Recurrent Amplifications and Deletions of Satellite DNA Accompanied Chromosomal Diversification in South American Tuco-tucos (Genus Ctenomys, Rodentia: Octodontidae): A Phylogenetic Approach

Claudio H. Slamovits, Joseph A. Cook, Enrique P. Lessa and M. Susana Rossi

Laboratorio de Fisiología y Biología Molecular, Facultad de Ciencias Exactas y Naturales, Departamento de Ciencias Biológicas, Pabellón II, Buenos Aires, Argentina;
Department of Biological Sciences, Idaho State University;
Laboratorio de Evolución, Facultad de Ciencias, Montevideo, Uruguay


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
We investigated the relationship between satellite copy number and chromosomal evolution in tuco-tucos (genus Ctenomys), a karyotypically diverse clade of rodents. To explore phylogenetic relationships among 23 species and 5 undescribed forms, we sequenced the complete mitochondrial cytochrome b genes of 27 specimens and incorporated 27 previously published sequences. We then used quantitative dot-blot techniques to assess changes in the copy number of the major Ctenomys satellite DNA (satDNA), named RPCS. Our analysis of the relationship between variation in copy number of RPCS and chromosomal changes employed a maximum-likelihood approach to infer the copy number of the satellite RPCS in the ancestors of each clade. We found that amplifications and deletions of RPCS were associated with extensive chromosomal rearrangements even among closely related species. In contrast, RPCS copy number stability was observed within clades characterized by chromosomal stability. This example reinforces the suspected role of amplification, deletion, and intragenomic movement of satDNA in promoting extensive chromosomal evolution.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Several hypotheses have been advanced to explain the apparent relationship between chromosomal rearrangements and speciation. Some investigators have focused on the meiotic viability of the heterokaryotype (King 1993Citation , pp. 126–168) or on highly structured populations that might facilitate the fixation of a new homokaryotype and hence production of new species (Bush 1975Citation ; Lande 1985Citation ; Nevo 1999Citation ). Other researchers have explored molecular mechanisms involved in the origin of rearrangements (Cáceres et al. 1999Citation ; Evgen'ev et al. 2000Citation ). In particular, satDNA has been purported to play a role in the dynamics of genomic rearrangements, with particular impact on chromosomal changes associated with speciation (Wichman et al. 1991Citation ; Bradley and Wichman 1994Citation ; Garagna et al. 1997Citation ).

A number of investigations have focused on the molecular properties and the chromosomal locations of the sequences involved in these rearrangements. Recently, two satellite DNAs (satDNAs) had been shown to be involved in Rb translocations in mice (Garagna et al. 2001)Citation and muntjac deer (Li et al. 2000)Citation . Another means of testing of ideas concerning the role of satDNA in chromosomal change would involve the assessment of the correlated evolution of chromosomal and genomic change through the evolutionary history of a clade. Such an approach would require the documentation of satellite DNA in a chromosomally diverse clade. In this paper, we characterize the evolutionary history of amplifications and deletions of a particular satDNA identified in the highly polymorphic tuco-tucos (genus Ctenomys). These subterranean rodents form one of the most karyotypically diverse clades of mammals known, with chromosomal diploid numbers ranging from 10 to 70 (Cook, Anderson, and Yates 1990Citation ; Gallardo 1991Citation ; Reig et al. 1992Citation ; Ortells 1995Citation ). Tuco-tucos belong to the family Octodontidae and are distributed throughout the southern cone of South America with more than 56 described species (Reig et al. 1990Citation ). Another member of this family, Tympanoctomys barrerae, has 102 chromosomes and apparently is the first tetraploid mammal known (Gallardo et al. 1999Citation ). This tremendous range in diploid numbers in a single mammalian clade has attracted speculation regarding the mechanisms that promoted high diversification (Nevo 1999Citation ). Reig and Kiblisky (1969)Citation first proposed that tuco-tucos were a prime example of chromosomal speciation. Diversification may have been facilitated by the isolation of small demes that characterize population structure in most species (Reig et al. 1990Citation ) and extensive chromosomal rearrangements (Reig and Kiblisky 1969Citation ; Cook 1990Citation ; Gallardo 1991Citation ; Ortells 1995Citation ). The apparent relationship between high karyotypic diversity and elevated rates of speciation in subterranean rodents has been explored by a number of investigators (White 1978Citation ; Reig 1989Citation ; Nevo 1999Citation ; Steinberg and Patton 2000Citation ).

