Received: 8 April 2020 | Revised: 7 June 2021 | Accepted: 2 August 2021 DOI: 10.1111/eva.13299 ORIGINAL ARTICLE Assessing the levels of intraspecific admixture and interspecific hybridization in Iberian wild goats (Capra pyrenaica) Tainã Figueiredo Cardoso1 | María Gracia Luigi-­Sierra1 | Anna Castelló1,2 | Betlem Cabrera1,2 | Antonia Noce3 | Emilio Mármol-­Sánchez1 | Ricardo García-­González4 | Alberto Fernández-­Arias5 | José Luis Alabart6 | Jorge Ramón López-­Olvera7 | Gregorio Mentaberre8 | José Enrique Granados-­Torres9 | Jesús Cardells-­Peris10 | Antonio Molina11 | Armand Sànchez1,2 | Alex Clop1 | Marcel Amills1,2 1 Department of Animal Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-­IRTA-­UAB-­UB, Campus de la Universitat Autònoma de Barcelona, Bellaterra, Spain 2 Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, Bellaterra, Spain 3 Leibniz-­Institute for Farm Animal Biology (FBN), Dummerstorf, Germany 4 Instituto Pirenaico de Ecología (IPE-­C SIC), Jaca, Spain 5 Servicio de Caza y Pesca, Departamento de Agricultura, Ganadería y Medio Ambiente, Gobierno de Aragón, Zaragoza, Spain 6 Unidad de Producción y Sanidad Animal, Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), Instituto Agroalimentario de Aragón -­IA2 (CITA-­Universidad de Zaragoza), Gobierno de Aragón, Zaragoza, Spain 7 Wildlife Ecology & Health Group and Servei d’Ecopatologia de Fauna Salvatge (SEFaS), Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra, Spain 8 Wildlife Ecology & Health Group and Departament de Ciència Animal, Escola Tècnica Superior d’Enginyeria Agraria (ETSEA), Universitat de Lleida (UdL), Lleida, Spain 9 Wildlife Ecology & Health Group and Espacio Natural Sierra Nevada, Granada, Spain 10 SAIGAS (Servicio de Análisis, Investigación y Gestión de Animales Silvestres) and Wildlife Ecology & Health Group, Faculty of Veterinary, Universidad Cardenal Herrera-­CEU, CEU Universities, Valencia, Spain 11 Departamento de Genética, Universidad de Córdoba, Córdoba, Spain Correspondence Marcel Amills, Department of Animal Abstract Genetics, Centre for Research in Iberian wild goats (Capra pyrenaica, also known as Iberian ibex, Spanish ibex, and Agricultural Genomics (CRAG), CSIC-­IRTA-­ UAB-­UB, Campus Universitat Autònoma Spanish wild goat) underwent strong genetic bottlenecks during the 19th and 20th de Barcelona, 08193 Bellaterra, Spain. centuries due to overhunting and habitat destruction. From the 1970s to 1990s, aug- Email: marcel.amills@uab.cat mentation translocations were frequently carried out to restock Iberian wild goat pop- Funding information ulations (very often with hunting purposes), but they were not systematically planned Ministerio de Economía y Competitividad, Grant/Award Number: AGL2016-­ or recorded. On the other hand, recent data suggest the occurrence of hybridization 76108-­R and SEV-­2015-­0533; European events between Iberian wild goats and domestic goats (Capra hircus). Augmentation Regional Development Fund (FEDER)/ Ministerio de Ciencia e Innovación—­ translocations and interspecific hybridization might have contributed to increase Agencia Estatal de Investigación, Grant/ the diversity of Iberian wild goats. With the aim of investigating this issue, we have Award Number: PID2019-­105805RB-­I 00; CERCA Programme/Generalitat de genotyped 118 Iberian wild goats from Tortosa-­Beceite, Sierra Nevada, Muela de Catalunya; Spanish Ministry of Economy Cortes, Gredos, Batuecas, and Ordesa and Monte Perdido by using the Goat SNP50 and Competitiveness for the Center of This is an open access article under the terms of the Creat​ive Commo​ns Attri​bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd. 2618 |  wileyonlinelibrary.com/journal/eva Evolutionary Applications. 2021;14:2618–2634. CARDOSO et al. | 2619 Excellence Severo Ochoa 2020–­2023, Grant/Award Number: CEX2019-­ BeadChip (Illumina). The analysis of genotypic data indicated that Iberian wild goat 000902-­S , CGL2012-­4 0043-­C02-­01, populations are strongly differentiated and display low diversity. Only three Iberian CGL2012-­4 0043-­C02-­02 and CGL2016-­ 80543-­P; Spanish Ministry of Education, wild goats out from 118 show genomic signatures of mixed ancestry, a result con- Grant/Award Number: BES-­C-­2017-­0 024 sistent with a scenario in which past augmentation translocations have had a limited and FPU15/01733; CAPES Foundation-­ Coordination of Improvement of Higher impact on the diversity of Iberian wild goats. Besides, we have detected eight Iberian Education, Ministry of Education of the wild goats from Tortosa-­Beceite with signs of domestic goat introgression. Although Federal Government of Brazil rare, hybridization with domestic goats could become a potential threat to the genetic integrity of Iberian wild goats; hence, measures should be taken to avoid the presence of uncontrolled herds of domestic or feral goats in mountainous areas inhabited by this iconic wild ungulate. KEYWORDS Capra pyrenaica, genetic diversity, high-­density SNP arrays, Iberian ibex, introgression 1 | I NTRO D U C TI O N native to the Iberian Peninsula which inhabits mountainous and rocky areas and feeds on shrubs, bushes, and grasses (Acevedo The genetic diversity of wild animal species has been modified by & Cassinello, 2009a; Granados et al., 2001, 2007). According to multiple factors related with human activity. Habitat destruction and Cabrera (1911, 1914), in the early 20 th century, there were four fragmentation combined with overhunting, climate change, and the Iberian wild goat subspecies, namely C. p. hispanica (CPH, south and introduction of invasive animals and plants have caused severe re- east of the Iberian Peninsula), C. p. victoriae (CPV, center and north- ductions in the genetic diversity and fitness of wild species, leading, west of the Iberian Peninsula), C. p. lusitanica (CPL, Galicia and north in some cases, to their extinction (Fahrig, 2003; Pimm et al., 2014). of Portugal, extinct in the 19th century), and C. p. pyrenaica (CPP, Human activities, either intentionally or not, might have also contrib- Pyrenees), which became extinct two decades ago (García-­González uted to increase the genetic diversity of wild species. Translocation, et al., 2021). The Iberian wild goat was abundant during the Middle which implies the deliberate release of animals from one location Ages but it experienced a sustained and strong demographic de- to another with the goal of reinforcing, introducing, or reintroduc- cline during the 19–­20 th centuries as a consequence of the growing ing a species within its indigenous range (Griffith et al., 1989), can hunting pressure (particularly during the 1940s–­1970s) and habitat be effective in enhancing genetic diversity (Chipman et al., 2008). loss and fragmentation (García-­González, 2011; Pérez et al., 2002). Obviously, it can also have adverse effects on resident animals at re- The strong reduction of genetic diversity produced by this process lease sites, including the spread of diseases that could cause drastic of demographic contraction has been previously reported (Amills population bottlenecks (Chipman et al., 2008). Moreover, increased et al., 2004; Angelone et al., 2018). Strong signs of genetic differ- stress and mortality of released animals may limit the potential ben- entiation among Iberian wild goat populations due to reproductive efits of translocations (Chipman et al., 2008). isolation and substantial genetic drift associated with severe genetic Hybridization between wild animals and livestock herds, which bottlenecks have also been described (Amills et al., 2004; Angelone is largely unintentional, can also increase