Chromosome Science 18: 59-62, 2015 Frolov et al. 59

Short Communication

Karyotypes of the lenok Brachymystax from the River basin – AgNORs are diff erent between sharp-snouted and blunt-snouted lenoks

Sergei V. Frolov, Harumi Sakai and Valentina N. Frolova

Received: October 29, 2015 / Accepted: February 5, 2016 © 2015 by the Society of Chromosome Research

Abstract problematic, and the genetic markers enabling unequivocal identifi cation from throughout their distribution ranges are Two sympatric forms of the Siberian lenok genus inconclusive (Osinov et al., 1990). Brachymystax, sharp-snouted lenok (B. lenok, SS) and Based on the morphological and genetic diff erences, the blunt-snouted lenok (B. tumensis is applied currently, names B. lenok (Pallas) for the SS lenok and B. tumensis BS), from the Amur River basin shared similar karyotype Mori for the BS lenok have been applied frequently (e.g. showing 2n = 90 and the formula of 8M+1SM+1SM- Shedko and Shedko, 2003; Bogutskaya and Naseka, 2004). ST+9ST+27A, with NF = 110. However, AgNORs were lo- However, the application of “tumensis” for BS lenok is cated on one each pair of acrocentric and subtelocentric questionable due to the mitochondrial DNA haplotype chromosomes in the SS lenok, and only one pair of sub- of specimens from the Tumen River, type locality of the telocentric chromosomes in the BS lenok. The observed species, approximating that of SS lenok (e.g. Froufe et al., karyotypic difference strongly supported these two 2008). Moreover, Ma et al. (2009) have even synonymized forms to be different species. Variations of previously B. tumensis with B lenok based on mitochondrial control published lenok karyotypes also were discussed. region analysis which indicated only slightly more inter- specifi c genetic divergence (2.2 %) than the intra-specifi c variation recorded for B. tumensis (1.2 %) in . Keywords: Brachymystax, lenok, Amur River, karyotype, In addition, B. l. savinovi Mitrofanov, described from NORs , has been sometimes synonymized with B. lenok (e.g. Shedko and Shedko, 2003; Bogutskaya and Naseka, 2004), and B. l. tsinlingensis Li, described from Introduction China, is of questionable taxonomic status (e.g. Zhao and Zhang, 2009; Xing et al., 2015). The Siberian lenok genus Brachymystax () Although karyological studies have made significant is known to include two morphologically diff erent forms, contributions to the identification and understanding of sharp-snouted and blunt-snouted lenoks (hereaft er referred relationships, origin and evolution in problematic species, to as SS and BS lenoks respectively), being different in such as salmonids (e.g. Frolov, 2000), and studies of the their head form, mouth position, osteological traits, gill karyotypes of lenoks have continued over more than 40 raker numbers and spot pattern on the body (Kifa, 1976; years, the data so far to hand have not yet resolved the Alekseyev et al., 1986, 2003; Chereshnev et al., 2002; Shedko taxonomic status of the latter. Chromosome numbers have and Shedko, 2003; Bogutskaya and Naseka, 2004; Mou et been reported as 2n (number of chromosomes) = 90-92 al., 2006; Ma et al., 2009). Genetic data, including allozyme in both SS and BS forms, but without any explanations (Osinov et al., 1990; Osinov, 1993) and microsatellite regarding the mechanisms of such variability. Moreover, analyses (Ma and Jiang, 2007; Froufe et al., 2008), indicated chromosome arm numbers (NF) are even more variable, that the SS and BS lenoks in the sympatric regions differ NF = 102-136 (see Table 1). genetically and are reproductively isolated from each other. The present study aimed to clarify the difference in However, identifi cation of the two forms in non-sympatric karyotypes, including nuclear organizer regions (NORs), regions based on morphological characters is frequently between SS and BS lenoks from the Amur River basin, as well as to summarize available data on the lenok karyotypes. Sergei V. Frolov and Valentina N. Frolova A.V. Zhyrmunsky Institute of Marine Biology, FEB RAS, Vladivostok Materials and methods 690041, Russia Harumi Sakai () Study materials were obtained from three SS and four BS National Fisheries University, Shimonoseki, Yamaguchi 759-6595, lenoks, caught in 1997 by casting nets or lure fi shing rods Japan from the Manoma, Kiya (tributaries of Manoma R.), Aniuy Tel: +81 83-227-3930 and Arigy (tributaries of Mukhen R.) rivers, in the Amur E-mail: sakaih@fi sh-u.ac.jp River basin (Table 2). 60 Brachymystax karyotype

