Molecular systematics of the South American caviomorph rodents: relationships among species and genera in the family Octodontidae

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 26 (2003) 476–489 www.elsevier.com/locate/ympev

Molecular systematics of the South American caviomorph rodents: relationships among species and genera in the family Octodontidae Rodney L. Honeycutt,a,b,* Diane L. Rowe,b and Milton H. Gallardoc b

a Faculty of Genetics, Texas A&M University, 2258 TAMUS, 210 Nagle Hall, College Station, TX 77843-2258, USA Department of Wildlife and Fisheries Sciences, Texas A&M University, 2258 TAMUS, 210 Nagle Hall, College Station, TX 77843-2258, USA c Instituto de Ecolog
ıa y Evoluci
on, Universidad Austral de Chile, Casilla 567, Valdivia, Chile

Received 13 March 2002; received in revised form 13 June 2002

Abstract Nucleotide sequences from mitochondrial (12S rRNA) and nuclear (growth hormone receptor) genes were used to investigate phylogenetic relationships among South American hystricognath rodents of the superfamily Octodontoidea, with special emphasis on the family Octodontidae. Relationships among most taxa were well resolved by a combined analysis of both genes, and the molecular phylogeny was used to address several long-standing phylogenetic problems. The family Abrocomidae was the most basal lineage within the superfamily Octodontoidea, sensu stricto, and the family Ctenomyidae was sister to the family Octodontidae, followed by a monophyletic group containing the families Myocastoridae and Echimyidae. A basic dichotomy was observed within the family Octodontidae. The Argentine desert specialists, Tympanoctomys and Octomys, grouped separate from Octodontomys, which was sister to a clade containing a monophyletic Octodon and a clade represented by species of Aconaemys and Spalacopus. Aconaemys was paraphyletic relative to Spalacopus. The phylogeny was used as an interpretive framework for an examination of variation in several non-molecular characters. The primitive diploid number for most of the octodontoids was determined to be between 46 and 56, and the primitive genome size 8.2 pg. Members of the Octodontidae appeared to be derived from an ancestral stock occupying lower elevations in scrub habitat. Furthermore, estimates of divergence time from the molecular data provided a temporal perspective for changes in plant communities, which demonstrated turnover and diversification in response to climatic and geologic events occurring in the Miocene through the Pleistocene. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Caviomorph rodents; Octodontidae; Molecular phylogeny; 12S rRNA; Growth hormone; Molecular clocks

1. Introduction The evolutionary history of South American flora and fauna is one characterized by isolation, extinction, and adaptive radiations (Marshall et al., 1982; Solbrig, 1976; Vrba, 1993). The South American hystricognath rodents (e.g., caviomorphs) are the result of an extensive Oligocene–Miocene radiation into an essentially vacant herbivorous niche (Mares and Ojeda, 1982). Representative taxa from the 14 extant families occupy nearly every major habitat in South America, including the high Andes. Phylogenetic information regarding relationships among families and genera of caviomorph * Corresponding author. Fax: 1-979-845-4096. E-mail address: [email protected] (R.L. Honeycutt).

rodents is scant and varied. For example, several superfamilies have been recognized, but designation of members to each superfamily has varied with the morphological characters selected for assessment (Patterson and Wood, 1982; Simpson, 1945; Woods, 1982). The Octodontoidea, containing the families Capromyidae, Ctenomyidae, Echimyidae, Myocastoridae, and Octodontidae, is probably the least contested monophyletic superfamily (Bugge, 1985; George and Weir, 1974; Nedbal et al., 1994; Sarich and Cronin, 1980; Woods, 1982; Woods and Hermanson, 1985). Nevertheless, except for the families Ctenomyidae and Echimyidae, which have received considerable attention as a result of their extensive chromosomal, morphological, and biochemical genetic variation (Gallardo and Palma, 1992; Patton et al., 1994; Reig et al., 1992), relationships

1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1055-7903(02)00368-8

R.L. Honeycutt et al. / Molecular Phylogenetics and Evolution 26 (2003) 476–489

among most octodontoid families and genera are still poorly resolved. From an evolutionary and ecological standpoint, the family Octodontidae is of particular interest. This family contains nine species assigned to four monotypic and two polytypic genera (Redford and Eisenberg, 1992), distributed in southwestern Peru, Chile, Argentina, and southwestern Bolivia (Contreras et al., 1987; Redford and Eisenberg, 1992). Seven of the nine species occur either exclusively or predominantly in Chile. Although the family is not speciose, it does contain an ecomorphologically diverse group of taxa ranging from above ground generalists to subterranean specialists (Contreras et al., 1987; Mares and Ojeda, 1982). There appears to be a geographic subdivision within the family, with generalists and fossorial forms occupying central Chile west of the Andes and more xeric adapted species residing in Argentina, southern Bolivia, and northeastern Chile (Contreras et al., 1987). Chromosomal diversity within the family is extensive, ranging from a diploid number (2N) of 38 and fundamental number (FN) of 64 for Octodontomys gliroides to a 2N ¼ 102 and FN ¼ 198 for Tympanoctomys barrerae (Gallardo, 1992). There is evidence from differential staining of chromosomes and estimates of total genome size that T. barrerae is potentially a polyploid (Gallardo et al., 1999). Monophyly of the family Octodontidae is well supported by unique figure eight-shaped molars (Reig et al., 1990), blood proteins (Woods, 1982), DNA reannealing (Gallardo and Kirsch, 2001), allozymes (K€ ohler et al., 2000), and nucleotide sequences (Nedbal et al., 1994). Nevertheless, relationships among genera and species within the family Octodontidae are not well resolved (Mares and Ojeda, 1982). In this paper we use nucleotide sequence data from both nuclear (growth hormone receptor; GHR) and mitochondrial (12S rRNA) genes to examine phylogenetic relationships among all genera and species within the caviomorph family Octodontidae. This molecular-based phylogeny is used to examine patterns of ecological, genetic, and geographical variation within the family.

