Journal of Heredity 2009:100(3):322–328 Ó The American Genetic Association. 2008. All rights reserved. doi:10.1093/jhered/esn105 For permissions, please email: [email protected]. Advance Access publication December 9, 2008 Divergence in (Rodentia: ) and Distribution of Amazonian Savannas

IBELE ONVICINO ABLO ONCALVES OA˜O DE LIVEIRA UIZ LAMARION DE LIVEIRA AND C R. B ,P R. G x ,J A. O ,L F B. O , Downloaded from https://academic.oup.com/jhered/article-abstract/100/3/322/867792 by guest on 06 November 2018 MARGARETE S. MATTEVI

From the Programa de Gene´tica, Instituto Nacional de Caˆncer, Rio de Janeiro, Brazil (Bonvicino and Goncxalves); the Laboratory de Biologia e Parasitologia de Mamı´feros Reservato´rios Sivestres, IOC-FIOCRUZ, Rio de Janeiro, Brazil (Bonvicino); the Departamento de Gene´tica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (Goncxalves); the Setor de Mamı´feros, Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (Oliveira and de Oliveira); and the Laboratory de Pesquisa em Biodiversidade , Universidade Luterana do Brasil (ULBRA), Programa de Po´s-graduacxa˜o em Gene´tica, Canoas, Brazil (Mattevi).

Address correspondence to Cibele R. Bonvicino at the address above, or e-mail: [email protected].

Abstract Northern South America presents a diverse array of nonforest or savanna-like ecosystems that are patchily distributed. The distribution of these open habitats has been quite dynamic during Quaternary glacial–interglacial cycles; yet, the relevance of climatically driven vicariance events to the diversification of nonforest Amazonian vertebrates remains poorly known. We analyzed karyologic and mitochondrial DNA sequence data of the genus Zygodontomys, a small cricetid distributed throughout nonforest habitats of northern Amazonia. Samples analyzed represented 4 Brazilian Amazonian localities and 2 French Guiana localities. Karyologic variation among Amazonian Brazilian Zygodontomys populations is high, with, at least, 3 karyomorphotypes. Molecular phylogenetic analyses recovered 3 major clades congruent with known karyotypes, a finding that suggests the existence of 3 , 2 of which currently undescribed. The French Guiana and Surumu´ clade, identified as microtinus, is characterized by 2n 5 86 and is sister to the clade formed by the 2 nondescribed forms. The Rio Negro–Rio Branco form is characterized by 2n 5 82, and the Ferreira Gomes–Itapoa´ form is characterized by 2n 5 84. The distribution of the 3 Zygodontomys lineages identified is in accordance with the geography of the open vegetation patches in Northern Amazonia, and divergence time estimates relate speciation events to the middle-upper Pleistocene, supporting the prominent role of Quaternary climatically driven vicariance events in the diversification of the genus. Key words: amazonian savanna, phylogeography, Zygodontomys

The northern Amazonian region of South America presents Anhuf et al. 2006), the effects of the climatic cycling on the a remarkably diverse array of nonforest or savanna-like diversification of its vertebrates are little known. Morpho- ecosystems that are patchily distributed from Panama to the logical studies of mammalian genera and species complexes Brazilian states of Amapa´, Roraima, and Amazonas. In the endemic to nonforest Amazonian ecosystems suggest northern region of the Brazilian Amazon, these enclaves are a major differentiation between western and eastern groups found intermingled by rainforests or restricted to the highest of populations (Voss 1991), but no further resolution has summits across the Guyanan shield, presenting varying been provided within these regions, specially regarding the levels of geographic isolation. The distribution of these levels of genetic divergence among populations. open habitats has been quite dynamic during the climatic The rodent genus Zygodontomys, a member of the tribe cycles of the Quaternary, with repeated episodes of sensu stricto (Voss and Carleton 1993; Steppan expansion during glacial periods and retraction on more 1995; Bonvicino et al. 2003), inhabits open vegetation humid interglacials (Haffer 1969; Vanzolini and Williams formations, including anthropogenic ones, in Central Amer- 1970; Haffer 2002). Although northern Amazonia has ica and northern South America (Voss 1991). In Brazil, this received an increasing attention from paleoecologists and species is a typical inhabitant of Amazonian ‘‘campinaranas,’’ geologists (e.g., Bush et al. 2004; Rosseti et al. 2005; a local designation of the grass savanna. The latest

