aqua, International Journal of Ichthyology

First report on the karyological analysis of the Churru snow trout, Schizothorax esocinus (Teleostei: Cyprinidae), from the River Jhelum, Kashmir
Farooq A Ganai1*, A. R. Yousuf 1, N. K. Tripathi2

1) Limnology and Fisheries Laboratory, Centre of Research for Development, University of Kashmir 190006, India. 2) Animal Cytogenetics Laboratory, P. G. Department of Zoology, University of Jammu, Jammu, India. *Corresponding author: farooqmd84@gmail.com
Received: 03 January 2011 Accepted: 09 May 2011

Abstract
The karyotype and chromosomal characteristics of the Churru snow trout, Schizothorax esocinus (Heckel), from the Jhelum River, Kashmir, were investigated. The analysis of 80 metaphase plates revealed that the chromosome number of this fish was 2n = 98 and the fundamental arm number (FN) = 150. The diploid complement comprised 15 metacentric pairs, 11 submetacentric pairs, 5 subtelocentric pairs and 18 telocentric pairs (30 m + 22 Sm + 10St + 36t). Total length of the haploid complement equalled 228.8 m with a range in the length of shortest and longest chromosome between 2.5-8.1 m. The arm ratio and the centromeric index ranged between 1- and 0-50 respectively. No heteromorphic sex chromosomes were found. The present study is the first to describe the chromosomal characteristics of Schizothorax esocinus from the Kashmir Valley.

la Jhelum River, Cachemire, ont t examins.Lanalyse de 80 images de mtaphase a rvl que le nombre de chromosomes de ce poisson est de 2n=98 et le nombre fondamental de bras (FN), de 150. Le complment diplode comprend 15 paires mtacentriques, 11 paires submtacentriques, 5 paires subtlocentriques et 18 paires tlocentriques (30m + 22Sm + 10St + 36t). La longueur totale du complment haplode quivaut 228,8 m avec une fourchette de 2,5-8,1 m pour la longueur respective du chromosome le plus court et le plus long. Le rapport des bras et lindex des centromres varie de 1- et de 0-50 respectivement.. On na pas trouv de chromosomes sexuels htromorphes. La prsente analyse est la premire dcrire les caractristiques chromosomiques de Schizothorax esocinus de la Kashmir Valley.

Sommario
Il cariotipo e le caratteristiche cromosomiche della trota delle nevi, Schizothorax esocinus (Heckel), del fiume Jhelum, Kashmir, sono stati indagati. Lanalisi di 80 vetrini in metafase ha rivelato che il numero di cromosomi di questo pesce 2n = 98 e il numero di braccia fondamentali (FN) pari a 150. Il corredo diploide comprende 15 coppie metacentriche, 11 submetacentriche, 5 subtelocentriche e 18 telocentriche (30 m + 22 Sm + 10 St + 36 t). La lunghezza totale del corredo aploide era pari a 228.8 m, con una oscillazione tra la lunghezza del pi corto e quella del pi lungo compresa tra 2.5 e 8.1 m. Il rapporto dei bracci e lindice centromerico nellintervallo 1- e 0-50, rispettivamente. Non sono stati rinvenuti cromosomi sessuali eteromorfici. Questo studio il primo che descrive le caratteristiche cromosomiche di Schizothorax esocinus della valle del Kashmir.

Zusammenfassung
Untersucht wurden Karyotypus und chromosomale Merkmale der Churru-Schneeforelle Schizothorax esocinus (Heckel) vom Jhelum-Fluss in Kaschmir. Die Untersuchung von 80 quatorialplatten (der Metaphase) ergab, dass dieser Fisch eine Chromosomenzahl von 2n = 98 und eine Fundamentalzahl von Chromosomenarmen (FN) = 150 aufweist. Der diploide Satz umfasste 15 metazentrische Paare, 11 submetazentrische Paare, 5 subtelozentrische Paare und 18 telozentrische Paare (30m + 22Sm +10St + 36t). Die Gesamtlnge des haploiden Satzes erreichte 228,8 m, mit einer Bandbreite von 2,5-8,1 m zwischen dem krzesten und dem lngsten Chromosom. Die Verhltniszahl der Arme reichte von 1-, der Zentromer-Index von 0-50. Heteromorphe Geschlechtschromosomen wurden nicht gefunden. Die vorliegende Studie ist die erste, in der die Chromosomenmerkmale von Schizothorax esocinus vom KaschmirTal beschrieben werden.

