PLOS ONE RESEARCH ARTICLE Genetic signature of blind reintroductions of Iberian ibex (Capra pyrenaica) in Catalonia, Northeast Spain Tânia Barros ID1☯*, Joana M. Fernandes ID1☯, Eduardo Ferreira ID1, João Carvalho1,2, Marta Valldeperes2, Santiago Lavı́n2, Carlos Fonseca1,3, Jordi Ruiz-Olmo4, Emmanuel Serrano ID2,5 1 Departamento de Biologia & CESAM (Centro de Estudos do Ambiente e do Mar) Universidade de Aveiro, Campus Universitário Santiago, Aveiro, Portugal, 2 Dept Medicina i Cirurgia Animals Facultat de Veterinària, Wildlife Ecology & Health group (WE&H) Servei d’ Ecopatologia de Fauna (SEFaS), Universitat Autònoma de a1111111111 Barcelona (UAB), Bellaterra, Barcelona, España, 3 ForestWISE—Collaborative Laboratory for Integrated a1111111111 Forest & Fire Management, Vila Real, Portugal, 4 Dirección General de Ecosistemas Forestales y Gestión a1111111111 del Medio (DARP), Barcelona, España, 5 Dipartimento di Scienze Veterinarie, Universitá di Torino, a1111111111 Grugliasco, Torino, Italy a1111111111 ☯ These authors contributed equally to this work. * taniabarros@ua.pt OPEN ACCESS Abstract Citation: Barros T, Fernandes JM, Ferreira E, The Iberian ibex is one of the most singular species of the Iberian Peninsula. Throughout Carvalho J, Valldeperes M, Lavı́n S, et al. (2022) Genetic signature of blind reintroductions of Iberian the years, this species suffered several threats which led the population to its decline. Many ibex (Capra pyrenaica) in Catalonia, Northeast reintroductions and translocations were made, however, none of those actions took into Spain. PLoS ONE 17(9): e0269873. https://doi.org/ account the genetic patterns of both reintroduced individuals and the target populations. In 10.1371/journal.pone.0269873 this paper, we explored the genetic traits of three populations of Iberian ibex in Catalonia, Editor: Tzen-Yuh Chiang, National Cheng Kung which experienced blind reintroductions in past years: The populations of Iberian ibex from University, TAIWAN Els Ports de Tortosa i Beseit National Game Reserve (TBNGR), Montserrat Natural Park Received: December 2, 2021 (Monserrat) and Montgrı́, les Illes Medes i el Baix Ter Natural Park (Montgrı́) Based on the Accepted: May 29, 2022 genetic patterns of the three populations coupled with the absence of genetic introgression Published: September 21, 2022 with domestic goats–inferred using mitochondrial and nuclear markers–we propose that these should be regarded as two different management units: TBNGR coupled with Mont- Peer Review History: PLOS recognizes the benefits of transparency in the peer review serrat, and Montgrı́. Montserrat population should be targeted as a population model for process; therefore, we enable the publication of ecology and evolution studies. Although we did not detect evidences of recent bottleneck all of the content of peer review and author events, this population seems to be monomorphic for the mtDNA haplotype. Our results responses alongside final, published articles. The suggest that the blind reintroductions from TBNGR to Montserrat failed on maximizing the editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0269873 genetic diversity of the latter. We enhance the importance of genetic monitoring of both the source population and the selected individuals to be re-introduced. We conclude that the Copyright: © 2022 Barros et al. This is an open access article distributed under the terms of the three studied population of Iberian ibex must be monitored to explore which strategy would Creative Commons Attribution License, which be advantageous for maintaining the genetic diversity. On the other hand, TBNGR should permits unrestricted use, distribution, and be monitored to assess the existence of its singular genetic variation, where stochastic reproduction in any medium, provided the original author and source are credited. events could preserve this lost genetic variation. Data Availability Statement: All data files are available from the GenBank database (ON724047- ON724060.). PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 1 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Funding: .B. is funded by national funds through Introduction CESAM/DBIO under the POSEUR project CP01- MARG-QUERCUS/2018. https://poseur. The endemic Iberian ibex (Capra pyrenaica) is one of the most iconic ungulate species in the portugal2020.pt/ E.F. is funded by national funds Iberian Peninsula [1]. The species was originally distributed throughout the Iberian range, (OE), through FCT—Fundação para a Ciência e a from sea level up to 3,400m, and also in the southwest of France. Currently the species is Tecnologia, I.P., in the scope of the framework extinct in the northernmost areas of its native range, namely in the Pyrenees [2]. Four Iberian contract foreseen in the numbers 4, 5 and 6 of the ibex subspecies have been historically recognized based on coat color and horn morphology article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. https:// [3–5]. Only two of these subspecies are currently extant: C. p. victoriae and C. p. hispanica, www.fct.pt/ J.F. was supported by a PhD grant with allopatric distribution in the Iberia Peninsula. The subspecies C. p. victoriae occurs in the from FCT/MCTES (PD/BD/150645/2020), co- central Spanish mountains (Sierra de Gredos) and has been reintroduced to a number of addi- financed by European Social Fund POPH-QREN tional sites in Spain (Batuecas, La Pedriza, Riaño, French Pyrenees) and naturally re-colonized program. https://www.fct.pt/ J.C. was supported northern Portugal (Peneda-Gerês National Park) [6, 7]. The subspecies C. p. hispanica occu- by a research contract (CEECIND/01428/2018) from the Fundação para a Ciência e a Tecnologia pies the arc of mountains that run along the Mediterranean coast, as well as Sierra Morena [8, (FCT). https://www.fct.pt/ FCT/MCTES financed 9]. The other two subspecies went extinct on different moments: C. p. lusitanica, from Iberian CESAM (UIDP/50017/2020+UIDB/50017/2020 NW mountains, in the end of the 19th century; and C. p. pyrenaica in the end of 20th century +LA/P/0094/2020), through national funds. https:// [10, 11]. The latter formerly occurred throughout much of the French, Spanish and Andorran www.fct.pt/ “Apoio à Contratação de Recursos Pyrenees, and persisted until recently in Ordesa and Monte Perdido National Park, in the Humanos Altamente Qualificados” (NORTE-06- Maladeta massif [2]. 3559-FSE-000045), supported by Norte Portugal Regional Operational Programme (NORTE 2020), During the 19th century, the Iberian ibex underwent a considerable demographic decline under the PORTUGAL 2020 Partnership caused by the destruction of its natural habitat, epidemics and unrestrained hunting activities Agreement. ForestWISE—Collaborative Laboratory [12]. The populations of Iberian ibex faced numerous threats, including contagious diseases, for Integrated Forest & Fire Management, was habitat fragmentation and poaching or unrestricted hunting plans that resulted on biased sex recognized as a CoLAB by the Foundation for ratio and age structure [13–16]. At the beginning of the 20th century, several conservation pro- Science and Technology, I.P. (FCT). https://www. forestwise.pt/pt/institution/ This article/publication grams were at the base of the recovery of the Iberian ibex populations [11, 17, 18]. The species is based upon work from COST Action, supported is currently widely distributed, however, the alteration and even loss of genetic diversity due to by COST (European Cooperation in Science and non-controlled translocations, re-introductions and natural