322 Bonvicino et al.  Divergence in Zygodontomys and Distribution of Amazonian Savannas comprehensive review of the genus Zygodontomys considered 2) Serra do Araca´, 00°54#05.7$N, 63°26#02.1$W (GPS), only 2 species: Zygodontomys brunneus and Zygodontomys near Igarape´ da Anta, a right-bank affluent of the Rio brevicauda, the latter with 3 subspecies. However, karyologic Araca´, Barcelos: female MN 69077 and male MN 6907; data (Gardner and Patton 1976; Reig et al. 1990) give support 3) Parque Nacional do Virua´, Caracaraı´: 1) Serra do Preto for the recognition of other species mentioned throughout (01°48#57$N61°07#40$W): females MN 70318 and this paper. Brazilian populations have not been allocated 70330; males MN 70316, 70329, 70330, 70336, 70337, to any described taxa and were referred to as Zygodontomys sp. 70347, 70351, 70352, and 70364; 2) Near Road Perdida, (Mattevi et al. 2002; Bonvicino et al. 2003). Parque Nacional do Virua´, Caracaraı´, female MN 70400; Whereas the karyotype of Z. brunneus remains unde- 3) Rio Branco, Campinho (0°57#34.7$S, 61°09#56.8$W): scribed, several chromosomal complements have been MN 70581 and 70582; 4) Lavrado Vista Alegre attributed to Z. brevicauda subspecies, with diploid numbers (01°42#18.1$N, 61°08#13.4$W) MN 70742 and 70747; of 84 or 88 and fundamental numbers ranging from 116 to and 5) Castanhal (01°30#34$N, 60°58#41$W) MN Downloaded from https://academic.oup.com/jhered/article-abstract/100/3/322/867792 by guest on 06 November 2018 118 (Gardner and Patton 1976; Perez-Zapata et al. 1984; 70777; Reig et al. 1990; Bonvicino et al. 2003). Several chromo- 4) Surumu´ (4°10#N and 60°30#W): male MN 65549 and somal complements have been associated with Zygodontomys female MN 65546; 2n 5 86; brevicauda cherriei, with diploid numbers ranging from 82 to 5) Fazenda Teimoso, Ferreira Gomes (01°17#N50°48#W): 84 (Gardner and Patton 1976; Voss 1991). However, the male AN386 and female AN381; 2n 5 84; actual chromosomal complement of this taxon is the one 6) Fazenda Itapoa´, Itapoa´ (km 380 of AP 156 road, reported for the Costa Rica population (Gardner and Patton 01°21#N50°56#W): males AN612 and 615. 1976). The karyotype of Zygodontomys brevicauda microtinus has The vegetation of all sampled areas, except locality 2, not been formally described or illustrated, but Tranier grows on seasonally flooded white sand and is composed of (1976) reported a diploid number of 78 for French Guiana natural grassland around 1-m high and sparse scrub specimens without describing the morphology of the vegetation of up to 3 m in height. Locality 2 is situated at autosomal complement. Other 2 karyotypes have been an altitude of 1300 m, in a tabular plateau of the geological attributed to this taxon but from localities far from its type formation of Mount Roraima, covered with grassland locality (Reig et al. 1990; Gardner and Patton 1976). Brazilian populations of Zygodontomys have, at least, 3 karyotypes reported up to date: the Rio Negro population (Amazonas state) with 2n 5 82 and FN 5 94; a population from Surumu´ (Roraima state) with 2n 5 86 and FN 5 96– 98; and a population from Tartarugalzinho (Amapa´ state) with 2n 5 84 and FN 5 96–98 (Mattevi et al. 2002; Bonvicino et al. 2003). This scenario suggests that the diversity of this genus is underestimated in Brazil, suggesting the existence of several evolutionary lineages. In this paper, we examine whether the geographic isolation of open vegetation enclaves have influenced population- and species-level diversification in the genus Zygodontomys in the northern Brazilian Amazon. Karyomor- photypes and mitochondrial DNA genealogies (23 Zygodontomys specimens) are here used to delineate species limits and phylogeographic patterns, which are mapped onto the distribution of open vegetation formations in northern South America, in an attempt to evaluate possible links between the fragmentation of these formations and the speciation events within Zygodontomys. Figure 1. Geographic provenance of samples analyzed in this study. Brazil, Amazonas state, Barcelos: (1) Igarape´ Materials and Methods Tucunare´ and (2) Serra do Araca´; Roraima state: (3) Caracaraı´ Zygodontomys specimens were collected in 6 Brazilian and (4) Surumu´; Amapa´ state: (5) Ferreira Gomes and (6) localities from the states of Amazonas (localities 1 and 2), Itapoa´; French Guiana: (7) Kourou and (8) Macouria. Black campinaranas Roraima (localities 3 and 4), and Amapa´ (localities 5 and 6, areas delimit , gray area delimits savannas, and Figure 1), as follows: spotted area mangrooves as defined by Brazil (1975) and Olson et al. (2001). White squares are localities of Zygodontomys sp.2 1) Igarape´ Tucunare´, 00°09#72$N, 63°30#72$W [global (2n 5 82 and FN 5 94, this study), black circles are localities of positioning system (GPS)], a right-bank affluent of the Zygodontomys brevicauda microtinus (2n 5 86 and FN 5 96–98, Rio Curuduri in tributaries of the Rio Araca´, a left-bank Mattevi et al. 2002), white circles are localities of Zygodontomys tributary of the middle Rio Negro, Barcelos: female sp.1 (2n 5 84 and FN 5 96–98, Mattevi et al. 2002), and black Museu Nacional (MN) 69027 and male MN 69030; and white circles are localities of sympatry.

