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) . 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) 
ranged from 17.16 to 24.85, showing little variation between
populations. The expected heterozygosity ranged from 0.35 to 0.93
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.
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.