Bidau, Et Al. 2003. Historia Natural, II (8), 53-72.

C. J. Bidau, D. A. Mart and M. D. Jimnez, 2003.
Two exceptional South American models for the study of chromosomal evolution: the tucura Dichroplus pratensis and the tuco-tucos of the genus Ctenomys.
Historia Natural (Segunda Serie) - Vol. II (8): 53-72. Buenos Aires, 2003.
ISSN 0326-1778
TWO EXCEPTIONAL SOUTH AMERICAN MODELS FOR THE STUDY OF CHROMOSOMAL EVOLUTION: THE TUCURA DICHROPLUS PRATENSIS AND THE TUCO-TUCOS OF THE GENUS CTENOMYS
TWO EXCEPTIONAL SOUTH AMERICAN MODELS FOR THE STUDY OF CHROMOSOMAL EVOLUTION: THE GRASSHOPPER DICHROPLUS PRATENSIS AND THE TUCO-TUCOS OF THE GENUS CTENOMYS
Claudio J. BIDAU1*, Dardo A. MART1 & Mabel D. GIMNEZ

Laboratorio de Gentica Evolutiva, Facultad de Ciencias Exactas, Qumicas y Naturales, Universidad Nacional de Misiones, Flix de Azara 1552, (3300) Posadas, Argentina. E-mail: genevol@fceqyn.unam.edu.ar 1. CONICET. *Present address: Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia, Departamento de Zoologia, Rio de Janeiro, Brazil. E-mail: bidau47@hotmail.com
Summary. In this work, we summarize the contributions that our research group has produced along more than 20 years, to the evolutionary biology of the grasshoppers of the genus Dichroplus (Orthoptera, Acrididae, Melanoplinae) y and the burrowing rodents of the genus Ctenomys (Rodentia, Ctenomyidae, Ctenomyinae). We especially emphasize their value as models for the study of the relevance of chromosomal rearrangements in microevolution (chromosomal polymorphisms) and macroevolution (chromosomal speciation), and the different approaches we have imprinted to their study along the years, such as population cytogenetics, karyotypic and meiotic studies, morphological and systematic analyses and finally, the macroecological and biogeographical research We also review the techniques that we have incorporated to our studies along these years, such as electron microscopy of syanptonemal complexes, analyses offemale meiosis, molecular cytogenetics techniques such as restriction enzyme banding and FISH (fluorescent in situ hybridization), and sequencing of mitochondrial genes.We conclude that it is only with a multidisciplinary approach that it is possible to ellucidate the complexities involved in the evolutionary biology of any group of living organisms.
Key words: chromosomal polymorphism, chromosomal speciation, Ctenomys, Dichroplus pratensis, evolutionary biology, molecular phylogeny, tuco-tuco, tucura.
Resumen. En este trabajo, resumimos las contribuciones que nuestro grupo de investigacin ha realizado a lo largo de ms de 20 aos, a la biologa evolutiva de los saltamontes del gnero Dichroplus (Orthoptera, Acrididae, Melanoplinae) y de los roedores excavadores del gnero Ctenomys (Rodentia, Ctenomyidae, Ctenomyinae). Enfatizamos especialmente su valor como modelos para el estudio de la importancia de los reordenamientos cromosmicos en la microevolucin (polimorfismos cromosmicos) y macroevolucin (especiacin cromosmica), y los diferentes enfoques que hemos impreso a su estudio a lo largo de los aos como son la citogentica de poblaciones, los estudios cariotpicos y meiticos, los anlisis morfolgicos y sistemticos, y finalmente, las investigaciones macroecolgicas y biogeogrficas. Asimismo, describimos diferentes tcnicas que hemos ido incorporando a nuestros estudios a lo largo de estos aos, como la microscopa de complejos sinaptonmicos, el anlisis de la meiosis femenina, tcnicas de citogentica molecular como el bandeo con enzimas de restriccin y FISH (fluorescent in situ hybridization) y la secuenciacin de genes mitocondriales. Concluimos que solamente con un esfuerzo multidisplinario es posible elucidar las complejidades invlucradas en el estudio de la biologa evolutiva de cualquier grupo de organismos vivos.
Palabras clave: biologa evolutiva, Ctenomys, Dichroplus pratensis, especiacin cromosmica, filogenia molecular, polimorfismos cromosmicos, tuco-tuco, tucura.
In fond memory of Osvaldo A. Reig, outstanding biologist whose influence on our ideas on Evolutionary Biology is still with us eleven years after his much lamented death.
I. Introduction: Chromosomal Evolution Chromosomal rearrangements have posed a continuous challenge to modern evolutionary theory and have raised a series of questions that, despite decades of population and evolutionary cytogenetic research, do not have yet clear answers (KING, 1993; WHITE, 1973, 1978): What is the meaning of the chromosomal differen-
53
C. J. Bidau, D. A. Mart and M. D. Jimnez, 2003. Two exceptional South American models for the study of chromosomal evolution: the tucura Dichroplus pratensis and the tuco-tucos of the genus Ctenomys.
ces between species? Did chromosomal rearrangements passively accompanied divergence between incipient species or could have they been causal factors of speciation? Why some taxa are chromosomally conservative while others are highly variable? What is the adaptive function of chromosomal polymorphisms, if any? Why certain chromosomal rearrangements are common in some groups of organisms and not in others? Many chromosomally-mediated speciation models have been put forward (BAKER & BICKHAM, 1986; BICKHAM, 1995; CAPANNA, 1982; JOHN, 1981; KING, 1993; SITES & MORITZ, 1987; SITES & REED, 1994; WHITE, 1978), although most are controversial. Furthermore, many hypotheses have been proposed to explain the meaning of extensive chromosomal polymorphisms even in intensively investigated organisms such as the genus Drosophila (BRUSSARD, 1984; POWELL, 1997). At present, an enormous body of data is available on these problems and the recent development of powerful molecular techniques for the comparative analysis of species and population structure, may shed new light on the old problem of chromosomes and evolution. In this paper we review several years of investigations of our research group on two exceptional South American models with that goal: the tucuras (grasshoppers) of the genus Dichroplus Stl (Acridoidea, Acrididae), and the subterranean rodents (tuco-tucos) of the genus Ctenomys Blainville (Octodontoidea; Ctenomyidae). II. The tucura`s case: Dichroplus pratensis and D. vittatus Robertsonian rearrangements have played a relevant role in chromosomal evolution as demonstrated de large number of cases in which the karyotypic differrences between species are due to centric fusions or fissions (BIDAU & MART, 1995, 2001; CAPANNA, 1982; BIDAU et al., 2001; JONES, 1990; KING, 1993; REDI & CAPANNA, 1988; SEARLE, 1986, 1993; WHITE, 1973, 1978). Robertsonian translocations may also occur as balanced polymorphisms (BIDAU, 1990; BIDAU & HASSON, 1984; BIDAU & MART, 1995; HEWITT, 1979; WHITE, 1973, 1978) or as spontaneous mutants (BIDAU, 1993; COLOMBO, 1990). Nevertheless, the factors that command the meiotic behavior of multiple Robertsonian configurations (trivalents and higher order multivalents) are in general poorly known (GROPP & WINKING, 1981; JOHN, 1981, 1983; 1987, 1990; KING, 1993; MIROL & BIDAU, 1992; BIDAU et al., 2001), partly because of the intrinsic individuality of all chromosomal rearrangements but also due to the lack of na adequate specific model in which the same translocation can be studied in a variety of natural situations (BIDAU & MART, 1995). Noteworthy exceptions are two different mammals, the murid rodent Mus musculus domesticus (domestic mouse) (BIDAU et al., 2001) and the insectivores of the genus Sorex (shrews) (SEARLE, 1986, 1993) and a species of South
American grasshopper, D. pratensis, which has proved to be na exceptional model for this kind of analysis. The neotropical genus Dichroplus (Melanoplinae, Acrididae) and its allies (Ronderosia, Scotussa, Leiotettix, etc.) exhibit an extraordinary inter- and intraspecific chromosomal variation, specially of the Robertsonian type, in number and morphology of both autosomes and sex-chromosomes (BIDAU & MART, 2001a); it represents a classical example of chromosomal evolution within the Acrididae (MESA et al., 1982). D. pratensis, has an extensive geographic distribution that includes most of Argentina, Uruguay and Southern Brazil. This eurytopic and polyphagous species occupies such diverse habitats as the Patagonian steppe, the Atlantic coast from Punta Indio to Caleta Olivia, the humid Pampas, hills and mountains of central-west Argentina, and the Jujuy Puna up to 2,500 metres above sea level (BIDAU et al., 1991; BIDAU & MART, 1995, 2001b, 2002). It is morphologically close to D. maculipennis and D. vittatus but is chromosomally unique and different from all other species in the genus (BIDAU, 1990). II.1. Nature and distribution of chromosomal variability in D. pratensis The majority of Dichroplus and related Melanoplines, share the telocentric 2n= 22 + X ()/2n= 22 + XX () karyotype, characteristic of the Cryptosacci Acridoids (BIDAU, 1990; BIDAU et al., 1991). The derived karyotypes, usually can be related to the ancestral one through one or several centric fusions that invokve both autosomes and sex-chromosomes. The standard karyotype of D. pratensis is exceptional in that it comprises nine pairs of telocentric autosomes and a telocentric X (2n= 18 + X ()/2n= 18 + XX (), and probably derives through two tandem fusions from the Cryptosacci complement (BIDAU, 1990). A similar situation seems to occur in a recently discovered species from Misiones province (Argentina) assignable to the related genus Scotussa, that shows a 2n= 21 telocentrico standard karyotype in the male (MART & BIDAU; unpublished results). The karyotype of D. pratensis is bimodal, with six pairs of large autosomal pairs (L1-L6) and three small (S7-S9). The X es slightly smaller than L4 (BIDAU & MART, 1995; MART & BIDAU, 1995). The standard karyotype is usually difficult to observe in nature (except in marginal popultions; see below) due to the extensive system of Robertsonian polymoprhisms and polytypisms that involve the six L autosomes (BIDAU, 1990, 1993; BIDAU et al., 1991; BIDAU & MART, 1995; BIDAU & MIROL, 1988). Until present, about 70 natural populations covering a wide range of habitats and latitudes have been examined chromosomally (BIDAU & MART, 2001a, 2001b, 2002; MART & BIDAU, 2001a, 2001b) and 8 different fusions have been recorded: L1/L2, L1/L4, L1/L6, L2/L4, L2/L5, L3/L4, L3/L5 and L5/L6. With few exceptions (see below), all populations
54
are usually polymorphic for one to four fusions which quailties and frequencies are very diverse. The frequency of fusion chromosomes is generally high except in marginal populations (see below) where between 77 and 100% of the individuals may be standard telocentric (BIDAU & MART, 2001a, 2001b; MART & BIDAU, 2001a, 2001b, 2002). Some fusions are widely distributed (L3/L4; L1/ L6), others restricted to small isolated areas (L1/L2) or even to a single population (L3/L5). Until today, a strict correlation between habitat preference and fusion system has not been established although fusions L1/L6 and L3/L4 characterise the populations of the temperate central area of the species` distribution while L5/L6 is only found in the austral portion of the distributional area of the Patagonian coastal populations. However, what is more relevant respect of the distribution of these polymorphisms, is that follows a strict central-marginal model. The central-marginal distribution of chromosomal polymorphisms has been extensively documented in several Drosophila species, but little is known about this pattern in other animal species. The distribution of D. pratensis covers a vasta area: from 23S to 46S and from the Andean slopes at more than 2,500 m to the Atlantic coast at sea level. In our studies we sampled 67 populations covering about 70% of the geographic range and extend along 23 of latitude and across 11 of longitude and comprise que comprenden 13 populations of the western margin (Andean), including the northernmost known population (Volcn, Jujuy province, 2300 masl) and seven from the Patagonian southern limit (such as Lago Musters and Villa Rada Tilly, Chubut province) (BIDAU & MART, 2002). The larger richness of fusions per population as well as their highest frequencies, occur in central-oriental Argentina that corresponds to the ecologically mor favourable habitats for the species, while towards North, West and South, the frequency and diversity of rearrangements decrease steadily until at the extreme margins of the geographic range, fusions practically disappear so that the majority of marginal populations are characterrised by possesing a 100% standard telocentric karyotype. Nevertheless, there exists a negative correlation between the mean number of different fusions per individual per population, and mean chiasma frequency per cell (taken as a measure of recombination; see below). Thus, marginal populations have higher levels of intraand interchromosomal recombination and different patterns of chiasma distribution. As we shall see, there exists a probable relationship between that situation and the degree of morphological variability within populations (BIDAU & MART, 2002, 2003a, 2003b; MART & BIDAU, 2001a, 2001b). These observations support the hypothesis of H. L. CARSON`s (BRUSSARD, 1984; CARSON, 1955, 1959; CARSON & HEED, 1964) on the meaning of genetic recombination in the centralmar-
ginal distribution of chromosomal rearrangements which is reinforced by that the fact that, in D. pratensis, marginal populations also show higher frequencies of B chromosomes (super-numeraries) than central ones; some of these Bs signifycantly increase chiasma frequency and thus, the potential ability for genetic recombination (BIDAU & MART, 2002; 2003c). II.2. Meiotic behavior of single and multiple Robertsonian heterozygotes Since all Rb polymorphisms of D. pratensis seem balanced and, since Rb translocations may in pronciple show irregular meiotic behavior when heterozygous (not compatible with a balanced polymoprphism unless compensating mechanisms that compensate for the negatevely heterotic effects of the rearrangements), in a series of publications we analysed the synaptic behavior, prometaphase I (PMI) and metaphase I (MI) orientation and anaphase I segregation in heterozygous males and females carrying seven of the eight known fusions. Of 8179 individual trivalents studied, only (0.44%) showed univalent formation (na indicator of chiasma or pairing failure) (BIDAU & MIROL, 1988; BIDAU & MART, 1995). This highly regular chiasmate behavior which is parallel to the telocentric and metacentric bivalents, is supported by the regularity of synapsis during zygotene and pachytene (see below). Anyway, although maximum multiple formation is normal, balanced segregation of trivalents is regulated by factors that ensure the correct convergent (disjunctional) orientation (CO) at MI. Some of these factors are: intercentromeric distances, size of the involved chromosomes, initial postion of the configuration within the meiotic cell, premeiotic architecture, relative time of activation of centromeres and genetic backgound (ARUNDHATI et al., 1986; BIDAU, 1993; 1996; JOHN, 1987, 1990; MIROL & BIDAU, 1992; NARASINGA RAO & SYBENGA, 1984; RICKARDS, 1983; SYBENGA, 1975; SYBENGA & RICKARDS, 1987). Our group studied in detail the orientation at PMI and MI of six heterozygous fusions in males and two in females, of D. pratensis. Non-convergent orientation (NCO) at PMI varied between 4% and 40% for any individual trivalent in different individuals but, the mean values for four different trivalents were not statistically significantly different (2= 1,64, gl= 3, 0.50<P<0.70). The NCOs in MI of single heterozygotes also show wide interindividual variation (BIDAU & MIROL, 1988; MIROL & BIDAU, 1991, 1992), but three points are worth of note: 1) Frequencies of NCOs at MI are invariably lower than those for PMI in all fusions and individuals analysed, indicating reorientation of the trivalent to attain disjunctional orienation before segregation. It must be assumed that the CO is the stablest configuration for all trivalents (see below); 2) A significant positive correlation between frequencies of NCO at PMI and MI at the individual level, exists (r=
