Napolitano Et Al 2014 - Phylogeo Guigna

Conserv Genet
DOI 10.1007/s10592-014-0566-3
RESEARCH ARTICLE
Phylogeography and population history of Leopardus guigna,

the smallest American felid
Constanza Napolitano • Warren E. Johnson • Jim Sanderson • Stephen J. O’Brien •
A. Rus Hoelzel • Rachel Freer • Nigel Dunstone • Kermit Ritland •

Carol E. Ritland • Elie Poulin
Received: 25 July 2013 / Accepted: 8 January 2014

Ó Springer Science+Business Media Dordrecht 2014
Abstract The guigna (Leopardus guigna) is the smallest from ATP-8 gene) and 15 microsatellite loci. Mitochon-
and most-restricted New World cat species, inhabiting only drial DNA data revealed a clear phylogeographic pattern
around 160,000 km2 of temperate rain forests in southern with moderate separation between northern and southern
South America and is currently threatened by habitat loss, Chilean populations supporting recognized subspecific
fragmentation and human persecution. We investigated partitions based on morphology. A recent demographic
phylogeographic patterns of genetic diversity, demographic expansion was inferred for the southern-most group (San
history and barriers to gene flow with 116 individuals Rafael Lake), presumably due to the complete coverage of
sampled across the species geographic range by analyzing this area during the last glacial period, 28000–16000 years
1,798 base pairs of the mtDNA (496 bp HVSI region, BP. Geographical barriers such as the Andes Mountains
720 bp NADH-5 gene, 364 bp from 16S gene and 218 bp and the Chacao Channel have partially restricted historic
and more-recent gene flow and the Chiloé Island popula-
tion has diverged genetically since being separated from
the mainland 7000 years BP. This is the first study of the
Data Accessibility Data available from the Dryad Digital genetic structure of this threatened species throughout its
Repository http://doi.org/10.5061/dryad.1035h, GenBank accession
numbers KF979174-KF979217, KF979218-KF979261, KF979262- whole geographic range.
KF979304 and TreeBASE accession number S15147 http://purl.org/
phylo/treebase/phylows/study/TB2:S15147. Keywords Leopardus guigna Felid Phylogeography
Electronic supplementary material The online version of this Demographic history Dispersal barriers
article (doi:10.1007/s10592-014-0566-3) contains supplementary
material, which is available to authorized users.
C. Napolitano (&) E. Poulin S. J. O’Brien

Laboratorio de Ecologı́a Molecular & Instituto de Ecologı́a y Theodosius Dobzhansky Center for Genome Bioinformatics,
Biodiversidad, Departamento de Ciencias Ecológicas, Facultad St. Petersburg State University, St. Petersburg, Russia
de Ciencias, Universidad de Chile, Las Palmeras 3425, Ñuñoa,
Santiago, Chile A. Rus Hoelzel R. Freer N. Dunstone
e-mail: [email protected] School of Biological and Biomedical Sciences, Durham
University, Durham, UK
W. E. Johnson
Laboratory of Genomic Diversity, National Cancer Institute, R. Freer
Frederick, MD, USA FPCR Environment & Design Ltd., Lockington, Derby, UK
Present Address: N. Dunstone

W. E. Johnson Natural History New Zealand, Dunedin, Otago, New Zealand
Smithsonian Conservation Biology Institute, National Zoological
Park, 1500 Remount Rd., Front Royal, VA, USA K. Ritland C. E. Ritland
Department of Forest and Conservation Sciences, University of
J. Sanderson British Columbia, Vancouver, BC V6T1Z4, Canada
Small Wild Cat Conservation Foundation, Campbell, CA, USA
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Introduction and vegetational contrast that formed between the eastern

