Comparative chromosomal analysis and phylogeny in four Ctenomys species (Rodentia, Octodontidae)

Biological Journal of the Linnean Society (2000), 69: 103–120 With 8 figures doi: 10.1006/bijl.1999.0314, available online at http:/ /www.idealibrary.com on

Comparative chromosomal analysis and phylogeny in four Ctenomys species (Rodentia, Octodontidae) ` 1,2∗, J. EGOZCUE1,2 AND M. GARCIA1,2 L. GARCIA1, M. PONSA 1

Departament de Biologia Cellular i de Fisiologia, and 2Institut de Biologia Fonamental ‘Vicent Villar Palasi’, Universitat Auto`noma de Barcelona, E-08193 Cerdanyola, Spain Received 5 January 1998; accepted for publication 12 January 1999

A cytogenetic chromosome study was carried out on specimens of four species of Ctenomys—C. talarum, C. rionegrensis, C. pearsoni and C. dorbignyi—from 10 different populations. The analysis of chromosomes was performed through sequential uniform stain, G and C-banding, and with restriction enzymes. The results obtained are discussed in relation to phylogeny. Chromosome evolution in the species studied suggests that the chromosome number has increased due to fissions, and that a reduction of the amount of constitutive heterochromatin has occurred. Different types of heterochromatin, with different patterns, have been added in parallel during evolution. Some taxonomic suggestions can be deduced from this cytogenetic study.  2000 The Linnean Society of London

ADDITIONAL KEY WORDS:—cytogenetics – heterochromatin – banding patterns – taxonomy. CONTENTS

Introduction . . . . . . Material and methods . . Results . . . . . . . Description of karyotypes Comparative analysis . Phylogenetic studies . . . Discussion . . . . . . Conclusions . . . . . . Acknowledgements . . . References . . . . . . Appendix . . . . . . .

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103 104 105 105 110 111 113 117 117 117 119

INTRODUCTION

Ctenomys is a genus of caviomorph subterranean rodents that includes the highest number of species in the Family Octodontidae. Colloquially known as ‘tuco-tucos’ because of their territorial noise, they are widely distributed in South America between parallels 15 and 54 in latitude south. ∗ Corresponding author. E-mail: [email protected] 0024–4066/00/010103+19 $35.00/0

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 2000 The Linnean Society of London

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T 1. Species, locality of recollection, number and sex of specimens studied Species C. talarum talarum

C. talarum recessus C. rionegrensis C. pearsoni C. dorbignyi

Locality

Province

No

Magdalena El Saladillo El Guanaco Camet Norte Necochea Ibicuy Parana´ Me´danos Mbariguı´ Sarandicito

Buenos Aires Buenos Aires La Pampa Buenos Aires Buenos Aires Entre Rios Entre Rios Entre Rios Corrientes Corrientes

2 6 4 4 8 6 3 6 1 1

No ℑ No∪ – 2 1 1 3 3 – 6 – –

2 4 3 3 5 3 3 – 1 1

Karyotypic differentiation among species of Ctenomys is one of the greatest found within a single genus of mammals, their diploid and fundamental numbers varying from 10 to 70, and from 16 to 84 respectively (Reig, 1989; Reig et al., 1990; Cook, Anderson & Yates, 1990). This is the reason why Ctenomys has been considered a typical example of explosive speciation related to a high number of chromosomal reorganizations; this radiation occurred during the Pleistocene (Reig et al., 1990). Furthermore, different groups of species behave differently with regard to the type of chromosomal repatterning and the amount and distribution of heterochromatin (Reig et al. 1992). There are species with the same karyotype, for example those designated by Massarini et al. (1991) as the mendocinus group with five species—C. mendocinus, C. azarae, C. australis, C. chasiquensis and C. porteousi—while other species show intraspecific chromosomal variations as has been described in C. flamarioni (Freitas, 1994) and in C. talarum (Massarini et al., 1995). In this paper, we describe the chromosomal characteristics: morphology, banding patterns (G, C and restriction enzyme bands) and heterochromatin variations, of four species of Ctenomys (C. talarum, C. rionegrensis, C. pearsoni and C. dorbignyi ) from ten different localities in four Argentinian provinces. We analyse chromosome homologies among species taking into account euchromatin and heterochromatin characteristics. We analyse chromosome evolution in the species of the genus and the possible implications of reorganizations and heterochromatin variations detected in the speciation processes of these species. We also describe phylogenetic relationships among the species of this group. Taxonomic implications from the cytogenetic study are suggested.

