Figs. 22-26 in Zavreliella inawaheia Sasa, Kitami & Suzuki,

© 2017 Academia Sinica, Taiwan Cytogenetics of Two Onychostoma Species in Taiwan by Ag-NOR and 18S rDNA Profiles Chiao-Chuan Han1,2, Tsair-Bor Yen3, Nian-Cih Chen4, and Mei-Chen Tseng4,* 1National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan. E-mail: [email protected].tw 2Graduate Institute of Marine Biology, National Dong Hwa University, Hualien 974, Taiwan 3Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung 912, Taiwan. E-mail: [email protected] 4Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 912, Taiwan (Received 5 June 2017; Accepted 5 September 2017; Published 25 September 2017; Communicated by Hin-Kiu Mok) Chiao-Chuan Han, Tsair-Bor Yen, Nian-Cih Chen, and Mei-Chen Tseng (2017) Both Onychostoma barbatulum and O. alticorpus are primary freshwater fish in Taiwan. The former has been developed as an aquaculture species with high economic value, while the latter is a native endemic species in Taiwan. Understanding the cytogenetic information of these two species is necessary for their selected breeding, recovery, and management. In this study, Giemsa staining, silver-binding nucleolar organizer region (Ag-NOR), C-banding, and fluorescence in situ hybridization (FISH) with 18S ribosomal (r)DNA probes were used to analyze the cytogenetic characteristics. Results of Giemsa staining showed that the two Onychostoma species shared the same number of chromosomes, 2n = 50. Respective karyotype formulas of the female and male were 10 m + 22 sm + 10 st + 8 t and 11 m + 22 sm + 10 st + 7 t in O. barbatulum, and 14 m + 18 sm + 8 st + 10 t and 15 m + 18 sm + 8 st + 9 t in O. alticorpus. Karyotypes of both species showed a pair of heteromorphic chromosomes in male fish. Their sex determination should be the XX/XY system. Two pairs of Ag-NORs were found in O. barbatulum, but only one pair occurred in O. alticorpus. C-banding areas were observed on centromeres or telomeres of some chromosomes. FISH revealed different cytogenetic characters between these two species. The above cytogenetic information will contribute to species identification, population recovery, and advantages for breeding and management in the future. Key words: Endemic species, Fluorescence in situ hybridization, Karyotype, Sex determination, Species identification. Chiao-Chuan Han and Tsair-Bor Yen contributed equally to this work. *Correspondence: Tel: +886-8-7703202 ext. 6227. E-mail: [email protected] BACKGROUND It is known that there are 23 Onychostoma species that are mainly found in eastern Asia, among which O. barbatulum and O. alticorpus are distributed on the main island of Taiwan. Both species mostly reside in rivers north of the Kaoping River in southwestern Taiwan and north of the Taimali River in eastern Taiwan (Tzeng 1986; Shen 1993). They dwell in rivers with good water quality. Algae attached to stones are their staple food; moreover, they also take small invertebrates. They were recorded breeding in January to July (Chang 1993). Onychostoma barbatulum has been developed as an aquaculture species, and females have a higher growth rate. Artificial breeding and selection efforts were carried out by Tseng et al. (2017). Onychostoma alticorpus is an endemic species in Taiwan. Because of depletion of wild fish resources by serious pollution of rivers and overexploitation, it is considered to require recovery efforts and management (Kottelat 1996; Jang-Liawn 2008). Both species have similar body shapes before reaching a length of about 7 cm. Consequently, it is difficult to distinguish young individuals of Zoological Studies 56: 25 (2017) doi:10.6620/ZS.2017.56-25 1 © 2017 Academia Sinica, Taiwan these two species by external morphological traits. It should be noted that in exploring