Natural Autotetraploid and Chromosomal Characteristics in the Subfamily Botiinae (Cypriniformes, Cobitinae) from Northeast Thailand

Puntivar Kaewmad1, Monthira Monthatong1, Weerayuth Supiwong2, Samnao Saowakoon3 and Alongklod Tanomtong1*

1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Kai Campus, Muang, Nong Kai 43000, Thailand 3 Faculty of Agriculture and Technology, Rajamangala University of Technology Issan Surin Campus, Muang, Surin 32000, Thailand

Received June 7, 2013; accepted October 17, 2013

Summary Here we report natural autotetraploid and chromosomal characteristics in the subfamily Botiinae from Northeast Thailand. Kidney cell samples were taken from tiger botia (Syncrossus helodes), red-ﬁnned loach (Yasuhikotakai modesta), silver botia (Y. lecontei) and skunk botia (Y. morleti). The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the tetraploid chromosome numbers of S. helodes, Y. modesta, Y. lecontei and Y. morleti were 4n (natural autotetraploid)=100 for all species, and the fundamental numbers (NF) were 122 for all species. The presences of metacentric, submetacentric, and telocentric chromosomes were 12-10-78 for all species. No cytologically distinguishable sex chromosome was observed. The nucleolar organizer regions (NORs) were observed at the region adjacent to the short arms of a pair of submetacentric chromosome for all species. These results show the evolutionary relationship between species of loach ﬁsh from Thailand. The karyotype formula was deduced as: m sm m sm t sm t S. helodes (4n=100): L8 +L4 +M4 +M4 +M26+S2 +S52 m sm t m sm t t Y. modesta (4n=100): L10+L8 +L22+M2 +M2 +M50+S6 m sm t m sm t t Y. lecontei (4n=100): L10+L8 +L22+M2 +M2 +M50+S6 m sm t t Y. morleti (4n=100): L12+L10 +L66+M12

Key words Loach ﬁsh, Tetraploid, Syncrossus, Yasuhikotakai, Karyotype, Chromosome.

Loach fishes (family Cobitidae) are classified into the order Cypriniformes which can be divided into 5 families, 279 genera and 2,662 species (Nelson 1994, Macdonald and Thoene 2008). Loaches can be found in Thailand and many other countries including China, Japan and Korea (Kottelat 2004). The main characteristics of loaches are a pair of razor-sharp spines under the eye sockets, sharp head and mouth without teeth, small lump around lips, three pairs of short antennae, a slim, flat-sided body with small fins, and short dorsal and deeply concave caudal fins as well as small embedded scales on the skin which is covered with sticky mucous (Supaporn 1999). They are divided into three subfamilies, namely, the Botiinae, Leptobotinae and Cobitinae. The subfamily Botiinae is divided into five genera including Botia, Chromobotia, Sinibotia, Syncrossus and Yasuhikotakia. These five genera possess a pair of razor-sharp spines under their eye sockets

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.79.299 300 P. Kaewmad et al. Cytologia 79(3)

Table 1. Review of loach ﬁsh cytogenetic reports in the genera Yasuhikotakia and Syncrossus (Cypriniformes, Cobitidae, Botiinae).

Species (loach ﬁshes) 4n NF Karyotype formula Locality NORs Reference

Yasuhikotakai modesta 100 128 14m+14sm/st+72a Thailand ̶ Suzuki and Taki (1996) 100 ̶ ̶ ̶ ̶ Ojima and Yamamoto (1990) 100 122 12m+10sm+78t Thailand S(TR)2 Present study Y. eos 100 ̶ ̶ Laos PDR ̶ Slechtova et al. (2006) 100 158 42m+16sm+42t Thailand ̶ Donsakul et al. (2010) Y. lecontei 100 130 14m+14sm/st+72a Thailand ̶ Suzuki and Taki (1996) 100 122 12m+10sm+78t Thailand S(TR)2 Present study Y. morleti 100 126 14m+12sm/st+74a Cambodia ̶ Suzuki and Taki (1996) 100 158 46m+6sm+48t Thailand ̶ Donsakul et al. (2010) 100 122 12m+10sm+78t Thailand S(TR)2 Present study Y. nigrolineata 100 ̶ ̶ China ̶ Slechtova et al. (2006) Y. sidthimunki 100 124 12m+12sm/st+76a Thailand ̶ Suzuki and Taki (1996) 100 138 30m+6sm+2st+62t Thailand ̶ Donsakul et al. (2010) Syncrossus helodes 100 120 8m+12sm/st+80a Cambodia ̶ Suzuki and Taki (1996) 100 122 12m+10sm+78t Thailand S(TR)2 Present study S. berdmorei 100 122 8m+14sm/st+78a Myanmar ̶ Suzuki and Taki (1996) 100 128 24m+4sm+72t Thailand ̶ Donsakul et al. (2010) S. hymenophysa 100 120 8m+12sm/st+80a Asia ̶ Suzuki and Taki (1996) 90 94 4m+86a India ̶ Rishi and Haobam (1984) S. reversa 100 ̶ ̶ Indonesia ̶ Slechtova et al. (2006)

Remark: 4 n=tetraploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric, st=subtelocentric chromosome, S=short arm, TR=telomeric region and ̶ =not available.

