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• D. aequipinnatus India (Assam) 50 96 14m + 32sm + 4t Khuda-Bukhsh et al. (1986)
• D. aequipinnatus Thailand (Nakhonphanom) 50 96 8m + 28sm + 10st + 4t Magtoon and Arai (1994)
• D. aequipinnatus India (Manipur) 50 96 6m + 34sm + 6st + 4t Present paper D. devario India (Orissa) 50 96 12m + 24 sm + 10st + 4t Khuda-Bukhsh et al. (1986)
• D. devario Asia 50 100 10m + 40st/A Fontana et al. (1970)
• D. malabaricus Asia 50 100 10m + 40st Fontana et al. (1970) Hardie and Hebert (2004)
• D. yuensis India (Manipur) 50 98 10m + 26sm + 12st + 2t Present paper chromosome preparations using hypotonic treatment in 0.56% KCl solution for 45 min, followed by fixation using fresh, chilled Carnoy’s fixative (methanol:acetic acid in 3:1 ratio). The fixative was used at least 3 times, or until a clear transparent cell suspension was obtained. A small quantity of cell suspension was taken in a Pasteur pipette and dropped onto a grease-free, pre-cleaned glass slide from a height of 45–60 cm. Then the slide was swiftly passed over a flame 2–3 times. The chromosome slides were aged in a dust-free place for 2–3 days before staining with 6% Giemsa solution (Sigma) in phosphate buffer (McGregor and Varley) of pH 6.8 for 15 min, washed with double distilled water, and air dried. The slides were inspected using a Leica DM3000 microscope coupled to a Leica digital camera, model
• DFC 310FX, and screened for good metaphase plates. The diploid number and characteristic morphology of these species were obtained from 100 chromosome plates from cells exhibiting the complete chromosome number. Selected chromosome plates were captured under a 100× oil immersion lens using Leica Application Suite software (LAS), version 4.0.0. Homologous pairs of chromosomes were arranged in order of decreasing length within each morphological group and, finally, a karyotype was constructed on the basis of centromere position of the 10 best metaphases. Mean lengths of the short arm (p) and the long arm (q), and arm ratio (the ratio of the long arm to the short arm length) of each chromosome were calculated to classify the chromosomes as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (t), following Levan et al. (1964). Fundamental arm number (NF) was established by assigning a value of 1 to all acrocentric chromosomes and a value of 2 to all metacentric, submetacentric, and subtelocentric chromosomes.
• Diagrammatic representations of haploid karyotypes, i.e. ideograms, were constructed according to short arm (p) length and long arm (q) length using Excel 2010 software
• (Microsoft), and evaluated for overall symmetry versus asymmetry in terms of centromere position and relative size differences. Results Analysis of 100 metaphase plates from the kidney and gill epithelial cells of 25 specimens each of the 2 species revealed that the modal chromosome number was 2n = 50, which was valid over 82% (D. aequipinnatus) and 85% (D. yuensis) of metaphase cells, respectively (Table 2). The karyotype consisted of 10 metacentric, 26 submetacentric, 12 subtelocentric, and 2 acrocentric chromosomes (Figure 2a), having the fundamental arm numbers NF = 98 for D. yuensis. Similarly, D. aequipinnatus karyotype consisted of 6 metacentric, 34 submetacentric, 6 subtelocentric, and 4 acrocentric chromosomes (Figure 2b), having arm numbers NF = 96. No morphologically different chromosomes related to sex were observed in any of the specimens examined in both species. The morphological and numerical data are summarized in Table 3. The averaged haploid ideograms of D. yuensis and D. aequipinnatus chromosome complements are represented in Figures 3a and 3b, respectively. Discussion The diploid chromosome numbers seem to be a rather conservative characteristic and are used as an indicator of the closeness of species interrelationships within families (Moyle and Cech, 2004). The karyotypic data for 4 species of the genus Devario are shown in Table 1. The apparent modal diploid number is 2n = 50 in the genus Devario. Cells lacking normal chromosome number (2n = 45, 47, 48, 49, 50, 51) were probably caused by losses during preparation or additions from nearby cells. Therefore, it can be concluded that chromosome number in this genus is conserved as in other cyprinid fishes of the subfamily Danioninae (e.g., Danio rerio, Rasbora rasbora, R. aurotaenia, R. daniconius, R. sumatrana, R. trilineata, Table Chromosome complements of Devario aequipinnatus and D. yuensis. Species