MATERIAL AND METHODS

Cytogenetic analyses were performed in 25 male and 16 female specimens of four species of Ctenomys (C. talarum, C. rionegrensis, C. pearsoni, and C. dorbignyi) from 10 different populations of the Argentinian provinces of Corrientes, Entre Rios, Buenos Aires and La Pampa (Table 1). These specimens were identified by the taxonomist Tito Sccaglia of the Museo de Ciencias Naturales, Mar del Plata, and voucher specimens were deposited in the Museo Nacional de Ciencias Naturales (CSIC, Madrid, Spain). Specimens were caught using tubular plastic traps or snaps (Oneida Victor no. 0), transported to the laboratory at the University of Buenos Aires (Buenos Aires, Argentina) and then processed. Bone marrow metaphase chromosomes were obtained as described by Ford & Hamerton (1956). Sequential uniform staining, G and C-bands were obtained using adapted procedures of Seabright (1971) and Sumner (1972) respectively.

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The restriction enzymes (REs) used for chromosome digestion were Alu I, Hae III and Sau 3A (Boehringer). Incubation solutions were prepared by dissolving 30 units of each enzyme in 100 ll of the corresponding incubation buffer (Gosa´lvez et al., 1989). The enzyme mixture was placed on the slide. Digestion was carried out in a humidified chamber at 37°C overnight. The preparations were stained for 4 min with Leishman (10%). Parsimony analysis (PAUP 3.1.1, Swofford 1993) was used to construct a phylogenetic tree. A heuristic search was conducted to find the most parsimonious constrained tree. Subsequently majority-rule consensus trees were computed for comparison of the different original trees. The study was based on the G, C and restriction enzymes banded karyotypes. The karyotype characters used were: (a) chromosome characters: whole chromosome homologies or whole arm homologies; (b) rearrangement characters: fusion-fission variants, presence or absence of pericentromeric inversions and translocations; (c) presence or absence of B chromosomes; (d) presence or absence of C-bands in homologous chromosomes of different species; (e) presence or absence of C-like bands with restriction enzymes in homologous chromosomes of different species; (f ) presence or absence of mosaicism in any of the previous characters. Data matrix (available from the authors on request) was established by assigning a value ‘1’ or ‘0’ for the presence or absence of each of 179 characters (Appendix). Characters referring to the heterochromatin in homologous chromosomes and mosaicisms had a weight of 0.25 in relation to the rest of the characters. Data from specimens of C. perrensi (Garcia et al., submitted) are also included in this analysis. Specimen #1072 has been considered as the outgroup. This specimen presents the cytogenetic characteristics described by Massarini et al. (1991) as the karyotype pattern for this C. talarum species. It has a stable karyotype with 2n=48 chromosomes and FN=84. If other species or other specimens of C. talarum with different chromosomal characteristics were considered as the outgroup, the phylogenetic tree presented longer distances.

RESULTS

Description of karyotypes In this paper we describe for the first time sequential uniform-G-C banded karyotypes in Ctenomys species. This technique allowed the identification and characterization of the chromosome variants present in the species of the genus. Ctenomys talarum, Thomas (1898) Two subspecies, with alopatric distribution, have been described: C. talarum talarum and C. talarum recessus (Contreras & Reig, 1965). In our study specimens of the subspecies C. talarum talarum from Magdalena, El Saladillo and Camet Norte had a diploid number of 2n=48 and FN=81–84 (Table 2) (Massarini et al., 1991). Specimens of C. talarum recessus from Necochea had a diploid number 2n=50 and FN=81–82. Specimens from El Guanaco were not taxonomically identified at the time of collection; morphological characters and cytogenetic characteristics indicated that the subspecies was C. t. talarum with 2n= 48 and FN=83–84 (Table 2). Differences in FN are not due to the presence or absence of heterochromatic arms as described in Reig et al. (1992).