323 Journal of Heredity 2009:100(3) vegetation intermingled with riparian forest at margins of approach were also estimated by bootstrap with 1000 creeks and depressions. replicates in GARLI 0.95. The topologies generated by The karyotypes of 4 specimens from Caracaraı´, Roraima different methods were compared through a Shimodaira– state, listed above are first reported here; those from other Hasegawa (SH) test (Shimodaira and Hasegawa 1999), using locality samples used in the molecular analyses have been full optimization with 1000 bootstrap replicates to generate previously described (Mattevi et al. 2002; Bonvicino et al. a null distribution of the likelihood difference statistic. 2003). Karyotyped specimens (2n 5 86 and FN 5 96–98) Kimura 2 parameters (Kimura 1980) were used to from Surumu´, Roraima, previously identified as Zygodontomys calculate pairwise sequence divergence between cytochrome b sp. (Mattevi et al. 2002) and here analyzed, are herein re- haplotypes using MEGA 4 (Tamura et al. 2007). In addition ferred to as Z. b. microtinus. Chromosome preparations were to genetic distances, summary statistics of intraspecific obtained in the field from bone marrow cultures in RPMI molecular diversity were also calculated. The nucleotide 1640 medium supplemented with 20% fetal calf serum, diversity (p) and the number of polymorphic sites were Downloaded from https://academic.oup.com/jhered/article-abstract/100/3/322/867792 by guest on 06 November 2018 ethidium bromide (5 g/ml), and 10À6 M colchicine for 2 h. computed directly from the sequence alignment entered Estimates of fundamental number were restricted to using ARLEQUIN 3.1 (Excoffier et al. 2005). The autosome pairs. Voucher specimens are deposited in the population diversity parameter h (h 5 Nel), representing collection of the Museu Nacional (Universidade the product of population size and neutral mutation rate, Federal do Rio de Janeiro) and Museu Paraense Emı´lio was estimated using the ML coalescent approach imple- Goeldi (field numbers AN reported above). mented in MIGRATE 2.4.3 (Beerli and Felsenstein 1999; DNA of 19 Zygodontomys specimens was isolated from Beerli 1997–2004). We estimated divergence times between livers preserved in 100% ethanol following the procedures sister species using the genealogical approach implemented of Sambrook et al. (1989). The complete cytochrome b gene in the program IMA (Hey and Nielsen 2007). The software was amplified with primers L14724 (Irwin et al. 1991) and employs a Markov chain Monte Carlo procedure to sample MVZ14 (Smith and Patton 1993), being sequenced with the across genealogies, generating a marginal distribution of the polymerase chain reaction primers plus 2 internal primers, posterior probabilities for the parameters of an isolation- MEU1 (5#-ACAACCATAGCAACAGCATTCGT-3#) and with-migration model of divergence (Hey and Nielsen MVZ16 (Smith and Patton 1993), in an ABI Prismä 377 2004). This model assumes that some gene flow may have automatic DNA sequencer. Sequences were assembled and occurred during the divergence of sister lineages from edited using the ChromasPro software (Technelysium Inc. a common ancestor and thus include parameters of time 2006) and deposited in GenBank (accession numbers since divergence scaled by the mutation rate (t 5 T/l), EU645578 and EU652749–EU652766). Sequence data gently population genetic diversity of contemporary sister species offered by Dr Francxois Catzeflis from 2 Z. b. microtinus and their ancestor (h1, h2, and hA), and asymmetric specimens from French Guiana (localities 7 and 8 in Figure 1, migration rates (m1 and m2). Our initial runs with the full which are nearer to the type locality of this species) and model parameters readily showed that parameter estimates 2 specimens (GenBank AY029478 and AY029479) from did not converge or were inconsistent across multiple runs Brazilian state of Amazonas were also included in this (flat probability distributions), probably because the cyto- study, as well as those of megacephalus (GenBank chrome b data set did not have enough sample sizes to AF108695), Neotoma albigula (DQ179858), and Scotinomys provide consistent estimates of the large number of xerampelinus (AF108706), used as outgroups in phylogenetic parameters of the isolation-with-migration model. There- analyses based on previous phylogenetic hypotheses for the fore, we simplified the model by excluding the migration group put forward by Steppan et al. (2004). parameters and run the analyses under a simpler isolation Maximum parsimony (MP) analyses were implemented model, which fitted the data better, providing more con- in PAUP 4.0b10 (Swofford 2001) with heuristic searches sistent and convergent estimates across multiple runs. using the tree bisection reconnection branch-swapping Furthermore, the geographic isolation of patch of savannah algorithm and starting from 100 random sequences of by extensive areas of forested areas reinforces the taxon addition. Confidence intervals for parsimony trees plausibility of the isolation model. We run 10 Metropolis- were estimated on the basis of 1000 bootstrap replications coupled Markov chains of 5 million steps each, repeating the (Felsenstein 1985), in which heuristic searches were based analysis multiple times and thoroughly investigating the on 10 taxon addition sequences. Maximum-likelihood (ML) effects of prior parameter values. We chose the rate of 0.023 analyses were carried out using GARLI 0.95 software substitution per site per million years (My), which was (Zwickl 2006), which implements a genetic algorithm to calibrated by Smith and Patton (1993) on the basis of the rapidly optimize the topology, branch lengths, and model fossil record for the subfamily Sigmodontinae (the splitting parameters. The most appropriate model of DNA evolution between and ), a calibration point phyloge- fitting the data was selected using ModelTest 3.7 (Posada netically closer to Zygodontomys. and Crandall 1998), and its corresponding estimated parameters were fixed during likelihood tree searches. A Results likelihood ratio test was performed to evaluate whether the best model differed significantly from a clock-like model. Karyotypic analyses of 9 specimens (females MN 70400 and Confidence intervals for trees found under this ML males MN 70330, MN 70337, MN 70347, MN 70351,