(Nelson 1994). In Thailand, loach fish are locally known as “Pla Moo” and “Pla Khao” where they are meshed for food and local trading. More recently some of them are popular as aquarium fish (Wittayanon 1999). In spite of the great interest in cobitid karyotype evolution, less than 40% of cobitid species have been karyotyped by conventional cytogenetic techniques, as well as Ag-NOR and C-banding techniques (Arai 2011). The diploid-tetraploid chromosome number varies from 2n (diploid)=40 to 4n (tetraploid)=100, with most of the karyotypes composed of 2n=50 in subfamilies Leptobotinae, Cobitinae and 4n=100 in subfamily Botiinae. Most cobitid chromosome complements consist of telocentric chromosomes which gradually decrease in size, and karyotype evolution within the group is accompanied by polyploidy, Robertsonian fissions, fusions and pericentric inversions (Arai 2011). In the present study, four species of loach fishes belonging to subfamily Botiinae will be surveyed from the river basins in northeast Thailand to study their genetic variation. There are three main river basins in northeast Thailand: the Chi, Moon and Mekong basins. There are five reports on cytogenetic studies of loach fishes in genera Syncrossus and Yasuhikotakai (Table 1), namely, Rishi and Haobam (1984), Suzuki and Taki (1996), Ojima and Yamamoto (1990), Donsakul et al. (2010) and Slechtova et al. (2006). The present study, cytogenetics of tiger botia (Syncrossus helodes), red-finned loach (Yasuhikotakai modesta), silver botia (Y. lecontei) and skunk botia (Y. morleti) (Fig. 1), provides the first report on the Ag-NOR banding technique, sizes of chromosome and standardized idiograms. Results obtained will increase our knowledge of the cytogenetics which will provide a basis to examine their taxonomy and evolutionary relationship. 2014 Natural Autotetraploid and Chromosomal Characteristics in the Subfamily Botiinae 301

Fig. 1. General characteristics of four species of loach ﬁsh in Thailand including tiger botia, Syncrossus helodes (A); red-ﬁnned loach, Yasuhikotakai modesta (B); silver botia, Y. lecontei (C) and skunk botia, Y. morleti (D). Scale bars indicate 3 cm.

Materials and methods

Samples Samples of S. helodes, Y. modesta, Y. lecontei and Y. morleti (20 males and 20 females) were obtained from Chi, Pao and Songkram river basins (three localities), Northeast of Thailand. The ﬁshes were transferred to laboratory aquaria and were kept under standard conditions for 7 d before experimentation.

Chromosome preparation Chromosomes were prepared in vivo (Chen and Ehbeling 1968, Nanda et al. 1995) by injecting phytohemagglutinin (PHA) solution into the abdominal cavity of the fish. After 24 h, colchicines were injected into the intramuscular and/or abdominal cavity and left for 2 to 4 h. Kidneys were cut into small pieces then mixed with 0.075 M KCl. After discarding all large cell pieces, 15 mL of cell sediments were transferred to a centrifuge tube and incubated for 25–35 min. KCl was discarded from the supernatant after subsequent centrifugation at 1,200 rpm for 8 min. Cells were fixed in fresh cool fixative (3 methanol : 1 glacial acetic acid) gradually added up to 8 mL before centrifuging again at 1,200 rpm for 8 min, whereupon the supernatant was discarded. 302 P. Kaewmad et al. Cytologia 79(3)

Fixation was repeated until the supernatant was clear and the pellet was mixed with 1 mL ﬁxative. The mixture was dropped onto a clean and cold slide by micropipette followed by the air-dry technique.

Chromosome staining Conventional staining by 20% Giemsa’s solution for 30 min and Ag-NOR banding were conducted (Howell and Black 1980) by adding two drops of 50% silver nitrate and 2% gelatin on slides, respectively. The slides were then sealed with cover glasses and incubated at 60°C for 5 min. After that, the slides were soaked in distilled water until cover glasses were separated. The slides were stained with 20% Giemsa’s solution for 1 min.