spreading is envisaged as a threat. Technology). https://www.cost.eu/ The funders had C. pyrenaica is a very popular game species and several translocations and restocking programs no role in study design, data collection and were conducted with hunting purposes, although many of these were poorly documented [19, analysis, decision to publish, or preparation of the 20]. These unmonitored translocations might have resulted in the admixture of animals from manuscript. distinct origins. Graver, though, is the possibility of hybridization with domestic goat (Capra Competing interests: The authors have declared aegagrus hircus), as recently reported [21, 22], threatening the integrity and fitness of the spe- that no competing interests exist. cies gene pool. Additionally, other studies using genotypic data suggest that the Iberian ibex is strongly differentiated among regions, and presents low genetic diversity and poor mixed ancestry, which may be a consequence of past augmentation translocations that resulted in a poor increase of genetic diversity of the species. Reintroductions have been widely used for several wild species and have been proven to be successful on the restoration of populations that are at risk of decline. It is considered a fruitful conservation tool, although its implementation needs to present consistent justification and it needs to reflect a balance between conservation benefits and its costs [23]. Management prac- tices targeting ungulate species–including translocations, captive-breeding, fencing–poten- tially lead to changes in the genetic patterns of populations [24–26] but can result on: the decline of genetic variation, increased inbreeding, low population viability and even loss of local adaptations [27–29]. Demographic restoration may not reflect genetic improvement of the target populations [30]. For these reasons, information on the phylogeographic origins and genetic affinities among populations may provide useful insights for the implementation of conservation and management strategies, although this is still underappreciated [31]. Hence, using genetic monitoring as a diagnostic tool, one can acquire valuable information regarding possible founder effects, success of supplementation, population dynamics and admixture, as well as other genetic-related parameters [32–34]. The use of genetic tools do play an important PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 2 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia a very essential role in reintroduction programs and should be implemented prior to the intro- duction of any specimens into the target population [35]. The populations of Iberian ibex from Els Ports de Tortosa i Beseit National Game Reserve (TBNGR), Montserrat Natural Park (Monserrat) and Montgrí, les Illes Medes i el Baix Ter Nat- ural Park (Montgrı́), in Catalonia (Spain), are three populations that have been under active management plans, that involved translocations and population reinforcement measures [36]. The Montserrat and Montgrı́ populations were originated from single founder events com- prising, respectively: (i) 46 individuals translocated from TBNGR to Montserrat, during the 90’s [36]; (ii) and five runaway individuals brought to Montgrı́ from the Sobrestany zoo, in Girona, that were originally from Andalusia. No population genetics studies have ever been envisaged for these three populations in specific, as genetic monitoring of the specimens involved in those translocation measures was not performed. The implementation of molecu- lar methods should be implemented as a tool for monitoring genetic changes in ungulate pop- ulations [37], especially in those that are explored for hunting purposes, which is the case of Montserrat, Montgrı́ and TBNGR populations. Such actions may affect genetic variability in a short period of time [14, 38]. Using both mitochondrial and nuclear markers, we aimed to characterize the genetic pat- terns of the three mentioned populations and evaluate if the translocation measures improved the genetic variability of the population (in the particular case of Montserrat). Specifically, we (1) assessed the presence and level of hybridization with domestic goat, as well as human- mediated introgression (through translocations or rearing in confined multi-species herds) of Alpine ibex (Capra ibex), as it may constitute a threat to the maintenance of genetic heritage of the Iberian ibex; (2) characterized the phylogeographic affinities with other C. pyrenaica popu- lations in the Iberian Peninsula; and (3) assessed the level of genetic diversity, structure and relatedness within and between the three Iberian ibex populations. The populations of Mont- serrat and Montgrı́ are a result of known historical founder events, thus, the assessment of diversity and endogamy levels are particularly relevant for these two populations. Additionally, given that the population of Montserrat was founded by individuals coming from TBNGR, these two populations may constitute an interesting case study and provide relevant insights into the microevolutionary processes at stake during translocation events. Thus, we also aim to (4) to discuss the genetic patterns observed in TBNGR and Montserrat at the light of the inti- mate relation among the two populations. These insights may highlight the importance of these and other reintroduced populations for conservation and management actions for this species in the Iberian Peninsula. Material and methods Study areas The Iberian ibex populations of TBNGR, Montserrat Natural Park and Montgrı́ mountain (northeastern Spain, Fig 1), experience a Mediterranean climate characterized by hot sum- mers, summer droughts, and cold winters [39]. The habitat is heterogeneous and encompasses holm oak (Quercus ilex) and pine (Pinus sp.) forests, evergreen sclerophyll shrublands and patches of pastures and crops. The Iberian ibex census in the TBNGR suggested a population decrease of approximately 25% over the last two decades [40]. Here, Iberian ibex males have been trophy-harvested over the last four decades. Selective harvesting towards individuals with undesired phenotypes is also carried out to reduce the intraspecific competition and to limit their reproduction [15]. The Iberian ibex population of Montserrat Natural Park resulted from a reintroduction programme conducted in the 90’s, when 46 individuals were translocated from the TBNGR. Currently, a natural increase in population density is taking place and a PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 3 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Fig 1. Location of the three studied populations of Iberian ibex. Elevation data used in the map were retrieved from the Shutter Radar Topography Mission (SRTM-DEM; [42]), that is publicly available at no charge, a courtesy of the U.S. Geological Survey. https://doi.org/10.1371/journal.pone.0269873.g001 restricted number of males can be hunted once the individual grows enough to be classified as a trophy. Since Iberian ibexes were accidentally released into Montgrı́ (2000–2007) [41], no further translocations were carried out. Current censuses point to a viable population with ca. 70 individuals [41]. DNA amplification and sequencing In this work, Iberian ibex (Capra pyrenaica) samples were collected from legally hunted indi- viduals harvested in different populations of the Catalan community. None of the animals were hunted for our scientific purposes and thus an ethic committee approval was not neces- sary. This sampling was performed within the framework of an official agreement between the