genetic differences between the two species, the possibility of hybridization of the two species cannot be ruled out. The development of cytogenetics with modern staining and microscopic techniques has resulted in a better understanding of chromosomal structures and provides a useful tool to study species evolution and efficiently characterize species (Rossi et al. 1997; Eichler and Sankoff 2003; Foresti de Almeida-Toledo et al. 2007). The cytogenetics of Onychostoma are currently not fully known, and only a few basic studies have been published simply comprising the chromosome numbers and karyotypes of several species (Table 1). Moreover, the cytogenetics of Onychostoma hybrids have never been studied. In general, numbers of chromosomes (2n) in fish range 16~446 (Yu and Yu 1990; Hallerman 2003). The majority of fish chromosome numbers are 2n = 48, which is considered to be an ancestral characteristic of bony fishes (Leggatt and Iwama 2003; Galetti et al. 2006). Nevertheless, numbers of chromosomes in the Cyprinidae range 42~446, among which Acheilognathus gracilis has the least and Diptycus dipogon the greatest numbers (Hong and Zhou 1985; Yu and Yu 1990). Numbers of chromosomes of 2n = 50 in Onychostoma lini, O. simum, O. gerlachi, and O. elongatum were described in previous reports (Gui et al. 1986; Li et al. 1986; Dai 2013; Han et al. 2015), whereas intraspecific and interspecific diversities in numbers of chromosomes in O. barbatulum and O. alticorpus still need to be explored. In the past, the cytogenetics of more than 1700 fishes were examined (Arkhipchuk 1995), but sex chromosomes were identified in only 176 species (10.4% of those). The reason that sex chromosomes are just found in a few fishes may be due to chromosomes of fish being too small to distinguish, or microscopic resolution being insufficient to examine them clearly (Devlin and Nagahama 2002). Sex determination systems of fish are divided into XX/XY and ZZ/ZW types, for which the heteromorphic chromosome respectively exists in the male and female (de Oliveira et al. 2007; Diniz et al. 2008). Therefore, to elucidate the sex determination systems of O. barbatulum and O. alticorpus, karyotypes of females and males of both species were analyzed in this study. In addition to chromosome numbers and karyotypes, some cytogenetic traits of both Onychostoma species still need to be carefully explored. In general, it is difficult to identify paired chromosomes before staining. Hence, many banding techniques of chromosomes have been developed using acid-base, heat, salt, enzyme, or dye treatments. Due to different DNA and protein compositions of each pair of chromosomes, these techniques show different banding patterns for distinguishing chromosomes. The C-banding technique can exhibit positions of constitutive heterochromatin, which plays an important role in the karyotype diversity of fish (Kavalco et al. 2004). The silver-staining nuclear organizer region (AgNOR) is the position of the major transcriptional activity of ribosomal (r)DNA (Reeder 1990). NOR patterns of the Salmonidae and Cyprinidae were reported to be polymorphic (Phillips and Ihssen 1985; Takai and Ojima 1992; Pendás et al. 1994; Castro et al. 2001). For example, the NORs of Leuciscus were interspecifically polymorphic, and those of Notropis chrysocephalus and Chondrostoma lusitanicum were intraspecifically polymorphic (Gold and Zoch 1990; Rodrigues and Collares-Pereira 1999; Boron et al. 2009). rRNA is the most abundant RNA in cells. Transcriptions of 18S, 5.8S, and 28S rRNAs are Table 1. List of karyotypic studies on six Onychostoma species from 1986 to 2017 Species Karyotype 2n NF NORs Reference O. barbatulum ♀ 10 m + 22 sm + 10 st + 8 t ♂ 11 m + 22 sm + 10 st + 7 t 50 82 83 2 pairs Han et al. 2015 this study