and western slopes of the Andes along the arid diagonal of
Contemporary geographical distribution and genetic South America, coincident with the gradual isolation of the
diversity of species are influenced by historical and eco- forests in southern South America, was one of the major
logical processes that cause population expansions and evolutionary and biogeographic events in southern South
contractions (Hewitt 2004). Historical climatic changes, America (Villagrán and Hinojosa 1997; Abraham et al.
specifically during the late Pleistocene glacial periods, 2000; Villagrán and Hinojosa 2005). This extended period
have had a large impact on current geographical distribu- of biogeographical isolation resulted in numerous endemic
tion and genetic diversity of vertebrates in regions of high flora and fauna species (Armesto et al. 1996; Arroyo et al.
and middle latitudes through habitat shifts, transient frag- 1996; Villagrán and Hinojosa 1997) and these isolated and
mentation of the ancestral range of a widely distributed unique temperate rainforests are recognized as the ‘‘Chil-
species, variation in gene flow, intensity of genetic drift, ean winter rainfall-Valdivian forests Hotspot’’ of biodi-
bottlenecks and loss of genetic diversity, genetic diver- versity (Myers et al. 2000; Arroyo et al. 2004), a ‘‘frontier
gence between refugial small isolated populations during forest’’ by the World Resources Institute (Armesto et al.
periods of increased ice cover and expansion of geographic 1996; Arroyo et al. 1996), and as a Global 200 priority
ranges of previously isolated populations during ice cover ecosystem of the World Wildlife Fund (Olson et al. 2001).
shrinkage (Klicka and Zink 1997; Taberlet et al. 1998; The guigna (Leopardus guigna), the smallest felid in the
Bennett 2004; Hewitt 2004). American continent and one of the smallest in the world
In southern South America, during the Last Glacial (weight 1.5–3.0 kg) (Nowell and Jackson 1996), is closely
Maximum (LGM) approximately 28000–16000 years BP, associated with this unique forest ecosystem. It has the
ice sheets covered most of upper elevation areas from 56° most-restricted distribution of New World cat species,
to 35°S and most of the current coastline south of 41°S inhabiting around 160,000 km2 of Chile (30°–48°S) and a
(Clapperton 1993; Heusser et al. 1999; McCulloch et al. narrow strip of south-western Argentina (39°–46°S west of
2000). Global sea levels dropped approximately 120 m 70°W), including some offshore islands such as Chiloé
below current levels, exposing much of the continental Island, from sea level to 2,500 meters (Nowell and Jackson
shelf and connecting Chiloé Island and the mainland 1996; Quintana et al. 2000) (Fig. 2). Two guigna subspe-
26000–7000 years BP (Villagrán et al. 1986; Moreno et al. cies are generally recognized based on morphological data
1994; Vidal et al. 2012). Chiloé Island is currently sepa- (Cabrera 1957). L. g. tigrillo (from 30° to 38°S in Chile)
rated from the mainland by the Chacao channel (2.3–6 km inhabits mediterranean matorral and sclerophyll woodlands
wide, 50–100 m deep) (Formas and Brieva 2000). There and forests in northern and central Chile and has a lighter
are no migration records of mammals across this channel. coat and larger body size (Figs. 1, 2; Table 1). L. g. guigna
Ice-free regions to the west, such as the continental (from 38° to 48° in Chile and from 39° to 46°S in
coastal area around 41°S, the northwest portion of Chiloé Argentina west of 70°W) inhabits more-dense Valdivian
Island and the intermediate area of exposed continental temperate rainforest and north Patagonian forest in south-
shelf were possible refugia for terrestrial biota during the ern Chile and the Andean Patagonian forest in southwest-
late Pleistocene glacial periods (Villagrán 1988; Sérsic ern Argentina and is darker and smaller (Osgood 1943)
et al. 2011; Vidal et al. 2012). Biological connections (Figs. 1, 2; Table 1). Melanistic individuals occur within
across the Andes may also have existed during the warmest the range of L. g. guigna (Dunstone et al. 2002; Sanderson
interglacial periods (Moreno et al. 1994; Moreno 2000), as et al. 2002).
has been suggested for a variety of trans-Andean mammals Vegetation cover is an important ecological requirement
(Smith et al. 2001; Palma et al. 2002, 2005; Himes et al. for this species (Sanderson et al. 2002; Acosta-Jamett and
2008). The evolutionary history and current genetic Simonetti 2004; Gálvez et al. 2013). Home range size of
diversity of many Patagonian species have also been linked guignas is 0.3–2.2 km2 in Torres del Paine and Queulat
with Quaternary glacial cycles and colonization from lower National Parks (Dunstone et al. 2002), and 1.3–22.4 km2 in
latitudes after glacial retreat (Lessa et al. 2010; Sérsic et al. a fragmented landscape on the northeastern coast of Chiloé
2011; Pardiñas et al. 2011). Island (Sanderson et al. 2002). The species’ maximum
The temperate rainforests of southern South America dispersal distance is 1.83 km in Torres del Paine and
(35–55°S) originated during the Middle Tertiary (Hinojosa Queulat National Parks (Dunstone et al. 2002), and
and Villagrán 1997) and were broadly impacted by the 13.9 km in a fragmented landscape on the northeastern
Miocene rise of the Andes (Garzione et al. 2008). The coast of Chiloé Island (Sanderson et al. 2002), revealing its
Andes became a barrier with sharp altitudinal climatic high and facultative dispersal ability (Napolitano 2012).
zones that restricted the distribution of most biota to val- Guignas are classified as Vulnerable with a decreasing
leys both east and west (Webb 1991). The stark climatic population on the IUCN Red List, and along with the
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Fig. 1 Morphological features of guigna subspecies. a L. guigna melanistic individual); d L. guigna guigna (Quetruleufu, Araucania
tigrillo (Molina, Maule Region, this study); b L. guigna guigna Region). Photo credits: a Luis Villanueva, b Andrés Charrier, c Jim
(Chiloé Island, this study); c L. guigna guigna (Chiloé Island, Sanderson, d Fauna Australis
Andean cat (Leopardus jacobita) is the most-threatened wild cat conservation issues worldwide, affecting over
South America cat species (Wozencraft 1993; Nowell and 75 % of the world’s felid species (Inskip and Zimmermann
Jackson 1996). In Chile, guignas are classified as Vulner- 2009). These conflicts for small cats usually involve live-
able (from Los Rı́os Region to the north) and as Near stock depredation and felids killed in retaliation (Woodr-
Threatened (from Los Lagos Region to the south) (CON- offe et al. 2005).
AMA 2011). Current threats include severe habitat loss, As reported by Zorondo (2005) and Silva-Rodriguez
fragmentation and direct persecution (Nowell and Jackson et al. (2007), most people in rural landscapes of central and
1996; Sanderson et al. 2002; Dunstone et al. 2002; Acosta- southern Chile had negative attitudes toward the guigna,
Jamett et al. 2003; Acosta-Jamett and Simonetti 2004; arguing livestock and poultry losses. Given this scenario,
Freer 2004; Silva-Rodriguez et al. 2007; Napolitano 2012; long-term conservation challenges for the guigna outside
Gálvez et al. 2013; Herrmann et al. 2013). protected areas will depend on the increase of local
Wild cats generally require large areas over which to awareness to reduce conflict in areas where they are con-
forage, thus are particularly affected by land use change sidered poultry pests, highlighting the services provided by
and the resulting loss of prey species and habitat (Lin- its role as controller of mice and European hares (Lepus
denmayer and Fischer 2006). The spatial extent of pro- europaeus) (Silva-Rodriguez et al. 2007; Gálvez et al.
tected areas alone is usually not enough for the long-term 2013) and also improving chicken coops (IUCN 2013).
viability of many cat species (Woodroffe and Ginsberg Another important challenge is preserving native vegetation
1998). Given this, the conservation of cats in private lands corridors to provide connectivity between forest fragments
outside protected areas has gained relevance (Simonetti or larger forested areas (Dunstone et al. 2002; Sanderson
and Acosta 2002). In the end, wild cat conservation hinges et al. 2002; Gálvez et al. 2013), since human populations
in how rural people perceive and know them, being human and deforestation are increasing in the Chilean temperate
attitudes among the most important issues when cat con- rainforest (Willson et al. 2005) and climate change may be
servation is the intended goal (Sillero-Zubiri and Lauren- an emerging additional threat (Malcolm et al. 2005).
son 2001; Silva-Rodriguez et al. 2007; Herrmann et al. From an evolutionary perspective, guignas are closely
2013). Human-felid conflicts are one of the most urgent related to six other small Neotropical cats of the genus
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Fig. 2 Mitochondrial haplotype network of guignas. Each circle in geographical groups shown in the map along with the sample size per
the network corresponds to a different haplotype, the size of the group. Sampling localities are shown with sample size per locality.
circles correspond to haplotype frequencies, the numbers associated The brown-shaded area in the map corresponds to the geographical
to each circle correspond to the number of individuals displaying that distribution of the northern subspecies L. g. tigrillo and the grey-
haplotype, and the colors of the circles correspond to the different shaded area corresponds to the southern subspecies L. g. guigna
Leopardus belonging to the Ocelot Lineage (Johnson et al. of geological events and areas of high conservation priority
2006). This exclusive Neotropical lineage diverged from a (Bermingham and Moritz 1998; Moritz 2002). In recent
common ancestor around the formation of the Panamanian years, phylogeographical knowledge in southern South
land bridge 2.8 million years (Ma) (Johnson et al. 1998, America has been accumulating for both aquatic and ter-
1999, 2006). Guignas’ sister species, the Geoffroy’s cat (L. restrial organisms (Sérsic et al. 2011). Concordant demo-
geoffroyi), which last shared a common ancestor less than graphic or spatial patterns across major organismal groups
1 Ma (Johnson et al. 2006), is found along the eastern side bring light into past geological events occurred in this
of the Andes mountain range, generally exhibiting a dis- region, and these types of comparisons require intraspecific
junct distribution (Nowell and Jackson 1996). data from multiple species (Lessa et al. 2010). This study
The objective of this study was to investigate population intends to build upon this knowledge and facilitate further
evolutionary history, uncover phylogeographic patterns of regional comparative phylogeographic hypotheses.
genetic diversity and to understand demographic partitions It is likely that the geological and environmental history
relevant to the conservation of guignas by addressing: of a certain region produced similar impacts on a regional
(i) Patterns of genetic structure relative to historic climatic biota with comparable natural history and ecological
events in southern South America and (ii) the influence of requirements. Thus, although species-specific, our results
historic and contemporary geographic barriers to gene might be extrapolable to other carnivores in southern South
flow, specifically testing the influence of the Andes and the America with vegetation cover as their main ecological
Chacao channel. requirement.
More generally, phylogeographical inferences, applied This study is the first to describe the genetic diversity
in a comparative framework across multiple species at a and demographic partitions of guignas, hence providing
regional scale, enables the detection of regional and land- much-needed information to guide suitable conservation
scape-level patterns of biodiversity, which are important strategies for the long-term preservation of these
for understanding macroecology, evolution, broad impacts populations.
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Table 1 Morphological measurements of guigna subspecies from captured individuals in this study
Subspecies n Locality Sex Coat Estimated age Age range Weight Body Tail Head Neck Hind foot Ear length
colour (months) classification (kg) lengtha length circumference circumference length (cm) (cm)
(cm) (cm) (cm) (cm)
L. g. 1 Molina, M Spotted 24 Adult 3.0 56.0 25.0 22.0 16.5 11.0 3.5
tigrillo Maule
Region
L. g. 10 Chiloé Island F Spotted 5 Juvenile 0.8 34.0 18.0 17.0 12.0 9.5 3.5
guigna
Chiloé Island F Spotted 12 Subadult 1.4 46.0 22.0 17.0 17.0 8.0 3.5
Chiloé Island F Spotted 8 Subadult 1.8 45.0 20.0 16.0 13.0 10.0 3.0
Chiloé Island M Spotted 12 Subadult 1.8 45.0 22.0 20.0 15.5 10.0 4.3
Chiloé Island M Spotted 18 Adult 1.8 47.0 22.0 19.0 17.0 9.5 4.0
a
Not including tail length. M male, F female. Individuals were classified as juvenile, subadult or adult on the basis of size, weight, dentition, and reproductive condition. Independent animals
that had not yet reached the age of full sexual maturity were classed as subadults
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Materials and methods strands were sequenced using an ABI 3730XL Analyzer by
Macrogen Inc. (Korea).
Sample collection Fifteen nuclear DNA microsatellite loci [twelve tetra-
nucleotide repeat loci (FCA441, F124, F41, FCA424,
Samples of 116 guignas were obtained from 22 sites F141, F146, FCA391, FCA453, F42, F98, F164, F27) and
(n = 1–20 samples per site) across most of its distribution three dinucleotide repeat loci (FCA008, FCA176,
(Fig. 2). To test barriers and subspecific partitions, indi- FCA698)] developed originally for the domestic cat
viduals were placed into six groups: Northern, Central, (Menotti-Raymond et al. 1999) and partially tested in
Lake District, Argentinian, Chiloé Island and San Rafael guignas (Johnson et al. 1999; Ruiz-Garcı́a et al. 2001) were
Lake. The Northern and Central groups correspond to the amplified separately by PCR in a 15 lL volume containing
range of L. g. tigrillo while the others correspond to the 1.5 lL 109 PCR Buffer, 1.5–2.0 mm MgCl2, 0.2 mm
range of L. g. guigna). Three L. geoffroyi and one L. wiedii each dNTP, 0.5 U of Taq DNA polymerase (Invitrogen),
were used as outgroups for the phylogenetic analyses. and 0.16 lm of the reverse primers, 0.064 lm of the for-
ward primer and 0.12 lm of the fluorescent dye-labeled
Laboratory procedures M13 tails (Schuelke 2000). Thermocycling parameters
consisted of an initial denaturation at 95 °C for 5 min, 35
Genomic DNA was extracted from whole blood from wild- cycles of 95 °C for 30 s, 53–63 °C for 30 s (annealing