55
0,44; P<0,01), suggesting a more or less fixed amount of prometaphasic reorientation that is independent of the fusion and the individual and pointing to the fact that the initial spatial relationships inside the cell, are important for the definition of trivalent orientation at MI; 3) All trivalents, despite large differences in size and symmetry, have similar frequencies of NCO in MI (2= 8,16, gl= 5, 0.10<P<0.20). Thus, single heterozygotes have on average, more than 85% of the MI cells with the trivalent orientated disjunctionally implying that less than 15% of the secondary spermatocytes would be potentially aneuploid due to unbalanced segregation (BIDAU & MART, 1995). Nevertheless, when the frequency of aneuplid and diploid secondary spermatocytes (which result from lagging chromosomes produced by non-disjunction and block cytokinesis) is compared with the frquency of NCO in MI, it is apparent that these values are invariably inferior to those expected from the MI orientations. Although some single heterozygotes show up to 14% anomalous spematocytes II (related to na unusual frequency of NCO), the mean values are well below 7% implying a negligible reduction in fertilty.This discrepance between the MI and MII data suggests that additional reorientation must occur during MI and that the effects on fertility of a given rearrangement must not be inferred exclusively from its MI behavior (BIDAU & MART, 1995). The problem of reorientation is controversial (SYBENGA & DE VRIES, 1987; SYBENGA & RICKARDS, 1987). There is little doubt that reorientation between PMI and MI occurs as observations on living spermatocytes demonstrate (WISE & RICKARDS, 1977). However, comparisons between MI-AI and MII that produce discrepant results have been doubted because cells in the different stages may belong to different cell populations (SYBENGA & RICKARDS, 1987). But, the constancy of the discrepance between MI and MII in D. pratensis, turns the former interpretation doubtful: the frequencies of abnormal spermatocytes II are always lower than those of NCOs of the trivalents independently of the arm combination or individual analysed. Moreover, there exists evidence that the reorientation of bivalents in Drosophila spermatocytes, may occur up to 10 min before AI (CHURCH & LIN, 1985). The regularity of reorientation is also confirmed by the behavior of higher order Rb multivalents (see below). In females (in which du to intrinsic technical complications, large samples of MI cells are difficult to obtain) we analysed the orientation behavior of fusions 1/2 y 5/6 in respectively 1 and 4 specimens from the Cerro Ceferino population (Sierra de la Ventana, Buenos Aires province) (BIDAU & MART, 1995; MART & BIDAU, 1995). In both cases, orientation was 100% convergent although in one female, in which only AI oocytes could be examined, 25% of the cells showed non-disjunction of trivalent 3/4.
In double and triple heterozygotes the situation is in principle, not different. As expected, the frequencies of NCO are higher than thos of single heterozygotes and roughly additive for all combinations of fusions analysed. The levels of MI NCO can be as high as 36% in some individuals (BIDAU & MIROL, 1988; MIROL & BIDAU, 1994; BIDAU & MART, 1995). Also, in these individuals, a considerable degree of metaphasic reorientation seems to occur and, the frequencies of anomalous spermatocytes II are relatively low although higher than those observed in single heterozygotes but probably not representing a substantial loss of haploid sperm (< 17%). It is relevant to note that in multiple heterozygotes, it is enough that a single trivalent experiences non-disjunction to produce aneuploid descendant gametes but the degree of aneuploidy and genetic imbalance will be increasingly higher as more bivalents undergo non-disjunction simultaneously. It is unlikely that highly aneuploid gametes are viable and able to fertilise. Our observations in double and triple heterozygotes (BIDAU & MIROL, 1994), have indicated that when there are two or three trivalents in the metaphasic celle, a mutual interaction occurs such that the number of MIs in which two or three trivalents undergo simultaneous NCO, is always significantly higher to the expected assuming independent behavior of the trivalents. This situation is probably related to the difficulties in synapsis experienced by the trivalents during zygotene-pachytene (MART & BIDAU, 2001c; see below). Nevertheless, the number of cells in which all trivalents are disjunctionally orientated, does not decrease significantly thus, no net increment of unbalanced gametes due to this factor would be expected. However, the gametes with multiple aneuploidy would be more frequent than those with single aneuploidy and this could produce consequences regarding the maintenance of the polymorphisms (see below). II.3. Factors affecting meiotic orientation of Robertsonian multiples Orientation of Rb multivalents is conditioned by the distance between centromeres which in turn, depends on the size of the involved chromosomes and the localisation and frequency of chiasmata, which determine the symmetry of the configuration (NARASINGA RAO & SYBENGA, 1984; AHRUNDATI et al., 1986; MIROL & BIDAU, 1991; BIDAU, 1991, 1996; BIDAU & MART, 1995). The number of centromeres in the multiple is also relevant. Multiple regression analysis involving the comparison of NCOs in four trivalent types (1/2, 3/4, 5/6 y 1/6) at PMI and MI in relation with arm length, arm ratio and frequency of proximal (P) chiasmata, revealed that the sole factor signifficantly affecting orientation in both stages was P chiasma frequency (MIROL & BIDAU, 1992). In fact, highly significant positive correlations between NCO and P chiasmata occur for both PMI (r= 0.869; P< 0.001)
56
and MI (r= 0.907; P< 0.01). Differences in total arm lengths and arm ratios were not significant respect to the NCOs, but it must be kept in mind that the longest chromosomes are in a sense, preadapted to orientate disjunctionally both because of a higher mechanical flexibility and because, if the size of the multivalent is large with respect to spindle size, linear orientations are not expected to be stable. Thus, P chiasmata affect trivalent orientation in two ways: 1) in bringing adjacent centromeres to close proximity they increase the probability of them functioning as a unit and orientating toward the same pole; 2) in effectively shortening total trivalent length they could favor stable linear orientations (de BOER & VAN BEEK, 1982; MIROL & BIDAU, 1992; BIDAU & MART, 1995; BIDAU, 1996). Higher order Rb multivalents also show a strong dependence on P and interstitial (I) chiasmata for their orientation and, also in this case, a highly significant positive correlation for all multivalent types occurs (r= 0.776; P< 0.05). Total length of the configuration is also important since NCO frequencies increase with multivalent size ("561"<"612"<"5612"; see below) (BIDAU, 1991, 1996; BIDAU & MART, 1995). II.4. Effects of Rb translocations on chiasma frequency and distribution Since the behavior of heterozygous Rb configurations is strongly affected by chiasmata we studied in detail the effects of the fusions on chiasma frequency and localisation. Our results may be summarised as follows: 1) L bivalents of standard individuals (telocentric) have na essentially Proximal-Distal (P-D) chiasma distribution (BIDAU, 1990, 1993; BIDAU & MART, 1995, 2001; MART & BIDAU, 1995, 2001a, 2001b.); 2) Fused males and females have significantly less total (T) and P chiasmata than standard ones and exhibit a shift of chiasmata towards distal chromosomal positions. No significant differents between fusion bivalents and trivalents exist, nor between sexes. The same chromosomes in standard homozygous state have a typical P-D chiasma distribution (BIDAU & MART, 1995; MART & BIDAU, 1995); 3) the effects of all fusions are intrachromosomal but, total reduction in choiasma frequency depends on the involved telocentrics since a highly significant positive correlation between telocentric length and individual chiasma frequency exists; 4) fusions have a homogeneising effect producing the same chiasma frequency in all chromosomes and chromosome combinations (about one D chiasmata per chromosome arm) in both sexes (BIDAU, 1990; BIDAU & MART, 1995, 2001a, 2001b; MART & BIDAU, 1995); 5) males and females with identical Rb karyotypes have indistinguishable chiasma frequencies and patterns with the exception of the X bivalents of females which has a typical P-D chiasma pattern; 6) intrachromosomal recombination within populations decreases as the number and frequency of fusions increases (see below). This effect is added to
the instant reduction of intrachromosomal recombination produced by the combination of two linkage groups in one as a consequence of each Rb translocation. II.5. Studies of meiotic chromosome pairing and synapsis In order to explain the parallel effects of all fusions on chiasma distribution and frequency in both sexes, a mechanistic model was put forward (BIDAU, 1990, 1993, 1996; BIDAU & MART, 1995). Our hypothesis had to assess the following questions: why evolutionary independent fusions involving 6 different chromosomes behave in the same way regarding chiasma repatterning (BIDAU, 1990, 1993)?, why the effects ocuur in parallel in both sexes independently of the fact that standard bivalents of males and females have different chiasma frequencies and distributions (MART & BIDAU, 1995; BIDAU & MART, 2002), and why standard bivalents retain their typical chiasma patterns in polymorphic populations (BIDAU & MART, 1995, 2002)?. Our model was based in the fact that in some Orthoptera species, meiotic synapsis at zygotene starts at chromosome ends attached to the inner nuclear envelope. Thus, in the case of D. pratensis, standard telocentric bivalents would start synapsis from both ends (centromeric and distal) towards the middle of the bivalent. If chiasmata are formed more readily in regions that pair firts and remain paired longer, then a P-D chiasma pattern is expected. When a fusion exists (either heterozygous or homozygous) four synapsis starting ends are converted into two, since the centromeric tips of chromosomes are no longer available as attaching sites to the nuclear envelope. Thus, using the previous argument, chiasmata will be readily formed at distal locations of trivalents and bivalents, and P and I chiasmata will be rare, producing large pericentromeric areas free of recombination. To test this model, we analysed prophase I synaptic patterns of homologous chromosomes with different Rb status by menas of Transmission Electron Microscopy (TEM) of surfacespread Synaptonemal Complexes (SC) (MART & BIDAU, 2001c). The study of nuclei at zygotene and early pachytene revealed that in the first stage, the pericentromeric regions of Rb bivalents and trivalents are the last to synapse and usually, remain unsynapsed in pachytene in the case of trivalents but complete synapsis in Rb bivalents. Standard telocentric bivalents usually show full synapsis at pachytene but different degrees of interstitial synapsis during zygotene, indicating that pairing starts ata opposite ends (centromeric and distal, respectively). The sequential nature of synapsis in the three types of configuration would be directly related to their patterns of chiasma distribution ata diplotene-MI and strongly supports our model. Furthermore, when two or more trivalents are present within the same nucleus, the unsynapsed chromosomal axes (lateral elements) of two different trivalents, may pair non-homologously; such behavior may be plausibly related to the distortion in the
57
frequency of simultaneous NCO observed in multiple heterozygotes (see section II.2) (MIROL & BIDAU, 1994). II.6. The hybrid zones: chromosomal and biochemical studies II.6.i. The chromosomes When a balanced chromosomal polymorphism is established, the polymorphic fusions would not have impact on speciation (however, see section II.7). But in D. pratensis, the same chromosomes engage in differrent arm combinations in different chromosomal races. Secondary contact between two races with distinct fusions may originate hybrid descendants of variable complexity depending on their chromosomal characteristics: from simple trivalent formation to the production of higher order multivalents in the case of monobrachial homologies. The latter have been considered relevant to the speciation model put forward for D. pratensis (BIDAU, 1991, 1996; BIDAU & MART, 1995; TOSTO & BIDAU, 1991). Na extensive and complex Rb polymorphism involving fusions L1/L2, L3/L4, L5/L6 and L1/L6 which occurs in Sierra de la Ventana (Buenos Aires province, Argentina) has been interpreted as the result of hybridisation between two chromosomal races sharing Rb chromosomes with monobrachial homologies. In this region, a Southern race, geographically restricted to the South of Buenos Aires and polymorphic for fusions L1/L2, L3/L4 y L5/L6, contacts the widespread Northern race polymorphic for fusions L1/L6 y L3/L4. Thus, because of reproductive interaction between individuals of both races, complex Rb heterozygotes with reduced fertilty are produced in the hybrid zone (BIDAU, 1991, 1996). Within the zone, chromosome frequencies vary widely over relatively short distances (<1000m) and altitudes (<550m), with fusions L1/L2 and L5/L6 generally associated to higher altitudes (rocky tops of hills) (TOSTO & BIDAU, 1991). This irregular distribution of both races within the hybrid zone possibly obeys to the complexity and heterogeneity of the Sierra de la Ventana environment that also constitutes a transition between radically different biomes. Thus, the hybrid zone has been interpreted as a mottled zone (Mirol & Searle, 1995) with very complex relationships between both races whose internal distribution reveals a notorious spatial heterogeneity (TOSTO & BIDAU, 1991). TOSTO & BIDAU (1991), discovered an intriguing exception to the latitudinal distribution of the chromosomal races specifically in the Cerro Ventana (> 1200 masl), where the pattern is inverted: the population on the top belongs to the Northern race while those from the slope and base of the mountain, to the Southern race. It is unknown if genetic flow occurs between them since no hybrids have been found to date (opposite to what occurs in the rest of analysed hills), and it has been suggested
