2009 Blackwell Verlag GmbH

Accepted on 23 April 2009 J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2009.00539.x

1 tedra de Genetica de Poblaciones y Evolucion, Facultad de Ciencias Exactas, Fsicas y Naturales, Universidad Nacional de Ca tedra de Introduccion a la Fsica y Qumica Biologicas, Facultad de Odontologa, Universidad Cordoba, Cordoba, Argentina; 2Ca Nacional de Cordoba, Cordoba, Argentina; 3Grupo de Investigaciones de la Biodiversidad, IADIZA, CONICET; Mendoza, Argentina

Patterns of speciation in two sibling species of Graomys (Rodentia, Cricetidae) based on mtDNA sequences
Juan J. Martnez1, Raul E. Gonzalez-Ittig1, Gerardo R. Theiler2, Ricardo Ojeda3, Cecilia Lanzone3, Agustina Ojeda3 and Cristina N. Gardenal1

Abstract
To increase our understanding of the speciation process occurred in the sibling species Graomys griseoavus and Graomys centralis, a phylogeographic study was conducted based on sequences of a hypervariable segment of the mtDNA D-loop region. The resulting haplotype phylogenetic network showed two well-dened clusters, one for each species. The clusters were connected by two haplotypes from localities that are almost 300 km apart, one situated in the Monte eco-region and the other, in the Chaco. This result is in agreement with a previous hypothesis about the geographical context in which the cladogenetic process occurred. A divergence time of 0.150.28 million years was estimated, which is consistent with a process of recent speciation. An amova test conrmed that at present gene ow between species does not exist. The mismatch distribution analyses suggest that the geographical and demographic expansion undergone by the species is related to the climatic events that occurred in the region during the Quaternary. Key words: Graomys speciation sibling species mtDNA sequences quaternary glaciations

Introduction
The role played by chromosomal rearrangements in evolution is strongly questioned because of the paradox that while only rearrangements that are strongly underdominant are likely to contribute to speciation, these are dicult to be xed in natural populations (see a review in Rieseberg 2001). In studies of chromosomal divergence, one of the main diculties that taxonomists may nd in identifying species is the occurrence of mosaic evolution: some populations may reach reproductive isolation with minimal morphological dierences, resulting in sibling species, whereas others present very dierent morphologies but do not acquire isolating mechanisms (Mayr 2000). According to Dobigny et al. (2005), sibling species constitute valuable biological models for empirical investigations about the eects of chromosomal changes on evolution, because phenotypic similarities could reect a recent dierentiation, thereby allowing the main events that promote their reproductive isolation to be identied. One of those biological models is provided by the South American grey leaf-eared mice, Graomys griseoavus (Waterhouse, 1837) and Graomys centralis Thomas, 1902, which form a pair of sibling species and in whose cladogenesis chromosomal rearrangements might have been involved. Wainberg and Fronza (1974a,b) reported karyomorphs of 2n = 38, 37 and 36 for the species, whereas Zambelli et al. (1994) described karyomorphs with 2n = 42, 41, 35 and 34. Analyses of chromosome G-banding determined that these dierences were originated by Robertsonian or centric fusions combined in some individuals with two pericentric inversions.

Corresponding author: Juan J. Mart nez (juan_jmart@yahoo.com.ar) Contributing authors: Raul E. Gonzalez-Ittig (regonzalez@efn. uncor.edu), Gerardo Raul Theiler (grtheiler@yahoo.com.ar), Ricardo Ojeda (rojeda@lab.cricyt.edu.ar), Cecilia Lanzone (celanzone@lab. cricyt.edu.ar), Agustina Ojeda (agustinao@lab.cricyt.edu.ar), Cristina N. Gardenal (ngardenal@efn.uncor.edu)

In laboratory crossing tests Theiler and Blanco (1996a) detected post-zygotic isolation between animals with 2n = 42 and 2n = 3836. This isolation was asymmetric, with matings between 2n = 42 males and 2n = 3836 females yielding almost all sterile hybrids. In addition, except for 20% of the females, which presented reduced fertility, reciprocal crosses were unviable. Theiler and Blanco (1996b) also found this post-zygotic isolation to be reinforced by pre-zygotic mechanisms, involving olfactory discrimination during female oestrus. Females were able to discriminate males of compatible chromosome complement from males that would produce non-viable descendent or sterile hybrids. These results led Theiler (1997) to validate a nominal species, G. centralis Thomas, 1902 for animals 2n = 42, previously considered to be G. griseoavus, and to propose that the form with 2n = 42 could be the ancestral karyotype which may have originated, by centric fusions, the karyotypes found in G. griseoavus. Phylogenetic studies based on the Cyt b and D-loop genes of the mtDNA showed that animals with 2n = 42 and 41 form a separate clade from individuals 2n = 3834 (Catanesi et al. 2002, 2006). The conclusion stated in Catanesi et al. (2006) that there is no phylogeographical structure in the populations analyzed may be incorrect, as they employed specimens of both species from General Belgrano and then stated that this sampling site was located in a mountain region in Cordoba province (Villa General Belgrano), where G. griseoavus has never been captured. In fact, we think that the true origin of these specimens was the department of General Belgrano in La Rioja province, where the presence of both species of Graomys is well documented (Theiler 1997). Individuals with 2n = 3834 (G. griseoavus) preferably inhabit the Monte and Patagonic steppe eco-regions, G. centralis (2n = 4241) occurring in the dry Chaco and Espinal eco-regions (D az et al. 2006). A series of nominal forms described for Chacoan localities have been found to fall within the G. griseoavus synonymy (e.g. medius and chacoensis). Lanzone et al. (2007b) considered that Graomys