caught individuals, tissues (liver, kidney or muscle) from temperature varied among loci, Table S1), 72 °C for 30 s,
recent road-kills, skin fragments from pelts found in local and a final extension of 72 °C for 10 min. To ensure that
communities and museums (Table S3) and faeces (only one allelic dropout or other genotyping errors have not com-
faeces sample; preserved in 95 % ethanol, 4° or -20 °C) promised our microsatellite data, we used: (i) multiple
using commercially available kits (DNeasy Blood and tubes approach, were each amplification was repeated
Tissue kit; QIAamp DNA Stool Mini Kit) following the twice per locus (Taberlet et al. 1996, 1999; Navidi et al.
manufacturer’s suggested protocol. Nucleotide sequences 1992; Creel et al. 2003; Frantz et al. 2003; Bellemain and
of four mtDNA gene segments encompassing 1,798 base Taberlet 2004; Smith et al. 2006); (ii) randomly reanalysed
pairs (bp), were obtained by PCR amplification (Saiki et al. 30 % of the samples per locus (Bonin et al. 2004; Smith
1985) from genomic DNA for: (i) NADH dehydrogenase et al. 2006); (iii) the program Micro-Checker 2.2.3 (Van
subunit 5 (NADH-5, 720 bp) using primers ND5-DF1 and Oosterhout et al. 2004) to identify genotyping errors, null
ND5-DR1 (Trigo et al. 2008), (ii) the 16S rDNA gene alleles or allele dropout in the data. PCR products were
(364 bp) as in Hoelzel and Green (1992) and Johnson et al. checked on an ethidium-bromide stained 1.5 % agarose gel
(1998), (iii) the adenosine triphosphate (ATP-8) and part of and sent for direct fragment analysis using an ABI Prism
the ATP-6 gene (275 bp) using primers ATP8-DF1 and 3730xl DNA Analyzer at the University of Illinois DNA
ATP6-DR1 (Trigo et al. 2008) and (iv) the 50 portion of the Core Sequencing Facility (USA).
Control Region (CR), containing the First Hypervariable
Segment (HVS-I) (439 bp) using primers CHF3 and CHR3
(Freeman et al. 2001). These four particular mtDNA gene MtDNA data analysis
segments were chosen because they are polymorphic, well
described, have a good collection of reference sequences Sequences (forward and reverse) were aligned using Pro-
and are broadly used in felid and mammal studies so rep- Seq 2.91 (Filatov 2002) against reference sequences and
resent an excellent source for comparative purposes. PCR checked by eye. The four mtDNA gene segments were
reactions were performed in a 25 lL volume containing concatenated (Huelsenbeck et al. 1996). The number of
1.5 lL 109 PCR buffer, 1.5–2.0 mm MgCl2, 0.2 mm each haplotypes and polymorphic sites, gene diversity, differ-
dNTP, 0.5 U of Taq DNA polymerase (Invitrogen), and ences between pairs of sequences (G) and nucleotide
0.2 lm of each primer. Thermocycling parameters con- diversity (p per nucleotide site) were estimated with Ar-
sisted of an initial denaturation at 94 °C for 5 min, 35 lequin 3.5.1.2 (Excoffier and Lischer 2010). Rarefaction
cycles of 94 °C for 1 min, 53–55 °C for 1 min (pending analysis with PAST (Hammer et al. 2001) was used to
primer sets), 72 °C for 1 min and a final extension of 72 °C adjust for unequal samples sizes for comparisons among
for 10 min. PCR amplifications of faecal samples were geographic groups.
repeated at least twice for each gene fragment to ensure A maximum-likelihood (ML) tree was constructed with
repeatability of species identification and haplotype MEGA 5.05 (Tamura et al. 2011) using the Tamura-Nei
assignment. PCR products were checked using ethidium- model of sequence evolution. Node support was obtained
bromide stained 1.5 % agarose gels. Forward and reverse by bootstrap analysis using 1,000 resampling steps.
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A haplotype network was generated using the median- to test for patterns of geographic distribution of genetic
joining approach method (Bandelt et al. 1999) imple- distances, a surface of genetic landscape was constructed
mented in Network 4.6. using Alleles in Space (Miller 2005; Miller et al. 2006),
Patterns of phylogeographic structure were tested using which applies a Delaunay triangulation connectivity net-
GST and NST coefficients (Burban et al. 1999) as imple- work to all sample sites and assigns a genetic distance to
mented in PERMUT (Pons and Petit 1995, 1996). Both of the middle point for each pair of sites. Genetic distances
these parameters will estimate the relationship between over the landscape surface were then interpolated (on a
average intrapopulation and total genetic diversity, but 50 9 50 grid of uniformly spaced cells) throughout the
only GST uses haplotype frequencies while NST uses dif- sampled geographic range, providing a qualitative graphic
ferences between haplotypes. The distribution of NST from representation of genetic distances.
1,000 permutations was compared with observed NST Inferences of population expansion or contraction were
values. NST values significantly larger than GST indicate based on mismatch distribution analyses (Rogers and
haplotype phylogeographic structure because similar hap- Harpending 1992) and estimates of neutrality tests such as
lotypes are more likely within a population than between Tajima’s D (Tajima 1989), Fu and Li’s F* and D* (Fu and
populations. Measures of genetic distance among geo- Li 1993), and Fu’s Fs (Fu 1997) were computed in DnaSP
graphic units (pairwise FST) were estimated with Arlequin 5.1 (Librado and Rozas 2009). Time estimates were cal-
3.5.1.2 (Excoffier et al. 1992). culated using a divergence rate of 0.67 %/MY, assessed
Two different clustering methods were used to infer the from a felid-specific mitochondrial divergence rate as in
spatial genetic structure of guigna populations. First, we Johnson et al. (1999). We acknowledge the possibility of
used SAMOVA 1.0 (Dupanloup et al. 2002), which defines some inaccuracy in estimated dates when using divergence
groups of populations that are geographically homoge- rates. Effective population sizes for each geographic group
neous and maximally differentiated from each other, to (Ne) and migration rates between groups (Nem) were
investigate population subdivision using analysis of estimated with LAMARC (Kuhner 2006).
molecular variance in a geographical context and to help
identify genetic barriers between groups. This method uses Microsatellite data analysis
a simulated annealing procedure, maximizing the propor-
tion of total genetic variance between groups of popula- Microsatellite genotypes were scored with GeneScan 3.7
tions. Statistical significance was tested using 1,000 (ABI) and Peak Scanner 1.0 (ABI) to precisely calibrate
permutations. Second, we estimated the number of clusters, allele sizes. Microsatellite data was analyzed with Genepop
as well as the spatial boundaries among them using a 4.0.10 (Raymond and Rousset 1995; Rousset 2008) for
Bayesian model computed with the GENELAND package, general diversity estimates including heterozygosity,
version 4.0.0 (Guillot et al. 2005) in the R environment (R, number and allele size range per locus and to test for
version 3.0.2; Ihaka and Gentleman 1996). This software deviations from Hardy–Weinberg equilibrium. Rarefaction
implements a Markov chain Monte Carlo (MCMC) pro- analysis was used to adjust for unequal samples sizes for
cedure to determine the best clustering of samples using comparisons among geographic groups. Genetic distances
genetic and geographical information. Geographical among geographic units (pairwise FST) were estimated with
information is taken into account at the Bayesian prior Arlequin 3.5.1.2. We used SAMOVA 1.0 to investigate
level. 5,000,000 MCMC iterations sampled each 1,000 population subdivision using analysis of molecular vari-
steps with a 4,900 burn-in period, using both the correlated ance in a geographical context, identifying the most likely
and uncorrelated frequency models and a maximum num- position (using 1,000 permutations) of inferred historical
ber of clusters K = 10 were run to estimate the model genetic barriers. Isolation by distance was assessed through
parameters and posterior probabilities of group member- correlation of genetic and geographic distances among
ship. The correlated frequency model sets a more realistic samples (mean genetic distance value for each unit) per-
scenario because most often, allele frequencies tend to be formed in Arlequin 3.5.1.2 with 100,000 permutations.
similar in different populations (Nicholson et al. 2002; We used Structure 2.3.3 (Pritchard et al. 2000) to define
Balding 2003). Described as a more biologically grounded the contemporary population structure through the defini-
way to make inference, it has been observed that using the tion of clusters or groups and assignment of individuals to
correlated frequency model could be more powerful at these clusters using 100,000 iterations, 100,000 MCMC
detecting subtle differentiations (Guillot et al. 2005). and an admixed ancestry model. We discarded individuals
To test for isolation by distance, we assessed the cor- with \50 % data (n = 16). We evaluated K = 1–8 popu-
relation between genetic and geographic distances among lations and results from 10 replicates to evaluate the vari-
samples (mean genetic distance for each unit), performed ance and stability of likelihood values. We also used
in Arlequin 3.5.1.2 with 100,000 permutations. In addition, BayesAss 1.3 (Wilson and Rannala 2003) to estimate
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recent migration rates (during the last several generations) the south (Lake District, Chiloé Island, Argentinian and
between populations, defined as the proportion of individ- San Rafael Lake groups). NST coefficients (0.421 ± 0.122)
uals in each generation that are not migrants. were significantly higher than GST values
(0.177 ± 0.0988) (p \ 0.001), suggesting that there was a
phylogeographic structuring of haplotypes and that within
Results a population it is more likely to find genetically related
than genetically distant haplotypes. There were fewer
We obtained 1,798 bp of mtDNA sequence for 87 indi- haplotypes in the Northern group (n = 7) compared with
viduals from four mitochondrial genes: NADH-5 (720 bp), Central, Lake District and Chiloé Island groups, which had
16S rDNA (364 bp), CR HVS-I (439 bp), ATP-8 and part a higher number of variable and divergent haplotypes
of the ATP-6 (275 bp), excluding 29 individuals without (n = 16). The four haplotypes from the Argentinian group
complete sequence data. Polymorphic sites (total = 55) are did not cluster independently, but grouped with Lake
mostly found in the CR HVS-I (37 sites, 67.3 %), followed District, Chiloé Island and most of San Rafael Lake
by NADH-5 (16 sites, 29.1 %) and 16S rDNA (2 sites, haplotypes.
3.6 %) genes. No polymorphic sites were found in the There is a shared haplotype among Northern and Central
ATP-8 and part of ATP-6 gene. To assess possible Numt groups, suggesting a possible historical connection (Fig. 2).
content, we checked the reading frames for the whole data There is a shared haplotype among Lake District and
set of protein coding (ND5) or RNA (16S) genes. No Chiloé Island groups, specifically between Puerto Montt
insertion/deletion or stop codons were detected. (locality 15) and Northern Chiloé Island (locality 20),
Regarding microsatellite loci, we obtained complete supporting historical connectivity across the land bridge
multilocus genotypes for 102 individuals. Locus F164 was during the last glacial period (Figs. 2, 3). Chiloé Island and
monomorphic and F27 resulted in unreliable genotyping Argentinian groups share their most common haplotype,
without concordance between independent amplifications, supporting a recent connection of the island with the con-
so they were not included in the subsequent analyses. There tinent and also suggesting that the Andes was not a com-
was 100 % concordance among replicates from the 13 plete barrier for gene flow (e.g. low-elevation pass exist at
microsatellite loci included in the analyses. With F124 and several points in this area). Also, the Argentinian and San
F98, a few groups deviated from Hardy–Weinberg Equi- Rafael Lake groups share their most common haplotype,
libirum with heterozygous deficiency, but since this was specifically between Southern Chubut Province (locality
not observed in most populations we included them in our 19) and San Rafael Lake (locality 22), suggesting a pos-
analyses. Micro-Checker 2.2.3 did not identify genotyping sible post-glacial recolonization route and glacial refugia
errors, null alleles or allele dropout in our data. The 102 (Figs. 2, 3).
individuals included in the analysis had [50 % loci data, The maximum likelihood tree revealed no significant
most of them displaying 80–100 %. phylogenetic separations among different geographic
All 87 individuals with mtDNA sequences correspond to groups (Fig. 4). A highly divergent haplotype from San
individuals with known and different microsatellite mul- Rafael Lake group was the most basal guigna haplotype,
tilocus genotypes, so there is no overrepresentation of with good bootstrap support (84 %). Other sequences
samples. Reasons for having less individuals with complete located in basal positions were from the Lake District and
concatenated mtDNA sequences (87 individuals) compared Chiloé Island groups. Haplotypes from the Northern group
to individuals with complete microsatellite multilocus were the most recently derived.
genotypes (102 individuals) may include longer sequences
in mtDNA gene segments (275, 364, 439, 720 bp after Population structure
editing) compared to microsatellite loci (129–304 bp),
which makes the former harder to amplify when DNA is To explore population subdivision and genetic structure in
degraded. Besides, only individuals with the four mtDNA a geographical context, we performed a spatial analysis of
gene segments were included in the final analysis to molecular variance (SAMOVA) using mtDNA and
improve definition (more information). microsatellite loci data (Table 2). The mtDNA variation
among groups was largest (48.8 %) and the percentage of
Phylogeography and evolutionary history variation within groups lowest (-0.70 %) when guigna
samples were divided into five groups (48.08 %):
The mtDNA haplotype network (Fig. 2) showed a non- (i) Northern, (ii) Central, (iii) Lake District, (iv) Chiloé
random association among the 45 haplotypes that corre- Island and Argentinian, and (v) San Rafael Lake (Table 2).
sponded with their geographic origin, with L. g. tigrillo in SAMOVA analysis for the microsatellite data showed
the north (Northern and Central groups) and L. g. guigna in that variation among groups was the largest (27.30 %) and
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Fig. 3 Maximum extension of