477

somal RNA (12S rRNA) gene (Table 1). These individuals represent all nine species of the family Octodontidae as well as four additional families (Abrocomidae, Ctenomyidae, Echimyidae, and Myocastoridae) within the superfamily Octodontoidea. Representatives from other caviomorph superfamilies served as outgroup taxa, including a caviid, an agoutid, an erethizontid, a chinchillid, and a dinomyid. When possible, both genes were sequenced from the same individual. The use of different individuals from a species did not compromise comparisons, because intraspecific variation for both genes was low (an average of 0.1% for GHR and an average of 0.3% for 12S rRNA). All taxa examined are listed in Table 1. 2.2. Nucleotide sequencing The complete 12S rRNA gene, representing approximately 1100 base pairs (bp), was amplified with the polymerase chain reaction (PCR) and two oligonucleotide primers (50 –30 ), L651: CATAGACACAGAGG TTTGGTCC and 12GH: TTTCATCTTTTCCTTG CGGTAC. In addition to these external primers, several internal primers were used to sequence both strands. These internal primers are reported in Nedbal et al. (1994). All heavy (H) or light (L) strand primer designations are to positions in the mouse mitochondrial genome (Bibb et al., 1981). PCR conditions are similar to those described in Nedbal et al. (1994, 1996). Exon 10 (888 bp) of GHR was PCR amplified with primers and conditions as reported in Rowe and Honeycutt (2002). Amplification products were cleaned using a Qiagen QIAquick PCR purification kit, and concentration of the product determined on an agarose gel containing a concentration standard. PCR products were subsequently sequenced using an ABI PRISM (Applied Biosystems, Perkin–Elmer Cetus, Norfolk, CT) dyeterminator cycle-sequencing kit. Excess dye and primers were removed from the samples on G-50 Sephadex spin columns, before being applied to an Applied Biosystems 377 automated sequencer (Foster City, CA). 2.3. Data analysis

2. Materials and methods 2.1. Specimens examined Mitochondrial DNA (mtDNA) and nuclear DNA were isolated from liver, kidney, or heart tissues. Mitochondrial DNA was purified using cesium chloride/ propidium iodide gradient centrifugation (Brown, 1980), and total genomic DNA was isolated using the method of Sambrook et al. (1989). Nucleotide sequence data were obtained from 23 individuals for exon 10 of the nuclear growth hormone receptor (GHR) gene and 32 individuals for the complete mitochondrial 12S ribo-

Sequences were aligned using Clustal W (Thompson et al., 1992), followed by visual inspection. In the case of 12S rRNA genes, a secondary structure model (Springer and Douzery, 1996) also was used for both alignment and the determination of stem and loop regions. Some regions of the12S rRNA gene were highly variable as a result of insertion/deletion (indel) events, thus making unambiguous alignment difficult. These ambiguous regions were excluded from any further analysis and included positions 79–88, 119–126, 224–239, 907–955, 1018–1025, 1026–1032, 1035–1049, and 1099–1135. For phylogenetic analyses with maximum parsimony (MP),

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Table 1 Specimens examined Species

Locality

Sourcea

Accession Numbersb 12S rRNA

GHR

Octodontoidea Family Abrocomidae Abrocoma cinerea

Bolivia: 8.5 km W San Andreas de Machaca

NK 30665

AF520666

AF520643

Family Ctenomyidae Ctenomys steinbachi

Bolivia: Santa Cruz Department; 2 km S Caranda

NK 15277

AF520667

AF520656

Family Echimyidae Hoplomys gymnurus

Brazil: 52 km SSW Altamira

AK 9671

AF520668

AF520661

Family Myocastoridae Myocastor coypu

USA: Texas, Lubbock, Lubbock

TK 23244

AF520669

AF520662

Family Octodontidae Aconaemys fuscus Aconaemys fuscus Aconaemys sagei Aconaemys sagei Aconaemys porteri Aconaemys porteri Octodon bridgesi Octodon bridgesi Octodon degus Octodon degus Octodon degus Octodon degus Octodon lunatus Octodon lunatus Octodon lunatus Octodontomys gliroides Octodontomys gliroides Octodontomys gliroides Octomys mimax Octomys mimax Octomys mimax Octomys mimax Spalacopus cyanus Spalacopus cyanus Spalacopus cyanus Spalacopus cyanus Tympanoctomys barrerae Tympanoctomys barrerae

Chile: Cueva los Pincheira Chile: Concepci
on; El Roble Ranch Chile: Malleco; Pedregosa, Caj
on de Agues Negras Argentina: Pampa Hui Hui, Lanin National Park Chile: Temuco; Sector Chinay, Parque Nac. Villarrica Chile: Cautin; Huerquehue National Park ~ uble; Las Heras, Quirihue Chile: N ~ uble; Las Heras, Quirihue Chile: N Chile: Santiago Chile: Elqui; Fray Jorge National Park Chile: Valpara
ıso; Pe~ nuelas National Reserve Chile: Valpar
ıso; Lago Pe~ nuelas Chile: Valpara
ıso, Lago Pe~ nuelas Chile: Valpara
ıso, Pe~ nuelas National Reserve Chile: Valpara
ıso, Lago Pe~ nuelas Argentina: Jujuy; 10 km W Purmaparca Argentina: Jujuy; 11 km E Humahauca Chile: Chile; Putre; Chusmiza Springs Argentina: San Juan Province Argentina: La Rioja, Ischigualsata Argentina: San Juan; Ischigualsto National Park Argentina: La Rioja, Ischigualsata Chile: Concon Province Chile: Choapa; Los Vilos Chile: Choapa; Huentelauqu
en ~ uble; Quirihue Chile: N Argentina: Mendoza Argentina: Mendoza; El Talp
on

K38 UACH 4181 MHG 1372 UACH 4400 DRL 2736 UACH 4389 UACH 4486 UACH 4487 K61 UACH 2745 UACH 6193 FML 617 JCT 1521 LCC 1077 LCC 1076 AK 15685 AK 15686 UACH 4338 AK 13474 MHG 1537 LCC 1622 MHG 1550 K50 UACH 4537 UACH 4552 H5626 AK 13811 LCC 1627