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Martnez, Gonzalez-Ittig, Theiler, Ojeda, Lanzone, Ojeda and Gardenal inferences about the area of speciation and the divergence time. From the analyses of the genetic structure, neutrality and mismatch distribution, we reconstructed the context of the demographic, climatic and geographical uctuations, which were probably associated with the speciation process.

medius should be synonymized with G. centralis, on the basis of cytogenetic and morphometric evidence in animals from the type locality (Catamarca). Regarding Graomys chacoensis, the type locality is situated in the Paraguayan Chaco, the animals from that area presenting the same 2n and FN as G. centralis from central-western Argentina (J. Patton, personal communication). This might indicate that both names would correspond to the same taxonomic entity. Ongoing studies conducted in our laboratory based on the geometric morphometrics of the skull indicate the existence of only one species in the Chaco eco-region (J.J. Mart nez, unpublished results). The genetic population structure of G. griseoavus and G. centralis inferred on the basis of allozymic data revealed that the both species have similarly high levels of genetic variability (Theiler and Gardenal 1994; Theiler et al. 1999). In some areas, Theiler et al. (1999) observed a very low interspecic dierentiation, even lower than that found in populations of the same species. Taking into account that no decrease in the levels of polymorphism (measured as heterozygosity and allelic richness) was observed, the absence of bottlenecks or a founder eect is suggested. Theiler et al. (1999) suggested that this cladogenetic event could have been rapid and relatively recent. However, this proposal has not yet been evaluated using a phylogeographic approach. When the phylogeny of haplotypes is associated with their geographical origin, past events that could have occurred during the time since divergence can be inferred. In this study we investigated the relationships between Dloop haplotypes of G. centralis and G. griseoavus with the aim of contributing to the knowledge of the speciation process in the genus Graomys. We analyzed the phylogeographic patterns in central-western Argentina to make

Materials and Methods

Specimens analyzed
Twenty-four specimens of G. griseoavus and 15 of G. centralis were analyzed. Graomys griseoavus was sampled in the following localities: Andalgala (n = 5), Mendoza (n = 2), Lujan de Cuyo (n = 2), Nacunan (n = 5), General Alvear (n = 4), Medanos (n = 1), Puerto Madryn (n = 1) and Comodoro Rivadavia (n = 4); Sampling of G. centralis was conducted in the localities of: Chumbicha (n = 3), Villa de Mar a (n = 4), Dean Funes (n = 2), Santiago Temple (n = 1), Chamical (n = 3) and Berna (n = 2) (Fig. 1). We assigned specimens to G. griseoavus or G. centralis based on previous cytogenetic studies (Theiler 1997; Lanzone et al. 2007b; N. Suarez, pers. comm.). Detailed information on vouchers, haplotypes, and GenBank accession numbers (EF672516 EF672540 and EF672542 EF672555) for D-loop sequences is presented as supporting information (Appendix S1) in the electronic version.

Nucleotide sequence analyses

Total genomic DNA was extracted from ethanol-preserved tissues following the standard phenolchloroform method (Maniatis et al. 1982). By analyzing the published sequences of the complete control region in Graomys (Catanesi et al. 2006; GenBank accessions: AY357923, AY359285, AY359679, AY359680, AY364006 AY364008 and AY398731 AY398744), we designed new primers because the complete D-loop has segments with unequal rates of substitution that may change the times of coalescence. The primers F1 Gra (5-TAC CCC CAG CAC CCA AAG CTG-3) and R1 Gra