Ministry of Climate Action, Food and Rural Agenda (Generalitat de Catalunya, Departament d’Acció Climàtica, Alimentació I Agenda Rural) and the Servei d’Ecopatologia de Fauna Sal- vatge (SEFaS). This agreement has as a main goal the surveillance of diseases from wildlife from the region of Catalonia, in Spain. None of the authors were directly involved in the sam- ple collection, and the samples were not purposefully collected for this study. Government employers delivered tissue samples to our laboratory. DNA was isolated from a total of 185 muscle samples (118 ibexes from TBNGR, 21 ibexes from Montserrat, 8 ibexes from Montgri, 31 domestic goats and 7 ibexes without known geographic coordinates) through a standard PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 4 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia salt-extraction protocol [43]–or using the QIAGEN1 Tissue and Blood extraction kit, for older samples–and quantified by spectrophotometry, using Nanodrop1. We amplified a fragment of 1140 bp from mitochondrial DNA (mtDNA; cytochrome b) using the universal primers L14841 and H15915 [44]. We amplified the target region, in a final volume of 25 microliters with 1x reaction buffer, 1.5 mM MgCl2, 1 μM of each primer, 10 mM of dNTP, 0.2 units of Taq polymerase (Invitrogen©). We performed a PCR amplification with 45 cycles (denaturation: 94˚C, 3 minutes; annealing: 48˚C, 45 seconds; polymerization: 72˚C, 120 sec- onds). Amplified (PCR) products were visualized in 2% agarose gels, purified and sequenced (Sanger) in an automatic sequencer ABIPRISM1 3730-XL DNA Analyzer, Applied Biosys- tems™, using BigDye™ sequencing kit. A total of 14 microsatellite markers were selected for this study (Supporting Material S1 File). DNA amplifications were performed using the QIAGEN1 Multiplex amplification kit, following manufacturer’s conditions. PCR products were visualized on 2% agarose gel and fragment analysis was performed using an ABIPRISM1 3730-XL DNA Analyser from Applied Biosystems™. In order to reduce the chance of mistype and minimize genotyping error, each sample was amplified and genotyped a minimum of three times for each marker. Data analysis Generated sequences were aligned and consensus sequences were inferred from the forward and reverse sequences. Sequences were then compared with previously published sequences of Iberian ibex, Alpine ibex, Nubian ibex (C. nubiana), Siberian ibex (C. sibirica) and domestic goat, retrieved from Genbank (Supporting Material S2 File). Mitochondrial DNA sequences were aligned using MEGA v.5 [45] with the CLUSTALW algorithm [46] and were further manually edited. Preliminary species diagnosis (for a preliminary screen of hybridisation with Capra a. hircus) was performed using BLAST queries to the GenBank1 online database. A haplotype network was constructed using a median-joining (MJ) algorithm implemented in POPART [47]. General diversity indices were calculated on DNAsp v5, using the generated mtDNA sequences: number of haplotypes, haplotype and nucleotide diversity and number of polymorphic sites [48]. Microsatellite genotyping was performed using Genemarker™ v2.4.1 (SoftGenetics™). Elec- tropherograms were analysed with this software but allele calling was performed through care- ful visual inspection. Identification of individual profiles was assessed only when at least 70% of the analysed microsatellites were successfully amplified. Mean genotyping error rate was estimated by assessing the ratio between the number of mistyped alleles relatively to the total number of genotyped alleles [49]. For detecting structure and possible hybridization between the Iberian ibex and domestic goat, we tested for evidence of genetic structure using Structure v2.3.4 [50]. We used the admixture model with correlated allele frequencies with no previous information about the original sampled location of each individual. The analysis was carried out for 2 million itera- tions of the Markov Chain Monte Carlo, with a burn-in of 100 thousand iterations. The puta- tive number of populations was simulated with K (number of putative subpopulations) varying between 1 and 6. Ten replicate runs were performed for each K. We then used Struc- ture Harvester [51] to summarize the results, by estimating the best K using both the Evanno method [52] and the original approach suggested by Pritchard et al. [50]. We then performed a Structure analysis within the Iberian ibex population, following the same conditions as described above. Evidence for significant deviations from Hardy-Weinberg equilibrium (HWE), and for linkage disequilibrium (LD), as well as allelic frequencies and patterns, hetero- zygosity levels and genetic differentiation through FST index [53] were assessed. Analysis of PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 5 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia molecular variance (AMOVA) using the allelic distances between individuals was also calcu- lated. All analyses were performed on Genalex v6.5 [54] and Arlequin v3.5 [55]. In order to search for evidence of recent bottlenecks, we estimated the M ratio between the number of alleles and the allele size range [56]. We estimated this ratio using ARLEQUIN, after we recoded the genotypes using the number of tandem repeats relatively to the smallest allele of each locus (which was coded with 1, for one repeat). Results Mitochondrial DNA data analysis We were able to successfully sequence a total of 164 amplified samples (102 samples from TBNGR, 21 samples from Montserrat, 8 samples from Montgrı́, 28 samples from domestic goats and 5 samples without geographic coordinates) for mtDNA, comprising 14 Iberian ibex haplotypes (Fig 3; Table 1; Accession numbers: ON724047-ON724060). All but two haplotypes (H10 and H12) were newly generated in this study and had not been detected in previous stud- ies. Haplotypes plotting across the sampling area evidences a wide distribution of haplotype H10, corresponding to the most frequent haplotype found in the studied area (present in ca. 64% of all analyzed samples). This haplotype is present in the population located in the TBNGR and it was the only haplotype detected in individuals sampled in Montserrat. Haplo- type H8 is present in 10% of the total samples and was reported only for individuals from TBNGR. Haplotype H12, also present in 10% of the total samples, was only detected in individ- uals sampled in Montgrı́ (Fig 2). With the aim of balancing the number of retrieved sequences and the amount of informa- tion per sequence (i.e., covering the maximum number of polymorphic sites found in previ- ously published sequences from the selected species available on Genbank), the haplotype network was built using 81 complete sequences with a minimum length of ca. 620 base pairs. All haplotypes generated in this study clustered with Iberian ibex individuals rather with domestic goat or Alpine ibex, as expected in the case of absence of hybridization between the wild and domestic populations. Nevertheless, we must call attention to the fact that mtDNA refers to matrilineal non-recombinant lineages and thus, evidence against hybridization, in Table 1. Correspondence between presence of the found haplotypes in the studied population of Iberian ibex, by sampling area and by genetic cluster (according to the Structure results for k = 2). By sampling area By genetic cluster Haplotypes Tortosa i Beseit Montserrat Montgrí Tortosa i Beseit + Montserrat Montgrí H1 1 1 H2 1 1 H3 1 1 H4 1 1 H5 1 1 H6 1 1 H7 1 1 H8 7 7 H9 1 1 H10 30 15 45 H11 1 1 H12 7 7 H13 1 1 H14 1 1 https://doi.org/10.1371/journal.pone.0269873.