O. alticorpus ♀ 14 m + 18 sm + 8 st + 10 t ♂ 15 m + 18 sm + 8 st + 9 t 50 82 83 1 pair Han et al. 2015 this study O. lini 12 m + 8 sm + 4 st + 26 t 50 70 - Dai 2013 O. simum 10 m + 16 sm + 16 st + 8 t 50 76 - Li et al. 1986 O. gerlachi 12 m + 12 sm + 14 st + 12 t 50 74 - Gui et al. 1986 O. elongatum 12 m + 12 sm + 14 st + 12 t 50 74 - Gui et al. 1986 2n, chromosome number; NF, fundamental arm number; NORs, nucleolus organizer regions; -, unknown; m: metacentric; sm: submetacentric; st: subtelocentric; t: telocentric. page 2 of 13Zoological Studies 56: 25 (2017) © 2017 Academia Sinica, Taiwan produced from 45S rDNA by RNA polymerase I (Doudna and Rath 2002). Therefore, the 18S rDNA gene is often used as a specific probe to locate the 45S rDNA region in cytogenetic studies (Gross et al. 2010). It is usually found at one or several different chromosomal loci with a tandem repeated arrangement in higher eukaryotes; nevertheless, the gene is often available in cytotaxonomic studies by fluorescence in situ hybridization (FISH) (Gornung et al. 1997; Nakajima et al. 2012; Maneechot et al. 2016). Both Onychostoma species have similar morphological traits. Therefore, the cytogenetic information will provide a useful tool for classification and hybrid identification. The aims of this study were to compare cytogenetic characters of these two species by Ag-NOR, C-banding, and FISH analyses using an 18S rDNA probe and provide genetic information for recovery of native populations and selected breeding in aquaculture farms in the future. In addition, a hybrid was also examined in the study. MATERIALS AND METHODS Sampling Onychostoma barbatulum and O. alticorpus were collected from Nanzixian Stream (22.15°N, 120.42°E) in southwestern Taiwan and were maintained in a 2-ton fiberglass-reinforced plastic (FRP) tank. Karyotype analyses of O. barbatulum and O. alticorpus were each performed on 15~18 random specimens. The probability of an interspecific hybrid was examined by experimental artificial breeding of a female O. barbatulum and male O. alticorpus. Feeding Some individuals of both species were fed in a 2-ton FRP tank until the chromosome preparation. Eight O. barbatulum and eight O. alticorpus individuals were separately quarantined in a 2-ton FRP tank for 2 weeks. After quarantine, healthy adults were moved to breeding tanks (0.5 ton each) for the hybridization test. The tanks were equipped with temperature control, filtration systems, and life support to maintain a good life quality (21.5°C for O. barbatulum; 23°C for O. alticorpus; dissolved oxygen (DO) of > 7.5 mg/L; and pH of 7.0~8.0). Each breeding tank contained one male and three female individuals. Fish were fed twice a day. Twelve hours of light and darkness were controlled by a timer setting. In terms of water quality, NH4+ (< 0.04 mg/L) and NO2- (< 0.2 mg/L) were monitored weekly. Artificial hybridization When the fish showed sand-stirring behavior, ova of O. barbatulum and sperm of O. alticorpus were stripped by gently pressing the abdomen. Fertilized eggs were evenly dispersed in two airsupplied tanks (40 L) under a rearing temperature of 21.5°C. One week after the eggs hatched, the fry were fed with freshly hatched brine shrimp for 3 weeks. Afterward, the proportion of commercial fish powder was gradually increased in the feed until it completely replaced the brine shrimp. The temperature was also gradually raised to 23°C. After 3 months, fish were transferred to an FRP tank (2 tons, with a water temperature of 23~25°C) for 1 year of rearing until the chromosome preparation. Chromosome preparation A mixture containing minimal essential medium (Eagle’s), 15% fetal bovine serum, and 0.0002% colchicine was filtered through an Acrodisc syringe filter with pore size of 0.45 μm (Pall, Ann