ice sheets during the last glacial
event in southern South
America shaded in brown,
modified from Denton et al.
(1999). Location numbers
correspond to sampling
localities in Fig. 2. Arrow
shows proposed recolonization
route
the percentage of variation within groups the lowest frequency model was more powerful at detecting subtle
(-3.37 %) with four groups: (i) Northern, (ii) Central, (iii) differentiations in a biologically realistic scenario.
Lake District, Argentinian, and San Rafael Lake, and (iv) Bayesian structure analyses of microsatellite data
Chiloé Island (Table 2). (Fig. 6) suggested that the existing population is composed
Based on the mtDNA data, the Bayesian clustering of 3 clusters or groups (K = 3). Log likelihood values for
algorithm implemented in Geneland using the correlated each K were the following: K = 2 LnP(D) = -2,604.3;
frequency model, detected five main clusters (K = 5): K = 3 LnP(D) = -2,500.0; K = 4 LnP(D) = -2,518.4;
(i) Northern, (ii) Central, (iii) Lake District, (iv) Chiloé K = 5 LnP(D) = -2,582.0; K = 6 LnP(D) = -2,535.6;
Island and Argentinian, and (v) San Rafael Lake (Fig. 5). K = 7 LnP(D) = -2,630.5. The 3 identified clusters cor-
The existence of these five clusters is supported by the respond to: (1) the Northern, Central, and Lake District and
results of the SAMOVA analysis. Using the uncorrelated some individuals from the Argentinian group, (2) Chiloé
frequency model, the Bayesian clustering detected only Island and some individuals from the Argentinian group,
two clusters (K = 2): (i) Northern and Central, (ii) Lake and (3) San Rafael Lake. Individuals from the Argentinian
District, Chiloé Island, Argentinian and San Rafael Lake group were assigned to the Northern, Central and Lake
(Figure S4). These two clusters coincide with subspecific District or to Chiloé Island. There were some individuals of
partitions, showing the boundary between them runs ‘‘mixed’’ heritage suggesting limited gene flow among
approximately across latitude 388S. The correlated these three geographical regions.
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Fig. 4 Maximum likelihood

