(Lama Guanicoe) in South America

Molecular Ecology (2013) 22, 463–482 doi: 10.1111/mec.12111

The inﬂuence of the arid Andean high plateau on the phylogeography and population genetics of guanaco (Lama guanicoe) in South America

JUAN C. MARIN,* BENITO A. GONZA´ LEZ,† ELIE POULIN,‡ CIARA S. CASEY§ and WARREN E. JOHNSON¶ *Laboratorio de Geno´mica y Biodiversidad, Departamento de Ciencias Ba´sicas, Facultad de Ciencias, Universidad del Bıó-Bıó, Casilla 447, Chillań, Chile, †Laboratorio de Ecologıá de Vida Silvestre, Facultad de Ciencias Forestales y de la Conservaciońdela Naturaleza, Universidad de Chile, Santiago, Chile, ‡Laboratorio de Ecologıá Molecular, Departamento de Ciencias Ecolo´gicas, Facultad de Ciencias, Instituto de Ecologıá y Biodiversidad, Universidad de Chile, Santiago, Chile, §Department of Biological Science, University of Lincoln, Lincoln, UK, ¶Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD, USA

Abstract A comprehensive study of the phylogeography and population genetics of the largest wild artiodactyl in the arid and cold-temperate South American environments, the guanaco (Lama guanicoe) was conducted. Patterns of molecular genetic structure were described using 514 bp of mtDNA sequence and 14 biparentally inherited microsatellite markers from 314 samples. These individuals originated from 17 localities throughout the current distribution across Peru, Bolivia, Argentina and Chile. This confirmed well-defined genetic differentiation and subspecies designation of populations geographically separated to the northwest (L. g. cacsilensis) and southeast (L. g. guanicoe) of the central Andes plateau. However, these populations are not completely isolated, as shown by admixture prevalent throughout a limited contact zone, and a strong sig- nal of expansion from north to south in the beginning of the Holocene. Microsatellite analyses differentiated three northwestern and 4–5 southeastern populations, suggesting patterns of genetic contact among these populations. Possible genetic refuges were identified, as were source-sink patterns of gene flow at historical and recent time scales. Conservation and management of guanaco should be implemented with an understanding of these local population dynamics while also considering the preservation of broader adaptive variation and evolutionary processes.

Keywords: camelids, contact zone, d-loop, Holocene, microsatellites, Patagonia Received 29 November 2011; revision received 14 September 2012; accepted 19 September 2012

most of Argentina (Torres 1985; Gonza´lez et al. 2006). Introduction The current distribution of guanacos corresponds to The guanaco (Lama guanicoe) is the most ecologically <30% of the range at the time of the arrival of the Europe- important and abundant native ungulate of the arid and ans to South America (Raedeke 1979). Currently fewer cold-temperate region in the southern cone of South than 5% of the guanaco population occupies <30% of America, occupying diverse habitats from sea level to their range, compared with when Europeans arrived to over 4000 m (Franklin 1982). West of the Andes, guana- South America in the XVII century (Raedeke 1979). cos are widely distributed from northern Peru to Tierra Uncontrolled hunting, habitat reduction and deteriora- del Fuego in Argentina and the Navarino Islands in tion, and displacement by introduced livestock have Chile. To the east, they extend southward from the Boliv- caused local and regional extinctions resulting in a ian and Paraguayan Chaco across the pampas throughout reduced and fragmented population (Torres 1992). In spite of the ecological importance of the guanaco, it’s past Correspondence: Juan C. Marı´n, Fax: 56 42 253153; and more recent evolutionary history and broad phyloge- e-mail: [email protected] netic biogeographic patterns are not well-described.

The genus Lama descended from the feeder-browser Holocene around 6000 years ago have been found along llama-like Hemiauchenia that originated in North America the Beagle Channel, between Tierra del Fuego and and subsequently migrated to South America around Navarino Island (Tivoli & Zangrando 2011), suggesting 2.5–3.0 million years ago (Ma) across the Isthmus of a more recent colonization of guanaco to these islands. Panama as part of the great American interchange Nevertheless, fossil records are abundant in the Pampas (Webb 1974). This migration was probably facilitated by from the late Pleistocene, but are scarce in Patagonia, a sub-tropical, relatively arid savannah-like corridor making it difficult to document their arrival time. How- from the Gulf of Mexico, across the Mexican plateau, ever, fossils are not unequivocal and are affected by and through the entire isthmus (Webb 1978, 1991). degradation, taphonomic processes and ease of discov- Through South America, this corridor divided into two ery (Borrero 2005, 2008; Fernańdez 2008), making local main routes: a ‘high road’ along the western slopes of geographic findings difficult to interpret on a broad the Andes and pacific coast, and a more eastern ‘low scale. road’ beginning in the northwestern low-lands of Traditionally, four subspecies of modern guanacos have Colombia through the Amazonian and Chaco regions been recognized (Wheeler 1995) based on their distribution (Webb 1978). Although it was initially postulated that and phenotype (body and skull size, and colouration): modern camelids evolving from Hemiauchenia arose in Lama guanicoe guanicoe (Mu¨ ller 1776); L. g. huanacus the Andes (Webb 1974; Webb & Stehli 1995), most Plio- (Molina 1782); L. g. cacsilensis (Lo¨nnberg 1913) and Pleistocene camelid fossils have been found in non-An- L. g. voglii (Krumbiegel 1944). However, Franklin (1982, dean locations (Menegaz et al. 1989; Ochsenius 1995; 1983) speculated that there were only two guanaco sub- Scherer 2009), suggesting that the early South American species, separated by the salt plains of southern Bolivia camelid radiation may have occurred elsewhere, such and the crests of the Andean chain: the smaller, lighter- as the pampas of Argentina, and that descendants later coloured L. g. cacsilensis, restricted to the north-western colonized Andean and subtropical regions (Menegaz slopes of the Andes between 8° and 41°S and the larger et al. 1989; Scherer 2009). and darker L. g. guanicoe located on the south-eastern Paleontological and zooarcheological evidence sug- side of the Andes between 18 and 55°S (Raedeke 1979; gests that guanacos expanded from north to south. The Gonza´lez et al. 2006). This hypothesis was partially earliest fossils, from the Pliocene and late Pleistocene, confirmed from an analysis of sequence variation in were found in the Pampas of Buenos Aires and the two mitochondrial genes (Marıń et al. 2008), but the ‘mesopotamic’ region of Argentina, in northern Uru- geographic limits for each taxon could not be clearly guay and northeastern and southern Brazil (Scherer defined. Additionally, substructure within these subspe- 2009). Subsequently, L. guanicoe is found along the cies has not been assessed, although analyses of micro- Reconquista River, east of the Pampean region, in strata satellite variation have suggested that low-levels of from the middle Pleistocene (<0.78 Ma and >0.126 Ma) genetic structure may occur between neighbouring pop- (Menegaz et al. 1989; Cajal et al. 2010). In the late Pleis- ulations, especially where these are isolated such as in tocene, guanaco have been found in Paso Otero (Neco- Tierra del Fuego (Sarno et al. 2001; Mate´ et al. 2005). chea County) in the La Chumbiada (35 000 radiocarbon Here we present a comprehensive assessment of the years BP, Cajal et al. 2010) and Guerrero Members molecular diversity of guanaco based on samples col- (c. 24 000 and c.10 000 radiocarbon years BP, Tonni lected throughout its distribution range and analyse et al. 2003) of the Lujań Formation. L. guanicoe was variation in14 microsatellite loci and sequence variation found in four sites in the Santa Cruz Province in Pata- in mtDNA control region sequence. We present evi- gonia: La Marıá (10 967 ± 55 radiocarbon years BP, dence of range-wide phylogeographic structure linked Paunero 2003), Los Toldos (12 600 ± 650 radiocarbon with guanaco evolutionary history to (i) define patterns years BP; Cardich & Hajduk 1973; Cardich 1987), El of molecular genetic structure among guanacos; (ii) link Ceibo (c.11 000 radiocarbon years BP; Cardich 1987) and patterns of genetic variation with phylogeographic his- Piedra Museo (units 6–4, dated between 12 890 ± 90 tory and barriers to gene flow; and (iii) describe and and 9230 ± 105 radiocarbon years BP; Miotti et al. 2003). contrast the evolutionary history and patterns of gene This radiocarbon datum is similar to other taxon-dates flow among these populations. obtained from L. guanicoe bones from the Cueva del Medio, 10 450 ± 100, 10 710 ± 190 and 10 850 ± 130 Materials and methods radiocarbon years before present (Nami & Nakamura 1995; Massone & Prieto 2004). In Tierra del Fuego Sample collection and DNA extraction L. guanicoe dates to 10 130 and 11 880 AMS years before present (Massone & Prieto 2004). Finally, guanaco bones Material for DNA analysis was collected from guana- associated with signs of human settlements from the cos throughout their range. DNA was extracted from