Arbor, MI, USA) for sterilization. The sterilized mixture was dispensed to 15-ml centrifuge tubes and stored in a -80°C freezer for further experiments. The stored mixture was allowed to equilibrate to room temperature before use. After the fish were cooled on ice as anesthesia, their head kidneys and renal tissues were excised, cut into small pieces, and cultured with the prepared mixture in centrifuge tubes. Chromosome slides were prepared following procedures of Han et al. (2015). Giemsa staining Slides of the two species and their hybrid were further stained with 5% Giemsa (SigmaAldrich, St. Louis, MO, USA) for 10 min, rinsed with distilled water, air-dried at room temperature, and finally mounted with gum arabic (Ledley et al. 1972). Ag-NOR analysis Slides of the two Onychostoma species were completed by silver staining; 2% (w/v) gelatin page 3 of 13Zoological Studies 56: 25 (2017) © 2017 Academia Sinica, Taiwan was prepared by thoroughly mixing 1 mL formic acid, 2 g gelatin, and 99 mL double-distilled (dd) H2O on a hot plate at 40~50°C. The solution was preserved in a dark glass bottle. A 50% silver nitrate solution was prepared by dissolving 5 g crystalline silver nitrate in 10 mL ddH2O. This solution was stored in a dark glass bottle at 4°C. Three drops of 2% gelatin and four drops of the 50% silver nitrate solution were added to the slide and covered with a cover glass. The slide was further placed on a 65°C hot plate for 2~3 min until it turned brown, rinsed with ddH2O, and airdried prior to being stained with Giemsa for 30 s (Dracopoli et al. 2001). The slide was observed at 1000× under a light microscope (Leica DM 2500 Microsystems, Wetzlar, Germany) with an oil lens. C-Banding analysis Slides of the two Onychostoma species with chromosome samples were maintained at 60°C overnight, and transferred into 0.2 N HCl at room temperature for 5~15 min. Afterward, a sample was rinsed with ddH2O, air-dried, incubated in 5% Ba(OH)2 for 0.5~5 min at 50°C, and rinsed with ddH2O. The sample was further rinsed with 2× SSC (0.3 M NaCl and 0.03 M sodium citrate, at pH 7.0) for 90 min at 60°C, washed with ddH2O, airdried, and stained with Giemsa for 90 min (Fujiwara et al. 1998). The stained sample was observed at 1000× under a light microscope (Leica DM 2500 Microsystems) with an oil lens. 18S rDNA subcloning and analysis DNA was extracted from 5~10 mg of muscle tissue of one specimen of both O. barbatulum and O. alticorpus using a Puregene Core kit A (Qiagen Sciences, Germantown, MD, USA). The 18S rDNA of the two species was amplified by polymerase chain reactions (PCRs) using 18S forward and reverse primers (White et al. 1990). Amplification was performed in a Px2 Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). The reaction solution consisted of approximately 50 ng genomic DNA, 50 pmol each of the forward and reverse primers, 2.5 mM dNTP, 0.1 mM MgCl2, 10× buffer, and 2 U Taq polymerase (Takara Shuzo, Shiga, Japan) brought up to 50 μL with sterile water. The PCR program included one cycle of 4 min at 94°C, 35 cycles of 30 s at 94°C, 1 min at 54°C, and 1 min at 72°C, followed by a single further extension of 5 min at 72°C. We evaluated 10 μL of the product on a 0.8% agarose gel to check the PCR success and confirm the product sizes. The remaining PCR products were run on 0.8% agarose gels and purified using a DNA Clean/Extraction kit (GeneMark, Taichung, Taiwan). Purified DNA was subcloned into a pGEM-T easy vector (Promega, Madison, WI, USA) and transformed into the Escherichia coli JM109 strain. Plasmid DNA was isolated using a mini plasmid kit (Geneaid, Taichung, Taiwan). Two clones from O. barbatulum and O. alticorpus were sequenced on an Applied Biosystems (ABI, Foster City, CA, USA) automated DNA sequencer ABI3730x1 using a