Phylogeography of the armadillo Chaetophractus villosus (Dasypodidae Xenarthra): post-glacial range expansion from Pampas to Patagonia (Argentina)

Molecular Phylogenetics and Evolution 55 (2010) 38–46

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Phylogeography of the armadillo Chaetophractus villosus (Dasypodidae Xenarthra): Post-glacial range expansion from Pampas to Patagonia (Argentina) Sebastián Poljak a, Viviana Confalonieri b, Mariana Fasanella a, Magalí Gabrielli a, Marta Susana Lizarralde a,* a

Laboratorio de Ecología Molecular, Centro Regional de Estudios Genómicos, Universidad Nacional de La Plata, Av. Calchaquí km 23.5 Piso 4, CP 1888 Florencio Varela, Buenos Aires, Argentina b Laboratorio de Investigación en Filogenias Moleculares y Filogeografía, Departamento de Ecologia, Genetica y Evolución, Fac. de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

a r t i c l e

i n f o

Article history: Received 21 April 2009 Revised 15 December 2009 Accepted 21 December 2009 Available online 6 January 2010 Keywords: Large hairy armadillo Colonisation mtDNA Nested Clade Analysis (NCA) South American mammals

a b s t r a c t We report a phylogeographic study of Chaetophractus villosus populations in Argentina. Control Region (CR) sequences (484 bp) were obtained for 76 C. villosus from 20 locations across the species whole distribution range. Seventeen new haplotypes were identified. The highest genetic variation and the earliest fossils were found in the Pampean Region, thus appearing as the most probable area of origin of the species. A general pattern of Contiguous Range Expansion (CRE) was revealed by Nested Clade Analysis (NCA) supported by mismatch analysis and Fu’s test. The Pampean Region would have been the preexpansion area, while Patagonia would have been the main dispersal route of contiguous expansion, possibly after the Pleistocenic glaciations. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction The order Xenarthra is one of the four major placental clades, despite their still unsettled phylogenetic relationships (Murphy et al., 2001; Kriegs et al., 2006; Nikolaev et al., 2007). The monophyly of the order has been classically recognised (McKenna and Bell, 1997), supported by morphological (Engelmann, 1985; Patterson et al., 1989, 1992; Rose and Emry, 1993; Gaudin, 1999) and molecular characters (de Jong et al., 1985; van Dijk et al., 1999; Delsuc et al., 2001). The evolution of xenarthrans was bound to South America, and they have been considered to be representatives of the initial mammalian stock in this continent (Patterson and Pascual, 1972). Taking into account the synchronicity between planetary and biological events, global changes may have played a crucial role in shaping the evolutionary history of extant xenarthrans (Delsuc et al., 2004), particularly those present in South America. The glacial events affected the southern region of this continent, especially in the Andes mountains and current Patagonia in southern Argentina. These events had a profound effect on the climate over the continent. The Pampean Region or ‘‘Pampas”, in Central Argentina, underwent major changes in the climatic, geological and ecological conditions during the Pleistocene (Tonni et al., 1999; Nabel et al., 2000; Rabassa et al., 2005).

* Corresponding author. Fax: +54 11 4275 8100. E-mail address: [email protected] (M.S. Lizarralde). 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.12.021