Chromosome examination Chromosome counting was performed on mitotic metaphase cells under a light microscope. Twenty clearly observable and well spread chromosomes of each male and female were selected and photographed. The length of the short arm chromosomes (Ls) and the length of long arm chromosomes (Ll) were measured with respect to the length of total arm chromosomes (LT, LT=Ls+Ll). The relative length (RL) and the centromeric index (CI) were estimated using Chaiyasut (1989). The CI (q/p+q) between 0.50–0.59, 0.60–0.69, 0.70–0.89 and 0.90–0.99 were described as metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. The fundamental number (number of chromosome arm, NF) was obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to telocentric chromosomes. All parameters were used in karyotyping and idiograming.

Results and discussion

Chromosome number, fundamental number and karyotype Cytogenetic comparisons were carried out on specimens of S. helodes, Y. modesta, Y. lecontei and Y. morleti from three localities in Northeast Thailand (Chi, Pao and Songkram river basins). The karyotypes of S. helodes, Y. modesta, Y. lecontei and Y. morleti were found to comprise of 100 chromosomes (4n, natural autotetraploid) as previously reported (Suzuki and Taki 1996, Ojima and Yamamoto 1990, Donsakul et al. 2010) (Figs. 2 and 3). Previous cytogenetical reports on the genera Yasuhikotakia and Syncrossus showed remarkable numerical (4n=100) and structural chromosomes with several acrocentric chromosomes (Suzuki and Taki 1996, Ojima and Yamamoto 1990, Donsakul et al. 2010). However, this was different from a previous study by Rishi and Haobam (1984), who reported that S. hymenophysa from India had 4n=90. The fundamental number (NF, chromosome arm numbers) of S. helodes, Y. modesta, Y. lecontei and Y. morleti was 122 for all species. The number of chromosome arms in the subfamily Botiinae varies from 94 to 136 (Rishi and Haobam 1984, Khuda-Bukhsh et al. 1986). Given the assumption that species with a larger NF are more advanced in evolutionary terms, such changes in chromosome arm number appear to be related to the occurrence of pericentric inversions. These are among the most common modifications contributing to karyotypic rearrangement in fish and other vertebrates (King 1993). The chromosome types of S. helodes, Y. modesta, Y. lecontei and Y. morleti consisted of 12 metacentric, 10 submetacentric, and 78 telocentric chromosomes for all species. It is inconsistent with the report of Suzuki and Taki (1996), which revealed that for the karyotypes of Y. modesta, Y. lecontei, Y. morlet, and S. helodes, the presences of metacentric, submetacentric/subtelocentric, and acrocentric chromosomes were 14-14-72, 14-14-72, 14-12-74, and 8-12-80, respectively. The differences between the studies may be due to the use of different criteria for classification of chromosome type. No cytologically distinguishable sex chromosome was observed which is similar 2014 Natural Autotetraploid and Chromosomal Characteristics in the Subfamily Botiinae 303

Fig. 2. Metaphase chromosome plates and karyotypes of tiger botia, Syncrossus helodes (A) and red-finned loach, Yasuhikotakai modesta (B); 4n (autotetraploid)=100 by conventional staining technique (scale bars indicate 10 μm). There are no irregularly sized chromosomes related to sex. to Y. eos, Y. nigrolineata, S. reversa (Slechtova et al. 2006), Y. sidthimunki, S. berdmorei (Suzuki and Taki 1996, Donsakul et al. 2010), S. hymenophysa (Suzuki and Taki 1996, Rishi and Haobam 1984) and other loach fishes in Thailand (Suzuki and Taki 1996, Ojima and Yamamoto 1990, Donsakul et al. 2010). It may be possible that sex chromosomes are at the initiation of differentiation and hence these chromosomes which contain the sex determination gene cannot be detected by cytogenetic analyses (Na-Nakron 2000). The fish in the order Cypriniformes are found worldwide in great numbers. Their karyotypes have been studied and fall into four types. The first type is an ancestor-related chromosome number. The fishes belonging in the family Cyrinidae carry a chromosome number which is 2n=50 or 48. For instance, the common silver barb (Barbodes gonionotus) chromosome number was reported as 2n=50. The second type, polyploid karyotype, is mostly found in the families Cyprinidae and Cobitidae (loach fish). Some loaches contain diploid-polyploid complexes (Kim and Lee 2000, Saitoh et al. 2000) and the occurrence of unisexual (all female) populations of hybrid origin in some of these complexes is emphasized to be a source of establishment of gonochonic tetraploid population (Vasil’ev et al. 1989). For example, goldfish (Carassius auratus) have a chromosome number of 2n=100–104, and goldfish langsdorffi (Carassius auratus langsdorffi) possess a triploid (3n=150) or tetraploid karyotype (4n=200). Suzuki and Taki (1988) reported that Catlocarpio siamensis (2n=98) which is native to Thailand might evolve from the close ancestor of the Indian carp Catlacatla (2n=50) by chromosome multiplication and rearrangement. The third type, reduction in chromosome number, is the outstanding characteristic of the families Characidae and Lebiasinidae which has remained as 2n=22–24. Finally, chromosome multiplication from 2n=48–50 of the ancestor to 2n=54–64 and metacentric chromosome is mostly 304 P. Kaewmad et al. Cytologia 79(3)