324 Bonvicino et al.  Divergence in Zygodontomys and Distribution of Amazonian Savannas Downloaded from https://academic.oup.com/jhered/article-abstract/100/3/322/867792 by guest on 06 November 2018

Figure 2. Conventional Giemsa staining of Zygodontomys sp.2 (male MN 70352), 2n 5 82 and FN 5 94, from Parque Nacional do Virua´, Caracaraı´, Roraima state, Brazil. Sex chromosomes are depicted to the right, the larger being the X chromosome and the smallest the Y chromosome.

MN 70352, MN 70742, MN 70747, and MN 70777) from analysis (bootstrap value 85%) has low nodal support in ML Caracaraı´ showed 2n 5 82 and FN 5 94 (Figure 2). The analysis. Zygodontomys sp.2 is further structured in 2 geographic autosome complement is composed by 7 pairs of biarmed chromosomes (2 large and 5 varying in size from medium to small) and 33 pairs of acrocentric chromosomes varying in size from medium to small (Figure 2). The X chromosome is a large submetacentric and the Y chromosome is a small chromosome. The complete cytochrome b (1.144 bp) of 17 specimens (16 haplotypes) and 1.026 bp of 3 specimens were here sequenced. Analysis of 20 Zygodontomys specimens sequenced here, 2 gently provided by Dr F. Catzflis and 2 from GenBank, showed 430 variable sites, 252 of which were parsimony informative. The MP and ML trees displayed basically the same topology with no statistically significant differences (SH test: ln L difference 5 21.31, P 5 0.102). The MP analyses recovered 63 equally parsimonious trees of 667 steps (consistency index 5 0.7991, retention index 5 0.7929), whose consensus differed from the ML only in the lack of resolution within more exclusive clades of haplotypes within localities. Therefore, only the ML tree is showed in Figure 3 portraying the bootstrap nodal support values of both analyses. The selected model fitting the data performed significantly worse when a clock-like pattern of evolution was enforced (K 5 24.4186, degrees of freedom 5 23, P , 0.005). All analyses consistently recovered the monophyly of Zygodontomys (bootstrap 100%, Figure 3), divided into 2 major clades. One clade (bootstrap values 99% and 96%, re- Figure 3. ML topology depicting phylogenetic relationships spectively), here ascribed to Z. b. microtinus, consists of among cytochrome b haplotypes in Zygodontomys from haplotypes from French Guiana and Surumu´ and one northeastern Amazon. The best tree (ln L 5 À4552.5297) was haplotype from Itapoa´. The second clade (bootstrap values selected with the following parameters of a general time 99% and 78%) is further divided into 2 subgroups, herein reversible model: mutation rate matrix 3.6758 (AC), 7.7132 referred to as Zygodontomys sp.1 and Zygodontomys sp.2. (AG), 2.1494 (AT), 1.1862 (CG), 13.6859 (CT), and 1 (GT); Zygodontomys sp.1 exclusively includes haplotypes from Amapa´ base frequencies A 5 0.3093, C 5 0.2944, G 5 0.1170, and state in the localities of Ferreira Gomes and Itapoa´, where it T 5 0.2794; gamma-distributed rates with a 5 0.3771. is sympatric with Z. b. microtinus, as revealed by molecular Numbers near nodes represent bootstrap support values for analyses (bootstrap values of 100% and 99%). Zygodontomys groups obtained under parsimony and likelihood analyses, sp.2 is formed by Rio Negro and Rio Branco (Caracaraı´) respectively (only values above 50% are shown). BR, Brazil, haplotypes, which although consistently recovered in MP FG, French Guiana. Symbols refer to Figure 1.

