Rodentia: Sigmodontinae) Based on Cytochrome B DNA Sequences Cibele Rodrigues Bonvicino and Miguel A

Home , Melanomys, Microryzomys minutus, Neacomys, Neacomys spinosus, Nectomys, Oecomys

Molecular Phylogenetics and Evolution Vol. 18, No. 2, February, pp. 282–292, 2001 doi:10.1006/mpev.2000.0878, available online at http://www.idealibrary.com on

Molecular Phylogeny of the Genus Oryzomys (Rodentia: Sigmodontinae) Based on Cytochrome b DNA Sequences Cibele Rodrigues Bonvicino and Miguel A. Martins Moreira Genetics Section, Instituto Nacional de Caˆ ncer—Ministe´ rio da Sau´ de, Prac¸a da Cruz Vermeiha, 23, 6°, 20230-130, Rio de Janeiro, RJ, Brazil

Received April 25, 2000; revised October 2, 2000; published online December 26, 2000

phyly of Oryzomys based on morphological studies The genus Oryzomys comprises 40 species arranged (Patton and Hafner, 1983; Weksler, 1996), karyological in several species groups. To test the monophyly of comparisons (Baker et al., 1983; Voss and Carleton, three Oryzomys species groups (“capito,” nitidus, and 1993), isozyme analyses (Dikerman and Yates, 1995), subflavus), we analyzed, by distance, parsimony, and and molecular data (Myers et al., 1995; Patton and da maximum-likelihood (ML), 801 bp of the mitochondrial Silva, 1995). Recent studies were concentrated in spe- gene cytochrome b. Our results did not sustain the cies complexes like nitidus (Weksler, 1996) and capito monophyly of Oryzomys nor of the nitidus and subfla- (Musser et al., 1998). vus species groups. Within the “capito” species group, The Oryzomys species recognized by Musser and Car- O. perenensis appeared as a valid species, as a sister leton (1993) were arranged by these authors in eight branch of the clade formed by O. megacephalus and O. species groups that were not cleary defined by morpho- laticeps. Within the nitidius species group, only the association between O. nitidus and O. lamia was logical criteria: albigularis, alfaroi, capito, melanotis, well supported. The subflavus species group split into nitidus, palustris, subflavus, and xantheolus. Musser et two clades: one with O. subflavus karyomorphotypes al. (1998), reviewing the “capito” complex, replaced it by and another grouping O. angouya with species of dif- four species groups: (1) “capito” (including O. megacepha- ferent genera in the parsimony, distance, and ML lus and O. laticeps), (2) yunganus (including O. yunganus trees. © 2001 Academic Press and O. tatei), (3) the trans-Andean species (O. talaman- cae and O. bolivaris), and (4) nitidus (including O. nitidus, O. macconnelli, O. emmonsae, and O. russatus). In INTRODUCTION this report, Musser et al. (1998) also considered another species group, viz. subflavus, with two species: O. subfla- The large number of species and subgenera assigned vus and O. angouya. to Oryzomys during its taxonomic history is good evi- A different arrangement was suggested by Patton et dence of the complexity of this taxon. Oryzomys was al. (2000) for some of the above-mentioned species: the initially defined as a subgenus of Hesperomys (Baird, megacephalus group (including O. megacephalus, O. 1859) and subsequently elevated to generic status perenensi, O. laticeps, and O. yunganus) and the mac- (Coues, 1890). The number of species included in it was connelli group (including O. emmonsae, O. maccon- initially 80 (Trouessart, 1897) but was later reduced to nelli, O. nitidus, and O. russatus). The inclusion of O. approximately 40 (Musser and Carleton, 1993). This lamia as a valid species, based on morphologic and led to the creation of several subgenera, such as Oeco- karyologic data (Weksler, 1996; Bonvicino et al., 1998), mys (Thomas, 1906), Oligoryzomys (Bangs, 1900), Mi- added a fifth species to this latter group. croryzomys (Thomas, 1917), and Melanomys (Thomas, The genus Oryzomys is distributed from the south of 1902), currently elevated to generic status. Informal North America through Central America to northern species groups were created for facilitating the study of Argentina. Species of the “capito” and nitidus groups Oryzomys but the number of species included within are distributed in the Atlantic Forest, Amazonian For- each group was controversial (Gyldenstolpe, 1932; est, and Cerrado, whereas species of the yunganus Tate, 1932; Ellerman, 1941; Hooper and Musser, 1964; group are distributed mainly in the Amazonian forest. Gardner and Patton, 1976; Weksler, 1996; Musser et The subflavus species group is the least known of all al., 1998). Consequently, the monophyly of Oryzomys groups, with O. angoya distributed in the Atlantic For- and the phylogenetic relations of its species lack ade- est and O. subflavus variants mainly in open vegeta- quate resolution, a reason that further analyses are tion areas of the Cerrado, Caatinga, and enclaves of necessary. Different authors questioned the mono- savanna in the Amazonian Forest.

