The Simultaneous Diversification of South American Echimyid Rodents (Hystricognathi) Based on Complete Cytochrome b Sequences

MOLECULAR PHYLOGENETICS AND EVOLUTION

Vol. 5, No. 2, April, pp. 403–413, 1996 ARTICLE NO. 0035

The Simultaneous Diversification of South American Echimyid Rodents (Hystricognathi) Based on Complete Cytochrome b Sequences MA´RCIA C. LARA,*,† JAMES L. PATTON,*

AND

MARIA NAZARETH F.

DA

SILVA*,‡

*Museum of Vertebrate Zoology, University of California, Berkeley, California 94720; †Department of Zoology, University of Queensland, Brisbane, 4072, Australia; and ‡Departamento de Ecologia, Instituto Nacional de Pesquisas da Amazoˆnia, CEP 478, 69011, Manaus, AM, Brazil Received April 3, 1995; revised July 26, 1995

Variation in the complete nucleotide sequence of the mitochondrial cytochrome b gene was examined for 32 individuals representing 12 supraspecific taxa of South American rodents of the family Echimyidae (Hystricognathi). Representative genera of four other New World hystricognath families, the Old World porcupine Hystrix, and the myomorph murid rodents Rattus and Mus were used as outgroups in phylogenetic reconstructions. Monophyly of the family Echimyidae is strongly supported, a result fully consistent with existing morphological and paleontological data relative to the taxa examined. However, relationships among most supraspecific taxa within the family are poorly resolved. Poor resolution appears not to result from lack of data, but to a rapid, nearly simultaneous divergence of most Recent taxa. Generic groupings that are moderately to strongly supported include the tree rats of the Brazilian Atlantic Forest (Nelomys) and Amazonia (Echimys, Makalata) and the Amazonian arboreal spiny rats Mesomys and Lonchothrix. However, the two subgenera of the terrestrial spiny rats, Proechimys, do not form a monophyletic unit, and elevation of the Atlantic Forest Trinomys to generic status is supported. The genus Hoplomys is closely related to Proechimys (sensu stricto), a finding supported by other molecular data.  1996 Academic Press, Inc.

INTRODUCTION With 15 Recent genera and about 70 living species (Woods, 1993), the family Echimyidae is one of the most speciose of living caviomorph rodents (Patton and Reig, 1989), and the most morphologically and ecologically diverse of all rodent families in the suborder Hystricognathi. Members range from Central America to northern Argentina, where they inhabit lowland and montane tropical forests, seasonally deciduous forests, and tropical savanna. Taxa may be semifossorial (Clyomys,

Euryzygomatomys), terrestrial (Carterodon, Hoplomys, Proechimys, Thrichomys), or arboreal (Dactylomys, Diplomys, Echimys, Isothrix, Kannabateomys, Lonchothrix, Makalata, Mesomys, and Olallamys). Commonly known as spiny rats and tree rats, echimyids are moderate-sized, with a body weight ranging from about 200 to 1000 g. All members have coarse fur and many taxa possess flexible and flat spines distributed mostly on the back (Emmons and Feer, 1990). Fossil echimyids are diverse in Deseadan deposits (Late Oligocene, about 25 Myr ago; MacFadden, 1985) of Patagonia and Bolivia, which have long been considered the earliest record for New World hystricognaths (Wood and Patterson, 1959; Patterson and Wood, 1982). However, Wyss et al. (1993) recently reported the presence of caviomorphs (cf. Dasyproctidae) from the pre-Deseadan Tinguirica fauna of Chile, with dates ranging from late Eocene to early Oligocene (37.5 to 31.5 Myr). In this paper we examine phylogenetic relationships among 10 of the 15 Recent genera of echimyids listed in Woods (1993) based on analyses of their mitochondrial cytochrome b (cyt b) gene sequences.1 Woods (1993) divided the modern Echimyidae into five subfamilies, four Recent and one extinct. The monotypic Chaetomyinae (genus Chaetomys) has been considered either an erethizontid or echimyid, most recently as the former based on enamel ultrastructure (Martin, 1994). The Dactylomyinae contains the three genera of cane, or bamboo, rats (Dactylomys, Kannabateomys, and Olallamys), which have been suggested (Reig, 1986) to have a relationship to the Capromyidae rather than the Echimyidae. The Echimyinae minimally contains four genera (Diplomys, Echimys, Isothrix, and Makalata). Echimys is a complex and diverse taxon within which boundaries are poorly defined; some authors include 1 Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. L23335, L23339, L23341, L23355-6, L23359, L23362-3, L23385, L23388, L23395, U34850-U34858, U35165-U35173, and U35412-U35415.