Fig. 3. Metaphase chromosome plates and karyotypes of silver botia, Yasuhikotakia lecontei (C) and skunk botia, Y. morleti (D); 4n (autotetraploid)=100 by conventional staining technique (scale bars indicate 10 μm). No cytologically distinguishable sex chromosome was observed. found (Na-Nakorn 2000). Ohno et al. (1967) proposed polyploidization of the origin vertebrate genome as a factor for vertebrate evolution; they found a diploid-tetraploid relationship in the family Cyprinidae on the basis of both the chromosome complement and DNA contents. It seems that S. helodes, Y. modesta, Y. lecontei and Y. morleti studied here may be natural autotetraploid species in the family Cobitidae because of their high chromosome number (4n=100).

Chromosome markers The cytogenetic study of S. helodes, Y. modesta, Y. lecontei and Y. morleti performed here by Ag-NOR banding technique has shown that the region adjacent to the short arms of a pair of submetacentric chromosome had clearly observable nucleolar organizer regions (NORs), hence marker chromosomes. Over 200 species of fishes have been investigated by the Ag-NOR banding technique, some of which have been applied to elucidate taxonomic and morphological affinities, e.g., in the family Cyprinidae (Gold et al. 1986). The Ag-NOR banding technique identifies transcriptionally active rDNA genes in the preceding interphase (Howell and Black 1980). Most of the other species belonging to the order Cypriniformes have one pair of the NORs located terminally on the uniarmed or submetacentric chromosomes (Takai and Ojima 1986, Birstain and Vasil’ev 1987, Sharma et al. 2002). The cyprinid species of polyploidy origin with the complete process of diploidization, such as carp, has also one pair of NORs. The other, for example, the genus Carassius has been shown to have two to four pairs of NORs bearing chromosomes (Takai and Ojima 1986, Buth et al. 1991, Boroń 1999). The four loach species from northeast Thailand were shown to have chromosome markers on 2014 Natural Autotetraploid and Chromosomal Characteristics in the Subfamily Botiinae 305

Table 2. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL) and centromeric index (CI) from 20 metaphases of male and female tiger botia (Syncrossus helodes), 4n (autotetraploid)=100.

