Rodentia, Cricetidae) and Their Hybrids
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_??_1995 The Japan Mendel Society Cytologia 60: 93-102 , 1995 Chromosomal and Synaptonemal Complex Analysis of Robertsonian Polymorphisms in Akodon dolores and Akodon molinae (Rodentia, Cricetidae) and their Hybrids P. Wittouck, E. Pinna Senn, C. A. Sonez, M. C. Provensal, J. J. Polop and J. A. Lisanti Departamento de Ciencias Naturales. Universidad Nacional de Rio Cuarto . (5800). Rio Cuarto, Argentina Accepted March 9, 1995 The complex cricetid genus Akodon, with its five subgenera and more than 40 species (Apfelbaum and Reig 1989, Reig 1987) is characterized by several features that make it very interesting for cytogenetical research, such as intraspecific variation of the autosomes and sex chromosomes, the existence of XY fertile females in some species, and the presence of different species that show apparently identical karyotypes (Bianchi and Merani 1984, Bianchi et al. 1971, 1979a, b, 1989, Gallardo 1982, Maia and Langguth 1981, Reig 1987, Vitullo et al. 1986, and references therein). Two closely related species of the genus, Akodon dolores and A. molinae, share the G-band pattern of their chromosomal arms (Bianchi et al. 1979a) and produce fertile hybrids, at least in laboratory conditions (Merani et al. 1978, Roldan et al. 1984). Their populations present Robertsonian polymorphisms affecting one chromosome pair in A. molinae (Bianchi et al. 1973), and this one and several other pairs in A. dolores (Bianchi et al. 1979a). We report here karyological analyses on some populations assigned to A. dolores on morphological grounds and on hybrid specimens, and show that one of these populations corresponds karyotypically to A. molinae. Craniometric studies fail to differentiate between populations with "dolores" or "molinae" karyotype . Pachytene trivalents of Robertsonian heterozygotes present a short side arm formed by the paired centromeric ends of the subterminal or terminal elements. Materials and methods Cytogenetical studies were carried out on 80 specimens, live-trapped or born in the laboratory. Standard air-dried preparations from bone marrow were made after injection of 0.5 ml of a 60ƒÊg/ml colchicine solution 2hr before sacrifice. G-banding was obtained by trypsin digestion (Seabright 1971). Karyotype order, terminology and tabular presentation of cyto genetical data are basically those of Bianchi et al. (1979a). Synaptonemal complex analysis was made on microdispersed preparations on plastic coated slides (Fletcher 1979). SDS was added to the fixing solution (Solari 1982). The preparations were stained with silver nitrate (Howell and Black 1980). In some cases, selected zones of the preparations were transferred to 100-mesh grids and examined in an Elmiskope 101 A electron microscope. For craniometric analysis, skulls of adult individuals were selected by means of dis criminant functions (Varela et al. 1991). Ten cranial characters (Table I) were measured with Prof. Elsa Pinna Senn Departamento de Ciencias Naturales, Universidad Nacional de Rio Cuarto (5800) Rio Cuarto, Pcia. Cordoba, Argentina. 94 P. Wittouck et al. Cytologia 60 caliper to the next 0.02mm. For these characters, normality (assymmetry and kurtosis) and variance homogeinity were tested (Varela and Polop 1991). To study phenetic variation between samples, a discriminant analysis of Mahalanobis' D2 was performed (STATISTICA), which gives an F-values matrix computed from D2 statistics. To visualize similarity relation ships between samples, a cluster analysis (UPGMA, NTSYS) was utilized, producing a D2-based phenogram. The localities sampled are the following: Coronel Baigorria (32•‹50•LS, 64•‹21•LW); Cruz del Eje (30•‹44•LS, 64•K48•LW); Chucul (32•‹55•LS, 64•‹10•LW); Laguna Larga (31•K46•LS, 63•K48•LW); Rio Tercero (32•K11•LS, 64•K06•LW); Villa de Maria del Rio Seco (29•K54•LS, 63•K43•LW); Villa Dolores, Yacanto (31•K56•LS, 65•K12•LW). Results The karyotypes found in the different populations examined belong to two clearly defined groups. The specimens from Yacanto (type locality of A. dolores) and Villa Dolores present high chromosome numbers (42, 43 or 44), and the karyotypes (Table 1, Fig. 1) are character ized by a polymorphism of pair 1. In effect, three animals with 42 chromosomes have a first pair formed by two large metacentric chromosomes (simple homomorphic, SH). In the nine animals with 43 chromosomes, one of the metacentric elements is replaced by two subterminal chromosomes, which correspond to the arms of the metacentric (heteromorphic, Ht). In the Table 1. Robertsonian polymorphisms in Akodon dolores-molinae populations and their hybrids. The number of specimens with each chromosome constitution is indicated, independently for each pair 1995 Chromosomal and Synaptonemal Complex Analysis of Robertsonian Polymorphisms in Akodon 95 Fig. 1. Representative karyotypes of Akodon dolores-molinae populations and their hybrids. A: Male from Villa Dolores (2n=44) with no metacentric