© 2012 Blackwell Publishing Ltd PHYLOGEOGRAPHY AND POPULATION GENETICS OF GUANACO 465 blood samples from 88 wild-caught adults following Microsatellites markers chemical immobilization (Sarno et al. 1996) at five localities, from muscle or skin samples from 55 dead Fourteen autosomal dinucleotide microsatellite loci, animals from six localities, and from 150 fresh faecal designated YWLL08, YWLL29, YWLL36, YWLL38, samples from different dung piles at eight localities YWLL40, YWLL43, YWLL46 (Lang et al. 1996), LCA5, (Table 1, Fig. 1A). DNA was also obtained opportu- LCA19, LCA22, LCA23 (Penedo et al. 1998), LCA65 (Pen- nistically from liver samples of 26 adult males in edo et al. 1999) and LGU49, LGU68 (Sarno et al. 2000) Valle Chacabuco (Valchac Ltd.), Chile, under a sus- were analysed. Amplification was carried out in a 10 lL tainable-use programme authorized by the Chilean reaction volume, containing 50–100 ng of template DNA, – l Government. Locations of sample sites and the geo- 1.5 2.0 mm MgCl2, 0.325 m of each primer, 0.2 mm graphic position of individuals collected at each site dNTP, 1X polymerase chain reaction (PCR) buffer are given in Fig. 1 and Table 1. All samples were (Qiagen) and 0.4 U Taq polymerase (Qiagen). All PCR stored at À70o C in the Laboratorio de Geno´mica y amplifications were performed in a PE9700 (Perkin Elmer Biodiversidad, Departamento de Ciencias Ba´sicas, Applied Biosystems) thermal cycler with cycling condi- Facultad de Ciencias, Universidad del Bio-Bıó, Chillań, tions of: initial denaturation at 95 °C for 15 min, followed Chile or at CONOPA in Lima, Peru. We followed by 40 cycles of 95 °C for 30 s, 52–57 °C for 90 s and 72 °C guidelines of the American Society of Mammalogists for 60 s, and a final extension of 72 °C for 30 min. Ampli- during the collection and handling of animals (Gannon fication and genotyping of DNA from faecal samples & Sises 2007). Total genomic DNA was extracted were repeated two or three times. One primer of each from blood and bone marrow using the Wizard pair was labelled with a fluorescent dye on the 5′-end Genomic DNA Purification Kit (Promega, Madison, and fragments analysed on an ABI-3100 sequencer (Per- WI, USA). DNA from liver and muscle samples was kin Elmer Applied Biosystems). Data collection, sizing of purified using proteinase K digestion and a standard bands and analyses were carried out using GeneScan phenol-chloroform protocol (Sambrook et al. 1989). software (Applied Biosystems). DNA from faeces was extracted using the QIAamp We identified multiple faecal samples that came from DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) in the same individual by searching for matching micro- a separate non-genetic-oriented laboratory. satellite genotypes using the Excel Microsatellite Toolkit

Table 1 Summary of the Lama guanicoe samples used in the analyses, including localities (ordered approximately from north to south), type of sample (B indicates blood; F, faecal; M, muscle; S, skin and L, liver), number of samples used from each locality for each genetic marker. Demarcation of regions were assisted by the empirical results of spatial analyses

Samples Samples mtDNA microsatellites Region population Localities, country (abbreviation) Sample type (N = 306) (N = 314)

Northwestern region Hiper-Arid Peru Huallhua, Peru (HU) F 10 18 Chilean Pre-altiplano Putre, Chile (PU) B, F 20 21 Arid Chile and Pan de Azucar National Park, Chile (PA) F 11 12 Chilean Peri-Puna Llanos de Challe National Park, Chile (LC) B, M, F 28 21 Illapel, Chile (IL) B, M 20 19

Southeastern region Bolivian Chaco Kaa-Iya National Park, Bolivia (KA) F 20 17 Northern La Payunia Reserve, Argentina (LP) B 10 10 and central Patagonia Loma Blanca, Argentina (LB) F 20 20 Telsen, Argentina (TE) S 13 15 Penı´nsula Valde´s, Argentina (PV) S 21 15 Camarones, Argentina (CA) S 13 15

Western Patagonia Valle Chacabuco, Chile (VC) L 20 26 Southern Patagonia Bosques Petriﬁcados, Argentina (BP) F 20 20 Monte Leon, Argentina (ML) F, M 20 22 Torres del Paine National Park, Chile (TP) B 20 23 Pali-Ayke National Park, Chile (PK) F 20 20

Fuegian zone Tierra del Fuego, Chile (TF) B 20 20

(A) (B)

PERU

L. g. cacsilensis 1.0 Hiper-Arid Peru BOLIVIA Huallhua (HU) 1.0 1.0 Chilean Bolivian

Putre (PU) Kaa-Iya (KA) Pre-altiplano Chaco

0.00086 0.00086 0.018 0.018 PARAGUAY Pan de Azucar (PA)

0.0025 Arid Llanos de Challe (LC) 0.98 Chile and

Chilean 0.0084 Contact Zone Peri- Puna Illapel (IL)

0.0065 ARGENTINA 1.0 La Payunia (LP)

Loma Blanca (LB) Northern and central

CHILE L. g. guanicoe Patagonia Telsen (TE) Valle 1.0 Deserts and Xeric Shrublands Chacabuco (VC) Camarones (CA) Western Flooded Grasslands and Savannas 0.018 Patagonia Inland Water Bosques Petrificados (BP) Mangroves 0.0097 Torres del Southern 0.97 Mediterranean Forest, Woodlands and Scrub Patagonia Paine (TP) Montane Grasslands and Shrublands 0.0051 Rock and Ice 1.0 Temperate Broadleaf and Mixed Forests Fuegian Temperate Grasslands, Savannas and Shrublands Pali-Ayke (PK) zone STRUCTURE clusters (K = 2) Tropical and Subtropical Dry Broadleaf Forests Tropical and Subtropical Grasslands, Savannas and Shrublands Tierra del Fuego (TF) BAPS clusters (K = 8) Tropical and Subtropical Moist Broadleaf Forests

Fig. 1 Geographic distribution of Lama guanicoe in South America (in dark-grey based on Gonza´lez et al. 2006) showing genetic divi- sions by subspecies determined by mitochondrial BAPS (red and green continuous lines) and STRUCTURE K = 2 (red and green-backed line) analyses, contact zone (yellow line) obtained by microsatellite assignations (STRUCTURE, see Table 3), and populations (blue lines) identified by microsatellite BAPS (A). Gene flow among populations identified by microsatellite BAPS analyses. The thickness of the arrow indicates the magnitude of gene flow (B).

(Park 2001) and eliminated samples from the study if 0.5 lM each primer and 0.1U/l Taq polymerase (Invit- ® they showed more than 85% overlap. We also evaluated rogenGibco, Life Technologies ). All PCR amplifications the existence of null alleles using the program Micro- were performed in a PE9700 (Perkin Elmer Applied Bio- Checker version 2.2.3 (Van Oosterhout et al. 2004). The systems) thermal cycler with cycling conditions as fol- software FSTAT 2.9.4 (Goudet 2005) was used to estimate lows: initial denaturation at 95 °C for 10 min, followed – ° ° allele frequency, observed heterozygosity (HO) and by 30 35 cycles of 94 C for 45 s, 62 C for 45 s and ° ° expected heterozygosity (HE). The inbreeding coefficient 72 C for 60 s, and a final extension of 72 C for 5 min.