that such situation could be due to a historical accident and not to a race-habitat correlation (TOSTO & BIDAU, 1991). Many populations include individuals from both races as well as complex structural chromosomal hybrids. The latter are of three types according to the involved monobrachial homologies: 1) type "561"; besides the chromosomes shared by both races, they have two Rb metacentrics (1.6 y 5.6) and two telocentrics (1 y 5) that engage in the formation of a quadrivalent (5-5.6-6.1-1); 2) type "612" (quadrivalent: 6-6.1-1.2-2), and 3) type "5612" (quinquivalent: 5-5.6-6.1-1.2-2). The frequency of NCOs in MI is invariably high for all multivalent types (BIDAU, 1991, 1996), specially for "612" yel "5612". Moreover, in all hybrids, the frequency of abnormal MIs is significantly higher than the combined NCO frequencies of the independent heterozygous fusions (BIDAU, 1991). Besides, desynapsis is very frequent (up to 18% of the cells in some individuals) and it always involve the telocentric chromosomes, particularly L5 and L6. NCOs of multivalents in PMI are even more frequent than in MI in all hybrids, reaching frequencies of 75% in same specimens (BIDAU, 1991, 1996; MART & BIDAU, 1995). Again, a positive significant correlation between frequencies of NCO in PMI and MI was found (r= 0,943; P<0,01) which suggets a fixed maximum of reorientation as for trivalents. The latter reinforces our argument on the importance of the initial position of the meiotic configuration on final (pre-disjunctional) orientation. Abnormal (aneuploid and diploid) secondary spermatocytes are significantly more abundant in complex hybrids than in regular heterozygotes and a highly significant positive correlation exists between their frequency and the frequencies of abnormal (non-disjunctional) MIs (r= 0,780; P<0,001). Nevertheless, the frequency of abnormal spermatocytes II is almost always lower than that of NCOs suggesting again, that further reorientation occurs at MI. II.6.ii. The allozymes In order to test the degree of genetic differentiation between both interacting races within the hybrid zone and thus, evaluate the importance of such zones as promoters of evolutionary divergence in D. pratensis, our group examined by means of allozyme electrophoresis, populations from both races and the zone (CHIAPPERO et al., 2001). No fixed alleles for any of the races were found among the studied loci; nevertheless, a cline of increasing frequency of allele d of the Ldh locus was observed. The cline could represent na introgression of Southern genes into the Northern race. The observed genotypic frequencies in all examined loci fitted the Hardy-Weinberg expectations and expected mean
58
heterozygosity per locus varied between 0.064 and 0.153. The Northern race populations showed the lower figures and those from the hybrid zone, the highest. Nei genetic distances between pais of populations were low, between 0.0011 and 0.0094, and in the neighbor-joining tree constructed on the basis of these distances, the clusters obtained were related to the geographic localisation and the chromosomal race of the populations. However, total genetic differentiation was low although statistically significant ( = 0.021; P<0.05). The paired matrices and the geographic distances between all populations were positively correlated. Also, a pattern of isolation by distance was obtained when the analysis was performed between the Northern race and the hybrid populations, but the correlation was not significant between the Southern race and the hybrid populations. Thus, the Northern race probably colonised the areas previously occupied by the Southern race. In this scenario, the populations from within the hybrid zone have been in contact with the northern race for a longer period of time than with those from the South and these results support the hypothesis that the Northern race invaded its present range displacing the Southern one that at present is only represented within the zone by relictual populations on the top of hills. II.7. The Central-Marginal model and the relationship between chromosomal and morphological variability II.7.i. Centrality, Marginality and Chiasma Frequency On the basis of our previous results (BIDAU, 1990; BIDAU et al., 1991), we had suggeted that the geographical distribution of chromosomal polymorphisms in D. pratensis follow a Central-Marginal pattern. This kind of distribution has been extensively studied in Drosophila species (BRUSSARD, 1984; POWELL, 1997) and also in Orthopterans (WESTERMAN, 1983). Hypotheses about the origin and evolutionary meaning of these distributions are several but, considering the role that chromosomal rearrangements play in the regulation of genetic recombination which determines the potential release of variability (BIDAU et al., 2001) and the observed distribution of chromosomal variants in Nature (high polymorphism in the central or ecollogically favorable areas and low or non-existent polymorphism in marginal or unfavorable habitats) we favor H. L. Carson's hypothesis (CARSON, 1955, 1959; CARSON & HEED, 1964): the decrease in number and frequency of fusions towrds marginal habitats would favor higher levels of intra- and interchromosomal recombination and the disruption of supergenes (maintained in central populations by the fusion systems that create recombination-free areas within fusion chromosomes and join previously independent limkage groups), implying higher release of genetic variability that could be adaptive in hostile and changing environments.
We sampled 67 populations of D. pratensis covering roughly 2/3 of its geographical range in the argentine provinces of Buenos Aires, Catamarca, Chubut, Crdoba, Jujuy, La Pampa, La Rioja, Mendoza, Ro Negro, Salta, San Juan, San Luis and Santa Fe. Males and females were collected for cytogenetic, morphological and molecular analyses. The frequency, quality and distribution of centric fusions and other chromosomal polymorphisms, such as B-chromosomes were studied, as well as their effects on recombination, morphological variability, sex dimorphism and the geographic location and ecological condition of the populations. Essentially, we analysed chiasma frequency (dependent on the frequency of centric fusions) en relation to the variation of six exomorphological characters (expressed as their Coefficients of Variation). This extensive analysis demonstrated without doubt that as suggested, chromosomal polymorphisms in D. pratensis are widespread and follow a typical centralmarginal distributional pattern. Central populations, that inhabit ecologically optimal regions exhibit mean frequencies of different fusions per individual (F) of up to F= 3.0 and show mean populational chiasma frequencies (Xt) as low as Xt= 8.98. On the pother hand marginal populations from central and Atlantic Patagonia and the Precordillera) have very low levels of chromosomal polymorphism (down to F= 0 in most sampled marginal localities), monomorphic karyotypes and high chiasma frequencies (Xt= 11.66 in the southernmost population [Villa Rada Tilly, Chubut, 4557S], and Xt= 12.01 in the northernmost one [Volcn, Jujuy, 2355S]). In this way, the progressive increase in recombination frequencies toward the margins of the geographic range is negatively correlated with the decrease in frequency of chromosomal polymorphisms but also (see below) positively with augmented levels of morphological variability. II.7.ii. Morphological variability, geographic clines and sexual size dimorphism: Bergmann's and Rensch's rules inverted We have hypothesised (BIDAU & MART, 2001a, 2001b; MART & BIDAU, 2001a, 2001b) that the decrease in fusion polymorphism toward marginal areas with the concomitant increase in intra- and interchromosomal genetic recombination, is the result of natural selection for higher levels of variability that could be adaptive in ecologically unfavorable and hostile environments. Many studies of geographic variation of body size in insects and other ectotherms have indicated that they follow the converse to Bergmann's rule (MOUSSEAU, 1997; BIDAU & MART, 2003) that is, individuals tend to be significantly smaller as the environment turns colder and hostile following higher latitudes and altitudes. We tested the hypothesis in 23 natural populations of D. pratensis in Argentina which comprise 22 degrees of latitude and are distributed between sea level and more
59
than 2000 masl. Six exomorphological characters were measured in ca. 25 individuals of both sexes in each population: Total Body Length, Femur 3 Length, Tibia 3 Length, Tegmina Length, Pronotum Length and Prontum Height. The species is sexually size-dimorphic with females larger than males; the dimorphism is constant along the whole range of the species (however, see below). Negative highly significant correlations occur between all six morphological characters and latitude and altitude. Extreme latitudes and altitudes correspond to the geographical margins of the distributional range and show comparable harsh environmental conditions (MART & BIDAU, 1998). The central populations were represented by Olavarra (Buenos Aires province, 36 55' S) where total body lengths of males and females were respectively 22.57 and 24.34 mm. The extreme marginal populations with respect to latitude and altitude exhibit significant lower means for all traits and represent the tips of a continuous morphological gradient: Rada Tilly in Patagonia (45 57' S, 10 masl), 18.91 mm (males) 23.17 mm (females) and Volcn in the Jujuy Prepuna (23 55 S; 2000 masl), 22.36 mm (males) and 23.34 mm (females) are the typical examples. Nevertheless, variability of the six morphological characters is positively correlated with latitude and altitude as the coefficients of variation of the six measurements in both sexes indicate (BIDAU & MART, 2003c). Considering that the latitudinal and altitudinal morphological gradients are themselves correlated to the above described chromosomal clines, it is possible on one hand that the higher morphological variability in marginal populations is due to their higher mean recombination frequencies (due to the absence of fusions) that would allow the release of more genetic and morphological variability. But other factors either genetic and /or environmental are probably involved in Bergmann's rule inversion and morphological diversity in D. pratensis. Na unexpected finding of this study was in regard of sexual-size dimorphism. Rensch's rule states that in species where males are larger than females, sexual size dimorphism increases with total size increase and this is true of, for example, mot mammals. Where females are larger, dimorphism decreases with size increase, and no satisfactory explanation has been developed for this observation (although possibly several mechanisms are involved in different taxa). D. pratensis follows the inverse Rensch's rule but since larger size is attained in central populations (thus, less dimorphism occurs) a hypothesis based on protandrous arrival timing and differential allometric growth between the sexes was put forwrd by us for this species (BIDAU & MART, 2003). Since the species is protandrous (males develop faster than females and reach adulthood earlier thus arriving earlier to breeding areas) and since developmental time is more protracted in central areas, both sexes attain larger sizes thus minimising size dimorphism. In marginal areas, time available for development is short thus, both sexes reach
adulthood with smaller mean sizes and due to differential allometry between the sexes, size dimorphism is increased. II.8. The detection of paracentric inversions Paracentric inversions are apparently very rare in Acriodoids (HEWITT, 1979) although very common in other organisms (BRUSSARD, 1984; POWELL, 1997). However, their frequency is probably higher than suspected and their lack of proper detection may be due to theri peculiar meiotic behavior (BIDAU & MART, 1998; MART & BIDAU, 2003). We studied the meiotic behavior of two spontaneous paracentric inversions. One was found in a male of the Sierra de la Ventana chromosomal hybrid zone. The mutant was 2n= 15 and was homozygous for fusion 3/4 and heterozygous for fusions 1/2 and 5/6. The inversion was located in arm 2 of the 1-1.2-2 trivalent and comprised the first half of its distal end involving about 25% of the length of the chromosome. The analysis of pairing at pachytne substages indicated that the main form of synapsis within the heterozygous ionverted zone, was homologous with a high frequency of complete reverse loop formation at early pachytene. No synaptic adjustment seemed to occur and homosynapsis was significantly correlated with crossing-over within the inverted region. As a result, a high frequency of bridge and fragment configurations at Anaphase I and Telophase, were observed. These produce significant levels of unreduced cells during sewcond meiotic division and later, macrospermatids (diploid or tetraploid) as well as microspermatids derived from lagging acentric fragments. The previous results suggested a deletereous effect of paracentric inversions in D. pratensis because of the production of aberrant recombinant chromosomes and very high levels of anomalous sperm. Besides, since this inversion induced a chiasma repatterning in the involved chromosome arm, it is evident a causal relationship between homologous pairing and crossing-over (BIDAU & MART, 1998). Although these observations resulted in the finding of an exceptional inversion because of its length and meiotic properties recently, with the aid of TEM we were able to detect other paracentric inversions that owing to their characteristics, woul pass unnoticed at the light microscopic level (MART & BIDAU, 2003). It is becoming obvious that at least in D. pratensis, paracentric inversions are more common than previously suspected although their evolutionary significance, if any, remains obscure. II.9. B chromosomes in D. pratensis BIDAU (1986, 1987), described two types of B chromosomes in Dichroplus pratensis; one was X-like and mitotically stable, carried an active nucleolus organising region (NOR) detected through silver-staining and showed non-Mendelian inheritance due to segregation-
60
distortion with respect to the X chromosome in males. The B chromosome associated preferentially with the X during zygotene and pachytene stages, determining that X and B passed to the same spindle pole during anaphase I thus producing XB and 00 spermatozoa in excess of the expected 50%, suggesting na accumulation mechanism (BIDAU, 1986). The other type of B was mitotically unstable (Bu) which means that different cells of the same individual (for example spermatogonia and spermatocytes) carried different numbers of B chromosomes (in this case, from 0 to 4) probably due to mitotic non-disjunction during pre-meiotic mitosis. This B had significant effects on chiasma frequency and very irregular behavior at meiosis (BIDAU, 1987). Although both B chromosomes are probably very old since they were found in geographically widely separated populations, their frequency is extremely low. Later (ROSATO et al., 2002), a second form of Bs was found also carrying a NOR but probably having resulted from a deletion of the first Bs. Extensive sampling allowed us to find several carriers of the three B types of B chromosomes in populations of Crdoba, Buenos Aires and Chubut to further the study of these extra elements. We were able to confirm the presence of active ribosomal genes in both classes of Bs by means of FISH (fluorescent in situ hybridisation) using a ribosomal DNA probe containing the 16S, 25S and 5.8S genes of wheat. We also confirmed the non-Mendelian nature of transmission of the Bs chromosomes in individuals of different populations, which probably explain their maintenance in nature despite their low frequencies. One surprising observation was that some marginal populations had very high frequencies of B chromosomes (up to 66.7% of the individuals), especially of Bu. Significantly, this element increases chiasma frequency and between-cell variance in the meiocytes in which they occur thus increasing genetic recombination. This fact reinforces our hypothesis to explain the central marginal distribution of chromosome polymorphisms: marginal populations would benefit from high recombination levels not only because of their lack of centric fusions (see above) but because of high frequencies of the Bu chromosome. II.10. Sex-chromosome systems: the case of Dichroplus vittatus The origin of neo-XY sex-chromosome systems in Acrididae, has traditionally been explained through the centric fusion of an X sex-chromosome with an autosome, both of them acrocentric or telocentric. Also, the meiotic behavior of sex-chromosomes had exclusively been studied in males. knowledge of the meiotic activities of neo sex-chromosomes in females was nil until we studied (BIDAU & MART, 2000) the neo-X neo-Y/neo-X neo-X system of males and females of Dichroplus vittatus, a species with wide distribution in Argentina. D. vittatus is phylogenetically related to D. pratensis y D. maculipennis. Its karyotype in all the geographic range comprises 2n= 20 chromosomes including 9 autosomal