AF520674 AF520675 AF520672 AF520673 AF520670 AF520671 AF520676 AF520677 AF520678 AF520679 AF520680

AF520657

Outgroup taxa Superfamily Cavioidea Caviidae Cavia aperea

Suriname: Sipaliwini

TK 17830

AF433908

AF433930

Agoutidae Agouti paca Agouti paca

Peru: Amazonas, Rio Cenepa, Huampami Bolivia: San Ramon

K7 NK 12997

AF520693 AF433906

AF433928

Erethizontidae Erethizon dorsatum Coendou bicolor

USA: Vermont Peru: Amazonas, Rio Cenepa, Huampami

K 1923 K5

AF520694 AF520695

AF520658 AF520663

Chinchilloidea Chinchillidae Chinchilla laniger Chinchilla laniger

USA: Florida Lab Colony Bolivia: Rio Tijamuchi

K94 NK 13161

AF520695

Dinomyidae Dinomys branickii

Peru: Amazonas, Rio Koska

K8

AF520697

a

AF520681 AF520682

AF520645 AF520644 AF520646 AF520648

AF520647 AF520650 AF520651

AF520683 AF520684 AF520685

AF520649 AF520664 AF520665

AF520686 AF520687 AF520688 AF520689 AF520690 AF520691 AF520692

AF520652 AF520654

AF520653 AF520655

AF520660 AF520659

Museum of Southwestern Biology, University of New Mexico—NK; The Museum, Texas Tech University—TK; Texas Cooperative Wildlife on, University de Austral de Chile, Casilla Collection, Texas A&M University—AK, H; University of Vermont—K; Instituto de Ecolog
ıa y Evoluci
567—MHG, DRL, UACH, FML, JCT. b GenBank accession numbers.

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gaps were treated as missing data and the indels then coded in a presence/absence matrix. All phylogenetic analyses were conducted using PAUP* 4.0b2 (Swofford, 1999). First, each gene was analyzed separately. Prior to combining both genes into a total evidence analysis (sensu Kluge, 1989), homogeneity between data sets was assessed using the partition homogeneity test (PHT) with 1000 replications and a a of 0.05 (PAUP*; Cunningham, 1997; Sullivan, 1996). MP analyses were conducted using the branch-andbound search option, and support for individual nodes was assessed with both bootstrap replication (1000 replicates; Felsenstein, 1985) and Bremer decay indices (Bremer, 1988). Both separate and combined maximum-likelihood (ML) analyses were conducted in PAUP*. Prior to initiating a ML analysis, it is important to select the most appropriate substitution model for the data being analyzed (Goldman, 1993). Therefore, the most optimal substitution model was determined using the Modeltest program (Posada and Crandall, 1998), which tests for significant changes in likelihood scores as model complexity increases in a nested manner. Once the appropriate model was determined, ML analyses were performed with all parameter values of the model and topology estimated. Bootstrap support for the ML tree was determined using 100 fast-addition replications (PAUP*). Potential rate heterogeneity for each gene was investigated using two approaches. First, TajimaÕs relative rate test (RRT; Tajima, 1993) was employed to assess rate heterogeneity via multiple pairwise comparisons of ingroup taxa to an outgroup taxon. Bonferroni correction was used to minimize effects from multiple testing (Rice, 1989). As a consequence, results from this test are generally conservative and can suffer from a lack of power without a high number of variable sites and an appropriate outgroup (Bromham et al., 2000). Second, a likelihood ratio test (LRT; Felsenstein, 1988) was used to compare likelihood scores, for the given tree topology, produced with and without an assumption of a molecular clock.

3. Results 3.1. Patterns of variation The entire mitochondrial 12S rRNA gene and exon 10 of GHR were sequenced for 32 and 23 species, respectively. Excluding ambiguous sites from the 12S rRNA gene, the total number of characters used in all phylogenetic analyses (including 12S rRNA and GHR) was 2026 (including 126 indels treated as present or absent in MP analyses). Both genes showed bias in base composition. Bias for the 12S rRNA gene was seen only

479

in loop regions, with an excess of adenine (A) relative to guanine (G). GHR showed a bias toward G over thymine (T) at the first codon position, A > G at the second position, and T > G at the third position. All taxa for both the 12S rRNA (v2 ¼ 24:7, P ¼ 1:0, df ¼ 60) and GHR (v2 ¼ 14:36, P ¼ 1:0, df ¼ 60) were similar in base composition as evidenced by v2 tests of homogeneity. The average transition/transversion (ts/tv) ratio among pairwise comparisons of taxa in the family Octodontidae was 7.1:1 for the 12S rRNA gene (stems 8.1:1.0 and loops 5.4:1.0). Loops showed an overall higher number of both transitions (ts) and transversions (tv) substitutions relative to stems. The ts/tv ratio for all three codon positions of GHR was 2.8:1.0, with the highest ratio found at the third codon position (2.6:1.0 for first position, 1.9:1.0 for second, and 3.7:1.0 for third). Comparisons of all members of the superfamily Octodontoidea showed a similar pattern of ts/tv ratios (1.7:1.0 for the 12S rRNA gene and 2.8:1.0 for GHR). Saturation plots of percent ts or tv as a function of pairwise distances (HKY 85 distances; Hasegawa et al., 1985) revealed a lack of saturation at all three codon positions of GHR (data not shown). Similar plots for separate analyses of 12S rRNA stems and loops revealed the beginning of saturation effects of transitions but not transversions in loops, especially between ingroup and outgroup taxa (data not shown). At least two individuals were examined for 12S rRNA variation (Table 1). Within species variation was low for nearly all comparisons, with the average HKY85 distance being 0.3%. GHR also showed low withinspecies variation, averaging 0.1%. Among species in the same genus, 12S rRNA averaged 2.3% and GHR 1.6%. For comparisons among genera in the Octodontidae, distances were 4.9% for 12S rRNA and 1.7% for GHR. Distances for comparisons among families within the superfamily Octodontoidea averaged 12.2% for 12S rRNA and 9.9% for GHR. After Bonferroni correction, the Tajima (1993) RRT detected no significant rate heterogeneity for GHR but did for 12S rRNA. Relative to some members of the family Octodontidae (both species of Aconaemys, Octodon bridgesi and Octodon lunatus, and Octodontomys), Abrocoma appeared to be evolving at a faster rate. The LRT, comparing unconstrained and clock-constrained likelihood scores, was not significant for GHR ( ln L ¼ 2847:31 with molecular clock and  ln L ¼ 2840:31 without a clock; df ¼ 15, P ¼ 0:05), also suggesting clock-like behavior. When all substitution classes were evaluated for the 12S rRNA gene, likelihood scores were significantly different under a clock-constrained model ( ln L ¼ 3783:70 with molecular clock and  ln L ¼ 3768:58 without a clock; df ¼ 15, P ¼ 0:05). However, when only tv substitutions in both stems and loops were considered, using a RRT and LRT ( ln L ¼ 1487:56 with molecular clock and  ln L ¼ 1478:36 without a