The major satDNA sequence characterized for tuco-tucos (named RPCS) varies in copy number among species (Rossi, Reig, and Zorzópulos 1990, 1993Citation ; Rossi et al. 1995bCitation ). RPCS has been located in chromosomal regions with positive C-heterochromatic bands in most species (Rossi et al. 1995bCitation ). The repetitive unit of RPCS shows high sequence and structural identity with the long terminal repeat (LTR) of retroviruses (Rossi et al. 1993Citation ; Pesce et al. 1994Citation ). This particular satDNA may promote chromosomal rearrangements by virtue of its capacity for amplification and movement through the genome. In turn, amplification is thought to facilitate rapid chromosomal evolution associated with speciation (Rossi et al. 1995a, 1995bCitation ).

We used an expanded phylogenetic framework based on complete cytochrome b sequences for Ctenomys to explore the evolution of the satellite RPCS. These phylogenies encompassed Bolivian, Chilean, Uruguayan, and Argentinean tuco-tucos from throughout the distribution of the genus and included representatives of the three named subgenera.

To further investigate the role of RPCS in chromosomal evolution in Ctenomys, we performed quantitative dot-blots of RPCS. We analyzed the copy number of the repetitive unit of RPCS in each species and inferred these values for the ancestral nodes to provide an assessment of the amplification/deletion process across the genus.

Because tuco-tucos have been considered an exemplary case of chromosomal speciation in mammals (Reig et al. 1990Citation ), we examined this clade as a case study of the role of satDNA in chromosomal change. We did this by (1) providing an expanded molecular phylogeny based on complete sequences of the mitochondrial cytochrome b gene, (2) estimating RPCS copy numbers in the genomes of the species represented in the phylogeny, and (3) using phylogeny-based methods to examine the relationship between the stability of the copy number of the RPCS satellite and the stability of chromosome numbers through the evolutionary history of the clade.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Specimens of Ctenomys Examined
We studied a total of 65 individuals belonging to 23 nominal species and 5 undetermined forms of Ctenomys and representatives of the sister taxa Octodon, Octodontomys, and Tympanoctomys. DNA for sequencing and dot-blot experiments was obtained from ethanol-preserved liver samples. Tissues used were from GIBE, Universidad de Buenos Aires, Argentina (I), and Laboratorio de Evolución, Facultad de Ciencias, Universidad de la República, Uruguay (CA). Other samples were courteously provided by Milton Gallardo (MHG) and D. Reize (DR), Angel Spotorno (AS), Carlos Quintana (CQ), M. Susana Rossi (MSR), the Museum of Vertebrate Zoology, Berkeley, University of California (MVZ), and the Field Museum of Natural History (FMNH), Chicago. Individuals included in the final phylogenetic trees are indicated with an asterisk. All localities, except those indicated otherwise, are in Argentina: Ctenomys porteousi (N = 3), Bonifacio, Buenos Aires province (I539*, I229*, I545); Ctenomys australis (N = 2), Necochea, Buenos Aires province (I1036*); Claromecó, Buenos Aires province (I1054); Ctenomys mendocinus (N = 3), Tupungato, Mendoza province (MSR2*, MSR33*); San Isidro, Mendoza province (MSR35); Ctenomys opimus (N = 2), Tres Cruces, Jujuy province (I464*), and Parinacota province, Chile (DR2326*); Ctenomys fulvus (N = 4), San Pedro de Atacama, El Loa province, Chile (DR2323*), Vegas de Turi, Antofagasta province, Chile (DR2324, MHG1042*), Salar de Atacama, Región de Antofagasta, Chile (AS872); Ctenomys talarum (N = 3), Necochea, Buenos Aires province (I734*, I733*, I695); Ctenomys argentinus (N = 1), Colonia Benítez, Chaco province (I702*); Ctenomys latro (N = 3), Tapia, Tucumán province (I998*, I995, I831); Ctenomys tucumanus (N = 3), Ticucho, Tucumán province (I987*, I988, I989); Ctenomys tuconax (N = 3), El Infiernillo, Tucumán province (I867*, I679, I990); Ctenomys magellanicus (N = 2), Tres Arroyos, Tierra del Fuego province (I308*, I307); Ctenomys haigi (N = 1), 13.5 km E Perito Moreno, Rio Negro province (MVZ184885); Ctenomys pearsoni (N = 1), Rocha, Uruguay (CA583); Ctenomys rionegrensis (N = 1), Río Negro, Uruguay (CA435); Ctenomys maulinus (N = 3), Rio Colorado, Caracautin, Malleco province, Chile (MHG1151*, MVZ183304), Pelechue, Chile (AS1019*); Ctenomys coyhaiquensis (N = 1), Region XI, Chile (FMNH134300); Octodontomys gliroides (N = 1), Tilcara, Jujuy province (CQ-1*); Octodon degus (N = 1), Región Metropolitana, Chile (I-89A). The following previously published sequences (Cook and Lessa 1998Citation ; Lessa and Cook 1998Citation ; D'Elía, Lessa, and Cook 1999Citation ) were used: C. boliviensis (GenBank accession numbers AF007037, AF007038*), Ctenomys goodfellowi (AF007050*, AF007051*), Ctenomys sp. ROBO (AF007039*, AF007040), Ctenomys steinbachi (AF007043*, AF007044), Ctenomys opimus (AF007042*), Ctenomys conoveri (AF007054, AF007055*), Ctenomys leucodon (AF007056*), Ctenomys frater (AF007045*, AF007046), Ctenomys sp. MINUT (AF007052, AF007053*), Ctenomys lewisi (AF007049*), Ctenomys sp. ita (AF007047*), Ctenomys sp. MONTE (AF007057*), Ctenomys sp. LLATHU (AF007048*), Ctenomys haigi (AF007063*), Ctenomys mendocinus (AF007062*), Ctenomys coyhaiquensis (AF119112*), Ctenomys rionegrensis (AF119114*), Ctenomys pearsoni (AF119108*), O. degus (AF007059*), and T. barrerae (AF007060*). New sequences have been deposited in GenBank under accession numbers AF370680AF370706 (see table 1 ).