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 6 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Fig 2. Haplotype distribution of the Iberian ibex samples across the studied area. Each haplotype (H1 to H14) is identified by a different color. Figure adapted from MDT05 2015 CC-BY 4.0 scne.es. https://doi.org/10.1371/journal.pone.0269873.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 7 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Fig 3. Haplotype network showing the haplotypes of Capra pyrenaica and Capra a. hircus identified in this study, and previously published haplotypes of Iberian ibex, Alpine ibex and domestic goat. (Cat–Catalonia; CS_Sp–Centre/Southern Spain). https://doi.org/10.1371/journal.pone.0269873.g003 this case, is limited. Contrarily to what we would expect, the inferred phylogeographic affini- ties within C. pyrenaica and with closely related species, did not support the monophyly of the species. In fact, Capra ibex appears as the sister group to all the C. pyrenaica subspecies, except the type subspecies, C. p. pyrenaica. We found shared haplotypes among TBNGR, Montserrat and previously published sequences of individuals from the same geographic range (Catalo- nia). Goats from Montgrı́, were found to share haplotypes with individuals from Central and Southern Spain (Fig 3). Microsatellite data analysis We found a clear structure between C. a. hircus and C. ibex sampled in the study area (Fig 4A), with percentages of attribution to each one of the clusters above 99% for each individual (Fig 4D) (Supporting Material S3 File). Regarding the Iberian ibex genotypes, we were able to retrieve a total of 96 individual genotypes (Supporting Material S4 File). We also found struc- ture among the Iberian ibex populations under study, in particular between the population from Montgrı́ and the populations of TBNGR and Montserrat (Fig 4E). According to the Evanno method, the most likely scenario within the Iberian ibex population from Catalonia comprises two genetic clusters (K = 2), with a Q proportion of the genome of each individual assigned to each of the inferred clusters above 96%. One cluster gathers all individuals from TBNGR and Montserrat, while all Montgrı́ individuals (Fig 4B) are grouped within the other genetic cluster. On the other hand, when inferring the most likely K number of genetic clusters PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 8 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Fig 4. Microsatellite analysis for both domestic goats (C. a. hircus) and the three Iberian ibex populations sampled in Catalonia. Individual Q proportion of the genome of each individual assigned to each of the genetic clusters inferred with STRUCTURE for (A) C. a. hircus and C. ibex sampled in the study area, and for (B) K = 2 and (C) K = 3, for the three Iberian ibex populations sampled in Catalonia. Each individual is represented by a vertical bar and the different genetic clusters are represented by different colors. Principal coordinates analysis (PCoA) plot for (D) C. a. hircus and C. ibex sampled in the study area and for the three Iberian ibex populations samples in Catalonia. https://doi.org/10.1371/journal.pone.0269873.g004 directly from likelihood values (following the original approach from Pritchard et al. 2000), the scenario with higher support includes with three genetic clusters (K = 3) of Iberian ibex, as follows: (i) one comprising all individuals sampled in Montgrı́; (ii) a second one comprising almost all individuals sampled in Montserrat and part of the individuals from TBNGR; and (iii) a third composed only by individuals from TBNGR, with exception to an individual from Monserrat (Fig 4C). PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 9 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Table 2. Genetic diversity, structure, levels of endogamy (FIS) and bottleneck results for the studied populations of Iberian ibex. Parameter Catalonia (n = 78) Tortosa i Beseit (n = 51) Montserrat (n = 18) Montgrí (n = 9) Mitochondrial DNA Genetic diversity π 0.003 0.004 - 0.001 Hd 0.573 0.604 - 0.524 S 14 14 - 2 Microsatellites Structure Loci in HWD 8/14 2/14 1/14 1/14 LD (pair of loci in LD) 11/91 3/91 0/91 1/91 Genetic Diversity NA 7 5 4 3 Ap - 24 9 8 Ar 3.073 5.286 4.143 2.714 Gene Diversity 0.652 0.641 0.560 0.421 HE 0.659 0.574 0.670 0.351 HO 0.569 0.627 0.629 0.485 Endogamy FIS 0.155 0.114 -0.157 0.219� Bold values indicate significance (p<0.001) or equivalent, after Bonferroni correction, applied for multiple testing. � —marginally significant. π–nucleotide diversity; Hd–haplotype diversity; S–number of polymorphic sites; HWD–Hardy Weinberg Disequilibrium; LD–Linkage Disequilibrium; NA–number of alleles (average among loci), Ap–number of private alleles; Ar–allelic richness (average among loci); HE–expected heterozygosity (average among loci); HO–observed heterozygosity (average among loci). Montserrat population was monomorphic for mtDNA. https://doi.org/10.1371/journal.pone.0269873.t002 FST values show a very strong and significant genetic differentiation between the population pairs TBNGR/Montgrı́ (FST = 0.25, p<0.001) and Montserrat/Montgrı́ (FST = 0.29, p<0.001). There was no evidence of a significant genetic differentiation between TBNGR and Montserrat (FST = 0.01, p<0.001). AMOVA results showed that for the three populations ca. 80% of the genetic variation is found within the individuals, ca. 5.5% among individuals and ca. 14% among populations. Number of polymorphic sites, nucleotide and haplotype diversity were calculated for the overall population of Iberian ibex and also for each population separately (Table 2). We have sampled 11 out of the 14 identified haplotypes in TBNGR, while in Montserrat we only identi- fied one haplotype. Three haplotypes, not shared with the other two populations, were sampled in Montgrı́ (Fig 3). Haplotype and nucleotide diversity was found to be higher in TBNGR. However, such results must be interpreted with caution, as both parameters are sensitive to the number of samples. As expected, microsatellite genotypes revealed a high level of departure from HWE for the whole population of Iberian ibex in Catalonia, as well as strong evidence of linkage disequilibrium (LD). When considering the three subpopulations individually, TBNGR carries the higher number of loci departing from HWE, as well as LD. Number of pri- vate alleles, allelic richness and gene diversity were also found to be higher in this population. We also found evidence of inbreeding in TBNGR and in Montgrı́, but only marginally signifi- cant in the latter (Table 2). The significant departures of HWE and presence of LD in the three populations might be related with natural variation (TBNGR and Montserrat are, in fact, one single genetic cluster, though not the same reproductive unit) and the reduced number of sam- ples, in this latter case the Montgrı́ population, and other stochastic causes. Since two of the microsatellite markers (BM1258, IDVGA30) had substantially more miss- ing data than the other twelve markers, and that this might result in the estimation of artifi- cially low values of M ratio, this ratio was estimated using only twelve markers. The values ranged from 0.623, in Monserrat, to 0.682, in Montgri. These values are very close to the theo- retical threshold of M = 0.68, inferred by the authors of the method [56] as providing evidence of recent population declines. These results are consistent with the known demographic PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 10 