CARYOLOGIA

Vol. 64, no. 4: 435-445, 2011

Cytotaxonomic status of Schizothoracine shes of Kashmir Himalaya (Teleostei: Cyprinidae)

Farooq1,* Ahmad Ganai, Abdul Rehman Yousuf1, Sabzar Ahmad Dar1, Narinder Kumar Tripathi2 and Samee Ullah Wani1
Limnology and Fisheries Laboratory, Centre of Research for Development, University of Kashmir, Hazratbal Srinagar-190 006, India. 2 Animal Cytogenetics Laboratory, Deptt. Of Zoology, University of Jammu.
1

Abstract Karyotypic study of ve Schizothorax species viz. S. niger, S. esocinus, S. labiatus, S. plagiostomus and S. curvifrons, belonging to subfamily Schizothoracinae, obtained from Dal lake and River Jhelum, Kashmir was carried out following the method of THORGAARD and DISNEY (1990).The diploid chromosome numbers recorded were 98 in S. niger (24m+32sm+22st+20t), 98 in S. esocinus (30m+22sm+10st+36t), 98 in S. labiatus (24m+20sm+2st+52t), 96 in S. plagiostomus (24m+18sm+54t) and 94 in S. curvifrons (26m+20sm+20st+28t). Pattern of chromosomal evolution and role of chromosomal rearrangements was discussed in addition to the cytotaxonomic status of these shes. The study conclusively conrmed the specic status of ve species of Schizothorax on the basis of their genetic material. Key words: Chromosome; Dal Lake; evolution; karyotype; Schizothorax.

INTRODUCTION Cytotaxonomy originated during the second half of the 19th century, when it was discovered that some animal and vegetal species may be classied according to their chromosomal characteristics (BERtOLLO et al. 1978). Taxonomy provides a vocabulary to discuss the world (KNAPP et al. 2002).It is a well established fact that without a detailed knowledge of cytogenetic make up, it is impossible to establish evolutionary relationships between various species, genera, families or orders of animals and plants. This holds true much more in shes than for any other group of vertebrates, as they occupy almost all types of aquatic habitats available on earth and have undergone a number of adaptational changes in the process. Chromosomes are the vehicle of the genetic constitution and, therefore, of paramount inter-

*Corresponding author: phone 09205139242; e-mail: farooqmd84@gmail.com.

est for understanding of evolutionary problems (FREDGA 1977).Genetic information assists in solving problems of identity and dening conservation units for species (FRANkHAM et al. 2004). Chromosomal studies in recent years gained a considerable importance, concerning species characterization, evolution and systematics (BARAt et al. 2002). Cytogenetic analysis in sh have allowed to determine sex chromosomes (MOREIRA-FILHO et al. 1993; DEVLIN and NAGAHAMA 2002; MOLINA and GALEttI 2007), the characterization of vertebrate models, like the zebra sh (SOLA and GORNUNG 2001), the evaluation of genetically modied lineages (PORtO-FOREStI et al. 2004), and to perform inferences on cytotaxonomic (BERtOLLO et al. 2000; BERtOLLO et al. 2004) and evolutionary issues (DEMIROk and UNLU 2001). Karyological studies have also provided basic information on the number, size and morphology of chromosomes (TAN et al. 2004) which is important to undertake chromosome manipulation in sh (kHAN ET AL. 2000). Since 1960 karyological studies in teleost sh have made noteworthy contributions in the eld of genetics, taxonomy