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Table 1 Individuals and Sequence Data

 
DNA Extraction, Sequencing, and Dot-Blot Analysis
Genomic DNA was extracted from ethanol-preserved liver samples, and complete mitochondrial cytochrome b genes from 27 individuals were PCR-amplified and sequenced following Lessa and Cook (1998)Citation . Tissue samples were ground in liquid nitrogen (-180°C), digested with proteinase K, and purified with phenol-chloroform-isoamylic alcohol, and DNA was precipitated with ethanol and salt. DNA concentration was measured using a UV spectrophotometer.

For dot-blot experiments, 10, 100, and 1,000 ng of genomic DNA were fixed to a nylon membrane using a Bio-Dot microfiltration unit (Bio-Rad) and hybridized to 32P-labeled RPCS under low-stringency conditions (6 x SSC, 60°C). Autoradiographs were scanned, and the density of dots was measured using NIH Image v1.62. RPCS copy number per genome was computed using pRPCS (Rossi, Reig, and Zorzópulos 1990Citation ) as standard.

Sequence and Phylogenetic Analysis
Alignment of DNA sequences was performed using the program Sequence Navigator, version 1.01 (Applied Biosystems). Nucleotide composition was computed with MEGA 1.02 (Kumar, Tamura, and Nei 1993Citation ). MacClade 3.0 (Maddison and Maddison 1992Citation ) was used to compute numbers of transitions, transversions, and changes per codon position. Genetic distances among all pairs of taxa were obtained with PAUP 4.0 beta (Swofford 1998Citation ). Phylogenetic signal was evaluated with the g1 value derived from 10,000 random trees (Hillis and Huelsenbeck 1992Citation ). All phylogenetic analyses used O. degus, O. gliroides, and T. barrerae as outgroups.

Maximum-parsimony (MP) trees were obtained using PAUP 4.0 beta (Swofford 1998Citation ). Fifty replicates of heuristic searches (with random addition of sequences) were performed using the weighting scheme 2, 5, and 1 for first, second, and third codon positions, respectively, and a stepmatrix giving a transition/transversion (Ts/Tv) ratio of 6. These parameters fit with biases associated with the molecular evolution of cytochrome b as analyzed here and elsewhere (Lessa and Cook 1998Citation ). Support for each clade was assessed by performing bootstrap analysis with 1,000 replicas (each repeated five times with random addition of sequences).

Maximum-likelihood (ML) analysis used PAUP 4.0 beta (Swofford 1998Citation ). The model that best fitted our data set was the general time reversible (GTR) model of evolution, whose parameters were calculated following the routine proposed by Sullivan, Markert, and Kilpatrick (1997)Citation . The proportion of sites assumed to be invariable was 0.44795, and the shape parameter of the gamma distribution was 0.94708. We performed a total of 10 runs with random addition of sequences.

Neighbor-joining (NJ) trees were constructed with PAUP 4.0 beta (Swofford 1998Citation ) using the same model and parameters used in the ML analysis. One thousand bootstrap replicas were performed to assess the robustness of the topology.

