Short Communication |
Corresponding author: Andrea Rezić ( arezic@agr.hr ) Academic editor: Jesus Maldonado
© 2022 Andrea Rezić, Toni Safner, Laura Iacolina, Elena Bužan, Nikica Šprem.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rezić A, Safner T, Iacolina L, Bužan E, Šprem N (2022) Traces of past reintroduction in genetic diversity: The case of the Balkan chamois (Mammalia, Artiodactyla). ZooKeys 1116: 57-70. https://doi.org/10.3897/zookeys.1116.84577
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The translocation of wild animal species became a common practice worldwide to re-establish local populations threatened with extinction. Archaeological data confirm that chamois once lived in the Biokovo Mountain but, prior to their reintroduction in the 1960s, there was no written evidence of their recent existence in the area. The population was reintroduced in the period 1964–1969, when 48 individuals of Balkan chamois from the neighbouring mountains in Bosnia and Herzegovina were released. The main objective of this study was to determine the accuracy of the existing historical data on the origin of the Balkan chamois population from the Biokovo Mountain and to assess the genetic diversity and population structure of the source and translocated populations 56 years after reintroduction. Sixteen microsatellite loci were used to analyse the genetic structure of three source chamois populations from Prenj, Čvrsnica and Čabulja Mountains and from Biokovo Mountain. Both STRUCTURE and GENELAND analyses showed a clear separation of the reintroduced population on Biokovo from Prenj’s chamois and considerable genetic similarity between the Biokovo population and the Čvrsnica-Čabulja population. This suggests that the current genetic composition of the Biokovo population does not derive exclusively from Prenj, as suggested by the available literature and personal interviews, but also from Čvrsnica and Čabulja. GENELAND analysis recognised the Balkan chamois from Prenj as a separate cluster, distinct from the populations of Čvrsnica and Čabulja. Our results thus highlight the need to implement genetic monitoring of both reintroduced and source populations of endangered Balkan chamois to inform sustainable management and conservation strategies in order to maximise the chances of population persistence.
Biokovo, genetic structure, microsatellite, Prenj, translocation
The reintroduction and translocation of wild species for various purposes became a common practice worldwide and was used as a conservation tool for rescuing and re-establishing extirpated populations (
Today’s populations of the Northern chamois in northern Dinaric Mountains in Croatia are descendants of successfully translocated individuals captured on mountain areas in Bosnia and Herzegovina (Rupicapra rupicapra balcanica) and Slovenia (Rupicapra rupicapra rupicapra) (
The Balkan chamois (Rupicapra rupicapra balcanica) is one of the seven recognised subspecies of the Northern chamois. It is found both in the mountainous regions of Croatia and in the mountain ranges of the eight other countries of the Balkan Peninsula, from north to south: Bosnia and Herzegovina, Serbia, Montenegro, Kosovo, North Macedonia, Albania, Bulgaria, and Greece. The lack of continuity of these habitats and overhunting in the post-Neolithic period have severely fragmented the subspecies’ present distribution (
The genetic structure of the Balkan chamois population on the Biokovo has been studied only by
The main objective of this study was to determine the accuracy of historical data on the origin of chamois in Biokovo, and to assess and document the genetic status of both the source and translocated populations, 56 years after reintroduction, by using microsatellite markers.
All samples used in this study were from hunted (regular hunting activities approved by the competent Ministry of Agriculture of the Republic of Croatia within the annual game management plans) and from remains of naturally dead animals (samples from Bosnia and Herzegovina).
We collected 20 samples from Biokovo and 29 samples from three areas which serve as source populations for reintroduction and possible recent recolonisation (Prenj, Čvrsnica, and Čabulja Mountains). Details of sampling locations are given in Fig.
Genetic diversity of four Balkan chamois populations assessed using sixteen microsatellite loci.
Population locality/ country | N | HO (SD) | HE (SD) | HWE | FIS (IC 95%) | A | AR | Npr | Ne (IC 95%) |
---|---|---|---|---|---|---|---|---|---|
Prenj 43°32'03"N, 17°54'12"E/BIH | 12 | 0.636 (0.274) | 0.637 (0.150) | 0.013* | 0.046 (-0.147–0.116) | 4.500 | 2.517 | 11 | 10.500 (6.500–18.600) |
Čvrsnica 43°38'18"N, 17°38'30"E/BIH | 12 | 0.552 (0.287) | 0.536 (0.206) | 0.011NS | 0.014 (-0.131–0.038) | 3.937 | 2.223 | 3 | 7.700 (4.100–13.600) |
Čabulja 43°29'11"N, 17°37'20"E/BIH | 5 | 0.575 (0.251) | 0.535 (0.187) | 0.913NS | 0.059 (-0.415–0.089) | 3.375 | 2.376 | 2 | – |
Biokovo 43°19'47"N, 17°63'05"E/HRV | 20 | 0.584 (0.132) | 0.597 (0.138) | 0.389NS | 0.048 (-0.050–0.084) | 4.625 | 2.356 | 6 | 57.200 (28.600–371.800) |
Results of the analysis of sixteen microsatellite loci in four Balkan chamois populations a geographical representation of results from STRUCTURE and GENELAND software. The pie charts show the results from STRUCTURE for K = 3. The different colours of the pie charts represent the proportions of each ancestral genotype per individual q (in %) in each of the four predefined Balkan chamois populations. The size of the pie charts indicates the number of samples collected at each location. The different shapes and colours of the chamois silhouettes represent the results of the spatial analysis under uncorrelated frequency model performed in GENELAND. The three spatial clusters are shown, while the assignment to the fourth ghost cluster was not shown because no individuals were assigned to it (see text for details). The dashed line indicates the national border, while the state road M17 in Bosnia and Herzegovina is marked with an orange line. The green lines represent connections with other main roads. The course of the river Neretva is marked by a blue line b genetic structure of the 49 Balkan chamois individuals analysed, shown as a bar plot from STRUCTURE at K = 3. Each vertical bar represents an individual, and the percentage of each colour corresponds to the percentage of the respective ancestral genotype. The studied populations are separated by a black line.