Fig. 1. Sampling location of Graomys in Argentina. Graomys griseoavus was sampled in the localities of: (1) Andalgala, (2) Mendoza, (3) Lujan de Cuyo, (4) Nacunan, (5) General Alvear, (6) Medanos, (7) Puerto Madryn and (8) Comodoro Rivadavia. Graomys centralis was sampled in the localities of: (9) Chumbicha, (10) Villa de Mar a, (11) Dean Funes, (12) Santiago Temple, (13) Chamical and (14) Berna. Eco-regions are delimited by continuous lines: I. Dry Chaco, II. Humid Chaco, III. Espinal, IV. Pampa, V. Monte and VI. Patagonic steppe. Shaded lines enclose the possible area of speciation proposed by Theiler et al. (1999)

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Patterns of speciation in Graomys

(5-TGG GCG GGT TGT TGG TTT CAC-3) amplied a segment of 384 bp, which encompassed the hypervariable segment 1 (ETAS domain), and also approximately 40 bp of the central domain. Polymerase chain reactions were performed in a Mastercycler, Eppendorf (Hamburg, Germany) according to the following conditions at a nal volume of 25 ll: 1x buer [75 mM TrisHCl pH 8.8, 20 mM (NH4)SO4, 0.01% Tween-20], 200 lM each dNTP (dATP, dCTP, dGTP and dTTP), 200 nM F1 Gra, 200 nM R1 Gra, 2.5 mM MgCl2, 0.4 mg ll)1 of BSA, 0.75 U Taq polimerase (Fermentas, Brazil), and 515 ng of template DNA. The cycling program consisted of an initial denaturation at 94C for 3 min, 35 cycles of denaturation at 94C for 30 s, annealing at 55C for 45 s, extension at 72C for 1 min and a nal extension at 72C for 7 min. Double-stranded PCR products were puried with Qiaquik (Qiagen, Germany) and sequenced by Macrogen Inc. (Seoul, South Korea) using only primer F1 Gra. These were aligned by hand according to previously published sequences of Graomys (Catanesi et al. 2006). Once aligned, these sequences were collapsed into haplotypes. The average haplotype diversity and the average nucleotide diversity for each species were calculated using the DNAsp 4.10 software (Rozas et al. 2003).

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(Huelsenbeck and Ronquist 2001) respectively. For MP, we employed a heuristic search with 250 random addition sequences, saving ve trees per replica with the TBR branch-swapping algorithm. Then we summarized the trees of MP in a strict consensus tree. The node supports were calculated with the Jacknife method (JK), based on 1000 replicates and a removal probability of 0.33. The modeltest 3.7 software (Posada and Crandall 1998) was used to select the best model of evolution, according to the Akaike information criterion (Akaike 1974) for which our haplotype dataset tted best. We employed HKY + I model for the Bayesian inference, based on a Markov Chain Monte Carlo for 2 000 000 generations (two simultaneous chains, one cold and one hot; sample frequency 100; burn-in 250). Bayesian analyses were run independently twice, beginning with dierent starting trees. Support for tree nodes was determined according to the values of Bayesian posterior probability obtained from a majority-rule consensus tree. In addition, a Minimum spanning network of haplotypes was constructed with the arlequin 3.11 program (Excoer et al. 2005). Three analyses of molecular variance (amova) (Excoer et al. 1992) were conducted using arlequin version 3.11 (Excoer et al. 2005). In the rst hierarchical analysis, individuals were grouped according to both their eco-regional origin and their cytotype, by comparing the two species of Graomys. To determine the intraspecic genetic dierentiation among populations, an amova analysis was performed for each species. Then, 1023 non-parametric permutations were used to test the signicance of variance components and xation indices.

Phylogeographic and genetic structure analyses

The phylogenetic relationships among haplotypes were estimated by two methods, maximum parsimony (MP) and a Bayesian method, using the TNT (Golobo et al. 2003, 2008) and MrBayes 3.1.2 software

Fig. 2. Majority rule consensus network of Bayesian analysis for 33 Graomys haplotypes. The values to the left and right of the crossbar represent the Bayesian posterior probability and the statistical support obtained with 1000 jacknife replicates in MP analysis respectively (only values above 50% are shown). Node A represents the clade of Graomys griseoavus haplotypes; node B indicates the clade of haplotypes belonging to Graomys centralis

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162 Neutrality test and mismatch distribution