FIS, which estimates the heterozygote deficit or excess PCR products were purified using the GeneClean Turbo within populations, was estimated following Weir & for PCR Kit (Bio101) following the manufacturer’s Cockerham (1984) using FSTAT 2.9.4. instructions. Products were sequenced in forward and reverse directions using BigDye chemistry on an ABI Prism 377 or 3100 semi-automated DNA analyser. The Mitochondrial DNA reactions were carried out in a 10-lL volume containing The left domain of the mitochondrial control region approximately 100 ng of purified DNA, 1 lL of either (514 bp) was amplified using the camelid and guanaco forward or reverse primer and 2 lL of BigDye Termina- specific primers LthrArtio (5′-GGT CCT GTA AGC tor Kit version 3.1 (PerkinElmer). Sequence reactions CGA AAA AGG A-3′), H15998V (5′-CCA GCT TCA were visualized using an ABI-3100 sequencer (Perkin ATT GAT TTG ACT GCG-3′), HLoop550G: 5′ ATG Elmer Applied Biosystems). GAC TGA ATA GCA CCT TAT G 3′ (Marin et al. 2007). Sequences were aligned using Clustal_X (Thompson Amplification was performed in a 50 lL reaction vol- et al. 1997) and were checked by eye. The number of ume with ~30 ng genomic DNA, 1x PCR buffer (8 mM segregating sites (S) and haplotypes (nh), haplotype Tris-HCl (pH 8.4); 20 mM KCl (InvitrogenGibco, Life diversity (h) (Nei 1987), nucleotide diversity (p) and l Technologies), 2 mM MgCl2,25M each of dNTP, average number of nucleotide differences between pairs

© 2012 Blackwell Publishing Ltd PHYLOGEOGRAPHY AND POPULATION GENETICS OF GUANACO 467 of sequences (k) was estimated using ARLEQUIN 3.5.1.2 –200 (A) (Excofﬁer & Lischer 2010). These estimates were calcu- 0 lated for each locality independently and for the two broad regions (as detected by Structure, see Results). 200 Intraspeciﬁc mtDNA genealogies were inferred using 400 the method of statistical parsimony (Templeton et al. 1992) implemented in the program TCS v1.21 (Clement 600 et al. 2000). TCS estimates phylogenetic relationships K = 2 when there are low levels of divergence and provides a 800

95% plausible set for all haplotype connections. Three 1000 polymorphic sites were deleted to reduce homoplasy 0 2 4 6 8 101214161820 among haplotypes for a better interpretation of the K network. 30,0 (B) 25,0 Estimation and delimitation of genetic units 20,0 The genetic structure of Lama guanicoe populations was investigated with a Bayesian clustering algorithm imple- 15,0 mented in STRUCTURE version 2.3.3 (Pritchard et al. 2000) 10,0 using the total microsatellite genotypes data set (n = 314) assuming that sampled individuals belong to an 5,0 unknown number of K genetically distinct clusters. For 0,0 each K (from 1 to 20), we performed 20 runs and calcu- 0 2 4 6 8 101214161820 lated the mean posterior probability of the data [‘log K probability of data’, L(K)]. We determined the most prob- Fig. 2 Support for defining of the number of guanaco popula- able number of populations, K, by evaluating the signifi- tion based on the microsatellite data set. (A) Mean L(K) ± SD cance of the posterior probabilities (Pritchard et al. 2007) over 20 runs as a function of K. (B) DK (Delta K = mean (Fig. 2A), and by using the method described by Evanno (∣L’’(K)∣)/SD(L(K))) following Evanno et al. (2005) as a func- et al. (2005) that examines DK,anad hoc quantity related tion of K. to the change in posterior probability between runs of different K (Fig. 2B). We ran the program using the The spatial method, implemented in the program BAPS admixture model and correlated allele frequencies option 5.2 (Corander et al. 2008), incorporates coordinate data for 500 000 iterations after a burn-in period of 30 000 to assign a non-uniform prior such that neighbouring iterations. These parameters are considered most appro- individuals are more likely to be assigned to the same priate for detecting structure among populations that are genetic cluster. We performed spatial mixture clustering probably to be similar due to migration or shared ances- of individuals for 10 runs with the maximum number try (Falush et al. 2003; Pritchard et al. 2007). In our data of populations (Kmax) set to 20. Results from each run set, the greatest amount of variation was explained by were stored and merged by the program, and the opti- separating two genetic groups (K=2) (see Results). To mal K value was selected as the partition with the detect potential substructure in each group, we assigned maximum likelihood (L(K)) and the highest posterior individuals based on their highest q-value—an estimate probability (P). The assignments from the mixture anal- of the proportion of an individual’s genome attributed to ysis were then used to perform admixture analysis with each of the identified genetic clusters—and then analysed the recommended parameter values, including 100 each group separately. iterations for individuals, 20 iterations for reference We assessed genetic differentiation using estimates of individuals and 200 reference individuals from each

FST (Weir & Cockerham 1984) from the microsatellite population. Similar to our Structure analyses, we data using ARLEQUIN 3.5.1.2 (Excoffier & Lischer 2010) assigned individuals to the group with the highest with 10 000 permutations for each of the eight popula- Q-value, flagging potentially admixed individuals tions and two regions. For comparison, genetic differen- (Q < 0.75). tiation between each pair of populations was also To investigate hierarchical levels of population struc- estimated using Jost’s D in GENODIVE version 2.0b22 ture of mitochondrial sequences, an analysis of molecu- (Meirmans & Van Tienderen 2004), a method which lar variance (AMOVA) was performed using ARLEQUIN is independent of the amount of within-population 3.5.1.2 (Excoffier & Lischer 2010), considering genetic diversity (Jost 2008). distances between haplotypes and their frequencies

© 2012 Blackwell Publishing Ltd 468 J. C. MARIN ET AL. among the defined populations, and with statistical sig- simultaneous estimation of population growth or nificance determined by 10 000 permutations. This anal- decline (Beerli & Felsenstein 1999). We ran 10 short ysis was based on the geographical division of the chains (using 500 of 10 000 sampled trees) and two long population as determinate by Structure analyses. chains (using 10 000 of 200 000 sampled trees), assuming two clusters with possible population growth and bi-directional migration. LAMARC searches were run Historical demography and migration with a starting value of h and with the Mixed K-Allele/ Deviations from a neutral Wright–Fisher model were Stepwise model (MixedKS) that considers both muta- estimated with Tajima’s D and Fu’s Fs statistics (Tajima tion to adjacent states (like the Stepwise model) and 1989; Fu 1997). A mismatch analysis was performed mutation to arbitrary states (like the K-Allele model). using ARLEQUIN 3.5.1.2 (Excoffier & Lischer 2010) to test for evidence of population expansion in the groups Results identified. ARLEQUIN uses a parametric bootstrap approach (10 000 simulations) to compute 95% confi- Genetic diversity dence intervals for τ (Tau), the time since the expansion in units of mutational time (τ = 2 lt, where l is the Of the 320 replicated microsatellite genotypes, we found mutational rate and t is the generation time in years), four pairs of faecal samples that were identified as h h = l h 0, the initial population size ( 0 2 N0) and 1, the being from the same individual and therefore were h = l final population size ( 1 2 N1). ARLEQUIN also com- removed from the analysis. We detected 209 alleles at putes the raggedness statistic (Harpending 1994) and 14 loci in 314 individuals. The number of alleles per the sum-of-squared deviations (SSD), which test the fit locus ranged from 9 to 30, and 43 alleles were unique of the observed data to the expected population growth to a single population. Within populations, we found model (Schneider & Excoffier 1999). To estimate t,we no significant departures from H-W equilibrium assumed a mutation rate of 13%/Myr (similar to used (P > 0.0011, the Bonferoni-adjusted critical value), no in water buffalo, Lau et al. 1998; hartebeest, Flagstad evidence for linkage among the five loci (P > 0.0005, the et al. 2001; African buffalo, Van Hooft et al. 2002; roan adjusted critical value, for each pair of loci across all antelope, Alpers et al. 2004; giraffe, Brown et al. 2007 populations) and low frequencies of null alleles per and other artiodactyls), with an average generation time locus (fo < 0.08). We found consistently high levels of of 3 years (Gonza´lez et al. 2006). genetic diversity (mean expected heterozygosity ranged To identify demographic scenarios that explain the from 0.56 to 0.84) and high values for allelic richness current diversity patterns in the two regions, we used (mean RA ranged from 4.41 to 9.33) (Table 2).Thirty- an Approximate Bayesian Computation approach nine variable positions (7.6%), 34 transitions, seven (Beaumont 2010) implemented in the popABC program transversions and one insertion from 514 nucleotides (Lopes et al. 2009) using mitochondrial sequences and and 35 haplotypes were identified in 306 sequences of microsatellite loci together for each of the two Structure the 5′ end of the control region. Haplotype (h) and clusters (Northwest and Southeast, see Results). We nucleotide diversity (p) is detailed in Table 2 and the adopted an ‘Isolation-Migration’ model (Nielsen & distribution of haplotypes within 17 localities is given Wakeley 2001) that assumes the two populations in Table 3. Sequences were deposited in GenBank with diverged from a common ancestral population and that accession numbers JX678291–JX678596. these populations may have been connected by migration The 35 Control Region sequences are connected since separation. The regression step was performed in R through a network with a maximum of 12 mutational using scripts by M. Beaumont (http://code.google.com/ steps (Fig. 4). Although the network did not completely p/popabc/model_choice.r) and additional scripts from delineate a genetic partition corresponding with a geo- the popABC package that were modified to fit our graphic separation between the northwest and south- analyses. Priors of population parameters were: uniform east, there is a clear trend that the most-divergent distributions bound between minimum and maximum haplotypes are found in the northwest. A single domi- values (see Table S6, Supporting information) with or nant haplotype (H35) was observed exclusively in the without gene flow. mitochondrial southern cluster, from which related Similarly, we also used LAMARC v. 2.1.8 (Kuhner 2006) sequences were separated by one or two mutational to estimate ML migration rates between two sub- steps. Up to six mutational steps separated the haplo- regions of L. guanicoe. This approach, based on coales- types of Northwest population from Southeast popula- cence using MCMC searches, takes both history and tion. Haplotype 35 was shared among localities asymmetrical gene flow into account, unlike classical throughout the southern group, including LC and IL, migration-drift equilibrium approaches, and allows two locations within the contact zone (Fig. 1A).

