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

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Kind regards,

Sven Winter

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0340580.r006

Acceptance letter

Sven Winter

PONE-D-25-16547R2

PLOS One

Dear Dr. Meléndez-Cal-y-Mayor,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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on behalf of

Dr. Sven Winter

Academic Editor

PLOS One

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Percentage of missing data for Emmental populations by Locus (on x-axis) and population (on y-axis).

The lowercase letters at the end of the population names indicates the population type (n = natural and c = colonized). Emmental region shows 0.8% missing alleles.

(EPS)

pone.0340580.s001.eps^{(64KB, eps)}

S2 Fig. Percentage of missing data for Lucerne populations by Locus (on x-axis) and population (on y-axis).

The lowercase letters at the end of the population names indicates the population type (n = natural and t = translocated). Lucerne region shows 0.78% missing alleles.

(EPS)

pone.0340580.s002.eps^{(46.5KB, eps)}

S3 Fig. Trace plot of Markov Chain Monte Carlo (MCMC) for inference of migration rate estimates in BayesAss for the Emmental populations.

(EPS)

pone.0340580.s003.eps^{(1.9MB, eps)}

S4 Fig. Trace plot of Markov Chain Monte Carlo (MCMC) for inference of migration rate estimates in BayesAss for the Lucerne populations.

(EPS)

pone.0340580.s004.eps^{(1.8MB, eps)}

(EPS)

pone.0340580.s005.eps^{(22.9MB, eps)}

S6 Fig. Principal components analysis (PCA) for the Emmental, natural and colonized, populations based on 344 individuals and 16 microsatellite loci.

(EPS)

pone.0340580.s006.eps^{(622.6KB, eps)}

(EPS)

pone.0340580.s007.eps^{(24.7MB, eps)}

S8 Fig. Principal components analysis (PCA) for the Lucerne, natural and translocated, populations based on 330 individuals and 16 microsatellite loci.

(EPS)

pone.0340580.s008.eps^{(470.7KB, eps)}

(EPS)

pone.0340580.s009.eps^{(16.9MB, eps)}

S10 Fig. Principal components analysis (PCA) for the Lucerne natural populations based on 170 individuals and 16 microsatellite loci.

(EPS)

pone.0340580.s010.eps^{(292.8KB, eps)}

S11 Fig. Circos plot showing the translocations.

(EPS)

pone.0340580.s011.eps^{(152.3KB, eps)}

(EPS)

pone.0340580.s012.eps^{(124.9KB, eps)}

S13 Fig. Final expected heterozygosity (H_e) for colonized and natural Emmental populations under various simulated scenarios (dispersal type, population size, and dispersal frequency).

(EPS)

pone.0340580.s013.eps^{(31.2KB, eps)}

(EPS)

pone.0340580.s014.eps^{(81.9KB, eps)}

S1 Table. New microsatellite DNA markers of Alytes obstetricans developed by ecogenics GmbH.

Repeat type is based on genomic DNA sequence analyzed on an Illumina MiSeq platform. Size bp (base pairs) is based on the fragment analysis of 15 individuals on an ABI373.

(DOCX)

pone.0340580.s015.docx^{(22.3KB, docx)}

S2 Table. Details for the simulations made in quantiNemo (v2.0.0) for Emmental and Lucerne populations.

This table shows eight simulated scenarios for dispersal frequency, population size, and dispersal type.

(DOCX)

pone.0340580.s016.docx^{(22.5KB, docx)}

S3 Table. Estimated deviation from Hardy-Weinberg equilibrium (HWE) and null allele frequencies per population and for the 16 microsatellite loci.

(DOCX)

pone.0340580.s017.docx^{(48.7KB, docx)}

S4 Table. Results on the Bottleneck analysis on natural, colonized and translocated populations from canton Lucerne and Emmental using three different calculation models.

(DOCX)

pone.0340580.s018.docx^{(24.6KB, docx)}

Attachment

Submitted filename: ResponseToReviewers.docx

pone.0340580.s020.docx^{(26.5KB, docx)}

Attachment

Submitted filename: ResponseToReviewers_auresp_2.docx

pone.0340580.s021.docx^{(23.9KB, docx)}

Data Availability Statement

The Zenodo DOI is already available: https://doi.org/10.5281/zenodo.15052694.

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PERMALINK

Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

José F Meléndez-Cal-y-Mayor

Jasmin Winkler

Ramon Müller

Beatrice Lüscher

Janine Bolliger

Arpat Ozgul

Benedikt R Schmidt

Roles

Abstract

Introduction

Materials and methods

Research permits and ethical considerations

Study species and sampling sites

Fig 1. Sampled populations located in central Switzerland.

Genetic data

Genetic diversity and bottleneck

Genetic population structure and migration rates

Projecting genetic diversity

Results

Genetic diversity and bottlenecks

Fig 2. Strip chart of the genetic diversity indicators for colonized, natural and translocated populations in the study areas Emmental and Lucerne.

Table 2.

Population structure

Fig 3. Pie charts (in map) and STRUCTURE plot showing the cluster assignments for Emmental populations (top; natural and colonized populations) and Lucerne population (bottom; natural and translocated populations).

Projecting genetic diversity

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Sven Winter

Roles

Author response to Decision Letter 1

Decision Letter 1

Sven Winter

Roles

Author response to Decision Letter 2

Decision Letter 2

Sven Winter

Roles

Acceptance letter

Sven Winter

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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