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clock; df ¼ 15, P ¼ 0:05), the 12S rRNA gene conformed to clock-like behavior. 3.2. Phylogenetic analyses Separate MP analyses were first performed for both the 12S rRNA and GHR genes. Preliminary analyses (both separate and combined; trees not shown), including additional representatives of all caviomorph superfamilies (Agouti, Cavia, Chinchilla, Coendou, Dasyprocta, Dinomys, and Erethizon), strongly supported a monophyletic Octodontoidea (containing Abrocomidae, Myocastoridae, Echimyidae, Ctenomyidae, and Octodontidae). Within the Octodontoidea, analysis of separate genes resulted in one and two most parsimonious trees for the 12S rRNA and GHR, respectively (Figs. 1 and 2). The family Abrocomidae was consistently basal to the octodontoid radiation. Analyses including representatives of other superfamilies consistently supported the monophyly of the superfamily Octodontoidea (families Octodontidae, Ctenomyidae, Echimyidae, and Myocastoridae), with an inclusive Abrocomidae and a sister-group relationship between the Myocastoridae and Echimyidae. However, the two genes differed with respect to relationships among the remaining octodontoid families. For instance, GHR strongly supported a sister-group relationship between Ctenomyidae and Octodontidae and a more basal position for Abrocom-

Fig. 1. 12S rRNA tree derived from maximum parsimony employing equal weighting and the branch-and-bound search option. Total tree length was 635, with CI ¼ 0:55 and RI ¼ 0:58. Numbers above the lines represent bootstrap values based on 100 replications, and those below the line are Bremer support values.

Fig. 2. GHR tree derived using maximum parsimony with equal weighting and the branch-and-bound search option. The tree represents a strict consensus for two trees of length 308, CI ¼ 0:68, and RI ¼ 0:75. Numbers above the lines represent bootstrap values based on 100 replications, and those below the line are Bremer support values.

idae, whereas 12S rRNA provided weak support for a sister-group relationship between the Octodontidae and Myocastoridae/Echimyidae with the Ctenomyidae being the basal octodontoid lineage. When a branch-andbound analysis, including all substitutions in stems and only transversions in loops (accommodating potential saturation effects of transitions in loops), was conducted for the 12S rRNA gene, relationships among other octodontoid families relative to the Octodontidae were identical to those observed for GHR (tree not shown). Both trees provided strong support for a monophyletic Octodontidae and a clade containing the endemic Chilean genera Octodon, Spalacopus, and Aconaemys. Although the semifossorial genus Aconaemys and the fossorial genus Spalacopus formed a monophyletic group, neither gene supported the monophyly of the Aconaemys species. In fact, the 12S rRNA gene strongly supported a sister-group relationship between A. porteri and Spalacopus. Contrary to the 12S rRNA gene, GHR did not provide strong support for the monophyly of Octodon. However, both genes supported the sistergroup relationship between O. bridgesi and O. lunatus. Octomys and Tympanoctomys, the desert specialists occurring east of the Andes in Argentina, grouped separately from Chilean taxa west of the Andes, with strong support from both genes. Octodontomys, a desert specialist occurring on both sides of the Andes in southern Bolivia and northern Chile and Argentina, was sister to the Chilean clade. The partition homogeneity test (1000 replications) suggested that the data sets were homogeneous

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(P ¼ 0:75), and the data sets were thus combined. The combined MP analysis resulted in a single most parsimonious tree (Fig. 3). The basic relationships were identical to those seen in the separate analyses (Figs. 1 and 2), but with stronger bootstrap support in the combined analysis. A monophyletic Octodontidae was strongly supported, as was the basic dichotomy separating the Chilean taxa plus Octodontomys from a clade containing Octomys and Tympanoctomys. The monophyly of Octodon, the sister-group relationship between the families Ctenomyidae and Octodontidae, and a relationship between the families Myocastoridae and Echimyidae also were supported. In addition, the family Abrocomidae appeared to be the most divergent octodontoid family, and the genus Aconaemys was paraphyletic relative to Spalacopus. The most optimal ML model for the 12S rRNA gene was the general time reversible (GTR) + C ða ¼ 0:45Þ and invariant sites ðinv ¼ 0:26Þ, whereas the GTR + C ða ¼ 0:78) was the most appropriate model for GHR. Given computational limitations, the number of outgroup and ingroup taxa was reduced. The ML tree for GHR was identical to the MP tree (Figs. 2 and 4), whereas the 12S rRNA tree derived from the ML analysis differed from that obtained in the MP analysis (Figs. 1 and 5). The primary differences included the placement of Octodontomys and relationships among families within the Octodontoidea. A combined ML

Fig. 3. Tree derived for a combined maximum parsimony analysis (branch-and-bound search option with equal weighting) of the 12S rRNA and GHR genes. The tree length is 944, CI ¼ 0:59, and RI ¼ 0:63. Numbers above the lines represent bootstrap values based on 100 replications, and those below the line are Bremer support values.

481

Fig. 4. Maximum likelihood tree for the 12S rRNA gene. The topology is based on the GTR (general time reversible) model + C ða ¼ 0:45Þ and invariant sites (inv ¼ 0:26).

Fig. 5. Maximum likelihood tree for the GHR gene. The topology is based on the GTR model + C ða ¼ 0:78Þ. Numbers on the tree represent bootstrap values obtained using 100 fast-addition replicates (PAUP*).

