Karyotypes and Banding Patterns of Tylototriton Andersoni Boulenger, a Newt Endemic to the Ryukyu Islands

Home , Notophthalmus, Tylototriton, Tylototriton verrucosus

Jpn. J. Genet. (1982) 57, pp. 527-534

Karyotypes and banding patterns of Tylototriton andersoni Boulenger, a newt endemic to the Ryukyu islands

BY Takeshi SETO, Yasuaki UTSUNOMIYA*and Taeko UTSUNOMIYA* Department of Biology, Faculty of Education, Shimane University, Matsue, Shimane 690 and *Faculty of Applied Biological Science , Hiroshima University, Fukuyama, Hiroshima 720

(Received April 3, 1982) ABSTRACT The karyotypes of Tylototriton andersoni Boulenger were studied. This species is known to be an endemic salamander of the Ryukyu islands and may be the most primitive species of the Salamandridae. Somatic chromosomes (2n=24) prepared from intestinal tracts of adult males and females were examined with both conventional Giemsa stain and the BSG technique. Karyological characteristics of the primitive species was recognized in comparison with other Japanese newts Cynops pyrrhogaster and C. ensicauda. The C-band pattern also indicated the dissimilarity with other examined species of more advanced salamandrids. No heteromorphic pair of chromosomes was detected between male and female karyograms prepared with either conventional Giemsa or the C-banding method.

1. INTRODUCTION Salamanders of the genus Tylototriton are thought to be the most primitive species in the family Salamandridae (Wake and Ozeti 1969). In the present time, six Tylototriton species are known. Although fossil specimens have been found widely throughout Europe, five of the six living species are restricted to eastern Asia, and a single species, T. andersoni, is found isolated on four islands lying in the central part of the Ryukyu Archipelago of southern Japan (Inger 1947; Utsunomiya et al. 1978) . Chromosomes of T. andersoni were previously observed by Sato (1941) and Makino (1951) based on the testis-sectioning method. Sato reported the diploid number (2n = 24) and suggested similarity of chromosome morphology to that of another common Japanese newt, Cynops (formerly Triturus) pyrrhogaster, while Makino (1951) described dissimilarities between Tylototriton and Cynops chromosomes. Because of the restricted distribution of Tylototriton andersoni and the difficulty of securing living specimens, detailed karyological studies have not been done since these earlier works. For the reason that its peculiar morpho- logical features and unique ecological and life history patterns, this endemic 528 T. SETO, Y. UTSUNOMIYA and T. UTSUNOMIYA species of newts has much of karyological interest. In this paper we report the karyotypes of both sexes of adult T ylototriton andersoni and the distribution of the constitutive heterochromatin on the somatic chromosomes.

2. MATERIALSAND METHODS Adult male and female Tylototriton andersoni were collected in Tokuno- shima island, Kagoshima Prefecture and bred in our laboratory. Fertilized eggs were obtained by artificial ovulation. Animals were raised to the adult stage about 120 mm total length as described by Utsunomiya and Utsunomiya (1977). These laboratory-reared animals were used for the chromosome study. Somatic chromosomes were prepared from the gut epithelium by the method of Kezer and Sessions (1979). Chromosomes were stained with conventional Giemsa stain. The C-band patterns of mitotic chromosomes were revealed by the BSG technique described by Sumner (1972). The slides were incubated for 15 to 20 seconds at 50°C in a saturated Ba (OH)2 solution. The staining was done in 4 % Giemsa (Gurr's, pH 6.8) for 5 minutes. Nomenclature and abbreviations indicating the shape of individual chromosomes are those pro- posed by Levan et al. (1964).

3. RESULTS The chromosome number of our specimens of T. andersoni was 2n = 24, confirming the previous reports of Sato (1941) and Makino (1951) for this species, and it is the same as that of T, verrucosus reported by Ferrier and Beetschen (1973) and Morescalchi (1973). The male and female karyotypes appear to be identical, as there was no consistent heteromorphic pair of chromosomes in either karyogram (Figs. 1 and 2). Quantitative data for the chromosomes of this species are presented in Table 1, based on the average measurements of 10 metaphasic cells of both sexes. The twelve pairs of somatic chromosomes were classified into three groups, which comprised four pairs of large metacentrics (Nos. 1-4) in the first group with similar morphology; two pairs of metacentric (Nos. 5 and 8) and two pairs of submetacentric elements (Nos. 6 and 7) of medium-sized in the second group; and a group of four pairs of small-sized chromosomes consisting of a metacentric pair (No. 9), two pairs of submetacentrics (Nos. 10 and 11) and a pair of subtelocentric elements (No. 12). Chromosomes of the first group were not easily matched with their homologues with conventional Giemsa staining, while the homo- logous chromosomes of the third group were much easier to identify, especially the smallest pair (No. 12), which have the most distinctive feature found in the complement. Occasionally, the homologues of pair No. 1 are of unequal size, especially in females. The variation, however, is not constant. Comparison of the present quantitative data (Table 1) for the chromosomes Karyotypes and C-banding of Tylototriton andersoni 529 530 T. SETO, Y. UTSUNOMIYA and T. UTSUNOMIYA

Figs. 1-4. Photomicrographs of metaphase chromosomes in the gut epithelial cells of Tylototriton andersoni. Fig. 1, karyotype of male. Fig. 2, karyotype of female. Figs. 3 and 4, C-banding karyotypes of male and female, showing differential staining of the centromere regions of all chromosome pairs. Fig. 5. C-banding karyotypes of Cynops pyrrhogaster (male). Main C-bands were found in the pericentric region of all chromosomes. Bars indicate 10 m. Karyotypes and C-banding of Tylototriton andersoni 531