Table 2 Genetic diversity indices from 14 microsatellite loci and mtDNA Control Region sequences by localities (deﬁned in Table 1 and Fig. 1A)

Microsatellites mtDNA

Region localities A ± SD Ho ± SD He ± SD nnph± SD p

Northwest 14.27 ± 4.85 0.70 ± 0.096 0.85 ± 0.05 17 15 0.87 ± 0.01 0.0079 ± 0.0044 HU 5.83 ± 1.95 0.72 ± 0.79 0.69 ± 0.13 5 5 0.64 ± 0.15 0.0100 ± 0.0060 PU 8.17 ± 2.66 0.76 ± 0.13 0.79 ± 0.09 5 4 0.76 ± 0.06 0.0074 ± 0.0044 PA 8.42 ± 2.40 0.76 ± 0.16 0.84 ± 0.06 3 0 0.54 ± 0.07 0.0087 ± 0.0052 LC 9.33 ± 2.89 0.60 ± 0.14 0.80 ± 0.08 5 1 0.67 ± 0.05 0.0053 ± 0.0032 IL 7.33 ± 3.08 0.68 ± 0.23 0.75 ± 0.11 6 3 0.66 ± 0.06 0.0018 ± 0.0014 Southeast 15.53 ± 5.50 0.70 ± 0.081 0.79 ± 0.09 20 18 0.33 ± 0.04 0.0008 ± 0.0008 KA 6.58 ± 2.35 0.58 ± 0.17 0.72 ± 0.13 2 0 0.00 ± 0.00 0.0000 ± 0.0000 LP 5.92 ± 2.97 0.64 ± 0.25 0.67 ± 0.18 5 2 0.53 ± 0.18 0.0024 ± 0.0018 LB 7.83 ± 3.51 0.89 ± 0.10 0.76 ± 0.11 4 1 0.18 ± 0.10 0.0003 ± 0.0005 TE 7.33 ± 3.28 0.73 ± 0.20 0.75 ± 0.13 5 2 0.29 ± 0.15 0.0009 ± 0.0009 PV 6.33 ± 3.31 0.76 ± 0.25 0.70 ± 0.14 4 2 0.40 ± 0.11 0.0008 ± 0.0008 CA 6.58 ± 2.42 0.64 ± 0.26 0.73 ± 0.12 4 2 0.15 ± 0.12 0.0003 ± 0.0005 VC 6.33 ± 2.49 0.63 ± 0.16 0.71 ± 0.10 10 6 0.64 ± 0.11 0.0015 ± 0.0013 BP 7.58 ± 3.29 0.80 ± 0.16 0.75 ± 0.09 4 2 0.28 ± 0.12 0.0006 ± 0.0007 ML 7.16 ± 2.91 0.69 ± 0.14 0.74 ± 0.11 6 2 0.41 ± 0.11 0.0008 ± 0.0009 TP 6.75 ± 3.27 0.62 ± 0.18 0.70 ± 0.12 5 2 0.42 ± 0.12 0.0018 ± 0.0014 PK 6.75 ± 2.76 0.69 ± 0.13 0.71 ± 0.10 6 1 0.35 ± 0.12 0.0007 ± 0.0008 TF 4.41 ± 1.97 0.57 ± 0.20 0.56 ± 0.18 4 2 0.18 ± 0.10 0.0003 ± 0.0005

A, Mean number of alleles per locus; He, mean expected heterozygosity; Ho, mean observed heterozygosity; n, number of haplotypes; np, number of private haplotypes; h, haplotype diversity; p, nucleotide diversity; p, number of polymorphic sites. Standard error values are in parentheses.

Genetic units and two were assigned to the Northwest cluster. Overall, Results of initial Structure analysis with all samples most of the individuals of mixed or incongruent heritage indicated the strongest support for K = 2 (Northwest were sampled in localities that were in relatively close vs. Southeast localities; Fig. 3), based on DK and the geographical proximity to the putative boundary between assignment plots, although the L(K) values increased the Northwest and Southeast (Fig. 3, Table 4). asymptotically with higher K (Fig. 2). Subsequent analy- Results of the BAPS analyses were concordant with sis of only the Northwest group provided evidence of the hierarchical Structure analysis. The optimal solution additional substructure (K = 2) between Hyper-Arid was obtained with K = 8 using all samples [L Peru (HU) and Chilean Pre-altiplano and arid Chile (K) = À18 299.36] and consisted of (blue line in Fig. 1A (PU + PA + LC). The Southeast group also showed evi- and dashed black line in Fig. 3): (i) Hyper-Arid Peru: dence of substructure (K = 5), delimiting the Bolivian HU; (ii) Chilean Pre-altiplano: PU; (iii) Arid Chile and Chaco and Chilean Peri-Puna (KA + IL), Northern Pata- Chilean Peri-Puna: PA, LC and IL; (iv) Bolivian Chaco: gonia (LP + LB), Central Patagonia, (TE + PV + CA), KA; (v) Northern and central Patagonia: LP, LB, TE, PV Southern and western Patagonia (VC + BP + ML + and CA; (vi) Western Patagonia: VC; (vii) Southern Pat- TP + PK) and the Fuegian zone (TF) (data not shown). agonia: BP, ML, TP and PK and (viii) Fuegian zone: TF. All runs at K = 2 produced identical clustering solutions. The BAPS analysis also infers a network based on rela- Of 314 individuals, 271 had a clear predominant heritage tive gene flow among clusters as indicated by weighted (i.e. Q > 75%). Of the 91 individuals sampled in the North- arrows reflecting the relative proportion of ancestry west region, 51 had a consistent genetic heritage, 26 were from the source cluster found among the individuals of mixed southeastern and northwestern origin and 14 had assigned to the target cluster. For example, our results clear Southeastern genetic heritages. The 14 of Southeastern suggest that the southern Patagonia population has had origin were all sampled in the northwestern localities of historic gene flow from five of the seven other clusters, PA and LC (Fig. 3) and also LC had the dominant south- and that southern Patagonia is the main source of gene western mtDNA haplotype (Haplotype 35, Fig. 4). Of the flow to northern and central Patagonia (Fig. 1B). 223 individuals sampled in the Southeast region, 206 had Most of the groups were significantly differentiated consistent genetic heritages and 15 were of mixed origin, from one another based on pairwise FST and D values

Table 3 Distribution of the 35 control region haplotypes observed in 307 guanacos from 17 localities. The vertical numbers indicate the position of polymorphic sites relative to haplotype 1. Localities are divided in Northern and Southern region based on AMOVA analyses.