Martnez, Gonzalez-Ittig, Theiler, Ojeda, Lanzone, Ojeda and Gardenal and 20 in G. griseoavus (n = 24). The average haplotype diversity was similar in both species (G. centralis, h = 0.981 0.031 and G. griseoavus, h = 0.982 0.018). However, the nucleotide diversity was higher in G. griseoavus (p = 0.01323) than in G. centralis (p = 0.00797). The average nucleotide divergence within species estimated using the HKY evolution model was 0.86 0.04% in G. centralis and 1.47 0.78% in G. griseoavus; between species this value was 3.31 0.95%. There was a marked overlap between the ranges of intraspecic distance detected in both species and the range of interspecic divergence. According to these data, we estimated that the lineages split about 0.15 0.28 million years ago. Phylogenetic network and genetic structure Both MP and Bayesian analysis showed two well-supported main clades, one comprising of all G. griseoavus haplotypes and the other, those of G. centralis (Fig. 2). The Minimum spanning network obtained is shown in Fig. 3. Each species forms an independent cluster of haplotypes without sharing any haplotype with the other species. Both clusters are interconnected by haplotype G10 (G. griseoavus), which is found in the locality of Andalgala, and also by haplotype C8 (G. centralis), present in Villa de Mar a and Dean Funes. These two haplotypes are separated by seven mutational changes, which are greater than the mutational steps observed within each species. Moreover, these two haploypes presented the lowest interspecic distance (1.71%). Neither species exhibited a signicant association between the phylogenetic relationships among the haplotypes and their geographical origin. The results of the amova comparison between species are shown in Table 1. The main source of variation was between species (57.61%; p < 0.001), whereas intraspecic dierentiation among populations explains 11.88% (p < 0.001) of the total genetic variation. Two additional amova were performed to assess the degree of genetic structure in each species (Table 2). Populations of G. griseoavus showed an almost identical degree of genetic

Two analyses of neutral evolution were conducted using arlequin 3.11 (Excoer et al. 2005): the Fu neutrality test (Fu 1997) and mismatch distribution (Rogers and Harpending 1992). Both analyses were performed separately on each species to test the hypothesis of neutral equilibrium and also to detect whether a recent or sudden range expansion had occurred. Signicant values of Fs statistics (Fu 1997) were tested by generating 1000 random samples using coalescent simulations. The validity of the estimated demographic and geographical expansion model was tested using 1000 parametric bootstrap replicates as well as the parameters s, h0, h1 and the raggedness index (r) of mismatch distribution (Harpending 1994).

Date of population divergence and expansion

The average amount of sequence divergence both within and between species was inferred by using the paup* 4.0b10 program (Swoord 1998) for the selected HKY + I evolution model (A = 0.3517, C = 0.1895, G = 0.0943, T = 0.3644 and I = 0.8315) (Hasegawa et al. 1985). The time of divergence between species was estimated using the average rate of nucleotide substitution of the ETAS domain (19.4 7.8 10)9 subssite year)1) calculated by Pesole et al. (1999); the interspecic distance was obtained using the HKY + I substitution model. We also calculated the expansion times according to Rogers and Harpending (1992) using the equation s = 2ut, where u is the mutation rate of the entire DNA fragment and t is the time of expansion in generations. The mutation rate for the entire stretch of DNA can be expressed as u = mTl, where mT is the sequence length and l is the divergence rate. In this paper, we adopted the divergence rate indicated above (19.4% sub site), which is very similar to the rate of 20% per million years estimated by Gunduz et al. (2005) in Mus musculus domesticus. We assumed two generations per year, on the basis of estimations reported for other species also belonging to the family Cricetidae (Crespo et al. 1970).

Results
Nucleotide divergence We analyzed 382 bp of the ETAS domain of the control region in 39 individuals belonging to the species G. centralis and G. griseoavus. A total of 37 variable sites were observed; 26 were polymorphic in G. griseoavus and 14 in G. centralis. We detected 33 dierent haplotypes: 13 in G. centralis (n = 15)

Fig. 3. Minimum spanning network showing the relationships and frequency of haplotypes found in Graomys, based on control region sequence variants. Branch lengths are proportional to the nucleotide differences among haplotypes. G and C denote Graomys griseoavus and Graomys centralis haplotypes respectively

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Patterns of speciation in Graomys

Table 1. Hierarchical analysis of the molecular variance (amova) in Graomys griseoavus and Graomys centralis Source of variation Between species, Among populations within species, rb2 Within populations, rc2 ra2 Variance 3.1274 0.6451 1.6560 % total 57.61 11.88 30.51 p-value <0.001 <0.001 <0.001

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Fixation indices 0.576 0.280 0.695

Neutrality test and mismatch distribution

Table 2. Analysis of molecular variance (amova) in (a) Graomys griseoavus and in (b) Graomys centralis Source of variation (a) Among populations, ra2 Within populations, rb2 (b) Among populations, ra2 Within populations, rb2 Variance 0.7316 1.8531 0.5320 1.3056 % total 28.30 71.70 28.95 71.05 p-value <0.01 Fixation indices 0.283

<0.05

0.289

dierentiation to that of the populations of G. centralis, 28.3% (p 0.01) and 28.95% (p 0.05) of the total variation respectively.