T. andersoni with that of Ferrier and Beetschen (1973) for T. verrucosus clearly indicates general similarity of both karyotypes. An exception was a minor difference found in pair No. 10, which is metacentric in T. verrucosus and submetacentric in T. andersoni. The most characteristic feature in C-banded chromosomes of T. andersoni is that most of the constitutive heterochromatin is localized in the centromeric 532 T. SETO, Y. UTSUNOMIYA and T. UTSUNOMIYA region. As illustrated in Figs. 3 and 4, dense centromeric C-spots appear in all metaphasic chromosomes. Besides the main band, when C-banding was clear enough, some large metacentric chromosomes (Nos. 1, 2, 3, 4 and 8) have an additional telomeric band on a single arm, and two pairs of submetacentric chromosomes in the third group (Nos. 10 and 11) have a pericentric band on both chromosome arms, which are not as dense as the centromeric band. No other pair of chromosomes has distinct pericentric or interstitial bands. Sex- specific chromosome was not seen using the differential staining technique of the present study.

4. DISCUSSION The karyotype analysis of the genus Tylototriton was done previously in only a single species, T. verrucosus. Chromosomes from larval epidermis and spermatogonia of this species were studied by Ferrier and Beetschen (1973) and Morescalchi (1973). No other study has been undertaken by modern techniques. The habitat as well as ecological behavior of T. verrucosus and T. andersoni are quite different. The former is rather aquatic and the latter is found on land. The distribution of T. verrucosus is relatively wide in east Asia in contrast to the endemic T. andersoni. Comparison of the karyotypes of male and female T. andersoni in the present study with that of T. verrucosus obtained by Ferrier and Beetschen (1973) indicated a general resemblance of both karyotypes. The relative lengths and arm ratios of the chromosomal complement in these species were similar ex- cept for the No. 10 pair, as described above. Another exception was the presence or absence of the secondary constriction. Ferrier and Beetschen (1973) indicated that all pairs but the No. 12 pair have secondary constrictions near the centromeres of T. verrucosus chromosomes. Our observations with both condensed and less-condensed somatic chromosomes in more than 40 metaphasic cells of both sexes did not reveal such distinctive secondary constrictions with consistency. Makino (1951) has pointed out, in spermatogonial cells of T. andersoni, that the karyotype was clearly distinguishable from those of other Japanese newts, Cynops pyrrhogaster and C. ensicauda. We confirmed the obvious difference in morphology between the species of Tylototriton and Cynops in two pairs of chromosomes of Nos. 6 and 12. The chromosomal dissimilarity in these newts was more remarkable in the C-band patterns of their karyotypes (Fig. 5). The specificity of C-band patterns in T. andersoni was easily recognized in comparison to those of examined species belonging to the Salamandridae; Triturus, Cynops, Notophthal- mus, and Pleurodeles (Nardi et al. 1973; Bailly et al. 1973; Hutchison and Pardue 1975; Rudak and Callan 1976; Schmid and Krone 1975; Schmid et al. Karyotypes and C-banding of Tylototriton andersoni 533

1979) . All of these examined species of newts revealed major constitutive heterochromatin localized in the pericentric region of every member of the karyotypes, and centromeric or telomeric C-bands appeared as less dense spots. In contrast, all chromosome pairs of T. andersoni were characterized by the occurrence of large and distinctive C-bands in the centromere region. Also, it is interesting to note that the C-band pattern of Pleurodeles waltlii, examined by Bailly et al. (1973), is different from that of T. andersoni, although Ferrier and Beetschen (1973) provided evidence for a considerable affinity between P. waltlii and T. verrucosus. They were able to produce viable inter- generic hybrids between these two species. Cytologically recognizable sex chromosomes were found in a few species in the Amphibia (Schmid et al. 1979; Kezer and Sessions 1979; Schmid 1980; Sessions 1980,1982; Schempp and Schmid 1981). In true salamanders, Schmid et al. (1979) reported the first definite examples of sex specific chromosomes identifiable by banding methods; an XX/XY-type of sex mechanism in Triturus a. alpestris and T. v. vulgaris. According to them, these species have heteromorphic pair of the telomeric constitutive heterochromatin in males, and the chiasma frequency in the bivalents having heteromorphic telomeres is strongly reduced. In contrast, T. h. helveticus, which was examined in the same time, did not provide evidence of a differentiated sex chromosome heteromorphism. These observation indicated the sex chromosome of the lower vertebrates still maintain initial stage of differentiation as Ohno (1967) described. We were concerned for detecting a heteromorphic pair of sex chromosomes in this primitive species of Salamandridae, paying special attention to the telomeric spot of C-band heterochromatin. Careful observation, however, failed to find unambiguously identifiable heteromorphism in either male or female C-band karyogram. Our attempt will be made to analyze the pairing configurations of bivalents in the male meiosis. We are deeply indebted to Doctor Ronald A. Nussbaum, Museum of Zoology,The University of Michigan, for critical reading and revision of the manuscript. Authors' thanks are due to Professor Emeritus Sajiro Makino,Hokkaido University, for his continuous encouragement and advice.

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Addendum While this paper was in press, a paper by Nussbaum and Brodie (Herpetologica, 38, 320-332, 1982) appeared, in which a new genus, Echinotriton, was described for Tylototriton andersoni and T. chinhaiensis. The remaining species (asperrimus, kweichowensis, taliangensis, verrucosus) were retained in Tylototriton. This taxonomic change, based on morphology and life history differences, agrees with our cytogenetic evidence.