chromosomes. B: Female from Cruz del Eje Ht for pairs 1 and 2, HS for pairs 3 and 5 and HD for pair 4. C: male from Chucul HS for pairs I and 2, Ht for pairs 3 and 5 and HD for pair 4. D: F, female Ht for pairs 1 and 2 and HD for pairs 3-5. E: F, female, Ht for pairs 1-5. F: F2 male HS for pairs 1-3 and HD for pairs 4 and 5. G: F2 male HD for pairs 1 and 5 and HS for pairs 2-4. B-G: Partial karyotypes. The scale represents 10ƒÊm. remaining nine animals (2n=44), the pair is represented by four subterminal chromosomes (double homomorphic, DH). Except a short metacentric pair common in the genus, the rest of the autosomes and the sex chromosomes are apparently telocentric. These karyotypes, however, are not coincident with those described in A. dolores (Bianchi et al. 1971, 1979a), but are identical with A. molinae karyotypes (Bianchi et al. 1969, 1971, 1973). The polymorphism of pair 1, shared also by A. dolores populations, may have arisen by centromeric breakage of the metacentric followed by pericentric inversion in the resulting elements (Bianchi et al. 1973, 1979b). The specimens from Chucul, Villa de Maria del Rio Seco, Cruz del Eje and Coronel Baigorria, on the other hand, have chromosome numbers from 34 to 39 (Table 1) and their karyotypes (Fig. 1) correspond to those of A. dolores (Bianchi et al. 1979a), except for pair 2 polymorphism, previously undescribed. The elements of five pairs (numbered 1 to 5) can be meta-submetacentric or be represented by two subterminal (in pair 1) or apparently telocentric chromosomes (pairs 2-5), corresponding to the biarmed chromosome arms. Biarmed chromo somes predominate in these pairs, but most karyotypes are heteromorphic, principally for pairs 96 P. Wittouck et al. Cytologia 60 4 and 5 (which are also the only pairs present in a DH condition in the Chucul population). Among the 21 animals from this locality, for instance, eight have one, six have two and two have three heteromorphic pairs; 76% of the animals are then heterozygous for at least one pair. Only five specimens present homomorphic karyotypes: in three, the chromosomes of pairs 1 to 5 are all biarmed, and the remaining two animals have four telocentric elements in pair 5. Among the five specimens from Villa de Maria, two are heteromorphic for three and one for one pair, the remaining animals being homomorphic. The small sample from Cruz del Eje includes one specimen heteromorphic for two pairs, another heteromorphic for one, and one homomorphic animal. Finally, the sole specimen from Baigorria has only biarmed chromo somes in the polymorphic pairs. In addition, we analyzed (Table 1, Fig. 1) 18 F1 and 11 F2 specimens resulting from crosses between animals from Yacanto or Villa Dolores and from Chucul; one female from Yacanto, DH for pair one, mated to two different males from Chucul, produced most (14 out of 18) of the F1 hybrids studied by us. Due to premature death of the animals or insatisfactory quality of the preparations, we obtained karyotypical information on both parents for only 15 of the F1 and 8 of the F2 hybrids, and only on one parent for the remaining specimens. Considering the data of Table 1, it can be noticed that, for each of the polymorphic pairs 1-5, all chromosome constitutions are observed. With respect to the F1 and F2 specimens, the only unexpected result is that DH karyotypes for pair 3 should have appeared in the F2: the karyologically characterized matings should have produced DH animals, and the karyotype of the known parent of the remaining three F2 specimens was compatible with the apparition of this constitution. However, the significance of these observations is severely limited by the small size of the sample and by the presence of several DH animals in the F1. We made craniometric comparisons (Tables 2, 3) between populations from the following localities, assigned to A. dolores on the basis of external morphology: in females, Laguna Larga, Table 2. Craniometric data on Akodon dolores-molinae populations a Samples sizes, males and females respectively. bMSL, maximum skull length; CBL, condylo basal length; BL basal length; CZL, condylo zygomatic length; IM3, incisive molar 3 length; D, diastema; ML, length of mandible; NL, nasal length; ZB, zygomatic breadth; SB, skull breadth. cMean•}standard deviation. dFirst row, males; second row, females. 1995 Chromosomal and Synaptonemal Complex Analysis of Robertsonian Polymorphisms in Akodon 97 Chucul (both with "dolores" karyotype; Table 3. Craniometric comparison between Bianchi et al. 1979 and this work), Rio Ter populations of Akodon dolores-molinae cero (karyotype unknown) and Villa Dolores ("molinae" karyotype); and in males, the same localities and Cruz del Eje ("dolores" karyotype). The population with "molinae" karyotype did not present significant differ ences with respect to one (in females) or two (in males) of the populations with "dolores" karyotype, while significant differences did appear in the comparison between the groups with "dolores" karyotype (Table 3).