0012-4966/01/0102- $25.00 © 2001 MAIK “Nauka / Interperiodica” 0086 Doklady Biological Sciences, Vol. 376, 2001, pp. 86–88. Translated from Doklady Akademii Nauk, Vol. 376, No. 5, 2001, pp. 707–709. Original Russian Text Copyright © 2001 by Borkin, Litvinchuk, Milto, Rosanov, Khalturin. The system of modern speciation states that species are genetically isolated population systems, although limited interspecies hybridization is possible in contact zones. Therefore, in population-genetic terms, species are determined by the level of reproductive, i.e., genetic, isolation rather than the degree of morpholog- ical and other differences. Striking evidence for this postulate is provided by the existence of the so-called twin species, i.e., the species that are almost indistin- guishable in appearance but obviously isolated geneti- cally [1]. Naturally, such species, also called cryptic (“hidden”), can only be identified by nonmorphological characteristics, such as differences in ecology, behav- ior, cytogenetics, or biochemistry. To date, twin species have been found in many groups of animals, including amphibians. In Europe, e.g., they were described for salamanders of the genus Hydromantes and in tailless amphibians of the genera Alytes, Discoglossus, Hyla, and Rana [2–7]. The occurrence (and, accordingly, fre- quency of finding) of twin species can form ideas of biodiversity whose preservation is now a priority. It is obvious that a reliable estimation of actual biodiversity is impossible without modern cytogenetic and molecu- lar methods. Twin species should occur most frequently in common widespread species, which live under dif- ferent ecological conditions and, hence, should be genetically diverse. Many species of amphibians in Eastern Europe and the former Soviet Union do have such vast ranges traversing different climatic and land- scape zones. In the course of long-term studies on the interspe- cies variability of the genome size in amphibians, we revealed two population types of the spade-footed toad Pelobates fuscus (Laurenti, 1768) from the family Pelobatidae differing in the DNA content. This species of tailless amphibians is typical of Europe; it is wide- spread from France to northern Kazakhstan. This spe- cies is common in many areas and has never attracted special interest from taxonomists. Except for northern Italy, inhabited by the isolated subspecies P. f. insubri- cus, the remaining enormous area of the species range is considered to be inhabited by the nominative subspe- cies, P. f. fuscus [8, 9]. In 1986–1999, we studied 143 animals from 25 P. f. fuscus populations in Russia, Latvia, Ukraine, and Moldova, using DNA flow cytometry [10] (figure). Peripheral blood of the brown frog Rana temporaria (Rt) was used as a reference sample. The size of its genome was assumed to be 1.0. According to the genome size, all the samples appeared to break down into two geographic groups with significantly different amounts of nuclear DNA. In the “eastern” group of spade-footed toads, the average genome size was 0.826 (0.811 to 0.842) Rt, whereas in the “western” group of spade-footed toads, it was only 0.782 (between 0.770 and 0.797) Rt. Thus, the variation ranges for these groups did not even overlap; the mean values differed by about 5.6%. In absolute units corrected for the genome size of the mouse Mus musculus (6.8 pg), the amount of nuclear DNA is 9.32 (9.17 to 9.43) pg in the “eastern” group and 8.84 (8.74 to 8.93) pg in the “west- ern” group. The “eastern” spade-footed toads were found in the Volga Region (Udmurtia and Nizhny Novgorod oblast), in the chernozem regions of Russia (Belgorod, Voron- ezh, and Tambov oblasts) and Ukraine (Kharkov oblast), as well as in central European Russia (Penza and Ryazan oblasts). Smaller spade-footed toads with a smaller genome were found in Latvia, Moldova, Ukraine (Odessa and Chernigov oblasts), as well as northwestern (Pskov and Yaroslavl oblasts) and central European parts of Russia (Tula oblast). It is obvious that these differences are not acciden- tal, since the region of the study covered a substantial part of Eastern Europe from the Baltic Sea to the Danube delta and the Volga, and the geographic distri- butions of the “larger” and “smaller” genomes fit a cer- tain pattern instead of being chaotic. Moreover, the dif- ferences between the “eastern” and “western” types were stable and reproducible when we analyzed the samples in different years. Cryptic Speciation in Pelobates fuscus (Amphibia, Pelobatidae): Cytometric and Biochemical Evidence L. J. Borkin*, S. N. Litvinchuk**, K. D. Milto*, J. M. Rosanov**, and M. D. Khalturin** Presented by Academician N.N. Nikol’skii March 20, 2000 Received April 19, 2000 * Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg, 199034 Russia ** Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St. Petersburg, 194064 Russia GENERAL BIOLOGY