pairs (one large metacentric and 8 telocentrics), and a pair of sex-chromosomes. The latter are peculiar: they include a very large metacentric X, and a very small telocentric neo-Y. This morphology strongly suggested that the traditional view of neo-system formation did not hold firmly in this case. Thus, we compared the meiotic behavior of the Sex-bivalent in both sexes as well as chiasma frequency and distribution in males and females in individuals from different Argentine localities. Mean autosomal chiasma frequency was low in both sexes and slightly but significantly higher in males. Female chiasma frequency increased significantly when the neoX/neoX sex-bivalent was included in the calculation. Nevertheless, chiasma distribution was essentially distal in both sexes. The behavior of the neo-XY pair of males was extremely complex as a priori suggested by its morphology which was analysed at mitosis and meiosis of diploid and polyploid cells. During meiosis orientation of the neo-XY bivalent at MI was highly irregular; only 21% of MI spermatocytes showed standard orientation. In the rest of the cells, the alternate activity of and extra kinetochore in the distal end of the short arm (XL) of the neo-X determines unusual MI orientations and a high frequency of non-disjunction and anaphase lagging of the sex chromosomes. In females, the neo-XX bivalent had a relatively more regular behavior but showed 17% asynapsis in the XL arm which in this case, orientated its distal ends towards opposite spindle poles again suggesting the activity of a second kinetochore. The dicentric nature and unstable meiotic behavior of the neo sexchromosomes of D.vittatus suggest a recent evolutionnary origin of the sex determination system, with pressumably adaptive benefits that could compensate its potential negative heterosis. Our observations also suggest that the neo sex-system originated through a tandem fusion of two original telocentric X chromosomes followed by a second tandem fusion with the small megameric bivalent, and a pericentric inversion of the neo-X. The remnant autosomal homolog became the neo-Y chromosome. Thus, the D. vittatus system shows completely novel features regarding origin and meiotic behavior. However, the dicentric nature of a chromosome (the neo-X in this case) that can behave relatively well, is not absolutely strange: our group discovered a dicentric B chromosome in another grasshopper species, Metalpetea brevicornis (GRIECO & BIDAU, 1999, 2000). III. The tuco-tucos case: genus Ctenomys Adaptation of mammals to subterranean life started about 45 MY ago following the gradual cooling and disseccation of the Earth and the biotic extinction in the transtion from middle Eocene to early Oligocene. It involved a spectacular natural evolutionary experiment of global convergence that involved three orders: insecti-
61
vores, rodents and marsupials and at present comprises 11 families, 50 genera and hundreds of species (NEVO, 1999). Subterranean mammals show convergent molecular and organismal adaptations to an environment characterised for being relatively simple, stable and discontinuous, which produces similar constraints and selective pressures in very different organisms. The existent divergent adaptations obey especially to their basic niches: herbivorism (rodents) and insectivorism (insectivores and marsupials), and to phylogeny (NEVO & REIG, 1990; NEVO, 1999). Subterranean mammals exhibit in general the most spectacular chromosomal variability yet known in mammals. The genera Thomomys, Spalax y Ellobius (from North America, Middle East and Asia, respectively) are characterised by many different karyotypes in differrent populations but in all of them, most of the chromosomal variation is confined within Linnean species that is, chromosomal diversity is centered in intraspecific polymorphisms and polytypisms (NEVO & REIG, 1990; NEVO, 1999; MASCHERETTI et al., 2000). The South American rodent genus Ctenomys is in this regard, very different and thus, unique: there seems to exist na almost 1:1 relationship between karyotype and Linnean species. The newest adaptive radiation of the subterranean mammals is represented by the South American Ctenomyidae whose explosive diversification is very recent: its more than 60 extant species of its sole genus, Ctenomys, plus 10 extinct species have originated geologically very recently in the early Pleistocene. The fossil record is good (VERZI, 1994) and the oldest fossils are no more than 1.8 MY old (REIG et al., 1990; CONTRERAS & BIDAU, 1999; NEVO, 1999). According to NEVO (1999) Ctenomys shows a subterranean life syndrome which involves patchy distributions, limited vagility, small population sizes, territoriality, small litter size, socially structured mating behavior and high karyotypic diversity. The syndrome probably explains its great diversification. Ctenomys shows an extraordinary chromosomal variability already evidenced in the pioneering work of REIG & KIBLISKY (1969). It comprises chromosomal numbers that range from 2n= 10in C. steinbachi from Bolivia to 2n= 70 in C. dorbignyi from Argentina and C. pearsoni from Uruguay), but most of the chromosomal forms are morphologically distinct and are pressumed to be reproductively isolated and thus, characterised as different Linnean species. Anyway, intraspecific chromosomal polymorphisms and polytypisms are known. Thus, Ctenomys seems to be one of the most rapidly evolving groups of mammals and is na excellent model to test the concept of chromosomal evolution. However, iit is worth mentioning that in the light of our research and that from other authors, it is possible that several modes of speciation may be operating within the genus and chromosomes may have a primary or a secondary role in different speciation events or even. No role at all (BIDAU
et al., 2000a; GIMNEZ et al., 2002). Our second interest in the genus Ctenomys is related to wildlife conservation. Ctenomys is a very important component of South American mammalian Biodiversity representing probably 100 or more species and several taxa within the genus are in severe risk of extinction or at least in a vulnerable status (BIDAU et al., 2000b) mostly because of loss of suitable habitats. There exist an imperative of establishing populational sizes and estimating the risks of genetic depletion and other dangers in the surviving populations in order to assess their present conservation status. Ctenomys is also interesting from the evolutionary point of view since species limits are usually contyroversial (LESSA & COOK, 1998; MASCHERETTI et al. 2000; GIMNEZ et al., 2002) and need establishing more firmly. Moreover, the speciesis the primary unit of biological conservation (MEFFE & CARROLL 1997; CALOW, 1998) thus, it is essential to know exactly what species are in Ctenomys if adequate conservation strategies are to be implemented. While the systematic and evolutionary appeal of Ctenomys has generated a voluminous literature, there are remarkably few studies on the ecology of its species (REIG et al. 1990; NEVO, 1999). But conservation of tuco-tucos requires genetic and ecological studies to go hand in hand. III.1. The systematic and biogeographical studies III.1.i. An evolutionary scheme Tuco-tucos have been known to science for about two hundred years. References to them are found in the writings Flix de Azara, Alcide D`orbignyi and Charles Darwin. The genus was erected by de Blainville in 1826, but it was Oldfield Thomas who described a large number of species mainly during the first two decades of the XXth century. It is worth noting that most of Thomas`s species still retain their status. Later, many new species where described in Argentina, Chile, Uruguay, Brasil, Bolivia and Per. Nevertheless, the systematic picture of the genus remained confusing during many years (REIG et al., 1990; CONTRERAS, 1996; CONTRERAS & BIDAU, 1999). CONTRERAS & BIDAU (1999), draw the main lines of a major hypothesis on the evolution of Ctenomys from a primordial stock of the middle Pleistocene based in the present-day Andean plains of Bolivia, that later would expand over an enormous geographic range and a diversifications that would transform Ctenomys in one of the most speciose genera of Caviomorph rodents. The hypothesis, meant to be an operative approach for the segmental work in different sections of the distributional scheme of the genus proved its validity to explain most of the intrageneric evolutionary relationships in Ctenomys, and has been supported and augmented by
62
successive molecular, parasitological, paleontological and anatomical auxiliary approaches. We postulated the existence of a primordial stock in the expansion of the genus, comprising symplesiomorphic forms allied to C. opimus and C. tucumanus that are distributed along the Andean axis extending from the Bolivian-Peruvian highlands, through the Sierras Pampeanas of Central Argentina reaching the Pampas plains, where species still primitive such as C. pundti y C. talarum occur. These species were the actors of a first dispersion wave that occurred about mid-Pleistocene times and still retain a symmetrical sperm. These species are later replaced by a second wave that covers the firsat one and is represented by the so-called mendocinus group sensu stricto and by the uruguayan, mesopotamic and riograndean species of the oriental mendocinus group. Towards the East and along all the primordial axis, emergencies of progenies have occurred. These expanded in the eastern lowlands and comprise at least, the Bolivian-Brazilian lineage that includes C. bolivianus, C. steinbachi, C. nattereri and C. rondoni; the northern Chacoan (Bolivian-Paraguayan) lineage with C. frater, C. sp. and C. conoveri; the central Chacoan lineage with C. scagliai, C. latro, C. argentinus and C. pilarensis, probably related to the mesopotamic dorbignyipearsoni group; and the southern Chacoan lineage including C. osvaldoreigi, C. rosendopascuali, C. yolandae, C. rionegrensis and C. bonettoi and is related to C. minutus and C. flamarioni (oriental mendocinus group). Additionally there is the mendocinus group sensu stricto with C. mendocinus, C. australis, C. porteousi, C. azarae and C. chasiquensis related to the patagonian forms from C. haigi to C. magellanicus. Besides, the derivative forms that reach Peru (C. peruvianus and C. leucodon) and others that are distributed in trans-Andean areas with several species in the Chilean territory, must be included in this account. This model speculates on the possible genetic, populational and structural aspects that accompanied- and could be acted as triggers- in the diversification process in relation to the major events of the paleogeographic and geomorphologic history of the continental area where Ctenomys is distributed. III.1.ii. The distribution of tuco-tucos analysed by means of Geographic Information Systems (GIS) Tuco-tucos are distributed between 10S and 56S, and from the Pacific to the Atlantic oceans. Their many species occupy divergent habitats and populations can be found from sea level to more than 4000m altitude. Our group decided to analyse the factors that may condition the distribution of Ctenomys species in South America using modern Geographic Information Systems (GIS). For that study 264 points of coordinates corresponding to 59 species and 12 unnamed forms were considered and data on body mass and sperm type obtained from the literature and personal records were included in the analysis. The GLOBTEX (Global Soil texture), ILASA Clima-
te Database, GRID (Global Resource Information Database) and Mathews (vegetation) databases were employed. The results obtained were interesting. The largest richness in species was found between 26 S and 38 S, abruptly decreasing towards South. A large hiatus between 26 S and 22 S exists and then, increases again up to 18 S. The correlation between body size and latitude was positive and statistically significant (this preliminary result prompted a study on the appliccation of Bergmann's rule to subterranean mammals that are, in principle, independent from the outer environment; this is an ongoing study involving the analysis of classical ecological rules in endothermic and ectothermic animal species). The effects of latitude, rainfall and soil were significant in a multiple regression test and the coefficient for fine soils was negative. The other predictors (vegetation and sperm type) were not significant; however, it was established that the symmetric sperm type (primitive) predominates in the north of the range (median, 27) while the asymmetric type (derived) is distributed towards south (median, 38). The relationships between the species and the types of soil, indicated that the correlation with fine soils was positive and significant, was non-significant for medium soils, and negative and significant for coarse soils. These observations indicated that most Ctenomys species inhabit fine and medium soils but avoid coarse-grained soils and this is probably related to their digging adaptations. III.2. The chromosomal studies As indicated above, one of the main sources of interest in tuco-tucos is their enormous chromosomal diversity (2n= 10 to 2n= 70), and because of their subterranean lifestyle chromosomal rearrangements could have been of primary importance in speciation as postmating isolating mechanisms or at least, rearrangements could have played a role in evolutionary divergence after speciation (ORTELLS et al., 1990: REIG et al., 1990, 1992; ORTELLS, 1995; BIDAU et al., 1996; CONTRERAS & BIDAU, 1999; NEVO, 1999; MASCHERETTI et al., 2000; GIMNEZ et al., 2002). Almost every analysed population shows a unique fixed karyotype upon which, chromosomal polymorphisms are frequently superimposed. In some cases however, the same karyotype (including the same polymorphisms) seems to be distributed across a wide geographical area and includes several Linnean species (see below). These observations raised two main questions. First, is karyotype a good species marker in Ctenomys? And second, is it possible that a large scale karyotypic repatterning occurred without leading to speciation (that is, without interruption of gene flow?). Both situations seem to occur in Nature which raises new questions: are there several modes of speciation within Ctenomys?, is it possible that chromosomal rearrangements were of prime relevance in some speciation events and not in others? The answer is, probably yes as the following cases demonstrate.