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1055-7903/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Makalata within this genus while separating the Atlantic Forest Nelomys (e.g., Emmons and Feer, 1990). Echimys is probably composite, but it is unclear at present how member taxa should be aligned. The Eumysopinae is the most diverse subfamily, exhibiting a notable diversification by the upper Miocene of Argentina, and including the presumptively most primitive of living South American echimyids (Verzi et al., 1994; Vucetich and Verzi, 1993). Two of its eight genera, Mesomys and Lonchothrix, have the type of brachyodont and pentalophodont molars that are hypothesized to be the ancestral condition for the New World Hystricognathi (Reig, 1986; Wyss et al., 1993). The other eumyopsine genera are Carterodon, Hoplomys, Proechimys (including Trinomys), Clyomys, Eurozygomatomys, and Thrichomys. Finally, the Heteropsomyinae is comprised of several extinct genera of West Indian and mainland rats somewhat intermediate in morphology between other echimyids and capromyids (Woods, 1993). MATERIALS AND METHODS The complete cytochrome b sequence (1140 bp) was obtained for 32 individual echimyid rodents, including the Amazonian Echimys, Isothrix, Dactylomys, Makalata, Mesomys, and Proechimys (sensu stricto) and the Atlantic Forest Proechimys (sugenus Trinomys), Nelomys, Euryzygomatomys, and Thrichomys. Partial sequences were obtained for the genera Lonchothrix (810 bp) and Hoplomys (675 bp). Sequences from genera of five other hystricognath families [Myoprocta (Dasyproctidae), Coendou (Erethizontidae), Ctenomys (Ctenomyidae), Cavia (Caviidae; Ma et al., 1993), and Hystrix (Hystricidae; Ma et al., 1993)], as well as the myomorph murids Rattus (Gadaleta et al., 1989) and Mus (Bibb et al., 1981), were used in varying combinations as outgroups in phylogenetic analyses. Extraction and Amplification DNA was extracted by the NaCl method of Miller et al. (1988) from liver tissues that were fixed in alcohol or frozen initially in liquid nitrogen. The entire cytochrome b gene (1140 bp in length) was amplified separately for both double and single strands by the polymerase chain reaction, following a protocol developed by S. dos Reis and given in Patton et al. (1996). The light strand template was sequenced directly with either primers MVZ05-M13RSP or MVZ05, and with the internal primers MVZ65, MVZ19, MVZ23, MVZ41, and MVZ67 (see Smith and Patton, 1993; da Silva and Patton, 1993; and Patton et al., 1996, for primer sequences). Sequence Analyses Alignment and translation of the sequences used either BIONET (IntelliGenetics, Inc.) on a Sun Sparc station or DNAsis v1.0 on a Macintosh computer. Pairwise

comparisons of observed sequence difference, number of transitions and transversions by codon positions, codon and amino acid frequencies, nucleotide composition, and nucleotide frequencies by codon position used MOSY (MOlecular SYstematics program) written by Christopher A. Meacham (U.C. Berkeley, unpublished). Percentages of sequence divergence corrected for multiple hits were calculated by the method of Brown et al. (1982) for comparison of divergence levels within and among the taxa examined. Maximum likelihood distances (from PHYLIP 3.4, Felsenstein, 1991; and PHYLIP 3.5c, Felsenstein, 1993) were used to evaluate the presence of saturation for both transversions (TV) and transitions (TS) at each codon position. For these, the empirically observed 2: 1:15 ratio of rate of change among codon positions was specified. Phylogenetic analyses employed the principle of maximum parsimony, using the PAUP computer package (version 3.1.1, Swofford, 1993). Critical values of skewness ( g1 statistics) of the tree length distribution (Hillis, 1991; Hillis and Huelsenbeck, 1992) were used to test for phylogenetic signal (as opposed to random noise), from a distribution of 10,000 randomly generated trees using PAUP. Support for specific nodes in the most parsimonious tree(s) was assessed both by bootstrap analysis (with 100 replicates and 10 random sequence additions with each bootstrap replicate) and by calculation of decay (5 Bremer) indices (Bremer, 1988; Donoghue et al., 1992). Finally, the log-likelihood ratio test of Kishino and Hasegawa (1989, as implemented by the DNAML routine in PHYLIP 3.4) was used to test the validity of specific taxon associations, both those obtained in the parsimony analyses and those expected by current taxonomy. Parsimony Weighting Schemes and Outgroup Choice Based on empirical evidence for saturation for third position TS (see below), and to accomodate for the high proportion (93%) of cytosine to thymine (C-T) changes at first positions that specify different codons of leucine, we employed several weighting schemes in parsimony analyses. The most simple of these ‘‘corrected’’ for third position transition saturation either by (a) specifying no third position TS or (b) using a 10:1 TV to TS weight at third positions with all first and second position substitutions given weights of 10. The most complex weighting scheme employed either (a) no third position TS and no first position C-T or (b) a 10: 1 TV to TS weight for third positions, a weight of 10 for all second positions, and weight of 1 for all first position C-T changes with all others at this position weighted 10. The 10:1 third position TV to TS ratio was the empirically observed rate difference between those types of substitutions (see below). Since outgroup choice can effect the assumption of ingroup monophyly (Swofford and Olsen, 1990), we performed all analyses specifying either (a) only the murid rodents, Rattus and Mus; (b)