After an intramuscular injection of 0.5 % colchicine The number of AgNOR-bearing chromosomes was solutions (1 ml/kg body weight, overnight), chromosomal counted in a total of 104 cells of SS and 65 cells of BS slides were prepared by the air-dry method from the specimens (Table 3). AgNORs were located in one to four kidney tissue after hypotonic treatment and fixation in chromosomes in the SS and one or two chromosomes in the 3:1 ethanol-acetic fixative (Frolov, 1989). Chromosomes BS, being most frequently observed in two chromosomes were conventionally stained with Giemsa (4 % solution in in both forms, except for one BS in which one AgNOR- distilled water). The silver staining method (Howell and bearing chromosome was frequently observed (Table 3). Black, 1980) was used to reveal active nuclear organizer The morphology of AgNOR-bearing chromosomes, on regions (AgNORs). the other hand, diff ered somewhat between forms, namely Metaphase plates were analyzed on a Zeiss Amplival A and/or ST being similar in length to the 5th pair of M microscope and photographed with an Olympus c4040 and much smaller than the long arm of the 9th pair of SM digital photographic camera. chromosomes in SS lenok (Fig. 1a), and ST being larger In the present study, lenok chromosomes were classifi ed than the 5th pair of M and nearly the same size as the long into the folowing categories: two-armed chromosomes arm of the 9th pair of SM chromosomes in BS lenok (Fig. including metacentric (M), submetacentric (SM) and 1b). In consequence, AgNORs were different between SS submeta-subtelocentric chromosomes (SM-ST), and one- and BS lenoks in length as well as in number (two pairs vs. armed chromosomes including subtelocentric (ST) and one pair at most). acrocentric chromosomes (A). Discussion Results The karyotypes of lenoks have been often studied. Karyotypes were observed in a total of 30 cells of SS and However, variable results have made comparisons diffi cult 99 cells of BS lenok specimens (Table 2). Conventionally (Table 1). For example, Kang and Park (1973) reported 2n stained karyotypes were similar in both lenoks, showing = 90 and NF = 116 in the Korean lenok, recognizing only modal 2n = 90 with two pairs of large М, two pairs of two morphological chromosome groups , A (meta- and smaller M, four pairs of very small M, one pair of SM submetacentrics) and B (telo- and acrocentrics). Dorofeeva (largest chromosomes in the karyotype), one pair of SM- (1977) documented 2n = 92 and NF = 102, without any ST (given as +2 in NF), eight pairs of ST, and 27 pairs of A, karyogram, and Viktorovsky et al. (1985) described 2n = giving NF = 110 (Table 1, 2 and Fig. 1). 92 and NF = 110-124 from the Amur River basin. None

Table 1. Published karyotypes of sharp-snouted (SS) and blunt-snouted (BS) lenok Snout type 2n NF Karyogram Locality Literature Note Not specifi ed 90 116 + Eastern Kang and Park (1973) as B. lenok ns 92 102 - ? Dorofeeva (1977) as B. lenok BS? 92 110-124 + Amur R. basin Viktorovsky et al. (1985) as B. lenok SS 92 120-122 + Amur R. basin Salimovskaya (1987) as B. lenok SS 90 - - Amur R. basin Ginatulina et al. (1998) as B. lenok BS 90 - - BS 92 - - SS 90-92 106-136 + Amur R. basin Makoedov (1999) as B. lenok SS 90 108 + Amur R. basin Chereshnev et al. (2002) as B. lenok SS 90 108 - Amur R. basin Frolov et al. (2002) as B. lenok BS 90 108 - ns 90 120 + Mudanjiang R. Xu et al. (2009) as B. lenok ns 90 110 + Yalu R. SS 90 106-126 + South Primorye Kartavtseva et al. (2013) as B. lenok BS 90 110-118 + as B. tumensis BS 92 110-124 + SS 90 110 + Amur R. basin present study B. lenok? BS 90 110 + B. tumensis? 2n, number of chromosomes; NF, number of arms; +, present; -, absent