the genetic diversity of et al., 2018). In the last decades, the creation of a network of national wild species, by introducing completely new alleles and genotypes, parks and protected areas, the absence of predators, reforestation at the expense of decreasing adaptive potential due to outbreeding policies, and the progressive abandonment of rural activities have depression and behavioral changes, for example, reduced predator contributed to the recovery and subsequent expansion of Iberian and human avoidance (Barbato et al., 2017; Goedbloed et al., 2013). wild goats (Acevedo & Cassinello, 2009b). Even in the cases in which interspecific hybridization is a rare event, Iberian wild goats constitute a valuable model to explore the it can lead to long-­lasting changes in the genomic architecture of the impact of translocation and hybridization on genetic diversity. affected wild species (Schwenk et al., 2008). While reduction of ge- Restocking/repopulation translocations have favored gene flow be- netic variation mediated by humans has been documented in wild an- tween distant populations (Acevedo & Cassinello, 2009a; Crampe, imals and its consequences have been thoroughly assessed (Abascal 1991). The most comprehensive report to date analyzing the vari- et al., 2016; Grossen et al., 2020), few reports have addressed the ability of 333 Iberian wild goats with a panel of 30 microsatellites potential impact of translocation and interspecific hybridization on did not show any evidence of genetic signatures typically associated the genetic diversity of wild species (Shackleton, 1997). with translocations and population admixture (Angelone-­Alasaad The Iberian wild goat (Capra pyrenaica, also known as Iberian et al., 2017). However, this outcome might be caused by the limited ibex, Spanish ibex, and Spanish wild goat) is a wild goat ungulate resolution of the microsatellite panel employed in such study. 2620 | CARDOSO et al. In addition, the impact of domestic goat introgression on the ge- reported in any of the five populations mentioned before. While netic diversity of Iberian wild goats is not well known yet. Alasaad the cohabitation of domestic goats and Iberian wild goats has been et al. (2012) reported the mating of one captive Iberian wild goat described as a risk factor for the transmission of certain diseases male with domestic goats and the obtaining of viable offspring. (Astorga Márquez et al., 2014), to the best of our knowledge the Hybrids between Alpine ibexes (Capra ibex) and domestic goats have spatial proximity between wild and domestic goat populations has been also described (Giacometti et al., 2004). Moreover, Angelone not been thoroughly investigated in Spain. Part of the translocations et al. (2018) reported the segregation, in Iberian wild goats from four among Iberian wild goat populations have been documented, and Southern Spain locations (Sierras de Cazorla, Segura and las Villas such information can be found in Figure 1. Natural Park, El Hosquillo in Serranía de Cuenca Natural Park, Sierra del Mencal, and Cabañeros National Park), of one major histocom- patibility complex class II DRB1 allele, MHC DRB1*7, identical to an- 2.2 | Isolation of genomic DNA from Iberian wild other one reported in domestic goats. They hypothesized that this goat samples result could be due to either the maintenance of ancient polymor- phisms by balancing selection or, alternatively, introgressions from We used two different batches of CPV and CPH samples. The first domestic goats through interspecific hybridization, and they con- batch was reported by Jiménez et al. (1999), as well as by Amills et al. cluded that this matter should be clarified in future (Angelone et al., (2004), and consisted of (1) CPV: liver samples from seven and 14 2018). By using a high-­throughput single nucleotide polymorphism Iberian wild goats from Batuecas and Gredos, respectively; (2) CPH: (SNP) genotyping approach, we expect to answer this question and blood samples from 27 and five Iberian wild goats from Tortosa-­ find out whether domestic goat introgression has had a significant Beceite and Sierra Nevada, respectively, and five liver samples from impact on the genetic diversity of Iberian wild goats. Iberian wild goats inhabiting Muela de Cortes. A second batch in- In summary, the main goal of the current work is to investi- cluded blood or solid tissue (muscle, spleen, or ear cartilage) samples gate the impact of intraspecific (translocations) and interspecific from 59 CPH individuals from Tortosa-­Beceite (N = 43, 2010–­2019), (hybridization between wild and domestic goats) gene flow on the Muela de Cortes (N = 7, 2017–­2019), and Sierra Nevada (N = 9, diversity of Iberian wild goats by genotyping 118 individuals with a 2006–­2014). Finally, one muscle sample from one of the last CPP SNP assay. representatives was collected in the location of Ordesa and Monte Perdido in 1996, before the extinction of this subspecies. Genomic DNA was isolated from blood samples as previously reported (Amills 2 | M ATE R I A L S A N D M E TH O DS et al., 1996), while a standard phenol–­chloroform protocol was used to purify genomic DNA from solid tissues (Sambrook & Russell, 2.1 | Study areas and historical description of 2006). The second batch of samples and the CPP sample had not populations been analyzed in previous genetic studies. In this work, we have investigated three CPH populations (Tortosa-­ Beceite, Muela de Cortes, and Sierra Nevada), two CPV populations 2.3 | Genotyping with the Goat SNP50 BeadChip (Gredos and Batuecas) and one CPP individual (Ordesa and Monte (Illumina) Perdido). All these Iberian wild goat populations underwent strong bottlenecks during the 19th and 20 th centuries but, as shown in Following the instructions of the manufacturer, we genotyped with Table 1, during the last six decades they have experienced an ac- the Goat SNP50 BeadChip from Illumina (Tosser-­Klopp et al., 2014) celerated demographic expansion due to the lack of predators, samples from 118 Iberian wild goats. The Goat SNP50 BeadChip human depopulation in rural areas, and protected status (Acevedo & (Illumina) includes 53,348 SNPs with an approximately uniform Cassinello, 2009b). Hybridization with domestic goats has not been distribution in the caprine genome (Tosser-­Klopp et al., 2014). The TA B L E 1 Current and past sizes of Iberian wild goat population investigated in the current work Sspa Population Past size Current size References CPH Tortosa-­Beceite 450 (1966) ~4000 (2008) Casanovas-­Urgell et al. (2008) and Angelone et al. (2018) Sierra Nevada 450 (1960) 15,000 (2020) Angelone et al. (2018) Muela de Cortes ? 