325 Journal of Heredity 2009:100(3) groups, one comprised by Rio Negro haplotypes (bootstrap Notwithstanding the above efforts in karyologic sampling, value 73%) and the other by Caracaraı´ haplotypes (bootstrap the correct chromosome complement of Z. brevicauda values 92% and 72%). A large concordance is found between brevicauda is still unknown (Bonvicino et al. 2003). Molecular karyotypes and mitochondrial clades (Figure 3). Karyotyped phylogenetic analyses depicted a perfect match between specimens with 2n 5 82 and FN 5 94 grouped in the mitochondrial clades and karyotypes, adding support to the Zygodontomys sp.2 clade, whereas specimens with 2n 5 84 and fact that at least 3 evolutionary lineages should be re- FN 5 96–98 grouped in Zygodontomys sp.1. Z. b. microtinus is cognized among Zygodontomys populations analyzed here. also karyologically distinct and characterized by 2n 5 86 and The molecular data also indicated sympatry between FN 5 96–98. Z. b. microtinus and Zygodontomys sp.1 from Itapoa´, represent- Interlineage pairwise genetic divergences vary from 2.4% ing direct evidence of species-level distinctiveness of these to 6.7% among the species analyzed. The deepest divergence lineages. However, the possibility of hybrids cannot be is presented in comparisons between Z. b. microtinus and the discarded, and karyologic and/or nuclear data analyses are Downloaded from https://academic.oup.com/jhered/article-abstract/100/3/322/867792 by guest on 06 November 2018 Brazilian species (Zygodontomys sp.1 and Zygodontomys sp.2), needed to confirm this hypothesis. Nevertheless, karyologic varying from 4.3% to 6.7%. Zygodontomys sp.1 and Zygodontomys differences between Z. b. microtinus (2n 5 86 and FN 5 96–98) sp.2 haplotypes differ by 2.4% to 3.5%. Sympatric individuals and Zygodontomys sp.1 (2n 5 84 and FN 5 96–98) are sufficient of Z. b. microtinus and Zygodontomys sp.1 at the locality of Itapoa´ for excluding the possibility of fertile hybrids. Deriving one differ by 5% of sequence. Intraspecific mean genetic karyotype from another requires one tandem fission (or distances were 1.7% (0.1–2.5%) for Z. microtinus haplotypes, fusion). This rearrangement is rather drastic and relevant for 0.5% (0.4–0.7%) for Zygodontomys sp.1, and 1.2% (0.1–2.7%) establishing reproductive barriers due to meiotic abnormal- for Zygodontomys sp.2. Despite the limited intraspecific ities in hybrids. The 2n 5 84 and 2n 5 82 lineages refer to 2 sampling, 26 haplotypes could be identified among the apparently undescribed species, in as much as the clade samples, revealing a moderate level of polymorphism within formed by Surumu´–French Guiana–Itapoa´ is referable to Z. b. species. The highest levels of molecular diversity were found microtinus, based on the proximity to the type locality of this in Z. b. microtinus (p 5 0.016, h 5 0.0285) and Zygodontomys species, in Suriname. sp.2 (p 5 0.012, h 5 0.0193), which were 2- to 4-fold higher The distribution of the genus Zygodontomys is closely than the polymorphism found in Zygodontomys sp.1 (p 5 linked to the current distribution of open phytophysiogn- 0.005, h 5 0.0065). omies of northern South America (Voss 1991). Detailed paleopalinological information covers only the last 30 000 years (encompassing the last glacial maximum—Flenley Discussion 1998; Anhuf et al. 2006), but an expansion–fragmentation cycle of savannas was a recurrent phenomenon during the Karyologic variation in Brazilian populations of Zygodontomys last 2 My (Bennett 1990), promoting repeated opportunities is high, with, at least, 3 karyomorphotypes reported so far for geographic isolation and differentiation of populations (Mattevi et al. 2002; Bonvicino et al. 2003). The 2n 5 82 and of Zygodontomys. The central question is whether species-level FN 5 94 karyotype here described for specimens from divergence in the genus is a legacy of Quaternary Caracaraı´ is similar to that reported for Rio Negro fragmentation episodes or was rather a consequence of specimens (Bonvicino et al. 2003). This karyotype differs previous events in the Tertiary. If speciation in Zygodontomys from the other 2 chromosome complements described for was prompted by climatically driven fragmentations of Brazilian populations with 2n 5 84 and FN 5 96–98 for savannas during warmer and more humid interglacial specimens from Ferreira Gomes and 2n 5 86 and FN 5 periods, the divergence between sister species should date 96–98 for specimens from Surumu´ (Mattevi et al. 2002). back to a specific interglacial period. In order to test this Moreover, these karyotypes are also different from those expectation, we estimated the divergence times between described from non-Brazilian populations, strengthening the sister species pairs following a genealogical–coalescent evolutionary uniqueness of the Brazilian populations. approach (Hey and Nielsen 2004). According to these The association of Zygodontomys karyotypes to nominal estimates, Zygodontomys sp.1 and Zygodontomys sp.2 diverged forms in this genus is not clear. Other karyotypes have been from a common ancestor approximately 670 thousand years attributed to Z. b. microtinus, also in Venezuela, 2n 5 88 from ago, as given by the highest probability point estimate Isla Guara, isolated from continental populations with 2n 5 (Figure 4). This estimate is associated with a wide 90% 84, suggesting that each of these karyotypes was fixed rather confidence interval of 633 750 years (348 550–982 300 than polymorphic (Reig et al. 1990). Karyotypes of 2n 5 78 years) but corroborates the splitting of the Brazilian species (Tranier 1976) and 2n 5 84 (Reig et al. 1990) have also been at the upper Pleistocene. The split between Z. b. microtinus referred to Z. b. microtinus in French Guiana. In the same and the common ancestor of the 2 Brazilian species is older, way, more than one chromosome complement have been dating approximately 1.16 Million years ago at the mid- attributed to Z. brevicauda,2n 5 84 for specimens from one Pleistocene (Figure 4b), and is associated with a broader locality of Costa Rica (Gardner and Patton 1976), 2n 5 82 90% confidence interval of 880 000 years (0.56–1.44 My). for specimens from another locality in the same country These results do corroborate a Quaternary divergence (Voss 1991), and 2n 5 82 and FN 5 116 for the Venezuelan among the species analyzed, most likely occurring between population of Misio´n Tukuko (Bonvicino et al. 2003). mid- and upper Pleistocene. However, the broad confidence

