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. niti- dus, 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. 282 1055-7903/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. MOLECULAR PHYLOGENY OF Oryzomys BASED ON CYTOCHROME b 283 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 Sa˜o 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, Joa˜o 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 Sa˜o Paulo (Sa˜o Paulo, Brazil); MVZ to Museum of with 5:1 and 10:1 ratios of transition:transversion Vertebrate Zoology (Berkeley, CA); USNM to United rates.