Comparative Studies of 5S rDNA Profiles and Cyt b Sequences in two Onychostoma Species (Cyprinidae)

Onychostoma barbatulum and O. alticorpus, two primarily freshwater cyprinid fish, have similar morphological characters and partially overlapping ecological habitats. In order to explore the genetic differences between these two species, chromosomal characteristics and genetic variations were examined by fluorescence in situ hybridization (FISH) of 5S rDNA and cytochrome (Cyt) b gene analysis. Ten specimens of O. barbatulum and O. alticorpus were collected from the Nanzihsian Stream in southern Taiwan. FISH revealed that the 5S rDNA loci of O. barbatulum and O. alticorpus were found at a pericentromeric and subtelomeric position, respectively, in a pair of submetacentric chromosomes. Cyt b genes were amplified and sequenced from five individuals of each species. Intraspecific genetic distances ranged from 0.001–0.004 in O. barbatulum and from 0.001–0.006 in O. alticorpus. Genetic distances between these two species ranged from 0.132–0.142. The phylogenetic tree showed these two species are not sister species. In conclusion, FISH cytogenetic information and Cyt b gene analyses indicated that these two species have significantly different genetic characteristics; nevertheless, their morphological similarities may be due to environmental adaptation.


Introduction
The genus Onychostoma contains about 20 species distributed widely in East Asia, and some of them are economically important. Both O. barbatulum (Pellegrin, 1908) and O. alticorpus (Oshima, 1920) reside on Taiwan Island and are cyprinid fish that have similar external morphological traits and almost identical body shapes before reaching a length of about 7 cm. Consequently, it is difficult to distinguish young individuals of these two species based on external traits. Upon reaching adulthood, their body shapes gradually become distinctive due to O. alticorpus expressing a greater body depth and a more obvious fusiform shape than O. barbatulum. In addition, they share partially sympatric habitats. Most individuals of O. barbatulum and O. alticorpus dwell along the stream bottom, feeding on algae and aquatic insects, but they prefer different micro-environments and have different distributions. O. barbatulum resides in upper stream but migrates to midstream or downstream for winter feeding [1]. It is found in all of Taiwan's rivers except the smaller streams of the southernmost Hengchun [2]. The Taiwan-endemic O. alticorpus resides in the midstream and downstream year-round, and has a narrower south-to-east distribution range that includes the Kaoping, the Hsiukuluan, the Beinan, and the Taimali Rivers ( Figure 1) [3,4]. However, previous studies considered the two species to have seasonal sympatric habitats. The development of cytogenetics with modern molecular techniques that has resulted in a better understanding in genome structure and organization provides a useful tool to study species evolution and efficiently characterize species and populations [5,6]. The cytogenetics of Onychostoma is currently not fully understood and only a few basic studies have been published simply comprising chromosome number and fundamental arm number [7][8][9]. The highly limited cytogenetic techniques applied to fish chromosomes have been greatly thrust by the convenience of fluorescence in situ hybridization (FISH) and FISH-derived procedures [10]. Repetitive DNAs represent a large fraction of vertebrate genomes and are the most frequently used probes to study the organization of fish genomes [11], particularly the multigene family of ribosomal DNAs (rDNAs) [12,13]. It is usually found at one or several different chromosomal loci with a tandem repeated arrangement in higher eukaryotes [14]. Molecular tools, especially the cytochrome b (Cyt b) gene, are routinely used in studies of species identification and systematics [15,16].
The aim of the present study was to explore the genetic differences and evolution of O. barbatulum and O. alticorpus by examining their 5S rDNA profiles using FISH and Cyt b gene analysis.