(a)

Populations of both species presented negative values of the Fs statistic; thus, the model of neutral evolution is rejected: Fs = )8.800 (p 0.001) and Fs = )13.516 (p 0.001) for G. centralis and G. griseoavus respectively. As this situation was likely to be in agreement with a recent population growth, mismatch analyses were performed to conrm and characterize these possible expansions. In G. griseoavus, the observed distribution was unimodal and adjusted to that expected assuming demographic or spatial expansion (Fig. 4a). The values of the raggedness statistic (r = 0.0185, non-signicant) and of h0 (1.211) and h1 (99999.0) indicate that the population increase in the species was probably abrupt. The analysis conducted on G. centralis (Fig. 4b) also revealed a population expansion, with a unimodal distribution. Moreover, raggedness was not signicant (r = 0.0824), with theta values also indicating an abrupt expansion (h0 = 0.0 and h1 = 99999.0). Finally, the values obtained for s suggest that expansion occurred more recently in G. centralis than in G. griseoavus (s = 3.32 and 3.879 respectively). This expansion converged to 0.11 million years in the former species and to 0.13 million years in the latter.

Discussion
In this study, we used 39 sequences of a segment of the mtDNA control region to analyze the speciation process in the sibling species G. griseoavus and G. centralis. The segment used includes the hypervariable region I (ETAS domain), which presents a very high evolutionary rate in mammals (Pesole et al. 1999) leading to the accumulation of mutations in a short period, and thereby favouring studies on the genetic composition of closely related species. However, the phylogenetic relationships among species of the genus Graomys are not clearly understood at present. In a previous study on the genus Phyllotis and close relatives, Steppan et al. (2007) found that the Cyt b sequence of one individual of Graomys domorum (a species with 2n = 28 and that is clearly recognizable by its morphological characters) was more closely related to G. griseoavus than to G. centralis. The support of the nodes for that molecular relationship, however, was low and nonsignicant. Further studies using other mtDNA regions (as the D-loop) might contribute to resolve the phylogeny of the genus. The average genetic variation between G. centralis and G. griseoavus obtained in this study was low (3.31 0.95%), and may be explained by a moderate to high current gene ow or by a relatively recent divergence between the species. Although hybrids can be obtained under laboratory conditions, Theiler and Blanco (1996a,b) proposed a lack of current gene ow between natural populations of G. griseoavus and G. centralis on the basis of the existence of post- and precopulatory mechanisms of isolation and the absence of hybrids in nature, even in populations occupying parapatric areas (Theiler 1997). The results on genetic structure here presented conrmed that barriers to gene ow are acting between them. J Zool Syst Evol Res (2010) 48(2), 159166 2009 Blackwell Verlag GmbH

(b)

Fig. 4. Frequency distribution of pairwise nucleotide dierences in haplotypes. (a) Graomys griseoavus and (b) Graomys centralis, obtained by mismatch analyses. The shaded bars represent the observed values, with the black diamonds showing the simulated values under the null hypothesis of demographic and spatial expansion (). In both cases there were no signicant departures from the sudden demographic and spatial expansion expected (p > 0.05)

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Martnez, Gonzalez-Ittig, Theiler, Ojeda, Lanzone, Ojeda and Gardenal (0.9 and 0.13 million years respectively). However, the condence intervals for these estimations clearly overlap, suggesting that the demographic and geographical expansions of both species occurred almost simultaneously, probably promoted by the spreading of open areas like the Chaco and Monte eco-regions during the Quaternary. Changes in this period were responsible for chorological alterations, thus expanding and contracting the distribution areas of taxa, communities and biomes (Ortiz-Jaureguizar and Cladera 2006). The present geographical distribution of G. centralis and G. griseoavus could be related to changes in the vegetation physiognomy occurred during the Pleistocene glacial and interglacial cycles. According to de Vivo and Carmignotto (2004), there is ample evidence indicating that these cycles were particularly intense and abrupt in South America; an expansion and interconnection of open biomes that took place during cold dry climates resulted in a concomicant contraction of the areas occupied by subtropical and tropical regions. On the other hand, when open areas retreated during interglacial periods of warm wet climates, the rainforest expanded (Ortiz-Jaureguizar and Cladera 2006). In their study, de Vivo and Carmignotto (2004) pointed out that the dry forests, such as the Chaco, turned into semi-evergreen forests, whereas in the present grassland region called the Pampa, the predominant physiognomy would have been that of dense savanna and tall forest savanna, similar to the physiognomy of some regions of the modern Chaco. At present, G. centralis is only distributed in the Chaco eco-region and does not extend to the Pampa, although Graomys sp. may have inhabited the Pampa during the Pleistocene (Pardinas et al. 2002; Voglino and Pardinas 2005). Expansions and retractions of the Chaco during the glacial and interglacial cycles could explain the present distribution of G. centralis. In north-western Argentina, the derived species G. griseoavus occurs in the Monte Desert, which is a relatively young biome evolved from the semiarid Chaco (Vuilleumier 1971; Ojeda and Tabeni in press), presently occupied by the ancestral G. centralis. In this study, we provide results that contribute to the understanding of how the cladogenetic event splitting G. centralis and G. griseoavus could have occurred: (a) the absence of shared haplotypes between species conrms that the post- and pre-zygotic barriers previously detected by Theiler and Blanco (1996a,b) in laboratory studies are also acting in the wild; (b) the low interspecic genetic dierentiation indicates a relative recent split between the two species of Graomys; (c) the haplotypes connecting both clusters of each species support previous hypotheses about the area of speciation and (d) present demographic and geographical distribution patterns could be associated with the climatic changes occurred in the Quaternary.