Xenarthrans are endemic of South and Central America, except for Dasypus novemcinctus, which dispersed into southern North America recently, after the rise of the Isthmus of Panamá between 3 and 2.5 million years (Myr) ago (Late Pliocene) (Coates and Obando, 1996). Armadillos (Cingulata, Dasypodidae) are the most diverse xenarthran lineage. They split after the isolation of South America from other continental masses at the end of the Cretaceous (Patterson and Pascual, 1972) or around the K/T boundary about 65 Myr ago (Delsuc et al., 2004). Previous cytogenetic and molecular studies have provided a view of intra-ordinal relationships and phylogeny of Xenarthra (Jorge et al., 1977, 1985; Cao et al., 1998; Delsuc et al., 2001, 2002, 2003; Lizarralde et al., 2005; Möller-Krull et al., 2007). Based on molecular clock analysis, Delsuc et al. (2004) proposed that the armadillos of the genus Chaetophractus may have split from other related genera about 6 Myr BP ago. However, the first paleontological record of this genus is represented by plates assignable to the living species Chaetophractus villosus (large hairy armadillo). These plates were found in the locality of Chapadmalal (Buenos Aires province, Argentina) in a stratum over the Atlantic coast of the Pampean Region that correspond to the Chapadmalalan mammal age defined by Cione and Tonni (1995) (4– 3.2 Myr, Late Pliocene, Carlini and Scillato-Yané, 1996). Chaetophractus villosus is endemic to southern South America, where it is one of the most widely distributed species. Its distribution range extends from the ‘‘Gran Chaco” of Bolivia and Paraguay to the Santa Cruz province in Argentina and to the Bio Bio and

S. Poljak et al. / Molecular Phylogenetics and Evolution 55 (2010) 38–46

Magallanes provinces in Chile (Azize Atallah, 1975; Tamayo, 1973; Wetzel, 1985; Wilson and Reeder, 1993) (Fig. 1a). C. villosus was also introduced in Isla Grande of Tierra del Fuego, the southernmost insular region of Argentina and Chile (Fig. 1) about 25 years ago (Poljak et al., 2007). Some anatomical and physiological peculiarities of living xenarthrans are considered by many authors as primitive compared with other placentals (e.g. Carlini et al., 1994; Carlini and Scillato-Yané, 1996; Thomas, 1887). The thermoregulatory responses and the life habits of armadillos are influenced by the environment (Roig, 1969, 1970) because of their low body temperature and metabolic rate in relation to body mass (Mc Nab, 1979, 1980, 1985). As reflected by its wide distribution, C. villosus shows high adaptive capacity to different climatic conditions and food sources. This can be achieved by having fossorial habits, which allow it to tolerate environmental temperature fluctuations (Mc Nab, 1979, 1985), and an unspecialised diet (Redford, 1985). According to Dr. S.F. Vizcaíno (personal communication), the factor constraining the southern distribution of C. villosus is not climate but rather the water barrier represented by the Magellan Strait to the south and the de La Plata river to the east, which prevents its dispersal to Tierra del Fuego Archipelago and Uruguay, respectively. In this paper, the population genetics of C. villosus from several locations across its whole distribution range in Argentina is analysed. We applied a phylogeographic approach using partial sequences of the Control Region (CR) in order to infer: (a) if populations show evidence of genetic structuring; (b) if so, the historical or recurrent processes that might explain its occurrence; (c) the most probable ancient area of the species’ distribution and if it is consistent with the fossil record; (d) range expansion patterns

39

across its distribution range and (e) the phylogeographic relationships between the introduced population of Tierra del Fuego and continental source populations.

2. Materials and methods 2.1. Specimens analysed and sampling locations Tissue samples (ethanol fixed or dried remains) of 76 C. villosus specimens were taken from 20 locations across Argentina (Fig. 1a and Table 2). Localities sampled are widely distributed, ranging the whole area of distribution of the species in Argentina. It is important to note that capture efforts were the same in northern, southern and central regions, so in this sense, the fewest samples from the northern part of the distribution implies much smaller population densities, typical of more marginal environments. C. villosus is a mammal of fossorial habits, difficult to capture alive with traps. Besides, it is nocturnal during the summer and diurnal during the winter, when they leave their burrows around noon, only at the warmest hours of the day, which reduces the possibility of capture. At the northern part of the distributions, the most abundant plant species where the hairy armadillo thrive are thorny shrubs, a fact that also makes the collection a challenging task. All the samples (liver, spleen, muscle, blood or skin) were obtained from armadillos collected by hand in the field (alive or dead on the roads) and from the collection of the Instituto Argentino de Investigaciones para las Zonas Áridas (IADIZA-CONICET), Mendoza. Blood samples were obtained by puncture of the tail ventral artery of anaesthetised animals (25–30 mg/kg of ketamine hydrochlorate

Fig. 1. (a) Distribution of 20 sampling locations of C. villosus in Argentina (see Table 1 for description of locations and sample sizes). The shaded area indicates the total current range of C. villosus. (b) Patagonian region (dotted line) and Pampean Region (solid line). Arrows indicate contiguous range expansion processes inferred by the nested clade analysis. Indexes 1.1, 1.2, 1.3 and 3.1 represent the inferred clades.