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia history of these populations, given that at least Montgri and Montserrat originated from known founder events. More details on microsatellite markers diversity indices are provided in supporting material (S5 File). Discussion Recent studies suggested that genetic monitoring is a key tool to be applied in reintroduction and translocation actions [57]. Unfortunately, the majority of monitoring programs do not use the full potential given by molecular genetic markers, which can provide information rele- vant to both ecological and evolutionary traits [58]. The case of these three Iberian ibex popu- lations constitutes a valuable opportunity not only to assess the current genetic patterns and detect possible admixture and genetic structure, but also if past translocation actions were effective for maximizing genetic diversity. Evolutionary affinities and current gene flow with other Capra species Our results suggest that there is no introgression from domestic goat or Alpine ibex gene pools in the three surveyed Iberian ibex populations. The patterns of assignment to genetic clusters inferred from the recombinant nuclear markers (microsatellites) corroborate the results of the phylogenetic analysis based on mtDNA and clearly differentiated Iberian ibex from domestic goat. We identified the presence of two clearly distinct genetic clusters separating Iberian ibex individuals from domestic goat individuals with a Q proportion of the genome of each individ- ual assigned to each genetic cluster, matching its putative source population by more than 99% (see Supporting Material S3 File). Both mitochondrial and microsatellite results indicate that there is no evidence of hybridization between both species in the populations under study (Montgrı́, Montserrat and TBNGR), a scenario that was already detected in other areas of the distributional range of this species [58]. On the other hand, the inferred evolutionary relationships reveal the non-monophyletic nature of C. pyrenaica, in relation to C. ibex. The possible presence of C. pyrenaica in the Ibe- rian Peninsula and its differentiation in the Late Pleistocene, or the fact that introgression by hybridization is a common process in the evolution of the Capra genus [59], may justify the paraphyletic or polyphyletic nature, respectively, of the Iberian ibex. In fact, either the histori- cal hybridization with C. ibex and other ancient Capra taxa [60], or the distinct times for the colonization of the Iberian by the ancestors of C. p. pyrenaica and the other Iberian ibex sub- species may help to understand these phylogenetic patterns. Also, skull morphology revealed an intermediate position of C. p. pyrenaica between the other 2 extant subspecies (C. p. victor- iae and C. p. hispanica) and the Alpine ibex (C. ibex) [60]. Genetic studies pointed that the Pyr- enean wild goat is clearly differentiated from the other Iberian populations [17]. Under the light of the evolutionary relations among Iberian ibex and Alpine ibex [61], our results leave room for questioning the taxonomic relations of European Capra species. In addition to pro- viding new data from Iberian goats geographically closer to the ancient range of C. p. pyre- naica, mtDNA phylogeography clearly supports the polyphyletic nature of what has been referred to as C. pyrenaica, by placing the Alpine ibex in-between the Iberian ibex and the Pyr- enean “bucardo”. Genetic diversity patterns and structure of the Iberian ibex in Catalonia Genetic structure patterns inferred with nuclear DNA, are in agreement with mtDNA observed patterns, not only at the level of separating domestic from wild goat, but also between the wild goat populations. The high nuclear genetic diversity found in TBNGR was expected, since this population did not suffer from an intense genetic bottleneck as the other populations PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 11 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia that were originated by a low number of individuals (46 individuals in Montserrat and 5 indi- viduals in Montgrı́). On the other hand, mitochondrial genetic diversity was found to be higher in Montgrı́, yet, this result should be interpreted with caution, as several diversity indi- ces are sensitive to sample size. The wild goat population from Montgrı́ was originated by a group of Iberian ibex individu- als translocated from Andalusia, that escaped from a zoo in Sobrestany, Girona province [41]. Hence, the presence of exclusive haplotypes, the sharing of certain haplotypes with goats from Centre and Southern Spain, the presence of private alleles and distinct allele patterns, that dif- fer from those observed in TBNGR and Montserrat. Further, the strong geographic isolation of Montgrı́ population and the absence of natural or artificial translocation of individuals from the other two populations might be related with the higher number of private alleles and the high level of endogamy found in this population. However, and most importantly, the strong founder effect that originated the Montgrı́ population might be the pivotal cause underlying its observed allelic patterns, which may raise questions regarding the management of this population. The observed structure between the Iberian ibex populations from Montgrı́ and from TBNGR and Montserrat are supported by the estimated high FST values. High FST values were already observed in other studies of within the Iberian ibex subspecies [58]. The genetic affini- ties between the source (TBNGR) and newly founded (Montserrat) populations is supported by either of the two most likely scenarios: K = 2 or K = 3 (Fig 4). Regarding K = 3 results, the individuals from both populations were not spatially segregated between Montserrat and TBNGR. Rather, there was high values of Q proportion of the genome of each individual, from both populations, assigned to both clusters. One of the two clusters, however, was more associated with the population from Montserrat. These results show the strong founder effect in Montserrat that resulted in a new random sample of alleles and allele frequencies and that, after 25 years since the reintroduction of the first individuals, Montserrat population might be in its way into becoming a new random mating population, genetically distinct from the population it was originated from. Thus, as previously reported [36], the pop- ulation of wild goats from Montserrat, in comparison with the source population of TBNGR, is likely to become a good model for studies in ecology and evolution. Reproductive dynamics may also reflect on the genetic composition, hence, the structuration of the populations after reintroduction can give important clues about those reproductive dynamics [61]. A detailed monitoring of this reintroduced population from Montserrat could provide the necessary baseline information to examine the effects of human-induced and/or environmental changes on phenotypic traits variation and quantify the effects of variable nutrition and dietary restric- tions on secondary sexual characters, such as horn growth and other traits. It is important to notice that, despite the absence of genetic differentiation between TBNGR and Montserrat–that derives from the fact that Montserrat population was originated by individuals from TBNGR–the evidences of independent reproductive dynamics of the two populations start to leave a genetic signature on both populations’ gene pools. However, due to the number of founder individuals in Montserrat and the matrilineal lineages in TBNGR, it was not expected that the Montserrat