Results and Discussion
Cytogenetic studies by Giemsa staining of Onychostoma barbatulum and O. alticorpus indicated that the diploid number for both species was 2n = 50 ( Figure 2). The Cyprinidae, which contains more than 2400 species, is the richest family of fish with very divergent chromosome patterns [9,17,18]  The development of cytogenetics with modern molecular techniques that has resulted in a better understanding in genome structure and organization provides a useful tool to study species evolution and efficiently characterize species and populations [5,6]. The cytogenetics of Onychostoma is currently not fully understood and only a few basic studies have been published simply comprising chromosome number and fundamental arm number [7][8][9]. The highly limited cytogenetic techniques applied to fish chromosomes have been greatly thrust by the convenience of fluorescence in situ hybridization (FISH) and FISH-derived procedures [10]. Repetitive DNAs represent a large fraction of vertebrate genomes and are the most frequently used probes to study the organization of fish genomes [11], particularly the multigene family of ribosomal DNAs (rDNAs) [12,13]. It is usually found at one or several different chromosomal loci with a tandem repeated arrangement in higher eukaryotes [14]. Molecular tools, especially the cytochrome b (Cyt b) gene, are routinely used in studies of species identification and systematics [15,16].
The aim of the present study was to explore the genetic differences and evolution of O. barbatulum and O. alticorpus by examining their 5S rDNA profiles using FISH and Cyt b gene analysis.

Results and Discussion
Cytogenetic studies by Giemsa staining of Onychostoma barbatulum and O. alticorpus indicated that the diploid number for both species was 2n = 50 ( Figure 2). The Cyprinidae, which contains more than 2400 species, is the richest family of fish with very divergent chromosome patterns [9,17,18] [8,9]. Most of their chromosomes were metacentric (m) and submetacentric (sm), which are the conservative characteristics of Onychostoma.
All these sequences were used to reconstruct the genealogic tree of 5S rDNA. The interspecific Kimura-2-parameter (K-2-P) genetic distance of 5S rDNA sequences between O. barbatulum and O. alticorpus (0.026) was significantly smaller than those (0.052-0.572) of Onychostoma to other referenced sequences in this study. A neighbor-joining analysis with an 87% bootstrapping value showed that O. barbatulum and O. alticorpus had a close relationship ( Figure 4).
Two different 5S rDNA probes were prepared separately and individually hybridized with the chromosomes of these two species by FISH, both representing a single pair of signals in each species. It is noteworthy that the 5S rDNA locus of O. barbatulum was found at a pericentromeric region and that of O. alticorpus was found on the subtelomeric position of a pair of submetacentric chromosomes ( Figure 5). One pair of 5S rDNA-bearing chromosomes has been presented as the plesiomorphic character state in fishes [23]. Nevertheless, the occurrence of multiple 5S rDNA sites had also been found in a few species, such as Acheilognathus tabira and Cyprinus carpio [24,25].  [8,9]. Most of their chromosomes were metacentric (m) and submetacentric (sm), which are the conservative characteristics of Onychostoma.
All these sequences were used to reconstruct the genealogic tree of 5S rDNA. The interspecific Kimura-2-parameter (K-2-P) genetic distance of 5S rDNA sequences between O. barbatulum and O. alticorpus (0.026) was significantly smaller than those (0.052-0.572) of Onychostoma to other referenced sequences in this study. A neighbor-joining analysis with an 87% bootstrapping value showed that O. barbatulum and O. alticorpus had a close relationship ( Figure 4).
Two different 5S rDNA probes were prepared separately and individually hybridized with the chromosomes of these two species by FISH, both representing a single pair of signals in each species. It is noteworthy that the 5S rDNA locus of O. barbatulum was found at a pericentromeric region and that of O. alticorpus was found on the subtelomeric position of a pair of submetacentric chromosomes ( Figure 5). One pair of 5S rDNA-bearing chromosomes has been presented as the plesiomorphic character state in fishes [23]. Nevertheless, the occurrence of multiple 5S rDNA sites had also been found in a few species, such as Acheilognathus tabira and Cyprinus carpio [24,25].          Table 1). A neighbor-joining analysis with 100% bootstrap support showed that specimens of O. barbatulum and O. alticorpus were on two different monophyletic branches of the phylogenetic tree ( Figure 6)

Fish Collection
Each of the five specimens of O. barbatulum and O. alticorpus were obtained from Nanzihsian Stream, a tributary of the Kaoping River in southwestern Taiwan (Figure 1). Live specimens were

Fish Collection
Each of the five specimens of O. barbatulum and O. alticorpus were obtained from Nanzihsian Stream, a tributary of the Kaoping River in southwestern Taiwan (Figure 1). Live specimens were transported to the laboratory and maintained in fresh water in 500 L aquaria according to the Guide for the care and use of laboratory animals [26] until being sacrificed.