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50 mg) (Poljak et al., 2004). After that, live specimens were released at the site of capture. Tissues and other data associated with each individual are referenced directly to each voucher specimen and stored along with a field catalogue number in the following collections belonging to Universidad Nacional de La Plata, Argentina: Centro Regional de Estudios Genómicos and Museo de Ciencias Naturales. 2.2. Control Region (CR) of mammalian mtDNA as phylogeographic tool The CR length of mammals varies between approximately 880 and 1400 bp and contains the main regulatory elements for mtDNA replication and expression (Shadel and Clayton, 1997). It is divided into three domains: Extended Termination Associated Sequences (ETAS), Central and Conserved Sequence Blocks (CSB) (Saccone et al., 1987; Sbisà et al., 1997). The ETAS and CSB domains show high mutation rates, therefore being called hypervariable regions, and are commonly used in studies of population genetics and phylogeography (Ducroz et al., 2005; Matson and Baker, 2001; Moraes-Barros et al., 2006; Reyes et al., 2003). In contrast, the Central Domain is conservative and especially useful in studies involving more divergent taxa (Brown et al., 1986; Douzery and Randi, 1997; Larissa et al., 2002; Saccone et al., 1987, 1991; Sbisà et al., 1997). The CR shows a strict maternal inheritance (Hutchinson et al., 1974), short length, different rates of evolution for the different domains, and great availability of sequences from several species. All these features make it an excellent model for studying the evolutionary relationships among mammals at different divergence levels (Brown et al., 1986; Saccone et al., 1987, 1991; Sbisà et al., 1997) and, in particular, for phylogeographic studies (Avise, 1994). 2.3. DNA extraction, amplification and sequencing DNA was extracted from fixed or dried tissues (liver, spleen, muscle and skin) using the sodium dodecyl sulphate–proteinase K/phenol/RNAse method (Sambrock et al., 1989), while DNA was extracted from blood following John et al. (1991). Samples were concentrated by ethanol precipitation. Partial CR sequences were amplified using the universal primers Thr-L15926: 50 -CAATTCCCC GGTCTTGTAAACC-30 located in the neighbouring tRNA-pro gene and DL-H16340: 50 -CCTGAAGTAGGAACCAGATG-30 (Vilà et al., 1999). Amplification of the double-stranded product was performed in 25 ll volume of PCR mix containing 1.25 U of Taq DNA polymerase (Invitrogen™), 2.5 ll of 10 Taq polymerase buffer with (NH4)SO4, 1.5 mM of MgCl2, 200 lM of DNTP’s and 5 pmol of each primer. The amplification was performed in a Hybaid MBS 0.2S thermocycler (Electron Corporation). The thermal profile consisted of an initial denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, at 55 °C for 30 s and at 72 °C for 45 s with a final extension step at 72 °C for 5 min. Double-stranded PCR products were purified and concentrated by ethanol precipitation, examined on 1% agarose gels and directly sequenced in both directions using the same primers as those used for amplifications. Sequencing was conducted using an ABI PRISM 3130 XL automated sequencer (Applied Biosystems™) at the Sequencing Services of Universidad de Buenos Aires (Buenos Aires, Argentina). Sequences were edited with Chromas 2.3 (Technelysium Pty. Ltd., 1998–2004, http://www.technelysium.com.au) and aligned using the computer software package CLUSTAL W (Thompson et al., 1994). 2.4. Phylogeographic analysis The package ANeCa (Panchal, 2007) was used to automate the phylogeographic analysis. The last version of this package includes