1400 (2014) Tinoco-­Torres et al. (2014) CPV Gredos 10 (1905) ~13,000 (2018) Angelone et al. (2018) Batuecas 200 (1980) 900 (2002) Pérez et al. (2002) CPP Ordesa and Monte Perdido 6–­14 (1990) Extinct (2000) García-­González and Herrero (1999) a Iberian wild goat subspecies: CPH = C. p. hispanica; CPV = C. p. victoriae; CPP = C. p. pyrenaica. CARDOSO et al. | 2621 F I G U R E 1 Documented translocations of Iberian wild goats: This description of Iberian wild goat translocations should be considered as partial because many historical translocations went unrecorded. With regard to Capra pyrenaica victoriae (CPV), 12 specimens from Gredos (Ávila) were translocated to Panticosa (Huesca, 1960s). A few years later, only 2 males and 4 females were left. While one of the males was transferred to Gredos and the other one died, the whereabouts of the females are unknown although it is highly unlikely that they reached Ordesa (García-­González, 1989). Iberian wild goats from Gredos were also translocated to Las Batuecas (Salamanca, 1970s), Regional Hunting Reserve of Riaño (León), and La Pedriza (current Sierra de Guadarrama National Park, Madrid-­Segovia) during the 90s. Before 1995, the introduction of specimens from Gredos to private farms in the Montes de Toledo is also documented (Acevedo et al., 2011). There were also translocations of individuals from Batuecas to Sierra de Guadarrama (Madrid) and to Riaño (León). The Natural Park of Invernadero also received Iberian wild goats from Batuecas (Prada & Herrero, 2013) and possibly from Gredos (Crampe, 2020), although this latter translocation event is not completely confirmed. An unsuccessful reintroduction attempt was made, between 1957 and 1962, in the Covadonga National Park (Asturias) with 14 individuals from Gredos and Cazorla (Jaen), as reported by Arenzana et al. (1964). At the end of the 90s, translocations from Riaño to Los Ancares (León) and, in 2005–­2007, to Mampodre (León) are also known. In 2018, CPV individuals from Guadarrama were transferred to the Pyrenees National Park and the Ariège Pyrenees Regional Park, both in France, and to the Valle de Arán (Lleida). Concerning Capra pyrenaica hispanica (CPH), Iberian wild goats from Cazorla were taken to private farms in the Montes de Toledo, Sierra Morena (Jaén-­Ciudad Real), Serranía de Cuenca (Hosquillo), Sierra de Guara (Huesca), Sierra de Baza (Granada), and Muela de Cortes (Valencia) during the 1960s and 1970s. More recently animals from Cazorla were transferred to an enclosure in the Serra del Montgrí (Girona), but they escaped and formed a population of more than one hundred specimens. At the end of the nineties, Iberian wild goats were taken from Tortosa to Montserrat (Barcelona). Finally, CPH from Sierra Nevada were introduced in the Serranía de Ronda (Málaga) and in the Sierra de Baza (Granada) during the 1970s and 1980s, and to Sierra de Mágina (Jaén). CPH from Sierra Nevada were also brought to enclosures in Almuñecar (Granada), Garcipollera (Huesca), and Cumbres Mayores (Huelva) at the end of the 90s. Finally, during the first decade of this century, Iberian wild goats from the Sierra Nevada have been brought to the Moratalla and Caravaca mountains in the Murcia region and Sierra de Orce in the north of the province of Granada GenomeStudio software (Illumina) was employed to call genotypes GenomeStudio, genotypes can be visualized as Genoplots. For each and to assess sample and genotype qualities by using a cluster file sample, genotypes are called by their signal intensity (norm R) and provided by the International Goat Genome Consortium (clus- allele frequency (Norm Theta) relative to canonical cluster positions ter file: GoatIGGC_Cons_60k.egt, Tosser-­Klopp et al., 2014). In for the SNP marker under study (https://www.illum​ina.com/techn​ 2622 | CARDOSO et al. iques/​micro​array​s/array​-­data-­analy​sis-­exper​iment​al-­desig​n/genom​ allele information from retrieved SNPs (–­cluster –­mds-­plot 2 eigen- estud​io.html). decomp eigvals). We did four MDS analyses: (1) only Iberian wild Preprocessing and filtering of data were carried out with the goat populations (1001 SNPs, N = 118); (2) only Iberian wild goat PLINK v.1.7 software (Purcell et al., 2007). More specifically, markers populations (N = 118) with a data set of 894 SNPs, that is, 1001 SNPs with a GenTrain score (Illumina descriptive statistic related to clus- minus the SNPs with missing values in the CPP sample (the individual tering quality) lower than 0.8, unmapped SNPs, as well as SNPs that with the highest genotype missingness rate). The reason for doing mapped to the X chromosome on the goat reference genome assem- this is that MDS analyses tend to "locate" samples close to the center bly Capra hircus—­ARS1 (Bickhart et al., 2017, https://www.ensem​ when missingness is high, so we wanted to test whether this circum- bl.org/Capra_hircu​s/) and those with a minimum allele frequency stance could affect our results; (3) Iberian wild goat and domestic (MAF) lower than 0.01 (-­maf 0.01) were filtered out. Markers with an goat populations (1001 SNPs, N = 118 Iberian wild goats, N = 50 individual missingness rate with more than 50% missing genotypes domestic goats); (4) eight hybrid Iberian wild goats from Tortosa-­ SNPs (–­mind 0.5) and SNP with a missingness across samples greater Beceite and 50 domestic goats (21,621 SNPs). MDS plots were built than 1% (–­geno 0.01) were also removed. After applying these filter- in R software by using the ggplot2 package (Wickham, 2016). ing criteria, 21,621 SNPs were retained for genetic analyses. Observed (Ho) and expected (He) heterozygosities, as well as the To compare the diversity of Iberian wild goats and domestic inbreeding coefficient Fhat2, were calculated by using the PLINK goats, we used a previously published caprine data set correspond- v.1.7 software (Purcell et al., 2007) and the data set of 1001 SNPs. We ing to 50 domestic goats (ten individuals per breed) from Northern chose Fhat2 as an estimate of inbreeding because, in a previous study Spain (Bermeya and Blanca de Rasquera) and Southern Spain focused on domestic goats, this statistic showed a high correlation (Florida, Malagueña, and Murciano-­Granadina) typed with the Goat (r = 0.88, p-­value = 1.00E-­0 4) with FROH (Cardoso et al., 2018). In SNP50 BeadChip (Illumina) by Manunza et al. (2016). contrast with other inbreeding coefficients, Fhat2 can take negative values (when the count of observed homozygotes is lower than the expected count of homozygotes) because it is not defined as a prob- 2.4 | Population genetics analyses ability but as an excess of homozygosity-­based inbreeding estimate (Purcell et al., 2007). The –­hardy command was used to compute Ho The majority of the 21,621 SNPs which passed the filtering criteria and He, while the –­ibc command was used to estimate the Fhat2 co- only segregated in eight Iberian wild goats from Tortosa-­Beceite, efficient. Nucleotide diversity was computed for each population on probably because of their introgression by domestic goats (see a per-­site basis (π, command: –­site-­pi) using the VCFtools software Section 3). Given that most Iberian wild goats were monomorphic for (Danecek et al., 2011). Confidence intervals (CI) for each parameter this set of SNPs (Figure S1), we generated a second set of SNPs by ex- were calculated according to the following formula: cluding the eight individuals referred above and considering the same filtering criteria defined in the previous section. After this, a set of CI = X ± 1.96*SE (1) 1001 SNPs was obtained. Population genetics analyses were based on both sets of SNPs depending on their goals. The analyses target- where X is the sample mean of the parameter for each population, 1.96 ing specifically the eight putative hybrid individuals were carried out is the Z-­score corresponding to a 95% confidence interval, and SE is the with the set of 21,621 SNPs, while analyses comprising the eight hy- standard error of the mean (Sim & Reid, 1999). brid and the 110 non-­hybrid Iberian wild goats were based on the set Genome-­wide identity by descent (IBD) between pairs of sam- of 1001 SNPs (because the remaining 20,620 SNPs are monomorphic ples was estimated with the PI-­HAT coefficient, which describes the in the non-­hybrid individuals so they cannot be used). For the sake of probability of sharing 0, 1, or 2 alleles IBD by pairs of individuals clarity, the dataset used for each one of the analysis carried out in our from the same homogeneous random-­mating population (Purcell study is specified below. We considered as putative hybrids