Chromosome Chromosome Chromosome Ls Ll Lt RL CI pair size type

1 0.6133 0.7016 1.3150 0.0331 0.5336 Large Metacentric 2 0.5647 0.6453 1.2099 0.0305 0.5333 Large Metacentric 3 0.5602 0.6047 1.1649 0.0294 0.5191 Large Metacentric 4 0.5130 0.5952 1.1082 0.0279 0.5371 Large Metacentric 5 0.4556 0.5292 0.9847 0.0248 0.5374 Large Metacentric 6 0.3656 0.4379 0.8034 0.0202 0.5450 Medium Metacentric 7 0.4070 0.6501 1.0571 0.0266 0.6150 Large Submetacentric 8 0.4026 0.6089 1.0116 0.0255 0.6020 Large Submetacentric 9 0.3747 0.5807 0.9554 0.0241 0.6078 Large Submetacentric 10 0.3553 0.5412 0.8966 0.0226 0.6037 Large Submetacentric 11 0.3164 0.4905 0.8069 0.0203 0.6079 Medium Submetacentric 12 0.0000 0.9975 0.9975 0.0251 1.0000 Large Telocentric 13 0.0000 0.9317 0.9317 0.0235 1.0000 Large Telocentric 14 0.0000 0.9236 0.9236 0.0233 1.0000 Medium Telocentric 15 0.0000 0.8883 0.8883 0.0224 1.0000 Medium Telocentric 16 0.0000 0.8882 0.8882 0.0224 1.0000 Medium Telocentric 17 0.0000 0.8747 0.8747 0.0220 1.0000 Medium Telocentric 18 0.0000 0.8224 0.8224 0.0207 1.0000 Medium Telocentric 19 0.0000 0.8044 0.8044 0.0203 1.0000 Medium Telocentric 20 0.0000 0.8005 0.8005 0.0202 1.0000 Medium Telocentric 21 0.0000 0.7894 0.7894 0.0199 1.0000 Medium Telocentric 22 0.0000 0.7822 0.7822 0.0197 1.0000 Medium Telocentric 23 0.0000 0.7804 0.7804 0.0197 1.0000 Medium Telocentric 24 0.0000 0.7803 0.7803 0.0197 1.0000 Medium Telocentric 25 0.0000 0.7758 0.7758 0.0196 1.0000 Medium Telocentric 26 0.0000 0.7679 0.7679 0.0194 1.0000 Medium Telocentric 27 0.0000 0.7629 0.7629 0.0192 1.0000 Medium Telocentric 28 0.0000 0.7584 0.7584 0.0191 1.0000 Medium Telocentric 29 0.0000 0.7517 0.7517 0.0189 1.0000 Medium Telocentric 30 0.0000 0.7447 0.7447 0.0188 1.0000 Medium Telocentric 31 0.0000 0.7182 0.7182 0.0181 1.0000 Medium Telocentric 32 0.0000 0.7178 0.7178 0.0181 1.0000 Medium Telocentric 33 0.0000 0.6884 0.6884 0.0174 1.0000 Medium Telocentric 34 0.0000 0.6774 0.6774 0.0171 1.0000 Medium Telocentric 35 0.0000 0.6625 0.6625 0.0167 1.0000 Medium Telocentric 36 0.0000 0.6536 0.6536 0.0165 1.0000 Small Telocentric 37 0.0000 0.6526 0.6526 0.0164 1.0000 Small Telocentric 38 0.0000 0.6486 0.6486 0.0163 1.0000 Small Telocentric 39 0.0000 0.6462 0.6462 0.0163 1.0000 Small Telocentric 40 0.0000 0.6457 0.6457 0.0163 1.0000 Small Telocentric 41 0.0000 0.6330 0.6330 0.0160 1.0000 Small Telocentric 42 0.0000 0.6292 0.6292 0.0159 1.0000 Small Telocentric 43 0.0000 0.6285 0.6285 0.0158 1.0000 Small Telocentric 44 0.0000 0.6185 0.6185 0.0156 1.0000 Small Telocentric 45 0.0000 0.6127 0.6127 0.0154 1.0000 Small Telocentric 46 0.0000 0.6007 0.6007 0.0151 1.0000 Small Telocentric 47 0.0000 0.5977 0.5977 0.0151 1.0000 Small Telocentric 48 0.0000 0.5884 0.5884 0.0148 1.0000 Small Telocentric 49 0.0000 0.5776 0.5776 0.0146 1.0000 Small Telocentric 50 0.0000 0.5394 0.5394 0.0136 1.0000 Small Telocentric 306 P. Kaewmad et al. Cytologia 79(3)

Table 3. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL) and centromeric index (CI) from 20 metaphases of male and female red-ﬁnned loach (Yasuhikotakai modesta), 4n (autotetraploid)=100.

