Previous research has shown that pampas cats present a large genetic diversity at the species level .
Results of the present study indicate that a large diversity is also
present at populations level and that it is comparable to other
non-endangered felid species, like the ocelot and margay . Although we reported a higher HVS-I genetic diversity for some pampas cat populations (P = 2.35 – 19.70; Table 2) than for those species (P = 3.71 – 14.73) 
this can be explained by bigger samples and the presence of highly
divergent clades in the most diverse pampas cat populations (Figure 6).
Genetic structure and geographic distribution
Along the Andean region, analysis of mitochondrial and nuclear
genomes revealed the presence of three groups of populations. These
groups show a strong geographical structure, with latitudinal
separations at 18°–20°S and 23°–25°S. Including results of previous
studies based strictly on mitochondrial DNA indicated the existence of
a fourth group in northern Chile , as well as the two groups out of the Andean region, in Brazil and central Chile . This genetic structure roughly corresponds to the distribution of morphological subspecies described by García Perea . Two of the museum samples, both from locality number 8, were identified as L. c. garleppi using the descriptions done by García-Perea ,
although it was not possible to directly assign a genotype to a
morphotype for other localities due to the nature of the samples.
However, according to the type localities of the described subspecies,
the pampas cat groups can be equivalent to garleppi (Clade A, 9°–18°S), budini (20°–23°S), pajeros (25°–38°S) and wolffshoni (Clade B northern Chile) subspecies (Figure 8).
The geographical location of populations 12 and 19 coincides with the subspecies steinbachi and crucinus, respectively, while population 1 is located between the supposed distribution ranges of thomasi and garleppi (Figure 8). These three localities are characterized by private HVS-I haplotypes
or ncDNA that differs from that of adjacent populations, and had
samples of only 3 individuals. Since a small sample size per locality
can affect the results of the STUCTURE program 
and other analyses, the correspondence between these localities and
subspecies needs to be validated by further investigations. The further
assignation of these localities to subspecies can be of conservation
value since many authors [i.e. [18,19]] do not recognise steinbachi and crucinus as
valid taxa. As this research focused in the central Andean region,
further research must be made to describe the genetic structure of the
The Andean area between 18° and 23°S, approximately, corresponds to
an extremely arid belt that separates a northern area, with summer
rains, from a southern one, with winter rains [32,33]. This zone is considered to be a barrier between subspecies of other land mammals such as the vicuna Vicugna vicugna  and the lesser grison Galictis cuja , and represents a distribution limit for other species, like the long-tailed weasel Mustela frenata . In contrast, the genetic structure of the puma Puma concolor from the high Andes is not affected by this barrier ,
probably due to a higher dispersal capacity. The pampas cat populations
distributed between 18°S and 25°S display three different phenotypes  and an important admixture of the clades typical of adjacent areas, suggesting the arid belt as a contact zone.
Influence of Pleistocene
The topology of the mtDNA trees enabled us to infer two periods of
prime importance for the demographic history of the pampas cat. The
lack of resolution for the relationships between pampas cat clades (as
well as among haplotypes within clades) suggests a rapid radiation.
A first episode comprised the split of the clades A, B, C and D and
occurred between the end of the Bramertonian Interglacial (1.30 – 1.55
MYA) and the beginning of the Pre-Pastonian glacial period (0.80 – 1.30
MYA; Figure 7). The Pre-Pastonian corresponds with the most extensive glaciations in southern South America . This period also coincides with the estimated date of the divergence between the major clades of the Andean bird genus Muscisaxicola , suggesting this period as an important phase in the diversification of the Andean fauna.
The very similar coalescence times of the current haplotypes of the
clades A, B, C and D suggest these events took place simultaneously,
during a second demographic episode. These splits were likely to be the
result of demographic expansions caused by geographically extended
phenomena associated with climate change. The calculated mean time for
these events overlapped the end of the Kansan glacial period (0.30 –
0.45 MYA) and the Aftonian interglacial (0.45 – 0.62 MYA), the
interglacial period being the more likely moment of occurrence for
these demographic events (Figure 7).
Interestingly, the divergence time between clades A, B, C and D is
similar or longer than between some felid species, such as the Iberian
lynx Lynx pardinus, Canadian lynx Lynx canadensis and Eurasian lynx Lynx lynx (between 1.18 and 1.61 MY), kodkod Leopardus guigna, little spotted cat Leopardus tigrinus andGeoffroy's cat Leopardus geoffroyi (0.74–0.93 MY) and ocelot and margay (1.58 MY) .
High diversity within clades, times of divergence and monophyletic
origin in most of the regions indicated long-term isolation into
distinct refuges (glacial)/regions (interglacial) rather than dispersal
from a unique refuge. No Pleistocene refugia were previously identified
in the central Andes for medium-sized mammals. However, considering the
current distribution of the clades and the zones apparently less
affected by the glaciations ,
clade A refuge probably existed in central Peru, while clade B and
clade D refuges were possibly located in northern Chile and eastern
Argentina, respectively. Clade C refuge was probably placed in eastern
Bolivia. Individuals with haplotypes of clades B, C and D would
subsequently migrate to a contact zone between 20 and 23°S during an
Implications for conservation
The concepts of "evolutionarily significant units" (ESUs)  and "management units" (MUs) 
were created with the objective of targeting operational units for
conservation below the species level. Although the use of the ESUs
concept and its importance for prioritising conservation efforts is
currently well accepted, its definition was strongly debated . Many authors consider reciprocal monophyly as mandatory for the recognition of ESUs, as proposed by Moritz , or subspecies, while for other definitions a different evolutionary history between populations is sufficient [41,44-46].
MUs, in the other hand, are defined as population units with
statistically significant differences of allelic frequencies at nuclear
or mitochondrial level .
The long divergence time among clades, as well as the
differentiation at the ncDNA level, highlight the importance of
recognizing the four pampas cat population groups identified here
(localities 2–10/11, 13–14/15–18/northern Chile) as different units for
conservation in the Andean region. Since they have different allelic
frequencies at both, mtDNA and ncDNA levels, all of these groups must
to be recognised as MUs. In addition, we recommend the recognition of
the localities 1, 12 and 19 as provisional MUs, since only private
haplotypes were found for them (localities 1 and 19) or they differ at
ncDNA level from adjacent MUs (locality 12; Figure 8).
The existence of an admixture zone in the central Andean region
results in a lack of reciprocal monophyly between all the four
population groups and makes their recognition as subspecies or ESUs
controversial. However, cases of hybridization between subspecies or
species are common in nature , of particular interest being the identification of hybridization areas for tigrinas, Geoffroy's cats and pampas cats [19,48].
In this sense, pampas cats from central Andes can be regarded as a
complex of at least four ESUs/subspecies, one of them being the result
of a past hybridization event.