STAR-PHYLOGENY OF ECHIMYID RODENTS

under the assumption of the monophyly of the New World hystricognath rodents (but see Woods, 1993), Rattus, Mus, and the Old World porcupine Hystrix; and (c) all nonechimyid genera, respectively, as outgroup taxa. Finally, all analyses were performed on the complete data set of 38 hystricognath taxa plus Rattus and Mus as well as on a reduced data set which excluded all but a single individual of each specific taxon. The latter included 25 taxa, 18 echimyids plus the 7 nonechimyid taxa. Specimens Examined Taxon names follow Woods (1993), except where noted, although use of a particular name is not meant to confirm any published taxonomic judgment. Elevation of Trinomys to generic status, as well as the infrageneric classification of this genus, follows Lara (1994). Voucher specimens are deposited in the collections of the Instituto Nacional de Pesquisas da Amazoˆnia (INPA), Manaus; the Museu Nacional (MN), Rio de Janeiro; the Museum of Vertebrate Zoology, University of California, Berkeley (MVZ); or the National Museum of Natural History, Smithsonian Institution (USNM). All other prefixes correspond to field catalog numbers of specimens collected by A. Langguth (AL), A. L. Gardner (ALG), L. Geise (LG), Y. Yonenaga (BIO), E. A. Lacey (EAL), M. Lara (ML), Y. Leite (YL), M. Mustrangi (MM), P. L. B. da Rocha (PEU), J. L. Patton (JLP), J. R. Malcolm (JUR), L. H. Emmons (LHE), L. M. Pessoˆ a (LMP), M. N. F. da Silva (MNFS), and R. Cerqueira, Federal University of Rio de Janeiro (JDM, MAM, MS, SU, XI). These will be deposited in INPA (MNFS, JLP, JUR), MVZ (MNFS, JLP, EAL, MM), MN (AL, JDM, LG, ML, LMP, MAM, MS, SU, XI), the Museu Paraense Emilio Goeldi, Bele´m (MNFS, JLP, JUR), the Museu de Zoologia, Universidade de Sa˜o Paulo (BIO, PEU), or the National Museum of Natural History (ALG, LHE). Dactylomys boliviensis (N 5 1)—Brazil. Acre: left bank Rio Jurua´, Fazenda Santa Fe´ (5 Flora), 72°51′W 8°36′S (MNFS 988). Dactylomys dactylinus (N 5 1).— Brazil. Amazonas: Municı´pio Beruri, right bank Rio Purus (INPA 2477). Echimys chrysurus (N 5 1)—Brazil. Para´: right bank Rio Xingu, 59 km SSW Altamira, 52°22′W 3°39′S (USNM-LHE 555). Euryzygomatomys spinosus (N 5 1)—Brazil. Rio de Janeiro: Sumidouro (SU 73). Hoplomys gymnurus (N 5 1)—Colombia. Valle: 6 km N Buenaventura (MVZ 162309). Isothrix bistriata (N 5 1)—Brazil. Amazonas: alto Rio Urucu, 65°16′W 4°51′S (MNFS 97). Lonchothrix emiliae (N 5 1)—Brazil. Para´: Alter do Chao, Rio Tocantins (INPA 2472). Makalata didelphoides (N 5 4)—Brazil. Amazonas: Rio Jurua´ , near Miranda, 70°00′W 6°45′S (JLP 15214); Municı´pio Beruri, right bank Rio Purus (INPA 2474). Para´: right bank Rio Xingu, 59 km SSW Altamira, 52°22′W 3°39′S (USNM-LHE 595, 600). Mesomys hispidus (N 5 2)—Brazil. Amazonas: left bank Rio

405

Jurua´, Barro Vermelho, 68°46′W 6°28′S (MNFS 745); right bank Rio Jurua´, Penedo, 70°45′W 6°50′S (MNFS 436). Mesomys sp (N 5 2)—Brazil. Amazonas: left bank Rio Jurua´, Igarape´ Arabidi, Colocac¸ a˜o Vira-Volta, 66°14′W 3°17′S (JUR 501); alto Rio Urucu, 65°16′W 4°51′S (MNFS 201). Nelomys cf. braziliensis (N 5 1)— Brazil. Minas Gerais: Mocambinho, Municı´pio de Jaı´ba (LMP 27). Proechimys amphichoricus (N 5 1)—Venezuela. Amazonas: San Carlos de Rio Negro, ca. 4 km N Isla Sarama (USNM-ALG 14040). Proechimys simonsi (N 5 1)—Peru. Depto. Madre de Dios: Aguas Calientes, Rio Alto Madre de Dios, ca. 1 km below Shintuya (MVZJLP 11051). Trinomys albispinus (N 5 1)—Brazil. Sergipe: Fazenda Cruzeiro, 13 km SSE Cristina´polis (MNAL 3072). Trinomys setosus (N 5 3)—Brazil. Sergipe: Fazenda Cruzeiro, 13 km SSE Cristina´polis (AL 3054); Minas Gerais: 30 km E & 4 km N (by road) Rio Casca, 430 m (MN 31448); Mata da Prau´na, 5 km N Conceic¸a˜o do Mato Dentro (MN 31441). Trinomys paratus (N 5 1)—Brazil. Espirito Santo: Grota da Aracruz Florestal, 12 km E Aracruz, 40 m, 19°49′S 40°10′W (YL 34). Trinomys eliasi (N 5 1)—Brazil. Rio de Janeiro: Restinga de Marica´, Aeronautic Trail (MN-ML 141). Trinomys dimidiatus (N 5 4)—Brazil. Rio de Janeiro: Nova Friburgo (LG 3); Mata da Rifa, Paraque Estadual do Desengano, 1.7 km N & 5.1 km NE (by road) Santa Maria Magdalena, 790 m (MN 31413); Mambucaba, Angra dos Reis (MAM 10); Fazenda Inglesa, Petro´polis (JDM 3). Trinomys iheringi (N 5 1)—Brazil. Sa˜o Paulo: Ilha da Sa˜o Sebastia˜o, Fazenda da Toca, 2.4 km E & 0.8 km NE (by road) Ilha Bela (MM 55). Trinomys species 1 (N 5 1)—Brazil. Bahia: dunes of the Rio Sa˜o Francisco, Vila Bonfim, Ibiraba (PEU 880027). Trinomys species 2 (N 5 1)—Brazil. Bahia: Estac¸ a˜o Ecolo´gica Pau Brasil, 16 km E (by road) Porto Seguro (MN 31459). Thrichomys apereoides (N 5 3)—Brazil. Alagoas: Vale do Rio Sa˜o Francisco, Delmiro Gouveia, Xingo´ (XI 012i, born in captivity); Bahia: Santo Ina´cio (BIO 872); Mato Grosso do Sul: Ribas do Rio Pardo (MS 011i, born in captivity). Myoprocta pratti (N 5 1)—Brazil. Amazonas: right bank Rio Jurua´, Altimira, 68°54′W 6°35′S (JLP 15972). Coendou bicolor (N 5 2)—Brazil. Acre: left bank Rio Jurua´, Fazenda Santa Fe´ (5 Flora), 72°51′W 8°36′S (MNFS 1016). Amazonas: Rio Jurua´ , Eirunepe´, 60°52′W 6°38′S (MNFS 439). Ctenomys sociabilis (N5 1)—Argentina. Neuquen Province: Reserva Nacional Nahuel Huapi, Estancı´a Rincon Grande (MVZ-EAL 1). RESULTS AND DISCUSSION Echimyid Cytochrome b Gene Sequences Variation in amino acid composition. The translated cytochrome b gene is 379 amino acids in length, terminating with an AGA or AGG stop codon (1140 bp), in all echimyid taxa examined. The role of cyt b in elec-