the eight et al., 2007). Heatmap plots were built in R software by using the individuals that did not collapse in the MDS plot shown in Figure S1 ggplot2 package (Wickham, 2016). and that, in addition, showed signatures of domestic goat introgres- sion in the admixture analyses (see below). The remaining 110 Iberian wild goats were considered as non-­hybrids, although we cannot com- 2.6 | Examining the ancestry of Iberian wild goats pletely rule out the possibility that a number of them may carry a with admixture domestic goat genetic component not detectable with our methods. We used the Admixture software (Alexander et al., 2009) to cal- culate maximum likelihood estimates of individual ancestries from 2.5 | Multidimensional scaling and estimation of SNP data generated with the Goat SNP50 BeadChip (Illumina) con- diversity parameters in Iberian wild goats sidering Iberian wild goat and domestic goat populations (1001 SNPs, N = 118 Iberian wild goats, N = 50 domestic goats). A cross-­ The PLINK v1.7 software (Purcell et al., 2007) was used to carry out validation fold at 5% and a block bootstrap with 2000 iterations were sample clustering based on the multidimensional scaling (MDS) of used to calculate SE for admixture proportions. Confidence intervals CARDOSO et al. | 2623 for admixture proportions were inferred with Equation 1. The opti- SNP markers were polymorphic (MAF > 0.01) in the non-­hybrid mal K-­value was the one with the lowest cross-­validation error, as Iberian wild goats as well as in the eight hybrids individuals from determined with the method of Alexander and Lange (2011). The Tortosa-­Beceite. In contrast, 20,620 markers were polymorphic only Pophelper package for R (Francis, 2017) was used to process the in the eight putative hybrid individuals. output results from the Admixture analysis. 3.2 | Population structure and diversity of Iberian 2.7 | Performance of an f3 test of admixture in wild goats eight putative hybrid Iberian wild goats We have investigated the population structure of 118 Iberian wild In the Admixture analysis, eight individuals from Tortosa-­Beceite goats with 1001 markers segregating in both non-­hybrid and puta- showed genomic signatures of introgression by domestic goats. We tive hybrid individuals. We did not use the remaining 20,620 mark- used the qp3pop program, included in the ADMIXTOOLS software ers because this would have caused a very strong bias in our package (Patterson et al., 2012), and the set of 21,621 SNPs to carry diversity estimates since all of them were monomorphic in the ma- out a 3-­population test in the form f3(admixed Tortosa-­Beceite; jority (N = 110) of Iberian wild goats. As expected, the MDS plot Tortosa-­Beceite, Malagueña), that is, we selected the non-­admixed based on the information provided by these 1001 SNPs showed Tortosa-­Beceite and Malagueña individuals as representatives of no signs of tight aggregation of Iberian wild goats in a single loca- Iberian wild goats and domestic goats, respectively. In the absence tion (Figure 3a). Instead, we observed the existence of three main of hybridization, f3 has a non-­negative mean, while a negative mean clusters comprising samples from (1) Tortosa-­Beceite, (2) Sierra is expected in the case of hybridization. The statistical significance Nevada and Muela de Cortes, and (3) Gredos and Batuecas, which of the result can be assessed by means of a Z-­score. In order to evalu- showed a close correspondence with the geographic distribution ate the robustness of the f3 results vs the choice of particular source of these populations (Figure 1). We also observed one individual populations, we did two additional analyses in the form f3(admixed from Sierra Nevada and two individuals from Tortosa-­Beceite lo- Tortosa-­Beceite; Gredos, Bermeya) and f3(admixed Tortosa-­Beceite; cated close to the Muela de Cortes cluster. The only representative Batuecas, Malagueña). of the CPP extinct population was also placed near to the Muela de Cortes cluster. We made a second analysis excluding 107 SNPs that were missing in the CPP sample (Figure 3b) to make sure that the 3 | R E S U LT S relatively high missingness rate of the CPP sample was not affect- ing its position in the MDS. This analysis, based on 894 SNPs, was 3.1 | About the polymorphism of domestic goat completely consistent with the one shown in Figure 3a. We made a SNP markers in Iberian wild goats third MDS analysis, also based on 1001 SNPs, and comprising both Iberian wild goat and domestic goat populations (Figure 3c). This The call rates of Iberian wild goats genotyped with the Goat SNP50 analysis evidenced eight Iberian wild goats from Tortosa-­Beceite BeadChip (Illumina) ranged between 0.94 and 0.99, with an average which were separated from the Tortosa-­Beceite cluster. These in- of 0.98 ± 0.04. The only exception was the CPP DNA extracted from dividuals correspond to the eight putative Iberian wild goat × do- a muscle sample, which was considerably degraded and displayed mestic goat hybrids which did not collapse in the MDS depicted in a call rate of 0.86. In Figure 2, we show four representative exam- Figure S1 (see previous section). Finally, we made a fourth analysis ples of the Genoplots generated with the GenomeStudio software. focused on the eight individuals mentioned above and 50 domes- The analysis of the Goat SNP50 BeadChip (Illumina) genotype data tic goats (Figure 3d) which revealed that these eight individuals are revealed the existence of 21,621 SNPs arrayed in the Goat SNP50 relatively close to the Malagueña goats, although such result should BeadChip (Illumina) that segregated (MAF > 0.01) in the 118 Iberian be taken with caution. wild goats under investigation. This high proportion (~40%) of SNPs As expected, the genetic diversity of Iberian wild goats was shared by domestic goats and Iberian wild goats is consistent with lower than that of domestic goats (Table 2). The observed (Ho) the introgression of Iberian wild goats by domestic goats. The MDS and expected (He) heterozygosities were not substantially differ- obtained with the 21,621 SNPs revealed that all Iberian wild goats, ent in the five sampled Iberian wild goat populations (Table 2). The with the exception of eight individuals from Tortosa-­Beceite, col- Batuecas and Sierra Nevada populations showed the lowest Ho and lapsed in a single location of the plot (Figure S1). This outcome is He values while Muela de Cortes and Tortosa-­Beceite had the high- produced by the fact that the majority of Iberian wild goats are est ones. The exclusion of the eight putative hybrid individuals from monomorphic for most of the Goat SNP50 BeadChip (Illumina) mark- the Tortosa-­Beceite population caused ~15% reductions in Ho and ers. The second SNP filtering procedure excluding the eight putative He despite the fact that they just represent 6.8% of the individuals hybrid individuals reported above yielded a drastic reduction of the sampled in this population (Table 2). Iberian wild goats showed lower number of domestic goat SNPs segregating (MAF > 0.01) in Iberian nucleotide diversity (π = 0.181) than domestic goats (π = 0.402). wild goats, that is, from 21,621 to 1001 markers. Thus, these 1001 Consistent with heterozygosity measurements, Sierra Nevada and 2624 | CARDOSO et al. F I G U R E 2 Four representative Genoplots obtained with the GenomeStudio software through the analysis of Goat SNP50 BeadChip (Illumina) data corresponding to Iberian wild goats (yellow) and domestic goats (red, purple, and blue). Genotypes are called for each sample (dots) by taking into account their signal intensity (Norm R, y-­axis) and