Bigdye sequencing kit (PerkinElmer, Wellesley, MA, USA). T7 or SP6 primer was used in the sequencing reaction, and the PCR cycle parameters for sequencing were 35 cycles of 30 s at 95°C, 30 s at 50°C, and 1 min at 72°C. The 18S rDNA sequences were checked using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov). The difference between the two 18S rDNA sequences was estimated by MEGA software (Tamura et al. 2007). FISH using 18S rDNA probes Labeled 18S rDNA probes of the two species were generated using a PCR DIG probe synthesis kit (Roche, Mannheim, Germany). The reaction solution contained 10× buffer, 4 mM MgCl2, 200 μM dNTP, 1 μM 18S rDNA primers, 50 ng plasmid DNA, and 2 U Taq polymerase, and was brought up to 100 μL with sterile water. DIG probes were purified by ethanol precipitation. Chromosome slides were processed with 100 μg/mL RNase A in 2× SSC buffer at 37°C for 1 h, and washed thrice in 2× SSC for 5 min each. Slides were then quickly immersed in a cold series of ethanol solutions (70%, 95%, and 100%) to dehydrate the chromatin. After air-drying, chromosome spreads were done by exposing chromosomes to 0.005% pepsin (Roche) in 10 mM HCl at 37°C for 10 min to remove residual proteins, and then washed in phosphate-buffered saline (PBS). Slides were then quickly dehydrated through a cold ethanol series and air-dried. Chromosomes were denatured at 80°C for 5 min in hybridization buffer (2× SSC, 10% dextran sulfate, and 50% deionized formamide). All slides were placed on ice for 3~5 min prior to the addition of 35 μL hybridization buffer containing 50 ng labeling probe. Hybridization occurred at 37°C for 12~16 h. Post-hybridization washes were carried out at page 4 of 13Zoological Studies 56: 25 (2017) © 2017 Academia Sinica, Taiwan 42°C for 15 min in 1× SSC with 50% deionized formamide, followed by 0.1× SSC at 60°C for 5 min thrice, and then rinsed thrice in PBS buffer with 0.2% Tween 20 at 37°C for 5 min each. Antidigoxigenin-rhodamine Fab fragments were diluted to 1:200 using TNB buffer (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% blocking reagent). An antibody solution (100 μL) was added to the slide at 37°C for 30 min and then washed for 5 min thrice in TNT buffer (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20) before being transferred to room temperature. The antibody solution at 100 µL was added to the slide and incubated at 37°C for 30 min, and was washed thrice in TNT buffer at room temperature for 5 min. The slide was immersed in a 70%~100% ethanol series to dehydrate the chromatin. Chromosomes were counterstained with 0.05 μg/mL of 4',6-diamidino-2-phenylindole (DAPI) and mounted in a 1,4-diazabicyclo [2.2.2] octane (DABCO) antifading solution (50% glycerol and 2% DABCO in PBS). Chromosome observation Chromosomes were observed using an optical microscope at 1000× with an oil lens. Digital images of the chromosomes were recorded and analyzed with a chromosome band analytical system (BandView 5.5, Applied Spectral Imaging, Migdal HaEmek, Israel). Chromosomes were classified according to relative lengths of their arms, and every chromosome could be identified into one of four groups (m, metacentric; sm, submetacentric; st, subtelocentric; t, telocentric). Each group was characterized by a defined amplitude of arm ratios (m: 1~1.7, sm: 1.7~3, st: 3~7, t: 7~∞) as described by Levan et al. (1964). In the FISH experiments, all slides were observed on a Leica DMLB fluorescence microscope (Leica Microsystems Wetzlar) equipped with a cooled CCD camera. RESULTS Numbers of chromosomes in the two Onychostoma species ranged ca. 40~50, and they mostly shared the same number, 2n = 50 (Tables 2, 3). The great majority of chromosomes were metacentric and submetacentric. The karyotype formulae of female and male