The low interspecic nucleotide divergence would be better explained by a recent split of G. griseoavus from G. centralis. Using as a reference the overall rate of rodent mitochondrial DNA evolution based on the DNADNA hybridization technique, which was estimated within the range of 4.89.7% substitution per million years (Catzeis et al. 1992), Catanesi et al. (2002) proposed that the separation of these species occurred about 1.5 million years ago. In this study, we detected an interspecic nucleotide divergence of 3.31 0.95%. Therefore, using the average rate of molecular substitution of ETAS domain proposed by Pesole et al. (1999) in mammals, we estimated that the splitting up of both lineages happened about 0.150.28 million years ago, a period compatible with a relatively recent divergence. The fossil record shows that in the early middle Pleistocene (20.5 million years ago), indeterminate forms of Graomys were already present in the Pampean region; the rst G. griseoavus recorded occurred in the Bonaerian stage (middle-upper Pleistocene, 0.50.13 million years ago; Pardinas et al. 2002). This nding is consistent with our molecular estimation. However, given the heterogeneity in the rates of molecular evolution observed for dierent genes, and even within a gene, a small bias in the estimation of the evolution rates could lead to very dierent conclusions about the timing of historical events (Avise 2004). Therefore, it is necessary to complement this estimation with additional evidence from other nuclear and mitochondrial DNA segments. Theiler et al. (1999) proposed that a cladogenetic event could have initiated in a marginal population of G. centralis, probably situated close to Chamical (Fig. 1) given the high genetic identity (even higher than that among conspecic populations) found in two populations proximal to that area, one belonging to G. centralis and the other to G. griseoavus. In this study, we observed that the lowest interspecic distance (1.71%) occurs between haplotype C8 (present in Villa de Mar a and Dean Funes, localities 10 and 11 respectively; Fig. 1) and G10 (Andalgala, locality 1, Fig. 1), situated in the same area proposed by Theiler et al. (1999) as the location of the original speciation event. This region was also considered a speciation centre for other species of Phyllotini (Braun 1993). Recently, Lanzone et al. (2007a) suggested that the barriers associated with the sub-Andean mountain uplift in north-western Argentina might have been a primary driver in the cladogenetic process of two species of the genus Eligmodontia, the lowland Monte desert silky mice. In G. griseoavus, the lack of correlation between Nm (eective number of migrants) and geographical distance among populations could be explained by a recent and fast colonization of the Monte eco-region (Theiler et al. 1999). The Minimum spanning network obtained presented a star-like pattern in G. griseoavus (Fig. 3), which is in agreement with a past exponential growth of the populations. In addition, the Fs value of the Fu test indicates that this species eectively underwent a population expansion. Furthermore, the analysis of mismatch distribution (Fig. 4a) showed that this historical population growth was signicant and abrupt (h0 = 1.211 versus h1 = 99999.0). In G. centralis, which is considered an ancestral species to G. griseoavus, our results (Fu test and mismatch distribution, Fig. 4b) also indicate an expansion of its geographical range. The parameter s, an estimator of the relative age of expansion (Rogers and Harpending 1992), was lower in G. centralis than in G. griseoavus (s = 3.32 and 3.879 respectively) (Fig. 4a) J Zool Syst Evol Res (2010) 48(2), 159166 2009 Blackwell Verlag GmbH

Acknowledgements
We thank V. Rodr guez from the Universidad Nacional de Cordoba for providing animals from Comodoro Rivadavia. We are grateful to A. Blanco for the critical review of the manuscript, to the IADIZA for providing institutional and infrastructural support, and to the Willi Hennig Society for sponsoring the TNT program. This work was funded by the Agencia Cordoba Ciencia, the SECYT of the Univers idad Nacional de Cordoba, and the Consejo Nacional de Investigac iones Cient cas y Tecnicas (CONICET), PIP 5944. JJM, CL and AO are fellows of CONICET. REG-I, RO and CNG are Career Investigators of the same institution. JJM and CL are graduate students of the Doctorado en Ciencias Biologicas, Universidad Nacional de Cordoba.