the program TCS version 1.21 (Clement et al., 2000a,b), GeoDis version 2.5 (Posada et al., 2000) and an automation for clade nesting and inference key steps. TCS built a haplotype network that was rooted using C. vellerosus sequence, the sister species of C. villosus (Frechkop and Yepes, 1949). Ambiguities in the net were resolved manually following Crandall and Templeton (1993). Nested Clade Analysis (NCA) (Templeton, 1998) was performed on the partial CR sequences of C. villosus to allow for the detection and biological interpretation of statistically significant phylogeographic patterns. The NCA tests for non-random geographical distribution of related haplotype groups and allows inferring if the geographical distribution of haplotypes was due to gene flow or population history. To test the effect of historical population events (e.g. fragmentation, contiguous range expansion), the NCA determines the departures from expectation under the null hypothesis of no geographical association for each clade at each hierarchical level. Calculations on geographical associations were performed with 10,000 permutations. Observed distance patterns were interpreted using the automated inference key. We used the NCA because it constitutes a useful analytical tool for the comprehension of the histories of the species. It is important to note that the debate about the statistical validation of the inferences of NCA became stronger in the past 2 years (see Knowles, 2008 and Templeton, 2009a,b). Studies by Petit (2008), based on independent analysis of Knowles and Maddison (2002), Petit and Grivet (2002) and Panchal and Beaumont (2007), found that the NCA tends to incur in ‘‘false positives”. Recent studies by Templeton (2008) concluded that there were mistakes of interpretation of the inference key or in the assumptions, so the problem of the NCA seems to be the tendency to the production of ‘‘false negatives”. From all this controversy, Garrick et al. (2008) concluded that there should be a cross-validation of the inferences of the NCA through the use of multiple independent loci and/or complementary analyses (statistical phylogeography), which had been already suggested by Templeton (2004). According to Beaumont and Panchal (2008) ‘‘There is a real demographic history that explains the data, but we do not yet have sufficient information to elucidate what it is” so NCA constitutes another analytical tool for the analysis of the histories of the organisms. In order to confront the interpretations of the NCA, we carry out Fu’s and Tajima’s neutrality tests and the goodness-of-fit test between the observed mismatch distribution and that expected under a sudden expansion model was also applied using the sum of squared deviations (Tajima, 1993; Fu, 1997). 2.5. Data analysis A median-joining (MJ) network (Bandelt et al., 1999) was constructed using Network 4 (www.fluxus-engineering.com) to calculate mismatch distributions, distance relationships, pairwise differences and relative frequencies among haplotypes. The interpretations of the NCA results were improved using Arlequin version 3.0 (Excoffier et al., 2005) to estimate the standard diversity index (p), and the mean number of pairwise differences between haplotypes (Nei, 1987; Tajima, 1993). 3. Results 3.1. Sequence variation Twelve C–T transition point mutations were observed in the sequence alignment and 17 new haplotypes were identified. There was no evidence of heteroplasmy. The haplotypes, their relative frequencies, polymorphic sites and GenBank accession numbers

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are given in Table 1. The sample locations, the number of individuals from each location and their corresponding haplotypes are listed in Table 2. The nucleotide composition was 26.54% C, 29.66% T, 30.68% A, 13.12% G and the mean nucleotide diversity (p) among all haplotypes was 0.003781 ± 0.002436.