O. barbatulum were 10 m + 22 sm + 10 st + 8 t and 11 m + 22 sm + 10 st + 7 t, respectively. The fundamental arm numbers (FNs) were 82 and 83. The karyotype formulae of the female and male O. alticorpus were 14 m + 18 sm + 8 st + 10 t and 15 m + 18 sm + 8 st + 9 t, respectively; the (FNs) were 82 and 83. Although obvious differences in the karyotypes were present between these two species, both species shared a pair of larger sm chromosomes. In addition, males of O. barbatulum and O. alticorpus had a pair of heteromorphic chromosomes which indicated that the sex determination system should be XX/XY (Fig. 1). Two pairs of NORs were located on telomeres of the no. 1 and 3 metacentric chromosomes in O. barbatulum, and only one pair of NORs was located on telomeres of the no. 2 metacentric chromosome in O. alticorpus, showing that there was a significant difference in the number of NORs between these two species (Fig. 2). However, there was no intraspecific difference in the number or positions of the Ag-NORs in males and females of these two species. While C-banding staining areas were distributed in the centromeres or telomeres of several chromosomes, no significant differences were observed between these two species (Fig. 3). 18S rDNA sequences were subcloned from O. barbatulum and O. alticorpus and were respectively 1844 and 1842 bp in length. Respective ratios of G+C in O. barbatulum and O. alticorpus were 56.3% and 56.6%. In total, 13 different nucleotides and two insertions/deletions were observed between these two sequences (Fig. 4). When two sets of 18S rDNA probes were individually hybridized to chromosomes of these two species using the FISH technique, an 18S rDNA locus was located on telomeres of the no. 10 submetacentric chromosome in both O. barbatulum and O. alticorpus (Fig. 5). All fertilized eggs of the hybrids had a very low hatching rate (< 1%). Most surviving individuals had a body shape or eye deformity (Fig. 6). The number of chromosomes in the hybrid was 75 and was triploid (Fig. 7). DISCUSSION Among 435 species of the Cyprinidae, chromosome numbers of 282 species were identified as being 2n = 50, those of 70 species were 2n = 48; and those of 52 species were 100 or 150. Therefore, 2n = 50 is the most common character of the Cyprinidae (Buth et al. 1991; Klinkhardt et al. 1995; Sola and Gornung 2001; Ueda et al. 2001). The same results were page 5 of 13Zoological Studies 56: 25 (2017) © 2017 Academia Sinica, Taiwan obtained for both O. barbatulum and O. alticorpus. Chromosomes of the Cyprinidae are generally characterized by a high fundamental arm number (FN); that is, there are more metacentric (m) and submetacentric (sm) chromosomes in the karyotype. So far, the study of the cytogenetics in Onychostoma fishes has been insufficient. The karyotypic formulae and FN of O. simum (Sauvage and Dabry de Thiersant 1874), O. elongatum (Pellegrin and Chevey 1934), O. lini (Wu 1939), and O. gerlachi (Peters 1881) are listed in table 1. Among these species, O. lini has the highest number of t chromosomes, because it is very difficult to distinguish between st and t chromosomes. In this study, two Onychostoma species also had similar characteristics to Cyprinidae, with both of their FNs being > 80. Onychostoma barbatulum consisted of 10 or 11 Table 3. Diploid chromosome counts of 15 Onychostoma alticorpus specimens Specimen number Sex Number of cells analyzed Diploid counts ≤ 40 41 42 43 44 45 46 47 48 49 50 1♀31 1 1 2 3 1 23 2♀31 1 1 1 2 1 1 2 22 3♀36 1 2 3 2 28 4 ♂ 34 3 1 30 5 ♂ 38 1 2 2 3 1 29 6 ♂ 34 1 3 1 1 28 7 ♂ 31 1 2 1 3 24 8♀34 1 2 2 2 27 9♀33 1 1 1 2 28 10 ♀33 1 1 3 3 25 11 ♂ 32 2 3 3 2 22 12 ♂ 34 1 1 2 3 1 1 25 13 ♀33 1 1 2 2 27 14 ♂ 36 1 1 3 31 15 ♂ 38 1 1 2 2 32 Table 2. Diploid chromosome counts of 18 Onychostoma barbatulum