Species belonging to each Oryzomys species group Museum of Southwestern Biology (University of New are morphologically very similar to one another, a rea- Mexico, Albuquerque, NM); CM to Carnegie Museum son that their systematic relationships are difficult to of Natural History (Pittsburgh, PA); and ROM to Royal discern. This is the case of O. subflavus, which proba- Ontario Museum (Toronto, Canada). The following ac- bly includes more than one species, as indicated by four ronyms refer to personal field numbers: CRB to C. R. different karyotypes (Bonvicino et al., 1999). These Bonvicino; JLP to J. L. Patton; MNFS to M. N. F. da occur in different geographic regions, with 2n ϭ 48–50, Silva; EDH to E. D. Hingst; ML to M. Lara; CEG to FN ϭ 56 in northeastern Brazil (Maia and Hulak, C. E. Grelle; EM to E. M. Vieira; AL to A. Langguth; 1981); 2n ϭ 54–56, FN ϭ 62–63 in Saõ Paulo state, and LHE to L. H. Emmons. CS and CCM refer to Brazil (Almeida and Yonenaga-Yassuda, 1985); 2n ϭ specimens deposited in the Instituto Nacional de Pes- 50, FN ϭ 64 in Bahia state, Brazil (Zanchin, 1988); and quisa da Amazoˆnia (INPA, Manaus, Brazil). 2n ϭ 58, FN ϭ 72 in Distrito Federal, Brazil (Svart- DNA Isolation, Amplification, Sequencing, and Data man, 1989). Additionally, two new karyomorphotypes (2n ϭ 56, FN ϭ 58 and 2n ϭ 56, FN ϭ 56) were found Analyses in Mato Grosso do Sul and Bahia States, respectively DNA samples of 13 specimens of Oryzomys, 1 speci- (unpublished). men of Nectomys squamipes, and 1 specimen of Delo- Mitochondrial DNA is adequate for studying taxa at mys collinus were isolated from livers preserved in low taxonomic levels like intrageneric or intraspecific ethanol or stored in liquid nitrogen (Tables 1 and 2). relationships (Hillis et al., 1996) due to its fast rate of DNA was obtained following the procedures of Smith et nucleotide substitutions. In this paper, we analyze the al. (1987) or with phenol–chloroform extractions (Sam- phylogenetic relationships within the subflavus species brook et al., 1989). Cytochrome b DNA (ca. 801 bp) was group and between this group and other Oryzomys amplified with primers MVZ 05 and MVZ 16 and se- species groups (yunganus, nitidus, “capito,” sensu quenced with an ABI Prism 377 automatic DNA se- Musser et al., 1998) as well as other Oryzomyini gen- quencer following Smith and Patton (1993). Sequence era. This is based on analysis of an 801-bp region of the data from 18 other Oryzomys specimens and 11 other cytochrome b gene in these taxa and comparisons with sigmodontine rodents were also included in this study previously reported sequence data. (see GenBank access in Tables 1 and 2) to evaluate the