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and environmental toxicology (CUccHI and BARUffALDI 1990). Chromosomal analysis is important for sh breeding from the view point of genetic control, the rapid production of inbred lines and evolutionary studies (KIRpIcHNIkOV 1981). Genetic divergences of populations and their local adaptations are a potential resource for breeding programs in aquaculture and for shery management (PHILLIpS and RAb 2001). The study of karyotype is also important in aquaculture in connection with the use of chromosome manipulation techniques including induction of polyploidy, gynogenesis, androgenesis and inter or intra-specic hybridization (WU et al. 1986; DItER et al. 1993). Karyological study can be useful for addressing a variety of evolutionary and genetic questions about animals (MAcGREGOR 1993) and may permit detection of changes that modied an ancestral karyotype as it evolved into new lines (WINkLER et al. 2004) and chromosomal analysis is important for genetic control, taxonomy and evolutionary studies (MAcGREGOR and VARLY 1993; FIStER et al. 1999; SULEYMAN et al. 2004) and is widely use in various investigations (PISANO et al. 2007). The study of sh chromosomes was initiated in India in 1960s and of about 2000 species of inland and marine sh analyzed for karyological information, over 200 species belong to India which include both fresh water and marine species (DAS and BARAt 1995) for instance, Schizothorax richardsonii (SHARMA et al. 1992; Lakara et al. 1997; BARAt et al. 1997), Schizothorichthys prograstus (RISHI et al. 1983), S. kumaonensis, (LAkARA et al. 1997; RISHI et al. 1998), Catla catla and Mystus vittatus (JOHN et al. 1992), Labeo (JOHN et al. 1993), Tor khudree and Tor mussullah (KUSHWAHA et al. 2001), Heteropneustes fos-

silis (KUSHWAHA et al. 2002), Labeo rohita (NAG1997 ), Clarias gariepinus (NAGpURE et al. 2000), Labeo rohita, Catla catla and Cirrhinus mrigala (NAGpURE et al. 2001). Labeo dussumieri, Horabagrus brachysoma and Puntius lamentosus (NAGpURE et al. 2004), Horabagrus nigricollaris, Puntius denisonii and Puntius sarana subnasutus (NAGpURE et al. 2004), etc.. Schizothorax Heckel (Cypriniformes: Cyprinidae) comprises many species that inhabit the reservoirs of Central Asia from Turkmenistan and Eastern Persia in the West to the far reaches of Mekong and Yangtzeking in the East. The genus is represented in Kashmir Himalaya by a number of species and though their taxonomy has been studied by many workers in the past (HEckEL 1838; MccLELLAND 1839; HORA 1936; SILAS 1960; TALWAR AND JHINGRAN 1991; KULLANDER et al. 1999), there is still a lot of confusion regarding the exact number of species occurring in different aquatic habitats of the valley which has actually hindered studies on this species complex. This is further complicated by the fact that hybrids of some of these species have also been reported (HEckEL 1838 AND HORA 1936).Consequently, despite being an important food shery; this species complex has not been studied for its nutritional and biochemical components and has not been cultured on commercial scale. This species complex being mostly herbivorous, can also act as an important biological control method for invasive aquatic vegetation like Myriophyllum, Potamageton etc. The present study has solved this problem and will pave the way for their further studies, manipulation and management. The study also gives an insight regarding their karyo-evolutionary status.
pURE

Fig. 1 Karyotype of S. niger.

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MATERIALS AND METHODS. Live sh (Thirty specimens, six of each species) were obtained from local shermen in the Dal lake and River Jhelum and transported live to the Limnology and Fisheries Laboratory of Centre of Research for Development, University of Kashmir and placed into ve (50 l) fully aerated aquaria at 20C for several days. Air dried chromosome preparation method with some modications was used as described by THORGAARD AND DISNEY (1990). Fish received two doses of phytohemagglutinin (PHA) injections (4gg-1 BW), in a 20-h interval at 20C. Eight hours after the second PHA injection, colchicine was injected intraperitoneally, 0.05% @ 1ml/100g BW to depress the mitotic division at the metaphase stage and left for 2-3 hours before sacricing. The sh 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 xed by cold fresh Carnoy xative (3:1 methanol and glacial acetic acid). This xation process was repeated three 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 - Leica DM LS2 trinoccular photomicroscope with 1000X magnication lens oil immersion was used for taking the photographs and analyzing the chromosomes. Eighty metaphase plates were counted per species and a proper plate was selected to obtain karyotype formulae. Microsoft Excel 2007 software was used to calculate the centromeric indices and to draw ideogram. For each chromosome Centromeric index, arm ratio and total length were calculated as described by LEVAN et al. (1964) and the fundamental arm number was also calculated. Chromosomes were classied into metacentric, sub-metacentric, sub-telocentric and telocentric following the method of LEVAN et al. (1964). RESULTS During the present study ve recognized species of Schizothorax viz., Schizothorax niger, S. esocinus, S. curvifrons, S. plagiostomus and S. labiatus were studied for various karyological features (Table 1). The somatic complement of Schizothorax niger revealed a diploid number of 98 comprising 12 metacentric pairs, 16 submetacentric pairs,

Fig. 2 Karyotype of S. esocinus.