specimens Specimen number Sex Number of cells analyzed Diploid counts ≤ 40 41 42 43 44 45 46 47 48 49 50 > 50 1♀55 1 2 1 1 2 2 5 41 2♀53 1 1 1 1 2 3 2 7 35 3 ♂ 50 1 1 1 5 42 4♀59 1 1 6 51 5 ♂ 43 1 1 2 1 2 36 6♀26 1 3 22 7♀34 1 2 1 1 29 8♀35 1 6 28 9 ♂ 34 1 1 1 1 3 27 10 ♂ 29 29 11 ♀30 1 2 2 25 12 ♂ 30 1 1 3 25 13 ♂ 30 1 3 6 20 1 14 ♀30 1 3 3 23 15 ♂ 30 2 1 27 16 ♂ 28 1 1 1 1 24 17 ♂ 21 1 3 17 18 ♂ 26 2 1 1 2 1 19 page 6 of 13Zoological Studies 56: 25 (2017) © 2017 Academia Sinica, Taiwan (a) (b) (c) (d) Fig. 1. Karyotypes of (a) female and (b) male Onychostoma barbatulum and (c) female and (d) male O. alticorpus. Fig. 2. NOR locations of (a) Onychostoma barbatulum and (b) O. alticorpus are indicated by arrows. Bars equal 5 μm (1000×). (a) (b) page 7 of 13Zoological Studies 56: 25 (2017) © 2017 Academia Sinica, Taiwan 1 30 O. barbatulum GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCAGGTC TAAGTACACA CGGCCGGTAC AGTGAAACTG CGAATGGCTC ATTAAATCAG TTATGGTCCC TTTGATCGCT CCACCCGGTA CTTGGATAAC O. alticorpus GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCAGGTC TAAGTACACA CGGCCGGTAC AGTGAAACTG CGAATGGCTC ATTAAATCAG TTATGGTCCC TTTGATCGCT CCACCCGGTA CTTGGATAAC 2 60 O. barbatulum TGTGGCAATT CCAGAGCTAA TACATGCAAA CGGGCGCCGA CCTGCCTCCC CCCCGGGGGT GGCGGGGACG CGTGCATTTA TCAGATCCAA AACCCAACCG GCGGCTTGGG CTCCGGCCCC TCCCCGGACC O. alticorpus TGTGGCAATT CCAGAGCTAA TACATGCAAA CGGGCGCCGA CCTGCCTCCC CCCCGGGGGG GGCGGGGACG CGTGCATTTA TCAGATCCAA AACCCATCCG GCGGTCGGGG CTCCGGCCCC GCCCCGGTCC 390 O. barbatulum CTTTGGTGAC TCTAGATAAC CTCGGGCCGA TCGCGCGCCC TCCGCGGCGG CGACGATTCT TTCGAATGTC TGCCCTATCA ACTTTCGATG GTACTTTAGG CGCCTACCAT GGTGACCACG GGTAACGGGG O. alticorpus CTTTGGTGAC TCTAGATAAC CTCGGGCCGA TCGCGCGCCC TCCGCGGCGG CGACGATTCT TTCGAATGTC TGCCCTATCA ACTTTCGATG GTACTTTAGG CGCCTACCAT GGTGACCACG GGTAACGGGG 5 20 O. barbatulum AATCAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAGGAAGGCA GCAGGCGCGC AAATTACCCA TTTCCGACTC GGAGAGGTAG TGACGAAAAA TAACAATACA GGTCTCTTTC O. alticorpus AATCAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAGGAAGGCA GCAGGCGCGC AAATTACCCA TTTCCGACTC GGAGAGGTAG TGACGAAAAA TAACAATACA GGTCTCTTTC 6 50 O. barbatulum GAGGCCCTGT AATTGGAATG AGCGTATCCT AAACCCATGG GTGAGGACCC ATTGGAGGGC AAGTCTGGTG CCAGCAGCCG CGGTAATTCC AGCTCCAATA GCGTATATTA AAGTTGCTGC AGTTAAAAAG O. alticorpus GAGGCCCTGT AATTGGAATG AGCGTATCCT AAACCCATGG GCGAGGACCC ATTGGAGGGC AAGTCTGGTG CCAGCAGCCG CGGTAATTCC AGCTCCAATA GCGTATATTA AAGTTGCTGC AGTTAAAAAG 7 80 O. barbatulum CTCGTAGTTG GATCTCGGGA GTGGGCTGGC GGTCCGCCGC GAGGCGAGCC ACCGCCTGTC CCGGACCCTG CCTCCCGGCG CCCCCCGGAT GCCCTTAACT GGGTGTCCGG TCACCTCGGG GCCCGGAGCG O. alticorpus CTCGTAGTTG GATCTCGGGA GTGGGCTGGC GGTCCGCCGC GAGGCGAGCC ACCGCCTGTC CCGGACCCTG CCTCCCGGCG CCCCCCGGAT GCCCTTAACT GGGTGTCCGG TCACCTCGGG GCCCGGAGCG 9 10 O. barbatulum TTTACTTTGA AAAAATTAGA GTGTTCAAAG CAGGCCGCCC GTCGCCGCTG AATACCGCAG CTAGGAATAA TGGAATAGGA CTCCGGTTCT ATTTTGTGGG TTTCTGGAAC CCGGGGCCAT GATTAAGAGG O. alticorpus TTTACTTTGA AAAAATTAGA GTGTTCAAAG CAGGCCGCCC GTCGCCGCTG AATACCGCAG CTAGGAATAA TGGAGTAGGA CTCCGGTTCT ATTTTGTGGG TTTCTGGAAC CCGGGGCCAT GATTAAGAGG 1 040 O. barbatulum GACGGCCCGG GGGCATTCGT ATTGCGCCGC TAGAGGTGAA ATTCTTGGAC CGGCGCAAGA CGGACGAAAG CGAAAGCATT TGCCAAGAAT GTTTTCATTA ATCAAGAACG AAAGTCGGAG GTTCGAAGAC O. alticorpus GACGGCC-GG GGGCATTCGT ATTGCGCCGC TAGAGGTGAA ATTCTTGGAC CGGCGCAAGA CGGACGAAAG CGAAAGCATT TGCCAAGAAT GTTTTCATTA ATCAAGAACG AAAGTCGGAG GTTCGAAGAC 1 170 O. barbatulum GACCAGATAC CGTCGTAGTT CCGACCGTAA ACGATGCCAA CCCGCGATCC GGCGGCGTTA TTCCCATGAC CCGCCGGGCA GCGTACGGGA AACCACGAGT CTTTGGGTTC CGGGGGGGAG TATGGTTGCA O. alticorpus GATCAGATAC CGTCGTAGTT CCGACCGTAA ACGATGCCGA CCCGCGATCC GGCGGCGTTA TTCCCATGAC CCGCCGGGCA GCGTGCGGGA AACCACGAGT CTTTGGGTTC CGGGGGG-AG TATGGTTGCA 1 300 O. barbatulum AAGCTGAAAC TTAAAGGAAT TGACGGAAGG GCACCACCAG