326 Bonvicino et al.  Divergence in Zygodontomys and Distribution of Amazonian Savannas

2006022308523714; Department of Tropical Medicine; FIOCRUZ (Fundacxa˜o Instituto Oswaldo Cruz); CNPq (Ministe´rio da Cieˆncia e Tecnologia, Brazil); research fellowships to C.R.B, J.A.O, L.F.B.O, and M.S.M.

Acknowledgments We are grateful to Dr F. Catzeflis for sequence of Zygodontomys specimens from French Guiana, to FUNASA (Fundacxa˜o Nacional de Sau´de, Ministry of Health) for permission to use laboratory facilities in Barcelos, Amazonas state, as well as Antonio Lisboa and Beatriz A. R. Lisboa from Instituto

Chico Mendes da Biodiversidade (ICMBio, Ministry of Environment) for Downloaded from https://academic.oup.com/jhered/article-abstract/100/3/322/867792 by guest on 06 November 2018 the invitation, resources, and logistic support for the field work in the Parque Nacional do Virua´. IBAMA granted us collecting permits (Licencxa para pesquisa em unidade de conservacxa˜o no. 055/2007, and Licencxa permanente para coleta de material cientı´fico no.11375/1). We are grateful to Fabrı´cio Escarlate, Jose´ Luis Passos Cordeiro, Igor Pfiffer, Julio Fernando Vilela, Fla´via Casado Dias da Silva, as well as local staffs of Funasa (Barcelos, Amazonas) and P. N. Virua´ (Caracaraı´, Roraima) for help during field parties.