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hydrogen bonding and base stacking involving G–C pairs release more energy, thus providing stem stability (Noller, 1984; Zuker, 1989). Therefore, one would expect to see an increase of G in stems. Compensatory mutations associated with the maintenance of secondary structure in rRNA genes have been documented (Wheeler and Honeycutt, 1988; Dixon and Hillis, 1993; Gatsey et al., 1994), and changes associated with base complementarity in stems may explain the increase in ts relative to tv substitutions (Springer et al., 1995). 4.2. Phylogenetic implications

Fig. 6. Maximum likelihood tree for the combined 12S rRNA and GHR data sets. The topology is based on the GTR model + C ða ¼ 0:62Þ þ inv (inv ¼ 0:29). Numbers on the tree represent bootstrap values obtained using 100 fast-addition replicates (PAUP*).

analysis, employing a GTR + C ða ¼ 0:62Þ + invariant sites ðinv ¼ 0:29Þ model, produced a tree that was identical to the combined MP analysis (Fig. 6).

4. Discussion 4.1. Heterogeneity in base composition and substitutions Although both genes showed base compositional bias, the bias was similar for all taxa examined. Compared to the GHR, the 12S rRNA gene showed more bias in both base composition and the frequency of ts and tv. This bias apportioned differently between stems and loops. The overall lack of compositional bias in stems compared to loops, which show an excess of A at the expense of G, is typical of the mammalian 12S rRNA gene (Springer et al., 1995; Nedbal et al., 1996). This among-site heterogeneity, apportioned into structurally different components of the 12S rRNA gene, is most likely the result of differences in selective constraint. For instance, the lack of base composition bias in stems may relate to enhanced stability through the maintenance of a free energy window. In this case, both

In addition to providing information on relationships within the Octodontidae, our molecular data provide several observations regarding relationships among families of caviomorph rodents (Figs. 3 and 6). First, the phylogenetic affinity of Abrocoma has been problematic. Some have placed it within either the family Echimyidae (Ellerman, 1940) or Octodontidae (Landry, 1957), whereas others have assigned familial status to Abrocoma (Abrocomidae; Patterson and Wood, 1982). A recent study (Glanz and Anderson, 1990) suggested a sistergroup relationship between Abrocomidae and Chinchillidae. Nevertheless, this conclusion was based on the examination of a small number of taxa and an assumption that the family Echimyidae was the appropriate outgroup. Our data, both separate and combined analyses of the 12S rRNA and GHR genes, suggest placement of abrocomids within a monophyletic Octodontoidea. GHR and combined analyses support a basal position of the Abrocomidae within the superfamily. This placement is consistent with an earlier paleontological study (Patterson and Pascual, 1972) and three recent molecular studies involving allozymes (K€ ohler et al., 2000), nucleotide sequences (Huchon and Douzery, 2001), and DNA annealing (Gallardo and Kirsch, 2001). Second, although the Echimyidae may be morphologically primitive, our data do not support a sistergroup relationship between this family and the Octodontidae, as suggested by Reig (1986). The combined 12S rRNA and GHR analyses, using both MP and ML, provide strong support for a sister-group relationship between the Ctenomyidae and Octodontidae, a finding similar to that suggested by several previous authors (Patterson and Pascual, 1972; Reig et al., 1990; Gallardo and Kirsch, 2001). Congruent with earlier 12S rRNA data (Nedbal et al., 1994), immunological results (Sarich, 1985), morphology (Woods and Hermanson, 1985), and nucleotide sequences (Huchon and Douzery, 2001), our data indicate a sister-group relationship between the families Echimyidae and Myocastoridae. Third, with respect to the arrangement of families within the Octodontoidea, our molecular phylogeny is most congruent with results from DNA annealing (Gallardo and Kirsch, 2001). Like the DNA annealing

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phylogeny, the 12S rRNA and GHR combined tree places the family Abrocomidae at the base of an octodontoid clade, with the Ctenomyidae sister to the Octodontidae. Nevertheless, our study provides a somewhat more convincing placement of these two families by the inclusion of more octodontoid taxa as well as other divergent caviomorph families. Our results are highly incongruent with other molecular data. For instance, based on a phenetic analysis of allozyme distances, K€ ohler et al. (2000) suggested a sister-group relationship between Abrocomidae and Octodontidae, with the remaining octodontoid families (Echimyidae, Myocastoridae, Ctenomyidae, and Capromyidae) being more divergent. To the contrary, sequences from exon 28 of the nuclear vWF gene (Huchon and Douzery, 2001) placed the Octodontidae at the base of the superfamily, with Abrocoma being sister to a clade containing the Ctenomyidae, Capromyidae, Echimyidae, and Myocastoridae (an observation similar to the ML analysis of the 12S rRNA gene shown in Fig. 4). Phylogenies based on both allozymes and vWF sequences were evaluated relative to our combined phylogeny. The number of taxa examined for vWF was considerably smaller than the number included in our study. Therefore, comparisons between data sets were conducted on a reduced data set, with fewer taxa. A K–H test (Kishino and Hasegawa, 1989) was used to evaluate changes in tree scores when the allozyme-derived and vWF-derived topologies were constrained and compared to the combined 12S rRNA/GHR data set. The allozyme topology was significantly (P < 0:0001) longer than our combined tree by 122 steps (MP), and was significantly less likely (P < 0:0001) than our ML tree topology. The vWF topology also was significantly longer (P ¼ 0:01; MP) by 19 steps, and our combined tree was significantly more likely (P ¼ 0:02; ML). It is possible that incongruence between the vWF and our combined tree is the result of either differences in taxonomic sampling or gene-tree effects in one or more of the data sets. As indicated earlier, a large amount of data supports the monophyly of the Octodontidae, and both the separate and combined 12S rRNA and GHR analyses are highly congruent with these earlier studies (Gallardo and Kirsch, 2001; K€ ohler et al., 2000; Nedbal et al., 1994; Reig et al., 1990; Woods, 1982). Nevertheless, few studies have addressed the details of relationships among species within the Octodontidae. Results from our study provide a well-resolved molecular phylogeny that can be compared to hypotheses of relationships proposed by several earlier studies. With two exceptions (Contreras et al., 1993; Verzi, 2001), little has been formally published on cladistic analyses of either morphological characters or differentially banded chromosomes to establish relationships among members of the Octodontidae. Contreras et al. (1993) performed a cladistic analysis of characters as-