4. Discussion 4.1. Phylogeography The NCA revealed a significant geographical association in the distribution of the genetic variation of C. villosus populations, which follows in most clades a general pattern of CRE. In the predicted pattern of CRE described by Cann et al. (1987), some of the older haplotypes are confined to the ancestral pre-expansion area while some of the younger haplotypes that arose in the expanding populations are geographically widespread (CRE) or distantly located from their ancestral (interior) haplotypes (long-distance colonisation). According to Templeton et al. (1995) these patterns should not be recurrent but can occur in more than one nested category due to ancestral polymorphism in the expanding populations and should be geographically congruent since they reflect the same historical event. Taking into account this simulation model and the geographical distribution of the older (interior) and younger (exterior or tip) haplotypes in the clades where CRE is inferred, it is reasonable to assume that the pre-expansion area is in the centre of Argentina and that the species would have undergone mainly a southwards, eastwards and northeastwards expansions (Fig. 1b). This is consistently evidenced in clades at different hierarchical levels 1.1, 1.3, 2.1 and 3.1 (Fig. 1b and Table 4). The clade 1.3 is composed of two haplotypes: HG at the interior position and HD at the tip position (Fig. 2). According to Castelloe and Templeton (1994), this result implies that HG (locations 6, 7 and 10 in Fig. 1a) is older than HD (locations 11 and 14 in Fig. 1a). Therefore, the most likely interpretation is that the CRE (Table 4) occurred from the centre to the east of Pampean Region Argentina, towards the Atlantic coast (Fig. 1b). It is also observed a slower southwards movement, as reflected by the presence of the HD haplotype at location 11 (Fig. 1a, b and Table 2). Following the same criteria, within clade 1.1 tip haplotype HM seem to have moved to the northwest of the Pampean Region (location 6, where is present) from localities 10, 14 and 15 (where interior haplotype HC is present) (Fig. 1a and b). At the next nesting level, the CRE of clade 2.1 it’s due to the presence of haplotype HF (tip clade 1.6) at locality 13 into the east, over the atlantic coast (Fig. 1a and b). Finally, the tip position of clade 2.1 within the clade 3.1 and in the whole haplotype network may indicate that its lineages are

3.2. Phylogeographic analysis Fig. 2 illustrates the network linking the 17 haplotypes of the CR from 76 C. villosus individuals and three hypothetical haplotypes (small empty circles) generated by the program in such a way that nearest haplotypes differ by a single point mutation. The groupings or clades were arranged in a nested hierarchical structure, so that the clades at a level or hierarchy become subclades at the next level. The haplotypes themselves represent the clades at level 0 and the network resulting from the present study is also composed of first-, second- and third-level clades. Table 3 shows the results of the nested contingency analysis of geographical associations; clades with no geographical or haplotype variation were not included. The null hypothesis of no geographical association was rejected in 6 of 11 clades with a confidence index of 0.05. The biological interpretations are described in Table 4. The contiguous range expansion (CRE) appears to be a general process in the history of the studied species (clades 1.1, 1.3, 2.1 and 3.1), whereas the second-level clade 2.3 and thirdlevel clade 3.2 are characterised by a restricted gene flow with isolation by distance (RGFID). The HA haplotype is the most frequently found among sample locations, as compared with other haplotypes of the network. This feature, together with its interior position (five haplotypes derive from it) makes it the most likely ancestral haplotype (Fig. 2 and Table 2).

3.3. Mismatch distribution and neutrality tests The mean pairwise differences were 1.829825 ± 1.063994 (S = 2.381) with differences between sequences ranging between 0% and 1.9% (i.e. 0–9 differences; Fig. 3). Observed vs. expected pairwise differences are also shown. Fu’s Fs was 8.63207 (p = 0.001) and Tajima’s D was 0.69809 (p = 0.269). The sum of squared deviations of the goodness-of-fit test under the sudden expansion model was 0.00092481 (p = 0.45).

Table 1 Haplotypes (HA–HQ), position of polymorphic sites in the CR 484 bp sequences (6–348) of C. villosus, number of individuals with each haplotype (n), their absolute frequencies (Abs. Freq.) and GenBank accession numbers (GB Acc. No.). Haplotype

6

13

20

177

204

214

238

289

295

311

335

348

n

Abs. Freq.

GB Acc. No.