Rsum
Le caryotype et les caractristiques chromosomiques de la truite des neiges de Churru Schizothorax esocinus (Heckel) de
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INTRODUCTION Cyprinid fishes belonging to the subfamily Schizothoracinae are widely distributed in mountain streams and lakes around the Himalayan Karakorum and Hindukush Ranges, the Tibet
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First report on the karyological analysis of the Churru snow trout, Schizothorax esocinus (Teleostei: Cyprinidae), from the River Jhelum, Kashmir

Plateau and Central Asia (Terashima 1984). The genus Schizothorax Heckel comprises many species that inhabit the reservoirs of Central Asia from Turkamenistan and Eastern Persia in the West to the far reaches of the Mekong and Yangtzekiang in the East. Schizothorax esocinus (Fig. 1), inhabiting cold streams, rivers and lakes (Menon 1999) is distributed in the inland waters of Kashmir viz. Dal Lake, Mansbal Lake, Jhelum River, Lidder Stream etc. (Kullander et al. 1999), Nepal (Shrestha 1990) and China (Wu & Wu 1992) besides Afghanistan and Pakistan. Hill-stream fish species constitute about 3.5% of the fish fauna recorded from India and all of these could be considered as threatened species on account of the adverse effect of increasing human activity (Rishi et al. 1998). Schizothorax esocinus is edible and is an important species for commercial and sport fishing and has not been cytologically investigated previously. Cyprinid karyotypes have not been without systematic implications (Joswiak et al. 1980) because comparative karyology has become a useful tool in fish systematic studies (e.g. Arai 1982; Buth et al. 1991). Chromosome number and morphology shows great promise for interpreting the evolution of fishes (Uyeno & Miller 1973) and permits the detection of changes that modified an ancestral karyotype as it evolved into new lines (Winkler et al. 2004) and are useful for addressing a variety of evolutionary, genetic and cytotaxonomic questions about animals (Kirpichnikov 1981; McGregor 1993). The present study was undertaken to investigate chromosomes and karyotype of S. esocinus to compare it to other members of the genus and generate information that can be utilized for management of the species. MATERIALS AND METHODS Live fish were obtained from local fishermen in the River Jhelum and transported live to the Limnology and Fisheries Laboratory of the Centre of Research for Development, University of Kashmir

and placed into 50 l fully aerated aquariums at 20 C for several days. Air-dried chromosome preparation method with some modifications was used as described by Thorgaard and Disney (1990). Fish received two doses of phytohemagglutinin (PHA) injections (4 gg-1 body weight), in a 20-h interval at 20 C. Eight hours after the second PHA injection, colchicine was injected intraperitoneally, 0.05% at 0.1 ml/100g body weight to depress mitotic division at the metaphase stage and left for 2-3 hours before sacrificing. The fish were anesthetized by 300 ppm clove oil for 40s, their anterior kidney was removed, homogenized and hypotonised simultaneously by potassium chloride 0.56% for 35 minutes at room temperature. Suspensions were centrifuged at 1000 rpm for 10 minutes. Supernatant was removed and the cells were fixed by cold fresh Carnoy (3:1 methanol and glacial acetic acid). This process was repeated three

Fig. 1. Schizothorax esocinus in lateral view. Photo by F. A. Ganai

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Fig. 2a-b: a. Chromosome preparation of Schizothorax esocinus. b. Karyotype of S. esocinus (m = metacentric; Sm = sub-metacentric; St =s ub-telocentric; t = telocentric).
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Table I. Frequency of chromosomes in the counted metaphase plates

Number of chromosomes in each metaphase plate Number of metaphase plates counted Frequency (%) 94 8 10 96.0 6.0 7.5 98 64 80 100.0 2.0 2.5

sub-metacentric, sub-telocentric and telocentric following the method of Levan et al. (1964). RESULTS Relatively small and high numbers of chromosomes were observed in Schizothorax esocinus. Eighty cells from the anterior kidney tissue were analysed in total. The majority (80%) of the metaphase complements contained 98 chromosomes, though the count varied between 94-100 in a few cells (Table I). The diploid complement (Fig. 2a) comprised 15 metacentric pairs, 11 submetacentric pairs, 5 subtelocentric pairs and 18 telocentric pairs (Fig. 2b). The total length of the haploid complement equalled 228.8 m with a range in the length of shortest and longest chromosome between 2.5-8.1 m. The arm ratio and the centromeric index ranged between 1- and 0-50 respectively. Using chromosomal indicators, an ideogram (Fig. 3) was drawn in MS Excel 2007. The chromosomal formula can be represented as: K (2n) = 98 = 30 m + 22 Sm + 10St + 36t. DISCUSSION The cytological analysis of Schizothorax esocinus in the present study revealed a high number of chromosomes: 2n = 98. Species with high numbers are considered to have resulted through polyploidy

times and the cold fresh Carnoy was replaced at 30 minute intervals. Smears were prepared on cold lamellae using splash method from 1m height and air dried for 24 h, then stained with 2% Giemsa. CHROMOSOMAL ANALYSIS A Leica DM LS2 trinocular photomicroscope with 100x 10x magnification lens oil immersion was used for taking the photographs and analysing the chromosomes. Eighty metaphase plates were counted and a proper plate was selected to obtain karyotype formulae. Microsoft Excel 2007 software was used to calculate the centromeric indices and to draw the ideogram. For each chromosome centromeric index, arm ratio and total length were calculated as described by Levan et al. (1964) and the fundamental number was also calculated. Chromosomes were classified into metacentric,

Fig. 3. Haploid ideogram of Schizothorax esocinus.