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11 subtelocentric pairs and 10 telocentric pairs of chromosomes (Fig. 1). Schizothorax esocinus showed a diploid complement of 98 comprising 15 metacentric chromosome pairs, 11 submetacentric pairs, 5 subtelocentric pairs and 18 telocentric pairs (Fig. 2). Schizothorax labiatus also revealed a diploid complement of 98 that comprised 12 metacentric pairs, 10 submetacentric pairs, 1 subtelocentric pair and 26 telocentric pairs (Fig. 3). Schizothorax plagiostomus showed a somatic complement of 96 comprising 12 metacentric pairs, 9 submetacentric pairs and 27 telocentric pairs (Fig. 4). The diploid chromosomal complement of Schizothorax curvifrons comprised 94 chromosomes; 13 metacentric pairs, 10 submetacentric pairs, 10 subtelocentric pairs and 14 telocentric pairs (Fig. 5). Various karyological features of these species are shown in table 2. DISCUSSION All the ve species of Schizothorax analyzed cytologically in the present study revealed a high number of chromosomes ranging from 94-98. Species with high numbers are considered to have resulted through polyploidy from 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 and HUGHES 2001; OHNO 1970; WANG and GU 2000), near the root of the phylogenetic tree of teleost shes (CHRIStOffELS et al. 2004; MEYER AND ScHARtL 1999; RObINSON-REcHAVI et al. 2001; WIttbRODt et al. 1998), and near the roots of several major teleostean clades [such as salmonids (ALLENDORf AND THORGAARD 1984), catastomids (FERRIS 1984; UYENO and SMItH 1972), acipenserids (VASILEV 1999) and some cyprinids (LARHAMMER and RISINGER 1994)]. Such genomic enlargements have

been hypothesized as key factors that enable or even drive diversication in various vertebrate groups (HOLLAND et al. 1994; MEYER and MALAGA-TRILLO 1999; NAVARRO and 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), tetraploids (96, 98 or 100) and hexaploids (148-150) have arisen through hybridization (DOWLING and SEcOR 1997). Amphiploidy of a sterile interspecic hybrid is now generally recognized as one of the commonest ways in which species arise (StEbbINS 1950). Numerous examples in the sheries literature infer species origins linked to hybridization events. Examples of asexual or Polyploid sh taxa of reported hybrid origin include the Poecilia complex (AVISE et al. 1991), the Poeciliopsis species complex (VRIjENHOEk 1993), and Cobitis species complex (SEZAkI et al. 1994). Examples of bisexual diploid taxa of hypothesized hybrid origin include Catostomus discobolus (CRAbtREE and BUtH 1987), Chasmistes brevirostris (MILLER and SMItH 1981) and Luxilus albeolus (MEAGHER and DOWLING 1991).This is well illustrated by a number of species of sh belonging to diverse orders. BUtH et al. (1991) noted 52 such taxa most of which belong to cyprinidae identied through karyological analysis (DOWLING and SEcOR 1997) and such forms are ancestral polyploids (OHNO et al. 1969). Polyploidy in shes has been associated with traits including large body size, fast growth rate, long life and ecological adaptability (UYENO and SMItH 1972; ScHULtZ 1980). Since Schizothorax shes are hill stream shes, it may be that polyploidy may have resulted on account of cold temperature of their habitat. The use of thermal shocks to eggs for induction of polyploidy (CHOURROUt 1988) provides support to the above assertion. The role of polyploidy in evolution and survival of sh is very important because it prevents from natural selection pressure (OELLERMAN and SkELtON 1990).