HA HB HC HD HE HF HG HH HI HJ HK HL HM HN HO HP HQ

G

T

G

T

C

C T

C

C

T

G

G

A

17 3 28 4 2 1 5 1 2 2 1 5 1 1 1 1 1

0.22368 0.03947 0.36842 0.05263 0.02631 0.01315 0.06578 0.01315 0.02631 0.02631 0.01315 0.06578 0.01315 0.01315 0.01315 0.01315 0.01315

DQ 136314 DQ 136315 DQ 136316 DQ 136317 EU 019190 EU 019191 EU 019192 EU 019193 EU 019194 EU 100942 EU 100943 EU 100944 FJ 544909 FJ 544910 FJ 544911 FJ 544912 FJ 544913

C C

A

A T

T

C C

C

T

T A A

C C

C C C C

T T C

A A

A

A A A A

A A A A

A A A A

A

G G

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Table 2 Sampling locations (1–20, see Fig. 1), province, locality, number of individuals (n) and their respective haplotypes. Locality No.

Province

Locality name

n

Haplotype (no. of individuals)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Chaco Tucumán Tucumán Santa Fé Mendoza San Luis Córdoba La Pampa San Luis La Pampa La Pampa Buenos Aires Buenos Aires Buenos Aires Buenos Aires Buenos Aires Chubut Chubut Santa Cruz Tierra del Fuego

Loro Hablador Aguilares Taco Ralo Lag. El Dientudo Lavalle Ea. El Centenario Laboulaye Nueva Galia Ea. La Luz Trenel Winifreda Navarro Uribelarrea Pip.-PtaPied-Ver Azul Bahia Blanca Pla. Valdés Garayalde Río Gallegos San Sebastián

3 1 3 1 1 4 1 1 2 15 2 1 1 10 3 2 2 1 2 20

HI(2) HK(1) HL(1) HQ(1) HO(1) HP(1) HL(1) HN(1) HG(1) HA(2) HM(1) HG(1) HA(1) HL(1) HJ(1) HA(7) HG(3) HB(1) HC(1) HL(1) HE(1) HJ(1) HD(1) HA(1) HA(1) HF(1) HB(2) HA(3) HD(3) HH(1) HC(1) HC(1) HL(1) HE(1) HA(2) HC(2) HC(1) HC(2) HC(20)

Table 3 Contingency analysis for each clade and geographical associations. Nesting clade

Chi-square

Probability

1.1 1.2 1.3 1.4 1.5 2.1 2.2 2.3 3.1 3.2

44.46 15.33 9 6 3 31 5.31 18 46.9 8.55

0.06 0.43 0.05 1 1 0.16 0.85 0.14 0.00 0.59

Total cladogram

60.02

0.00

Table 4 Nested contingency analysis of geographical associations for the mitochondrial CR haplotypes of Chaetophractus villosus, identified from 20 sampling locations. Numbers in the chain of inference refer to the series of steps undertaken in Templeton’s key. The Table only includes clades in which the null hypothesis of no geographical association between haplotype variation and geographical distribution is rejected.

Fig. 2. Haplotype network of the Control Region (CR) of C. villosus. Each haplotype is represented by two letters (e.g. haplotype C: HC) enclosed in a circle of size proportional to the amount of individuals with that haplotype. The small rectangle (Ch) corresponds to a sequence of Chaetophractus vellerosus used as reference (outgroup) and the arrow indicates the root location of the network. The ancestral haplotype HA is represented by a rectangle. The small empty circles represent hypothetical haplotypes needed to join the haplotypes in a way that each haplotype differs from the nearest one by one nucleotide. Thin black lines delimit first-level clades (1.1–1.9), light grey lines second-level clades (2.1–2.4) and thick dark grey lines third-level clades (3.1 and 3.2).