population would be monomorphic for mtDNA. Despite the absence of inbreeding and bottleneck in Montserrat, both molecular markers tell distinct stories: while mtDNA reveals a low genetic diversity (only one haplotype), microsatellites reveal an allelic richness and heterozygosity values similar with those from original population (TBNGR). This raises questions regarding if this outcome is an effect of the selection of specific features associated with particular genetic lineages (particularly matrilineal ones), since the founder event, or if it is the result of random genetic drift processes during and after the rein- troduction event. This possible current differentiation process between the populations of PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 12 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Tortosa i Beseit and Montserrat is in part evidenced by the STRUCTURE results and, eventu- ally, by the monomorphic aspect of the mtDNA diversity of Montserrat population. Other studies on extant populations of C. pyrenaica subspecies showed that the Iberian ibex has suffered recent genetic bottlenecks in some locations, which could ultimately and negatively affect the future conservation of this taxon [e.g. 59, 62]. A recent genetic study of the Iberian ibex in southern Spain suggested that severe bottlenecks may have occurred, based on the low mtDNA haplotype and nucleotide diversity estimations [63]. In our study, the values of the M ratio, between the number of alleles and the allele size range estimated for the three populations are close to the threshold for populations that gone through recent population bottlenecks [56]. In fact, these values are similar or lower to those estimated for other populations of ungulates or carnivores known to have gone through recent population reductions [64–66]. Conservation and management implications The authors of previous studies argued that there are independent local populations of the Iberian ibex in Spain that should be considered as harbours of unique portions of the total genetic variation of the species and they recommended that they should be managed sepa- rately, as single management units [15, 67]. Based on the genetic patterns detected in each of the three populations of Iberian ibex, together with the absence of genetic introgression with domestic goats (which could raise concerns), we suggest that these populations should be viewed as different management units: (a) TBNGR together with Montserrat, and (b) Mon- tgrı́. However, Montserrat should be regarded as a population model for ecology and evolu- tion studies. Despite the bottleneck events (through the studied nuclear markers), the population seems to be monomorphic for one of the mtDNA haplotypes. These results stress the importance of genetic monitoring of both the source population and the selected individ- uals to be re-introduced, as the translocation actions from TBNGR to Montserrat failed on maximizing the genetic diversity of the latter. We also point out the case of Montgrı́, which is known to have a distinct and “allochthonous” origin, which reflects on the haplotypes and alleles present in the population. Regarding TBNGR and Montserrat populations, due to their identical origin, we found that both populations may be managed equally, however, special conservation measures should be taken in account for Montserrat population due to their poor genetic diversity concerning mtDNA. We believe that this population will gradually differ from Tortosa i Beseit, even if occasional natural translocations might occur. In fact, translocations may be necessary and can decrease inbreeding and increase genetic variation [31], but those should be performed under a careful inspection of the genetic diversity of the translocated individuals to improve the genetic structure of the population. Under the light of the usage of genetic tools for wild species management, we suggest that in the future these populations may be seen as Manage- ment Units [64], as they may play an important role for management and conservation of the Iberian ibex in the Iberian Peninsula, aiding on short-term management of the more inclusive Evolutionary Significant Units [64]. In a nutshell, we believe the three studied population of Iberian ibex should be continuously monitored to test which strategy would be beneficial for the maintenance of genetic diversity (e.g. release of different breeding lines at the same time versus ongoing supplementation) [57], to assess the magnitude of admixture when transloca- tions occur, and assess genetic diversity and population size through time. Finally, we must look for the existence of a part of the lost or “forgotten” genetic variation of the goats from TBNGR, in the neighbouring territories of northern Castellón and Eastern Teruel, where sto- chasticity could have preserved this lost genetic variation. PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 13 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Supporting information S1 File. Microsatellite markers used in the study, with reference, Multiplex, GenBank Accession number (GB acc.numb.) and Annealing Temperature (Ta). (DOCX) S2 File. GeneBank accession numbers (GB acc. numb.) for the cyt b sequences from domes- tic goat (C. a. hircus), Alpine ibex (C. ibex) and Iberian wild goat (C. pyrenaica) used in both the haplotype network and phylogenetic tree. (DOCX) S3 File. Q proportion of the genome of Iberian ibexes (C. pyrenaica) and domestic goats (C. a. hircus) to each of the two genetic clusters (Cluster 1 and Cluster 2) inferred using STRUCTURE, in the assessment of hybridization among wild and domestic goats. (DOCX) S4 File. Multilocus genotypes of Capra hircus and Capra pyrenaica (from individuals sam- pled at Montgrı́, Montserrat and Tortosa I Beseit areas) generated in this study. (XLSX) S5 File. Number of alleles and level of heterozygosity for the Iberian ibex from Catalonia assessed in this study. (n = number of individuals; NA = number of alleles; Ho = Observed heterozygosity; He = Expected heterozygosity; � —monomorphic locus for that population). (DOCX) Acknowledgments We are grateful Josep Vicens Jovani, Xavier Oliver, Maria Josep Vargas, Xavier Sempere, Jordi Xifra and Ignasi de Dalmases, from the Tortosa and Beseit National Game Reserve, the Con- trolled Game Area of Montserrat, the Game and Fish Section of Girona (DARP) and the Rural Agents corp, for their advice and samples provided. Author Contributions Conceptualization: Tânia Barros, Joana M. Fernandes, Eduardo Ferreira, João Carvalho, Car- los Fonseca, Jordi Ruiz-Olmo, Emmanuel Serrano. Data curation: João Carvalho, Marta Valldeperes, Santiago Lavı́n, Jordi Ruiz-Olmo, Emman- uel Serrano. Formal analysis: Tânia Barros, Joana M. Fernandes, Eduardo Ferreira. Investigation: Tânia Barros, Joana M. Fernandes, Eduardo Ferreira, João Carvalho, Santiago Lavı́n, Emmanuel Serrano. Methodology: Tânia Barros, Joana M. Fernandes, Eduardo Ferreira, João Carvalho, Marta Valldeperes, Jordi Ruiz-Olmo, Emmanuel Serrano. Project administration: Emmanuel Serrano. Supervision: Emmanuel Serrano. Validation: Tânia Barros, Eduardo Ferreira, João Carvalho, Marta Valldeperes, Santiago Lavı́n, Carlos Fonseca, Jordi Ruiz-Olmo, Emmanuel Serrano. Visualization: Marta Valldeperes. PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 14 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia Writing – original draft: Tânia Barros, Joana M. Fernandes, Eduardo Ferreira, João Carvalho, Marta Valldeperes, Santiago Lavı́n, Carlos Fonseca, Jordi Ruiz-Olmo, Emmanuel Serrano. Writing – review & editing: Tânia Barros, Joana M. Fernandes, Eduardo Ferreira, João Car- valho, Marta Valldeperes, Santiago Lavı́n, Carlos Fonseca, Jordi Ruiz-Olmo, Emmanuel Serrano. References 1. Sarasa M, Alasaad S, Pérez JM. Common names of species, the curious case of Capra pyrenaica and the concomitant steps towards the ’wild-to-domestic’ transformation of a flagship species and its ver- nacular names. Biodivers Conserv. 