monophyly of Oryzomys and its species group. Cyto- chrome b sequences from Rattus norvegicus (GenBank MATERIAL AND METHODS Accession No. X14848) and Mus musculus (GenBank Species Identification Accession No. V00711) were used as outgroups. Sequences were aligned with XESEE (Cabot, Species names and species groups followed the tax- 1994); parsimony analyses were carried out with onomic criterion of Musser et al. (1998) except for O. PAUP 3.1.1 (Swofford, 1993) and distance and max- lamia and O. perenensis herein considered valid spe- imum-likelihood analyses with PHYLIP 3.5c cies and for the different karyomorphotypes of the sub- (Felsenstein, 1995). Different procedures were car- flavus group herein denominated O. subflavus sensu ried out to avoid saturation effects due to the high stricto (2n ϭ 54–56, FN ϭ 62–63), Oryzomys sp.n.1 rate of nucleotide substitutions of mitochondrial (2n ϭ 48–50, FN ϭ 56), Oryzomys sp.n.2 (2n ϭ 50, DNA. Parsimony analyses were carried out consid- FN ϭ 64), Oryzomys sp.n.3 (2n ϭ 58, FN ϭ 72), Oryzo- ering (1) all codon positions, (2) only transversions at mys sp.n.4 (2n ϭ 56, FN ϭ 58), and Oryzomys sp.n.5 third codon position, and (3) weighting transversions (2n ϭ 56, FN ϭ 56). All animals whose cytochrome b 5 or 10 times higher than transitions. Distance anal- gene was sequenced were karyotyped for confirming yses were carried out with the program DNADIST; morphological identification (Tables 1 and 2). Chromo- distances were estimated with the Kimura two-pa- some preparations were obtained from bone marrow rameter method with two different ratios of transi- cultures in RPMI 1640 with 20% foetal calf serum, tion:transversion rates (5:1 and 10:1), and trees were ethidium bromide (5 ␮g/ml), and colchicine (10Ϫ6 M) for constructed by neighbor-joining using the Neighbor 2h. program. Maximum-likelihood (ML) trees were ob- Skins and skulls of sequenced specimens were depos- tained using the DNAML program with two different ited at the Museu Nacional (MN, Rio de Janeiro, Bra- ratios of transition/transversion rates (5:1 and 10:1), zil) and the Mammalian Collection of the Universidade and the topologies obtained with parsimony, dis- Federal da Paraı´ba (UFPB, Joaõ Pessoa, Brazil). tance, and ML analyses were tested by the Kishino MZUSP refers to Museu de Zoologia da Universidade and Hasegawa’s test (Kishino and Hasegawa, 1989), de Saõ Paulo (Saõ Paulo, Brazil); MVZ to Museum of with 5:1 and 10:1 ratios of transition:transversion Vertebrate Zoology (Berkeley, CA); USNM to United rates. Bootstrap values were obtained from 1000 rep- States National Museum (Washington, DC); MSB to licates. 284 BONVICINO AND MOREIRA