Clade

Chain of inference

Demographic event inferred

1.1 1.3

Contiguous range expansion Contiguous range expansion

2.1 2.3

1–2–11–12 NO 1–19–20–2–11–12 NO 1–2–11–12 NO 1–2–3–4 NO

3.1 3.2

1–2–11–12 NO 1–2–3–4 NO

Contiguous range expansion Restricted gene flow with isolation by distance Contiguous range expansion Restricted gene flow with isolation by distance

younger than those contained in clade 2.2, at an interior position (Fig. 2). HC was found in the centre of the Pampean Region (locations 10 and 15) but also it was exclusively found in all Patagonian locations (including the Isla Grande of Tierra del Fuego where the species was introduced (Poljak et al., 2007). Therefore, HC would represent the younger haplotype geographically widespread that arose in the expanding populations from the Pampean Region to the Patagonia (Fig. 1b), representing the inferred CRE of clade 3.1. The climate changes in the Pampean Region were historically closely related to the glacial and interglacial periods in Patagonia over the last 5 Myr, especially during the Pleistocene (Rabassa et al., 2005). This resulted in a series of changes in the ecological conditions which correlated with changes in the fauna (Tonni

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Fig. 3. Pairwise differences. The values above and below the bars indicate the amount of sequence pairs among the total possible pairs between the 76 sequences and the number of differences (0–9) of each group, respectively. The up-right graphic shows the observed vs. expected differences distribution among possible sequence pairs.

et al., 1999; Nabel et al., 2000; Rabassa et al., 2005). After the definitive retreat of the ice masses to the Andes due to climatic changes (Rabassa et al., 2005), new available areas in Patagonia may have had an impact on the biogeography of South American biotas. Its fossorial habits that enable thermal regulation allowed C. villosus to extend its distribution to areas with adverse climatic conditions such as southern Patagonia. The lower availability of feeding resources in these areas related to the less favourable climate conditions probably compelled C. villosus to extend its home-range. This process may have promoted a rapid post-glacial colonisation of the region, most probably favoring the expansion of HC across the Patagonia. Evidences of a similar process of rapid range expansion with severe loss of genetic variability in the populations at the colonisation front, have been previously reported by Huchon et al. (1999) for the armadillo species D. novemcinctus into southern North America. The CRE process evidenced at different nested levels by the NCA analysis was further supported by the mismatch distribution test (Fig. 3), which showed an unimodal distribution of the pairwise differences among all sequences of C. villosus. This model is predicted for populations that have experienced a recent demographic expansion and/or an increase in the distribution range, implying departures from strict neutrality (Slatkin and Hudson, 1991; Rogers and Harpending, 1992; Schneider and Excoffier, 1999). Con-

sidering the introduction of C. villosus in Tierra del Fuego (the southernmost insular part of Argentina), the positive effect of human activity on its establishment (Poljak et al., 2007) and the null population genetic variability in all Patagonia, the possibility that colonisation of the abovementioned area could be linked to human occupation seems reasonable. The restricted gene flow with isolation by distance (RGFID) detected in clade 2.3 reflects the wider distribution of the interior (ancestral) subclade 1.4 (HN–HL) compared with the restricted distribution of the tip subclades (derivates) 1.7 and 1.5. Besides genetic differentiation, the geographic ranges of the last two mentioned subclades are totally separated (1.7) and partially overlap (1.5) with that of interior subclade 1.4 (Table 2 and Fig. 1a). In the case of clade 3.2, the same phylogeographical inference can be made from the confinement of subclade 1.8 to locality 3, while the distribution of the entire clade 3.2 comprises localities 1, 2, 3, 4, 5, 9, 10 and 15. This type of process is observed in low vagility species (Frankham et al., 2002; Moraes-Barros et al., 2006), as seems to be the case for C. villosus. The RGFID inference would be due to the geographical separation of localities in the northern sector from the central area of the distribution range. But, on the other hand, although we collected almost half the amount of armadillos in the northern part compared with the central one, it is important to note that capture efforts were the same in both areas, which

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implies much smaller population densities in the first one, typical of marginal environments. Based on the geographical location of haplotype HA, its position in the network, and its frequency in the populations sampled it is proposed that the most probable area of origin of the species is the Pampean Region. The following reasons support this hypothesis: (1) HA is the dominant haplotype in the interior clade 1.2 and it shows the largest number of derived haplotypes (at least five), suggesting an ancestral condition; (2) it has a high frequency of occurrence at different locations in the Pampean Region (Table 2), suggesting a long term process of lineage spreading; (3) its distribution area coincides with the region of highest haplotype variation across the species’ range (Fig. 1a and Table 2) and (4) the earliest fossil record is from the locality of Chapadmalal, on the Atlantic coast of the Pampean Region (Fig. 1b) (Carlini and Scillato-Yané, 1999).