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close to its site of origin: studies in Turkey. Biol J Linn Soc 84:473 485. Harpending RC (1994) Signature of ancient population growth in a low-resolution mitochondrial DNA mismatch distribution. Hum Biol 66:591600. Hasegawa M, Kishino K, Yano T (1985) Dating the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol 22:160174. Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogeny. Bioinformatics 17:754755. Lanzone C, Ojeda RA, Gallardo MH (2007a) Integrative taxonomy, systematics and distribution of the genus Eligmodontia (Rodentia, Cricetidae, Sigmodontinae) in the temperate Monte Desert of Argentina. Mamm Biol 72:299312. Lanzone C, Novillo A, Suarez NS, Ojeda RA (2007b) Cytogenetics and redescription of Graomys (Rodentia, Sigmodontinae) from Chumbicha, Catamarca, Argentina. Mastozool Neotrop 14:249 255. Maniatis T, Fritsh EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. Mayr E (2000) The biological species concept. In: Wheeler QD, Meier R (eds). Species Concepts and Phylogenetic Theory. A debate. Columbia University Press, New York. Ojeda RA, Tabeni S (2009) The mammals of the Monte desert revisited. J Arid Environ 73:173181. Ortiz-Jaureguizar E, Cladera GA (2006) Paleoenvironmental evolution of southern South America during the Cenozoic. J Arid Environ 66:498532. Pardinas UFJ, DElia G, Ortiz PE (2002) Sigmodontinos fosiles (Rodentia, Muroidea, Sigmodontinae) de America del Sur: Estado actual de su conocimiento y prospectiva. Mastozool Neotrop 9: 209252. Pesole G, Gissi C, De Chirico A, Saccone C (1999) Nucleotide substitution rate of mammalian mitochondrial genomes. J Mol Evol 48:427434. Posada D, Crandall K (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817818. Rieseberg LH (2001) Chromosomal rearrangements and speciation. Trends Ecol Evol 16:351358. Rogers AR, Harpending H (1992) Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol 9:552569. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:24962497. Steppan SJ, Ramirez O, Banbury J, Huchon D, Pacheco V, Walker LI, Spotorno AE (2007) A molecular reappraisal of the systematics of the leaf-eared mice Phyllotis and their relatives. In: Kelt DA, Lessa EP, Salazar-Bravo J, Patton JL (eds). The Quintessential Naturalist: Honoring the Life and Legacy of Oliver P. Pearson. University of California Publications in Zoology, Berkeley Los Angeles, CA 134: 1981. Swoord DL (1998) PAUP*. Phylogenetic analysis using parsimony (* and other methods) Version 4. Sinauer Associates, Sunderland. Theiler GR (1997) Patrones de evolucion en Graomys griseoavus (Rodentia, Muridae). Un caso de especiacion rapida. PhD dissertation. Facultad de Ciencias Exactas, F sicas y Naturales; Univers idad Nacional de Cordoba, Argentina. Theiler GR, Blanco A (1996a) Patterns of evolution in Graomys griseoavus (Rodentia, Muridae). II. Reproductive isolation between cytotypes. J Mamm 77:776784. Theiler GR, Blanco A (1996b) Patterns of evolution in Graomys griseoavus (Rodentia, Muridae). III. Olfactory discrimination as a premating isolation mechanism between cytotypes. J Exp Zool 274:346350. Theiler GR, Gardenal CN (1994) Patterns of evolution in Graomys griseoavus (Rodentia, Cricetidae). I. Protein polymorphism in populations with different chromosome numbers. Hereditas 120:225229. Theiler GR, Gardenal CN, Blanco A (1999) Patterns of evolution in Graomys griseoavus (Rodentia, Muridae). IV. A case of rapid speciation. J Evol Biol 12:970979.

Resumen
n Patrones de especiacio en dos especies crpticas del genero Graomys (Rodentia, Cricetidae) basados en secuencias de ADNmt. Con el objetivo de contribuir al conocimiento del proceso de separacion de las especies Graomys griseoavus y Graomys centralis, se realizo un estudio logeograco basado en secuencias de un segmento de la region de D-loop del ADNmt de 39 ejemplares. La red logenetica de haplotipos mostro dos agrupamientos bien soportados, uno para cada especie. Los grupos estan conectados por dos haplotipos presentes en localidades separadas por 300 km, una de ellas situada en la eco-region del Monte y la otra, en el Chaco. Este resultado apoya una hipotesis previa acerca del contexto geograco del proceso cladogenetico. La separacion de G. griseoavus y G. centralis podr a haber ocurrido hace 0,150,28 millones de anos, estimacion que concuerda con una especiacion relativamente reciente. El test de amova conrmo la ausencia de ujo genico actual entre las especies. Los analisis de distribucion de diferencias apareadas entre haplotipos sugieren expansiones geogracas y demogracas para ambas especies, probablemente relacionadas con los cambios climaticos ocurridos en la region durante el Cuaternario.