Chromosome Preparations and Staining
Before the experiment, fish were anesthetized using 140 µg/mL tricaine methanesulphonate (MS222, Sigma-Aldrich, St. Louis, MO, USA). Mitotic chromosomes were prepared from the cephalic kidney of each specimen. Briefly, cells were cultured in Minimum Essential Medium (MEM) with 15% fetal bovine serum (GIBCO, New York, NY, USA) and mitotic inhibitor (0.0001% Colchicine) using a Rota-mixer (Firstek Scientific Co., Taipei, Taiwan) at 100 rpm and room temperature for 2 h. A hypotonic solution (0.075 M KCl) was added to the cells for 30 min at room temperature and then centrifuged (3000 rpm, 5 min). Supernatant was decanted. Cells were fixed in a methanol-acetic acid solution at room temperature for 15 min followed by centrifugation (3000 rpm, 5 min), and supernatant was decanted. The fixation process was repeated thrice. One to two drops of cell suspension were dripped onto hot slides from a height of 10-30 cm and stained in 5% Giemsa solution for 10 min. Ten metaphase plates for each species were observed and photographed under a Leica DM2500 light microscope (Leica Biosystems, Wetzlar, Germany) with a BASLER charge-coupled device (CCD) scA1400-17gmASI camera (Basler AG, Ahrensburg, Germany). Metaphase displaying distinct chromosome morphology was used for preparing karyograms. Chromosome morphology was defined according to Levan et al. [27]. Chromosome numbers of species were counted and manually classified with BandView karyotyping software (ASI, Migdal Haemek, Israel).

5S rDNA Purification, Subcloning and Analysis
DNA was extracted from 5 to 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 5S rDNA of the two species was amplified by a polymerase chain reaction (PCR) using 5S forward 5 1 -TACGCCCGATCTCGTCCGATC-3 1 and reverse 5 1 -CAGGCTGGTATGGCCGTAAGC-3 1 primers [23]. Amplification was performed in a Px2 Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). The reaction solution consisted of approximately 5 ng genomic DNA, 50 pmol each of the forward and reverse primers, 25 mM dNTP, 0.05-0.1 mM MgCl 2 , 10ˆbuffer, and 5 U Taq polymerase (Takara Shuzo, Shiga, Japan) brought up to 125 µL with sterile water. The PCR program included one cycle of 4 min at 94˝C, 35 cycles of 30 s at 94˝C, 30 s at 62˝C, and 1 min at 72˝C, followed by a single further extension of 5 min at 72˝C. We evaluated 10 µL of this product on a 1.0% agarose gel to check the PCR success and confirm product sizes. The remaining PCR products were run on 1.0% agarose gels and purified using a DNA Clean/Extraction kit (GeneMark, Taichung, Taiwan). Purified DNA was subcloned into 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 (Perkin-Elmer, Wellesley, MA, USA). T7 and SP6 primers were 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 genealogic analysis of 5S rDNA sequences depended on the neighbour-joining (NJ) method [28] and the significance of the clusters was assessed using bootstrap analysis with 1000 replications. The phylogeny analyses were carried out using MEGA software version 4.0 [29]. Several reference sequences, Tor putitora (EU621853), Leporinus elongatus (AF284729), Liza ramada (AM706452), and Dicentrarchus labrax (HM014367), from NCBI were added to this analysis.