Chromosome Chromosome Chromosome Ls Ll Lt RL CI pair size type

1 1.713 2.150 3.863 0.029 0.557 Large Metacentric 2 1.485 1.688 3.173 0.024 0.532 Large Metacentric 3 1.501 1.664 3.165 0.023 0.526 Large Metacentric 4 1.411 1.692 3.103 0.023 0.545 Large Metacentric 5 1.456 1.620 3.076 0.023 0.527 Large Metacentric 6 1.241 1.442 2.683 0.020 0.538 Medium Metacentric 7 1.211 1.901 3.112 0.023 0.611 Large Submetacentric 8 1.138 1.931 3.069 0.023 0.629 Large Submetacentric 9 1.196 1.827 3.023 0.022 0.604 Large Submetacentric 10 1.149 1.739 2.889 0.021 0.602 Large Submetacentric 11 1.077 1.640 2.717 0.020 0.604 Medium Submetacentric 12 0.000 3.255 3.255 0.024 1.000 Large Telocentric 13 0.000 3.106 3.106 0.023 1.000 Large Telocentric 14 0.000 3.101 3.101 0.023 1.000 Large Telocentric 15 0.000 2.988 2.988 0.022 1.000 Large Telocentric 16 0.000 2.984 2.984 0.022 1.000 Large Telocentric 17 0.000 2.957 2.957 0.022 1.000 Large Telocentric 18 0.000 2.936 2.936 0.022 1.000 Large Telocentric 19 0.000 2.875 2.875 0.021 1.000 Large Telocentric 20 0.000 2.826 2.826 0.021 1.000 Large Telocentric 21 0.000 2.794 2.794 0.021 1.000 Large Telocentric 22 0.000 2.792 2.792 0.021 1.000 Large Telocentric 23 0.000 2.771 2.771 0.021 1.000 Medium Telocentric 24 0.000 2.728 2.728 0.020 1.000 Medium Telocentric 25 0.000 2.718 2.718 0.020 1.000 Medium Telocentric 26 0.000 2.691 2.691 0.020 1.000 Medium Telocentric 27 0.000 2.683 2.683 0.020 1.000 Medium Telocentric 28 0.000 2.676 2.676 0.020 1.000 Medium Telocentric 29 0.000 2.664 2.664 0.020 1.000 Medium Telocentric 30 0.000 2.661 2.661 0.020 1.000 Medium Telocentric 31 0.000 2.659 2.659 0.020 1.000 Medium Telocentric 32 0.000 2.592 2.592 0.019 1.000 Medium Telocentric 33 0.000 2.589 2.589 0.019 1.000 Medium Telocentric 34 0.000 2.562 2.562 0.019 1.000 Medium Telocentric 35 0.000 2.553 2.553 0.019 1.000 Medium Telocentric 36 0.000 2.552 2.552 0.019 1.000 Medium Telocentric 37 0.000 2.535 2.535 0.019 1.000 Medium Telocentric 38 0.000 2.510 2.510 0.019 1.000 Medium Telocentric 39 0.000 2.464 2.464 0.018 1.000 Medium Telocentric 40 0.000 2.451 2.451 0.018 1.000 Medium Telocentric 41 0.000 2.442 2.442 0.018 1.000 Medium Telocentric 42 0.000 2.400 2.400 0.018 1.000 Medium Telocentric 43 0.000 2.380 2.380 0.018 1.000 Medium Telocentric 44 0.000 2.309 2.309 0.017 1.000 Medium Telocentric 45 0.000 2.244 2.244 0.017 1.000 Medium Telocentric 46 0.000 2.229 2.229 0.017 1.000 Medium Telocentric 47 0.000 2.144 2.144 0.016 1.000 Medium Telocentric 48 0.000 1.790 1.790 0.013 1.000 Small Telocentric 49 0.000 1.777 1.777 0.013 1.000 Small Telocentric 50 0.000 1.689 1.689 0.013 1.000 Small Telocentric 2014 Natural Autotetraploid and Chromosomal Characteristics in the Subfamily Botiinae 307

Table 4. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL) and centromeric index (CI) from 20 metaphases of male and female silver botia (Yasuhikotakia lecontei), 4n (autotetraploid)=100.

Chromosome Chromosome Chromosome Ls Ll Lt RL CI pair size type

1 1.706 2.142 3.848 0.029 0.557 Large Metacentric 2 1.477 1.680 3.158 0.023 0.532 Large Metacentric 3 1.493 1.656 3.149 0.023 0.526 Large Metacentric 4 1.404 1.684 3.088 0.023 0.545 Large Metacentric 5 1.448 1.612 3.061 0.023 0.527 Large Metacentric 6 1.233 1.434 2.667 0.020 0.538 Medium Metacentric 7 1.204 1.893 3.097 0.023 0.611 Large Submetacentric 8 1.131 1.923 3.053 0.023 0.630 Large Submetacentric 9 1.188 1.819 3.007 0.022 0.605 Large Submetacentric 10 1.142 1.732 2.873 0.021 0.603 Large Submetacentric 11 1.069 1.632 2.701 0.020 0.604 Medium Submetacentric 12 0.000 3.247 3.247 0.024 1.000 Large Telocentric 13 0.000 3.098 3.098 0.023 1.000 Large Telocentric 14 0.000 3.093 3.093 0.023 1.000 Large Telocentric 15 0.000 2.981 2.981 0.022 1.000 Large Telocentric 16 0.000 2.976 2.976 0.022 1.000 Large Telocentric 17 0.000 2.950 2.950 0.022 1.000 Large Telocentric 18 0.000 2.928 2.928 0.022 1.000 Large Telocentric 19 0.000 2.867 2.867 0.021 1.000 Large Telocentric 20 0.000 2.818 2.818 0.021 1.000 Large Telocentric 21 0.000 2.786 2.786 0.021 1.000 Large Telocentric 22 0.000 2.784 2.784 0.021 1.000 Large Telocentric 23 0.000 2.763 2.763 0.021 1.000 Medium Telocentric 24 0.000 2.721 2.721 0.020 1.000 Medium Telocentric 25 0.000 2.710 2.710 0.020 1.000 Medium Telocentric 26 0.000 2.683 2.683 0.020 1.000 Medium Telocentric 27 0.000 2.675 2.675 0.020 1.000 Medium Telocentric 28 0.000 2.669 2.669 0.020 1.000 Medium Telocentric 29 0.000 2.656 2.656 0.020 1.000 Medium Telocentric 30 0.000 2.653 2.653 0.020 1.000 Medium Telocentric 31 0.000 2.651 2.651 0.020 1.000 Medium Telocentric 32 0.000 2.584 2.584 0.019 1.000 Medium Telocentric 33 0.000 2.581 2.581 0.019 1.000 Medium Telocentric 34 0.000 2.554 2.554 0.019 1.000 Medium Telocentric 35 0.000 2.545 2.545 0.019 1.000 Medium Telocentric 36 0.000 2.544 2.544 0.019 1.000 Medium Telocentric 37 0.000 2.527 2.527 0.019 1.000 Medium Telocentric 38 0.000 2.502 2.502 0.019 1.000 Medium Telocentric 39 0.000 2.456 2.456 0.018 1.000 Medium Telocentric 40 0.000 2.443 2.443 0.018 1.000 Medium Telocentric 41 0.000 2.434 2.434 0.018 1.000 Medium Telocentric 42 0.000 2.392 2.392 0.018 1.000 Medium Telocentric 43 0.000 2.372 2.372 0.018 1.000 Medium Telocentric 44 0.000 2.301 2.301 0.017 1.000 Medium Telocentric 45 0.000 2.236 2.236 0.017 1.000 Medium Telocentric 46 0.000 2.221 2.221 0.017 1.000 Medium Telocentric 47 0.000 2.136 2.136 0.016 1.000 Medium Telocentric 48 0.000 1.782 1.782 0.013 1.000 Small Telocentric 49 0.000 1.769 1.769 0.013 1.000 Small Telocentric 50 0.000 1.681 1.681 0.013 1.000 Small Telocentric 308 P. Kaewmad et al. Cytologia 79(3)

