2004 Asiatic Herpetological Research Vol. 10, pp. 282-294 Intraspecific and Interspecific Genome Size Variation in Hynobiid Salamanders of Russia and Kazakhstan: Determination by Flow Cytometry SPARTAK N. LITVINCHUK1,*, LEO J. BORKIN2 AND JURY M. ROSANOV1 1Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 194064, Russia; *E-mail: [email protected] 2Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia Abstract. - The amount of DNA per diploid nucleus in Salamandrella keyserlingii and Onychodactylus fischeri from Russia, as well as in Ranodon sibiricus from Kazakhstan was determined by flow cytometry. Onychodactylus fischeri had the highest genome size, Ranodon sibiricus the lowest, and Salamandrella keyserlingii was intermediate. Obvious geographic variation in genome size was revealed for Salamandrella keyserlingii and Ranodon sibiricus. The com- parison of nuclear DNA content in eleven hynobiid species supported the genus Onychodactylus as a separate line- age. Key words. - nuclear DNA content, genome size, flow cytometry, Caudata, Hynobiidae, Onychodactylus fischeri, Salamandrella keyserlingii, Ranodon sibiricus, Russia, Kazakhstan. Introduction 2002; Litvinchuk et al., 2003). Many authors have noted a relationship between genome size and some biological Diploid genome size, measured in picograms (pg; 1 pg parameters, such as cellular and nuclear sizes, replica- = 10-12 g) of DNA per nucleus, varies widely in verte- tion time, cell-cycle length, cell division rate, metabolic brates. Living amphibians represent the largest range of rate, longevity, morphological complexity in the brain, genome size variability among terrestrial vertebrates, etc. (Van't Hoff and Sparrow, 1963; Goin et al., 1968; with a minimum of 1.7 pg in male frogs of Olmo and Morescalchi, 1975; Bachmann and Nishioka, Eleutherodactylus shrevei (Schmid et al., 2002) and 1978; Sessions and Larson, 1987; Shakhbazov and maximum of 241 pg in Necturus lewisi (Olmo, 1973). Gapchenko, 1990; Licht and Lowcock, 1991; Nevo and Some extinct amphibians may have had maximum DNA Beiles, 1991; Roth et al., 1994; Vinogradov, 1999; contents greater (approximately 300 pg) than the maxi- Gregory, 2001b, 2001c; Griffith et al., 2003). However, mum known in living forms (Thoson and Muraszko, an adaptive value of genome size is most clearly shown 1978). in its relationship with the timing of embryonic develop- Various methods are used for the measuring of ment in amphibians (Bachmann, 1972; Oeldorf et al., genome size in amphibians (for example, biochemical 1978; Horner and Macgregor, 1983; Pagel and Jonstone, analysis, Feulgen densitometry, static cell fluorometry, 1992; Jockusch, 1997; Chipman et al., 2001; Gregory, ultraviolet microscopy, etc.). However, modern studies 2002, 2003). are based on a comparatively new and very precise This research is focused on salamanders of the fam- method of flow DNA cytometry. As a rule, standard ily Hynobiidae, which consists of approximately 42 errors of this method (peak mean ratios) are less than species. Among modern amphibians, hynobiids are rec- 0.5% (Rosanov and Vinogradov, 1998). Among approx- ognized to be one of the primitive urodelans (e.g., imately 415 amphibian species examined, nuclear DNA Duellman and Trueb, 1986; Larson and Dimmick, contents were studied by flow cytometry in about 143 1993). Hynobiids inhabit various mountain regions of species only (Gregory, 2001a). Palaearctic Asia, where they have quite restricted Among some specific applications, DNA flow ranges. The territory of Russia and Kazakhstan are only cytometry has been used to examine ploidy levels inhabited by three hynobiid species. Other family mem- (Borkin et al., 1986, 1996, 2001a; Vinogradov et al., bers, such as the Siberian Salamander (Salamandrella 1990; Sharbel et al., 1997; Litvinchuk et al., 1998, keyserlingii), is widely distributed from Northeastern 2001), to identify morphologically similar species European Russia to the Kamchatka Peninsula. The (Sharbel et al., 1995; MacCulloch et al., 1996; Ussuri Clawed Salamander (Onychodactylus fischeri) Litvinchuk et al., 1997, 1999; Murphy et al., 1997; can be found in the southern part of Russian Far East and Borkin et al., 2001b, 2003; Khalturin et al., 2003), and in the Korean Peninsula. The Semirechensk salamander to search for hybrid individuals (Borkin et al., 1987, (Ranodon sibiricus) inhabits the Dzhungarsky Alatau © 2004 by Asiatic Herpetological Research Vol. 10, p. 283 Asiatic Herpetological Research 2004 Figure 1. Distribution of Salamandrella keyserlingii (grayish area), Ranodon sibiricus (dark square), and Onychodactylus fischeri (dark-grayish area), with localities studied (locality number as in Table 1). Dark circles desig- nate the "Euro-Siberian" type of Salamandrella keyserlingii, white ones - "Primorsky" type. Mountains in southeastern Kazakhstan