The ancestral condition of RPCS content was reconstructed by estimating the haploid copy number of RPCS at internal nodes using the program ANCML (Schluter et al. 1997Citation ). This analysis was based on an ML tree computed under the GTR model assuming a molecular clock.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Phylogeny of Tuco-tucos
Complete sequences (1,140 bp) of the cytochrome b gene from 54 individuals, representing 23 species and 5 undetermined forms, plus 3 octodontine genera were used and resulted in 461 (40%) variable sites (26.3% in first, 19.9% in second, and 53.7% in third codon positions). The compositional bias was similar to that published for other mammals (e.g., Irwin, Kocher, and Wilson 1991Citation ) and other caviomorph rodents (Lara, Patton, and Silva 1996Citation ). For example, G is the least common nucleotide (12.4%). Uncorrected genetic divergence (p distance) was computed for all pairs of individuals in order to evaluate inter- and intraspecific variability. The highest values of intraspecific divergence were found in C. mendocinus (0.7%–2.8%; N = 3), C. talarum (0.9%–2.1%; N = 2), C. maulinus (1.8%; N = 3), C. opimus (1.1%–1.8%; N = 4), C. porteousi (1.6%), and C. fulvus (0.1%–1.6%; N = 4). The least divergent pairs of species had levels of divergence comparable to those of some intraspecific comparisons. Ctenomys argentinus and C. latro differed by 1.2%–1.4%, C. porteousi and C. australis differed by 1.2%, and divergence between C. opimus and C. fulvus ranged from 1.2% to 2.2%. The most divergent pair of species was C. conoveri and C. leucodon (12.5%).

To facilitate computationally intensive analyses, only one individual of each taxon was retained in phylogenetic searches, except for those taxa exhibiting values of intraspecific divergences higher than 1.5%. The g1 value, -2.887236, computed from 10,000 random trees was highly significant (P < 0.01), indicating that this data matrix has strong phylogenetic signal (Hillis and Huelsenbeck 1992Citation ).

The 50% majority-rule consensus of 80 equally parsimonious trees, as well as the corresponding bootstrap support values, reveals a polytomy at the base of the tuco-tuco radiation (fig. 1 ). This basal polytomy of cytochrome b–based trees of ctenomyine evolution has been observed in previous analyses (Lessa and Cook 1998Citation ; D'Elía, Lessa, and Cook 1999Citation ; Mascheretti et al. 2000Citation ). The persistence of this polytomy, even with our larger sample size, provides additional support for an early burst of cladogenesis in the evolution of the genus (Lessa and Cook 1998Citation ). The molecular signature of a rapid cladogenetic event corresponds to the burst of speciation found in the fossil record, recently reviewed by Cook, Lessa, and Hadly (2000)Citation . The phylogenetic relationships of the well-supported clades of tuco-tucos are discussed below.



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Fig. 1.—Maximum-parsimony trees for ctenomyines with transitions/transversions weighted 6:1 and weights of 2:5:1 for first, second, and third codon positions, respectively. The lineages with derived asymmetrical sperm type are indicated. For Ctenomys maulinus, the figure of asymmetrical sperm type is faint because the phylogenetic position of this species is uncertain (see text). A, Fifty percent majority-rule consensus tree of 80 most-parsimonious trees (2,490 steps). B, Bootstrap analysis of 1,000 pseudoreplicates with five random-addition sequences each

 
Generally, relationships among Bolivian species and forms agree with previous morphological (Anderson, Yates, and Cook 1987Citation ), allozymic (Cook and Yates 1994Citation ), and DNA sequence (Lessa and Cook 1998Citation ) analyses and include (1) a clade of C. boliviensis, C. goodfellowi and Ctenomys sp. ROBO, with which C. steinbachi tends to be associated (fig. 1B ); and (2) a clade composed of C. frater, C. lewisi, Ctenomys sp. LLATHU, and C. conoveri. The relationship between C. frater and C. lewisi was previously shown by cytochrome b sequences and other molecular, karyologic, and morphologic characters (Lessa and Cook 1998Citation and references therein). The association between C. conoveri and Ctenomys sp. LLATHU appeared stronger in this study than in previous ones (Lessa and Cook 1998Citation ) and confirmed that C. conoveri is not different enough to constitute a subgenus of Ctenomys to the exclusion of other species, as previously proposed (Osgood 1946Citation ).

Another well-characterized clade is composed of C. opimus and C. fulvus. These species have similar standard (2n = 26 and FN = 48) and C-banded karyotypes (Gallardo 1991Citation ). The close phylogenetic relationship based on cytochrome b between these two northern species from the highland deserts of Chile, together with their karyologic similarities, indicate that these two taxa may be conspecific (Gallardo 1979Citation ; Reig et al. 1992Citation ).