63
III.2.i. One karyotype, one species In many cases, the relationship species-karyotype is 1:1. We shall describe two clear examples studied by us. In the first case, we studied the karyotypes of two recently described Ctenomys species from Crdoba province. C. osvaldoreigi is only known from its type locality, Estancia San Luis in the Sierras Grandes at more than 2000 masl. Its karyotype consists of 2n= 52 chromosomes (FN= 56) and includes 22 telocentric, 1 subtelocentric and 2 small metacentric autosomal pairs plus a pair of Sex chromosomes. At the same time three populations of C. rosendopascuali, a new species from the northe-eastern plains of Crdoba with morphological and molecular (see below) affinities with C. osvaldoreigi. All analysed individuals were 2n= 52 but the FNs of the three populations were different. Individuals from Los Mistoles, had FN= 62 and the karyotype consists of a large subtelocentric, a small subtelocentric, 20 telocentric and three metacentric autosomal pairs, and the seX chromosomes. The specimens from Candelaria were FN= 64 and their karyotype included a second large subtelocentric pair replacing a large telocentic while the rest of the karyotype is simlar to that of Los Mistoles. A third subtelocentric pair occurs in Mar Chiquita (the type locality) thus FN= 66 (GIMNEZ et al., 1999). To compare the new species with previously known taxa from the same general area, we karyotyped individuals of C. bergi from northeastern Crdoba. All specimens were 2n= 48, FN= 90, a clearly divergent karyotype. The three karyomorphs found in C. rosendopascuali are remarkably similar and obviously related through simple fixed rearrangements while that of C. osvaldoreigi, although clearly related to the former reinforcing the morphological and molecular proximity of both species, is unique so that the species can be easily distinguished by their karyotypes. It is worth noting that the finding of divergent karyotypes in these populations led to detailed morphological studies and the description of two new species. Another interesting case is that of some Patagonian populations of Ctenomys in which chromosomal characterrisation coupled with molecular studies, allowed us to discover the existence of three new species. Until present, 8 Patagonian species have been described in Argentina: C. colburni, C. coyahiquensis, C. emilianus, C. fodax, C. sericeus, C. magellanicus, C. haigi and C. sociabilis. However, coastal forms are poorly known. Thus, we analysed karyotypically a series of Patagonian populations from Chubut and Santa Cruz provinces. Three of the populations belonged to known species (C. colburni, 2n= 34, FN= 64; C. fodax, 2n= 28, FN= 42, and C. sericeus, 2n= 28-30, FN= 44-46). The other 5 were coastal isolates in Chubut province from the Puerto Madryn/Pennsula Valds area. Two widely separated populations showed a 2n= 28, FN= 40. Although several Ctenomys species exhibit 2n= 28 karyotypes,
the one found in the former populations is unique regarding chromosomal morphology. The same is true for the two populations (also widely separated) that showed 2n= 38, FN= 40-44. A very isolated population from Punta Delgada in Pennsula Valds, had 2n= 48, FN= 72, again a previously undescribed karyotype despite the fact that 2n= 48 is a relatively common chromosome number in tuco-tucos. All species had asymmetrical sperm. We thus found a 1:1 relationship between three grups of populations and unique karyotypes and this fact, coupled to the vast distances that separate these froms from known Ctenomys species, could represent taxa (probably species) new to science (MONTES et al., 2001). This hypothesis was tested using molecular methods (sequencing of the mitochondrial cytochrome b gene) as described in section III.3.ii. III.2.ii. Several karyotypes, the same species In other cases, species are chromosomally polytypic since different populations represent chromosomal races with distinct fixed karyotypes although related through chromosomal rearrangements (GIMNEZ et al., 1999; MASCHERETTI et al., 1999). The analysis of such cases is useful to understand the origin and dispersal of new chromosomal variants and their impact on microevolution and speciation. Two cases studied by us are revealing in this respect. The first corresponds to C. pilarensis a species from eastern Paraguay in which GIMNEZ et al. (1997) described three distinct chromosomal races in four population nuclei of the eembuc Department. The second example corresponds to C. dorbignyi. This species was originally described on the basis of four well-delimited groups of populations in the north of Corrientes province to which, a further three isolated and disjunct groups were later incorporated (CONTRERAS et al., 1985). Recently, a new and distant group of C. dorbignyi populations was found by us in Entre Ros province. All known populations are 2n= 70 although the extreme isolation between the different population nuclei suggested the possibility of chromosomal divergence. We analysed (ARGELLES et al., 2000) karyotypically specimens of C. dorbignyi from Mbarig (type locality) and Sarandicito (both from Corrientes), and Tiro Federal and Paso Vera (Entre Ros). Thre different fixed karyomorphs were described. While Mbarig and Sarandicito exhibited 6 pairs of bibrachial autosomes and 28 pairs of telocentrics (pair 20 carries aNOR) thus, FN= 80). However, pair 2 was metacentric in Sarandicito and submetacentric in Mbarig. The third karyomorph characterrised the Entre Ros populations and was significantly different from the previous two. It showedcon 8 pairs of biarmed chromosomes and 26 telocentric pairs. Here, pair 3 (metacentric) carries the NOR and FN= 84. Pair 3 is also polymorphic for the length of the short arm due to differences in heterochromatin content. The polytypic nature of C. dorbignyi is reasonable due to the extreme isolation of the different population nuclei which favors
64
chromosomal divergence. It is worth noting that recently in our laboratory, specimens from the Contreras Cu population (the oriental nucleus) revealed a 2n= 42 karyotype, completely divergent from the 2n= 70 forms (unpublished results) suggesting that its inclusion in C. dorbignyi is at least, doubtful. It is important to note that the obseved 2n= 70 karyotypes are strongly related to that of C. pearsoni (2n= 70; Novello & Lessa, 1978) an Uruguayan species isolated from C. dorbignyi by the formidable barrier of the Uruguay river which suggest a vast paleodistribution of an ancestral 2n= 70 form whose fragmentation gave rise to both species and the races of C. dorbignyi. The close relationship between both species was recently confirmed by with the use of sequencing of the mitochondrial cytochrome b gene (BIDAU et al., 2000; MONTES et al., 2001). Cases of intrapopulation polymorphisms are relatively frequent but the involved rearrangements may be of a very different nature. In some cases, heterochromatin polymorphisms detected by C-banding (MASSARINI et al., 1991a, 1991b; FREITAS, 1994) or restriction Enzyme (RE) banding (IPUCHA et al., 2003) although there is a consensus that this kind of variation would not have a significant impact on speciation (KING, 1993). Anyway, heterochromatic changes have accompanied the evolutionary divergence of the different Ctenomys lineages as molecular cytogenetic studies have demonstrated (IPUCHA et al., 2001, 2003; ROSSI et al., 1995; SLAMOVITS et al., 2001). In other cases, the chromosomal polymorphisms have resulted from hybridisation petween polytypic races that have accumulated in isolation, fixed chromosomal differences. C. talarum is mainly distributed in Buenos Aires province and neighboring areas and 3 subspecies are recognised at present: C. t. talarum, C. t. recessus y C. t. occidentalis. The standard chromosome number of the species is 2n= 48 but 2n= 49 and 2n= 50 karyotypes have been cited for some individuals of C. t. recessus. We studied 56 individuals of 12 populations: 1) Cerro de la Gloria, 2) San Clemente del Tuy, 3) Santa Teresita, 4) Mar del Tuy, 5) Costa del Este, 6) Aguas Verdes, 7) La Lucila, 8) San Bernardo, 9) Mar de Aj, 10) Punta Mdanos, 11) Pinamar and 12) Villa Gesell, that belong to the geographic range of C. t. talarum. As reference we used as the karyotype of the Magdalena population (KIBLISKY & REIG, 1966), considered standard for C. t. talarum, because it is the nearest population to the type locality (Los Talas) which has become extinct. Along this transect a continuous chromosomal variation was evident and it was due to the presence of two polymorphic asymmetric Rb translocations that produces (from North to South) a decrease in the mean 2n of the populations from 2n= 47 (1, Cerro de la Gloria) through 2n= 45.7 (2), 2n= 44.0 (3), 2n= 44.8 (4), 2n= 45.3 (5), 2n= 44.8 (6) and 2n= 44.2 (7); from that locality south-
ward a new increase of the mean diploid number starts: 2n= 44,6 (8), 2n= 44.4 (9), 2n= 46.5 (10), 2n= 47.8 (11) and 2n= 48, in the southernmost population of the transect (12, Villa Gesell). This distribution, that involved two opposite chromosomal gradients shows a high concentration of 2n= 44 karyotypes in the area between Santa Teresita to the North and Mar de Aj to the South. This karyotype corresponds to individuals homozygous for the two Rb translocations (BRAGGIO et al., 1999). To explain the observed distribution we proposed the existence of a double hybrid zone between a population with 2n= 44 (with two fixed Rb translocations), and two groups of populations with the standard 2n= 48 karyotype at the south and the north of the analysed geographic area. The putative ancestral 2n= 44 could have appeared in an isolated deme within the distributional area of C. t. talarum during a period of strong populational retraction and fragmentation of the geographic range, followed by a stage of expansion that could have generated two secondary contacts with the standard forms with the subsequent formation of two hybrid zones that generated the polymorphisms observed today (BRAGGIO et al., 1999). The production of hybrid zones may in fact promote speciation if the heterozygous rearrangements are negatively heterotic since reinforcement of the postzygotic barrier could occur (BIDAU, 1991, 1996; KING, 1993). III.2.iii. Many species, the same karyotype The central region of Argentina is inhabited by a group of karyotypically stable Ctenomys species (2n= 47-48; FN= 71-82) that constitute the so-called "mendocinus" group (C. australis, C. azarae, C. mendocinus, C. porteousi and C. chasiquensis). The group contacts with populations of C. talarum and C. pundti and intermediate karyomorphs between both species (the socalled "pundti complex") (MASSARINI et al., 1991a, 1991b, 1998; BRAGGIO et al., 2000). Our group analysed several populations of this region to contribute to the ellucidation of the systematic and evolutionary problems posed by these groups of species (BRAGGIO et al., 2000). Our work complement and confirm previous results of MASSARINI et al. (1991a, 1991b, 1998). The 4748 polymorphism seems to have a common origin in all populations analysed from a complex rearrangement that involves the largest pair of biarmed autosomes and the second largest acrocentric, producing a large metacentric element. This hypothesis is based in the patterns of Gbanding and was supported by observations of male meiosis. Besides other heteromorphic chromosome pairs are common to all popualtions and species. They result from deletion or addition of heterochromatic short arms. Some general conclusions can be drawn from these data. The populations from La Pampa can be separated in two main groups a) El Guanaco popualtion which, considering its chromosomal morphology, banding patterns and sperm morphology can be ascribed to C. tala-
65
rum talarum, b) the rest of the popualtions are included in the "mendocinus group", and within it, would form part of C. azarae y C. porteousi. However, the geographic delimitation of the species of the "mendocinus group". This circumstance has frequently led to the extrapolation of supposed distributions on the basis of the correspondence of the taxa with certain biotopes. For this reason, a study of reproduction between these populations is needed in order to establish the correct taxonomic status of these forms. Such karyotypic constancy accompanied by an ancestral polymorophism that has apparently trascended the barriers of speciation, does not occur in any other group of Ctenomys species. However, it is known that remotely related species may have almost identical karyotypes as with C. bonettoi from Chaco and C. yolandae from Santa Fe (GIMNEZ et al., 2003). III.2.iv. Too many karyotypes: one species, three or a superspecies? The case of the Mesopotamic Ctenomys populations, especially those from Corrientes province, is puzzling. Although three Linnean species have been described in Corrientes (C. perrensi (2n= 50), C. dorbignyi (2n= 70) and C. roigi (2n =48) there exist a large number of populations which have not been assigned to any known species but are morphologically closely related with the previously mentioned taxa. Moreover the chromosomal variability within this group is outstanding (2n= 40 to 2n= 70) (ORTELLS et al., 1990; ORTELLS, 1995; GIMNEZ et al., 2002). This assemblage of chromosomal forms and species has been called either the "Corrientes group" or the "perrensi species complex" and is phylogenetically allied to species from Paraguay such as C. pilarensis (GIMNEZ et al., 1997; CONTRERAS & BIDAU, 1999; MASCHERETTI et al., 2000). The extreme chromosomal variability cooupled to the low morphological differentiation exhibited by these forms, suggested that the group might be undergoing rapid and explosive speciation aided or promoted by the special features of the habitat (short humid and dry periods of irregular alternance that possibly condition population structure producing populational expansions and retractions related to changes in the size of the numerous water bodies). This situation plus a probably very high rate of chromosomal mutation may favor the fixation of novel chromosomal rearrangements through genetic drift and inbreeding when population numbers are severely reduced (CONTRERAS & BIDAU, 1999). It is thus possible that the "perrensi species complex" is in fact a superspecies (as suggested by previous molecular data obtained by us (MASCHERETTI et al., 2000) in which stochastic and deterministic processes are probably promoting intense speciation. The joint results of our cytogenetic and molecular analyses are described in section III.3.iii.