tree for guigna haplotypes, three
sequences of L. geoffroyi (GEO)
and one sequence of L. wiedii
(WIE) are used as outgroups.
Bootstrap support values are
shown and the colors on
haplotype labels correspond to
different geographic groups
We explored the distribution of genetic diversity Argentinian groups. Genetic differentiation between the
through an analysis of landscape shape interpolation of Northern and Central groups (L. g. tigrillo)
genetic distances in geographic space (Figure S2) which (FST = 0.26494, p \ 0.001) was lower than between the
illustrated two peaks of genetic distance (greatest genetic Central and Lake District groups (representing the transi-
discontinuity in the landscape). These correspond with tional zone between L. g. tigrillo and L. g. guigna,
areas near Concepción (36°460 S, 72°530 W) and Los respectively) (FST = 0.34856, p \ 0.001) (Table 3).
Angeles (38°110 S, 72°170 W) which are in the transition Patterns of pairwise FST estimates using microsatellite
area between the two subspecies. To the north, maximum loci were similar (Table 3), but the San Rafael Lake and
genetic distances decreased with decreasing latitude, sug- Chiloé Island groups had a significant degree of differen-
gesting increased genetic connectivity. tiation with most of the other geographical groups, except
The mtDNA genetic differentiation increased with with the Argentinian and San Rafael Lake groups. Genetic
geographic distance between groups. There were low levels differentiation between the Central, Lake District and Ar-
of genetic differentiation between the Argentinian and gentinian groups was low and not significant. Genetic
Chiloé Island groups, and between the Lake District and differentiation between the Northern and Central groups of
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Table 2 Spatial analysis of molecular variance of mtDNA and microsatellite loci for different guigna geographic groups
Clusters mtDNA Microsatellite loci
% Variance % Variance between Clusters % Variance % Variance between
between groups populations within groups between groups populations within groups
2 groups 37.25* 18.45** 2 groups 20.50 n.s 12.34 n.s