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tron transport is well known in mammals, and its functional property constrains evolution at the amino acid level. It is not surprising, therefore, that the distribution of variable amino acids in the cytochrome b gene of echimyid rodents is nonrandom, with the most conserved regions located at the outer surface [11.8% of all variable amino acid positions in the entire gene (residues 136–178 and 253–288)] and the most variable regions in the transmembrane domain [58.1% of variable positions (residues 179–201, 230–252, and 349–371)]. This pattern of amino acid variation is in complete agreement with those obtained from other taxa of placental mammals (Edwards et al., 1991; Irwin et al., 1991; Martin and Palumbi, 1993). Base composition and sequence variation. Animal mitochondrial DNA is characterized by strand-specific base compositional biases both at different codon positions and within codons (Brown et al., 1982; Kocher et al., 1989; Irwin et al., 1991; Holmes, 1991; Martin and Palumbi, 1993). The base composition of the cyt b gene of echimyids does not differ from the patterns found in this gene for mammals in general (see Irwin et al., 1991; Ma et al., 1993). In the light strand sequence, there is a deficit of guanine residues (12.5%, on average across all taxa examined), adenine and thymine are the most common nucleotides (30.7 and 29.6%, respectively), and cytosine makes up 27.3% of the total. Although guanine is the least common nucleotide, its frequency differs greatly among the three codon positions, being present in 3.3% of the third positions, 13.7% of second positions, and 20.4% of first positions. First and third positions are richer in adenine (30.1 and 41.3%), and second positions have more thymine (40.7%). First positions of codons have a less biased composition, as expected since substitutions at this position result in more conservative amino acid replacements than those at second positions (Irwin et al., 1991). The opportunity for evolution at the nucleotide level differs among codon positions. Of the 1140 bp of the echimyid cyt b gene, 535 sites were variable, 450 of which were potentially phylogenetically informative changes (present in two or more taxa). Of the latter, 90 changes were at first positions, 33 at second positions, and 327 at third positions. The evolutionary constraints at the amino acid level are reflected in the distribution of nucleotide variation along the gene. Because variation is not random along the gene, it is expected that some regions will contain more phylogenetic information than others. We examined the pattern of variability along the cyt b gene as a moving window of seven amino acid positions, beginning with the start codon. The numbers of phylogenetically informative nucleotide substitutions were scored for each seven-codon block and apportioned to first, second, and third positions in each codon. Groups of nucleotide sites

that contain most phylogenetically informative substitutions at first positions are from sites 693 to 735 and 903 to 942; those for second positions are likewise from site 693 to 735 as well as between 1029 and 1137. Third position informative sites are scattered rather evenly across the entire gene sequence. Importantly, the number of potentially informative sites along the last third of the cyt b gene is nearly double that along the first two-thirds. Since most of the changes at the third position result in no amino acid substitutions (silent changes), third positions are more free to vary and, as a consequence, change faster. Transitions are more frequent than transversions, both in tRNA and in protein-coding genes, which may reflect a bias in the mutation process (Brown et al., 1982). However, multiple transitions at a single site will appear as if only a single transition had occurred, and a single transversional event will erase the record of all previous transitions at that site. Since transversional history is not obscured by subsequent transitions in the same position, through time the observed frequency of transitions is expected to decrease relative to the observed number of transversions, even though the actual total number of transitional changes is much higher. We examined the empirical relationship between TS and TV at each codon position relative to the maximum likelihood distances among taxon haplotypes to determine the degree of saturation for both types of changes. Relationships between the number of either TS or TV with distance are rather linear for both first and second positions. At third positions, however, transitions increase rapidly and reach an asymptote at distances above 0.2, while transversional changes remain relatively linear (Fig. 1). Comparisons of the slopes derived by fitting a straight line before the inflection point (before saturation) give a 10: 1 transition to transversion ratio for third positions. Levels of Sequence Divergence A matrix of the percent sequence divergence (corrected for multiple hits by the method of Brown et al., 1982), among all taxa of echimyids and the other hystricognath outgroups, is given in Table 1. Divergence values among conspecific individuals are always relatively low, at least in comparison to intergeneric comparisons. Nevertheless, several of what are currently considered single species are composed of quite differentiated geographic units. For example, the three individuals of Thrichomys apereoides, sampled across a 2000-km range, differ by an average of 8.4% among themselves, and specimens of the Amazonian Makalata didelphoides, also separated by some 2000 km, differ by an average of 19.3% (see also da Silva and Patton, 1993). Similarly, the various species of Trinomys, as currently recognized (Moojen, 1948; Pessoˆa and dos Reis, 1991,

STAR-PHYLOGENY OF ECHIMYID RODENTS

407

bamboo rat Dactylomys are most similar (8.8% divergence), with those of the spiny tree rat Mesomys next (15.3%, Table 1; see also da Silva and Patton, 1993). On the other hand, species within each of the two genera of terrestrial spiny rats, Proechimys and Trinomys, are quite divergent. The Amazonian P. simonsi differs from P. amphichoricus by 19.7%, and those of the Atlantic Forest Trinomys differ by an average of nearly 20% (see Lara, 1994, for details). These values are about as great as are those between some genera, which range from 19.9% (Amazonian Echimys and Atlantic Forest Nelomys) to 40.0% (Mesomys and Euryzygomatomys). The average degree of sequence divergence among all genera of echimyids is substantial, at 31.9% (SD 3.08). Phylogenetic Analysis of the Echimyidae

FIG. 1. Relationships between the number of transitions (above) and transversions (below) at each codon position and the maximum likelihood distance between all pairs in the comparisons of the complete cytochrome b sequences of echimyid and nonechimyid hystricognath rodents (Rattus and Mus excluded). Lines are fitted by regression analysis and are for visualization only.