allelic intensity ratio (Norm Theta, x-­axis) relative to canonical cluster positions (dark shading). (a) Genoplot showing the genotypes obtained for SNP 58771. It can be seen that this SNP is successfully called and it segregates in both the Iberian wild goat and domestic goat populations. (b) Genoplot showing the genotypes obtained for SNP 27056. This SNP is successfully called and it segregates in domestic goats (two genotypes are called) but not in Iberian wild goats (a single genotype is called). The intensity of the signal (norm R) is similar in domestic goats and Iberian wild goats. (c) Genoplot showing the genotypes obtained for SNP 2756. This SNP is successfully called and it segregates in domestic goats (three genotypes are called) but not in Iberian wild goats (a single genotype is called and with a weaker intensity than the one corresponding to domestic goats). (d) Genoplot showing the genotypes obtained for SNP 52788. This SNP is not successfully called in Iberian wild goats (norm R is much lower than that observed in domestic goats) Batuecas populations had the lowest nucleotide diversity values domestic goat breeds (FST < 0.072), with the only exception of the (π ≅ 0.160) among the investigated populations. Gredos and Batuecas populations which had a weak genetic differ- As shown in Table 2, the inbreeding Fhat2 coefficient calculated entiation (FST = 0.035). The magnitude of the genetic differentiation with PLINK v.1.7 (Purcell et al., 2007) reached high values in the did not correlate well with the assignment of populations to the CPH Sierra Nevada (Fhat2 = 0.590) and Batuecas (Fhat2 = 0.539) popu- or CPV subspecies. For instance, the FST coefficient between the lations, while in the Muela de Cortes population the magnitude of Sierra Nevada and Tortosa-­Beceite populations (FST = 0.379), which the Fhat2 coefficient was 20%–­28% lower (Fhat2 = 0.442). The values belong to the same CPH subspecies, was similar to FST coefficients of the Fhat2 coefficient in the Tortosa-­Beceite population were no- measured between CPH and CPV populations. In addition, genetic tably different depending on whether the eight hybrid individuals differentiation among Spanish domestic goat and non-­hybrid Iberian were included (Fhat2 = 0.516) or not (Fhat2 = 0.593) in the calculation wild goat was higher (FST = 0.337) than between Spanish domestic (Table 2). In contrast, inbreeding Fhat2 coefficients values were close goat and the eight putative hybrid Iberian wild goats (FST = 0.183). to zero in all five domestic goat breeds (Table 2). It could be argued The FST coefficient between the non-­hybrid Iberian wild goat popu- that 1001 SNPs are not enough to obtain reliable estimates of diver- lations and the eight Tortosa-­Beceite hybrids was 0.122. sity parameters. To check this issue, we estimated Ho, He, π, and Fhat2 We also inferred the degree of genome-­wide IBD among Iberian in our data set of 50 domestic goats by using a set of 53,325 SNPs. wild goats (Figure 4a) and among domestic goats (Figure 4b). PI-­HAT By inspecting Table S1, it can be seen that diversity parameters es- coefficients took zero values for most pairwise comparisons among timated with either 53,325 SNPs or 1001 SNPs display fairly con- Iberian wild goats from different populations (except for the Gredos sistent values, with the only exception of Fhat2 coefficients, which vs Batuecas comparison), indicating the absence of relatedness. display negative (Table 2) or close to zero (Table S1) values when In contrast, when these pairwise comparisons were made at the using sets of 1001 SNPs or 53,325 SNPs, respectively. within-­population level (Figure 4a), the degree of genetic similarity As shown in Table 3, genetic differentiation among Iberian wild among individuals increased substantially. In domestic goats, PI-­HAT goat populations (FST > 0.287) was much higher than among Spanish coefficients reached values close to zero for the majority of pairwise CARDOSO et al. | 2625 F I G U R E 3 Multidimensional scaling (MDS) plot of Iberian wild goats. (a) MDS plot including 118 Iberian wild goat samples. This analysis is based on 1001 SNPs from the Goat SNP50 BeadChip (Illumina), (b) MDS of Iberian wild goat individuals. This MDS plot includes 118 Iberian wild goat samples and it is based on 894 SNPs from the Goat SNP50 BeadChip (Illumina), that is, 1001 SNPs minus the SNPs showing missing values in the CPP sample (the individual with the highest genotype missingness rate). We carried out this analysis because we wanted to test whether the centrality of the CPP sample in the MDS is caused by its high missingness rate (MDS analyses tend to "locate" samples close to the center when missingness is high). By comparing a and b, it becomes clear that this is not the case. (c) MDS plot including Iberian wild goat and domestic goat populations (1001 SNPs, N = 118 Iberian wild goats, N = 50 domestic goats), and (d) MDS plot including the eight hybrid Iberian wild goats from Tortosa-­Beceite and domestic goat populations (N = 50). This analysis is based on 21,621 SNPs from the Goat SNP50 BeadChip (Illumina). Iberian wild goat individuals were sampled in Batuecas (N = 7, Capra pyrenaica victoriae), Gredos (N = 14, Capra pyrenaica victoriae), Tortosa-­Beceite (N = 70, Capra pyrenaica hispanica), Muela de Cortes (N = 12, Capra pyrenaica hispanica), and Sierra Nevada (N = 14, Capra pyrenaica hispanica) and National Park of Ordesa and Monte Perdido (CPP, Capra pyrenaica pyrenaica, N = 1). Goats belonged to the Bermeya, Blanca de Rasquera, Florida, Malagueña and Murciano-­Granadina Spanish breeds (N = 10 for each breed) comparisons, and even when they comprised individuals drawn from to the Admixture analysis, two individuals from Tortosa-­Beceite the same breed (Figure 4b). (Tortosa-­Beceite_22 and Tortosa-­Beceite_23) showed evidence of having Sierra Nevada ancestry, while one individual from Sierra Nevada (Sierra_Nevada_9) displayed signs of Muela de Cortes ances- 3.3 | Examining the ancestry of Iberian wild try (Figure 5). We did not calculate f3-­statistics for these three po- goats and detecting genomic signatures of admixture tentially admixed Iberian wild goats because they cannot be reliably estimated with just 1001 SNPs. The results of the Admixture analysis corresponding to 118 Iberian wild goats and 50 domestic goats characterized with 1001 SNPs are shown in Figure 5 (K = 2–­7, K = 7 is the number of clusters with the 3.4 | Performance of an f3 test of admixture in lowest cross-­validation error, Figure S2). The percentages of ances- eight putative hybrid Iberian wild goats try for each one of the Iberian wild goat populations are displayed in Figure S3 for K = 7. In the majority of Iberian wild goat populations, The eight putative hybrids from Tortosa-­Beceite displayed negative admixture was low or non-­detectable (Figure 5 and Figure S3). The f3 values, indicative of admixture between the two Tortosa-­Beceite only exception were Iberian wild goats from Gredos and Batuecas, and Malagueña source populations (Figure 6). The Z-­scores were which showed signs of a common ancestry even at K = 7 (Figure 5). high and significant (Table S2). These results were consistent even The CPP sample also seemed to have different ancestries (Figure 5 when different source populations were selected, for example, with and Figure S3) but in this case, results are not reliable because allelic Gredos and Bermeya, and Batuecas and Malagueña as source popu- frequencies cannot be inferred from a single individual. According lations (Table S3). 