Table 5. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL) and centromeric index (CI) from 20 metaphases of male and female skunk botia (Yasuhikotakia morleti), 4n (autotetraploid)=100.

Chromosome Chromosome Chromosome Ls Ll Lt RL CI pair size type

1 0.4783 0.4992 0.9775 0.0277 0.5107 Large Metacentric 2 0.3890 0.4387 0.8277 0.0234 0.5300 Large Metacentric 3 0.3629 0.4391 0.8019 0.0227 0.5475 Large Metacentric 4 0.3857 0.3433 0.7290 0.0206 0.4709 Large Metacentric 5 0.3624 0.3624 0.7247 0.0205 0.5000 Large Metacentric 6 0.3480 0.3242 0.6723 0.0190 0.4823 Large Metacentric 7 0.3624 0.6106 0.9730 0.0276 0.6276 Large Submetacentric 8 0.3624 0.5541 0.9164 0.0260 0.6046 Large Submetacentric 9 0.3438 0.5563 0.9001 0.0255 0.6180 Large Submetacentric 10 0.3433 0.5340 0.8773 0.0248 0.6087 Large Submetacentric 11 0.3265 0.4963 0.8227 0.0233 0.6032 Large Submetacentric 12 0.000 0.8446 0.8446 0.0239 1.0000 Large Telocentric 13 0.000 0.8401 0.8401 0.0238 1.0000 Large Telocentric 14 0.000 0.8256 0.8256 0.0234 1.0000 Large Telocentric 15 0.000 0.8210 0.8210 0.0233 1.0000 Large Telocentric 16 0.000 0.8013 0.8013 0.0227 1.0000 Large Telocentric 17 0.000 0.7829 0.7829 0.0222 1.0000 Large Telocentric 18 0.000 0.7639 0.7639 0.0216 1.0000 Large Telocentric 19 0.000 0.7448 0.7448 0.0211 1.0000 Large Telocentric 20 0.000 0.7441 0.7441 0.0211 1.0000 Large Telocentric 21 0.000 0.7288 0.7288 0.0206 1.0000 Large Telocentric 22 0.000 0.7288 0.7288 0.0206 1.0000 Large Telocentric 23 0.000 0.7258 0.7258 0.0206 1.0000 Large Telocentric 24 0.000 0.7080 0.7080 0.0201 1.0000 Large Telocentric 25 0.000 0.7067 0.7067 0.0200 1.0000 Large Telocentric 26 0.000 0.7067 0.7067 0.0200 1.0000 Large Telocentric 27 0.000 0.6890 0.6890 0.0195 1.0000 Large Telocentric 28 0.000 0.6877 0.6877 0.0195 1.0000 Large Telocentric 29 0.000 0.6877 0.6877 0.0195 1.0000 Large Telocentric 30 0.000 0.6869 0.6869 0.0195 1.0000 Large Telocentric 31 0.000 0.6869 0.6869 0.0195 1.0000 Large Telocentric 32 0.000 0.6829 0.6829 0.0193 1.0000 Large Telocentric 33 0.000 0.6719 0.6719 0.0190 1.0000 Large Telocentric 34 0.000 0.6678 0.6678 0.0189 1.0000 Large Telocentric 35 0.000 0.6585 0.6585 0.0187 1.0000 Large Telocentric 36 0.000 0.6487 0.6487 0.0184 1.0000 Large Telocentric 37 0.000 0.6305 0.6305 0.0179 1.0000 Large Telocentric 38 0.000 0.6297 0.6297 0.0178 1.0000 Large Telocentric 39 0.000 0.6297 0.6297 0.0178 1.0000 Large Telocentric 40 0.000 0.6115 0.6115 0.0173 1.0000 Large Telocentric 41 0.000 0.6106 0.6106 0.0173 1.0000 Large Telocentric 42 0.000 0.6106 0.6106 0.0173 1.0000 Large Telocentric 43 0.000 0.6106 0.6106 0.0173 1.0000 Large Telocentric 44 0.000 0.6061 0.6061 0.0172 1.0000 Large Telocentric 45 0.000 0.5340 0.5340 0.0151 1.0000 Medium Telocentric 46 0.000 0.5340 0.5340 0.0151 1.0000 Medium Telocentric 47 0.000 0.4974 0.4974 0.0141 1.0000 Medium Telocentric 48 0.000 0.4577 0.4577 0.0130 1.0000 Medium Telocentric 49 0.000 0.4403 0.4403 0.0125 1.0000 Medium Telocentric 50 0.000 0.4387 0.4387 0.0124 1.0000 Medium Telocentric 2014 Natural Autotetraploid and Chromosomal Characteristics in the Subfamily Botiinae 309