1992a,b, 1993, 1994), are individually composed of quite divergent cyt b haplotypes. Again, for example, samples allocated to the broadly distributed T. iheringi (see Pessoˆa and dos Reis, 1994) differ by an average of 19.5% (see Lara, 1994, for details of within-Trinomys variation). Apart from these cases, however, sequences from multiple individuals of single species are otherwise quite similar, exhibiting maximum divergence values of 4 to 5%. We will not consider relationships among conspecific samples further, except to note that the levels of sequence divergence in the examples cited immediately above suggest these ‘‘species’’ are probably composite. The degree of divergence among species within given genera also spans a large range. The two species of the

For the sake of simplicity, phylogenetic analyses presented below are based on a reduced data set composed of 18 echimyids and 7 nonechimyid genera. Analyses based on the entire data set did not differ qualitatively either in tree topology or in relative branch lengths. The 18 echimyids included each of the currently recognized species examined [with the exception of Trinomys, for which only one of each of the three haplotype clades identified by Lara (1994) was used] as well as the highly divergent haplotypes uncovered within currently recognized single species (e.g., Makalata didelphoides). The different weighting schemes also produced qualitatively similar ingroup topologies, tree statistics, and relative branch lengths regardless of whether all 7 nonechimyids or only Rattus and Mus were designated as outgroups. Consequently, only results that ignored first position C-T changes and all third position transitions with Rattus and Mus as outgroups are presented. Since only limited data were available for Lonchothrix (810 bp) and Hoplomys (675 bp), the phylogenetic placement of these taxa was based on separate analyses. Assessment of phylogenetic information. The strength of phylogenetic signal in the data set was assessed from the distribution of the lengths of a sample of random trees, following Hillis (1991) and Hillis and Huelsenbeck (1992). Significant skewness can result from the inclusion of only one pair of strongly related taxa, thus giving false confidence to the measured phylogenetic signal. Consequently, skewness was investigated using subsets of taxa that represent all hierarchical levels in the tree (see Schneider, 1993). An initial analysis contained all sequences, but subsequent ones prunned taxa sequentially until only those representing the outgroups remained. The minimum corrected sequence divergence for any pair of taxa in these analyses was 23.5%, a degree of divergence unlikely to be solely responsible for strong signal. Tree length distributions were analyzed under four specifications of character state change: (1) considering all characters

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TABLE 1 Estimated Percentage Nucleotide Sequence Divergence among Pairwise Comparisons of Taxa Based on Mitochondrial Cytochrome b Sequences HYS COE CAV MYO

CTE DAC-d DAC-b ECH EUR

ISO

44.4 52.8 48.9 57.1 —

MAKw MAKe MES-s MES-h LON

NEL PRO-a PRO-s HOP THR TRI-a TRI-s TRI-i

I HYS COE CAV MYO CTE

—

43.9 0.4

56.6 49.9 —

56.7 48.9 44.4 —

46.1 46.1 52.1 54.0 43.6

44.9 45.0 53.3 49.9 40.1

47.7 48.2 51.2 53.2 41.7

50.6 47.7 51.6 51.2 47.2

50.5 47.1 51.6 53.6 46.2

55.1 53.8 51.1 58.5 44.1

50.0 48.8 48.8 55.4 45.4

51.2 43.9 45.2 48.3 38.4

51.0 46.1 49.9 46.1 42.2

52.6 52.6 43.0 52.0 39.7

47.2 47.7 51.6 54.7 40.7

54.2 48.3 52.8 46.6 44.0

47.2 48.3 47.1 45.5 44.1

53.5 45.8 50.5 49.5 43.1

51.4 43.7 45.6 46.7 44.8

46.2 42.3 47.3 50.6 40.1

46.6 42.8 47.1 48.9 43.5

43.0 45.5 44.0 48.4 38.1

—

8.8 —

26.8 26.7 —

34.4 35.3 31.0 —

29.5 28.8 29.2 34.6 —

28.8 28.7 29.5 35.4 30.7 5.6

28.5 27.4 25.4 32.9 31.9 20.7 8.9

30.8 31.9 30.8 38.4 33.0 35.7 33.2 3.7

31.5 30.5 29.3 40.0 30.7 33.9 32.0 15.8 1.4

27.8 28.1 29.6 32.5 27.6 26.2 26.3 23.2 21.4 —

27.9 26.6 20.2 33.3 29.5 30.5 25.0 31.6 30.9 30.3 —

36.8 34.4 36.3 36.3 36.2 39.0 39.0 39.0 36.2 33.0 35.4 —

34.5 32.2 34.0 31.9 32.8 35.3 32.4 36.4 33.2 29.6 34.9 20.3 —

35.4 33.5 31.0 30.7 32.1 39.6 33.6 30.9 29.3 30.1 32.3 29.1 25.5 —

36.4 36.6 31.6 34.4 30.1 36.5 35.5 32.3 33.6 31.8 34.7 32.3 31.4 27.1 —

27.2 27.7 25.3 30.9 30.0 32.9 29.9 30.4 31.1 28.1 32.6 30.9 30.9 33.3 30.8 —

28.3 30.8 30.6 27.8 30.8 35.1 29.2 30.2 30.8 32.3 30.8 29.7 29.7 30.4 29.2 23.5 14.1