The monophyly of C. mendocinus, C. porteousi, C. australis, and Ctenomys azarae (not included here) of central and western areas of Argentina, referred to as the mendocinus group, has been noted since their karyotypes were first described (Massarini et al. 1991Citation ). Other investigations supported the inclusion of C. flamarioni and C. rionegrensis in this group (D'Elía, Lessa, and Cook 1999Citation ). Ctenomys rionegrensis also showed a strong association with the mendocinus group in our MP (fig. 1A and B ) and NJ analyses. The relationship between this last group and C. pearsoni is suggested but not strongly supported (fig. 1B ). Finally, C. talarum is possibly related to this clade. Ctenomys talarum shares its chromosomal number with species of the mendocinus group (Massarini et al. 1991Citation ), although differences in G-banding patterns and morphological characters have been noted (Ortells and Barrantes 1994Citation ).

This analysis extends previous assessments of the mendocinus group (Rossi, Reig, and Zorzópulos 1990, 1993Citation ) that supported the monophyly of the clade and suggested a close association with C. rionegrensis (D'Elía, Lessa, and Cook 1999Citation ). Our three specimens of C. mendocinus do not form a monophyletic group (figs. 1–3 ). Ctenomys mendocinus 3 exhibited a closer relationship with other species of the group than with the two other specimens of C. mendocinus. It is possible that some of the numerous tuco-tuco populations that have been assigned to C. mendocinus represent distinct but closely related forms (C. Borghi, personal communication). The northern Argentine C. tucumanus, C. argentinus, and C. latro form a monophyletic group in all analyses (figs. 1B, 2, and 3 ).

Ctenomys haigi, C. coyhaiquensis, and C. magellanicus are all distributed in southern Argentina and Chile and form a well-supported clade (figs. 1B, 2, and 3 ). These species all have asymmetric sperm morphology and may be closely related (Kelt and Gallardo 1994Citation ). This southern clade is distinct from the mendocinus group, which is the other major clade with asymmetric sperm, indicating that the derived sperm morphology (Vitullo and Cook 1991Citation ) may have evolved twice in the history of the genus, as suggested by D'Elía, Lessa, and Cook (1999)Citation . The position of C. maulinus, which also has this type of sperm, cannot be determined with certainty because this taxon collapsed to the basal polytomy (fig. 1B ). Further analysis of several new species from Patagonia (southern Argentina and Chile) may resolve the position of C. maulinus.

Evolution of RPCS Copy Number
Dramatic differences in RPCS copy numbers among species were previously demonstrated in a group of Argentine species (Rossi, Reig, and Zorzópulos 1993Citation ). We extended these analyses to Uruguayan, Bolivian, Chilean, and additional Argentinean species. Quantitative dot-blots, displayed together with the ML tree (fig. 2 ), showed species with high, medium, low, and undetectable RPCS copies. In C. opimus, C. fulvus, C. magellanicus, C. tucumanus, and C. maulinus, RPCS is virtually absent. Other species and forms, like C. steinbachi, C. minut, Ctenomys sp. ITA, Ctenomys sp. MONTE, C. argentinus, C. latro, and C. tuconax, range from low to medium copy numbers of RPCS. In the remaining species, RPCS seemed to be present in high numbers. Ctenomys haigi, a species from Argentinean Patagonia, displayed the highest RPCS content (6.60 x 106 copies), as did the species of the mendocinus group, with 3.3 x 106 to 4.69 x 106 copies.



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Fig. 2.—Maximum-likelihood tree for the ctenomyines. The numbers on the internal nodes are the RPCS copy numbers inferred by the ANCML program (see Materials and Methods) assuming a molecular clock for the cytochrome b gene. Values differing by >25% from neighbors are displayed. The first column includes the haploid value of RPCS copy number in each species obtained from dot-blot experiments. The second column displays dot-blot experiments corresponding to 10, 100, and 1,000 ng of genomic DNA hybridized to RPCS under low-stringency conditions (ND = not done). Deletions (empty triangles) and amplifications (filled triangles) of RPCS satellites greater than twofold are marked along tree branches. Species with derived asymmetrical sperm type are marked according to the criterion employed in figure 1 . The third column displays diploid and fundamental numbers (2n/FN). References are as follows: (1) Cook (1990)Citation ; (2) Gallardo (1991)Citation ; (3) Reig et al. (1992)Citation ; (4) Cook, Anderson, and Yates (1990)Citation ; (5) Anderson, Yates, and Cook (1987)Citation ; (6) Kelt and Gallardo (1994)Citation ; (7) Reig and Kiblisky (1969)Citation ; (8) Novello and Lessa (1986)Citation ; (9) Massarini et al. (1991)Citation ; (10) Ortells, Contreras, and Reig (1990)Citation ; (11) Contreras, Torres-Mura, and Spotorno (1990)Citation ; (12) J. A. Cook (unpublished data)

 
These results attest to the high variability in copy numbers of RPCS among species of Ctenomys but do not uncover the amplification or deletion events of this satellite sequence during its evolution.