GAGTGGAGCC TGCGGCTTAA TTTGACTCAA CACGGGAAAC CTCACCCGGC CCGGACACGG AAAGGATTGA CAGATTGATA GCTCTTTCTC O. alticorpus AAGCTGAAAC TTAAAGGAAT TGACGGAAGG GCACCACCAG GAGTGGAGCC TGCGGCTTAA TTTGACTCAA CACGGGAAAC CTCACCCGGC CCGGACACGG AAAGGATTGA CAGATTGATA GCTCTTTCTC 1 430 O. barbatulum GATTCTGTGG GTGGTGGTGC ATGGCCGTTC TTAGTTGGTG GAGCGATTTG TCTGGTTCAT TCCGATAACG AACGAGACTC CGGCTTGTTA AATAGTTACG CGGCCCCGTG CGGTCGGCGT TCAACTTCTT O. alticorpus GATTCTGTGG GTGGTGGTGC ATGGCCGTTC TTAGTTGGTG GAGCGATTTG TCTGGTTCAT TCCGATAACG AACGAGACTC CGGCTTGCTA AATAGTTACG CGGCCCCGTG CGGTCGGCGT TCAACTTCTT 1 560 O. barbatulum AGAGGGACAA GTGGCGTTCA GCCACGCGAG ATGGAGCAAT AACAGGTCTG TGATGCCCTT AGATGTCCGG GGCTGCACGC GCGCCACAAT GGGCGGATCA GCGTGTGTCT ACCCTGCGCC GAGAGGCGCG O. alticorpus AGAGGGACAA GTGGCGTTCA GCCACGCGAG ATGGAGCAAT AACAGGTCTG TGATGCCCTT AGATGTCCGG GGCTGCACGC GCGCCACAAT GGGCGGATCA GCGTGTGTCT ACCCTGCGCC GAGAGGCGCG 1 690 O. barbatulum GGTAACCCGC TGAACCCCGC TCGTGATCGG GACTGGGGAT TGAAACTATT TCCCATCAAC GAGGAGTTCC CAGTAAGCGC GGGTCATAAG CTCGCGTTGA TTAAGTCCCT GCCCTTTGTA CACACCGCCC O. alticorpus GGTAACCCGC TGAACCCCGC TCGTGATCGG GACTGGGGAT TGAAACTATT TCCCATCAAC GAGGAATTCC CAGTAAGCGC GGGTCATAAG CTCGCGTTGA TTAAGTCCCT GCCCTTTGTA CACACCGCCC 1 820 O. barbatulum GTCGCTACTA CCGATTGGAT GGTTTAGTGA GGTCCTCGGA TCGGCCCCGC CGGGGCTCCT CGCGGGCCCT GGCGGAGCGC CGAGAAGACG ATCAAACTTG ACTATCTAGA GGAAGTAAAA GTCGTAACAA O. alticorpus GTCGCTACTA CCGATTGGAT GGTTTAGTGA GGTCCTCGGA TCGGCCCCGC CGGGGCTCCT CGCGGGCCCT GGCGGAGCGC CGAGAAGACG ATCAAACTTG ACTATCTAGA GGAAGTAAAA GTCGTAACAA 1844 O. barbatulum GGTTTCCGTA GGTGAACCTG CGGA O. alticorpus GGTTTCCGTA GGTGAACCTG CGGA Fig. 4. Cloned 18S rDNA sequences from Onychostoma barbatulum (NCBI accession no. MF598161) and O. alticorpus (acc. no. MF598162). Gray highlights are variable sites. Fig. 3. C-Banded metaphase of Onychostoma barbatulum (a: female, b: male) and O. alticorpus (c: female, d: male). Darker staining on the chromosomes are where C-positive signals occurred. Bars equal 5 μm (1000×). (a) (c) (b) (d) page 8 of 13Zoological Studies 56: 25 (2017) © 2017 Academia Sinica, Taiwan metacentric and 22 submetacentric chromosomes, which differed from O. alticorpus which had a larger number of metacentric chromosomes (14 or 15 m). However, O. barbatulum and O. alticorpus had slightly larger NFs (82~83) than those of the others (70~76). Onychostoma barbatulum had two pairs of NORs on the m chromosome, while O. alticorpus had one pair of NORs on the m chromosome short arm, illustrating a difference in chromosomal characters between these two species. Buth et al. (1991) reported that 69 species of the Cyprinidae in North America possessed one or two pairs of NORs, and only three species had three pairs of NORs. Klinkhardt et al. (1995) pointed out that nearly 100 species of the Cyprinidae in their study contained only one pair of NORs which was located in telomeres of either the m or sm chromosome short arm. Most of the NOR pairs in the Cyprinidae of North American fishes were polymorphic with 15 or more types (Buth et al. 1991). Similar results were reported by Takai and Ojima (1992). They also found that NORs of more than 30 species of Japanese carp revealed high variations among species. These results from previous studies indicate that the morphological position of NORs can be an effective tool for cytogenetic classification in the Cyprinidae. For example, there are three types of NORs present Fig. 5. FISH results of chromosomes of Onychostoma species using 18S rDNA probes; (a) O. barbatulum and (b) O. alticorpus. Bars equal 5 μm (1000×). Arrows indicate FISH signals. Fig. 6. Body shape and eye deformities in (a) a male and (b) female hybrid of Onychostoma barbatulum × O. alticorpus. Bars equal 3 cm. (a) (b) (a) (b) page 9 of 13Zoological Studies 56: 25 (2017)