28.6 29.2 31.6 28.2 29.3 32.6 32.4 31.7 28.8 31.9 31.0 30.1 30.1 30.5 29.8 26.5 24.4 8.7

II DAC-d DAC-b ECH EUR ISO MAK-w MAK-e MES-sp MES-h LON NEL PRO-a PRO-s HOP THR TRI-a TRI-s TRI-i

Note. Divergence values are corrected for multiple hits by the method of Brown et al. (1982). Comparison with Lonchothrix are based on 810 bp; those with Hoplomys on 675 bp. Values on the diagonal are those between multiple sequences of the same species or species clade (see text). Taxon abbreviations: I. Nonechimyids: HYS, Hystrix africaeaustralis; COE, Coendou bicolor; CAV, Cavia porcellus; MYO, Myoprocta pratti; CTE, Ctenomys sociabilis. II. Echimyids: DAC-d, Dactylomys dactylinus; DAC-b, Dactylomys bolivianus; ECH, Echimys chrysurus; EUR, Euryzygomatomys spinosus; ISO, Isothrix bistriata; MAK-w, Makalata didelphoides (Rio Jurua´ ); MAK-e, Makalata didelphoides (Rio Xingu); MES-sp, Mesomys sp.; MES-h, Mesomys hispidus; LON, Lonchothrix emiliae; NEL, Nelomys brasiliensis; PRO-a, Proechimys amphichoricus; PRO-s, Proechimys simonsi; HOP, Hoplomys gymnurus; THR, Thrichomys apereoides; TRI-a, Trinomys albispinus; TRI-s, Trinomys setosus; TRI-i, Trinomys iheringi.

as unordered, (2) using only third position transitions, (3) including all substitutions except third position transitions, and (4) weighting third position transversions ten times transitions. The results are presented in Table 2. When all characters and sequences are considered, there is a highly significant skew in the tree-length distribution ( g1 5 20.911; P , 0.01) indicating that the information content is significantly more structured than are random data (Hillis and Huelsenbeck, 1992). The most parsimonious tree with all characters unordered (3583 steps) was over 1300 steps shorter than the shortest one obtained by the random generation method (4907 steps). Not surprisingly, third position transitions, which are the fastest type of change and reach saturation quickly, show phylogenetic signal mainly among the terminal relationships (closely related taxa) and lose utility among more distant relationships (Table 2). When third position transitions are excluded, or when a 10: 1 stepmatrix is applied to weight transversions relative to transitions, significant phylogenetic signal is found among the deepest nodes of the tree as well as more terminal relationships.

Phylogenetic relationships among echimyid rodents. A heuristic search with 100 replicates of random addition of taxa, and using the specified weighting scheme, resulted in a single most parsimonious tree 1201 steps in length (Fig. 2). Monophyly of the family Echimyidae is supported by a decay index of 26 (that is, the shortest tree without echimyids as a monophyletic group is 26 steps longer than the most parsimonious one) and a bootstrap value of 88%. Consequently, the monophyly of this family appears well supported. There is, however, surprisingly little support for any relationship at the deeper nodes linking most of the echimyid taxa. Decay indices at all basal nodes within the Echimyidae are quite small, with most being 1, the minimum possible number (Fig. 2). With three exceptions, all other supraspecific taxa, including each of the lineages of Atlantic Forest Trinomys (discussed below), collapse to a basal polytomy in the bootstrap analysis (i.e., all pairings have bootstrap values less than 50%; Fig. 2). The exceptions are those of the arboreal spiny rats Mesomys and Lonchothrix (bootstrap 5 82; decay index 5 11), the tree rats Echimys and Nelomys (bootstrap 5 94; decay index 5 8) coupled with Makalata

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STAR-PHYLOGENY OF ECHIMYID RODENTS

TABLE 2 Phylogenetic Signal Using Taxa to Represent Different Levels of Divergence and with Different Types of Character State Changes, as Indicated by the g1 Statistics of Tree-Length Distributions Using 10,000 Random Trees Type of character

Taxa included

All unordered

No third position TS

10 :1 TV :TS

Only third, position TS

All sequences TRI-a, TRI-s, TRI-i, PRO-s, DAC-b, ECH, MAK-w, ISO, MES-s, THR 1 outgroups TRI-a, TRI-s, TRI-i, PRO-s, ECH, ISO, MES-s, THR 1 outgroups TRI-a, ECH, MES-s, THR 1 outgroups TRI-a, MES-s, THR 1 outgroups TRI-a, THR 1 outgroups TRI-a 1 outgroups Outgroups only

20.911** 20.717** 20.408** 20.248** 20.266** 20.212ns 20.188ns 20.114ns

20.655** 20.612** 20.548** 20.382** 20.486** 20.508** 20.518** 20.291ns

20.762** 20.668** 20.609** 20.500** 20.461** 20.482** 20.389** 20.036ns

20.613** 20.362** 20.083ns 20.089ns 20.057ns 20.025ns 20.013ns 20.051ns

Note. ( ns ) Nonsignificant; (**) P , 0.01. Taxon abbreviations are as in Table 1. Outgroups included Rattus, Hystrix, Coendou, Cavia, Myoprocta, and Ctenomys.