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T 2. Details of the karyotypes of the twenty specimens of C. talarum studied. Differences are due to the presence of chromosome pair types 1a/1b/1c, 2a/2b, 4a/4b/4d, 7a/7b and 11a/11b/11c′ Locality Magdalena

El Saladillo

Camet Norte

Necochea

El Guanaco

No 1056 1057 1071 1072 1073 1074 1076 1077 1107 1108 1109 1110 1097 1098 1099 1100 1101 1103 1104 1106 1080 1081 1082 1085

Karyotypes ∪ ∪ ℑ ∪ ∪ ∪ ℑ ∪ ℑ ∪ ∪ ∪ ℑ ∪ ∪ ∪ ∪ ℑ ℑ ∪ ∪ ∪ ∪ ℑ

2n=48 2n=48 2n=48 2n=48 2n=48 2n=48 2n=48 2n=48 2n=48 2n=48 2n=48 2n=48 2n=50 2n=50 2n=50 2n=50 2n=50 2n=50 2n=50 2n=50 2n=48 2n=48 2n=48 2n=48

(1c 2a 4a 7a 11b) (1a 2a 4a 7a 11b) (1c 2a 4a 7a 11a) (1a 2a 4a 7a 11a) (1a 2a 4a 7a 11a)+t (5;13) (1a 2a 4a 7a 11a) (1a 2a 4a 7a 11a); (1a 2a 4b 7a 11a) (1b 2b 4d 7b 11a) (1a 2a 4a 7a 11a) (1a 2a 4a 7a 11a) (1a 2a 4a 7a 11a) (1a 2a 4a 7a 11a) (1c 2a 4b 7a 11c′) (1c 2a 4b 7a 11c′) (1c 2a 4a 7a 11c′) (1c 2a 4a 7a 11c′) (1c 2a 4a 7a 11c′) (1c 2a 4a 7a 11c′) (1c 2a 4a 7a 11c′) (1c 2a 4a 7a 11c′) (1a 2a 4a 7a 11a) (1a 2a 4a 7a 11a)/(1a 2a 4b 7a 11a) (1a 2a 4a 7a 11a) (1a 2a 4a 7a 11a)

FN 81 83 82 84 83 84 83–84 82 84 84 84 84 81 81 82 82 82 82 82 82 84 83–84 84 84

a - pairs are supposed to be the original homozygotic forms b - pairs present a fission in one member of the pair c - pairs present a fission in two members of the pair c′ - pairs present a fission in two members of the pair but the short arms are maintained as a telocentric chromosome pair d - pericentric inversion in one chromosome of pair 4 Specimen 1073 shows a translocation 13/5.

In C. talarum the diploid number (2n), FN and chromosome morphology are variable; polymorphisms are due to chromosomal reorganizations. Chromosomal variations observed in C. talarum are intraindividual, intrapopulational and interpopulational. The polymorphisms and chromosomal reorganization detected by us are summarized in Table 2 and illustrated in Figure 1. The only cytogenetic characteristic found in this study that can identify individuals of the subspecies C. talarum recessus from C. talarum talarum is the presence of a fission affecting pair 11 (pair 11c′) producing two telocentric chromosome pairs (Fig. 1) (in disagreement with Massarini et al. [1991]) who considered the difference due to the presence of two microchromosomes). The existence of specimens with two different cell lines can be explained by a post-zygotic mechanism of somatic segregation (Garcia et al., submitted). The original zygote is supposed to be heterozygotic for chromosome #4 and after a mechanism of differential segregation; one cell line with the original form and a second cell line with one of the newly produced forms, coexist in mosaic. Reig et al. (1992) described variations in the heterochromatin of C. talarum; in our study intraindividual, intrapopulational and interpopulational heterochromatin variability is observed in Ctenomys talarum. With regard to chromosomes presenting fissions, in specimens of C. talarum talarum from Magdalena heterochromatin is maintained, while in specimens from El Saladillo (as in C. talarum recessus specimens from Necochea) heterochromatin is not present. G+ C− heterochromatin is observed in pairs 1b, 2b, 4b, 7b and 11b, G+ C+ is observed in pairs 2b, 7b and 1c.