2012; 21:1–12. https://doi.org/10.1007/s10531-011-0172-3 2. Herrero J, Acevedo, P, Arnal MC, Fernández de Luco D, Fonseca C, Garcı́a-González R, et al. Capra pyrenaica (amended version of 2020 assessment). In: The IUCN Red List of Threatened Species 2021: e.T3798A10085397. 2021. https://dx.doi.org/10.2305/IUCN.UK.2021-1.RLTS. T3798A195855497.en 3. Cabrera A. The subspecies of the Spanish ibex. Proc Zool Soc London. 1911; 81: 963–977. https://doi. org/10.1111/j.1096-3642.1911.tb01967.x 4. Cabrera A. Fauna Ibérica: Mamı́feros. Madrid, Spain: Museo Nacional de Ciencias Naturales; 1914. 5. Shackleton DM, IUCN/SSC Caprinae Specialist Group, editors. Wild Sheep and Goats and their Rela- tives. Status Survey and Conservation Action Plan for Caprinae. IUCN, Gland, Switzerland, and Cam- bridge, UK; 1997. 6. Moço G, Guerreiro M, Ferreira AF, Rebelo A, Loureiro A, Petrucci-Fonseca F, et al. The ibex Capra pyr- enaica returns to its former Portuguese range. Oryx. 2006; 40: 351–354. https://doi.org/10.1017/ S0030605306000718 7. Fonseca C, Migueis D, Fernandes T, Carvalho H, Loureiro A, Carvalho J, et al. The return of the Iberian wild goat Capra pyrenaica to Portugal: From reintroduction to recolonization. J Nat Conserv. 2017; 38: 56–61. https://doi.org/10.1016/j.jnc.2017.05.006 8. Grubb P. Order Artiodactyla. In: Wilson DE, Reeder DM, editors. Mammal Species of the World: A Tax- onomic and Geographic Reference, 3rd ed. Baltimore, Maryland: Johns Hopkins University Press; 2005. pp. 637–722. 9. Acevedo P, Real R. Biogeographical differences between the two Capra pyrenaica subspecies, C. p. victoriae and C. p. hispanica, inhabiting the Iberian Peninsula: Implications for conservation. Ecol Mod- ell. 2011; 222: 814–823. https://doi.org/10.1016/j.ecolmodel.2010.10.006 10. Alados CL. Distribution and status of the Spanish ibex (Capra pyrenaica, Schinz). In: Lovari S, editor. The Biology and Management of Mountain Ungulates. Croom-Helm, London: IUCN/SSC Caprinae Specialist Group; 1985; pp. 204–211. 11. Pérez JM, Granados JE, Soriguer RC, Fandos P, Márquez FJ, Crampe JP. Distribution, status and con- servation problems of the Spanish Ibex, Capra pyrenaica (Mammalia: Artiodactyla). Mammal Rev. 2002; 32: 26–39. https://doi.org/10.1046/j.1365-2907.2002.00097.x 12. Fandos P. La cabra montés (Capra pyrenaica) en el Parque Natural de las Sierras de Cazorla, Segura y las Villas. Madrid, Spain: Colección Técnica—ICONA. 1991. 13. León-Vizcaı́no L, Ruiz de Ybáñez MR, Cubero MJ, Ortiz JM, Espinosa J, Pérez L, et al. Sarcoptic mange in Spanish ibex from Spain. J Wildl Dis. 1999; 35: 647–659. https://doi.org/10.7589/0090-3558- 35.4.647 PMID: 10574523 14. Granados JE, Pérez JM, Márquez FJ, Serrano E, Soriguer RC, et al. La cabra montés (Capra pyre- naica, Schinz 1838). Galemys. 2001; 13: 3–37. 15. Pérez JM. Distribución, genética y estatus sanitario de las poblaciones andaluzas de la cabra montés. Jaén: Universidad de Jaén, Consejerı́a de Medio Ambiente; 2001. 16. González-Candela M, Cubero MJ, Martin-Atance P, León-Vizcaı́no L. Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain. J Wildl Dis. 2006; 42: 325–334. https://doi. org/10.7589/0090-3558-42.2.325 PMID: 16870855 17. Manceau V. Polymorphisme des séquences d’ADN mitochondrial dans le genre Capra. Application à la conservation du bouquetin des Pyrénées (C. pyrenaica pyrenaica). Doctoral Thesis, University Joseph Fourie. 1997. 18. Alasaad S, Granados JE, Cano-Manuel FJ, Meana A, Zhu XQ, Pérez JM. Epidemiology of fasciolosis affecting Iberian ibex (Capra pyrenaica) in southern Spain. Parasitol Res. 2008; 102: 751–755. https:// doi.org/10.1007/s00436-007-0830-2 PMID: 18092179 PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 15 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia 19. Crampe JP. Le bouquetin ibérique, éléments pour une réintroduction au versant nord des Pyrénées Occidentales. Tarbes: Parc national des Pyrénées. 1990. 20. Acevedo P, Cassinello J. Biology, ecology and status of Iberian ibex Capra pyrenaica: a critical review and research prospectus. Mammal Rev. 2009; 39: 17–32. https://doi.org/10.1111/j.1365-2907.2008. 00138.x 21. Alasaad S, Fickel J, Rossi L, Sarasa M, Benı́tez-Camacho B, Granados JE, et al. Applicability of major histocompatibility complex DRB1 alleles as markers to detect vertebrate hybridization: a case study from Iberian ibex × domestic goat in southern Spain. Acta Vet Scand. 2012; 54: 1–6. https://doi.org/10. 1186/1751-0147-54-56 PMID: 23006678 22. Cardoso TF, Luigi-Sierra MG, Castelló A, Cabrera B, Noce A, Mármol-Sánchez E, et al. Assessing the levels of intraspecific admixture and interspecific hybridization in Iberian wild goats (Capra pyrenaica). Evol Appl. 2021; 14: 2618–2634. https://doi.org/10.1111/eva.13299 PMID: 34815743 23. IUCN/SSC. Guidelines for Reintroductions and Other Conservation Translocations. Version 1.0. Gland, Switzerland: IUCN Species Survival Commission; 2013. 24. Linnell JDC, Zachos FE. Status and distribution patterns of European ungulates: genetics, population history and conservation. In: Putman R, Apollonio M, Andersen R, editors. Ungulate management in Europe:problems and practices. Cambridge: Cambridge University Press; 2011. pp 12–53. https://doi. org/10.1017/CBO9780511974137.003 25. Niedziałkowska M, Jędrzejewska B, Wójcik JM, Goodman SJ. Genetic structure of red deer population in north-eastern Poland in relation to the history of human interventions. J Wildl Manage. 2012; 76: 1264–1276. https://doi.org/10.1002/jwmg.367 26. Fernández-Garcı́a JL, Carranza J, Martı́nez JG, Randi E. Mitochondrial D-loop phylogeny signals two native Iberian red deer (Cervus elaphus) lineages genetically different to western and eastern European red deer and infers human-mediated translocations. Biodivers Conserv. 2014; 23: 537–554. https://doi. org/10.1007/s10531-013-0585-2 27. Zachos FE, Althoff C, von Steynitz Y, Eckert I, Hartl GB. Genetic analysis of an isolated red deer (Cer- vus elaphus) population showing signs of inbreeding depression. Eur J Wildl Res. 2007; 53: 61–67. https://doi.org/10.1007/s10344-006-0065-z 28. Zachos FE, Hartl GB. Phylogeography, population genetics and conservation of the European red deer Cervus elaphus. Mammal Rev. 2011; 41: 138–150. https://doi.org/10.1111/j.1365-2907.2010.00177.x 29. Galarza JA, Sanchez-Fernandez B, Fandos P, Soriguer R. The genetic landscape of the Iberian red deer (Cervus elaphus hispanicus) after 30 years of big-game hunting in southern Spain. J Wildl Man- age. 2015; 79:500–504. https://doi.org/10.1002/jwmg.854 30. White LC, Moseby KE, Thomson VA, Donnellan SC, Austin JJ. Long-term genetic consequences of mammal reintroductions into an Australian conservation reserve. Biol Conserv. 2018; 219: 1–11. https://doi.org/10.1016/j.biocon.2017.12.038 31. Whiteley AR, Fitzpatrick SW, Funk WC, Tallmon DA. Genetic rescue to the rescue. Trends Ecol Evol. 2015; 30: 42–49. https://doi.org/10.1016/j.tree.2014.10.009 PMID: 25435267 32. De Barba M, Waits LP, Garton EO, Genovesi P, Randi E, Mustoni A, et al. The power of genetic moni- toring for studying demography, ecology and genetics of a reintroduced brown bear population. Mol Ecol. 2010; 19: 3938–3951. https://doi.org/10.1111/j.1365-294X.2010.04791.x PMID: 20735733 33. Frosch C, Kraus RHS, Angst C, Allgöwer R, Michaux J, Teubner J, et al. The genetic legacy of multiple beaver reintroductions in Central Europe. PloS One. 2014; 9: e97619. https://doi.org/10.1371/journal. pone.0097619 PMID: 24827835 34. Jamieson IG. Significance of population genetics for managing small natural and reintroduced popula- tions in New Zealand. N Z J Ecol. 2015; 39: 1–18. 