_??_1995 The Japan Mendel Society Cytologia 60: 93-102 , 1995

Chromosomal and Synaptonemal Complex Analysis of Robertsonian Polymorphisms in dolores and Akodon molinae (Rodentia, ) 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,

Accepted March 9, 1995

The complex cricetid genus Akodon, with its five subgenera and more than 40 (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 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 . 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). The results of the cluster analyses (Fig. 2) showed, in addition, that the patterns of var iation are not correlated with geographical distances. The observation of microdispersed pa chytene spermatocytes after silver staining allowed the analysis of synaptonemal com plexes (Fig. 3). At the light microscope, preparations from 20 animals, including a Group sizes in table II. b*: significant, P<0.05; n. s.: nonsignificant. cRIII: Rio Tercero; LL: Laguna members of the populations studied and F1 Larga; VD: Villa Dolores; CH: Chucul; CE: Cruz del and F2 animals, were analyzed. Trivalent Eje. dUpper row: males; lower row: females. complexes could be characterized by the pre sence of a perpendicular projection in a posi tion consistent in each case with that of the centromere; the side arm of the trivalent corresponding to pair 1 was noticeably longer. In all cases, the number of synaptonemal complexes and of trivalent ones were as expected from the mitotic karyotype. At the electron microscope (five animals), the short lateral arm of the trivalents could be shown to consist of the paired terminal portions of the axes of the two telocentric chromosomes (Fig. 4). Kinetochore position could be not determined in our silver-stained preparations.

Discussion

The cytogenetical data on the populations of Chucul, Villa de Maria, Cruz del Eje and Baigorria are in accordance with the considerable chromosomal polymorphism described in A.

Fig. 2. Phenograms obtained by cluster analysis of Akodon dolores-molinae populations. 98 P. Wittouck et al. Cytologia 60

Fig. 3. Synaptonemal complexes in Robertsonian heterozygotes of Akodon dolores-molinae. A: F2 specimen Ht for pairs 1, 3 and 4, HS for pair 2 and HD for pair 5; light microphotograph. B: F2 specimen HD for pairs 1 and 4 HS for pairs 2 and 3 and Ht for pair 5; light micro