Recently, a phylogenetic approach was successfully employed to analyze chromosomal number evolution in muntjac deers (Wang and Lan 2000)Citation . However, a comprehensive integration of a phylogenetic framework with chromosomal evolution, as well as copy number assessments of satDNA data, has been lacking.

In the present work, we considered RPCS copy number as a continuous character and inferred ancestral values by using an ML approach. MP-based methods, which produce estimates requiring the smallest number of changes through time, are among the most popular for inferring ancestral character states (Maddison and Maddison 1992Citation ; Schluter et al. 1997Citation ). When characters evolve rapidly and the probabilities of gains and loses are unequal, however, MP-based methods may be problematic (Cunningham, Omland, and Oakley 1998Citation ). Inevitably, uncertainties of ancestral-state estimation appear when stochastic elements are present in the evolutionary process (Schluter et al. 1997Citation ).

New approaches for reconstructing ancestral states based on ML algorithms have been developed for discrete (Schluter 1995Citation ) and continuous traits (Schluter et al. 1997Citation ). The advantages and weaknesses of ML-based methods are detailed in Schluter et al. (1997)Citation and reviewed by Cunningham, Omland, and Oakley (1998)Citation . An important feature of ML-based methods is that they provide an opportunity to quantify uncertainty. The substantial degree of variation in RPCS content suggests high rates of change in this trait. Therefore, we chose an ML approach to infer RPCS copy number for internal nodes of the ctenomyine radiation (fig. 2 ), an approach that required branch lengths to be proportional to time. We used the DNAMLK program (Felsenstein 1993Citation ) but had to assume a molecular clock due to constraints imposed by this program, even though cytochrome b does not fit constant rates of evolution in this group (Cook and Lessa l998Citation ). It is worth noting that the molecular-clock assumption affects only branch lengths, and not the topology of the ML tree (fig. 2 ), which was obtained without that constraint. Finally, errors associated with ancestral nodes were high, but they were lower than the range of variation of the values in the tips (see fig. 2 ). This relationship contrasts with values of the examples studied by Schluter et al. (1997)Citation , indicating that this method should provide a reasonable overview of the evolutionary history of RPCS.

We used our ML phylogenetic hypothesis, in combination with RPCS copy number (a continuous character), to estimate changes in RPCS through the evolutionary history of the ctenomyine. Our ML reconstruction of the history of RPCS contrasts the relatively high copy numbers found in the deep branches of the ctenomyine radiation with the comparatively low copy numbers among the octodontines (fig. 2 ). The ML tree also suggests that RPCS remained relatively stable (at ca. 1,500 x 103 copies) along most other basal branches of the tuco-tuco clade. Although this is an area of poor phylogenetic resolution, it appears that little variation in RPCS took place along these branches. Subsequently, substantial increases in the number of copies of RPCS are suggested along the branch leading to the mendocinus group, followed by relatively constant values along most of the terminal branches. Ctenomys talarum may be related to the mendocinus group (fig. 1 ); therefore, we cannot rule out the possibility that a single amplification accounts for greater copy numbers in these taxa.

Deletions also occurred throughout internal branches. For example, the ancestor of the C. opimusC. fulvus clade is inferred to have experienced a 17-fold reduction in RPCS copy number. Low amounts of RPCS were conserved along the terminal branches.

In contrast, several well-defined clades show substantial variation internally in RPCS copy numbers, implying relatively recent and rapid changes. Specifically,

  1. A reduction in RPCS copy numbers in the branch leading to C. steinbachi, in contrast to other members of the C. boliviensis clade, which retain relatively high copy numbers (fig. 2 ).
  2. Amplification of RPCS in C. haigi, in contrast to a reduction in the other members of this southern clade, C. magellanicus and, especially, C. coyhaiquensis (fig. 2 ).
  3. A reduction in copy numbers in the lineage leading to C. tucumanus, whose sister taxa, C. latro and C. argentinus, show somewhat reduced, but nonetheless greater, copy numbers of RPCS (fig. 2 ).

The broadened comparative framework provided by these analyses suggests that the history of the RPCS repeat has been extremely complex, with multiple events of gains and losses in copy number.