We extracted DNA from tissue samples (N = 49) using the commercial peqGOLD Tissue DNA Mini Kit (PEQLAB Biotechnologie GmbH) following the manufacturer´s protocol in a final volume of 150 µL. DNA concentrations were measured with Qubit dsDNA BR Assay Kit (Invitrogen BR Assay Kit, Carlsbad, CA, USA) on a 3.0 Qubit Fluorimeter (Life Technologies, Carlsbad, CA, USA). Sixteen microsatellites were amplified using PCR multiplex sets previously investigated in studies with chamois (
We used the Expectation-Maximization (EM) algorithm implemented in FREENA (
We considered each sampling location as a separate population due to limited dispersal of subspecies between mountain ranges (see Table
GENETIX ver. 4.05.2 (Belkhir et al. 1996–2004) was used to calculate the mean number of alleles, observed (HO) and expected (HE;
The Bayesian clustering program STRUCTURE ver. 2.3.4. (
The modal proportions of ancestral genotypes for each individual in each sampled area from the run with the highest log-likelihoods was plotted on a map using QGIS ver. 2.18.21 (
The robustness of the results of STRUCTURE was estimated by analysing the same data with the spatial Bayesian clustering model implemented in GENELAND software (
The sixteen microsatellite loci yielded a total of 95 alleles, which varied between 2 (for locus ETH10 and SR-CRSP-6) and 10 (for locus BM1258) with an average value of 5.937 alleles per locus (see Suppl. material
The values of null allele frequencies were low for most analysed loci, except for loci ETH10, SY434, TGLA53, and SR-CRSP-6, whose frequencies were estimated to be r ≥ 0.20 (see Suppl. material
The Prenj population deviated from Hardy-Weinberg equilibrium (HWE) but the deviation was significant at the 0.05 level only for locus SY434 after applying sequential Bonferroni adjustment (Table
The Prenj population had the highest values of observed (0.636) and expected (0.637) heterozygosity, and allelic richness (2.517). A similar pattern was recorded in the study of
Effective population size was estimated for three sampled sites, excluding the Čabulja population due to small sample size (Table
The lowest FST value was found between Čvrsnica and Čabulja (FST = 0.024), while the highest and significant FST value (0.084) was observed between two neighbouring populations from Bosnia and Herzegovina (Prenj and Čvrsnica; Table
Populations | Čvrsnica | Čabulja | Biokovo |
---|---|---|---|
Prenj | 0.084* | 0.047NS | 0.074* |
Čvrsnica | 0.024NS | 0.072* | |
Čabulja | 0.027NS |
The algorithm developed by
To improve the previous analyses, the spatial context of individuals was taken into consideration and tested with GENELAND. This analysis revealed a similar pattern of clustering of individuals as STRUCTURE, but suggested an additional fourth spatial cluster along the MCMC chain (see Suppl. material
Future studies will need to incorporate non-invasive genetic sampling, telemetry and behavioural patterns to confirm possible migration and gene flow between these populations. In the available literature there is no indication of the exact location where the animals released on Biokovo were caught. It is only known that the individuals came from the Prenj hunting district, which included two game reserves called “Čvrsnica” and “Prenj” (established in 1893 by the Austro-Hungarian Empire) and which were declared protected areas (
Non-invasive monitoring of genetic parameters of both reintroduced and source populations of endangered Balkan chamois, together with demographic monitoring, is crucial for sustainable management practices and improving conservation strategies to maximise the chances of population persistence. Our genetic diversity results show that the Balkan chamois population from Biokovo can serve as a potential source for future translocations, especially to the source habitats, Čvrsnica and Čabulja, that are currently threatened by loss of genetic diversity due to unsustainable hunting and poaching, leading to inbreeding and genetic drift.
We thank Luca Corlatti for permission to use his drawing (the silhouettes of the chamois yearling). This work was supported by the Croatian Science Foundation [grant number IP 2016-06-5751] and the ResBios European Union’s Horizon 2020 Research and Innovation Program [grant number 872146].
Tables and figures
Data type: Population genetics.
Explanation note: Table S1. Information about microsatellite primer sequences used for the analysis of Balkan chamois population genetics. Table S2. Locus/population matrix containing null alleles identified by the FreeNA software. Null allele frequencies were estimated using the EM algorithm. Frequencies with values higher than r ≥ 0.20 were indicated. Figure S1. The comparison of two groups of FST values (original pairwise FST values and ENA-corrected pairwise FST values) using the Wilcoxon Two Sample test. Figure S2. Evanno method (