FIG. 2. Phylogenetic relationships among 12 genera and 18 species of echimyid rodents based on complete cytochrome b sequences (1140 bp), performed with 100 replicates of random addition of taxa and a weighting scheme that ignored first position C-T changes and all third position transitions (see text). Species representing the South American hystricognath families Ctenomyidae (Ctenomys), Caviidae (Cavia), Dasyproctidae (Myoprocta), Erethizontidae (Coendou), as well as the Old World Hystricidae (Hystrix) are included; the murid rodents Rattus and Mus were used as outgroups. (Left) The single maximum parsimony tree generated from a heuristic search (length 5 1201 steps; CI 5 0.563; RI 5 0.507). Numbers at nodes are decay indices, the number of additional steps before a particular nodes collapses. (Right) Bootstrap analysis of the complete cytochrome b sequences, with bootstrap values above 50% indicated at all nodes; those below 50% are indicated by polytomies. Values below the branches are average corrected percent sequence divergences (Brown et al., 1982) for the terminal members of that branch relative to all other echimyid taxa. The decay indices, bootstrap values, and positions of Lonchothrix and Hoplomys (indicated by dashed lines in both trees) are based on separate analyses with 810 and 675 bp, respectively.

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(bootstrap 5 57; decay index 5 6), and the terrestrial spiny rats Proechimys and Haplomys (bootstrap 5 84; decay index 5 8). Systematic conclusions stemming from these observations are directed to the following considerations: (1) Monophyly of Trinomys and Proechimys. These two taxa are usually included as subgenera in the genus Proechimys (Moojen, 1948; Woods, 1993). If this is correct, they should form a monophyletic unit. Three major clades within the Atlantic Forest terrestrial spiny rats, Trinomys, are identified (see Lara, 1994, for details), each of which is highly divergent (average of 25.3%; Table 1). These taxa are neither demonstrably monophyletic among themselves nor with the two species of Amazonian Proechimys (simonsi and amphichoricus). However, there is no statistical difference between tree topologies that treat Trinomys as a monophyletic clade compared to those depicting it as paraphyletic relative to other echimyid taxa, based on log-likelihood tests (P . 0.05). Similarly, these tests also fail to distinguish between trees depicting either a genus Proechimys (including Trinomys) as monophyletic to the exclusion of other echimyids, or a genus Proechimys as paraphyletic (P . 0.05 in all cases). Failure of log-likelihood tests to differentiate between these alternative sets of relationships is not surprising, given the similar overall levels of sequence divergence and general lack of any resolution among most supraspecific taxa of echimyids (Fig. 2). However, even if future studies indicate monophyly between Trinomys and Proechimys, the relationships between them must remain quite deep and near the base of the radiation of the modern genera. Consequently, much of the general morphological similarity that underlies their current taxonomy likely results from the retention of ancestral, pleisiomorphic characters. To continue to recognize Trinomys as a subgenus of Proechimys would thus be misleading, and we argue for generic separation. (2) Monophyly of Proechimys and Hoplomys. Although fewer data are available for the terrestrial spiny rat Haplomys (675bp), the sister relationship of this genus with Proechimys is well supported (bootstrap value 5 84; decay index 5 8). However, the divergence between these taxa is large, averaging 27.3%, eventhough divergence between species within Proechimys is nearly 20% (Table 1). These analyses are insufficient to examine the suggestion stemming from protein electrophoresis indicating that Proechimys is paraphyletic relative to Hoplomys and thus that the two genera should be combined (Patton and Reig. 1989). (3) A Mesomys and Lonchothrix clade. These two arboreal spiny rats share many similar morphological features of the skin and skull, and a close relationship has been recognized by most previous authors (Ellerman, 1940; Emmons and Feer, 1990; Moojen, 1952). This relationship is well supported by the available cy-

tochrome b sequence data. Over the 810 bp compared, these two genera differ by only 22.3% corrected sequence divergence, on average, in comparison to the 15.8% degree of differentiation between the two species of Mesomys (Table 1). All phylogenetic analyses place them in a sister relationship, with a bootstrap of 82 and decay index of 11. The possible basal position of the two genera relative to the subfamilies Echimyinae and Eumysopinae (Patton and Reig, 1989; Vucetich and Verzi, 1991) cannot be evaluated with the cyt b data. (4) The Makalata, Echimys, and Nelomys clade. The monophyly of Echimys and Nelomys is well supported, with a bootstrap of 96% and a decay index of 8 (Fig. 2). Moreover, their degree of corrected sequence divergence (19.9%; Table 1) is the lowest for any intergeneric comparison and virtually the same as the between species within well-supported supraspecific taxa, such as Proechimys, for example (19.7%). Makalata is marginally supported as the sister taxon to Echimys 1 Nelomys by a decay index of 7 and a bootstrap value of 57 (Fig. 2). Based on log-likelihood tests, both Echimys or Makalata are equally likely to be the sister taxon to Nelomys. However, there is a statistical difference between which taxon is the sister to Makalata, since the tree depicting Echimys 1 Makalata is significantly worse (P , 0.05) than those depicting Echimys 1 Nelomys as the sister to Makalata or Echimys as the sister to Makalata 1 Nelomys. Husson (1978) erected the genus Makalata as distinct from Echimys, while Emmons and Feer (1990) argued that the Atlantic Forest Nelomys comprised a phylogenetic unit separate from the Amazonian Echimys, in which they included Makalata. Phylogenetic analysis of the cyt b sequences offers some resolution to this conflict of opinion. If the branching relationships between these three entities remain as depicted in Fig. 2, then the choices as to their classification are limited to the following: (1) include all in the same genus (Echimys); (2) give separate generic status to all three; or (3) separate Makalata from an Echimys that would include both Amazonian and Atlantic Forest representatives. Separation of Echimys and Nelomys to the exclusion of Makalata cannot be supported as it would make the expanded Echimys paraphyletic. However, both the Atlantic Forest Nelomys and Amazonian Echimys are speciose taxa composed of several highly divergent morphological entities. Additional samples of this diversity may alter topological relationships of those taxa examined here, and thus require other taxonomic solutions. (5) Monophyly of the extant subfamilies Dactylomyinae, Echimyinae and Eumysopinae. None of the currently recognized subfamilies of echimyids can be supported by phylogenetic analysis of the cyt b data, although in some cases neither can their monophyly be firmly rejected. For example, members of the Eumysopinae (including Euryzygomatomys, Mesomys, Proechi-