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Figure 1. Polymorphic pairs of C. talarum sequentially G–C-banded. Chromosome pairs 1a, 2a, 4a, 7a and 11a are supposed to be the originals; pairs 1b, 2b, 4b, 7b and 11b and 1c appear to be the result of one (b) or two (c) chromosomes fissioning respectively. Chromosome pair type 11c′ would result from both chromosome-fissioned short and long arms becoming telocentric chromosomes. In chromosome pair type 4d one of the chromosomes that has lost its short arm shows a pericentric inversion.

Treatment of metaphases with restriction enzymes produced different C-like patterns (Fig. 2). Using Sau 3A intraindividual polymorphisms are observed and with Hae III, a terminal C-like band can be observed in the short arm of pair 1 (Fig. 2). According to these results different types of (REs+) heterochromatin can be observed in this species: four correspond to constitutive C+ heterochromatin regions and two are C− (Table 3). Ctenomys rionegrensis Langguth and Abella (1970) Nine specimens were collected in two different regions of the province of Entre Rios (Table 1). No chromosomal differences or polymorphisms were observed when comparing specimens from both localities. The diploid number observed is 2n=50 and FN=72 (Fig. 3). Ortells et al. (1990) described 2n=50 FN=68–70 with heteromorphisms and 2n=52 FN=72 specimens analysing, in both cases, unbanded chromosomes. Centromeric C bands are only observed in pairs 3, 6, 7, 8, 9, 10, 13, 14 and in the X chromosome. Pairs 1, 2, 4, 5 have a large centromeric band that can affect the short arms. Only non centromeric C bands are polymorphic in size (Fig. 4). Reig et al. (1992) described two specimens

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Figure 2. C. talarum karyotype after (A) Alu I treatment, (B) Sau 3A treatment and (C) Hae III treatment. Alu I C-like bands are observed in pairs 1, 13, 14, 15, 20 and chromosome Y. Sau 3A and Hae III C-like bands are observed in almost all chromosome pairs.

with 2n=50 and NF=68–70, they have a higher number of chromosomes presenting heterochromatin. Digestion with restriction enzymes Alu I and Sau 3A produces C-like bands in the same chromosomes that are C+ (Fig. 4). According to our results C. rionegrensis has two different types of C+ heterochromatin (Table 3).

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T 3. Heterochromatin patterns in Ctenomys species Species

C

Alu I

Sau 3A

Hae III

C. talarum

+ + + + − − + + + + + + −

+ − − − − + + − + − ? + +

+ + + − − − + − + − + ? ?

+ + − + + − ? ? ? ? ? ? ?

C. rionegrensis C. perrensi C. pearsoni C. dorbinyi ?=no data available.

Figure 3. G-banded C. rionegrensis karyotype. The basic karyotype includes seven pairs of submetacentrics (pairs 1–7), three pairs of metacentrics (pairs 8–10) and 14 pairs of medium and small sized (11–24) acrocentrics. The X chromosome is metacentric, and the Y is medium submetacentric.

Ctenomys pearsoni Lessa and Langguth (1983) Ctenomys dorbignyi Contreras and De Contreras (1984) C. pearsoni and C. dorbignyi have a constant karyotype with the same diploid number 2n=70 and FN=88 and identical G banding patterns (Fig. 5). The diploid number of C. dorbignyi, but not the morphology of the chromosomes, coincides with the description of Ortells et al. (1990). From the cytogenetic point of view both species are equivalent. The only difference observed is in C bands; where the C+ band present in 3p of C. pearsoni is C– in C. dorbignyi. In C. pearsoni we observe a polymorphism in pair 3 (Fig. 5). In C. dorbignyi sequentially stained C banded chromosomes, all centromeric regions are C−; while if C bands are obtained in chromosomes not treated previously all centromeres are faintly C+ and Yq is strongly stained. In specimens of C. dorbignyi studied by Reig et al. (1992) results are different. In C. pearsoni digestion with restriction enzyme Sau 3A produces C-like bands in the same C+ banded chromosomes with the same polymorphism in pair 3 (Fig. 5). According to these results only one type of C+ heterochromatin is detected (Table 3). In C. dorbignyi digestion with restriction enzyme Alu I produces C-like centromeric