References Anhuf D, Ledru M-P, Behling H, da Cruz F Jr, Cordeiro RC, Van der Hammen T, Karmann I, Marengo JA, de Oliveira PE, Pessenda L, et al. 2006. Paleo-environmental change in Amazonian and African rainforest during the LGM. Palaeogeogr Palaeoclimatol Palaeoecol. 239:510–527. Bates J. 2001. Avian diversification in Amazonia: evidence for historical Figure 4. Marginal distributions of posterior probabilities for complexity and a vicariance model for a basin diversification pattern. In: Vieira divergence time intervals between (A) Zygodontomys sp.1 and IC, Silva JMC, Oren DC, DIncao MA, editors. Diversidade Biologica e Cultural da Amazoˆnia. Bele´m (Brazil): Museu Paraense Emılio Goeldi. p. 119–139. Zygodontomys sp.2 and (B) Zygodontomys brevicauda microtinus and the ancestor of Zygodontomys sp.1–Zygodontomys sp.2. Estimates Beerli P. 1997–2004. MIGRATE:documentation and program, part of LAMARC. Version 2.0. Revised December 23, 2004 [Internet]. Florida are based on the cytochrome b gene mutation rate of 2.3% State University. Available from: http://evolution.gs.washington.edu/ divergence per My and were calculated from genealogies lamarc.html. Accessed Jan 2008. sampled in 10 Metropolis-coupled Markov chains of 5 million Beerli P, Felsenstein J. 1999. Maximum-likelihood estimation of migration steps each run in the program IMA. rates and effective population numbers in two populations using a coalescent approach. Genetics 152:763–773. intervals associated with divergence estimates do not allow Bennet KD. 1990. Milankovitch cycles and their effects on species in the identification of specific interglacial periods. Recent ecological and evolutionary time. Paleobiology 16:11–21. studies have questioned the influence of Quaternary climatic Bonvicino CR, Maroja LS, de Oliveira JA, Coura JR. 2003. Karyology and oscillations on the origin of vertebrate species diversity in morphology of Zygodontomys (Rodentia, Sigmodontinae) from the Brazilian Amazonia, dating speciation events to older periods in the Amazon, with a molecular appraisal of the phylogenetic relationships of the Tertiary and relating them to geological rather than climatic genus. Mammalia 67:119–132. changes (e.g., Patton et al. 2000; Bates 2001; Rosseti et al. Brazil. 1975. Projeto RADAMBRASIL FolhaNA.20 Boa Vista e parte das 2005). These studies, however, are focused on the forest Folhas NA.21 Tumucumaque, NB.20 Roraima eNB21; geologia, geo- fauna of western Amazonia, a region that has experienced morfologia, pedologia, vegetacxa˜o e uso potencial daterra. Rio de Janeiro little ecological instability during the glacial–interglacial (Brazil): Departamento Nacional de Producxa˜o Mineral. cycles (Colinvaux et al. 1996; Bush et al. 2004; Anhuf Bush MB, de Oliveira PE, Colinvaux PA, Miller MC, Moreno JE. 2004. et al. 2006). The scenario for northern Amazonia seems to Amazonian paleoecological histories: one hill, three watersheds. Palae- be remarkably different, as evidenced by the present study, ogeogr Palaeoclimatol Palaeoecol. 214:359–393. which indicates that Quaternary climatic changes had Colinvaux PA, Oliveira PE, Moreno JE, Miller MC, Bush MB. 1996. A long a major influence in the species-level diversification in the pollen record from lowland Amazonia: forest and cooling in glacial times. Science 274(5284):85–88. Amazonian campinaranas, probably giving rise to a still overlooked diversity of vertebrates. Excoffier L, Laval G, Schneider S. 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinform Online. 1:47–50. Funding Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791. Fundo Brasileiro para a Biodiversidade (Funbio) and Flenley JR. 1998. Tropical forests under the climates of the last 30,000 Amazon Region Protected Areas Project (ARPA), proc. years. Clim Change 39:177–197.