1111122222 12223333334466778892789911134 Haplotypes 152560456890459072516511628953

1AG– GTTTAAGT TGTACACACATTCCCATCA 2..– ...... 3..– .CCC...CC..G....T....T..... 4..– ...... G...... 5..– .CCC...CC..G....T....T..... 6..– ...C...C...... T....TTG... 7..– ...C..A...... T..... 8..– ...C..A...... C..T..... 9..– ...... T..C.. 10 ..T...CGGA...... T..... 11 . . – A..C...C.T...... T....T..... 12 . . – ...C...C...... T....T.G... 13 . . – ...C...C...... T....TT.... 14 . . – ...C...C...... T....T..... 15 . . – ...C...C...... T....TT.... 16 . . – ...C...C...... T.T....T...T. 17 . . – ...C...C..C...... T..... 18 . . – ...C...C...... T....T..C.. 19 . . – ...C...C.A...... T....T...T. 20 . . – ...C...C...... GT....T..... 21 . . – ...C...C....T...T....T..... 22 . . – ...C...C...... T..C.T...T. 23 C . – ...C...C...... T....T..... 24 T . – ...C...C...... T....T..... 25 . A – ...C...C...... T....T..... 26 . . – ...C...C...... T.C..T..... 27 . . – ...C...C...... T....T..... 28 . . – ...C...C.....G..T....T..... 29 . . – ...C...C...... T....T....G 30 . . – ...C...C...... T...TT..... 31 . . – ...C...C...... TG...T..... 32 . . – ...C...C...... T...TT..... 33 . . – ...C...C...... T....T..... 34 . . – ...C...C...... T....T..... 35 . . – ...C...C...... T....T.....

Φ = = using microsatellites data and concordant results were ( CT 0.404, P 0.00136) when the sample sites were achieved whether BAPS vs. Structure or eight vs. two divided into the two groups: (i) HU, PU, PA; and (ii) populations were considered (Table 5). Pairwise-popu- LC, IL and all sites of the Southeastern Region. This lation differentiation estimated as FST values were the result supports the differentiation between the North- – modest (FST 0.06 0.424), with the Hyper-Arid Peru west and Southeast, but further reduces the number of and Fuegian Zone (Tierra del Fuego) clusters being locations assigned to the Northwest Region. = most distinct from the other populations (FST 0.424). However, pairwise population differentiation estimated Historical demography and migration as D (Jost 2008) was more pronounced (D 0.142– 0.738) and the Hyper-Arid Peru and Bolivian Chaco Tajima′s D and Fu’s F values were negative and statisti- = À clusters were most differentiated from the other popula- cally significant for the Southeast region (Fs 3.032, tions (D = 0.738). P < 0.02; D = À1.832, P < 0.05) but not significant for = À > = À Similarly, the mitochondrial DNA data showed a the Northwest region (Fs 0.628, P 0.10; D 0.242, hierarchical AMOVA pattern with significant structure P > 0.10), hence, we inferred a scenario of expansion for

22223345 Localities 57882530 91471618Region HUPUPALCKAILLPLBTEPVCAVCBPMLTPPKTFN

GTCCCCAGN 1 1 ..T.....O 2 2 ..T.....R 5 5 .....T..T 1 1 ..T....CH 1 1 ...... W 1 1 ...... E 4 4 ...... S 5 5 A...... T 8 5 13 ...... R 2 6 6 14 ...... N 1 1 ...... S 8 8 ...... G.O 1 1 ..T.....U 1 1 ...... T 9 9 ...... H 11 2 T...... E 1 1 ...... A 2 2 ...... S 1 1 ...... T 1 1 ...... R 4 4 ...... N 1 1 ...... 11 ...... 11 ...... 22 ...... 11 ...G.... 11 ...... 11 2 ...... 33 ....T... 22 ...... 1 1 1 4 3 10 ...... 11 .C...... 1 2 25 ...... 11 ...... 132087 18111612121715151617197

the Southeast region and non-expansion for the Northwest Although Structure is very efficient at assigning indi- region. Furthermore, Fu’s test, which is considered a pow- viduals to genetic clusters, it fails to detect historical erful test to detect past population expansion (Fu 1997; gene flow events (Pritchard et al. 2000). Therefore, the Ramos-Onsins & Rozas 2002), was negative and significant programs LAMARC and popABC were used to investi- for the Southeast region, indicating an excess of lower gate relative patterns of population size and gene flow frequency haplotypes compared with predicted values between the Northwest and Southeast regions. Rates of under the Wright-Fisher model. Mismatch distributions gene flow and effective population size were jointly were consistent with a model of a rapidly expanding estimated with popABC using both mitochondrial and population for the Southeast region (τ = 0.516, h = 0.255) nuclear markers. Estimated posterior distributions for (Fig. 5B), but not the Northwest region (τ = 7.555, all parameters are shown in Fig 6. Effective population h = 0.004) (Fig. 5A). Based on the expansion parameter size (Ne) of the Southeast population was estimated to (τ), sudden growth of the South-eastern population would be around 17 000 individuals (Fig. 6B), although the τ = have occurred around 11 583 years ago (t Low bound 0 estimated posterior distribution was substantially differ- τ = and t Up bound 50 508). ent between the Southeast and Northwest (Fig. 6A).

Contact zone

Northwestern region Southeastern region 1.00

0.80

0.60

0.40

0.20

0.00 Illapel (IL) Putre (PU) Telsen (TE) Kaa-Iya (KA) Huallhua (HU) Pali-Ayke (PK) La Payunia (LP) Camarones (CA) Loma Blanca (LB) Pan de Azucar (PA) Tierra del Fuego (TF) Torres del Paine (TP) Torres del Llanos de Challe (LC) Valle Chacabuco (VC) i ii iii iv v vi vii viii Bosques Petrificados (BP)

Fig. 3 Plot of posterior probability of assignment for 319 guanacos (vertical lines) to two genetic clusters based on Bayesian analysis of variation at 14 microsatellite loci. Individuals are grouped by locality, and localities are indicated along the horizontal axis. Light grey = Genetic Cluster 1: Northwest group; dark grey = Genetic Cluster 2: Southeast group. The tables contain the locations, in light grey and dark grey, show the seven clusters obtained from STRUCTURE analyses (citation). The numbers in the tables indicates BAPS grouping for comparisons; where i: Hyper-Arid Peru, ii: Chilean Pre-altiplano; iii: Arid Chile and Chilean Peri-Puna, iv: Bolivian Chaco, v: Northern and central Patagonia, vi: Western Patagonia, vii: Southern Patagonia, and viii: Fuegian zone.

Prior to the divergence of the two populations around Patagonian grasslands, parts of the Southern Andean 50 000 years ago (Fig. 6D), the models predict an esti- steppe in the montane grasslands and shrub lands eco- mated population of between 8000 and 12 000 individu- system, and portions of temperate broadleaf and mixed als (Fig. 6C). The effective migration rate between the forest represented by the sub-polar Nothofagus forest Northwest and Southeast populations was low, but (Dinerstein et al.1995; Gonza´lez et al.2006). The Central slightly higher from north to south (Fig. 6E and F). Andes Plateau formed by the ‘altiplano and puna’ Results from multiple Maximum Likelihood appears to have been the most important geographic LAMARC runs were mostly consistent with popABC barrier, significantly reducing gene flow between gua- results, with largely concordant confidence intervals. naco populations on either side of this portion of the Diversity in the Northwest (h = 0.3355) was higher than Andes, which is 1800 km long and 300–400 km wide. in the Southeast region (h = 0.1534). Migration rates, Paleontological evidence indicates that guanacos occu- expressed as the number of migrants per generation, pied the regions surrounding the altiplano since at least were higher from the Northwest to the Southeast 0.78 Ma (see review in Cajal et al. 2010) and their distri- (M12 = 8.5279) compared with from the Southeast to bution has certainly been influenced by repeated peri- the Northwest (M21 = 5.3502). ods of glacial build-up and melting even as recently as the Last Glacial Maximum near 25 000 years ago (Hoff- stetter 1986; MacQuarrie et al. 2005; Rabassa et al. 2011). Discussion During glacial maxima glaciers were found as low as 3800 meters above sea level (masl), over 1000 m below Genetic structure the current glacial line. Further, during inter-glacial Microsatellite and mtDNA sequence variation revealed periods large lakes (Hoffstetter 1986) may have also two relatively distinct genetic groups of guanaco that been physical barriers to guanaco displacement. were segregated geographically by the Central Andes The climatic conditions and vegetative cover in the Plateau. In the North-western region guanacos range Andean plateau during the Holocene probably contrib- over arid ecosystems such as desert and xeric shrub uted greatly to this hypothesized longitudinal ecological lands that include the Sechura and Atacama deserts barrier limiting guanaco dispersal. A warmer and drier between Peru and northern Chile. Southern guanacos climate was established around 7000–8000 years ago inhabit a more diverse array of ecosystems distributed with a brief interlude of increased humidity around from Bolivia southward, including tropical and subtrop- 4000–5000 years ago (Baied & Wheeler 1993), which ical dry broadleaf forests represented by Chaco savan- would have favoured microthermic vegetation adapted nas, temperate grasslands, savannas and shrub lands to arid conditions and high solar radiation, by develop- that include Argentinean Monte, Patagonian steppe, ing hard silica cuticles and other water and energy