1?2; 3?4?5?6 1; 2?3?4?5?6
3 groups 37.06* 14.37** 3 groups 22.24 n.s 6.45 **
1?2; 3?4?5; 6 1; 2; 3?4?5?6
4 groups 37.58* 10.29** 4 groups 27.30* 23.37 n.s
1?2; 3; 4?5; 6 1; 2; 3?5?6; 4
5 groups 48.08* 20.70** 5 groups 26.95 n.s -4.28 n.s
1; 2; 3; 4?5; 6 1; 2; 3?5; 4; 6
1 Northern group, 2 Central group, 3 Lake District group, 4 Chiloé Island group, 5 Argentinian group, 6 San Rafael Lake group
* Significant (p \ 0.05); ** significant (p \ 0.001); n.s. non significant (p [ 0.05)
In bold, largest variation among groups and lowest variation within groups
Fig. 5 Spatial output from Geneland using the correlated frequency belong to the different clusters. Black circles indicate the relative
model for mtDNA data of all guigna samples. a Number of position of the sampled populations. Darker and lighter shading are
populations (K = 5); b relative position of sampled populations; proportional to posterior probabilities of membership in clusters, with
c map of population membership; d map of posterior probabilities to lighter (yellow) areas showing the highest probabilities of clusters
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Fig. 6 Genetic population structure and assignment of individuals to color represents the probability of assignment to the cluster of that
clusters for different guignas geographic groups based on microsat- color. Log likelihood values, K = 2 LnP(D) = -2,604.3; K=3
ellite loci information. Each vertical line represents an individual. LnP(D) = -2,500.0; K=4 LnP(D) = -2,518.4; K=5
Individuals are grouped according to their location of origin. Clusters LnP(D) = -2,582.0; K=6 LnP(D) = -2,535.6; K=7
are encoded with 3 different colors and the fraction of each individual LnP(D) = -2,630.5
Table 3 Measures of genetic distance (pairwise FST) using mtDNA and microsatellite loci between guigna geographic groups
Geographic groups Northern Central Lake District Argentinian Chiloé Island San Rafael Lake
Northern (32.5–35° S) – 0.101** 0.132* 0.158* 0.203** 0.334**

Central (36–37.5° S) 0.26494** – 0.027 n.s 0.011 n.s 0.110** 0.153**
Lake District (38.5–41.5° S) 0.61506** 0.34856** – 0.063* 0.053* 0.215**
Argentinian (39–44° S) 0.71665** 0.35911** 0.16568* – 0.078* 0.003 n.s
Chiloé Island (41.7–43.5° S) 0.64245** 0.45663** 0.26057** 0.03932n.s. – 0.202 **
San Rafael Lake (46.5° S) 0.75299** 0.46474** 0.36240** 0.36646** 0.42667** –
mtDNA information below diagonal, microsatellite loci information above diagonal
* Significant (p \ 0.05); **significant (p \ 0.001); n.s. non significant (p [ 0.05)
L. g. tigrillo were higher (FST = 0.101, p \ 0.001) than the Microsatellite analyses reflected more-contemporary
differentiation between Central and Lake District groups barriers to gene flow and migration (Table 5). Rates from
(corresponding to the transitional zone between L. g. tig- the Lake District to the Northern and Central groups were
rillo and L. g. guigna, respectively) (FST = 0.027, the highest. San Rafael Lake and Chiloé Island groups had
p [ 0.05). very low rates of migration with the other geographic
There was a highly significant positive correlation groups and very high intra-population migration rates.
between geographic distance and genetic distance among Migration rates were unequal between the Northern and
all guigna samples for both mtDNA (r = 0.8084, Lake District groups, between the Central and Lake District
p = 0.001) and microsatellite data (r = 0.762, p = 0.006). groups, and among Lake District, Argentinian and San
Genetic differentiation increased with geographic distance Rafael Lake groups.
(Fig S1) throughout the species’ distribution. Differences in bidirectional migration rates may be
affected by smaller sample sizes (e.g. between Argentinian
Gene flow among populations group and all the other groups), because they increase the
variance of the posterior probabilities distribution,
The Central group had the highest effective population size decreasing its accuracy when compared to larger sample
and the Northern and San Rafael Lake groups the lowest sizes (Wilson and Rannala 2003).
using mtDNA data (Table 4). Migration rates from the
Northern group to the Central group (Nem = 0.000424) Intrapopulation diversity and bottleneck events
were approximately one order of magnitude higher than in
the opposite direction (Nem = 0.0000127). There were Overall, guignas had medium to high mtDNA genetic
high migration rates from the Lake District to the Central diversity, with 45 haplotypes, 55 polymorphic sites, hap-
group and from the Argentinian to Chiloé Island group. lotype diversity of 0.94 (±0.02) and an average 7.014
Low migration rates were observed from most geographic number of nucleotide differences between pairs of
groups to the Northern and the San Rafael Lake groups. sequences (Table 6). The Central, Lake District and
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Table 4 Effective population size (Ne) and bidirectional migration rates (Nem) among guigna geographic groups using mtDNA information
Geographic groups Ne To
From Northern Central Lake District Chiloé Island Argentinian San Rafael Lake
Northern 90,000 – 0.000424 2.245 0.00016 0.000035 0.506