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Figure 4. C. rionegrensis karyotype after (A) Alu I treatment and (B) Sau 3A treatment. Observe that Alu I and Sau 3A produces C-like bands in the same chromosomes.

bands in all chromosomes and the Y chromosome is fully stained. According to these results two types of C+ heterochromatin are detected (Table 3). Comparative analysis Karyotypes of the different species studied have been compared in order to analyse chromosome evolution in the genus Ctenomys. C. perrensi (Garcia et al., submitted) has been included in the study of chromosome homologies (Fig. 6). Comparisons are always referred to C. talarum (Table 4). The results of comparative studies show that chromosomes 2, 4, 5, 11 and X of C. talarum are present in all species studied. Different reorganizations have been detected in pair 2. Morphologies observed in C. perrensi and C. rionegrensis can be related with the morphology of C. azarae (unpublished results) by different inversions. Morphologies observed in C. talarum and in C. dorbignyi can be obtained from C. rionegrensis by different rearrangements (Fig. 7). There is a considerable variability in the distribution and in the amount of heterochromatin (Table 4). C. talarum has centromeric C bands in all chromosomes, C. rionegrensis in 50% of the chromosomes, and in C. pearsoni and C. dorbignyi C+ bands are rare. In the Y chromosome the heterochromatin has different distributions;

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Figure 5. C. pearsoni karyotype sequentially stained. (A) uniform, (B) G-banded, (C) C-banded, (D) shows the polymorphism observed in pair 3.

in C. dorbignyi the Y chromosome is fully heterochromatic, the centromere and the short arm are C+ in C. talarum, heterochromatin is telomeric in C. perrensi (Garcia et al., submitted), and is absent in the Y chromosome of C. rionegrensis and C. pearsoni. The X chromosome shows centromeric heterochromatin in C. talarum, and C. dorbignyi; no C+ centromeric band is present in C. pearsoni. Analysing the localization and the subtype of heterochromatin in different species, intraindividual (C. rionegrensis) and intraspecific (C. rionegrensis, C. talarum and C. pearsoni) variability is observed. Taking into account Alu I and Sau 3A bands, the subtypes of heterochromatin observed in C. rionegrensis are also found in C. talarum and in C. perrensi, showing that they are common in these species (Table 3). Homologous chromosomes show different patterns of heterochromatin localization—presence or absence of C bands—(Tables 4 and 5) showing that while G bands are conserved the heterochromatic bands are not, thus homologous chromosomes that conserve G bands (Table 4) do not always conserve the localization of the heterochromatin; even in C. pearsoni and C. dorbignyi, which have equivalent G banded karyotypes, the distribution of the heterochromatin in the sex chromosomes is different. Results obtained after RE treatments show that homologous chromosomes in different species do not conserve the same RE pattern. PHYLOGENETIC STUDIES

Phylogenetic relationships among specimens of different species have been studied using PAUP (Swofford, 1993).

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Figure 6. Comparative haploid karyotype of C. t=Ctenomys talarum and homologous chromosomes of C. r=Ctenomys rionegrensis, C. p=Ctenomys perrensi, C. pe=Ctenomys pearsoni and C. d=Ctenomys dorbignyi.