References
Akaike H (1974) A new look at the statistical model identication. IEEE Trans Automat Control 19:716723. Avise JC (2004) Molecular Markers, Natural History and Evolution. Sinauer Associates, Sunderland, Massachusetts. Braun JK (1993) Systematic Relationships of the Tribe Phyllotini (Muridae: Sigmodontinae) of South America. Spec Publ Oklahoma Mus Nat Hist, Norman, pp 150. Catanesi CI, Vidal-Rioja L, Crisci JV, Zambelli A (2002) Phylogenetics relationships among Robertsonian karyomorphs of Graomys griseoavus (Rodentia, Muridae) by mitochondrial cytochrome b DNA sequencing. Hereditas 136:130136. Catanesi CI, Vidal-Rioja L, Zambelli A (2006) Molecular and phylogenetic analysis of mitochondrial control region in Robertsonian karyomorphs of Graomys griseoavus (Rodentia, Sigmodontinae). Mastozool Neotrop 13:2130. Catzeis FM, Aguilar JP, Jaeger JJ (1992) Muroid rodents: phylogeny and evolution. Trends Ecol Evol 7:122126. Crespo J, Sabattini M, Piantanida M, de Villafane G (1970) Estudios ecologicos sobre roedores silvestres. Observaciones sobre densidad, reproduccion y estructura de comunidades de roedores silvestres en el sur de la provincia de Cordoba. Ministerio de Bienestar Social, Secretar a de Estado y Salud Publica, Buenos Aires, Argentina. D az MM, Teta P, Pardinas UFJ, Barquez RM (2006) Tribu Phyllotini Vorontsov, 1959. In: Barquez RM, D az MM, Ojeda RA (eds). Mam feros de Argentina. Sistematicay distribucion. Sarem, Tuc uman, Argentina. Dobigny G, Aniskin V, Granjon L, Cornette R, Volobouev V (2005) Recent radiation in West African Taterillus (Rodentia, Gerbilinae): the concerted role of chromosome and climatic changes. Heredity 95:358368. Excoer L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131:479491. Excoer L, Laval G, Schneider S (2005) Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinform 1:4750. Fu YX (1997) Statistical test of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147:915925. Golobo P, Farris J, Nixon K (2003) TNT: tree analysis using new technology. Program and documentation, available at: http:// www.zmuc.dk/public/phylogeny/tnt (accessed June, 2009). Golobo P, Farris J, Nixon K (2008) TNT, a free program for phylogenetic analysis. Cladistics 24:774786. Gunduz I, Rambau RV, Tez C, Searle JB (2005) Mitochondrial DNA variation in the western house mouse (Mus musculus domesticus)

J Zool Syst Evol Res (2010) 48(2), 159166 2009 Blackwell Verlag GmbH

166

Martnez, Gonzalez-Ittig, Theiler, Ojeda, Lanzone, Ojeda and Gardenal

Zambelli A, Vidal-Rioja L, Wainberg R (1994) Cytogenetic analysis of autosomal polymorphism in Graomys griseoavus (Rodentia, Cricetidae). Z Zaugetierk 59:1420.

de Vivo M, Carmignotto AP (2004) Holocene vegetation change and the mammal faunas of South America and Africa. J Biogeogr 31:943957. Voglino D, Pardinas UFJ (2005) Roedores sigmodontinos (Mammalia: Rodentia, Cricetidae) y otros micromam feros pleistocenicos del norte de la provincia de Buenos Aires (Argentina): reconstruccion paleoambiental para el Ensenadense cuspidal. Ameghiniana 42:143158. Vuilleumier B (1971) Pleistocene changes in the ora and fauna of South America. Science 173:771780. Wainberg RL, Fronza TG (1974a) Autosomic polymorphism in Phyllotis griseoavus griseoavus Waterhouse, 1837 (Rodentia, Cricetidae). Boll Zool 41:1924. Wainberg RL, Fronza TG (1974b) Autosomic polymorphism in the South-American (Argentinean) rodent Phyllotis griseoavus griseoavus Waterhouse, 1837 (Cricetidae) with karyotypes 2n = 38, 37 and 36. II. The complement 2n = 36. First Inter Theriol Congr Moscow 2:293294.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Appendix S1. List of specimens of Graomys sequenced. Locality numbers are shown on map (Fig. 1); haplotype numbers were assigned in order of appearance. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

J Zool Syst Evol Res (2010) 48(2), 159166 2009 Blackwell Verlag GmbH