TABLE 1 Oryzomys Specimens Used in Phylogenetic Analyses

Taxon (GenBank Accession No.) 2n FN Number Locality of collection

O. capito group O. perenensis (U03538) MVZ166676 Riteni, Rio Urubamba, Cuzco, Peru O. megacephalus 1 (AF251516) JLP16731 Macaco, left bank rio Jau, Amazonas, Brazil O. megacephalus 2 (AF251517) CM 76933 Geyskes Creek, Tafelberg, Saramacca, Suriname O. megacephalus 3 (AF251518) ROM 97979 30 km NE, Surama, Rupununi, Guyana O. megacephalus 4 (AF251519) LHE 510 52 km SWW Altamira, left bank rio Xingu, Para´, Brazil O. laticeps 1 (AF251520) EDH 22 Faz. Beijo Grande, 12 km S 81 km E Itabuna, Bahia, Brazil O. laticeps 2 (AF251521) ML 101 CEPLAC, 6 km E Itabuna, Bahia, Brazil O. yunganus group O. yunganus 1 (U58380) MNFS 1101 Ig. Porongaba, rio Jurua´, Acre, Brazil O. yunganus 2 (AF251522) JLP 15535 Seringal Condor, left bank of the rio Jurua´, Amazonas, Brazil O. nitidus group O. russatus 1 (AF251523) ML 48 Faz. Patoca, Ilha Bela, Saõ Paulo, Brazil O. russatus 2 (AF251524) ML 94 Parque Estadual do Desengano, 1.7 km N, 5.1 km W Santa Maria Madalena, Rio de Janeiro, Brazil O. russatus 3 (AF181271)* AL 3466 Faz. Aldeia, 7 km NNW Valença, Bahia, Brazil O. russatus 4 (AF181272)* 80 84 ORG 67 Guapimirim, Rio de Janeiro, Brazil O. emmonsae 1 (AF251525) CS 37 Floresta Nacional Tapirape´-aquiri, Maraba´, Para´, Brazil O. emmonsae 2 (AF251526) MZUSP 27150 52 km SW Altamira, left bank of the rio Xingu, Para´, Brazil O. macconnelli 1 (U58379) MNFS 156 Alto rio Urucu, Amazonas, Brazil O. macconnelli 2 (AF251527) MNFS 747 Barro Vermelho, left bank rio Jurua´, Amazonas, Brazil O. macconnelli 3 (AF251528) CCM 44-2 82 km N Manaus, Amazonas, Brazil O. lamia (AF181273)* 58 84 CRB 968 Faz. Fiandeira, 65 km SSW Cavalcante, Goia´s, Brazil O. nitidus 1 (U58383) MNFS 1419 Ig. Porongaba, right bank rio Jurua´, Acre, Brazil O. nitidus 2 (AF251529) 52 MSB 166027 Cuzco Amazonico, rio Madre de Dios, Peru O. subflavus group O. subflavus (AF181274)* 54 62 CEG 42 P.N. do Rio Doce, Minas Gerais, Brazil Oryzomys sp.n.2 (AF181275)* 50 54 EDH 60 Itabuna, Bahia, Brazil Oryzomys sp.n.2 (AF181275)* 50 54 FC148 Faz. Canoas, Juramento, Minas Gerais, Brazil Oryzomys sp.n.1 (AF181276)* 50 56 AL 3066 Joaõ Pessoa, Paraı´ba, Brazil Oryzomys sp.n.3 (AF181277)* 58 72 CRB 962 Faz. Fiandeira, 