483

sociated with the glans penis, where relationships were inferred based on the number of spikes on each side and at the base of the intromitent sac. Spike number varied from 1–1 to 4–5. Although most taxa were polymorphic for number of spikes, these authors arbitrarily grouped the arrangement of spikes into three classes, 1–1, 2–3, and >3, with the 1–1 pattern being pleisiomorphic and the 2–2 pattern apomorphic. Based on this interpretation, two major clades could be diagnosed, one containing Octomys and Tympanoctomys (plesiomorphic) and a clade containing Octodontomys, Octodon, Aconaemys, and Spalacopus. This basic dichotomy is congruent with both separate and combined analyses of the 12S rRNA and GHR genes (Figs. 3 and 6). Optimization of the three spike states on the molecular tree (as initiated in MacClade 3.1; Maddison and Maddison, 1992) yielded the same distribution of primitive and derived characters suggested by Contreras et al. (1993). Nevertheless, unlike Contreras et al. (1993), our data do not support the placement of Octodon lunatus closer to Spalacopus cyanus than to its congeners, O. bridgesi and O. degus. A consideration of the polymorphism associated with the entire range of spike variation resulted in the 2–2 pattern being ambiguous and the 1–1 pattern remaining ancestral. Clearly, some of the incongruence associated with this morphological feature relates to the inherent homoplasy associated with spike distribution and the level of polymorphism observed for this character. Verzi (2001) used characters associated with craniomandibular and dental morphology to determine the placement of a fossil taxon, Abalosia, as well as relationships among extant species of Octodontidae. Although our molecular data are congruent with a sister-group relationship between Tympanoctomys and Octomys, the combined tree (Figs. 3 and 6) is not congruent with VerziÕs (2001) data regarding relationships of Octodon, Octodontomys, Spalacopus, and Aconaemys. We reanalyzed the morphological data in PAUP* with the characters treated as unordered and using a bootstrap analysis. Two equally parsimonious trees were found, with the strict consensus showing the placement of Octodontomys to be unresolved. The topology presented in Fig. 8 of Verzi (2001) was compared to our molecular phylogeny. A KH-test, comparing the two topologies, suggested that the morphological topology is significantly less likely (P ¼ 0:0006), given our molecular data. Under MP analysis, the morphological tree is significantly longer by 20 steps (P ¼ 0:0003). Using our molecular tree as an interpretive framework, the 28 characters examined by Verzi were mapped individually using MacClade 3.1. The results are as follows: (1) Seven character states (6, 7, 20, 22, 25, 26, and 28) appear to have arisen independently in the Aconaemys and the Tympanoctomys/Octomys clade. (2) Two (9 and 24) arose in parallel between Octomys and Aconaemys. (3) Ten

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(1, 2, 4, 12, 14, 16, 19, 21, 23, and 27) and possibly 11 (15) are phylogenetically uninformative. (4) Two (3 and 8) represent reversals to a plesiomorphic condition in Spalacopus and/or Aconaemys. (5) Four (11, 13, 17, and 18) are synapomorphies supporting the Tympanoctomys/ Octomys clade, one (5) is a synapomorphy for Octodon/ Spalacopus/Aconaemys, and one (10) is a synapomorphy for Spalacopus/Aconaemys. According to the molecular phylogeny, the informative morphological data appear to have a high level of homoplasy, possibly as a consequence of parallel changes in response to similar ecological constraints or some other life history trait. As indicated earlier, with the exception of a sistergroup relationship between Octomys and Tympanoctomys, our combined tree is not congruent with results from a previous allozyme study (K€ ohler et al., 2000). However, our combined analysis and the results from DNA annealing (Gallardo and Kirsch, 2001) are congruent in several respects. First, the desert specialists from Argentina, Octomys and Tympanoctomys, are distinct from a clade containing the remaining primarily Chilean taxa, with the genus Octodontomys being basal to this clade (Figs. 3 and 6). Second, the genus Octodon is monophyletic and, as suggested earlier based on morphological characteristics (Contreras et al., 1987), O. lunatus and O. bridgesi are closely related to O. degus. Although the specific-level status of O. lunatus and O. bridgesi has been questioned (Contreras et al., 1987), results of these molecular data and the distinct diploid number characterizing O. lunatus (Spotorno et al., 1988) provide support for these two taxa being recognized as distinct species.Third, semifossorial species in the genus Aconaemys are sister to the fossorial species, Spalacopus cyanus, with Aconaemys being paraphyletic relative to Spalacopus. Pearson (1984) recommended the recognition of only two species of Aconaemys, A. sagei and A. fuscus, with the latter containing two subspecies (A. f. fuscus and A. f. porteri). Contrary to this suggestion, recent morphometric and chromosomal studies (Gallardo and Reise, 1992; Gallardo and Mondaca, 2001) have suggested that the two subspecies of A. fuscus deserve specific level status. Results from DNA annealing (Gallardo and Kirsch, 2001) and our combined analysis add support to recognition of three species. The molecular results differ, however, in the placement of A. f. porteri. DNA annealing places A. f. porteri at the base, with Spalacopus more closely related to a clade containing A. f. fuscus and A. sagei. Our combined tree indicates two clades, one containing Spalacopus and A. f. porteri and the other A. f. fuscus and A. sagei. The combined analysis is compatible with information from standard karyotypes (Gallardo and Reise, 1992; Gallardo and Mondaca, 2001). A. sagei and A. f. fuscus have diploid numbers (2N) of 54 and 56, respectively, and Spalacopus and A. f. porteri have a 2N of 58.