Evolution of RPCS Copy Number and Chromosomes in Tuco-tucos
Amplification and movement of satDNA is well documented (e.g., Hamilton, Honeycutt, and Baker 1990Citation ; Modi 1993Citation ) and has been suspected to play a role in karyotypic evolution (Wichman et al. 1991Citation ; Hamilton, Hong, and Wichman 1992Citation ; Bradley and Wichman 1994Citation ; Garagna et al. 1997, 2001Citation ; Yang et al. 1997Citation ). These processes may have dramatic consequences for chromosome architecture. For example, Garagna et al. (2001)Citation elucidated the organization of a satDNA at fusion points involved in Rb translocations in mouse chromosomes. Li et al. (2000)Citation demonstrated the involvement of two satDNAs of the muntjac deer genome in tandem fusions of chromosomes; these fusions resulted in drastic reduction in diploid numbers. These studies are in agreement with an early hypothesis of Wichman et al. (1991)Citation that suggests that intragenomic movements of repetitive sequences (studied by in situ hybridization in ancestral and derived karyotypes) promote rapid chromosomal evolution. This hypothesis was consistent with several cases of chromosomal evolution, including Microtus (Modi 1993Citation ), Reithrodontomys (Hamilton, Honeycutt, and Baker 1990Citation ), Peromyscus (Hamilton, Hong, and Wichman 1992Citation ), and Equus (Bradley and Wichman 1994Citation ). A phylogenetic context provides an opportunity to investigate the impact of satDNA dynamics on chromosomal evolution.

The ctenomyine radiation has been recognized as an exceptional model for investigating the dynamics of chromosome rearrangements (Reig et al. 1990Citation ). RPCS, a retroviral satDNA present in the genome of these rodents (Rossi et al. 199Citation 3; Pesce et al. 1994Citation ), was proposed to be associated with the extreme chromosomal variability documented for the group (Rossi et al. 1995aCitation ).

Analysis of the evolution of RPCS copy number together with chromosomal variability in lineages of Ctenomys showed two distinctive patterns. One pattern was characterized by high variability in copy numbers between closely related lineages, accompanied by substantial chromosomal variability. The other pattern consisted of rather stable copy numbers of RPCS within karyotypically stable clades. The first pattern was mirrored by three well-defined clades. RPCS copy number in the clade formed by C. tucumanus, C. latro, and C. argentinus (figs. 1–3 ) exhibited deletions, particularly conspicuous in the terminal branch leading to C. tucumanus (fig. 2 ). Many rearrangements differentiate the karyotype of C. tucumanus (2n = 28) from that of C. latro (2n = 40–42) and C. argentinus (2n = 44).

More impressive is the case of a clade of species from Patagonia Argentina composed of C. haigi, C. coyhaiquensis, and C. magellanicus (figs. 1–3 ). Both large amplifications and large deletions occurred within this group. Ctenomys haigi experienced a 2.5-fold amplification, whereas C. magellanicus and C. coyhaiquensis underwent 91- and 17-fold deletions, respectively (fig. 2 ). Once again, these amplifications and deletions were accompanied by high levels of chromosomal evolution. In this case, a largely bi-armed karyotype of low chromosomal number (C. coyhaiquensis, 2n = 28) (Kelt and Gallardo 1994Citation ) evolved into primarily uni-armed karyotypes with higher chromosomal numbers (C. haigi, 2n = 50) (Gallardo 1991Citation ). These rearrangements would have been primarily fissions and would have been accompanied by significant RPCS expansion (fig. 2 ). Conversely, a hypothesis implying the loss of RPCS and a series of chromosomal fusion events in the lineage leading to C. coyhaiquensis is also plausible.

Although the close relationship between C. steinbachi (2n = 10) and the group formed by Ctenomys sp. ROBO, C. goodfellowi, and C. boliviensis is not strongly supported (figs. 1 and 2 ), previous studies (Cook and Yates 1994Citation ; Cook and Lessa 1998Citation ) indicate that these species are related. Ctenomys steinbachi is thought to have undergone rapid and extensive chromosomal evolution from karyotypes of higher diploid numbers (Lessa and Cook 1998Citation ). This dramatic chromosomal evolution is associated with a 46-fold RPCS deletion (fig. 2 ).

Stasis in copy number associated with karyotype stability characterized the second pattern of RPCS evolution and was observed in the mendocinus group and in the C. opimusC. fulvus clade. RPCS content is uniformly high in all members of the mendocinus group. Ancestral character state analysis suggested that significant amplification in RPCS occurred in the ancestor of this group, followed by stasis throughout subsequent branches (fig. 2 ). The clade containing C. opimus and C. fulvus also displayed chromosomal and RPCS stability. Both species have 2n = 26, as well as similar C-banded karyotypes (Reig and Kiblisky 1969Citation ; Gallardo 1979, 1991Citation ) and almost undetectable amounts of RPCS. The inferred RPCS value for the base of this clade is large (85,000 copies; fig. 2 ), and it is therefore possible that a larger deletion occurred prior to this node, maintaining the RPCS low copy number constant throughout all of the branches of this clade (fig. 2 ). However, the high ancestral value for the clade may be overestimated because the method estimates ancestor state as a weighted average of the neighboring tips (Schluter et al. 1997Citation ).