Central 1,800,000 0.0000127 – 1.32 0.271 0.000018 0.000006
Lake District 500,000 0.7154 9.892 – 0.181 0.1448 0.000007
Chiloé Island 360,000 0.00032 1.127 0.314 – 0.7253 0.182
Argentinian 290,000 0.0000844 0.00016 3.9 7.0124 – 0.188
San Rafael Lake 97,000 0.000029 0.00029 0.000073 0.00006 4.345 –
Table 5 Recent bidirectional migration rates (Nem) among guigna geographic groups using microsatellite loci information
From To
Northern Central Lake District Argentinian Chiloé Island San Rafael Lake
Northern 0.750 – 0.05 0.024 ± 0.03 0.008 ± 0.01 0.024 ± 0.03 0.002 ± 0.00 0.005 ± 0.01
Central 0.014 ± 0.02 0.719 – 0.04 0.009 ± 0.02 0.028 ± 0.03 0.003 ± 0.01 0.007 ± 0.01
Lake District 0.201 ± 0.05 0.203 ± 0.05 0.917 – 0.07 0.089 ± 0.06 0.002 ± 0.00 0.006 ± 0.01
Argentinian 0.011 ± 0.02 0.012 ± 0.02 0.008 ± 0.01 0.713 – 0.04 0.002 ± 0.00 0.004 ± 0.01
Chiloé Island 0.014 ± 0.02 0.014 ± 0.02 0.014 ± 0.02 0.100 ± 0.06 0.989 – 0.01 0.005 ± 0.01
San Rafael Lake 0.010 ± 0.01 0.029 ± 0.03 0.044 ± 0.06 0.046 ± 0.04 0.002 ± 0.00 0.973 – 0.03
Main diagonal (in bold): Migration rates from one population to the same population corresponds to the proportion of individuals in each
generation who are not migrants
Argentinian groups had the highest genetic variability and around 89000 years BP. The mismatch distribution for all
Chiloé Island and San Rafael Lake groups, the lowest. The guigna sequences had a unimodal distribution representing
Northern group had the lowest average number of differ- a demographic expansion event an estimated 318000 years
ences between pairs of sequences, while the Central group BP (Tau = 4.115) (Fig. 7d).
had the highest number of pairwise differences. Guignas To further explore our results, we unlinked the sequence
had both moderate levels of microsatellite loci heterozy- data and carried out mismatch distribution analysis exclu-
gosity and number of alleles per locus (Table 6). Average sively with D-loop, the most informative segment. We also
heterozygosity decreased from north to south. The Argen- performed a Bayesian skyline plot (BSP) analysis imple-
tinian group had no private alleles and the Central group mented in BEAST, version 1.7 (Drummond and Rambaut
had the most. 2007) exclusively for D-loop sequences, using the
We inferred historic demographic changes for each GTR?G?I model previously estimated with MrModeltest
geographical group. The San Rafael Lake group showed version 2.3 (Nylander 2004). The D-loop mismatch dis-
evidence of a contraction–expansion demographic event tribution analysis showed similar patterns of population
supported by Tajima (D = -1.93, p \ 0.05) and Fu and Li expansion as for the populations in the total concatenate
(D = -2.21, p \ 0.05; F = -2.42, p \ 0.05) tests (Table analysis (Figure S3). In the case of the BPS analysis, it did
S2). Results for all other geographic groups were not sig- not show clear signals of population size change in any of
nificant. The Central, Lake District, Argentina and Chiloé the populations (Figure S3). We believe that low sample
Island groups had multimodal mismatch distribution pat- size and sampling strategy (local or scattered sampling)
terns (Fig. 7), suggesting populations in demographic may be affecting the BPS analysis, impeding us to detect
equilibrium (Rogers and Harpending 1992). In contrast, the recent population size changes, as has been recorded in
San Rafael Lake group had a main peak (Fig. 7a) sugges- simulations by Heller et al. (2013). Recent (Holocene or
tive of a recent demographic expansion around 9,800 years Pleistocene) population size changes were unobservable in
BP (after removing the highly divergent haplotype from the scenarios under local sampling or scattered sampling,
analysis, Tau = 0.2) (Fig. 7b). The Northern group had a leading to the false negative of failing to detect a true
unimodal mismatch distribution pattern (Fig. 7c), sug- population expansion towards the present (Heller et al.
gesting a demographic expansion event (Tau = 1.82) 2013). Other studies have also highlighted the danger of
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Table 6 Measures of genetic diversity for mtDNA and microsatellite loci in different guigna geographic groups
Geographic n Number of Number of Haplotype Average number of nucleotide Nucleotide Rarefactiona:
groups haplotypes polymorphic diversity differences between pairs of diversity Number of
(K) sites (S) (H) sequences (G) (p) haplotypes (Ka)
mtDNA
Total 87 45 55 0.94 ± 0.02 7.014 0.00461
Northern 12 7 7 0.89 ± 0.06 1.818 0.00119 6.59 ± 0.52
(32.5–35°S)
Central 10 10 23 1.00 ± 0.03 8.026 0.00527 10.00 ± 0.00
(36–37.5°S)
Lake District 12 9 14 0.91 ± 0.08 5.667 0.00372 7.96 ± 0.64
(38.5–41.5°S)
Argentinian 5 4 10 0.90 ± 0.16 4.800 0.00315
(39–44°S)
Chiloé Island 37 16 20 0.76 ± 0.08 4.204 0.00276 8.32 ± 0.92
(41.7–43.5°S)
San Rafael 11 3 14 0.35 ± 0.17 2.691 0.00177 3.00 ± 0.00
Lake (46.5°S)
Geographic groups n Average Average number Average allele Number of Rarefactiona: Average
Heterozygosity of alleles per locus size range private alleles number of alleles per locus
Microsatellite loci
Total 102 0.49 ± 0.1 6.54 ± 2.0 21.39 ± 11.5
Northern (32.5–35°S) 16 0.58 ± 0.2 4.31 ± 1.6 14.77 ± 10.0 5 3.99 ± 0.1
Central (36–37.5°S) 17 0.52 ± 0.2 4.08 ± 1.3 14.77 ± 11.2 7 3.99 ± 0.1
Lake District (38.5–41.5°S) 12 0.51 ± 0.2 4.08 ± 1.7 11.69 ± 6.3 2 4.08 ± 1.7
Argentinian (39–44°S) 5 0.48 ± 0.3 3.17 ± 1.1 9.67 ± 7.0 0 _
Chiloé Island (41.7–43.5°S) 38 0.49 ± 0.1 4.54 ± 1.7 13.54 ± 8.1 4 3.94 ± 0.2
San Rafael Lake (46.5°S) 14 0.39 ± 0.2 2.92 ± 0.8 9.0 ± 4.3 1 3.0 ± 0
a
Rarefaction curves to compare the average number of alleles per locus in geographical groups with different sample sizes
violating the panmixia assumption when inferring popula- modern molecular techniques is not uncommon (Burbrink
tion size changes for BSP inference in natural populations et al. 2000; Zink 2004). Reciprocal monophyly is less
(Peter et al. 2010; Ho and Shapiro 2011). likely to be observed in species with high gene flow in
natural environments (e.g. birds or medium to large
mammals) (Waits et al. 1998; Wayne et al. 1992; Haig
Discussion et al. 2001), or in recently diverged subspecies at an early
stage of differentiation (Phillimore and Owens 2006).
Subspecific partitions Discordant results may also suggest that morphological
differences used to define subspecies could represent phe-
A lack of reciprocal monophyly was revealed in the notypic plasticity in differing environments rather than
mtDNA phylogenetic tree, with no major partitions or long-term genetic isolation (Waits et al. 1998), or may be
statistically different lineages within guignas. This pattern due to historical processes such as introgression or
reflects the relatively recent evolutionary history of guignas incomplete lineage separation (Funk and Omland 2003).
(400000 BP, Johnson et al. 1999; 318000 BP, this study), Despite the lack of reciprocal monophyly, a phyloge-
which is likely insufficient time to cause complete sepa- ographic structuring of haplotypes according to their
ration of intraspecific lineages. Moreover, the large home geographic origin is shown in the mtDNA haplotype
ranges and dispersal ability of guignas (Dunstone et al. network. Individuals from the northern (L. g. tigrillo) and
2002; Sanderson et al. 2002), exemplified by evidence of southern (L. g. guigna) subspecies are generally distrib-
gene flow between major mainland populations, might also uted into distinct groups, although with a low degree of
have contributed to less differentiation among groups. genetic differentiation (1–11 substitutions). Geneland
Contrasting patterns between subspecies defined phe- analysis (uncorrelated model, Fig S4), landscape shape
notypically and their phylogenetic clusters identified using interpolation analysis (Fig S2) and population
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Fig. 7 Mismatch distribution of guigna sequences in different e central; f Lake District; g Argentinian; h Chiloé Island. Dotted black
geographical groups. a San Rafael Lake group, all sequences; line observed values; continuous red line expected values under a
b San Rafael Lake group, all sequences after removing the highly model of population growth-decline
divergent haplotype; c northern group; d all guigna sample sequences;
distinctiveness analysis to define Management Units for Incipient divergence between both guigna subspecies
Conservation (Fig. 8) spatially separate the two subspe- may reflect physiological adaptation of ecotypes to differ-
cific groups. ent environmental conditions, given that the patterns of
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Fig. 8 Categories of population