III.2.v. Meiotic studies in Ctenomys The autosomes When chromosomal rearrangements are central to speciation processes, the study of meiosis is essential for the understanding of their effects in evolution, as seen in section II. However, meiotic studies in tuco-tucos are almost non-existent. The "perrensi species complex" with its extraordinary level of chromosomal variability mainly based on Rb translocations (see sections III.2.iv and III.3.iii) is a very favorable group for the study of the impact of chromosomal heterozygosis. We analysed male meiosis in individuals from different populations of Corrientes province in which 2n varied between 42 and 66. The maximum number of heterozygous Rb translocation found in a given individual, was 2. In the translocation heterozygotes, trivalents were formed with high regularity (> 90%) corresponding to the observed regular pachytene synapsis analysed with light and electron microscopy. All trivalents show the same behavior: the short arms of the acrocentrics may synapse completely between them or with the pericentromeric region of the metacentric. When no SC is formed, the unsynapsed region never extends beyond the pericentromeric area. The same regular behavior was observed for C. talarum individuals heterozygous for a chromosomal fusion. Association of unsynapsed regions of trivalents with the sex chromosomes (a possible source of meiotic arrest and thus of germ-cell lethality) or with autosomal bivalents, and univalency, were observed only very rarely. Zygotene observations revealed that points of initiation of synapsis always coincided with chromosome ends and could be uni- or bidirectional. All these data indicate and orderly meiotic behavior of heterozygous configurations in Ctenomys at least at the synaptic level and are not suggestive of negative heterosis. Nevertheless, the distribution of chiasmata deserves attention; it has been demonstrated for other, nonmammalian species that formation of proximal chiasmata interferes in the balanced segregation of trivalents (MIROL & BIDAU, 1992 and section II.2). The trivalents we analysed in Ctenomys showed high frequencies of P and I chiasmata with respect of metacentric bivalents and those chiasmata could be involved in the induction of non-disjunctional segregation. Nevertheless, in two species of rodents polymorphic for Rb fusions (Mus domesticus and Holochilus brasiliensis) although P and I chiasmata are also formed in excess of expectations in trivalents, such an effect on segregation was not observed (BIDAU et al., 2001; NACHMAN, 1993) and at least for M. domesticus, a role of the Rb fusions and the associated chiasma repatterning on the regulation of recombination has been suggested (BIDAU et al., 2001). At present, the meaning of Rb variation in Ctenomys remains obscure.
66
The sex chromosomes Synaptic behaviour and the progression of morphological differentiation of the XY chromosome pair during pachytene was studied for the first time in three species of the South American subterranean rodents of the genus Ctenomys. In general, synapsis progression in the sex pair could be subdivided into four substages: i) initial partial synapsis of the X and Y axes and beginning of the differentiation of the unsynapsed regions ii) complete or almost complete synapsis of the Y axis accompanied with morphological differentiation of the unsynapsed region of the X chromosome, iii) a novel stage exclusive to C. perrensi consisting in a retraction of the free X axis, associated with the formation of a homogeneous and dense structure along the synaptic region, which leads to the achievement of full synapsis between sex chromosomes, iv) an increase in morphological complexity involving extreme splitting of the XY pair. The implications of the peculiar synaptic behaviour displayed by sex chromosomes in C. perrensi, a species complex highly polymorphic for Robertsonian translocations could be related to both the triggering of the pachytene checkpoint and the avoidance of non-homologous associations between sex chromosomes and the asynaptic pericentromeric regions of trivalents in translocation heterozygotes which, as stated before, could be a source of germ-cell death and thus, sterility (KING, 1993; LANZONE et al., 2002). III.3. The molecular studies: phylogeny and systematics based on mitochondrial DNA III.3.i. A Ctenomys phylogeny based on sequences of the mitochondrial cytochrome b gene The sequences of mitochondrial DNA (mtDNA) offer and excelent opportunity to generate solid phylogenies. We used such approach to produce a molecular phylogeny of 27 Linnean species of Ctenomys from Argentina, Bolivia and Paraguay in order to test our evolutionnary model for the genus (see section III.1.i) and to confirm and/or clarify the preexisting taxonomy (MASCHERETTI et al., 1999). We compared DNA sequences of the mitochondrial cytochrome b gene in specimens of the following species collected during several field trips: C. argentinus, C. azarae, C. bergi, C. boliviensis, C. bonettoi, C. dorbignyi, C. juris, C. latro, C. nattereri, C. occultus, C. opimus, C. pilarensis, C. pundti, C. roigi, C. rosendopascuali, C. scagliai, C. talarum, C. tuconax, C. tucumanus and C. yolandae. We also analysed the following Ctenomys from the GenBank database: C. boliviensis, C. conoveri, C. goodfellowi, C. haigi, C. leucodon, C. mendocinus, C. opimus, C. sociabilis y C. steinbachi. Octodon degus, Spalacopus cyanus y Tympanoctomys barrerae (Octodontidae) were used as extra-group taxa.
The main results of the phylogenetic analyses revealed that: a) The grouping of C. argentinus, C. latro, C. occultus, C. pilarensis and probably C. juris, is supported by our results; b) C. bergi, C. bonettoi and C. yolandae form a natural group apparently related to C. azarae and C. mendocinus, group which probably includes C. rosendopascuali; c) C. pundti and C. talarum form a lineage strongly supported by our analysis; d) C. dorbignyi and C. roigi also form a strongly supported but reveals a certain uncertainty regarding the specific status of both taxa (see below); e) C. conoveri, C. frater and C. lewisi form a lineage with a high bootstrap value, confirming in part the results of LESSA & COOK (1998); f) C. haigi and C. sociabilis were considered sister species by LESSA & COOK (1998); our analysis support such relationship; g) A strong relationship seems to exist between C. boliviensis and C. goodfellowi, a group that, according to our phylogenetic trees would also include C. steinbachi and C. nattereri; h) The molecular data on C. bergi and C. yolandae reinforce our previous observations about their similarity in terms of cytochrome b sequences; these Linnean species (with very divergent karyotypes) do not form monophyletic groups on the basis of molecular evidence. In all, mtDNA comparisons strongly suport our evolutionary model. III.3.ii. Species delimitation in Patagonian tuco-tucos The recently discovered populations from the Patagonian Atlantic coast comprise three karyotypically differrenttiated groups with 2n= 28, 2n= 38 and 2n= 48 chromosomes (see section III.2.i). The three karyotypes are unique within the genus and show little affinities with those of other Patagonian Ctenomys species. This suggested us that the three karyotypic forms may consitute three species new to science. We used molecular methodolgy to test the hypothesis. The comparison of 412 bp of the mitochondrial cytochrome b gene of the three karyotypic forms and four Patagonian and one Pampasic species, revealed the existence of three major clades that correspond exactly with the chromosomally differentiated populations (MONTES et al., 2001). These three clades very probably define three different species. The 2n= 28 and 2n= 38 forms show molecular affinity and are also related to C. haigi (2n= 50), a Patagonian species with uncertain relationship to other Ctenomys and a geographic distribution that does not overlap with the coastal populations. The 2n= 48 form from Punta Delgada, is distinctive from other Ctenomys analysed so far and has a closer relationship with C. colburni (2n= 34). It also seems to be morphologically close to C. avellanedae, a recently described species from northern Patagonia (2n= 48) whose geographic distribution is completely disjunct from the population analysed by us. Both forms are probably the remnants of a 2n= 48 species with a vast paleodistribution. Surprisingly C. fodax (2n= 28) and C. sericeus (2n= 28-30) share the same cytochrome b haplotype. The molecular and chromosomal comparison of the coastal Ctenomys po-
67
pulations with the other Patagonian and Pampasic species (C. colburni, C. fodax, C. sericeus, C. haigi and C. mendocinus) indicated as we proposed, that they are different species. III.3.iii. The "perrensi" complex: genes and chromosomes evolving at different rates The specific diversity and the large chromosomal variation exhibited by the genus Ctenomys are outstanding. According to JUSTO & CONTRERAS (1999) the genus comprises almost 70 recognised species. Diploid chromosome numbers range from 10 in C. steinbachi (ANDERSON et al. 1987) to 70 in C. pearsoni (NOVELLO & LESSA 1986) and C. dorbignyi (ARGELLES et al. 2001) The North-Eastern Argentine Mesopotamia is inhabited by three Linnean species of Ctenomys (C. roigi, C. dorbignyi and C. perrensi), and a large number of populations of uncertain taxonomic status morphologically close to the former three species. These tucotucos are characterised by low morphological divergence and extreme karyotypic variation (from 2n= 40 to 2n= 70) and they are informally referred to as the "Corrientes group" (ORTELLS 1995) or the "C. perrensi species complex". Twenty-five Ctenomys populations from Corrientes and Entre Rios provinces in the Argentine Mesopotamia were analysed and 59 individuals were obtained over the period 1994 to 2001. These Ctenomys are characterised by low morphological divergence and extreme karyotypic variation (2n= 40-70) and may be demonstrating active chromosomal speciation. Variation in the nucleotide sequence of the mitochondrial cytochrome b gene (427 bp) was analysed for 59 individuals representing three species of Ctenomys (9 populations) and 16 Ctenomys sp. populations from Corrientes and Entre Rios provinces. Fourteen haplotypes were recorded. Haplotype 4 was present in 16 individuals of several populations (30.8%). The second most frequent haplotype was haplotype 1 present in 10 individuals (19.2%). The most divergent haplotype (7 substitutions relative to haplotype 4) was haplotype 12 from the Rincn de Ambrosio population, which is distinctive enough to be considered a new species. In general, the cytochrome b sequences showed little variation and did not discriminate among C. roigi, C. dorbignyi, C. perrensi and the 16 populations without specific names.
Acknowledgements
On the basis of cytochrome b it is difficult to define the limit between these three species and to identify species among the different karyomorphs. The relatively poor genetic differentiation among the three species and other populations may be the result of either genetic exchange between populations or a recent divergence time and in this case we are probably in the presence of a rapidly evolving superspecies. It is thus of importance to reanalyse the extraordinary karyotypic variation within this lineage in the light of these new findings IV. Conclusion Biological models that can be analysed with realtive ease are of extreme relevance in Evolutionary Biology since they allow the formulation of general hypotheses which can be tested in the field and in the laboratory. Moreover, such models should be the subject of a multidisciplinary, not segmental approach to their study since knowledge gained within one particular field, invariably leads to new questions that often can be answered within the realm of a different area of science or a different methodology. In this review, we intended to show that some South American taxa of wide geographic distribution are exceptional candidates to serve as models for evolutionary research. Although we concentrated on Ctenomys and Dichroplus, many other species and genera remain that can be studied in this way. Most of our present knowledge on evolution derives from research on organisms from the northern hemisphere while Neotropical taxa, which were of paramount importance for Darwin's formulation of the modern evolutionary theory are in many cases neglected, subjected to fragmentary analyses or in the worst of cases, studied in laboratories of remote parts of the world. Evolutionary biological studies of our fauna and flora are of extreme relevance in order that we have first hand information on the Biodiversity of our land. That Biodiversity is, as everybody knows, undergoing one of the worst challenges in history. It is our duty as biologists, to aim our efforts to the knowledge and conservation of the species richness in this part of the world. We must know how species originated, evolved and change and what is the nature of their adaptations, in order to preserve as much as we can of such a marvelous assembalge of living forms. We are sure that Darwin would be happy with this line of thought. And Osvaldo Reig too.