Table 4 Percentage of individuals assigned to the Northwest 10 or Southeast genetic clusters with nuclear (Q > 75%, STRUCTURE analysis). The number of individuals from each locality is in parentheses. Localities containing animals from of both > 5 clusters, considered hybrids localities (Q 25%), are in bold 4 (localities are deﬁned in Table 1 and Fig. 1A) 7 1 3 Microsatellites 8 2 17 6 19 Localities Northwestern Southeastern Hybrids

16 11 HU 100.0 (18) 0.0 (0) 0.0 (0) PU 95.2 (20) 0.0 (0) 4.8 (1) 28 14 PA 33.3 (4) 8.3 (1) 58.4 (7) 9 LC 42.8 (9) 4.8 (1) 52.4 (11) 34 12 IL 0.0 (0) 63.2 (12) 36.8 (7) KA 11.8 (2) 64.7 (11) 23.5 (4) 26 15 13 LP 0.0 (0) 60.0 (6) 40.0 (4) LB 0.0 (0) 90.0 (18) 10.0 (2) 24 35 20 29 TE 0.0 (0) 93.3 (14) 6.7 (1) PV 0.0 (0) 100.0 (15) 0.0 (0) 23 21 CA 0.0 (0) 86.7 (13) 13.3 (2) VC 0.0 (0) 96.2 (25) 3.8 (1) 25 18 BP 0.0 (0) 95.0 (19) 5.0 (1) 32 22 ML 0.0 (0) 100.0 (22) 0.0 (0) TP 0.0 (0) 100.0 (23) 0.0 (0) 31 27 33 100 PK 0.0 (0) 100.0 (20) 0.0 (0) 30 TF 0.0 (0) 100.0 (20) 0.0 (0)

Northwestern region 20 Although the altiplano is not a biogeographical bar- Southeastern region 5 rier for all species, the environmental conditions found 1 in the area have helped isolate and differentiate some Sample size species, such as the Sigmodont rodents. For example, Fig. 4 Minimum spanning network representing the relation- Phyllotis xantophygus chilensis lives in the Puna and ships among 35 control region haplotypes (see Table 5). Circle Phyllotis limatus inhabits lowlands/pre-Puna area, sizes correspond to haplotype frequencies. where climate and the composition of vegetation differ because of the altitudinal effect (Steppan 1998; Palma saving strategies (Menegaz et al. 1989; Adler et al. 2004). et al. 2005). In contrast, Abrothryx olivaceus is character- This vegetation would not have been very palatable for ized by having one of the largest ranges in Chile, the guanaco, in spite of its generalist foraging strategy. In encompassing most of the north, the central valley and contrast, the vicunã(Vicugna vicugna) was better adapted the southern tip of the country, whereas A. andinus is to these extreme environments with its smaller body-size, restricted to high elevations in the pre-Puna and lower energy requirements, hair that was better adapted Altiplano regions of the Andes (Palma et al. 2005). to low temperatures and intense solar radiation and con- Although these small-sized mammals have smaller tinuous tooth growth better adapted to abrasive vegeta- home-ranges and generational time than guanacos, they tion (Franklin 1983; Lucherini 1996; Arzamendia et al. are indicative of the strength of selection and isolation 2006; Weinstock et al. 2009; Borgnia et al. 2010; Mosca pressures on this region’s biota. Torres & Puig 2010). Currently there is a relatively nar- Although geographically fairly well defined by sev- row portion of the Altiplano and Puna where guanacos eral analyses, the two groups of guanaco are clearly not and vicunãs are both found, but where they segregate completely isolated as there were several examples of spatially in different habitats. This pattern suggests that populations with individuals of both north-western and direct competition for food may occur periodically south-eastern heritage, as well as individuals of mixed (Lucherini 1996; Lucherini & Birocho 1997; Cajal et al. genetic heritage. However, our results suggest that gene 2010) and that competition for limited resources with flow between groups is asymmetric and is mostly from vicunã in the Altiplano and the Puna may have also the Northwest to Southeast, as shown by the LAMARC helped limit movement of guanacos across this region. and popABC analyses. Prevalent migration from Northwest

Table 5 Pairwise population differentiation based on allelic frequencies of 14 polymorphic microsatellites estimated as Jost D values

(below the diagonal) and Slatkin linearized FST values (above the diagonal) for each of the eight (top) and two (bottom) values used in microsatellite population scenarios. All comparisons were statistically signiﬁcant (P < 0.005) based on 10 000 permutations

Northwestern Southeastern

Arid Chile Northern Region (K = 2) and Hiper-Arid Chilean and Chilean Bolivian and central Western Southern Fuegian population (K = 8) N Peru Pre-altiplano Peri-Puna Chaco Patagonia Patagonia Patagonia zone

Hyper-Arid Peru 18 — 0.184 0.199 0.295 0.208 0.265 0.249 0.424 Chilean Pre-altiplano 21 0.552 — 0.072 0.123 0.111 0.165 0.115 0.278 Arid Chile and Chilean 52 0.662 0.322 — 0.060 0.058 0.086 0.051 0.191 Peri-Puna Bolivian Chaco 17 0.739 0.395 0.232 — 0.087 0.119 0.080 0.265 Northern and central 75 0.599 0.413 0.249 0.286 — 0.081 0.061 0.143 Patagonia Western Patagonia 26 0.626 0.505 0.283 0.317 0.245 — 0.053 0.242 Southern Patagonia 85 0.682 0.408 0.196 0.264 0.221 0.143 — 0.145 Fuegian zone 20 0.724 0.610 0.492 0.500 0.333 0.457 0.339 —

Northwest 91 — 0.042

Southeast 223 0.200 —

(A) 160 for easier detection of migrants using assignment tests 140 τ = 7.555 and this difference in level of genetic diversity has 120 potentially resulted in an underestimate of the migra- 100 tion rate into the Northwest. Nevertheless, migration 80 rates in both directions are low and the previous con- clusion that these populations should be considered 60 demographically independent is supported by the 40

Relative frequency migration rates estimated from our popABC analysis. 20 A good example was the population from Illapel (IL), 0 0 1 2 3 4 5 6 7 8 9 10 11 which is located in the Chilean mountains at the south Pairwise difference of the altiplano-puna zone. Throughout this contact zone, evidence of admixture was prevalent, but dissi- (B) 25 000 pated in populations further north and south based on τ = 0.516 microsatellite assignation. For example, in the popula- 20 000 tions of Pan de Azucar (PA), Llanos de Challe (LC),

15 000 Illapel (IL) and La Payunia (LP) just south of the Ata- cama Desert and the Altiplano, 36 to 58% of the indi- 10 000 viduals were of mixed origin. Levels of admixture decreased in localities farther away, as was observed in

Relative frequency 5000 Kaa-Iya (KA). This contact region is consistent with the absence of complete reciprocal monophyly between 0 0 1 2 3 4 5 6 7 8 L. g. cacsilensis and L. g. guanicoe using mtDNA control Pairwise difference region and CytB haplotypes in a smaller sampling of guanacos (Gonza´lez et al. 2006; Marıń et al. 2007, 2008). Fig. 5 Mismatch distribution of pairwise nucleotide differences Several mutually non-exclusive factors may have con- among control region sequences of northwestern guanacos (A), tributed to the fairly large numbers of individuals of and southeastern guanacos (B). Gray histograms represent the observed differences and the thin lines represent the ideal northwest genetic heritage in the south-eastern popula- distribution as predicted by the model. tions of La Payunia (LP), Loma Blanca (LB) and Cama- rones (CA). This includes closer geographic and to Southeast agrees with the expansion history of gua- ecological affinity among these localities than is evident naco. However, the Northwest population has more today because of the extirpation of intermediate popu- unique alleles than the Southeast population, allowing lations, long range dispersal patterns, or significant

(A) (D)

(B) (E)