Finally, the clade composed of C. frater, C. lewisi, Ctenomys sp. LLATHU, and C. conoveri shows relatively stable amounts of RPCS (except in Ctenomys sp. LLATHU). Diploid numbers are also fairly constant (52 for C. frater, 56 for C. lewisi, and 48–50 for C. conoveri), but a comprehensive picture of this clade is lacking because Ctenomys sp. LLATHU has not been karyotyped.

Several models relating chromosomal evolution to amounts of heterochromatin have been proposed for the distinctive patterns that have been described. For example, Patton and Sherwood (1982)Citation investigated the patterns of heterochromatin variation in rodents of the genus Thomomys and argued that large amounts of heterochromatin do not appear to promote rearrangements, as proposed by Hatch et al. (1976)Citation for kangaroo rats (genus Dipodomys). Other investigators have suggested that heterochromatic differences might cause reproductive isolation (Yunis and Yasmineh 1971Citation ), although Patton and Sherwood (1982)Citation and others (Rice and Straus 1973Citation ; Dev et al. 1975Citation ; John and Miklos 1979Citation and papers therein) provided examples in which this does not occur. We found that clades that exhibited karyotypic stability contained high, as well as low, RPCS content. On the other extreme, the clade from Corrientes Province with high chromosomal variability showed few C-bands (Reig et al. 1992Citation ). The existence of many different scenarios of satDNA and chromosome variation seems to indicate that the influence of repetitive DNA on chromosomal evolution does not rely simply on copy number. Rather, our analyses of tuco-tucos suggest an association between changes in karyotypes and changes in RPCS copy number (either increase or decrease). Conversely, we found that the stability of RPCS copy number was tied to chromosomal stability. We speculate that only satDNA that is actively involved in processes of expansion, contraction, and mobilization is likely to promote changes in karyotype morphology. If so, an "active" satDNA could promote chromosomal change even if it is present at a low copy number. Thus, the molecular nature of each particular type of satDNA probably determines its potential for promoting chromosomal rearrangements. The potential distinction between active and stabilized satDNA and their differing roles in relation to chromosomal change is, as noted, speculative. Further analyses should include both detailed reconstructions of actual chromosomal events (using chromosome banding or in situ methods) and more detailed characterization of the status of satDNA.

We have shown that a phylogenetic perspective has improved our understanding of not only chromosomal evolution, but also the related and complex history of RPCS across this diverse mammalian clade. Indeed, inferences concerning radical changes (or stability) across the phylogeny were key to assessing the relationship between amplifications and/or deletions of RPCS and chromosomal evolution in tuco-tucos. Clearly, further phylogenetic work is needed to explore uncertainty at or near the base of the tuco-tuco tree, which could not be resolved by cytochrome b sequences. Other markers should be incorporated to test the clades proposed by these analyses and to investigate the early evolutionary history of this chromosomally diverse clade.



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Fig. 3.—Neighbor-joining tree for ctenomyines. Numbers above the nodes are the bootstrap values of 1,000 pseudoreplicates (single random-addition sequence), with values >50% shown. Clades with bootstrap values above 50% are highlighted. Lineages with derived asymmetrical sperm type are marked according to the criterion employed in figure 1

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
We thank Milton Gallardo, Eileen Lacey (from the Museum of Vertebrate Zoology, Berkeley, University of California), Carlos Quintana, Angel Spotorno, the Field Museum of Natural History, Chicago, and the Museo Municipal de Historia Natural "Lorenzo Scaglia," Mar del Plata, Argentina, for providing of tissues and for identification of specimens. Brandy Jacobsen and Tom LeCroy provided excellent assistance in the sequencing laboratory. This work was supported by grants from the University of Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina; the National Science Foundation, United States; and Consejo Nacional de Investigaciones Científicas y Técnicas, and CSIC-Universidad de la República, Uruguay. The authors wish to dedicate this work to the memory of Professor Osualdo A. Reig, a dear teacher and a great evolutionary biologist who died in 1992.


    Footnotes
 
Simon Easteal, Reviewing Editor

1 Keywords: satellite DNA chromosomal evolution cytochrome b phylogenetics Back

2 Address for correspondence and reprints: M. Susana Rossi, Laboratorio de Fisiología y Biología Molecular, Facultad de Ciencias Exactas y Naturales, Departamento de Ciencias Biológicas, Pabellón II, Ciudad Universitaria, C1428EHA, Buenos Aires, Argentina. srossi{at}bg.fcen.uba.ar Back


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Accepted for publication May 17, 2001.