65 km SSW Cavalcante, Goia´s, Brazil Oryzomys sp.n.3 (AF181277)* 58 72 CRB 404 Corumba´ de Goia´s, Goia´s, Brazil Oryzomys sp.n.4 (AF181278)* 56 58 MN44178 Faz. da Mata, Maracaju´ , Mato Grosso do Sul, Brazil Oryzomys sp.n.5 (AF181279)* 56 56 CRB 1560 Faz. Sertaõ Do Formoso, Jaborandi, Bahia, Brazil O. angouya 1 (AF181281)* 58 CRB 1271 Tereso´polis, Rio de Janeiro, Brazil O. angouya 2 (AF181280)* 58 EM 1207 Faz. Intervales, Saõ Paulo, Brazil

Note. 2n, diploid number; FN, autosomal fundamental number; Number, ﬁeld or museum number; asterisks (*) indicate specimens sequenced in the present work.

RESULTS pologies with a high number of homoplasies. However, as exclusion of transitions at third codon positions or Cytochrome b DNA sequence data of the species exclusion of third codon positions could imply loss of herewith sequenced were deposited in GenBank (Ta- phylogenetic information of recently diverged taxa, dif- bles 1 and 2). The two specimens of Oryzomys sp.n.3 ferent parsimony analyses were carried out. shared the same nucleotide sequence as both speci- Parsimony analyses resulted in different arrange- mens of Oryzomys sp.n.2 (see Table 1). Conversely, O. ments, but in all of them the genus Oryzomys was angouya, O. russatus, O. macconnelli, O. emmonsae, O. paraphyletic due to association of some Oryzomys spe- laticeps, O. yunganus, and O. megacephalus showed cies with species of other genera in arrangements sup- intraspeciﬁc differences and each individual sequence ported by low bootstrap values (Ͻ50%). Analysis with was included in the analyses. all codon positions and transitions and transversions Parsimony Analyses at third positions resulted in six most-parsimonious Genetic distance estimates for each codon position trees (Fig. 1; steps ϭ 2132, C.I. ϭ 0.273), indicating the when plotted against total genetic distance indicated monophyly of species included in the “capito” and sub- that transitions at third codon positions were satu- ﬂavus groups (bootstraps values of 83 and Ͻ50%, re- rated. Consequently, phylogenetic analysis considering spectively). However, the monophyly of the nitidus transitions at third codon positions may result in to- group could not be demonstrated because the (O. MOLECULAR PHYLOGENY OF Oryzomys BASED ON CYTOCHROME b 285

