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In this study, the genetic structure of the pampas cat (Leopardus colocolo), …

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- Influence of ancient glacial periods on the Andean fauna: the case of the pampas cat (Leopardus colocolo)

Species identification and mtDNA variability

A total of 39 skin samples and 532 faecal samples from 19 localities were analyzed (Figure 1; Table 1). Of the faecal samples, 406 were unambiguously assigned to pampas cats according to the mtDNA 16S region. The remaining samples were assigned to Andean cats Leopardus jacobita (40), domestic cats (11) or canid species (32). A small number of faecal samples (43) failed to be amplified and could not be identified. All skins were assigned to pampas cats.

The length of two mitochondrial control region segments combined varied between 336 and 351 bp. Two variable mononucleotide repeats (T)3–7 (C)3–11 were identified but not considered for the analyses. Without considering the RS2 region composed of a variable number of large tandem repeats, the pampas cat HVS-I region contains between 178 and 190 bp (Figure 2). The range known for other felid species is 231–245 bp [26-28].

SSCP analyses revealed a single allele per sample, excluding the presence of mtDNA sequences transferred into the nuclear genome (Numts) [28]. The SSCP survey of the 406 samples and the sequencing revealed a total of 41 HVS-I haplotypes and 94 variable sites. Phylogenetic relationships inferred among these haplotypes were consistent for both NJ and ML methods. The haplotypes clustered into four major clades, named hereafter A to D, strongly supported by bootstrap values (Figure 3). However, relationships among clades were not completely resolved.

Sequencing of the NADH-5 and ATP-8 genes revealed 29 sites of additional variation for the 19 individuals selected from our sample. These individuals were selected randomly, with the constraints of choosing individuals with different HVS-I haplotypes and covering all the major HVS-I clades. The phylogenetic tree inferred using both the NADH-5 and ATP-8 genes was fully congruent with the one performed with HVS-I and allowed the four major clades to be recovered, while the resolution was lower. When individuals from previous studies [20,29] were included into the present dataset, a total of 41 variable sites was detected for NADH-5 and ATP-8 genes. Most of these additional individuals clustered within clade B (15) and D (3) (Figure 4) while individuals geographically distant from the Andean region, located in central Chile and Brazil, formed two additional clades. Interestingly, individuals presenting HVS-I haplotypes of the clades A and C formed two clusters not identified by previous studies.

Individual identification and population diversity

Microsatellite amplifications provided results for 290 (71%) of the 406 pampas cat faecal samples and for the 39 skin samples (100%) for a total of 329 samples. The microsatellites were highly variable, with 10–21 alleles per locus (Table 2) and the probability of sampling two different individuals with the same genotype ranged between 4.80 × 10-3 and 2.88 × 10-16. Unique multilocus genotypes were recorded for 99 faecal samples (30%). However, in several cases, two or more samples displayed the very same multilocus genotype indicating that the same individual had been sampled several times. According to the low probability of obtaining the same genotype in different individuals, samples with the same genotype were assigned to a unique individual, providing a final sample size of 199 pampas cat individuals. The number of individuals per locality varied from 5 to 30, except for 6 localities that had a sample size of 4 individuals or less (Table 1). Localities with sampling size lower than four individuals were not included in the following analyses, unless mentioned in the text.

The total number of alleles per population ranged from 17 to 48 (Table 2), but was correlated to sampling size (Pearson's r = 0.915, P < 0.0001). The total number of alleles estimated for 5 individuals per population (allelic richness, FSTAT 2.9.3) [30] ranged from 17.16 to 24.85, showing little variation between populations. The expected heterozygosity ranged from 0.35 to 0.93 (Table 2) and was not correlated to sampling size (r = 0.013, P = 0.960). None of the localities displayed deviation from HW expectations, indicating that no more than one population was sampled per locality.

For the mtDNA control region, the number of haplotypes per population ranged from 2 to 14 (Table 2) and was also correlated to sampling size (r = 0.884, P < 0.0001). Haplotype diversity values ranged from 0.60 to 0.93 between the sampled localities, and nucleotide diversity varied by one order of magnitude between 0.0059 and 0.0519.

Population structure

The best value of the ln Pr(X|K) obtained with the program STRUCTURE on microsatellite data corresponded to K = 3 population groups. These groups included the localities (2–6, 8, 9), (11, 14) and (15–18). Relationships among populations inferred from the microsatellite data supported this grouping, although bootstrap values between populations 2–6, 8 and 9 were low (Figure 5A). AMOVA analysis also supported this structure as the one displaying the highest variation among groups on either mitochondrial or microsatellite data (Table 3).

The distribution of the groups of populations appeared to be clearly correlated to latitude (Figure 6). The first group occupies the area north to 18°S and is formed almost exclusively by individuals of the clade A. The second group, distributed between 20° and 23°S, presents a great proportion of individuals of clade C, although clades B and D are present too. The third group, distributed south to 25°S, contains principally clade D individuals, with some clade B and C individuals in its northern region. The northernmost and the southernmost sampled localities are represented by private haplotypes of the clades A and D, respectively.

Including all localities did not modify the results provided by STRUCTURE: (1–10), (11, 13–14) and (12, 15–19) (Figure 5B) and the population structure remains strongly correlated to geography except for population 12. While geography and mtDNA data suggested including this population in the group (11–13–14), the microsatellite data suggested a closer affiliation with the southernmost group (15–19).

Evolutionary and demographic history

Based on the HVS-I sequences, the coalescence times of the clades [A, B, C and D], [A – B], and [C – D] were estimated to 1.52 MY ago (with a confidence interval from 1.05 to 2.21 MYA), 1.37 MY ago (0.95 – 2) and 1.17 (0.81 – 1.7) MY ago respectively. Interestingly, the radiation of the clades A, B, C and D occurred almost simultaneously, with coalescence times of 0.43, 0.39, 0.54 and 0.44 MY ago, respectively. The results for Tamura-Nei distances between clades, coalescence times and confidence intervals are presented in Figure 7.

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