2626 | TA B L E 2 Means of diversity parameters (and their 95% confidence intervals) estimated in five Iberian wild goat populations and five domestic goat breeds on the basis of 1001 SNPs typed with the Goat SNP50 BeadChip (Illumina)a Species Population N Ho He π Fhat2 Iberian wild goat Tortosa-­Beceite (without 8 hybrid individuals) 62 0.167 (0.156–­0.178) 0.174 (0.162–­0.186) 0.176 (0.174–­0.177) 0.593 (0.588–­0.598) (Capra pyrenaica) Tortosa-­Beceite (with 8 hybrid individuals) 70 0.194 (0.183–­0.204) 0.198 (0.187–­0.209) 0.199 (0.198–­0.201) 0.516 (0.501–­0.0.530) Muela de Cortes 12 0.202 (0.187–­0.216) 0.192 (0.180–­0.205) 0.211 (0.209–­0.213) 0.442 (0.440–­0.445) Sierra Nevada 14 0.153 (0.140–­0.166) 0.157 (0.145–­0.169) 0.160 (0.150–­0.180) 0.590 (0.570–­0.610) Gredos 14 0.181 (0.168–­0.195) 0.169 (0.157–­0.182) 0.176 (0.174–­0.177) 0.489 (0.487–­0.491) Batuecas 7 0.165 (0.151–­0.179) 0.153 (0.141–­0.165) 0.165 (0.163–­0.166) 0.539 (0.537–­0.541) Total 117 0.177 0.174 0.181 0.528 Domestic goat Bermeya 10 0.404 (0.392–­0.415) 0.385 (0.378–­0.393) 0.406 (0.404–­0.406) −0.184 (−0.186 to −0.182) (Capra hircus) Blanca de Rasquera 10 0.391 (0.380–­0.402) 0.377 (0.369–­0.385) 0.397 (0.396–­0.398) −0.139 (−0.144 to −0.133) Florida 10 0.408 (0.396–­0.420) 0.373 (0.365–­0.382) 0.393 (0.392–­0.394) −0.188 (−0.190 to −0.186) Malagueña 10 0.412 (0.401–­0.424) 0.395 (0.387–­0.402) 0.414 (0.414–­0.416) −0.205 (−0.207 to −0.203) Murciano-­Granadina 10 0.395 (0.384–­0.407) 0.382 (0.374–­0.3990) 0.402 (0.401–­0.403) −0.164 (−0.167 to −0.161) Total/mean 50 0.402 0.382 0.402 −0.176 a The single CPP sample has not been included in this analysis. N = number of individuals, Ho = observed heterozygosity, He = expected heterozygosity, π = nucleotide diversity estimated on a per-­site basis, Fhat2 = excess homozygosity-­based inbreeding coefficient (it takes negative values when the observed homozygote count is lower than the expected homozygote count). CARDOSO et al. CARDOSO et al. | 2627 TA B L E 3 Estimates of FST coefficients between five Iberian wild goat populationsa and between five domestic goat breeds on the basis of 1001 SNPs typed with the Goat SNP50 BeadChip (Illumina) Iberian wild goats Population Batuecas Gredos Muela de Cortes Sierra Nevada Tortosa-­Beceite Batuecas 0.000 Gredos 0.035 0.000 Muela de Cortes 0.390 0.371 0.000 Sierra Nevada 0.465 0.445 0.287 0.000 Tortosa-­Beceite 0.415 0.400 0.306 0.379 0.000 Domestic goats Population Bermeya Florida Malagueña Murciano-­Granadina Blanca de Rasquera Bermeya 0.000 Florida 0.072 0.000 Malagueña 0.047 0.0509 0.000 Murciano-­Granadina 0.054 0.0709 0.034 0.000 Blanca de Rasquera 0.049 0.067 0.044 0.058 0.000 a The single CPP sample has not been included in this analysis. F I G U R E 4 Heatmaps representing genome-­wide identity by descent (IBD) between pairs of samples as estimated with the PI-­HAT coefficient. (a) Heatmap of PI-­HAT coefficients calculated from all Iberian wild goat pairs of samples (CPP is not included in this analysis because it is represented by just 1 sample). Iberian wild goat individuals were sampled in Batuecas (N = 7, Capra pyrenaica victoriae), Gredos (N = 14, Capra pyrenaica victoriae), Tortosa-­Beceite (N = 70, Capra pyrenaica hispanica), Muela de Cortes (N = 12, Capra pyrenaica hispanica), and Sierra Nevada (N = 14, Capra pyrenaica hispanica). (b) Heatmap of PI-­HAT coefficients calculated from all domestic goat pairs of samples. Domestic goats belonged to the Bermeya, Blanca de Rasquera, Florida, Malagueña and Murciano-­Granadina breeds (N = 10 for each breed) 2628 | CARDOSO et al. F I G U R E 5 Admixture analysis of the 118 Iberian wild goats and 50 domestic goats. Each individual is represented by a single column divided into K colored segments, where K is the number of assumed clusters. Populations are separated by white lines. This analysis is based on the information provided by 1001 SNPs. The K-­value with the lowest cross-­validation error is 7. Iberian wild goat individuals were sampled in Batuecas (N = 7, Capra pyrenaica victoriae), Gredos (N = 14, Capra pyrenaica victoriae), Tortosa-­Beceite (N = 70, Capra pyrenaica hispanica), Muela de Cortes (N = 12, Capra pyrenaica hispanica), Sierra Nevada (N = 14, Capra pyrenaica hispanica) and National Park of Ordesa and Monte Perdido (CPP, Capra pyrenaica pyrenaica, N = 1). Goats belonged to the Bermeya, Blanca de Rasquera, Florida, Malagueña and Murciano-­Granadina breeds (N = 10 for each breed) 4 | DISCUSSION the Goat SNP50 BeadChip (Illumina) were consistent with previous estimates obtained by Miller et al. (2012), who reported call rates 4.1 | Reduced diversity in Iberian wild goats of 0.98 when using the Ovine SNP50 BeadChip (Illumina) in wild sheep species. In our study, only 1.8% of the SNPs contained in the In the current work, we have evaluated the applicability of a high-­ Goat SNP50 BeadChip (Illumina) were polymorphic (MAF >0.01) density SNP array, the Goat SNP50 BeadChip (Illumina), to investi- in the 110 non-­hybrid Iberian wild goats. This result was expected gate the diversity of Iberian wild goats. The call rates obtained with because call rates and shared polymorphic sites decrease at linear CARDOSO et al. | 2629 Diversity across the five Iberian wild goat populations studied was similar, a result that is consistent with previous microsatellite data (Amills et al., 2004; Angelone-­Alasaad et al., 2017). These find- ings indicated that the bottlenecks suffered by this species during the 19th and 20 th centuries had widespread effects on its overall genetic variation (Amills et al., 2004; Pérez et al., 2002). Moreover, Fhat2 coefficients reached values of 0.442–­0.593 in the Iberian wild goats, while in the domestic goats they were close to zero (Table 2), a result consistent with previous reports (Cardoso et al., 2018). The PI-­ HAT values calculated in pairwise comparisons were also substan- tially higher in Iberian wild goats than in domestic goats, reflecting a significant proportion of IBD between pairs of Iberian wild goats coming from the same population (Figure 4a). As a reference, the PI-­HAT values estimated at the within-­population level in domestic goats (Figure 4b) were comparable to those reported in domestic sheep from Switzerland (Burren et al., 2014) as well as in domestic goats from South Africa (Visser et al., 2016). As we have discussed previously, it could be argued that as- certainment bias might have distorted to some extent the estima- F I G U R E 6 Measurement of f3-­statistics. This analysis has been tion of Fhat2 and PI-­HAT coefficients in Iberian wild goats. Indeed, carried out in the eight Iberian wild goats from Tortosa-­Beceite Angelone-­Alasaad et al. (2017) reported inbreeding FIS coefficients showing signatures of domestic goat introgression in the Admixture analysis. We computed f3-­statistics of the form f3(hybrid Tortosa-­ close to zero in Iberian wild goat populations from Maestrazgo, Beceite; Tortosa-­Beceite, Malagueña) with the set of 21,621 SNPs. Sierra Nevada, and Gredos. Similarly, Bozzuto et al. (2019) described Error bars represent the standard errors. It can be seen that all FIS coefficients close to zero in Alpine ibex populations, but they at- eight f3-­statistics are negative, thus providing evidence that the tributed this finding to the inherent lack of power of this individual eight individuals under investigation are hybrids between Iberian inbreeding coefficient when estimated with limited molecular data. wild goats and domestic goats Through a RAD-­Seq approach, Grossen et al. (2018) demonstrated that the median total length of long ROH (>5 Mb) was much higher and exponential rates, respectively (Miller et