TABLE 2 Sigmodontinae Specimens (Except Oryzomys) from Which Cytochrome b DNA Data Were Used for Phylogenetic Analyses

Taxon (GenBank Accession No.) 2n FN Number Locality of collection

Rhipidomys leucodactylus (U03550) MVZ-RMW09 Albaque, Madre de Dios, Peru Delomys collinus (AF181282)* 80 84 CRB 1341 Brejo da Lapa, Itamonte, Minas Gerais, Brazil Akodon azarae (U03529) UNMZ134443 5.8 km by rd NE Pilar, Neembacu, Paraguay Calomys lepidus (U03544) MVZ171562 6 km N Putina, Puno, Peru Thomasomys aureus (U03540) MVZ166714 75 km NE Paucartambo, Cuzco, Peru MVZ170076 Other Oryzomyini Oecomys bicolor (U58382) MNFS1499 Brazil Oecomys trinitatus (U58390) MNFS1250 Brazil Nectomys garleppii (U03539) MVZ166700 Rio Urubamba, Kiteni, Cuzco, Peru Nectomys squamipes (AF181283)* 56 56 CRB 540 Faz. Da Mata, Maracaju´ , Mato Grosso do Sul, Brazil Microryzomys minutus (U58387) MVZ173957 Peru Neacomys spinosus (U58391) MNFS1262 Brazil Oligoryzomys longicaudatus (U03535) MVZ15582 12 km W Bariloche, Prov. Rio Negro, Argentina Oligoryzomys microtis (U58381) MNFS1321 Brazil

Note. 2n, diploid number; FN, autosomal fundamental; Number, museum or field number; asterisks (*) indicate specimens sequenced in the present work. lamia, O. nitidus) clade was dissociated from the re- of 89.7% (with a 10:1 transition:transversion ratio) and maining species of this group. 97.4% (with a 5:1 transition:transversion ratio, Fig. 2), Exclusion of transitions at third codon positions re- as was the nitidus group, showing the same internal sulted in two most-parsimonious trees (991 steps, CI ϭ topology in both analyses. However, bootstrap values 0.291, RI ϭ 0.591), showing the monophyly of the niti- sustaining the nitidus group and the majority of inter- dus and “capito” groups sustained by bootstrap values specific arrangements were below 50%, except for the of Ͻ50 and 75.6%, respectively. The association of O. association between O. lamia and O. nitidus. yunganus with the “capito” species group, although Maximum-Likelihood Analyses sustained by a bootstrap value below 50%, corre- sponded to the megacephalus group sensu Patton et al. ML topologies (Fig. 3) did not support the monophyly (2000). With respect to the subflavus species group, O. of Oryzomys, although the “capito” and nitidus species angouya did not associate with O. subflavus and the groups were shown to be monophyletic in both ML five different karyomorphotypes which grouped in a analyses. In the subflavus species group, O. angouya clade supported by a bootstrap value of 97.7%. was associated with Nectomys and with the remaining Different weights of transversion:transition events O. subflavus taxa, including the five karyomorpho- (5:1 and 10:1) resulted in similar parsimony trees. Spe- types, representing a monophyletic arrangement. cies belonging to the “capito” group formed a well- The different topologies obtained with parsimony, sustained clade (bootstrap values 85 and 78%, respec- distance, and ML analyses were tested by the Kishino tively) and were associated with O. yunganus, forming and Hasegawa test (Kishino and Hasegawa, 1989), the megacephalus species group sensu Patton et al. with 5:1 and 10:1 ratios of transition:transversion (2000), although supported by low bootstrap values rates. The trees tested were not significantly different (Ͻ50%). In the subflavus species group, O. angouya did from one another. not group with the remaining karyomorphotypes. These karyomorphotypes were closely associated, with DISCUSSION a bootstrap value of 98% with both 10:1 and 5:1 transition:transversion ratios. The monophyly of the Oryzomyini could not be supported by parsimony, distance, or ML analyses of cyto- Distance Analyses chrome b DNA sequences despite the apparent mono- Neighbor-joining trees did not support the monophyly observed by analysis of morphological data (Voss phyly of Oryzomys. Both topologies showed O. angouya and Carleton, 1993) and molecular characters (Myers associated with Nectomys with bootstrap values below et al., 1995; Smith and Patton, 1999). Molecular topol- 50%. The association among the remaining taxa of the ogies, resulting from analysis of 401 bp (Myers et al., subflavus group was well sustained, with a bootstrap 1995) and 801 bp (Smith and Patton, 1999) of cyto- value of 99.9% in both analyses. The “capito” group was chrome b DNA, showed low decay index and bootstrap monophyletic in both analyses, with bootstrap values values. Our results, however, differed from the former 286 BONVICINO AND MOREIRA