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T 4. Chromosome homologies showing the presence (+) or absence (−) of C bands in homologous chromosomes observed in the species studied. C. t=Ctenomys talarum; C. r=Ctenomys rionegrensis; C. p=Ctenomys perrensi; C. pe=Ctenomys pearsoni; C. d=dorbignyi C. t

C. r

No

C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 X

+ + + + + + + + + + + + + + + + + + + + + + + +

Y

+

C. p

C. pe

C. d

No

C

No

C

No

C

No

C

1

+

−

10

−/+

+ + +

9 1

− −

9 1

−/+ −/+

13 9

− +

− − + + + − −

10

4 3 2

5 10 1 3 2 8 7

8

+

14 4q

− +

6 2

± −

6 2

±/+ −/+

12 13 15 16

− − − −

7

±

7

±/+

12 19

− −

12 19

−/+ −/+

X

−

X

−/+

18

−

18

−/+

Y

−

Y

+/+

10 11 17 19

+ − − −

X 6 18

+ + −

Y

−

X 6

Y

− −

+

Three clusters are observed (Fig. 8). (I) C. talarum, (II) C. rionegrensis, C. pearsoni and C. dorbignyi, (III) C. perrensi. In cluster I we observe that all specimens of C. talarum recessus are included in the same subcluster. Clusters II and III have the same phylogenetic distance to (I). The species C. pearsoni and C. dorbignyi are closer than any other species analysed in this work, followed by C. talarum and C. perrensi. The longest distance observed is between C. dorbignyi and C. talarum. Cluster III accumulates the greatest number of variations inside a group; the number of variations is even higher than in cluster II, which includes specimens from three different species. DISCUSSION

Our results confirm the extensive chromosomal variability already described in the species of Ctenomys (Reig & Kiblisky, 1969; Kiblisky et al., 1977; Freitas & Lessa, 1984; Anderson et al., 1987; Cook et al., 1990; Ortells et al., 1990; Gallardo, 1991; Massarini et al., 1991; Freitas, 1994). The species studied show an important heterogeneity in relation to the number of chromosomes, FN, chromosome morphology and banding patterns.

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Ctenomys azarae (cr. 1) Paracentric inversion

Ctenomys rionegrensis (cr. 1)

Pericentric inversion, p (–)

Ctenomys perrensi (cr. 1)

Pericentric inversion, p (–)

Ctenomys talarum (cr. 2) p (–)

Ctenomys pearsoni (cr. 10)/Ctenomys dorbignyi (cr. 10)

Figure 7. Chromosome reorganizations affecting the homologous of pair 2 of C. talarum in Ctenomys rionegrensis, C. azarae, C. perrensi, C. pearsoni and C. dorbignyi species.

Two patterns of cytogenetic behaviour are present in Ctenomys species; one corresponds to the species with a stable karyotype, C. rionegrensis, C. pearsoni and C. dorbignyi, and the other to species with intraspecific and intraindividual chromosome variations as observed in C. talarum and C. perrensi. C. talarum shows different types of chromosomal reorganizations: mainly fissions and occasionally inversions, translocations, and deletions. We found eight different karyotypes in five different localities. In C. perrensi we only observed fissions (Garcia et al., submitted). Deletions of short arms occur after fissions in C. talarum while in C. perrensi short arms are maintained as small telocentric chromosomes. In conclusion, fissions are a common chromosomal rearrangement in C. talarum, affecting pairs 1, 4 and 11. Changes in chromosomes 2 and 7 may be specific

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T 5. Heterochromatin patterns in the homologous pairs #2, 4, 5, 11 and X of C. talarum also present in the other species analysed C. talarum

C. rionegrensis

C. perrensi

C. pearsoni

C. dorbignyi

C Alu I Sau 3A

#2 + − +

#1 + + −

#5 − − +

#10 − ? −

#10 − + ?

C Alu I Sau 3A

#4 + − +

#4 + + +

#1 + + +

#9 − ? −

#9 − + ?

C Alu I Sau 3A

#5 + − +

#3 + + +

#3 + + +

#1 − ? −

#1 − + ?

C Alu I Sau 3A

#11 + − +

#8 + + +

#4q + + +

#2 − ? −

#2 − + ?

C Alu I Sau 3A

#X + − +

#X + + +

#X − − −

#X − ? −

#X − + ?