photograph. C: Same specimen as A, partial electron microphotograph. D: Male from Chucul Ht for pairs 3 and 5 (karyotype in Fig. 1, C); partial electron microphotograph. The scale represents 10ƒÊm in A, B and 5ƒÊm in C, D. dolores (Bianchi et al. 1979a). In addition to the known variability of pairs 1, 3, 4 and 5, polymorphism is also apparent in pair 2; all metacentric chromosomes, except small pair 16, are then polymorphic in A. dolores. The predominance of animals with heteromorphic karyotypes in A. dolores natural populations, and the fact that hybrid individuals with up to five heteromorphic pairs can breed, address the question of the fertility of heterozygotes for Robertsonian translocations, and of the persistence of these polymorphisms. Robertsonian variation has been repeatedly described in , either characterizing geographically localized karyomorphs or races or, less frequently, as intrapopulational poly morphisms (for review see Searle 1993). Early studies on meiosis I non-disjunction in Robertsonian heterozygotes of Mus musculus, generally done in laboratory mice with metacentrics of laboratory or feral origin (mostly summarized in Gropp and Winking 1981) showed great variation in non-disjunction rates between male bearers of different Robertsonian translocations. However, the range of non-disjunction rates is lower in heterozygotes for metacentrics of spontaneous laboratory origin (3-6%) than in heterozygotes for metacentrics of feral origin introduced in laboratory strains (2-28%), indicating the importance of genic factors in meiotic disturbances. Non disjunction values are generally higher in female meiosis (Gropp and Winking 1981). Robertsonian heterozygotes for a single translocation are usually fertile. In males heterozygous for two or more pairs without brachial homology, non-disjunction values are 1995 Chromosomal and Synaptonemal Complex Analysis of Robertsonian Polymorphisms in Akodon 99 higher, but fertility can still be normal in heterozygotes for three Robertsonian translocations (Grao et al. 1989). Fertility is generally reduced in mice heterozygous for a higher number of chromosomes. If brachial homology exists, male sterility or semisterility is observed in many cases. In addition, several studies on wild mice from different European regions have shown that the fertility of heterozygotes for single Robertsonian translocations is similar to that of homozygotes (Bauchau et al. 1990, Britton-Davidian et al. 1990, Wallace et al. 1992, Winking 1986). Accordingly, in the marsh rat Holochilus brasiliensis, no significant differences in non disjunction frequencies exist between homozygotes and heterozygotes for up to four Robertson ian translocations, including cases of monobrachial homology; single, double and monobrachial heterozygotes seem to be completely fertile in laboratory crosses (Nachman 1992). Also, simple Robertsonian heterozygotes from a hybrid zone between two karyotypic races of the common shrew Sorex araneus have normal fertility (Searle 1984, 1986, 1990, Garagna et al. 1989). Remarkably, meiotic and gametogenic studies on chain VII-forming complex male heterozygotes suggest that their fertility can be only slightly reduced in comparison to homozygous or simple heterozygous shrews (Mercer et al. 1992). Likewise, fertility does not seem to be greatly compromised in Sigmodon fulviventer heterozygotes (Elder and Pathak 1980). As we have seen, the individuals from Yacanto and V. Dolores show "molinae" karyo types. The thoroughly studied polymorphism of A. molinae has very interesting features. In effect, the DH form is extremely rare in the populations examined until now, corresponding to about 1.5% of trapped individuals, a value which diverges from that expected (6%) from the frequencies of SH and Ht forms (Merani et al. 1980, Redi et al. 1982). The proportion of DH animals in laboratory Ht X Ht crosses is well below the expected value and, moreover, the fertility of DH and Ht individuals is only 0.58 and 39.7% of that of SH animals, respectively (Bianchi et al. 1979b). In a meiotic analysis, a very high MII aneuploidy in the DH form (over 40%) and a milder aneuploidy in the Ht specimens were found (Merani et al. 1980). This is reflected in the frequencies of euploid sperm in each form, although considerable heterogeneity within each type is present; in addition, a process of epididymal selection against aneuploid sperm, already described in M. musculus, is operative in A. molinae (Redi et al. 1982). Strikingly, among the animals from Yacanto and Villa Dolores, DH individuals constitute the predominant form, one of the predominant forms. This, together with the fact that a DH female from Yacanto produced the majority of the F1 hybrids here studied, and with the abundance of DH individuals for pair 1 in the F2 animals, indicate the viability and fertility of this chromosome constitution in the specimens from Cordoba, and exclude a possible meiotic instability of the subterminal elements. The meiotic and fertility studies previously discussed (Bianchi et al. 1979b, Merani et al. 1980, Redi et al. 1982) were carried out on animals from a laboratory colony started with specimens captured in the area of Chasico, Province of Buenos Aires. Other factors, possibly of genic nature, should be considered to explain the high degree of meiotic abnormality and low fertility of the DH form in the Buenos Aires populations. With respect to the populations that, additionally, manifest Robertsonian polymorphism in pairs 2 to 5 ("dolores" karyotypes), it seems that, as in the cases previously discussed, no serious fertility compromise of Ht individuals (or of any of the homozygous forms) exists, although no detailed analyses have been made. Otherwise, such high levels of heterozygosity would be unbearable. A similar situation seems to be present in the populations of the related species, Akodon simulator, polymorphic for three Robertsonian translocations (Liascovich et al. 1990). The pachytene trivalents of individuals with "dolores" or "molinae" karyotype and their 100 P. Wittouck et al. Cytologia 60 hybrids are characterized by the presence of a lateral arm which corresponds to the paired centromeric ends of the acrocentrics. The pachytene pairing of pair 1 trivalent of A. molinae has been previously described by Fernandez-Donoso and Berrios (1985). This configuration of the trivalents has been also observed in laboratory (Grao et al. 1989, Gropp and Winking 1981, Haaf et al. 1989, Mahadevaiah et al. 1990). and wild mice (Wallace et al. 1992), in Sigmodon fulviventer (Elder and Pathak 1980), in Lemur interracial hybrids (Moses et al. 1979) and in Siberian common shrews (Borodin 1991), although in British shrews, most pachytene trivalents lack side arms (Wallace and Searle 1990). It has been suggested that this disposition of the centromeric ends of the acrocentrics at pachytene may favour balanced segregation (Borodin 1991, Elder and Pathak 1980, Fernandez-Donoso and Berrios 1985, Moses et al. 1979); see however Wallace et al. (1992). A. molinae was described as a new species on the basis of animals trapped in the south of Buenos Aires province (Contreras 1968). Several observations must be taken into account when considering the taxonomic status of A. dolores and A. molinae. 1) Fertile hybrids have been obtained in crosses between animals from laboratory colonies of both nominal species (Merani et al. 1978, Roldan et al. 1984) and between specimens from natural populations karyotypically assigned to these species (this report). 2) They share the G-banding patterns of their chromosomal arms (Bianchi et al. 1979a). 3) Nei's coefficient of genetic similarity estimated between specimens obtained from laboratory colonies of the two species and from a wild population of A. dolores gave high values, in the range usually observed between conspecific populations (Apfelbaum and Blanco 1984). 4) External morphology is apparently insufficient to differentiate the two species clearly (Apfelbaum and Blanco 1984). Similarly, we were unable to discriminate between specimens with "molinae" or "dolores" karyotypes by external characteristics; and craniometric analysis revealed significant differences in comparisons between groups with "dolores" karyotype and their absence in some compari sons between the group with "molinae" and the groups with "dolores" karyotype. 5) As we have seen,specimens trapped in the type locality of A. dolores (Yacanto) and the near area of Villa Dolores show "molinae" karyotypes (but historical factors may be here involved). These observations favor the view (Apfelbaum and Blanco 1984, Apfelbaum and Reig 1989) that the populations with 42-44 chromosomes ("molinae" karyotypes) may represent a chromosomal race of A. dolores.

Summary

A cytogenetical study was performed on several populations assigned to Akodon dolores by external morphology. The specimens from Yacanto (type locality of A. dolores) and the near Villa Dolores, however, showed the karyotype of Akodon molinae, presenting the Robertsonian polymorphism of pair 1 described in this species. In the populations with "dolores" karyotype, most specimens were heterozygous for at least one Robertsonian polymorphism in pairs 1 to 5. A discriminant analysis of craniometric values failed to differentiate between groups with "dolores" or "molinae" karyotype. In the F1 and F2 individuals obtained by crossing specimens from Yacanto or V. Dolores with specimens from Chucul ("dolores" karyotype) all the possible chromosomal constitutions in pairs 1 to 5 were seen. These results favor the possibility that A. molinae represents a chromosomal race of A. dolores. In male Robertsonian heterozygotes for one or more pairs, the corresponding number of trivalent complexes was observed in micro dispersed pachytene meiocytes; these were characterized by the presence of a short perpendic ular segment formed by the pairing of the centromeric ends of the subterminal or terminal chromosomes. 1995 Chromosomal and Synaptonemal Complex Analysis of Robertsonian Polymorphisms in Akodon 101

Acknowledgements

We wish to thank M. I. Ortiz for her continuous help and her comments on the manuscript, and M. Torres for assistance in the field. This work was partly supported by grants from CONICOR and CONICET.

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