Acknowledgments

4.2. Paleobiogeographic and phylogenetic considerations of genus Chaetophractus

References

The appearance of Chaetophractus in the fossil record coincides with that of many species of family Dasypodidae and other Xenarthra (Carlini and Scillato-Yané, 1999). Diversification was likely to be related to the formation and climax of the widespread Southern plains (‘‘Age of Southern Plains”) during the Pliocene. This occurred after the large-scale sea transgression termed ‘‘Paranense Sea” in the Miocene and the uplifting of the Andes mountains during the Quechua Phase (Ortiz-Jaureguízar and Cladera, 2006 and references therein). According to Delsuc et al. (2004), the lineage giving rise to the genus Chaetophractus separated from the rest of subfamily Euphractinae during the late Miocene about 6 Myr ago. However, the oldest fossils assigned to C. villosus (and to genus Chaetophractus) are younger. They come from sediments in the Pampean Region (specifically in the Buenos Aires province), dated between 4 and 3.2 Myr (early Pliocene) (Scillato-Yané, 1982; Carlini and ScillatoYané, 1999). These sediments contain mammal fauna characterising the Chapadmalalan age (Cione and Tonni, 1995). The oldest fossil assignable to C. vellerosus also came from Buenos Aires province, in the coastal locality of Punta Hermengo (Fig. 1b). It was found in sediments of the Ensenadan mammal age (2–0.5 Myr), in a stratum dated between 0.9 and 0.78 Myr (Soibelzon et al., 2006 and references therein). Currently, C. villosus and C. vellerosus coexist and are considered sister species, whether by sharing a common ancestor which may have existed at least 6 Myr ago or by sympatric speciation of the latter. There is no conclusive evidence supporting either of these hypotheses but the fact that the oldest fossils of both species were found in Pampean Region, support it as an ancestral area of distribution of the genera. Summarising, contiguous range expansion would be the most important historical process characterising the C. villosus population. This was confirmed by both the NCA and the statistics phylogeographic approaches. The lineage giving rise to genus Chaetophractus possibly comes from the Pampean Region, hypothesis supported by the fossil record and our phylogeographic results. Finally, the fact that all the individuals from Patagonia analysed have a single haplotype (HC) suggests that C. villosus dispersed from the Pampean Region (after the retraction of glaciers during the Pleistocene) to Patagonia, where a rapid colonisation involving a single wave took place. The lack of genetic structure in C. villosus Patagonian populations are in agreement with other data from the same area small mammals populations (Dr. U.F.J. Pardiñas, personal communication) and from D. novemcinctus in the south of North America, reinforcing the idea of a recent colonisation of the Patagonia. Actually, we are carrying out new prospects to test this hypothesis.

This study was supported by a grant from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT 0074-2002) to M.S.L. We thank Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) of Argentina, Instituto Argentino de Investigaciones de las Zonas Áridas (IADIZA), Parque Nacional Los Glaciares seccional El Chaltén and Museo de Ciencias Naturales de San Cristóbal (Santa Fé). Special thanks to Mr. Julio Escobar and Dr. Guillermo Deferrari for participation in the collection of material studied in this project. We also thanks Lic. Igor Berkunsky, Lic. Jimena Bustos, Lic. Alfredo Carlini, Dr. Mariano Merino, Lic. Marcela Nabte, Dr. Ulyses Pardiñas, Lic. Mercedes Santos and Lic. Martín Ciancio for provide samples. Thanks to Gorda Papota Scotto for her revivifying joy.

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