?=no data available.

to one locality (El Saladillo). According to our findings (Fig. 8), C. talarum specimens from Necochea corresponding to the subspecies C. talarum recessus are all grouped in the same subcluster. C. pearsoni and C. dorbignyi specimens are all grouped in the same cluster. While we have made no conventional morphological comparisons among the members of this group to test its homogeneity, we observe that all of them have the same chromosomal complements and karyotype, as also observed by Ortells et al. (1990); consequently, from the cytogenetic point of view there are insufficient reasons to consider them different taxa. Species with a high number of chromosomes (C. rionegrensis, C. pearsoni and C. dorbignyi) have a high number of acrocentrics (75%, 75% and 56% respectively); this can be adduced to support the hypothesis that fissions are among the most important reorganizations in the chromosome evolution of Ctenomys (Ortells, 1995). C. rionegrensis, C. pearsoni and C. dorbignyi, after a period of chromosome rearrangement, now have a stable karyotype. By contrast, C. talarum and C. perrensi could now be in a speciation process. Species of Ctenomys behave quite differently as regards the amount and type of heterochromatin in their chromosomal complement. A reduction of the amount of constitutive heterochromatin C+ has occurred in parallel with fissions during evolution. RE treatments indicate that the characteristics of the heterochromatin are not conserved in the homologous chromosomes even when G bands are conserved (Table 5); this means that heterochromatin variations are independent of euchromatin evolution, as has already been demonstrated, in our laboratory, in primate species by Garcia et al. (1999). In C. talarum specimens it is not possible to associate a specific RE banding pattern of heterochromatin with a taxonomic subspecies or with a special geographic localization, because some patterns are widely expanded among different populations. It cannot be discounted that the different behaviour of

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100

99 100

96 61

100

100 100 100 100 100 100

100 100

100 90 100

100

100

100 100

87

100

91 100 91

1056 1097 1098 1099 1100 1101 1103 1104 1106 1071 1073 1077 1057 1072 1074 1076 1085 1107 1108 1109 1110 1080 1081 1082 1058 1059 1060 1061 1062 1068 1160 1162 1164 1063 1064 1065 1145 1166 1066 1067 1069 1134 1137 1140 1132 1141 1133 1149 1147 1152 1156 1153 1146 1148 1151 1159

C. talarum recessus

I

C. talarum talarum

C. rionegrensis

C. pearsoni

II

C. dorbignyi C. pearsoni

C. perrensi

III

Figure 8. Phylogenetic tree obtained by PAUP 3.1.1. (Swofford, 1993). A total of 179 characters has been used in the analysis: 103 chromosome, 11 rearrangement, 3 B chromosome, 57 heterochromatinrelated and 5 mosaic. We used specimen #1072 of C. talarum as the outgroup. Numbers at the end of the branches indicate specimens and on the branches indicate phylogenetic distances.

heterochromatin may have a significance in chromosomal evolution in these species. C. dorbignyi, extant in Corrientes province in areas which are periodically flooded, has a stable karyotype. C. talarum from Buenos Aires and La Pampa provinces shows an important number of variations (Table 2); in this case, chromosomal variations are not related to the stability of the habitat. Reig and Kiblisky (1969) proposed that chromosomal reorganizations associated with the special characteristics of the structure of populations could be responsible for the strong diversification of Ctenomys. Chromosomal reorganization can play an

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important role in future speciations, although we cannot rule out the possibility that the polymorphisms observed can subsist without acting as reproductive barriers. No data are available with respect to the structure of the populations of these species or to the reproductive success in Ctenomys, and no meiotic studies have been made. As Patton & Sherwood (1982) and Rahn et al. (1996) have suggested, we cannot exclude the possibility that some changes could be related to speciation while others could be maintained as stable polymorphisms.

CONCLUSIONS

(1) Different cytogenetic patterns are observed in Ctenomys. One group of species presents a stable karyotype: C. rionegrensis, C. pearsoni and C. dorbignyi. C. talarum, together with C. perrensi, presents an unstable karyotype, Those characteristics are not related to the stability of the habitat. (2) The heterochromatin distribution in Ctenomys presents interspecific, intraspecific, interpopulational, intrapopulational and intraindividual variations. The use of restriction enzymes reveals that the heterochromatin characteristics are not uniform. (3) Fissions seem to have played an important role in chromosome speciation of the genus Ctenomys. (4) From the cytogenetic point of view there are insufficient reasons to consider C. pearsoni and C. dorbignyi as different taxa; the latter probably has to be included within the former. (5) Cytogenetic characteristics that differentiate C. talarum talarum and C. talarum recessus support the taxonomic classification that consider them as two different subspecies.