35. Schwartz MK. Guidelines on the use of molecular genetics in reintroduction efforts. In: The EU LIFE- Nature Projects to guidelines for the reintroduction of threatened species. Caramanico, Italy; 2005. pp. 51–58. 36. Serrano E, Carvalho J, Mentaberr G, Ferreira E, Fonseca C, Lavı́n S. La Muntanya de Montserrat: Un laboratori natural per a la cabra salvatge. Cinegeticat, gestió caça i equilibri. 2017; 22: 27–31. 37. Apollonio M, Belkin VV, Borkowski J, Borodin OI, Borowik T, Cagnacci F, et al. Challenges and science- based implications for modern management and conservation of European ungulate populations. Mam- mal Res. 2017; 62: 209–217. https://doi.org/10.1007/s13364-017-0321-5 38. Ryman N, Baccus R, Reuterwall C, Smith MH. Effective population size, generation interval, and poten- tial loss of genetic variability in game species deer under different hunting regimes. Oikos; 1981; 36: 257–266. https://doi.org/10.2307/3544622 39. Kottek M, Grieser J, Beck C, Rudolf B, Rubel F. World map of the Köppen-Geiger climate classification updated. Meteorol Zeitschrift. 2006; 15: 259–263. https://doi.org/10.1127/0941-2948/2006/0130 PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 16 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia 40. Carvalho J, Büntgen U, Pettorelli N, Mentaberre G, Olivé-Boix X, Eizaguirre O, et al. Habitat and Har- vesting Practices Influence Horn Growth of Male Ibex. J Wildl Manage. 2020; 84, 651–665. https://doi. org/10.1002/jwmg.21830 41. Surroca GP. La Cabra Salvatge Al Montgrı́. Seguiment de La Població de Capra Pyrenaica Del Montgrı́ (2013–2016). Annals de l’Institut d’Estudis Empordanesos. 2018; 49: 249–264. 42. Farr TG, Kobrick M. Shuttle radar topography mission produces a wealth of data. Eos, Trans Am Geo- phys Union. 2000; 81, 583–585. https://doi.org/10.1029/EO081i048p00583 43. Bruford MW, Hanotte O, Brookfield JFY, Burke T. Single-locus and multilocus DNA fingerprint. In: Hoel- zel AR, editor. Molecular Genetic Analysis of Populations: A Practical Approach. 1st ed. Oxford, England: IRL Press; 1992. pp. 225–270. 44. Irwin DM, Kocher TD, Wilson AC. Evolution of the cytochrome b gene of mammals. J Mol Evol. 1991; 32: 128–144. https://doi.org/10.1007/BF02515385 PMID: 1901092 45. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genet- ics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28: 2731–2739. https://doi.org/10.1093/molbev/msr121 PMID: 21546353 46. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 199422: 4673–4680. https://doi.org/10.1093/nar/22.22.4673 PMID: 7984417 47. Leigh JW, Bryant D. PopART: Full-feature software for haplotype network construction. Methods Ecol Evol. 2015; 6:1110–1116. https://doi.org/10.1111/2041-210X.12410 48. Librado P, Rozas J. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bio- informatics. 2009; 25: 1451–1452. https://doi.org/10.1093/bioinformatics/btp187 PMID: 19346325 49. Pompanon F, Bonin A, Bellemain E, Taberlet P. Genotyping errors: causes, consequences and solu- tions. Nat Rev Genet. 2005; 6: 847–859. https://doi.org/10.1038/nrg1707 PMID: 16304600 50. Pritchard SD. Inference of Population Structure Using Multilocus Genotype Data. Genetics. 2000; 155: 945–959. https://doi.org/10.1093/genetics/155.2.945 PMID: 10835412 51. Earl DA, VonHoldt BM. STRUCTURE HARVESTER: a website and program for visualizing STRUC- TURE output and implementing the Evanno method. Conserv Genet Resour. 2012; 4: 359–361 https:// doi.org/10.1007/s12686-011-9548-7 52. Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol. 2005; 14: 2611–2620. https://doi.org/10.1111/j.1365- 294X.2005.02553.x PMID: 15969739 53. Wright S. The relation of livestock breeding to theories of evolution. J Anim Sci. 1978; 46, 1192–1200. https://doi.org/10.2527/jas1978.4651192x 54. Peakall R, Smouse PE. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teach- ing and research–an update. Bioinformatics. 2012; 28: 2537–2539. https://doi.org/10.1093/ bioinformatics/bts460 PMID: 22820204 55. Excoffier L, Lischer HEL. Arlequin suite ver 3.5: A new series of programs to perform population genet- ics analyses under Linux and Windows. Mol Ecol Resour. 2010; 10: 564–567. https://doi.org/10.1111/j. 1755-0998.2010.02847.x PMID: 21565059 56. Garza J, Williamson E. Detection of Reduction in Population Size Using Data from Microsatellite Loci. Mol Ecol. 2001; 10: 305–318 https://doi.org/10.1046/j.1365-294x.2001.01190.x https://doi.org/10. 1046/j.1365-294x.2001.01190.x PMID: 11298947 57. La Haye MJJ, Reiners TE, Raedts R, Verbist V, Koelewijn HP. Genetic monitoring to evaluate reintro- duction attempts of a highly endangered rodent. Conserv Genet. 2017; 18: 877–892. https://doi.org/10. 1007/s10592-017-0940-z 58. Angelone-Alasaad S, Biebach I, Pérez JM, Soriguer RC, Granados JE. Molecular analyses reveal unexpected genetic structure in Iberian ibex populations. PloS One. 2017; 12: e0170827. https://doi. org/10.1371/journal.pone.0170827 PMID: 28135293 59. Ropiquet A, Hassanin A. Hybrid origin of the Pliocene ancestor of wild goats. Mol Phylogenet Evol. 2006; 41: 395–404. https://doi.org/10.1016/j.ympev.2006.05.033 PMID: 16837213 60. Garcı́a–González R, Herrero J, Nores C. The names of southwestern European goats: is Iberian ibex the best common name for Capra pyrenaica? Anim Biodiv Conserv. 2020; 44.1: 1–16, https://doi.org/ 10.32800/abc.2021.44.0001 61. Muller LI, Murrow JL, Lupardus JL, Clark JD, Yarkovich JG, Stiver WH, et al. Genetic structure in elk persists after translocation. J Wildl Manage. 2018; 82: 1124–1134. https://doi.org/10.1002/jwmg.21482 PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 17 / 18 PLOS ONE Genetic signature of the Iberian ibex in Catalonia 62. Grossen C, Guillaume F, Keller LF, Croll D. Purging of highly deleterious mutations through severe bot- tlenecks in Alpine ibex. Nat Commun. 2020; 11: 1–12. https://doi.org/10.1038/s41467-020-14803-1 PMID: 32081890 63. Márquez FJ, Granados JE, Caruz A, Soriguer RC, Fandos P, Cano-Manuel FJ, et al. Genetic diversity of cytochrome b in Iberian ibex from Andalusia. Mamm Biol. 2020; 100: 675–684. https://doi.org/10. 1007/s42991-020-00077-z 64. Velickovic N, Ferreira E, Djan M, Ernst M, Vidakovic DO, Monaco A, et al. Demographic history, current expansion and future management challenges of wild boar populations in the Balkans and Europe. Heredity 2016; 117: 348–357. https://doi.org/10.1038/hdy.2016.53 PMID: 27436523 65. Gregório I, Barros T, Pando D, Morante J, Fonseca C, Ferreira E. Paths for colonization or exodus? New insights from the brown bear (Ursus arctos) population of the Cantabrian Mountains. PLoS ONE 2020; 15: e0227302. https://doi.org/10.1371/journal.pone.0227302 PMID: 32004321 66. Barros T, Ferreira E, Rocha RG, Brotas G, Carranza J, Fonseca C, et al. The Multiple Origins of Roe Deer Populations in Western Iberia and Their Relevance for Conservation. Animals 2020; 10: 2419. https://doi.org/10.3390/ani10122419 PMID: 33348694 67. Moritz C. Defining ‘evolutionarily significant units’ for conservation. Trends Ecol Evol. 1994; 9: 373–375. https://doi.org/10.1016/0169-5347(94)90057-4 PMID: 21236896 PLOS ONE | https://doi.org/10.1371/journal.pone.0269873 September 21, 2022 18 / 18