Table 2. Number of cells indicating chromosome numbers (2n) of Table 3. Number of cells having 1 to 4 NOR-bearing chromosomes 90 or greater or less than 90 in 3 individuals of sharp-snouted (SS) in 2 individuals of sharp-snouted (SS) and 3 individuals of blunt- and 4 individuals of blunt-snouted (BS) lenoks from the Amur River snouted (BS) lenoks from the Amur River basin basin Number of NOR-bearing Chromosome numbers chromosomes Individual 2n < 90 2n = 90 2n > 90 Tributary Individual 1 2 3 4 Tributary SS 1 0 11 1 Manoma R. SS 1 1 42 8 1 Manoma R. SS 2 0 14 2 Arigy R. SS 2 2 32 14 4 Arigy R. SS 3 0 1 1 Arigy R. BS 1 1 24 0 0 Manoma R. BS 1 6 24 4 Manoma R. BS 2 5 20 0 0 Aniuy R. BS 2 1 14 0 Aniuy R. BS 3 12 3 0 0 Kiya R. BS 3 0 21 0 Kiya R. Individual as in Table 2 BS 4 3 26 0 Manoma R. Frolov et al. 61

Figure 1. Karyograms and AgNORs (in boxes with 5th and 9th chromosomes for comparison) of sharp-snouted lenok (A) and blunt-snouted lenok (B) from the Amur River basin. 1000х. Karyogram and AgNORs in each lenok form are from diff erent cells. Chromosomes in each morphological cat- egory are arranged according to their centromere position and size. of these papers specified the snout type, SS or BS, but especially of NF (Table 1). In fact, only the reports of according to Salimovskaya (personal communication) and Kang and Park (1973) and Makoedov (1999) included a Makoedov (1999), the karyotype given by Viktorovsky et description of the principles followed for chromosome al. (1985) was for a BS lenok. classifi cation. Ginatulina et al. (1998) noted 2n = 90 or 92 in BS lenok Although the conventional karyotypes of both lenok from Primorye, but failed to give NF or a karyogram. forms from sympatric habitats in the Amur River basin Makoedov (1999) also noted variability in SS lenok from (Table 2) gave very similar results as described (Table 2 and the Amur River basin as 2n = 90-92 and NF = 106-136, Fig. 1), the silver-staining exhibited a certain difference but did not explain the mechanism of the karyotype between SS and BS lenok karyotypes. AgNORs were located variability, NF in particular, and presented only a single in two chromosome pairs at most in SS specimens and one karyogram (2n = 90, NF = 108). Kartavtseva et al. (2013) chromosome pair at most in BS specimens (Table 3, Fig. 1). also reported 2n = 90 or 92 in BS lenok. All the other Moreover, the length and morphology of AgNOR-bearing authors documented each lenok karyotype as 2n = 90, chromosomes were very different between the SS and BS with supporting karyograms (Table 1). Thus, recorded forms (Fig. 1). karyotypes vary from 2n = 90-92 and NF = 102-136. The present results clearly indicated the karyological Because reports of lenok karyotype with 2n = 92 were difference between SS and BS lenoks in the Amur River. not confirmed by karyograms or were based on blurred Th e obtained fi ndings strongly suggest that the two lenok karyograms, the variations may have been due to artifacts. forms examined herein are different species, currently Moreover, the large variation in NF (102-136) must have identified as B. lenok and B. tumensis, respectively (e.g. accentuated by the differing principles of chromosome Shedko and Shedko, 2003; Bogutskaya and Naseka, 2004). classifi cation adopted by the authors. However, their and phylogenetic relationships, In the karyotypes of both lenoks, there is a group of including those with other nominal species (or subspecies), nine pairs of chromosome with short second arms of should be clarified with further comprehensive studies, equivocal length were unable to be classified as submeta- including comparisons of more materials collected from a or subtelocentric chromosomes, according to traditionally wide distribution range. used principles (e.g. Levan et al., 1964). This may be a reason for the variations in reported lenok karyotypes, 62 Brachymystax karyotype

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