al., 2012). Based on the in Iberian wild goats from Sierra Nevada (228 Mb) and Maestrazgo data presented by Miller et al. (2012), domestic goats and Iberian (179 Mb) than in domestic goats (<10 Mb). This result was further wild goats, two species that diverged 1.5 Mya (Lalueza-­Fox et al., confirmed by Grossen et al. (2020), who demonstrated that the me- 2005), should share, on average, less than 10% polymorphic sites. dian of the proportion of the genome covered by ROH >2.5 Mb is In accordance with the data reported by Amills et al. (2004), we approximately twice higher in Iberian wild goats than in domestic have detected reduced observed and expected heterozygosities goats. These findings indicate that the decreased diversity and in- in Iberian wild goats when compared to domestic goats (Table 2). creased inbreeding of Iberian wild goats, as compared to domestic Nucleotide diversities were also lower in Iberian wild goats than in goats, observed in our study is not an artifact entirely produced by domestic goats. Moreover, nucleotide diversities of domestic goats ascertainment bias. Indeed, the existence of inbreeding and low were similar to those observed in other livestock populations (Luigi-­ variation in Iberian wild goats is consistent with the dramatic genetic Sierra et al., 2020; Wang et al., 2018). Nucleotide diversity estimated bottlenecks suffered by this ungulate species (Amills et al., 2004). on a per-­site basis (–­site-­pi), as we have done in the current work, yields values that are different from those obtained by considering windows of 1000 bp (–­window-­pi), as previously discussed by Luigi-­ 4.2 | Iberian wild goat populations are strongly Sierra et al. (2020). Obviously, the reduced diversity of Iberian wild differentiated goats when compared to domestic goats could be due, at least in part, to ascertainment bias. Noteworthy, Grossen et al. (2018) geno- The classification of Iberian wild goats in four subspecies proposed typed more than 100,000 SNPs in Alpine ibexes, Iberian wild goats, by Cabrera (1911, 1914) raised controversy because it relied ex- and domestic goats and found that Ho and He were twofold higher clusively on a limited number of highly variable phenotypic traits in domestic goats when compared to Alpine ibexes or Iberian wild recorded in a low number of individuals (Angelone-­Alasaad et al., goats, a result fully consistent with ours. Moreover, Grossen et al. 2017). Our study demonstrated that the FST coefficients among (2020) reported that Iberian wild goats have twice lower genome-­ Iberian wild goat populations were much higher (FST > 0.287) than wide heterozygosity (expressed as the number of heterozygous those measured among domestic goat populations (FST < 0.072), and autosomal SNPs per kb) than domestic goats, a result that again the latter were similar to those reported by Manunza et al. (2016) matches very well ours. for the same populations using a data set of 39,257 SNPs. Moreover, 2630 | CARDOSO et al. genetic differentiation between the CPV and CPH populations had evidence of admixed individuals was obtained in any of the three a magnitude similar to that observed between the Tortosa-­Beceite Iberian wild goat populations under study. In our work, resolution and Sierra Nevada CPH populations (Table 3). and sensitivity to identify introgressed individuals were expected to Overall, these results indicated that the classification of Cabrera be much higher because our set of 1001 SNPs would be roughly (1911, 1914) is not well supported by genetic evidence. Consistently, equivalent to a panel of ~300 microsatellites (Fernández et al., Angelone-­Alasaad et al. (2017) analyzed 333 Iberian wild goats with 2013; Herráeza et al., 2005). We have detected two individuals a panel of microsatellites and found that the CPH populations from from Tortosa-­Beceite showing evidence of Sierra Nevada ancestry Sierra Nevada and Maestrazgo had a degree of genetic differentia- (Figure 5), and one individual from Sierra Nevada harbored a ge- tion similar to that observed among CPH and CPV specimens. The nomic signature of Muela de Cortes ancestry. Landscape fragmen- strong genetic differentiation between Iberian wild goat popula- tation and large geographic distances between Sierra Nevada versus tions, irrespective of their assignment to one subspecies or another, Muela de Cortes (300 km) and Tortosa-­Beceite (more than 500 km) is probably due to intense drift associated with past genetic bottle- make difficult to attribute this result to a natural expansion of Iberian necks combined with prolonged geographic isolation (Pérez et al., wild goats. More probably, the presence of admixed individuals was 2002). The only exception to this general trend were the CPV popu- the consequence of human-­mediated translocations. To the best of lations of Gredos and Batuecas, which had a weak degree of genetic our knowledge, no translocation between these three geographic differentiation (FST = 0.035, Table 3), probably because the Batuecas areas is documented (Figure 1), thus supporting the notion that population originated from restockings with individuals from Gredos translocation events involving Iberian wild goats went often unre- (Pérez et al., 2002). corded (Angelone-­Alasaad et al., 2017). This lack of recording might In the MDS analyses shown in Figure 3a,b, the only CPP indi- reflect that many translocations involving Iberian wild goats were vidual did not cluster with its CPV and CPH counterparts, although not carefully planned or executed to achieve a long-­term goal. In any it was located close to the Muela de Cortes population (CPH). This case, this lack of reliable data makes it difficult to infer the impact result also needs to be interpreted with caution because allele fre- of the officially registered restocking/repopulation translocations by quencies cannot be estimated from just one individual. However, comparing the genetic diversity of populations before and after they CPP suffered strong population bottlenecks and an irreversible ge- were implemented. Despite this important caveat, the low propor- netic erosion that enhanced genetic differentiation before extinc- tion of individuals (~3%) with genomic signatures of mixed ancestry tion (García-­González & Herrero, 1999). Moreover, there was no is consistent with a scenario in which past restocking/repopulation recent gene flow between CPP and the other CPV and CPH sub- translocations did not have a strong impact on the genetic variability species, which strengthened the progressive genetic differentiation of Iberian wild goats. We do not think that this finding is produced by of CPP. The analysis of additional museum CPP samples would be the limited ability of the panel of 1001 SNPs to detect admixed indi- needed to accurately characterize the genetic relationships between viduals because Iberian wild goat populations are highly differenti- this extinct subspecies and CPH and CPV. ated, a feature that compensates to some extent the limited number of available markers, thus making it possible to detect recent admix- ture with enough confidence. 