Native European Mouflon populations Ovis gmelini musimon live in natural state only in Sardinia and Corsica, but in the last decades direct and indirect human activities determined habitat fragmentation and their isolation in few isolated areas. Our aim was to investigate whether population fragmentation and isolation determine genetic structuring in Sardinian mouflon using both nuclear and mitochondrial molecular markers. The current Sardinian mouflon populations are mainly located in the central area of the island. We collected blood samples from individuals coming from three distinct sites of this area: at the North (N; n°=22) where mouflons were reintroduced 40 years ago; in the middle (C; n=13) and at the South Est (S; n=18) where still live native free mouflon populations. We assayed 16 microsatellite loci length and the mitochondrial D-Loop region polymorphisms to genetically characterize each group and to clarify the genetic relationships among them. To genotype microsatellites, DNA fragments containing the polymorphisms were amplified by PCR according to standard protocols. Microsatellite products were analysed on a ABI PRISM 3100 DNA Analyser (Applied Biosystems) and data were processed by GENESCAN v3.1 and GENOTYPER v2.5 software. Standard intra-population indices, such as allele frequency, gene diversity and deviation from Hardy-Weinberg Equilibrium (HwE), were tested with the software package Arlequin 1.1 (http://lgb.unige.ch/arlequin/). Genetic differentiation (Fst) and the test of genotype assignment have been chosen as inter-population indices to assess the relationships among the three sample groups. The whole D-loop region of the mitochondrial DNA was amplified by standard PCR. PCR product was sequenced by Dye-terminator chemistry and sequencing reaction was run on ABI PRISM 3100 DNA Analyser (Applied Biosystems). Clustal X software was used for the sequence multi-alignment. Phylogenetic analyses were conducted using DnaSP, Tree Puzzle, MEGA and Network softwares. Fourteen of the sixteen genotyped microsatellite loci are polymorphic in at least one of the three groups with a mean of four alleles/locus (7 to 9). The observed heterozigosity shows, for every marker, high variability among the three populations. Five microsatellite loci deviated from HwE in at least one mouflon group due to heterozygote deficiency. The exact test for population differentiation (Fst) revealed strong genetic differentiation among the three populations, since almost all the relative P values are significant (P<0.05). The high Fst values are largely due to private alleles (12 loci had private alleles). The test of Genotype Assignment affirmed the effective belonging of every individual to the population it was originated and Book of Abstracts, 2nd Edition. VIth World Congress on Mountain Ungulates and Vth International Symposium on Mouflon. Lefkosia, Cyprus, 28 August -1 September 2016 73 individuals in each population appeared clustered separately from the other two populations. A total of 11 single-nucleotide polymorphism (SNPs) at the level of the hyper variable domain I of the mitochondrial D-loop region were detected. Based on maximum likelihood and neighbor joining tree analysis, sequences were grouped in three distinct clusters specific to each geographic area where samples were collected. The highest genetic variability was detected within the clade including all the individuals from the C population, as confirmed by the occurrence of three distinct haplotypes. In addition, two individuals from the C population were located in a central position, as inferred by the network analysis, likely representing the ancestral type who gave rise to all the current populations. Our results evidence a clear phylo-geographic structuring among the samples analyzed and support the hypothesis that the mouflons currently living in the C area could represent the remnant of the native Sardinian population. The loss of genetic variation detected into the N and S populations could be the results of a founder effect or a population bottleneck. Furthermore, the genetic differentiation observed among the mouflons living in the three different areas could be due to the fragmentation of the original genetic pool caused by long term isolation. This study permits the reintroduction of this species into new habitats, taking into consideration the selection of the best genotypes, to minimize founder effect and to maximize genetic diversity.
HABITAT FRAGMENTATION CAUSES GENETIC DIFFERENTIATION OF SARDINIAN MOUFLON
Doro MG;Casula S;Casu G;
2016
Abstract
Native European Mouflon populations Ovis gmelini musimon live in natural state only in Sardinia and Corsica, but in the last decades direct and indirect human activities determined habitat fragmentation and their isolation in few isolated areas. Our aim was to investigate whether population fragmentation and isolation determine genetic structuring in Sardinian mouflon using both nuclear and mitochondrial molecular markers. The current Sardinian mouflon populations are mainly located in the central area of the island. We collected blood samples from individuals coming from three distinct sites of this area: at the North (N; n°=22) where mouflons were reintroduced 40 years ago; in the middle (C; n=13) and at the South Est (S; n=18) where still live native free mouflon populations. We assayed 16 microsatellite loci length and the mitochondrial D-Loop region polymorphisms to genetically characterize each group and to clarify the genetic relationships among them. To genotype microsatellites, DNA fragments containing the polymorphisms were amplified by PCR according to standard protocols. Microsatellite products were analysed on a ABI PRISM 3100 DNA Analyser (Applied Biosystems) and data were processed by GENESCAN v3.1 and GENOTYPER v2.5 software. Standard intra-population indices, such as allele frequency, gene diversity and deviation from Hardy-Weinberg Equilibrium (HwE), were tested with the software package Arlequin 1.1 (http://lgb.unige.ch/arlequin/). Genetic differentiation (Fst) and the test of genotype assignment have been chosen as inter-population indices to assess the relationships among the three sample groups. The whole D-loop region of the mitochondrial DNA was amplified by standard PCR. PCR product was sequenced by Dye-terminator chemistry and sequencing reaction was run on ABI PRISM 3100 DNA Analyser (Applied Biosystems). Clustal X software was used for the sequence multi-alignment. Phylogenetic analyses were conducted using DnaSP, Tree Puzzle, MEGA and Network softwares. Fourteen of the sixteen genotyped microsatellite loci are polymorphic in at least one of the three groups with a mean of four alleles/locus (7 to 9). The observed heterozigosity shows, for every marker, high variability among the three populations. Five microsatellite loci deviated from HwE in at least one mouflon group due to heterozygote deficiency. The exact test for population differentiation (Fst) revealed strong genetic differentiation among the three populations, since almost all the relative P values are significant (P<0.05). The high Fst values are largely due to private alleles (12 loci had private alleles). The test of Genotype Assignment affirmed the effective belonging of every individual to the population it was originated and Book of Abstracts, 2nd Edition. VIth World Congress on Mountain Ungulates and Vth International Symposium on Mouflon. Lefkosia, Cyprus, 28 August -1 September 2016 73 individuals in each population appeared clustered separately from the other two populations. A total of 11 single-nucleotide polymorphism (SNPs) at the level of the hyper variable domain I of the mitochondrial D-loop region were detected. Based on maximum likelihood and neighbor joining tree analysis, sequences were grouped in three distinct clusters specific to each geographic area where samples were collected. The highest genetic variability was detected within the clade including all the individuals from the C population, as confirmed by the occurrence of three distinct haplotypes. In addition, two individuals from the C population were located in a central position, as inferred by the network analysis, likely representing the ancestral type who gave rise to all the current populations. Our results evidence a clear phylo-geographic structuring among the samples analyzed and support the hypothesis that the mouflons currently living in the C area could represent the remnant of the native Sardinian population. The loss of genetic variation detected into the N and S populations could be the results of a founder effect or a population bottleneck. Furthermore, the genetic differentiation observed among the mouflons living in the three different areas could be due to the fragmentation of the original genetic pool caused by long term isolation. This study permits the reintroduction of this species into new habitats, taking into consideration the selection of the best genotypes, to minimize founder effect and to maximize genetic diversity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