327 Journal of Heredity 2009:100(3)

Gardner AL, Patton JL. 1976. Karyotypic variation in oryzomyine Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory () with comments on chromosomal evolution in the Neotropical manual. 2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. cricetinae complex. Occas Pap Lousiana State Univ Mus Zool. 49:1–48. Shimodaira H, Hasegawa M. 1999. Multiple comparisons of log- Haffer J. 1969. Speciation in Amazonian forest birds. Science 165:131–137. likelihoods with applications to phylogenetic inference. Mol Biol Evol. 16: Haffer J. 2002. A rare hybrid manakin (Aves, Pipridae) and the origin of 1114–1116. vertebrate species in Amazonia. Rudolstadter naturhistorische Schr. Smith MF, Patton JL. 1993. The diversification of South American murid 4(Suppl):47–73. rodents: evidence from mitochondrial DNA sequence data for the Hey J, Nielsen R. 2004. Multilocus methods for estimating population sizes, Akodontine tribe. Biol J Linn Soc. 50:149–177. migration rates and divergence time, with applications to the divergence of Steppan SJ. 1995. Revision of the tribe Phyllotini (Rodentia: Sigmodontinae) Drosophila pseudoobscura and D. persimilis. Genetics 167:747–760. with a phylogenetic hypothesis for the Sigmodontinae. Field Zool. 80:1–112. Hey J, Nielsen R. 2007. Integration within the Felsenstein equation for Steppan SJ, Adkins R, Anderson J. 2004. Phylogeny and divergence-date

improved Markov chain Monte Carlo methods in population genetics. Proc estimates of rapid radiations in muroid rodents based on multiple nuclear Downloaded from https://academic.oup.com/jhered/article-abstract/100/3/322/867792 by guest on 06 November 2018 Natl Acad Sci USA. 104:2785–2790. genes. Syst Biol. 53(4):533–553. Irwin DM, Kocher TD, Wilson AC. 1991. Evolution of the cytochrome b Swofford DL. 2001. PAUP: phylogenetic analysis using parsimony (*and gene of . J Mol Evol. 32:128–144. other methods), 4.0 b10. Sunderland (MA): Sinauer Associates. Kimura M. 1980. A simple method for estimating evolutionary rate of base Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: molecular substitutions through comparative studies of nucleotide sequences. J Mol evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. Evol. 16:111–120. 24:1596–1599. Mattevi MS, Haag T, Nunes AP, Cordeiro JLP, Andrades-Miranda J, de Technelysium Pty Ltd. 2006. Chromas Prosoftware, version 1.34 [Internet]. Oliveira LF. 2002. Karyotypes of Brazilian representatives of genus Queensland (Australia). Available from: http://www.technelysium.com.au/ Zygodontomys (Rodentia, Sigmodontinae). J Neotrop Mammal 9:5–10. ChromasPro.html. Olson DM, Dinerstein E, Wikramanayake ED, Burgess ND, Powell GVN, Tranier M. 1976. Nouvelles donne´es sur l’e´volution non paralle`le du Underwood EC, D’amico JA, Itoua I, Strand HE, Morrison JC, et al. 2001. karyotype et de la morphologie chez les phyllotine´s (Roungeurs, Crice´tide´s). Terrestrial Ecoregions of the World: a New Map of Life on Earth. C R Acad Sci Paris D. 283:1201–1203. Bioscience 51:933–938. Vanzolini PE, Williams EE. 1970. South American anoles: the geographic Patton JL, Silva MN, Malcolm JR. 2000. Mammals of the Rio Jurua and the differentiation and evolution of the Anolis chrysolepis species group (Sauria, evolutionary and ecological diversification of Amazonia. Bull Am Mus Nat Iguanidae). Arq Zool Sa˜o Paulo. 19:1–298. Hist. 244:1–306. Voss RS. 1991. An introduction to the Neotropical muroid rodent genus Perez-Zapata A, Aguilera M, Ferrer A, Reig OA. 1984. El cariotipo de una Zygodontomys. Bull Am Mus Nat Hist. 210:1–113. poblacio´n de Zygodontomys sp. (Rodentia, Cricetidae) del Delta del Orinoco. Voss RS, Carleton MD. 1993. A new genus for Hesperomys molitor Winge and Acta Cient Venez. 35(Suppl 1):227. magnus Hershkovitz (Mammalia, Muridae) with an analysis of its Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA phylogenetic relationships. Am Mus Novit. 3085:1–39. substitution. Bioinformatics 14(9):817–818. Zwickl DJ. 2006. GARLI manual (version 0.95). [Internet]. University of Reig OA, Aguilera M, Perez-Zapata A. 1990. Cytogenetics and karyosys- Texas. Austin (TX) Available from: URL http://www.bio.utexas.edu/ tematics of South American oryzomyine rodents (Cricetidae: Sigmodonti- faculty/antisense/garli/GARLI.html. Accessed 22 Jan 2008. nae). II. High numbered karyotypes and chromosomal heterogeneity in Venezuelan Zygodontomys. Z Saugetierkd. 55:361–370. Received June 3, 2008; Revised November 12, 2008; Rosseti DF, de Toledo PM, Goe´s AM. 2005. New geological framework for Accepted November 17, 2008 Western Amazonia (Brazil) and implications for biogeography and evolution. Quat Res. 63:78–89. Corresponding Editor: William Modi

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