STAR-PHYLOGENY OF ECHIMYID RODENTS

mys, Trinomys, and Thrichomys) clearly do not comprise a monophyletic assemblage (Fig. 2). This may not be surprising, since the subfamily is recognizable in the Miocene fossil record prior to the presumptive origin of the Echimyinae and Dactylomyinae (Verzi et al., 1995). Nevertheless, the log-likelihood topology with the Eumysopinae monophyletic is not significantly worse than that of the most parsimonious tree depicting subfamily members as paraphyletic (P . 0.05). Three of the four sampled genera of the Echimyinae (Echimys, Nelomys, and Makalata) belong to a single clade in the most parsimonious tree, but their purported sister-taxon is the dactylomyine Dactylomys, not to the fourth echimyine Isothrix (Fig. 2). As with the Eumysopinae, there is also no statistical difference between either the paraphyly or monophyly of the subfamily Echimyinae (P . 0.05). While the status of the Echimyinae remains debatable, if the relationship of Dactylomys within this group (as depicted in Fig. 2) remains intact with additional data, then the concept of a subfamily Dactylomyinae will lack validity. Although we have not examined sequences for members of the family Capromyidae, the suggestion of Reig (1986) that the Dactylomyinae might have a closer relationship with that family than to the Echimyidae is not supported by the cyt b data (unless the capromyids actually comprise a clade with a relationship to the bamboo and tree rats within the Echimyidae). Do Extant Echimyids Represent a Star-Phylogeny? The lack of resolution for relationships among the genera and subgenera of echimyids is due, in part, to their very high and rather similar levels of sequence divergence (Fig. 2). Does this reflect simply the poor resolving power of the cytochrome b gene sequences (i.e., the molecule is too constrained to provide phylogenetic resolution at deep nodes) or was the divergence of the modern supraspecific taxa of echimyid rodents essentially simultaneous (i.e., a true ‘‘star-phylogeny’’)? The star-phylogeny hypothesis is potentially falsifiable with additional data (both sequences and taxa), but it is supported by three lines of current evidence. For one, while there is virtually no phylogenetic resolution among genera of echimyids, monophyly of the family is well supported relative to other caviomorphs. The decay index of 26 for the node unifying all echimyids is the highest value for any on the tree, including those that link conspecific taxa (Fig. 2). The bootstrap value of 88 at this node is likewise one of the highest on the tree. Indeed, there is both stronger decay index and bootstrap support for all nodes linking the various nonechimyid taxa at the base of the entire tree than there is between nearly all echimyid genera. Secondly, strong decay index and bootstrap support is also present for several terminally placed taxa, so that weak support is found only in the region of the tree reflecting intergeneric relationships. Finally, all tree-length distribu-

411

tions generated for the weighted character analysis are strongly and significantly skewed, indicating high phylogenetic linformation content in the data for each analysis (Table 2). Because these analyses were done stepwise at successively deeper nodes by removing taxa with similar sequences at the terminal twigs, significant skew is not an artifact of the inclusion of single pairs of related sequences. The sparse and spotty fossil record for echimyids offers inadequate, but tantalizing support for the starphylogeny hypothesis for living members of the family. Although the family is old, with members recognized from the Oligocene (Wood and Patterson, 1959; Patterson and Wood, 1982), the diversification of modern taxa apparently did not begin until the late Miocene (Verzi et al., 1994, 1995). Of the taxa sampled herein, these authors recognize Euryzygomatomys and Thrichomys as living members of that radiation, but note that the changing climates of that time likely generated cladogenic events that also led to the rich diversity of the more tropical members of the modern family. In this context, even if phylogenetic position cannot be established with the cyt b data, it is noteworthy that Euryzygomatomys is the most basal echimyid genus in the parsimony tree, with the highest sequence divergence relative to all other genera examined (Fig. 2). Thus, the extant members of the family Echimyidae may well represent a star-phylogeny, with an origin in the late Miocene resulting in a set of polytomous relationships reflecting that cladogenetic history rather than to inadequate data. True polytomous relationships, as opposed to demonstrably dichotomous ones, are an expected outcome of rapid and near-simultaneous divergence of multiple lineages. As a consequence, resultant taxa are likely to be composites of sharedprimitive and uniquely derived characters, and relationships based on any character set will be difficult to establish. These features seem to fit well with the complexities of extant echimyid rodent taxa. ACKNOWLEDGMENTS We are especially grateful to those individuals who helped procure many of the specimens used in this report, specifically Rui Cerqueira, Louise Emmons, Gustavo da Fonseca, Monica Fonseca, Alfred Gardner, Lena Geise, Erika Hingst, Yuri Leite, Jay Malcolm, Meika Mustrangi, Alexandre Percequillo, Leila Pessoˆa, and Pedro da Rocha. Permits for field work were provided by agencies of the Brazilian and Peruvian governments, to whom we are thankful. Anne Walton and Guiomar Vucetich reviewed the manuscript and offered extremely helpful insights on the echimyid fossil record; they, Chris Schneider, Margaret Smith, and two anonymous reviewers greatly improved our efforts, although they should not be held responsible for the final product. Financial support for field work was obtained from the National Geographic Society, the Museum of Vertebrate Zoology, and the Wildlife Conservation Society. Laboratory analyses were funded by a grant from the National Science Foundation (to J.L.P). Both M.C.L. and M.N.F.S. were supported by fellowships from the Brazilian Conselho Nacional de Desenvolvimento Cientı´fico e Technolo´gico (CNPq).

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