Fig 6 (A) Plot of prior (black) and posterior (gray) distributions of Northwest effective population sizes, (B) Southeast and (C) ancestral of Lama guanicoe. (D) Prior (black) and posterior (gray) distributions of divergence time (in years) between Northwest and South- east clusters. (E) Northwest to Southeast migration rates and (F) Southeast to Northwest migration rates. anthropic translocation by Amerindians or early Euro- units observed in Patagonian sigmodontine rodents peans of individuals from the Andes to the coast (Poli- (Lessa et al. 2010) and probably reflects incipient regio- tis et al. 2011). nal barriers to gene flow as well as behavioural phylop- There was significant structure of microsatellite varia- atry exhibited by guanacos (Ortega & Franklin 1995; tion at the regional level, suggesting only moderate Young & Franklin 2004). This is consistent with other levels of genetic connectivity. Although slightly fewer molecular studies carried out at a regional spatial scale populations were recognized when the spatial compo- where populations from the Argentine provinces of nent was included in genetic variation (BAPS) relative Chubut and northern Santa Cruz southward to south- to the non-spatial Bayesian analysis (Structure), the ern Patagonia had little genetic differentiation and low results were largely comparable. In the north-western genetic structure (Mate´ et al. 2005) while remote isolated cluster there were 2–3 populations, one encompassing populations separated by geographical barriers such as Huallhua (HU) (Peru) and possibly unsampled areas Torres del Paine National Park and Tierra del Fuego had further to the north and one to two populations increased genetic uniqueness (Sarno et al. 2001). Broad between Putre (PU), and Llanos de Challe (LC) and Illa- geographic differences do not correlate well with guanaco pel (IL) in Chile. Among south-eastern guanacos there population structure, suggesting that more studies are is a less-well-defined gradient of related populations needed at the landscape scale to better understand the approximately consisting of: those from the central processes influencing guanaco population dynamics. Andean corridor from the Bolivan Chaco and peri-puna Locally, patterns of microsatellite genetic variation in Chile to the northern and/or central Patagonia, suggest that the relatively low levels of gene flow southern and/or western Patagonia, and the Fuegian among populations has occurred mostly from north to zone. This pattern corresponds with the phylogeographic south, and that guanacos probably occupied several

© 2012 Blackwell Publishing Ltd 476 J. C. MARIN ET AL. glacial refuges during the recent ice age (Fig. 1B). The Fu’s F values were significantly negative. The combina- southern Patagonian population shows evidence of tion of these two observations suggests a recent expan- gene flow from most of the other areas, including the sion 11 500 years ago, at the start of the Holocene distant populations of Bolivia and Peru, suggesting that deglaciation process in southern South America (McCul- gene flow and interconnectivity among guanacos loch et al. 2000). The uniformity of mitochondrial haplo- during the Holocene was very high. However, gene types across a large geographic range, with only one flow among populations is uneven, and generally in common haplotype that is shared in guanacos from the only one direction. Western Patagonia and the Fuegian Bolivian Chaco to the Fuegian zone, provided further populations also contributed to the genetic variation in support for a past demographic expansion. This pattern Southern Patagonia, suggesting that these regions may is expected when there are high levels of gene flow or have been reservoirs of genetic variation and a manifes- small effective size in populations that have not been tation of ice age refuges. The apparent gene flow from affected by long-term biogeographic barriers (Avise Southern Patagonia towards north and central Pata- 2000) and is common in populations where numbers gonia probably reflects more-recent animal movements increased rapidly as they expanded their ranges. This and gene flow, perhaps associated with habitat degra- pattern would result in genealogical trees with shallow dation, persecution and an associated ‘source/sink’ branches, many of which are dispersed widely across phenomenon. Further north, there is less evidence of the species current ranges, as we observed with south- connectivity between geographically proximate popula- ern guanacos. It is also possible that the shallow diver- tions, such as between Hyper-Arid Peru and Chilean gence levels reflect the historical mixing of populations pre-altiplano, and between these populations and the resulting from climatic and landscape changes that Bolivian Chaco. Populations in this region have proba- affected Patagonia throughout the Quaternary. bly been stable for longer periods of time (Marıń et al. During the late Quaternary it is likely that the south- 2008) due to more enduring geographic barriers to gene ward expansion of guanacos on the eastern side of the flow caused by the Andean altiplano. Andean plateau in Peru and northern Chile (Scherer Genetic interchange among populations is also evident 2009) was influenced and limited by the Atacama Desert, from the significant number of individuals with discor- which has persisted, except for brief interludes, for over dant mtDNA and microsatellites patterns and/or incon- 200 million years (Hartley & Chong 2002; Dunai et al. gruent (<75%) population assignment with Structure 2005; Clarke 2006). The severe climate of the Atacama and/or BAPS. This asymmetric pattern may reflect continues to impede guanaco movements in the region, sexual differences in dispersal through generations. limiting dispersion southwards to more humid habitats. Although males and females in family groups are territo- Similarly, the Atacama desert separates the two vicunã rial during the reproductive period and show phylopatry (Vicugna vicugna) subspecies, V. v. vicugna to the arid since they use the same territory for several years (Young area of the altiplano and puna under the Dry Diagonal, & Franklin 2004; Bank et al. 2003), adult males coming and V. v. mensalis to the more-humid altiplano at the from non-territorial male groups are more likely to dis- north (Marıń et al. 2007). Other mammalian genera have perse longer distances looking for new territories (Frank- similar distributional patterns, with sister species occu- lin 1983; Young & Franklin 2004). These differences in pying different sides of this xeric diagonal. These dispersal between genders may be facilitated at the pop- include north-south disjunct distributions between Abro- ulation-level by long-range movements of groups looking coma cinerea and Abrocoma bennetti (Abrocomidae), Chin- for forage and better weather conditions in the autumn- chilla brevicaudata and Chinchilla lanigera (Chinchillidae) winter. Although most guanaco populations are largely (Hershkovitz 1972), and Phyllotis magister and Phyllotis sedentary with small annual home ranges (Franklin 1982; darwini (Cricetidae)(Steppan 1998). Although these Marino & Baldi 2008), facultative migration has also been small- and medium- sized mammals have smaller home- documented in the mountains of central Chile/Argentina ranges than guanacos, they are indicative of the strength and parts of Patagonia (Ortega & Franklin 1995; Gonza´lez of different selection and isolation pressures that influ- et al. 2008; Moraga pers. comm.). These patterns would enced this regions biota. The small and restricted North- facilitate contact among neighbouring populations and western guanaco cluster of mixed genetic heritage, facilitate genetic interchange. which is seemingly confined to the Sechura and Atacama Deserts, may have been established and reconnected sporadically with populations to the south and east Population expansion during relatively brief periods of increased moisture, The mismatch distribution of Southeast guanacos was such as those that occurred 16.2–10.5 Ma and between close to the theoretically expected unimodal distribu- 8–3 Ma when the amount of vegetation east of the desert tions for population expansion, while Tajima′s D and increased (Betancourt et al. 2000).