• No. of metaphase plates No. of chromosomes Percentage D. aequipinnatus 100 45 47 49 50 51 2% 9% 4% 82% 3% D. yuensis 100 45 48 49 50 51 4% 6% 3% 85% 2% sm t a sm m t st b 11 16 18 18 Figure 2. Karyotypes of (a) Devario yuensis and (b) D. aequipinnatus (Bar = 10 µm). Table Chromosome measurements (in µm) and classification of Devario aequipinnatus and D. yuensis from 10 best metaphase chromosomes each (CN: chromosome number; T: chromosome type; m: metacentric; sm: submetacentric; st: subtelocentric; t: acrocentric). D. aequipinnatus D. yuensis CN Long arm Mean ± SD Short arm Mean ± SD Arm ratio Mean ± SD T CN Long arm Mean ± SD Short arm Mean ± SD Arm ratio Mean ± SD T 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 95 ± 0.06 63 ± 0.88 53 ± 0.41 70 ± 0.20 88 ± 0.25 08 ± 0.97 38 ± 0.75 00 ± 0.00 68 ± 0.43 28 ± 0.21 05 ± 0.25 25 ± 0.87 60 ± 0.45 13 ± 0.25 60 ± 0.54 05 ± 0.10 60 ± 0.23 00 ± 0.00 40 ± 0.49 05 ± 0.84 20 ± 0.59 75 ± 0.29 48 ± 0.79 00 ± 0.00 14 ± 0.00 95 ± 0.06 50 ± 0.71 00 ± 0.71 05 ± 0.33 63 ± 0.25 25 ± 0.40 55 ± 0.40 50 ± 0.58 38 ± 0.25 53 ± 0.61 60 ± 0.71 38 ± 0.48 30 ± 0.23 65 ± 0.18 00 ± 0.28 05 ± 0.06 30 ± 0.23 00 ± 0.00 88 ± 0.10 50 ± 0.58 90 ± 0.28 30 ± 0.12 65 ± 0.29 50 ± 0.00 0.00 41 ± 0.03 49 ± 0.18 12 ± 0.13 42 ± 0.16 06 ± 0.10 61 ± 0.06 65 ± 0.12 63 ± 0.43 59 ± 0.30 41 ± 0.43 31 ± 0.50 46 ± 0.14 31 ± 0.08 96 ± 0.14 55 ± 0.27 31 ± 0.03 01 ± 0.21 00 ± 0.00 46 ± 0.20 51 ± 0.60 40 ± 0.34 27 ± 0.20 64 ± 0.13 20 ± 0.00 ∞ m m m m m sm sm sm sm sm sm sm sm sm sm sm sm sm st st st st st st t Esomus danricus, Barilius bendelisis, B. gatensis, B. vagra, B. naseeri, B. pakistanicus, and B. tileo) (Ryoichi, 2011). Thus, the conservative nature of diploid chromosome number in the subfamily Danioninae also suggests the monophyly of this group. In addition, the subfamily Danioninae shows similarity to many of the fish species of the subfamily
• Cyprininae (Collares-Pereira, 1989; Rishi, 1989; Al-Sabti, 1991; Gül et al., 2004) belonging to different genera, such as Chagunius, Cirrhinus, Labeo, Puntius, and Osteobrama, whose diploid numbers are 50. This finding suggests the close relationship between the 2 subfamilies of the family
• Cyprinidae and supports the conservative nature of the karyotype macrostructure within the group, especially the 2n = Though chromosome numbers of Devario species are conserved despite different geographical locations, the fundamental arm numbers (NF) are different. This divergence may be attributed to differences in the karyotype macrostructure, reflecting a real geographical variation common to widespread species (Thaís et al., 2010), or may be the result of differences in the scoring of subtelocentric or acrocentric chromosomes in different species of Devario. Devario aequipinnatus (from the Nakhonphanom region, Thailand, and Assam and Manipur, India) exhibit cytological closeness with D. devario of Orissa (India) by having the same chromosome number and fundamental arm numbers, compared with other species reported from different regions. The only difference observed is in the karyotype formula, indicating that pericentric inversions might have played a substantial role during the evolutionary pathway of these species. Moreover, differences in the karyotype formula may be limited to cryptic chromosome rearrangements, such as those involving the heterochromatin segments and/or the nucleolus organizer regions (Thaís et al., 2010). Alternatively, it can be attributed to different degrees of chromosome condensation, leading to differences in chromosome classification among authors
• (Ryoichi, 2011). On the other hand, the differences in the fundamental arms within the same species of D. devario from different geographical locations suggest that the structural rearrangement in chromosome complements causes changes in chromosome morphology without change in chromosome number (Rishi et al., 1998). This intra-individual similarity in diploid chromosome number but dissimilarity in fundamental arm numbers and karyotype formula in Devario species cannot be fully explained by pericentric inversion alone, though it is considered to be the main mechanism of karyotypic evolution resulting in the variations in NF within the group (Galetti et al., 2000). Karyotypes of other native Devario species (D. acuticephala, D. assamensis, D. devario, D. naganensis, Devario sp. 1, Devario sp. 2, and Devario sp. 3 listed by Viswanath et al. [2007] from northeast India) have not been investigated so far. As a result, chromosomal evolution of this group is not fully understood.
• There was no evidence of sexual dimorphism of the chromosomes in either of the 2 species, which agrees with the reports on D. devario and D. malabaricus (Khuda-Bukhsh et al., 1986; Hardie and Hebert, 2004). Similarly, sex chromosomes were indistinguishable in several cyprinid fishes reported so far (Ergene et al, 2010;
• Kılıç-Demirok and Ünlü, 2001; Kilic-Demirok and Ünlü, 2004; Esmaeili et al., 2007, 2008, 2009, 2010; Kalbassi et al., 2008). The occurrence of cytologically differentiated sex chromosomes in a large number of living marine fish species appears to be rare (Galleti et al., 2000), although it has been described in some catfishes (Alves et al., 2006) and in platyfish (Devlin and Nagahama, 2002).
• Considering the difficulties in identifying several of the Devario species and their unclear phylogeny, cytogenetics may prove itself as an important tool in understanding the systematics of the genus. Thus, karyotype characteristics may contribute towards a better systematic interpretation, especially in the case of cryptic species, which are difficult to define (Artoni et al., 2009). The data of the present study on chromosome composition would contribute toward clarifying the karyotypic evolution and phylogenetic relationships in this group. Further analysis, including additional species of Devario of different regions and different staining techniques, will provide a better 5 10 15 20 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425 Total (µm) a
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