Fig. 4. Standardized idiogram showing lengths and shapes of chromosomes of the tiger botia (Syncrossus helodes) 4n (autotetraploid)=100 by conventional staining technique.

Fig. 5. Standardized idiogram showing lengths and shapes of chromosomes of thered-ﬁnned loach (Yasuhikotakai modesta), 4n (autotetraploid)=100 by conventional staining technique. 310 P. Kaewmad et al. Cytologia 79(3)

Fig. 6. Standardized idiogram showing lengths and shapes of chromosomes of the silver botia (Yasuhikotakia lecontei), 4n (autotetraploid)=100 by conventional staining technique.

Fig. 7. Standardized idiogram showing lengths and shapes of chromosomes of the skunk botia (Yasuhikotakia morleti), 4n (autotetraploid)=100 by conventional staining technique. 2014 Natural Autotetraploid and Chromosomal Characteristics in the Subfamily Botiinae 311

Fig. 8. Metaphase chromosome plates of tiger botia, Syncrossus helodes (A); red-ﬁnned loach, Yasuhikotakai modesta (B); silver botia, Y. lecontei (C) and skunk botia, Y. morleti (D) 4n (autotetraploid)=100 by Ag-NOR banding technique. The nucleolar organizer regions/NORs (arrows) were observed at the region adjacent to the short arms of a pair of submetacentric chromosomes for all species. Scale bars indicate 10 μm. chromosome pair 1, which are the largest metacentric chromosomes, and on chromosome pair 50, which are the smallest telocentric chromosomes. The important karyotype features are the asymmetrical karyotype, which has three types of chromosomes (metacentric, submetacentric and telocentric chromosome). The largest chromosome is three times larger than the smallest chromosomes. The data from the chromosomal examination of mitotic metaphase cells of S. helodes, Y. modesta, Y. lecontei and Y. morleti are shown in Tables 2–5. Figures 4–7 show the standardized idiogram from conventional staining technique. The idiogram of four loach species showed the gradual decreasing length of the chromosomes. Thus, in order to gain a more thorough understanding of the chromosomal evolutionary history of relationships within the loach ﬁshes, further cytogenetic investigation of the remaining species in this subfamily is required. The karyotype formulas are as follows: m sm m sm t sm t S. helodes (4n=100): L8 +L4 +M4 +M4 +M26+S2 +S52 m sm t m sm t t Y. modesta (4n=100): L10+L8 +L22+M2 +M2 +M50+S6 m sm t m sm t t Y. lecontei (4n=100): L10+L8 +L22+M2 +M2 +M50+S6 m sm t t Y. morleti (4n=100): L12+L10 +L66+M12

Acknowledgements

This work was supported by the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant ATRC-R5304 and grant of Faculty of Science, Khon Kaen University. We wish to acknowledge the support of the Khon Kaen University Publication Clinic, Research and 312 P. Kaewmad et al. Cytologia 79(3)

Technology Transfer Affairs, Khon Kaen University, for their assistance.

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