and adjacent Peripheral blood cells of specimens of the Ribbed China. The last two species are included in the Russian Newt, Pleurodeles waltl, received from Prof. J. C. and Kazakhstan Red Data Books, respectively. Ranodon Lacroix (Université Paris VI, Paris, France) were used sibiricus is also included in the IUCN Red List of as a reference standard. Details of the technique have Threatened Animals under the category "vulnerable" been published previously (Vinogradov et al., 1990; (Borkin, 1998; Kuzmin, 1999). The goal of present Rosanov and Vinogradov, 1998; Borkin et al., 2001). paper is to evaluate the intra- and interspecific genome The relative genome size differences (RD) were calcu- size variation in three hynobiid species of Russia and lated with use of formula: RD=(m1-m2)/(m1+m2) × Kazakhstan, with flow cytometry. 200%, where m1 and m2 are the sample means. Materials and methods Results Eighty-eight metamorphosed specimens from 13 popu- The genome size variation in three hynobiid species var- lations of three species distributed in Russia and ied between 54.5 and 109.0 pg (Table 1). The lowest Kazakhstan were used in this study of genome size vari- nuclear DNA content was recorded in Ranodon sibiricus ation. Blood was taken from the clipped tail tip. The (in average 55.4 pg), whereas the highest was in samples of Salamandrella keyserlingii and Onychodactylus fischeri (107.7 pg). Salamandrella key- Onychodactylus fischeri studied are kept at the collec- serlingii was intermediate in size (66.4 pg). tions of the Department of Herpetology, Zoological Differences between populations were studied in Institute, Russian Academy of Sciences. All specimens two species, represented by more than one sample. In S. of Ranodon sibiricus were obtained from the Almaty keyserlingii, ten samples were allocated to two main, Zoo (Kazakhstan), and after preparation of blood sam- distinct groups with different genome sizes (Table 1 and ples, the live salamanders were sent to the Moscow Zoo. Fig. 1). The geographic distribution of these two groups 2004 Asiatic Herpetological Research Vol. 10, p. 284 Table 1. Locality of origin ("Lat" is latitude, and "Lon" is longitude), sample size, genome size (in picograms; SD is standard deviation) with the coefficient of variation (CV; in percents) for 13 populations of three hynobiid species. Province/Region Locality Lat Lon n Mean SD Range CV Salamandrella keyserlingii (Russia) The "Euro-Siberian" group 1 Nizhny Novgorod Pizhma 57°52' 47°05' 5 66.7 0.2 66.5 - 66.9 0.3 2 Udmurtia Chur 57°06' 52°59' 10 67.5 0.4 66.7 - 68.1 0.6 3 Ekaterinburg Ekaterinburg 56°51' 60°43' 15 66.6 0.5 65.5 - 67.7 0.8 4 Tomsk Tomsk 56°31' 84°58' 11 66.6 0.6 66.0 - 68.0 0.9 5 Yakutia Khandyga 62°39' 135°33' 2 67.8 67.5 - 68.0 6 Kamchatka Petropavlovsk 53°02' 158°38' 1 66.8 7 Kurile Islands Paramushir Island 50°41' 156°07' 2 67.1 66.8 - 67.4 8 Sakhalin Island Aniva 46°43' 142°31' 2 66.5 66.3 - 66.6 TOTAL (for the group) 48 66.9 0.6 65.5 - 68.1 0.9 The "Primorsky" type 9 Primorsky Kedrovaya Pad' Reserve 43°07' 131°32' 11 64.8 0.3 64.4 - 65.2 0.4 10 Primorsky Ussuriysk Reserve 43°38' 132°09' 2 64.8 64.7 - 64.8 TOTAL (for the group) 13 64.8 0.2 64.4 - 65.2 0.4 TOTAL (for the species) 61 66.4 1.0 64.4 - 68.1 1.5 Onychodactylus fischeri (Russia) 11 Primorsky Ussuriysk Reserve 43°38' 132°09' 7 107.7 0.7 106.7 - 109.0 0.7 Ranodon sibiricus (Kazakhstan) 12 Taldy-Kurgan Oy-Saz River 44°51' 79°02' 10 54.8 0.2 54.5 - 55.1 0.3 13 Taldy-Kurgan Borokhudzir River 44°30' 79°28' 10 56.0 0.6 55.1 - 56.7 1.0 TOTAL 20 55.4 0.7 54.5 - 56.7 1.3 Table 2. The genome size (GS; in picograms) of hynobiids, referred by some authors. Species Locality GS Reference Hynobius (Hynobius) dunni unknown 33.762 Olmo, 1973 H. (H.) nebulosus unknown 38.402 Olmo, 1973 H. (H.) tsuensis unknown 32.962 Olmo, 1973 H. (Satobius) retardatus unknown 38.312 Olmo, 1973 H. (Pseudosalamandra) naevis unknown 40.902 Olmo, 1973 Onychodactylus fischeri Ussuriysk Reserve 45.52 Mazin, 1978 -//- 95.081 Vinogradov, 1998 -//- 107.71 Present paper O. japonicus unknown 102-1062 Olmo, 1983 Paradactylodon gorganensis Shirabad cave 34.771 Stöck, 1999 P. mustersi unknown 43.32 Morescalchi et al., 1979 Ranodon sibiricus Tekeli 50.72 Morescalchi et al., 1979 -//- 45.5911 Vinogradov, 1998 2 localities 55.41 Present paper Salamandrella keyserlingii Yakutia 42.52 Mazin, 1978 Ekaterinburg 42.32 Morescalchi et al., 1979 unknown 382 Grafodatsky and Grigoriev, 1982 Ekaterinburg 33.23 Vladychenskaya et al., 1988 -//- 55.4811 Vinogradov, 1998 10 localities 66.411 Present paper 1Flow cytometry; 2Feulgen's densitometry; 3Kinetics reassociation. Vol. 10, p. 285 Asiatic Herpetological Research 2004 Table 3. Genome size (2C), range of egg diameters (mm), and time of embryonic ("Embr.") and larval ("Larv.") devel- opment (days) in some hynobiids. Taxon 2C Reference Egg Embr.