4.3. Evolutionary implications The phylogeny obtained from our combined analysis and the DNA annealing data provide an interpretive framework for evaluating several hypotheses related to evolutionary processes. The standard karyotype of octodontoid rodents ranges from a diploid number (2N) of 38–102 and a fundamental number (FN) of 64–198 (Gallardo, 1992). According to George and Weir (1974), the basic process of chromosomal evolution in the group involved a series of Robertsonian centric fusions from an ancestral stock with a high diploid number (2N ¼ 98). Spotorno et al. (1988) also suggested a similar process for the Octodontidae, with lower diploid numbers derived via fusions from a presumed primitive 2N of 78. In contrast to the hypothesis of Spotorno et al. (1988), Gallardo (1992) proposed an ancestral 2N ¼ 58 for the Octodontidae. The linear parsimony method (Swofford and Maddison, 1987) in MacClade 3.1 (Maddison and Maddison, 1992) was used to reconstruct changes in a continuous character, 2N, by using the combined phylogeny as the interpretive framework. The range (minimum and maximum values) for 2N is 64 for Octodontoidea and a 2N between 46 and 56 for the Octodontidae (Fig. 7), thus supporting GallardoÕs (1992) hypothesis. Rather than a series of centric fusions from a high diploid ancestor, chromosomal evolution within the Octodontoidea and Octodontidae, in particular, has been complex, involving possible Robertsonian fusions (e.g., Ctenomys, Myocastor, Aconaemys sagei, and Octodontomys), centric fissions (Octodon, Aconaemys porteri, and Spalacopus), and polyploidy (Tympanoctomys; Gallardo et al., 1999). In comparison to changes in 2N, patterns of genome size variation among octodontoid rodent lineages are considerably less complex (Gallardo et al., 1999). Although genome size variation within the Octodontoidea has a broad range of 7.2–16.8 picograms (pg), the overall amount of interspecific variation is considerably less than that seen for chromosome number. The ancestral genome size for Octodontoidea is estimated to be 7.2–8.2 pg. Ctenomyidae shows an increase in genome size, and the ancestral genome size for the Octodontidae is 8.2 pg (Fig. 7). Several taxa within the Octodontidae show both decreases and increases from the ancestral condition, with Tympanoctomys showing the most drastic increase in genome size. Mares et al. (2000) recently described two new octodontid taxa, Pipanacoctomys aureus and Salinoctomys loschalchalerosorum, that appear sister to Tympanoctomys. Given the recent suggestion that Tympanoctomys is a possible polyploid (Gallardo et al., 1999), it will be interesting to see if these two new taxa also have high 2N and genome sizes, characteristic of Tympanoctomys. The family Octodontidae is comprised of an ecologically diverse array of species that occupy a broad array

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485

Fig. 7. Diploid numbers (above the line) and genome size (below the line) optimized using the linear parsimony method (Swofford and Maddison, 1987) in MacClade 3.1 (Maddison and Maddison, 1992). Numbers represent minimum and maximum values. Estimates of genome sizes are from Gallardo et al. (1999).

of habitats, and demonstrate a wide range of specializations (Mares and Ojeda, 1982; Reig, 1986; Contreras et al., 1987). For example, some species west of the Andes are specialized for a fossorial (Spalacopus) and semi-fossorial (Aconaemys) lifestyle, while members of the genus Octodon occur at lower elevations and are habitat generalists (Contreras et al., 1987; Begall and Gallardo, 2000). Octodontomys gliroides, a species consisting of allopatric populations occurring east and west of the Andes, displays specializations for xeric habitats in the Andes, and east of the Andes. Both Octomys and Tympanoctomys are highly adapted to desert environments. Several hypotheses, derived primarily from interpretations of historical trends associated with changes in climate, vegetation, and geology (e.g., the Andean uplift), have been proposed to account for the current distribution and ecological diversity of the Octodontoidae (Contreras et al., 1987; Mares, 1975; Reig, 1986). Mares (1975) and Reig (1986) hypothesized that adaptations by octodontids to montane habitats occurred during the Miocene and Pliocene, with Andean regions colonized more recently, in the Middle Pliocene. Mares (1975) also suggested that the association of Octomys and Tympanoctomys with arid regions is probably old, dating to at least the Pliocene. Some authors (Reig, 1986; Contreras et al., 1987) have argued that caviomorphs and octodontids, in particular, evolved from ground-dwelling forms occurring in more forest and scrub habitat, with higher Andean regions being secondarily invaded in more recent times. In an effort to test these evolutionary hypotheses, several traits were traced, using MacClade 3.1, on the combined phylogeny

(Table 2). Based on these comparisons, the ancestral octodontid occurred in more mesic scrub habitats at low elevations (< 500 m). Furthermore, fossoriality arose twice in the families Ctenomyidae and Octodontidae (Aconaemys and Spalacopus), and adaptation for arid environments occurred early in the Octodontidae, as can be seen by the positions of Octodontomys and the Octomys/Tympanoctomys clade (Figs. 3 and 6). Temporal changes in vegetation in southern South America have been characterized by Solbrig (1976), and diversification within the family Octodontidae can be compared to the various geological and climatic events responsible for vegetative changes. According to Solbrig (1976), major vegetational changes occurred during the Miocene in response to the Andean uplift and increasing aridity. By the end of the Pliocene, the landscape was similar to that seen today, with high montane flora, more humid coastal regions west of the Andes in Chile and drier areas located east of the Andes in Argentina. In terms of plant diversity, habitats during the Pleistocene became more fragmented, ranges of species shifted, and barriers to dispersal appeared. This temporal sequence of changes in plant communities can be compared with the timing of events associated with the octodontid radiation. According to our tests for rate homogeneity, both the 12S rRNA gene (tv only in both stems and loops) and the GHR gene demonstrate clocklike behavior. Therefore, divergence times for the nodes (A–H) shown in Fig. 6 were determined based on rates of molecular divergence derived from two calibration points (Table 3). We realize that estimates of divergence time should be considered to have a broad range.

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Table 2 Ecological traits evaluated on combined tree Taxa

Habitsa

Habitatb

Elevationc

Moistured

Caviidae Cavia apera

Scansorial

Scrub, grassland

Low, medium, high

Mesic

Abrocomidae Abrocoma cinerea

Scansorial

Scrub

Low, medium, high

Semiarid and arid

Echimyidae Hoplomys gymnurus

Ground dwelling

Forest, grassland

Low

Mesic

Myocastoridae Myocastor coypu

Semiaquatic

Scrub, forest, grassland

Low, medium

Mesic

Ctenomyidae Ctenomys steinbachi

Fossorial

Scrub, forest, grassland

Low, medium, high

Mesic, semiarid

Octodontidae Aconaemys porteri Aconaemys fuscus Aconaemys sagei Octodon bridgesi Octodon degus Octodon lunatus Octodontomys gliroides Octomys mimax Spalacopus cyanus Tympanoctomys barrerae

Fossorial Fossorial Fossorial Scansorial Scansorial Scansorial Scansorial Scansorial Fossorial Ground dwelling

Forest Scrub, forest Scrub, forest Scrub, forest Scrub Scrub Scrub Scrub Scrub Scrub