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First report on the karyological analysis of the Churru snow trout, Schizothorax esocinus (Teleostei: Cyprinidae), from the River Jhelum, Kashmir

from an ancestral 2n = 48 or 50 (Rishi et al. 1998). Large-scale genomic expansions or whole-genome duplication events have been documented in early vertebrate evolution (Friedman & Hughes 2001; Ohno 1970; Wang & Gu 2000), near the base of the phylogenetic tree of teleost fishes (Christoffels et al. 2004; Meyer & Schartl 1999; RobinsonRechavi et al. 2001; Wittbrodt et al. 1998), and near the basal roots of several major teleostean clades, such as salmonids (Allendorf & Thorgaard 1984), catostomids (Ferris 1984; Uyeno & Smith 1972), acipenserids (Vasilev 1999) and some cyprinids (Larhammer & Risinger 1994). Such genomic enlargements have been hypothesised as key factors that enable or even drive diversification in various vertebrate groups (Holland et al. 1994; Meyer & Malaga-trillo 1999; Navarro & Barton 2003a,b; Ohno 1970; Stephens 1951). Chromosome counts in nearly all cyprinid polyploids occur in multiples or combinations of the most common karyotype (48-50) and tetraploids (96, 98 or 100) and hexaploids (148-150) have arisen through hybridisation (Dowling & Secor 1997). This is well illustrated by a number of species of fish belonging to diverse orders. Buth et al. (1991) noted 52 such taxa belonging to the Cyprinidae identified through karyological analysis (Dowling and Secor 1997) and such forms are ancestral polyploids (Ohno et al. 1969). Polyploidy in fishes has been associated with traits that include large body size, fast growth rate, long life and ecological adaptability (Uyeno & Smith 1972; Schultz 1980). Since Schizothorax fishes are hill stream fishes, it may be that polyploidy may have resulted on account of the cold temperature of their habitat. The use of thermal shocks to eggs for induction of polyploidy (Chourrout 1988) provides support to the above assertion. It is interesting that the Kashmir Valley Schizothorax esocinus showed a diploid number similar to that recorded for other species inhabiting different geographical locations eg., Schizothorax richardsonii, 2n = 98 (Sharma et al. 1992; Lakara et al. 1997), Schizothoracichthys prograstus, 2n = 98 (Rishi et al. 1983) and S. kumaonensis, 2n = 98 (Rishi et al. 1998; Lakara et al. 1997). A different fundamental arm number, which may be attributed to the intra-chromosomal changes involving pericentric and paracentric inversion, suggests an origin from the same primitive ancestor. The overall similarity in the chromosome number and morphology implies that Schizothorax species are very
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closely related in that they have not been isolated as evolving entities long enough for random chromosome changes to have taken place and become fixed. That a particular karyotype would be selected implies an adaptive advantage for that particular configuration. This hypothesis has been suggested for chromosome differences found in Fundulus (Chen 1971) and rivulines (Scheel 1972). The latter study followed the suggestion of Stebbins (1958) in that co-adapted gene sequences in plants have become closer linked via chromosome structural rearrangements and thus is less likely to be disrupted by normal exchange events. Similar arguments for chromosome changes paralleling species evolution in frogs and mammals can be found in Wilson et al. (1974) and for other animals in White (1978). No heteromorphic sex chromosomes were found in Schizothorax esocinus. Cells lacking normal value (2n = 94-100) were also encountered in the preparations and these probably resulted from losses during the preparation or addition from the neighbouring cells or hypotonic overtreatment (Nanda et al. 1995). The present study is the first to describe the chromosomal characteristics of Schizothorax esocinus from the Kashmir Valley. The results of the study can be used for the genetic manipulation and management and conservation of the species. ACKNOWLEDGEMENT The authors wish to thank the Director of the department for research facilities. We are also thankful to Dr. Farooz Ahmad Bhat, Assistant Professor Division of Fisheries SKAUST-K for his help in the identification of the fish and CSIR, New Delhi for providing financial assistance in the form of JRF to Farooq Ahmad. REFERENCES
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