FIG. 1. Consensus parsimony tree of six most-parsimonious trees with 2132 steps (CI ϭ 0.273; RI ϭ 0.525). Numbers at nodes are bootstrap values above 50%. MOLECULAR PHYLOGENY OF Oryzomys BASED ON CYTOCHROME b 287

FIG. 2. Neighbor-joining distance tree with distances estimated by the Kimura two-parameter method with a 5:1 ratio of transition:transversion rates. Numbers at nodes are bootstrap values above 50%. Asterisk (*) indicates a grouping that was not found with a 10:1 ratio. 288 BONVICINO AND MOREIRA

FIG. 3. Maximum-likelihood tree obtained with a 5:1 transition:transversion ratio. Asterisks indicate groups that were not found with a 10:1 ratio. MOLECULAR PHYLOGENY OF Oryzomys BASED ON CYTOCHROME b 289 report in sequence length and from both reports in ported. Most intraspecific clades were well sustained respect to the species herein analyzed. (bootstrap values above 95%) but the clade grouping O. Oryzomyini genera represented by more than one macconnelli specimens was sustained by bootstrap val- species, except Oryzomys (Nectomys, Oecomys, Oligo- ues ranging from 60 to 89%. Internal arrangements ryzomys), were found to be monophyletic in all analyses supported the interspecific clade grouping O. lamia and supported by bootstrap values above 60%. The with O. nitidus but the positions of the remaining three cohesive group formed by Oligoryzomys species was species (O. russatus, O. emmonsae, and O. macconnelli) already demonstrated (Myers et al., 1995), as was the varied in different analyses and were poorly supported. monophyly of Oecomys (Smith and Patton, 1999). The strong association between O. lamia and O. niti- However, the monophyly of Oryzomys, the largest dus clearly indicated that O. lamia is a valid species and most complex genus of the tribe Oryzomyini, could rather than a synonymous junior of Oryzomys russa- not be demonstrated because the different analyses tus, as suggested by Musser et al. (1998). carried out in the present study grouped Oryzomys The monophyly of the nitidus species group was also species with species of different genera. This was also evident by morphological data (Weksler, 1996) and by observed by Myers et al. (1995) with cytochrome b DNA molecular analysis using O. yunganus and species of (ca. 401 bp) and by Voss and Carleton (1993) with the “capito” group as outgroups, showing O. russatus, karyologic comparisons based on data of Baker et al. O. emmonsae, and O. macconnelli in a trichotomy (Pat- (1983). These two latter reports, however, included the ton et al., 2000). type species of the genus (Oryzomys palustris), which The O. subflavus Species Group was not included in this study. Of the three informally defined Oryzomys species The monophyly of this group was found only in the groups (“capito”, nitidus, and subflavus) herein stud- parsimony analysis considering all character states ied, only “capito” was shown to be monophyletic in all with the same weight, although it was poorly sup- analyses. ported. In the remaining analyses, these species were divided into two groups: a monophyletic well-sustained The “capito” Species Group group comprising O. subflavus sensu stricto and the All analyses resulted in similar topologies (Figs. five karyomorphotypes and another group with O. 1–3), in correspondence to the geographic distribution angouya associated with different Oryzomyini genera. of the species belonging to this group. These arrange- Contrary to our results, however, morphological stud- ments grouped species from the Atlantic Forest (O. ies placed O. angouya and O. subflavus in the same laticeps) with species from the Cerrado and Amazonian species group (Musser et al., 1998), an association that Forests (O. megacephalus) but apart from the species was also demonstrated by karyotypic comparisons from Western Amazonia (O. perenensis). This arrange- (Zanchin, 1988). ment differed from that reported by Patton et al. The position of Nectomys with respect to the O. sub- (2000), which indicated a trichotomy among O. mega- flavus group could not be established despite molecular cephalus, O. perenensis, and O. laticeps. Conversely, analyses (Smith and Patton, 1993; Myers et al., 1995) our data did not support the monophyly of the mega- and morphological studies also showing an association cephalus complex sensu Patton et al. (2000), which between Nectomys and Oryzomys (Weksler, 1996). In grouped O. yunganus, O. megacephalus, O. perenensis, this latter report, the clade formed by Nectomys, O. and O. laticeps. The association between the “capito” palustris, O. xantheolus, O. subflavus, and O. angouya species group and O. yunganus was obtained with par- was sustained by three synapomorphies (1, absence of simony and distance analyses excluding transitions at flexus in the procingulum of M1; 2, carotid circulation third positions and with parsimony analyses with dif- pattern derived, small opening of the stapedial fora- ferent weights for transversion:transition events but men and the carotid channel, and absence of squamo- with bootstrap values below 50%, whereas, in the re- sal–alisphenoide groove and alisphenoid foramen; 3, maining analyses, O. yunganus was associated with posterior palatal pits in fossa). other species. The association of O. laticeps with O. The five O. subflavus karyomorphotypes (see Mate- megacephalus apart from O. perenensis suggested that rial and Methods and Table 1) and O. subflavus sensu O. perenensis, considered a junior synonym of O. mega- stricto were strongly grouped. The components of the cephalus by Musser et al. (1998), is a valid species, as clade formed by Oryzomys sp.n.1, Oryzomys sp.n.2, and proposed by Patton et al. (2000). O. subflavus sensu stricto were very similar to one another in cranial and external morphologic charac- The O. nitidus Species Group ters, although they were karyotypically different from The nitidus species group formed a monophyletic one another (see Table 1). The sister branch of this arrangement in all topologies except in parsimony clade, Oryzomys sp.n.3, was morphologically distin- analysis considering all character states with the same guishable from them by its dark lined-yellow dorsal weight, although this association was weakly sup- coloration and cranial characters (e.g. presence of alis- 290 BONVICINO AND MOREIRA