Low, medium High Low, medium Low, medium Low, medium Low, medium Medium, high Medium Low, medium Medium

Mesic Mesic Mesic Mesic Semiarid Mesic Arid Arid Mesic Arid

a

Mares and Ojeda (1982). Solbrig (1976), Mares and Ojeda (1982), and Redford and Eisenberg (1992). c Elevation categories obtained from Redford and Eisenberg (1992); low: <500 m, medium: >500 m, high: >3000 m. d Mares and Ojeda (1982) and Solbrig (1976). b

Table 3 Estimates of divergence times Nodesa

Date of divergence (million years) 12S rRNA b

Cav/Oct A B C D E F G H

GHR Myo + Ech/Oct/Cte

c

Cav/Octb

Myo + Ech/Oct/Ctec

—

40.4

—

35.0

23.1–27.6 16.7–20.0 10.8–12.9 2.9–3.5 1.1–1.4 1.4–1.7 1.1–1.3

—

26.6–31.6 18.9–22.5 5.0–5.9 3.4–4.1 4.4–5.3 5.0–5.9 2.2–2.7

21.2 5.6 3.8 4.9 5.6 2.5

21.7 14.1 3.8 1.5 1.8 1.4

—

a

The combined ML tree (Fig. 6) shows location of nodes A–H. Rates (0:85  109 and 1:016  109 for 12S rRNA and 1:6  109 and 1:9  109 for GHR) based on fossil dates of 31–37 million years for separation of Cavioidea and Octodontoidea (node A; Wyss et al., 1993). c Rates (0:78  109 for 12S rRNA and 1:7  109 for GHR) based on fossil date of 30 million years for separation of Myocastoride/Echimyidae from Octodontidae/Ctenomyidae (node A; Vucetich et al., 1999). b

Nevertheless, the estimates presented here exhibit an overall consistency relative to the calibration points used and previous suggestions from the fossil record. Dates estimated from both genes are very similar for nodes A through C, with rates derived from each calibration point being directly comparable. These estimates of divergence time also are similar to those determined from the fossil record (Vucetich et al., 1999; Wyss et al.,

1993). For instance, first appearance of several superfamilies occurs in the late Eocene to early Oligocene (approximately 31–37 mya; Wyss et al., 1993), and the calibration point at node B supports this estimate. Similarly, rate estimates derived from node A overlap with the estimated fossil divergence time of 30 mya for node B (Vucetich et al., 1999). The two genes, however, diverge in time estimates beginning at node D, with 12S

R.L. Honeycutt et al. / Molecular Phylogenetics and Evolution 26 (2003) 476–489

rRNA divergence appearing more clock-like in comparison to estimates for GHR. Based on estimates of divergence times derived from the 12S rRNA gene, diversification within the Octodontidae is very similar to events experienced by plant communities in southern South America. For example, Vucetich et al. (1999) indicated on the basis of fossil evidence that caviomorph rodents experienced a second major radiation in response to climatic changes occurring during the Middle to Late Miocene. Both the 12S rRNA and GHR dates (Table 2) for major divergences among families of octodontoids are similar to timing of the radiation documented by Vucetich et al. (1999). Mares (1975) hypothesized that the arid adapted clade containing Tympanoctomys/Octomys represents an early divergence (Miocene) within the Octodontidae, and the Middle to Late Miocene estimate obtained from the 12S rRNA gene supports this hypothesis. Most diversification within the Octodontidae is more recent and tends to coincide with changes in the landscape and fragmentation events occurring in the Pliocene and Pleistocene, respectively. For instance, Octodontomys (occurring in Argentina and Chile) appears to have separated from a Chilean clade containing Octodon, Aconaemys, and Spalacopus during the early to late Pliocene, and diversification in the Chilean clade occurred almost simultaneous and in response to Pleistocene events. The observed discrepancies between the 12S rRNA and GHR genes at several nodes are interesting in that the GHR dates appear too young for node D and too old for nodes F–H (Fig. 6). In their broader survey of caviomorph rodents, Huchon and Douzery (2001) observed a minimum of five local molecular clocks for the vWF gene, and it is possible that GHR is showing a similar pattern. Rowe and Honeycutt (2002) have revealed rate heterogeneity in GHR evolution within the caviomorph superfamily Cavioidea, and this heterogeneity appears to be associated with body size alone and perhaps correlates of body size such as generation time. Using the Ctenomyidae as an outgroup, the two cluster test (TCT) of the linearized tree method (Takezaki et al., 1995) was used to further evaluate evidence of rate heterogeneity in more recently derived octodontids. Based on this comparison, the clade containing Aconaemys, Octodon, and Spalacopus appears to be evolving faster than Octodontomys (PLINTRE < 0:01). This may account for the apparent discrepancies associated with more recent divergences (e.g., nodes F–H). Nevertheless, the pattern may be more complicated than our current analyses can detect. The observation of a reoccurring pattern of rate heterogeneity in some nuclear genes showing possible correlates with life history traits clearly needs further investigation. Given the overall diversity in ecology, behavior, reproductive and other life history strategies, and body size, octodontoid rodents and South America caviomorph rodents, in particular, offer

487

valuable models for detailed evaluations of molecular evolutionary processes, especially as they relate to diversification and adaptive radiation.

Acknowledgments We thank two anonymous reviewers for their constructive comments. We also thank Michael Mares and Janet Braun for providing access to tissues of some Argentine rodents. Support for Dr. MaresÕ fieldwork was provided by grants from the National Science Foundation (BSR-8906665) and National Geographic Society (4820-92). We thank and acknowledge individuals and institutions for providing samples from their frozen tissue collections. These include: (1) Robert J. Baker, The Museum, Texas Tech University; (2) William C. Kilpatrick, University of Vermont; (3) James L. Patton, Museum of Vertebrate Zoology, University of California at Berkeley; (4) Terry L. Yates, Museum of Southwest Biology, University of New Mexico. Field assistance was kindly provided by F. Mondaca, R. Ojeda, J. Gonnet, and G. D
ıaz. This research was funded by Fondecyt Grants 1010727 and 1970710 to MHG and National Science Foundation Grant DEB 9615163 to RLH. This paper represents contribution #105 of the Center for Biosystematics and Biodiversity at Texas A&M University.

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