The authors wish to express their gratitude to the institutions that along these years have financed their projects on Dichroplus and Ctenomys: CONICET, Agencia Nacional de Promocin Cientfica y Tecnolgica, Universidad de Buenos Aires, Universidad Nacional de Misiones, British Council, Fundacin Antorchas, Agencia Espaola de Cooperacin Internacional (AECI), The Wellcome Trust, The University of York, Universidad Complutense de Madrid, Universidad Nacional de Crdoba, Universidade do Estado do Rio de Janeiro y Fundaao de Amparo a Pesquisa do Rio de Janeiro (FAPERJ). CJB is especially grateful to Dr. Lena GEISE in whose laboratory and through her kindness, this manuscript was completed.
68
Bibliography ARGELLES, C. F., P. SUREZ, M. D. GIMNEZ and C. J. BIDAU, 2001. Intraspecific chromosome variation between different populations of Ctenomys dorbignyi (Rodentia, Ctenomyidae) from Argentina. Acta Theriologica, 46: 363373. Wialowieza. ARUNDHATI, A., P. S. R. L. NARASINGA RAO and J. SYBENGA, 1986. Posible causes of variation in trivalent orientation frequencies in pearl millet and rye. Genetica, 70: 81-87. Dordretch. BAKER, R. J. and J. W. BICKHAM. 1986. Speciation by monobrachial centric fusions. Proc. Nat. Acad. Sci. USA, 83: 8245-8248. New York. BICKHAM, J. W., 1995. The birth of new species. Bioscience, 45: 725-727. Washington. BIDAU, C. J., 1986. A nucleolar-organising B chromosome showing segregation-distortion in the grasshopper Dichroplus pratensis (Melanoplinae, Acrididae). Can. J. Genet. Cytol., 28: 138-148. Ottawa. BIDAU, C. J., 1987. Influence of a rare unstable B-chromosome on chiasma frequency and non-haploid sperm formation in Dichroplus pratensis (Melanoplinae, Acrididae). Genetica, 73: 201-210. Dordretch. BIDAU, C. J., 1990. The complex Robertsonian system of Dichroplus pratensis (Melanoplinae, Acrididae). II. Effects of the fussion polymorphysms on chiasma frequency and distribution. Heredity, 64: 145-159. Cambridge. BIDAU, C. J., 1991. Multivalents resulting from monobrachial homologies within a hybrid zone in Dichroplus pratensis (Acrididae): meiotic orientation and segregation. Heredity, 66: 219-232. Cambridge. BIDAU, C. J., 1993. Causes of chiasma repatterning due to centric fusions. Brazil. J. Genet., 16 (2): 283-296. Ribeiro Preto. BIDAU, C. J., 1996. Chiasma reparttening in hybrids between chromosomal races of the grasshopper Dichroplus pratensis (Melanoplinae, Acrididae). Cytobios, 85: 91-110. Cambridge. BIDAU, C. J., C. BELINCO, P. M. MIROL and D. S. TOSTO, 1991. The complex Robertsonian system of Dichroplus pratensis (Melanoplinae, Acrididae) .I. Geographic distribution of fusion polymorphysms. Gntique, Slection, Evolution, 23: 353-370. Toulouse. BIDAU, C. J., M. D. GIMENEZ and J. R. CONTRERAS, 1996. Especiacin cromosmica y la conservacin de la variabilidad gentica: el caso del gnero Ctenomys (Rodentia, Caviomorpha, Ctenomyidae). Mendeliana, 12: 25-37. Buenos Aires. BIDAU, C. J., M. D. GIMNEZ, J. R. CONTRERAS, C. F. ARGELLES, E. BRAGGIO, R. D'ERRICO, C. IPUCHA, C. LANZONE, M. MONTES and P. SUREZ, 2000a. Variabilidad cromosmica y molecular inter- e intraespecficas en Ctenomys (Rodentia, Ctenomyidae, Octodontoidea): mltiples patrones evolutivos? Actas IX Congreso Iberoamericano de Biodiversidad y Zoologa de Vertebrados, Buenos Aires, pp. 127-130. BIDAU, C. J., M. D. GIMNEZ, C. LANZONE, P. SUREZ, C. ARGELLES, E. BRAGGIO, M. MONTES and R. D'ERRICO, 2000b. Ctenomyidae. In (G.B. DAZ. & R.A. OJEDA, R.A. eds.). Libro Rojo de Mamferos Amenazados de la Argentina. Sociedad Argentina para el Estudio de los Mamferos. Buenos Aires, pp. 70-75. BIDAU, C. J., M. D. GIMNEZ, CH. L. PALMER and J. B. SEARLE, 2001. The effects of Robertsonian fusions on chiasma frequency and distribution in the house mouse (Mus musculus domesticus) from a hybrid zone in northern Scotland. Heredity, 87 (3): 305-313. Cambridge. BIDAU, C. J. and E. R. HASSON, 1984. Population cytology of Leptysma argentina BRUNER (Leptysminae, Acrididae). Genetica, 62: 165-175. Gravenhage. BIDAU, C. J. and D. A. MART, 1995. Male and female meiosis in Robertsonian heterozygotes of Dichroplus pratensis (Acrididae). In (P. E. BRANDHAM and M. D. BENNETT, eds.). Kew Chromosome Conference IV. Royal Botanic Gardens, Kew, pp. 381-396. BIDAU, C. J. and D. A. MART, 1998. Pairing and crossing-over within a paracentric invertion asociated to a heterozygous Robertsonian translocation in Dichroplus pratensis (Melanoplinae, Acrididae). Cytologia, 63: 49-63. Tokyo. BIDAU, C. J. and D. A. MART, 2000. Meiosis and the Neo-XY system of Dichroplus vittatus (Melanoplinae, Acrididae): a comparison between sexes. Genetica, 110: 185-194. Dordretch. BIDAU, C. J. and D. A. MART, 2001. The Central-Marginal Model in Acrididae: The case of the Robertsonian system of Dichroplus pratensis (Melanoplinae). Chromosome Research, 9: 105-106. Dordretch. BIDAU, C. J. and D. A. MART, 2002. Geographic distribution of Robertsonian fusions in Dichroplus pratensis (Acrididae): the central-marginal hypothesis reanalysed. Cytogenetic and Genome Research, 96: 66-74. Basel. BIDAU, C. J. and D. A. MART, 2004. B chromosomes and Robertsonian fusions of Dichroplus pratensis (Acrididae): intraspecific support for the centromeric drive theory. Cytogenetic & Genome Research (Basel), 106: 347-350. BIDAU, C. J. and D. A. MARTI, 2005. Variability of Chiasma Frequency and Morphological Characters along a Latitudinal Gradient in Dichroplus pratensis (Orthoptera: Acrididae). European Journal of Entomology (Ceske Budejovice), 102: 1-12. BIDAU C. J. and D. A. MART, 2005. Clinal variation of body size in Dichroplus pratensis (Orthoptera: Acrididae): inversion of Bergmanns and Rensch's Rules. Annals of the Entomological Society of America, 97: en prensa.
69
BIDAU, C. J. and A. MEDINA, 2005. Subterranean rodents of the genus Ctenomys (Caviomorpha, Ctenomyidae) follow the converse to Bergmanns rule. Journal of Biogeography: en prensa. BIDAU, C. J. and P. M. MIROL, 1988. Orientation and segregation of Robertsonian trivalents in Dichroplus pratensis (Melanoplinae, Acrididae). Genome, 30: 947-955. Ottawa. BIDAU, C. J., M. ROSATO and D. A. MARTI, 2004. FISH detection of ribosomal cistrons and assortment-distortion for X and B chromosomes in Dichroplus pratensis (Acrididae). Cytogenetic and Genome Research. Basel, 106: 295-301. BRAGGIO, E., M. D. GIMNEZ, J. R. CONTRERAS, E. JUSTO and C. J. BIDAU, 1999. Karyotypic variation in populations of Ctenomys (Rodentia, Ctenomyidae) from La Pampa Province, Argentina. Caryologia, 52 (3-4): 131140. Firenze. BRAGGIO, E., C. IPUCHA, M. D. GIMNEZ and C. J. BIDAU, 1999. Una posible doble zona hbrida cromosmica en Ctenomys talarum de la provincia de Buenos Aires, Argentina (Rodentia, Ctenomyidae). XXIX Congreso Argentino de Gentica, Rosario, Santa Fe: p. 182. BRUSSARD, P. F., 1984. Geographic patterns and environmental gradients: The central-marginal model in Drosophila revisited. Ann. Rev. Ecol. Syst., 15: 25-64. Palo Alto. CALOW, P. (ed.), 1998. The Encyclopedia of Ecology and Environmental Management. Blackwell Science, London. CAPANNA, E., 1982. Robertsonian numerical variation in animal speciation: Mus musculus, an emblematic model. In (C. BARIGOZZI, ed.) Mechanisms of Speciation. Alan R. Liss, New York, pp. 155-177. CARSON, H. L., 1955. The genetic characteristics of marginal populations of Drosophila. Cold Spring Harbor Symp. Quant. Biol., 20: 276-287. Cold Spring Harbor. CARSON, H. L., 1959. Genetic conditions which promote or retard the formation of species. Cold Spring Harbor Symp. Quant. Biol., 24: 87-105. Cold Spring Harbor. CARSON, H. L. and W. B. HEED, 1964. Structural homozygosity in marginal populations of neartic and Neotropical species of Drosophila in Florida. Proc. Nat. Acad. Sci. USA, 52: 427-430. Washington. CHIAPPERO, M., C. PARISE, D. A. MART, C. N. GARDENAL and C. J. BIDAU, 2003. Distribution of allozymic genetic variability in populations from two chromosomal races and their hybrid zone of Dichroplus pratensis (Melanoplinae, Acrididae). Journal of Evolutionary Biology. Heidelberg: 17 (1): 76-82. CHURCH, K. and H. P. P. LIN, 1985. Kinetochore microtubules and chromosome movements during prometaphase in Drosophila melanogaster spermatocytes studied in life and with the electron microscope. Chromosoma, 92: 273282. Heidelberg. COLOMBO, P. C., 1990. Effects of centric fusions on chiasma frequency and position in Leptysma argentina (Acrididae: Orthoptera). II. Intra- and interchromosome effects. Caryologia, 43: 131-147. Firenze. CONTRERAS, J. R., 1996. Acerca de la distribucin geogrfica de la morfologa espermtica en el gnero Ctenomys (Rodentia: Ctenomyidae). Ntulas Faunsticas, 88: 1-5. Corrientes. CONTRERAS, J. R. and C. J. BIDAU, 1999. Lneas generales del panorama evolutivo de los rodeores excavadores del gnero Ctenomys (Rodentia: Ctenomyidae). Ciencia Siglo XXI, 1: 1-22. Buenos Aires. FREITAS, T. R. O. DE., 1994. Geographical distribution of heterochromatin in Ctenomys flamarioni (RodentiaOctodontidae) and its cytogenetic relationship with other species of the genus. Cytogenet. Cell. Genet., 67: 193-198. Basel. GIMNEZ, M. D., J. R. CONTRERAS and C. J. BIDAU, 1997. Chromosomal variation in Ctenomys pilarensis, a recently described species from eastern Paraguay (Rodentia, Ctenomyidae). Mammalia, 61(3): 385-398. Paris. GIMENEZ, M. D., C. J. BIDAU, C. F. ARGELLES and J. R. CONTRERAS, 1999. Chromosomal characterization and relationship between two new species of Ctenomys (Rodentia, Ctenomyidae) from northern Crdoba province, Argentina. Z. Sugetierkunde, 64: 91-106. Kiel. GIMNEZ, M. D., C. J. BIDAU, Y. E. DAVIES and J. R. CONTRERAS, 2003. New Ctenomys karyotypes from Lower Chaco (Argentina) (Rodentia, Ctenomyidae). Mastozoologa Neotropical: in press. Mendoza. GIMNEZ, M. D., C. J. BIDAU and J. B. SEARLE, 2001. Chromosomal and molecular delimitation of Ctenomys (Rodentia, Ctenomyidae) from Argentine Mesopotamia. Chromosome Research, 9: 109-110. Dordretch. GIMNEZ, M. D., P. M. MIROL, C. J. BIDAU and J. B. SEARLE, 2002. Molecular analysis of populations of Ctenomys (Caviomorpha, Rodentia) with high karyotypic variability. Cytogenetic and Genome Research, 96: 130-136. Basel. GRIECO, M. L. and C. J. BIDAU, 1999. Chiasma frequency and distribuution in males and females of Metaleptea brevicornis adspersa (Acridinae, Acrididae) with and without B chromosomes. Hereditas, 131: 101-107. Lund. GRIECO, M. L. and C. J. BIDAU, 2000. The dicentric nature of the metacentric B chromosome of Metaleptea brevicornis adspersa (Acridinae, Acrididae). Heredity, 84: 639-646. Cambridge. GROPP, A. and H. WINKING, 1981. Robertsonian translocations: cytology, meiosis, segregation patterns and biological consequences of heterozygosity. Symp. Zool. Soc. Lond., 47: 141-181. London. HEWITT, G. M., 1979. Orthoptera. In (B. JOHN, ed.). Animal Cytogenetics 3. Insecta 1. Gebrder Borntraeger, BerlinStuttgart.