ACKNOWLEDGEMENTS

This paper is dedicated to the memory of the late Professor Osvaldo Reig. Financial support was received from DGICYT (PB/0144), CSIC no. 072-1991 and ICI 1992-93-94 (Ministerio de Educacio´n y Ciencia, Spain). We thank the Grupo de Investigaciones en Biologia Evolutiva (GIBE, University of Buenos Aires, Argentina) for its help in collecting and preparing the samples for this study.

REFERENCES

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CHROMOSOMAL ANALYSIS OF CTENOMYS

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APPENDIX

Description of 179 characters used in the matrix to construct the phylogenetic tree in Figure 8. Matrix data available from the authors on request. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 38 40 41 42 43 44 45 46 47 48 49 50 51 52

Character C.t. 1p C.t. 1q C.t. 1p C.t. 1q C.t.1,p+1q fusion C.t. 2p C.t. 2q C.t. 2, 2p+2q fusion C.t 2, inversion and loss of the short arms of R C.t 2, metacentric by pericentric inversion C.t. 3, 3p+3q fusion C.t. 4p C.t 4q C.t 4, 4p+4q fusion mosaicism 4a, 4b C.t 4, inversion and loss of the short arm C.t 5 C.t 5q translocation with chromosome 13 C.t 6 C.t 7p C.t 7q C.t 7 C.t 8 C.t 9 C.t 10 C.t 11p C.t 11q C.t 11p C.t 11q C.t 11, 11p+11q fusion C.t 12 C.t 13 C.t 13 translocated C.t 14 C.t 15 C.t 16 C.t 17 C.t 18 C.t 19 C.t 20 C.t 21 C.t 22 C.t 23 C.t X C.t Y C.t 5 C.r 6 C.r 7 C.r 12 C.r 14 C.r 15 C.r 16

No. 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104

Character C.r 18 C.r 20 C.r 21 C.r 22 C.r 23 C.r 24 C.r Y C.r 1p C.p 1q C.p 1p C.p 1q mosaicism for chromosome 1 C.p 1p C.p 2q C.p 2p C.p 2q C.p 4p C.p 4q C.p 4p C.p 4q C.p 4q+p C.p 7p C.p 7q C.p 7p C.p 7q C.p mosaicism for chromosome 7 C.p 8q mosaicism for chromosome 8 C.p 9 C.p 11 C.p 17 C.p 18 C.p 19 C.p 20 C.p 21 C.p 22 C.p 23 C.p 24 C.p Y C.p +1 chromosomes B C.p +2 chromosomes B C.p +4 chromosomes B mosaicism with/without chromosome B C.pe 3 C.pe with rearrangement C.pe 4 C.pe 5 C.pe 8 C.pe 11 C.pe 13 C.pe 4 C.pe 15

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No. 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179

Character C.t 8 Sau 3A+ C.t 9 C+ C.t 9 Alu I+ C.t 9 Sau 3A+ C.t 10 C+ C.t 10 Alu I+ C.t 10 Sau 3A+ C.t 11 C+ C.t 11 Alu I+ C.t 11 Sau 3A+ C.t 14 C+ C.t 14 Alu I+ C.t 14 Sau 3A+ C.t 15 C+ C.t 15 Alu I+ C.t 15 Sau 3A+ C.t 16 C+ C.t 16 Alu I+ C.t 16 Sau 3A+ C.t 17 C+ C.t 17 Alu I+ C.t 17 Sau 3A+ C.t 18 C+ C.t 18 Alu I+ C.t 18 Sau 3A+ C.t 19 C+ C.t 19 Alu I+ C.t 19 Sau 3A+ C.t X C+ C.t X Alu I+ C.t X Sau 3A+ C.r 6 C+ C.r 6 Alu I+ C.r 6 Sau 3A+ C.r 18 C+ C.r 18 Alu I+ C.r 18 Sau 3A+