Fluorescence in Situ Hybridization of 5S rDNA Probe
Labeled 5S rDNA probes of the two species were generated using a PCR fluorescein labeling mix kit (Roche, Mannheim, Germany). The reaction solution contained 10ˆbuffer, 4 mM MgCl 2 , 200 µM dNTP, 1 µM 5S rDNA primers [23], 50 ng plasmid DNA, and 2 U Taq polymerase, and was brought up to 100 µL with sterile water. Fluorescein-labeled probes were purified by ethanol precipitation.
Chromosomes were prepared with 100 µg/mL RNase A in 2ˆSSC buffer at 37˝C for 1 h, and washed thrice in 2ˆSSC for 5 min. Slides were then quickly immersed in a cold series of ethanol solutions (70%, 95% and 100%) for dehydrating the chromatin. After air-drying, chromosome spreads were done by exposing chromosomes to 0.005% pepsin (Roche, Mannheim, Germany) 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 (2ˆSSC, 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 42˝C for 15 min in 1ˆSSC with 50% deionized formamide, followed by 0.1ˆSSC 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. Fluorescent signals were amplified using a rabbit anti-FITC antibody (Invitrogen, Carlsbad, CA, USA) which was diluted to 1:200 using the TNB buffer (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Blocking reagent). The 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, 0.05% Tween 20) before being transferred to room temperature. A FITC-labeled goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was diluted to 1:250 using the TNB buffer. Then 100 uL of the antibody solution 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 70%-100% ethanol series for dehydrating chromatin. Chromosomes were counterstained with 0.05 µg/mL of 4 1 ,6-diamidino-2-phenylindole (DAPI) and mounted in 1,4-diazabicyclo[2.2.2]octane (DABCO) anti-fading solution (50% glycerol and 2% DABCO in PBS). All slides were observed on a Leica DM 2500 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CCD camera. FISHView EXPO 4.0 software (Applied Spectral Imaging, Migdal HaEmek, Israel) was used for image analysis.

Cytochrome b Gene Cloning and Analysis
DNA was extracted from 5 to 10 mg of muscle tissue of each of the five specimens of O. barbatulum and O. alticorpus.
The complete Cyt b gene was amplified using the FishcytB-F primer 5 1 -ACCACCGTTGTTATTCAACTACAAGAAC-3 1 and TruccytB-R primer 5 1 -CCGACTTCCGGATTACAAGACCG-3 1 [30]. The reaction solution consisted of approximately 5 ng genomic DNA, 50 pmol each of the forward and reverse primers, 25 mM dNTP, 0.05-0.1 mM MgCl 2 , 10ˆbuffer, and 5 U Taq polymerase (Takara Shuzo, Shiga, Japan), and brought up to 125 µL with Milli-Q water (Millipore, Billerica, MA, USA). The PCR program included one cycle of 4 min at 95˝C, 38 cycles of 1 min at 94˝C, 50 s at 54˝C, and 1 min at 72˝C, followed by a single further extension of 10 min at 72˝C. We evaluated 10 µL of each product on a 0.8% agarose gel to check PCR success and confirm 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 the pGEM-T easy vector (Promega, Madison, WI, USA) and transformed into Escherichia coli JM109 strain. Plasmid DNA was isolated using a mini plasmid kit (Geneaid, Taichung, Taiwan). Different clones from each of five individuals of 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 (Perkin-Elmer, Wellesley, MA, USA) as mentioned above.

Conclusions
Onychostoma barbatulum and O. alticorpus have identical chromosome numbers 2n = 50, but variable karyotypes. The 5S rDNAs of these two Onychostoma species were cloned, sequenced, and mapped by FISH on metaphase plates. The 5S rDNA sequences showed differences in nucleotide lengths of NTRs with the chromosome location being pericentromeric in O. barbatulum and subtelomeric in O. alticorpus. The Cyt b gene sequences of O. barbatulum and O. alticorpus clearly revealed that these two species are not sister species and the Onychostoma genus is not monophyletic; nevertheless, their morphological similarities may be due to environmental adaptation.