70
IPUCHA, M. C., C. J. BIDAU and M. D. GIMNEZ, 2001. Divergencia evolutiva de la heterocromatina en dos linajes de Ctenomys (Rodentia, Ctenomyidae) evidenciada con bandeo Re. XXX Congreso Argentino de Gentica. J. Basic. Applied Genet., 14 (2) (suplemento): 89-90. Buenos Aires. IPUCHA, M. C., C. J. BIDAU and M. D. GIMNEZ. 2003. Heterogeneity of heterochromatin in the genus Ctenomys revealed by means of a combined analysis of C- and RE-banding. Genetica: submitted. Dordretch. JOHN, B., 1981. Chromosome change and evolutionary change: a critique. In: (W. R. ATCHLEY and R. S. WOODRUFF, eds.) Evolution and Speciation. Cambridge University Press, Cambridge. pp. 23-51. JOHN, B., 1983. The role of chromosome change in the evolution of Orthopteroid insects. In (A.K. SHARMA and A.S. SHARMA, eds.). Chromosomes in the Evolution of Eukaryotes. 1. CRC Press, Boca Raton, pp. 1-110. JOHN, B., 1987. The orientation behaviour of multiple chromosome configurations in Acridid grasshoppers. Genome, 29: 292-308. Ottawa. JOHN, B., 1990. Meiosis. Cambridge University Press, Cambridge. JONES, K., 1990. Robertsonian changes in allies of Zebrina (Commelinaceae). Pl. Syst. Evol., 172: 263-271. KING, M., 1993. Species Evolution. The Role of Chromosome Change. Cambridge University Press, Cambridge. LANZONE, C., C. J. BIDAU, M. D. GIMNEZ and J. L. SANTOS, 2002. Synaptic behaviour and morphological modification of the X and Y chromosomes during pachytene in species of Ctenomys (Rodentia, Caviomorpha, Ctenomyidae). Genome. Ottawa, 45 (6): 1110-1115. LESSA, E. P. and J. A. COOK, 1998. The molecular phylogenetics of tuco-tucos (genus Ctenomys, Rodentia: Octodontidae) suggests an early burst of speciation. Molecular Phylogenetics and Evolution, 9: 88-99. MART, D. A. and C. J. BIDAU, 1995. Male and female meiosis in a natural population of Dichroplus pratensis (Acrididae) polymorphic for Robertsonian translocations: A study of chiasma frequency and distribution. Hereditas, 123: 227-235. Lund. MART, D. A. and C. J. BIDAU, 2001a. Changes in recombination, morphological variation and the central marginal distribution of Dichroplus pratensis (Melanoplinae: Acrididae). Metaleptea-Special Meeting Issue. VIII International Meeting of the Orthopterists Society, Montpellier, France: 66. MART, D. A. and C. J. BIDAU, 2001b. Variation of body size in Dichroplus pratensis (Melanoplinae: Acrididae): A case of the converse Bergmanns Rule. Metaleptea-Special Meeting Issue. VIII International Meeting of the Orthopterists Society, Montpellier, France: 66-67. MART, D. A. and C. J. BIDAU, 2001c. Synapsis in Robertsonian heterozygotes and homozygotes of Dichroplus pratensis (Melanoplinae, Acrididae) and its relationship with chiasma patterns. Hereditas, 134: 245-254. Lund. MARTI, D. A. and C. J. BIDAU, 2003. A heterozygous paracentric inversion only detectable through electron microscopy, in the grasshopper Dichroplus pratensis (Melanoplinae, Acrididae). Caryologia. Firenze, 56 (2): 175-180. MASCHERETTI, S., P. M. MIROL., M. D. GIMNEZ, J. R. CONTRERAS, C. J. BIDAU and J. B. SEARLE, 2000. Phylogenetics of the speciose and chromosomally variable rodent genus Ctenomys, based on mitochondrial cytochrome b sequences. Biological Journal of the Linnean Society, 70: 361-376. London. MASSARINI, A. I., M. A. BARROS, M. O. ORTELLS and O. A. REIG, 1991a. Chromosomal polymorphism and small karyotypic differentiation in a group of Ctenomys species from central Argentina (Rodentia, Octodontidae). Genetica, 83: 131-144. Dordretch. MASSARINI, A. I., M. A. BARROS, V. ROIG and O. A. REIG, 1991b. Banded karyotypes of Ctenomys mendocinus (Rodentia, Octodontidae) from Mendoza, Argentina. J. Mammal., 72: 194-197. Lawrence, KS. MASSARINI, A. I., F. J. DYZENCHAUZ and S. I. TIRANTI, 1998. Geographic variation of chromosomal polymorphism of Ctenomys azarae, tuco-tuco of the Ctenomys mendocinus group (Rodentia, Octodontidae). Hereditas, 128: 207211. Lund. MEFFE, G. K. and C. R. CARROLL, 1997. Principles of Conservation Biology. Sinauer, Sunderland. MESA, A., A. FERREIRA and C. S. CARBONELL, 1982. Cariologa de los acridoideos neotropicales: estado actual de su conocimiento y nuevas contribuciones. Ann. Soc. Entomol. Fr. (NS), 18: 507-526. Paris. MIROL, P. M. and C. J. BIDAU, 1991. Meiotic behaviour of Robertsonian heterozygotes in populations of Dichroplus pratensis (Acrididae) with different fusion frequencies. Genetica, 84: 171-178. Dordretch. MIROL, P. M. and C. J. BIDAU, 1992. Proximal chiasmata induce non-disjunctional orientation of Robertsonian trivalents in a grasshopper. Heredity, 69: 268-278. Cambridge. MIROL, P. M. and C. J. BIDAU, 1994. Non-random patterns of non-disjunctional orientation in trivalents of multiple Robertsonian heterozygotes of Dichroplus pratensis (Acrididae). Genetica, 84: 171-178. Dordretch. MIROL, P. M. and J. B. SEARLE, 1994. Dichroplus pratensis and Sorex araneus: to examples of chromosomal hybrid zones in the context of speciation. Folia Zool., 43 (Suppl. 1): 29-36. MONTES, M. A., M. D. GIMNEZ, C. J. BIDAU and J. B. SEARLE, 2001. Molecular delimitation of three new species of the genus Ctenomys from Patagonia (Argentina). Chromosome Research, 9: 79-80. Dordretch. MYERS, N., 1988. Threatened biotas: "hot spots" in tropical forests. The Environmentalist, 10: 243-256. NARASINGA RAO, P. S. R. L and J. SYBENGA, 1984. Linear orientation of trivalents and quadrivalents in the metaphase I pollen mother cells of pearl millet. Can. J. Genet. Cytol., 26: 506-510. Toronto.
71
NEVO, E., 1999. Mosaic Evolution of Subterranean Mammals. Oxford Science Publications, Oxford. NEVO, E. and O. A. REIG, 1990. Evolution of Subterranean Mammals at the Organismal and Molecular Levels. WileyLiss, New York. ORTELLS, M. O., 1995. Phylogenetic analysis of G-banded karyotypes among the South American subterranean rodents of the genus Ctenomys (Caviomorpha: Octodontidae). Biological Journal of the Linnean Society, 54: 45-70. London. ORTELLS, M. O., J. R. CONTRERAS and O. A. REIG, 1990. New Ctenomys karyotypes (Rodentia, Octodontidae) from north-eastern Argentina and from Paraguay confirm the extreme chromosomal multiformity of the genus. Genetica, 82: 189-201. Dordretch. POWELL, J. P., 1997. Progress and Prospects in Evolutionary Biology: The Drosophila Model. Oxford University Press, New York, Oxford. REIG, O. A., C. BUSCH, M. O. ORTELLS and J. R. CONTRERAS, 1990. An overview of evolution, systematics, population biology, cytogenetics, molecular biology and speciation in Ctenomys.In (NEVO, E. and O.A. REIG, eds.). Evolution of Subterranean Mammals at the Organismal and Molecular Levels. Wiley-Liss, New York. pp. 71-96. REIG, O. A. and P. KIBLISKY, 1969. Chromosome multiformity in the genus Ctenomys (Rodentia, Octodontidae). Chromosoma, 28: 211-244. Berlin. REIG, O. A., A. L. MASSARINI, M. O. ORTELLS, M. A. BARROS, S. I. TIRANTI and F. I. DYZENCHAUZ, 1992. New karyotypes and C-banding patterns of the subterranean rodents of the genus Ctenomys (Caviomorpha, Octodontidae) from Argentina. Mammalia, 56: 603-623. Paris. RICKARDS, G. K., 1983. Orientation behaviour of chromosome multiples of interchange (reciprocal translocation) heterozygotes. Ann. Rev. Genet., 17: 443-498. Palo Alto. ROSATO, M., D. A. MART and C. J. BIDAU, 2003. Meiotic behavior of the B chromosomes of Dichroplus pratensis (Melanoplinae: Acrididae). Cytogenetic and Genome Research: in press. Basel. Rossi, M. S., C. G. Pesce, A. R. Kornblihtt and J. Zorzpulos, 1995. Origin and evolution of a major satellite DNA from South American rodents of the genus Ctenomys. Revista Chilena de Historia Natural, 68: 171-183. Santiago. SEARLE, J. B., 1986. Factors responsible for karyotypic polymorphism in the common shrew, Sorex araneus. Proc. R. Soc. Lond. B, 229: 277-292. London. SEARLE, J. B., 1993. Chromosomal hybrid zones in eutherian mammals. In (HARRISON, R. G., ed.) Hybrid Zones and the Evolutionary Process. Oxford University Press, Oxford. pp. 309-353. SITES, J. W. and C. MORITZ, 1987. Chromosomal evolution and speciation revisited. Syst. Zool., 36: 155-174. SITES, J. W. and K. M. REED, 1994. Chromosomal evolution, speciation and systematics: some relevant issues. Herpetologica, 50: 237-249. SLAMOVITS, C. H., J. A. COOK, E. P. LESSA and M. S. ROSSI, 2001. Recurrent amplifications and deletions of satellite DNA accompanied chromosomal diversification in South American tuco-tucos (Genus Ctenomys, Rodentia: Octodontidae): A phylogenetic approach. Mol. Biol. Evol., 18 (9): 1708-1719. Oxford. SYBENGA, J., 1975. Meiotic Configurations. Springer Verlag, Berlin. SYBENGA, J. and J. M. DE VRIES, 1987. Variation in orientation of a multivalent formed by a reciprocal and a Robertsonian translocation in rye. Biol. Zentralbl., 106: 409-415. SYBENGA, J. and G. K. RICKARDS, 1987. The orientation of multivalents at meiotic metaphase I: a workshop report. Genome, 29: 612-620. Ottawa. TOSTO, D. S. and C. J. BIDAU, 1991. Distribution of chromosome frequencies within a hybrid zone of Dichroplus pratensis (Melanoplinae, Acrididae). Heredity, 67: 299-307. Cambridge. VERZI, D., 1994. Origen y Evolucin de los Ctenomyinae (Rodentia, Octodontidae): Anlisis de la Anatoma CrneoDentaria. Tesis doctoral, Universidad de La Plata. WESTERMAN, M., 1983. Chiasma frequency and variability of morphological characters in populations of two grasshopper species. Heredity, 51: 501-506. Cambridge. WHITE, M. J. D., 1973. Animal Cytology and Evolution. 3rd Edit. Cambridge University Press, Cambridge. WHITE, M. J. D., 1978. Modes of Speciation. W. H. Freeman, San Francisco. WISE, D. and G. K. RICKARDS, 1977. A quadrivalent studied in living and fixed grasshopper spermatocytes. Chromosoma, 63: 305-315. Berlin.
Recibido: 28 de enero de 2003. Aceptado: 3 de marzo de 2003.
72

Bidau, Et Al. 2003. Historia Natural, II (8), 53-72.

Încărcat de

Informații document

Descriere originală:

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Bidau, Et Al. 2003. Historia Natural, II (8), 53-72.

Încărcat de

Drepturi de autor:

Formate disponibile

C. J. Bidau, D. A. Mart and M. D. Jimnez, 2003.

Claudio J. BIDAU1*, Dardo A. MART1 & Mabel D. GIMNEZ

S-ar putea să vă placă și