FIG. 4. Localities of occurrence of O. subflavus karyomorphotypes (F) and O. angouya (}). Delimited areas of Oryzomys subflavus sensu stricto (A), Oryzomys sp.n.1 (B), Oryzomys sp.n.2 (C), Oryzomys sp.n.3 (D), Oryzomys sp.n.4 (E), and Oryzomys sp.n.5 (F). phenoid strut). Karyotypic analysis also suggested the sp.n.2, and Oryzomys sp.n.1) were strongly related to monophyly of these four taxa, with partial homologies one another. These species can also be found in the between their chromosome complement (Svartman, Atlantic forest, whereas another related species 1989). (Oryzomys sp.n.3) is endemic to the Cerrado biome. The congruent topology of all analyses strongly Oryzomys sp.n.4 and Oryzomys sp.n.5 occur in separate grouped Oryzomys sp.n.4 and Oryzomys sp.n.5 apart enclaves of the Cerrado, partially overlapping the dis- from the other karyomorphotypes, suggesting that this tribution of Oryzomys sp.n.3. O. subflavus karyomor- clade includes different species, as indicated by mor- photypes are allopatric with respect to O. angouya, phological and karyological studies. Morphological which occurs in the Brazilian Atlantic forest up to data showed that they differed in cranial characters, Espı´rito Santo State. such as absence of mastoid fenestra in Oryzomys sp.n.5 The monophyly of O. angouya with the remaining and presence in Oryzomys sp.n.4, a wider zygomatic subflavus karyomorphotypes was not supported by any plate producing a deep zygomatic notch in Oryzomys analyses. Most topologies showed that O. angouya sp.n.5, and a more narrow zygomatic plate with a more grouped with Nectomys and in some trees grouped with shallow zygomatic notch in Oryzomys sp.n.4. Neacomys and Microryzomys. Therefore, our analyses Bonvicino et al. (1999) suggested that three of these showed different arrangements for O. angoya and did karyomorphotypes (1, 2, and 3), in addition to O. sub- not indicate a clear position with respect to other flavus sensu stricto, represented separate species Oryzomyini species. rather than populations, indicating that O. subflavus sensu Musser et al. (1998) includes more than a single CONCLUSION taxon. O. subflavus sensu stricto is allopatric with respect to the other five karyomorphotypes (Fig. 4). Two Analyses of cytochrome b DNA sequence data did not karyomorphotypes (Oryzomys sp.n.2 and Oryzomys support the monophyly of the genus Oryzomys and the sp.n.1) are also allopatric, whereas the distributions of monophyly of the tribe Oryzomyini was found only in the other karyomorphotypes show partial overlaps. the distance tree. Of the informally defined Oryzomys The geographic distribution of these forms and cyto- species groups, only the monophyly of the “capito” spe- chrome b variation were coincident in showing that cies group (O. megacephalus, O. laticeps, and O. eastern species (O. subflavus sensu stricto, Oryzomys perenensis) was observed. In the nitidus group, only MOLECULAR PHYLOGENY OF Oryzomys BASED ON CYTOCHROME b 291 the clade formed by O. lamia and O. nitidus was sup- Avise, J. C. (1993). “Molecular Markers, Natural History and Evo- ported, and in the subflavus species group, only the lution,” Chapman & Hall, New York. monophyly among O. subflavus sensu stricto, Oryzomys Baird, S. F. (1859). “Mammals of North America,” Lippincott, Phil- sp.n.1, Oryzomys sp.n.2, Oryzomys sp.n.3, Oryzomys adelphia. sp.n.4, and Oryzomys sp.n.5 could be established. Fur- Baker, R. J., Koop, B. F., and Haiduk, M. W. (1983). Resolving systematic relationships with G-bands: A study of five genera of thermore, our analyses suggested that a new taxo- South American cricetine rodents. Syst. Zool. 32: 403–416. nomic arrangement (at the generic level) is necessary Bangs, O. (1900). List of the mammals collected in the Santa Marta for understanding the association among species region of Colombia by W. W. Brown Jr. Proc. N. E. Zool. Club 1: herein investigated. O. palustris, the type species of 87–102. the genus Oryzomys, appears to be more closely related Bonvicino, C. R., Otazu, I., and Weksler, M. (1998). Oryzomys lamia to the O. subflavus species group than to the nitidus Thomas, 1901 (Rodentia, Sigmodontinae): Karyotype, geographic and “capito” species groups, as previously demon- distribution and conservation status. Mammalia 62: 253–258. strated by morphological analysis (Weksler, 1996). Bonvicino, C. R., Otazu, I. B., and Borodin, P. M. (1999). Chromo- This suggests that the generic status of taxa belonging some variation in Oryzomys subflavus species group (Sigmodonti- nae, Rodentia) and its taxonomic implication. Cytologia 64: 327– to species groups other than subflavus needs to be 332. reevaluated. Cabot, E. L. (1994). “The Eyeball Sequence Editor, version 3.0,” The phylogenetic relationships within the O. subfla- Department of Ecology and Evolution, Univ. of Chicago, Chicago. vus species group herein analyzed (excluding O. an- Coues, E. (1890). Oryzomys (verbete). In “The Century Dictionary: goya) were coincident with biogeographic data. This is An Encyclopedic Lexicon of the English Language” (W. D. Whit- because three tightly related species of the southeast- ney, Org.), Vol. 1, p. 4164. Century, New York. ern Cerrado and Caatinga (O. subflavus, Oryzomys Dikerman, A. W., and Yates, Y. (1995). Systematics of Oligoryzomys: sp.n.2, and Oryzomys sp.n.1) grouped with the most Protein electrophoretic analyses. J. 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