distinctiveness to define
management units for
conservation in guignas. Null
hypotheses of genetic and
ecological exchangeability are
scored as rejected (?) or not
rejected (-) for both recent and
historical time frames, as in
Crandall et al. (2000)
genetic differentiation between subspecies coincide with mismatch distribution analysis (89000 BP). Low intraspe-
underlying ecosystem discontinuities. The northern bio- cific nucleotide differentiation and the smallest estimated
geographical macroregion (31°–38°S), is temperate and effective population size (Ne = 90,000) of all guigna
mesomorphic, has a mediterranean climate with a distinct geographic groups also points out to this more recent
dry season (Kottek et al. 2006), and is mainly dominated by demographic history of the Northern group. This south to
sclerophyll scrub and forest vegetation (Gajardo 1994). north latitudinal pattern may be linked to the consecutive
The southern biogeographical macroregion (38°–43°S), is glacial events during the late Pleistocene (Mercer 1983),
temperate and higromorphic, with humid and cold rainy which may have promoted population migration to lower
climates (Kottek et al. 2006), dominated by dense ever- latitudes in search of non-glaciated more favorable areas
green temperate rainforests and north-Patagonian forests (Mercer 1983; Moreno et al. 1999). Habitat tracking has
(Gajardo 1994). been exemplified by various studies (Eronen and Rook
2004; Parmesan 2006; Devictor et al. 2008; Hofreiter and
Phylogeographic and evolutionary patterns Stewart 2009; Reside et al. 2013), even though a lack of
postglacial habitat tracking was shown for a mid-sized
A south to north latitudinal pattern is present in the mtDNA Northern Hemisphere carnivore (Dalén et al. 2007). The
phylogenetic tree, with the most-basal and most-divergent LGM-to-present climate-change velocity exhibits marked
haplotypes found in the southern regions (San Rafael Lake, geographic variation, with peaks in northeast North
Chiloé Island and Lake District groups), while the most America and north-central Eurasia, while velocities tended
derived haplotypes belong to northern regions (Northern to be lower in the Southern Hemisphere (Sandel et al.
and Central groups). The most ancestral lineage within 2011). Thus, a certain pattern for a Northern Hemisphere
guignas is the highly divergent haplotype from San Rafael species may not be comparable to a Southern Hemisphere
Lake group, which would be consistent with the divergence species. In addition, strong dispersers like the guigna may
of guignas from Geoffroy’s cat haplotypes following track climate fairly closely avoiding extinction (Graham
migration from the west and subsequent isolation by the et al. 2010; Sandel et al. 2011). The individualistic
Andes. Movement across the Andes would have been response of species to climate change suggests that it is
facilitated at higher latitudes given that the Andes average very difficult to precisely predict the responses of indi-
elevation decreases from around 5,000 m to around vidual species to rapid climate change, as each species
2,000 m southwards (Aragón et al. 2011). The most responds differently (Bennett and Provan 2008; Stewart
derived haplotypes from the Northern group suggest a 2008, 2009; Hofreiter and Stewart 2009; Graham et al.
more-recent geographic expansion, as was also inferred by 2010).
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A major phylogeographic break was inferred around mtDNA SAMOVA, correlated frequency model Geneland
38°S, corresponding with the transitional area between analysis and historical migration-rate estimates. Since
biogeographical macroregions and subspecies geographic elevation of the Andes decreases at higher latitudes (Ar-
distributions. For guignas, the major phylogeographical agón et al. 2011), movement across the Andes would have
break separated clades latitudinally, rather than longitudi- been facilitated in this region. Similarly, other authors have
nally from east to west as would be suggested by the reported the existence of connectivity and dispersal of
organization of macrohabitats or segregating units east and individuals between both sides of the Andes through low-
west of the Andes (Patterson 2010). With prevailing elevation mountain passes in southern latitudes (Smith
westerlies at temperate latitudes, the north–south orienta- et al. 2001; Palma et al. 2002, 2005; Himes et al. 2008;
tion of the Andes creates temperate and sub-antarctic Victoriano et al. 2008; Sérsic et al. 2011). On the other
rainforests on the Pacific slopes but a rain shadow on the hand, recent high genetic connectivity between both sides
eastern side of Patagonia. Thus, major floristic associations of the Andes is supported by microsatellite SAMOVA,
in southern South America are often oriented in north– migration-rate estimates, and genetic assignment analysis
south strips, rather than the latitudinal bands observed in in Structure, where individuals from the Argentinian group
most other regions. The general latitudinal organization of do not constitute an independent cluster.
the phylogeographical breaks in southern South America, The lack of private alleles or distinct genetic structure of
as observed with guignas, suggests the predominance of the Argentinian group supports a scenario of high levels of
historical signal over ecological determinism (Patterson connectivity between the Andes, perhaps combined with
2010); although a mosaic of phylogeographical patterns the recent establishment of Argentinian populations from
occurs in Patagonia (Sérsic et al. 2011). Similar phyloge- Chile. Either way, during the last glacial event, populations
ographic breaks have been observed in other species, of guignas persisted in ice-free regions in Argentina, par-
including the marsupial monito del monte (Dromiciops ticipating later in the recolonization of southern regions
gliroides) (39°S; Himes et al. 2008), several Patagonian- (i.e. San Rafael Lake) after the retreat of the ice sheets. The
Fueguian rodents (39–42°S; Lessa et al. 2010), three lizard sharing of the most common haplotype between the
species of the genus Liolaemus (38°S; Victoriano et al. southernmost Argentina locality and San Rafael Lake
2008), and several Patagonian terrestrial vertebrates (33°S, supports this postglacial recolonization route. Peripheral
35°S and 38°S; Sérsic et al. 2011). Similar patterns are also glacial refugia have been described in the eastern foothills
apparent in some plant species in the temperate rainforests of the Andes between 39°S and 43°S (Sérsic et al. 2011).
of southern Chile, including the tineo (Weinmannia At those latitudes, the ice sheet was confined to the higher-
trichosperma) (39°–40°S; Montenegro 2011), the ulmo elevation summits with most of the foothill forests
(Eucryphia cordifolia) (40°S; Segovia et al. 2012), the tepa remaining unglaciated, thus allowing persistence of dif-
(Laureliopsis philippiana) (40°S; Bosshard 2011) and the ferent taxa through Quaternary climate shifts (Heusser
lenga beech (Nothofagus pumilio) (38°S, 40°S and 43°S; et al. 1999). Similar southward postglacial colonization
Mathiasen and Premoli 2010). routes have been proposed from the east towards the
We acknowledge that additional sampling within the southwest at different latitudes for terrestrial vertebrates
unsampled area (40°S–45°S in continental Chile) of guigna such as the rodents Loxodontomys micropus (Sérsic et al.
geographic range may contribute to complete the evolu- 2011), Abrothrix olivaceus (Lessa et al. 2010) and Oli-
tionary patterns described here. Limited sampling can lead goryzomys longicaudatus (Palma et al. 2005), and the liz-
to the erroneous diagnosis of distinct populations when ards Liolaemus pictus and Liolaemus lemniscatus (Sérsic
sampling intermediate populations would show ongoing et al. 2011), all potential or described guigna prey-species
gene flow (Crandall et al. 2000). Therefore, we believe the (Correa and Roa 2005).
main genetic patterns showed in this study are unlikely to
change significantly, given that the unsampled area belongs The Chacao channel as a recent barrier to gene flow
to the same ecoregion as other thoroughly sampled areas
like Lake District and Chiloé Island, the Valdivian forest During the last glacial event in southern South America,
(Cofre and Marquet 1999). between 0.026 and 0.007 Ma, a land bridge was formed
between Chiloé Island and the mainland, allowing the
The Andes range is not a barrier to gene flow movement of many fauna species and effective gene flow
(Vidal et al. 2012). High historical connectivity between
The Andes mountain range was neither a historical nor a Chiloé Island and mainland populations is supported by the
current effective barrier to gene flow for guignas. Histori- mtDNA haplotype network, mtDNA SAMOVA, correlated
cally, high genetic connectivity between both sides of the frequency model Geneland analysis and historical migra-
Andes is supported by the mtDNA haplotype network, the tion rate estimates. The modern insularity of Chiloé Island
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was reached only about 7000 BP. The current isolation of categorization of population distinctiveness based on con-
Chiloé Island is evident from the assignment analysis in cepts of ecological and genetic exchangeability (Fig. 8).
Structure, where all individuals from Chiloé Island group This approach provides better insights into the conserva-
to a unique, independent cluster. Recent limited migration tion units that can best maintain evolutionary processes and
rates between Chiloé Island and all other geographic the potential for evolutionary change in the future
groups, along with microsatellite SAMOVA results also (Crandall et al. 2000).
support this demographic isolation scenario. Overall, the We propose at least two Management Units for Con-
Chacao channel is a recent effective barrier to gene flow for servation: (i) Northern group ? Central group, (ii) Lake
guignas, but was not a historical effective barrier to gene District group ? Argentinian group ? Chiloé Island
flow in the past. group ? San Rafael Lake group (Fig. 8). Each Manage-
ment Unit warrants separate priority conservation and
San Rafael Lake should be monitored and managed independently (Taylor
and Dizon 1999; Crandall et al. 2000; Palsbøll et al. 2006).
The San Rafael Lake group experienced a significant Within Management Units, populations should be treated
demographic bottleneck during the last glacial event in as connected by various degrees of gene flow (Crandall
southern South America, when much of its current geo- et al. 2000). The two identified Management Units roughly
graphic range was covered by ice. This is likely reflected in coincide with the current conservation status classification
the relatively low levels of genetic diversity compared with groups for guignas in Chile: Vulnerable from Los Rı́os
the other geographic groups, one of the smallest estimated Region to the north, and Near Threatened from Los Lagos
effective population sizes (Ne = 97,000), and in both Region to the south (CONAMA 2011).
demographic inference tests (Tajima, Fu and Fu and Li) The two identified Management Units correspond to the
and the mismatch distribution analysis suggesting a con- traditional morphological subspecies. Evidence for rejec-
traction and expansion event around 9800 BP. Genetic tion of recent or historical genetic exchangeability alone is
footprints of demographic expansions following the retreat not sufficient to warrant separate priority conservation
of ice sheets during the last glacial event have been unless it is accompanied by adaptive divergence (Crandall
described in species located along previously glaciated et al. 2000) (Case 1 and 2, Fig. 8). Evidence for recent
areas in the Patagonian Andean cordillera (41°S–52°S) ecological nonexchangeability is indicative of the adaptive
(Sérsic et al. 2011), in several Patagonian rodent species divergence necessary for distinct population persistence
(Lessa et al. 2010), and also the rodents A. olivaceus (Crandall et al. 2000) (Case 3, Fig. 8).
(Smith et al. 2001; Rodrı́guez-Serrano et al. 2006) and O. Overall, levels of genetic diversity in guignas are rela-
longicaudatus (Palma et al. 2005; Belmar-Lucero et al. tively high for mtDNA data and moderate for microsatel-
2009) along the southern Andes. lites, compared to other South American felids (Eizirik
More generally, the observed genetic footprints of late et al. 1998, 2001; Johnson et al. 1999; Culver et al. 2000;
Quaternary climate change in the genetic diversity patterns Sinclair et al. 2001; Uphyrkina et al. 2001; Cossios et al.
of guignas coincide with other fauna and plant population 2009). However, patterns of genetic variation, biogeo-
genetic patterns, supporting a common geological history graphic history, and conservation threats vary significantly
in southern South America. among guigna geographic groups, suggesting different
particular situations for each population within the two
Conservation implications and definition Management Units.
of management units The Northern group, situated in the northernmost limit
of the distribution range of guignas, is quite isolated from
Population management should aim to preserve adaptive other groups, displaying genetic uniqueness. The rich
diversity and evolutionary processes across the geographic Chilean Matorral ecosystem it inhabits, where more than
range of a species by maintaining the natural network of half of the country’s total human population inhabits, has
genetic connections between populations (gene flow) been dramatically reduced by habitat conversion to pine
(Taylor and Dizon 1999; Crandall et al. 2000, Palsbøll plantations and agricultural lands (Nowell and Jackson
et al. 2006). 1996). Moreover, direct human pressure over native fauna
Criteria based exclusively on molecular phylogenies are is not uncommon. Guigna populations in these areas have
not adequate for determining appropriate Management been negatively impacted, currently subsisting in frag-
Units. As suggested by Crandall et al. (2000), incorporating mented and restricted populations of variable size, con-
both ecological data and genetic variation of adaptive sidered to be severely endangered (Nowell and Jackson
significance, are more relevant for conservation. Following 1996). Connectivity between these groups inhabiting
Crandall et al. (2000) criteria, we used a broader fragmented landscapes through habitat corridors is a
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Conserv Genet
critical issue to maintain and secure viable long term suggesting it may be going through a current population
populations, favoring metapopulation dynamics to assist size reduction (Table S4).
demographic and genetic interchange between populations The San Rafael Lake group, the southernmost limit of
(Taylor et al. 1993; Hanski et al. 1995; Hanski and Sim- guignas, is geographically isolated on the Taitao Peninsula
berloff 1997). flanked to the east by extensive ice fields. This group dis-
The Central group displays the highest genetic vari- plays unique genetic identity and demographic isolation.
ability, the highest pairwise differences, the highest num- Conservation challenges for the long-term viability of
ber of private alleles, and the highest estimated effective guigna populations are the maintenance of adequate pop-
population size among all guigna geographic groups. ulation sizes and effective dispersal, especially in human
Coinciding with our results, a pattern of higher vertebrate dominated landscapes. Future research should focus on
richness at mid-latitudes in Chile (33–43°S) has been new hypothesis and molecular approaches, widening the
proposed by Samaniego and Marquet (2009), attributed to studied area and using different molecular markers for a
the interaction between historical processes associated with clearer picture. Most importantly, future directions should
desertification in the north and ice ages in the south. The consider comparative phylogeographic perspectives in
location of the Central group just north of the last glacial southern South America, as a way to bring light into more
icefield probably determined its persistence throughout the general questions in biogeography, evolution and conser-
last glacial maxima, thus harbouring a longer demographic vation in the region.
history. In addition, high latitude populations may have
migrated to lower latitudes avoiding areas covered by ice Acknowledgments Guignas were captured with permission from
the Agriculture and Livestock Service (SAG) in Chile, capture permit
during the last glacial period, probably resulting in the numbers 814/13-Feb-2008, 109/9-Jan-2009, 1220/22-Feb-2010 and
admixture of different genetic lineages in this ice-free area. 1708/26-Mar-2010. Samples were obtained in Argentina with per-
We estimated the number of guignas that could theo- mission from the National Parks Administration (APN), permit
retically inhabit the total surface area for each studied zone number 1002/8-May-2009. Samples were imported with permission
from the Agriculture and Livestock Service (SAG) in Chile, permit
(Table S4). The Central group displays a pattern of number 30/09. C.N. acknowledges support from the doctoral fel-
Ne N, suggesting it may be going through a current lowship ICM P05-002 from the Instituto de Ecologı́a y Biodiversidad
population size reduction (Table S4). In practice, the Ne is (IEB, Facultad de Ciencias, Universidad de Chile), the Emerging
usually less than the number of breeding adults, because Leaders in The Americas Program (ELAP, Canadian Bureau for
International Education, Government of Canada) and Becas Chile
they deviate from the assumptions of an idealized popu- para pasantı́as doctorales 2010 (CONICYT, Chile). This study was
lation (Frankham et al. 2005). When population size funded by the Instituto de Ecologı́a y Biodiversidad (Facultad de
(N) decreases, the effective population size (Ne) also Ciencias, Universidad de Chile), Panthera Kaplan Awards Program
decreases but remains higher than N during a bottleneck (Panthera Foundation, New York, USA), Scott Neotropical Fund
Award (Cleveland Metroparks Zoo & the Cleveland Zoological
event. This area has a high prevalence of human-related Society, Cleveland, USA) and the Eric York Scholarship (Felidae
threats: habitat conversion, fragmentation and direct human Conservation Fund, California, USA). This research was supported in
persecution (Acosta-Jamett et al. 2003; Acosta-Jamett and part by the Intramural Research Program of the NCI, NIH. We wish to
Simonetti 2004; Silva-Rodriguez et al. 2007; Herrmann thank Benito González, Emma Elgueta, Milton Gallardo (Universidad
Austral), Jaime Rau (Universidad de Los Lagos), Oscar Skewes
et al. 2013). (Universidad de Concepción), Bárbara Zentilli (CODEFF), Denise
The Argentinian group shows a lack of genetic structure Donoso, Nicolás Gálvez, Felipe Hernández, Elke Schüttler, Cristián
in relation to populations from the western side of the Bonacic, Martı́n Monteverde (Centro de Ecologı́a Aplicada del
Andes in Chile, along with its current high connectivity Neuquén—Argentina), José Luis Brito (Museo Municipal de Ciencias
Naturales y Arqueologı́a de San Antonio), Juan Carlos Torres (Museo
with Lake District group. Inhabiting only a narrow strip of de Historia Natural Santiago), José Yáñez (Museo de Historia Natural
land in southwestern Argentina, these populations are Santiago), Osvaldo Rojas (Museo de Historia Natural Calama), Fer-
likely exposed to competition from its more abundant and nando Soto (Museo de Historia Natural de Valparaı́so), Elvira Solar
bigger sized sister species, L. geoffroyi, a frequent resident (Museo de Zoologı́a Universidad de Concepción), Franklin Troncoso
(Museo de Historia Natural de Concepción), Luis Villanueva (SAG
on the eastern side of the Andes. VII Región), Cecilia González (SAG RM), David Flores (Museo de
The Chiloé Island group, situated in complete isolation Ciencias Naturales Bernardino Rivadavia—Argentina), Diego Verzi
from mainland populations, harbours a unique genetic (Museo de La Plata—Argentina), Manuel Valdés (Parque Tantauco),
identity. Although the land may be sparsely populated in Bernardita Silva, Lito Quezada, Maximiliano Sepúlveda, Javier Ca-
bello, Alejandro Bravo (Universidad Austral), Jorge Valenzuela
Chiloé Island, native forests have been largely cleared to (CECPAN), Andrés Charrier, Juan Luis Celis, Buin Zoo and Fidel
support domestic fowl, grazing, and farming (Sanderson Ovidio Castro (Banco de Recursos Genéticos, Facultad de Ciencias
et al. 2002) and genetic diversity has been negatively Veterinarias, Universidad de Concepción, Campus Chillán) for gen-
impacted by habitat loss and fragmentation, and also erous collaboration with samples for this study. We specially thank
Magdalena Bennett for map elaboration. We thank Sylvain Faugeron
human persecution (Napolitano 2012). This may be reason and Juliana Vianna de Abreu for help with molecular markers. Special
why the group also displays a pattern of Ne N,
123
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thanks to Jennifer Hetz, Mónica Mora, Valentina Sánchez, Tatiana Bonin A, Bellemain E, Bronken Eidesen P et al (2004) How to track
Vuskovic, Verónica Solé, Rousset Palou, Yuri Zúñiga and Juan Vidal and assess genotyping errors in population genetics studies. Mol
for assisting in the capture of guignas. We thank Raleigh International Ecol 13:3261–3273
for logistical support in collection of samples at San Rafael Lake. We Bosshard M (2011) Evidencias genéticas de la historia glacial de
thank Parque Ahuenco, especially to Alberto Carvacho, for kindly Laureliopsis philippiana. Dissertation, Universidad de Chile,
letting us work in their lands. We thank the Sindicato de Pescadores Santiago, Chile. p 33
Mar Adentro from Chepu, especially to Carlos Villarroel, for their Burban C, Petit RJ, Carcreff E et al (1999) Rangewide variation of the
kind help during field work. We thank Parque Tantauco, especially to maritime pine bast scale Matsucoccus feytaudi Duc (Homoptera:
Alan Bannister, for their kind support and for letting us work in their Matsucoccidae) in relation to the genetic structure of its host.
lands. We thank the Senda Darwin Foundation, Inés Hanning from Mol Ecol 8:1593–1602
Caulı́n, and many other local land owners in Chiloé Island who kindly Burbrink FT, Lawson R, Slowinski JB (2000) Mitochondrial DNA
let us work in their lands. phylogeography of the polytypic North American rat snake
(Elaphe obsoleta): a critique of the subspecies concept. Evolu-
tion 54:2107–2118
Cabrera A (1957) Catálogo de los mamı́feros de América del Sur.
Parte 1, Metatheria-Unguiculata-Carnivora. Revista del Museo
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Conserv Genet

Phylogeography and population history of Leopardus guigna,

A. Rus Hoelzel • Rachel Freer • Nigel Dunstone • Kermit Ritland •

Received: 25 July 2013 / Accepted: 8 January 2014

C. Napolitano (&) E. Poulin S. J. O’Brien

Present Address: N. Dunstone

Introduction and vegetational contrast that formed between the eastern

Fig. 3 Maximum extension of

Fig. 4 Maximum likelihood

2 groups 37.25* 18.45** 2 groups 20.50 n.s 12.34 n.s

Northern (32.5–35° S) – 0.101** 0.132* 0.158* 0.203 0.334

Northern 90,000 – 0.000424 2.245 0.00016 0.000035 0.506

Fig. 8 Categories of population

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