The western slopes of the Andes, perhaps to the in Valle Chacabuco (VC), where estimates of mtDNA northwest of their current distributions, probably variation are the highest (Table 2). The Late Holocene served as refugia for guanacos during the Pleistocene was a period of low humidity or dry climate, which glacial periods, as evidenced by patterns of genetic was favourable for guanaco populations (Stine & Stine diversity, the network of mitochondrial haplotypes and 1990). Similar patterns have been proposed for austral the mismatch distributions. Current populations in Peru huemul deer (Marıń et al. 2012) and several rodent spe- and northern Chile have the highest mtDNA and cies (Lessa et al. 2010; Pardinãs et al. 2011). However, microsatellite diversity and multiple private and derived timing of the demographic expansion in rodent gener- mtDNA haplotypes, indicating that guanaco popula- ally exceeds 21 000 years, to be older than the LGM tions have existed in these areas for extended time peri- (Lessa et al. 2010). ods and with little suggestion of population expansions Finally our data also reflect the precipitous reduction or extended contractions. A similar multimodal mis- in population sizes that occurred as intensive sheep match distribution pattern was observed in populations ranching spread throughout the region during the last described as L. g. cacsilensis (Marıń et al. 2008). Glacial centuries and guanacos were harvested for their pelts or ecological refugia perhaps also existed east of the (Dennler de La Tour 1954; Mares & Ojeda 1984), proba- Andean plateau, but may not have been sampled bly resulting in the loss of rare haplotypes. Chile because past extinctions/extirpations and because cur- exported around 35 000 pelts during the 20th century rent guanaco populations in this region are severely (Iriarte & Jaksic 1986), but far fewer than Argentina, reduced and isolated. Habitat changes from the LGM to which exported around 223 000 units between 1976 and the beginning of the Holocene, when open savannah 1979 (Mares & Ojeda 1984). habitats and grasslands were replaced by dense savannah and evergreen Amazon forests (De Vivo & Carmi- Taxonomic, conservation and management implications gnotto 2004), probably led to the local extinction of guanacos from north-eastern and southern Brazil, Uru- Given the dynamic history of the guanaco during the guay and the Pampeana region of Argentina towards last 20 000 years, we suggest that both clusters should the end of the Pleistocene (Couto 1983; Gue´rin & Faure be considered to be incipient subspecies, with a broad 1999; Scherer 2009). Moreover, in some areas of the contact area with individuals with different degrees of Pampas, guanacos inhabited drier and cooler conditions genetic heritage from each subspecies. The recognition until the late Holocene (between c.AD 800–1200), when of two subspecies is supported by the presence of local extinctions occurred following environmental unique derived haplotypes and significant differences changes produced by the Medieval Thermal Maximum in microsatellite structure in each. L. g. cacsilensis would (Politis & Pedrotta 2006; Politis et al. 2011). encompass populations from Peru to northern Chile, The evolution and expansion of guanacos in southern including guanacos from Llanos de Challe (LC) and South American was probably also affected by the Illapel (IL) and L. g. guanicoe would include all areas changing availability of ecological niches left following further south and east. These two groups could also be the extinction of Megatherium, mylodones, gliptodontes, considered as different Evolutionary Significant Units Hippidion, Lama gracilis, vicunãs and other herbivores, (ESU) as defined by Moritz (1994). Currently, the gua- through the end of the Pleistocene (Markgraf naco is classified as a species of ‘Least Concern’ by the 1985; Weinstock et al. 2009; Barnosky & Lindsey 2010). IUCN (Baldi et al. 2008), and the recognition of two dif- By the end of Quaternary, guanacos had increased their ferent subspecies would facilitate more precise assess- effective population size sharply and had probably col- ments of population threats and management needs for onized much of southern South America by the Holo- both. This would also be the first step for defining man- cene. They are theorized to have already colonized and agement units and to better understanding whether the been isolated on the island of Tierra del Fuego by the observed genetic structure is due in part to climatic, formation of the Straits of Magellan around that time topographic/geographic or ecological factors (PasbØll (Sarno et al. 2001). et al. 2006). Patterns of local extinction and colonization are com- Overall, our analysis has provided estimates of demo- mon in Patagonian terrestrial mammals during the graphic parameters that fit well with our current under- repeated glacial advances and retreats, with some spe- standing of guanaco demographic history and population cies surviving glacial periods in one or a few southern status. Our results confirm the recent splitting of the refugia while others inhabited and subsequently recol- Northwest and Southeast populations, the low migra- onized from northern areas (Pardinãs et al. 2011; Sersic tion rates between the two populations and the low et al. 2011). It is therefore reasonable that some guana- effective population size of the Northwest guanacos. cos may have survived in Patagonian refugia, perhaps The results provide new insights that are important to

© 2012 Blackwell Publishing Ltd 478 J. C. MARIN ET AL. guanaco conservation management. Although the Gustavo Suarez de Freitas and Antonio Morizaki (INRENA), Northwest population appears to have been relatively Wilder Trejo, Daniel Rivera, Daniel Arestegui, Leonidas Rodri- stable during the last 20 000 years, and second, our esti- guez, Carlos Flores and Dirky Arias at CONACS, Carlos Ponce (Conservation International). In Chile, we thank the Servicio mates of effective population size suggest the census Agrıćola y Ganadero, SAG (Permit 447, 2002), the Corporacioń size of this population is possibly larger than is cur- Nacional Forestal, CONAF (permit 6/02, 2002), for granting rently believed. These findings reinforce the importance other collection permits and helping in collecting samples. We of incorporating better monitoring and management of especially thanks C. Bonacic (Pontificia Universidad Cato´lica the Northwest population into the guanaco recovery de Chile), O. Skewes (Universidad de Concepcioń), F. de Smet plan and the maintenance of healthy guanaco popula- (Valchac Ltd.), F. Novoa (Centro de Ecologıá Aplicada Ltda), tions throughout its distribution. and Minera Los Pelambres, R. Baldi and V. Burgi (CENPAT, Argentina), E. Cuellar (WCS-Bolivia), A. Duarte (Zoolo´gico de Conservation and management actions should be Mendoza, Argentina) for sharing samples; Homero Pezoa, implemented at a local scale, taking into consideration Nicola´s Aravena, for support in the laboratory and Pablo Oro- the preservation of adaptive variation and evolutionary zco-teWengel (Cardiff University), for support in the software process throughout the entire range of the species, analyses. Special thanks are due to Jane C. Wheeler (CONOPA) while correcting or mitigating adverse effects of human for facilitating samples and collaboration, and Michael Bruford activities (Crandall et al. 2000; Frankham et al. 2003). (Cardiff University) and Angel Spotorno (Universidad de Therefore it is important to: (i) protect populations with Chile) for useful information, discussions, advice and support. Samples were transported under CITES authorization large amounts of genetic diversity; (ii) promote the con- numbers 6282, 4222, 6007, 5971, 5177, 5178, 23355, 22967 and nectivity among localities within the same populations 22920. detected in the Structure/BAPS analyses by improving corridors for dispersal; (iii) translocate individuals if necessary among localities with animals of similar References genetic heritage to limit extreme effects of inbreeding Adler PB, Milchunas DG, Lauenroth W, Sala O, Burke IC (Franklin & Grigione 2005); and (iv) carefully monitor (2004) Functional traits of graminoids in semi-arid steppes: populations being legally harvested (Marıń et al. 2009). a test of grazing histories. Journal of Applied Ecology, 41, Several guanaco populations need close demographic 653–663. and genetic monitoring, including (i) Peruvian popula- Alpers DL, Van Vuuren BJ, Arctander P, Robinson TJ (2004) Hippotragus equi- tions, which are heavily impacted by hunting and habi- Population genetics of the roan antelope ( nus) with suggestions for conservation. Molecular Ecology, 13, tat deterioration, (ii) the small fragmented populations 1771–1784. in Bolivia and Paraguay, (iii) the isolated and frag- Arzamendia Y, Cassini MH, Vila´ VL (2006) Habitat use by mented populations of northern Chile and Argentina vicunã Vicugna vicugna in Laguna Pozuelos Reserve, Jujuy, and (iv) the large and legally harvested, but genetically Argentina. Oryx, 40,1–6. less diverse and more isolated population of Tierra del Avise JC (2000) Phylogeography: The History and Formation of Fuego (Zapata et al. 2008). For naturally isolated popu- Species. Harvard University Press, Cambridge, Massachusetts. lations or for population fragmented by human activi- Baied CA, Wheeler JC (1993) Evolution of high Andean Puna Ecosystems: environment, climate, and culture change over ties, the introduced guanacos on the Falkland Islands the last 12,000 years in the Central Andes. Mountain Research may serve as an example of founder effects and bottle- and Development, 13, 145–156. necks on the genetic and reproductive features of this Baldi R, Lichtenstein G, Gonza´lez B et al. (2008) Lama guanicoe. species (Franklin & Grigione 2005). In: IUCN 2010. IUCN Red List of Threatened Species. Version 2010.1. www.iucnredlist.org and http://www.iucnredlist. org/apps/ redlist/details/11186/0 Acknowledgements Bank MS, Sarno RJ, Franklin WL (2003) Spatial distribution of Financial support from the following organizations is gratefully guanaco mating sites in southern Chile: conservation impli- acknowledged: CONICYT, Chile (Beca de Apoyo a Tesis Doc- cations. Biological Conservation, 112, 427–434. toral); FONDECYT, Chile Grant 101105 and Postdoctoral Grant Barnosky AD, Lindsey EL (2010) Timing of quaternary megafa- 3050046; Darwin Initiative for the Survival of Species (UK), unal extinction in South America in relation to human arri- The British Embassy (Lima), NERC (UK) grant GST/02/828, val and climate change. Quaternary International, 217,10–29. Darwin Initiative grant 12/022, European Commission INCO- Beaumont MA (2010) Approximate Bayesian computation in DC ICA4-2000-10229, MACS. This research was supported in evolution and ecology. Annual Review of Ecology, Evolution, part by the Intramural Research Program of the National Insti- and Systematics, 41, 379–406. tutes of Health (NIH) and the National Cancer Institute (NCI) Beerli P, Felsenstein J (1999) Maximum-likelihood estimation of (US). This work was written by Benito A. 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Additional supporting information may be found in the online ver- J.C.M. developed the ideas, obtained funding, and conducted sion of this article. the DNA analyses; C.S.C., B.A.G., and J.C.M. collected the sam- Table S1 Prior distributions used for the analysis of the Lama ples; J.C.M., E.P., and C.S.C. analysed the data; and J.C.M., B. guanicoe data (popABC analysis). A.G., and W.E.J. wrote the paper. All authors read, commented on and approved the ﬁnal manuscript..