4.3 | Genomic signatures of past restocking Many factors may explain why augmentation translocations left translocations are rarely detected in current Iberian a scarce genomic footprint on the genomes of Iberian wild goats. For wild goat populations instance, chronic stress produced by the capture, handling, trans- port, captivity, and release of wild animals to a new location might As pointed out by Acevedo and Cassinello (2009a), Iberian wild goat result in substantially reduced reproductive success and increased distribution is the result of both natural and artificial expansion pro- mortality (Dickens et al., 2010). Another factor could be competition cesses. Most translocations were carried out after 1970, particu- for food resources between the released animals and the residing larly during the 1980s and 1990s (Acevedo & Cassinello, 2009a). conspecific population, or endemic diseases that might have deci- Although there is a general consensus indicating that Iberian wild mated the incoming individuals. Avian translocations have a high goats were the subject of numerous restocking/repopulation trans- failure rate (Dickens et al., 2009), and documented success in car- locations (movement of individuals into a population of conspecif- nivores is also low (Macdonald, 2009). In the case of the American ics), the majority of them are poorly documented (Angelone-­Alasaad marten population of Wisconsin, augmentation provided minimal et al., 2017). In Figure 1, we provide a description of translocations genetic and demographic rescue contributions (Manlick et al., 2017). that have been reported so far. A failed reintroduction of 14 Iberian wild goats from Gredos and One of the main goals of the current work was to infer whether Cazorla to the National Park of Covadonga in 1957–­1962 has been past restocking translocation processes have left genomic signa- also reported (Arenzana et al., 1964). Despite their low diversity, tures that can be identified in current Iberian wild goat populations, Iberian wild goat populations are increasing in numbers at a fast pace as well as to ascertain the impact of restocking/repopulation on (Acevedo & Cassinello, 2009a; Acevedo et al., 2007). In light of this, the within-­population genetic diversity. In a previous study based we consider that gene flow between naturally expanding popula- on a panel of 30 microsatellites (Angelone-­Alasaad et al., 2017), no tions could be established without the need of human intervention. CARDOSO et al. | 2631 4.4 | Hybridization with domestic goats as a can adapt quite successfully to mountainous habitats (Herrero, potential threat to the conservation of Iberian Fernández-­Arberas, Prada, García-­Serrano, & García-­González, wild goats 2013). For instance, in the Sierra de Guara, a population of Iberian wild goats coexists with almost one thousand feral goats (Herrero, We detected eight Iberian wild goats from Tortosa-­Beceite with Fernández-­Arberas, Prada, García-­Serrano, & García-­González, genomic signatures of domestic goat introgression. The f3 test 2013) descending from individuals probably abandoned by their agreed with the results of the Admixture analysis by showing that owners. Extensive field surveys based on SNP markers should be domestic goat introgression was significant (Figure 6). The absence conducted to evaluate the presence and frequency of hybrid individ- of hybrid individuals in Sierra Nevada, Gredos, Batuecas, or Muela uals in current Iberian wild goat populations, with special emphasis de Cortes might indicate that the introgression of Iberian wild goats on those inhabiting geographic areas in which the presence of un- by domestic goats is a sporadic event. However, the sampling of sev- controlled herds of feral goats is well documented. eral of these locations was quite limited and quantitative inferences on the abundance of hybrid individuals could not be made with high AC K N OW L E D G M E N T S confidence. This research was funded by the European Regional Development Angelone et al. (2018) detected the segregation of a domestic Fund (FEDER)/Ministerio de Ciencia e Innovación—­A gencia Estatal goat MHC-­DRB1*7 allele in Iberian wild goats from the Sierras de de Investigación/Project Reference grant: PID2019-­105805RB-­I 00 Cazorla, Segura, and Las Villas (Jaén, Andalusia). They concluded and by the CERCA Programme/Generalitat de Catalunya. We ac- that this could be due to either the maintenance of ancient poly- knowledge the support of the Spanish Ministry of Economy and morphisms by balancing selection or, alternatively, introgressions Competitiveness for the Center of Excellence Severo Ochoa from domestic goats through interspecific hybridization (Angelone 2020–­2023 (CEX2019-­0 00902-­S) grant awarded to the Centre for et al., 2018). Our results support this latter interpretation. Research in Agricultural Genomics (CRAG). We also acknowledge Giacometti et al. (2004) documented the existence of free-­ranging José Folch, of the Centro de Investigación y Tecnología Agroalimentaria Alpine ibexes in the Bregaglia Valley of southern Switzerland which de Aragón for his help in the development of his research. MGLS displayed signs of domestic goat introgression. The occurrence of was funded with a FPI Ph.D. grant from the Spanish Ministry of such interspecific hybridization events was confirmed by Grossen Education (BES-­C-­2017-­079079). TFC was funded with a fellow- et al. (2014) by demonstrating that one of the two major histocom- ship from the CAPES Foundation-­Coordination of Improvement of patibility complex class DRB alleles that segregates in Alpine ibexes Higher Education, Ministry of Education of the Federal Government was identical to another one described in domestic goats. When of Brazil. EMS was funded with a FPU Ph.D. grant from the Spanish the National Game Reserve of Tortosa-­Beceite was created in Ministry of Education (FPU15/01733). GM is a Serra Húnter fel- 1966, there was an estimated population of 200 Iberian wild goats. low (professor) of the Generalitat de Catalunya. Thanks also to the Although domestic goats were less abundant than domestic sheep, CERCA Programme of the Generalitat de Catalunya for their support. the presence of both domestic and feral goats combined with the Sample collection benefitted from the research grants CGL2012-­ low number of Iberian wild goats (Viñas-­Borrell et al., 1993) origi- 40043-­C02-­01, CGL2012-­4 0043-­C02-­02, and CGL2016-­8 0543-­P, nated a window of opportunity for the occurrence of interspecific also from the Spanish Ministry of Economy and Competitiveness. hybridization events. Many thanks to Laura Botigué for her help and assistance in carrying According to our data, the introgression of Iberian wild goats by out the f3 tests of admixture. domestic goats does not seem to be widespread, probably because the interbreeding of these two species in the wild is a rare event, C O N FL I C T O F I N T E R E S T S and moreover, some degree of reproductive incompatibility may The authors declare that they have no competing interests. exist (Herrero, Fernández-­Arberas, Prada, & García-­Serrano, 2013). However, hybridization cannot be disregarded as a potential threat DATA AVA I L A B I L I T Y S TAT E M E N T to the genetic conservation of Iberian wild goats, since its preva- The data that support the findings of this study are openly available lence might increase as a result of the rapid expansion, in numbers in Figshare at https://doi.org/10.6084/m9.figsh​are.11955​981.v2. and geographic range, of Iberian wild goat populations (Acevedo & Cassinello, 2009; Acevedo et al., 2007; Perea et al., 2015). The in- ORCID trogression of Iberian wild goats by domestic goats could imply a Tainã Figueiredo Cardoso https://orcid.org/0000-0002-6038-7521 decrease of reproductive potential and fitness, the introduction of Marcel Amills https://orcid.org/0000-0002-8999-0770 maladaptive alleles, a reduction or loss of genetic integrity, and it may also have legal implications regarding individual or population REFERENCES conservation status (Leonard et al., 2013). 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