TAbLE 1 Chromosome morphology of different Schizothorax species worked out cytologically in Kashmir (m=metacentric; Sm = submetacentric; St = subtelocentric; t = telocentric; NF = fundamental arm number).
S.No. 1 2 3 4 5 Name of the species Schizothorax niger Schizothorax esocinus Schizothorax labiatus Schizothorax plagiostomus Schizothorax curvifrons 2n 98 98 98 96 94 m 24 30 24 24 26 Sm 32 22 20 18 20 20 St 22 10 2 t 20 36 52 54 28 NF value 154 150 142 138 140 Author and Year Present work Present work Present work Present work Present work

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Three out of the ve species analyzed in the present study viz., S. niger, S. esocinus and S. labiatus revealed a diploid number of 98 and a fundamental arm number of FN of 154, 150 and 142, respectively. The variation in the fundamental arm number without change in the 2n may be attributed to the intrachromosomal changes involving pericentric and paracentric inversion and centromeric shifts (RISHI et al. 1998). Variation in the karyotypic conguration of S. niger and S. esocinus can be easily explained by centric fusion and ssion events. Both centric ssion and fusion probably provide important mechanisms to explain the diverse range of chromosome numbers observed in many mammalian and non-mammalian animal taxa (TODD 1970; IMAI et al. 1986, 1988; KOLNIckI 2000; IMAI et al. 2001). Centric fusion has usually been believed to be much more common in evolution than centric ssion (StURtEVANt and NOVItSkI 1941; MAttHEY 1973; BAkER et al. 1975) but there are also well documented examples of centric s-

sion (YOSIDA et al. 1979; FRYNS et al. 1980). It is evident from the karyotype of these two species that there has been simultaneous fusion of telocentric and ssion of metacentric chromosomes in S. esocinus which resulted in the karyotype of S. niger. This is because S. niger is having more biarmed chromosomes than S. esocinus and karyotype with biarmed chromosomes are generally regarded to represent a derived condition (OHNO et al. 1968; OHNO 1970; DENtON 1973; GOLD 1979). Same types of chromosomal rearrangements seem to have framed the karyotype of S. labiatus. Other two species viz., S. plagiostomus and S. curvifrons revealed a diploid number of 96 and 94 and fundamental number FN of 138 and 140 respectively. Decrease in the 2n in these species may be attributed to the Robertsonian arrangements and change in FN to pericentric inversion (CHOUDHURY et al. 1982). The chromosomes of all the ve species of Schizothorax except S. plagiostomus, were cat-

Fig. 3 Karyotype of S. labiatus.

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egorized into four groups i.e., metacentric, submetacentric, subtelocentric and telocentric following LEVAN et al. (1964). In S. plagiostomus none of the chromosomes could be recognized as subtelocentric because cyprinid shes are characterized by presence of relatively small chromosomes with their centromeric positions ranging gradually from median to nearly terminal, making it difcult to assign some chromosomes to particular chromosomal categories and thus making correct identication of individual chromosomes nearly impossible (RAb AND COLLARES-PEREIRA 1995). The karyological study of teleost sh presents technical difculties which are not encountered in the study of other vertebrates and these difculties are due to small size and high number of chromosomes (CUccHI AND BARUffALDI 1990). Further, differ-

ential arm contraction can alter a chromosome classication in a karyotypic formula (JOSWIAk et al. 1980). A more plausible reason seems to be that the two subtelocentric chromosomes of S. labiatus (Table 1) underwent centric ssion and yielded two perfect telocentric chromosomes of S.plagiostomus, whereas the smaller fragments could not be incorporated into the nucleus and disintegrated. These two shes are morphologically closely related to each other. Highest number of biarmed chromosomes was found in S. niger followed by S. esocinus, S. curvifrons, S. plagiostomus and S. labiatus whereas highest number of subtelo-telocentrics were found in the order of S. plagiostomus and S. labiatus (equal), S. curvifrons, S. esocinus and S. niger. The primitive teleost karyotype is thought to have consisted of 46-48 acrocentrics (NAYYAR

Fig. 4 Karyotype of S. plagiostomus.

TAbLE 2 Karyological features of various species of Schizothorax (CI= Centromeric Index; FN= Fundamental arm numer).
Species Schizothorax niger Schizothorax esocinus Schizothorax labiatus Schizothorax plagiostomus Schizothorax curvifrons 2n 98 98 98 96 94 Range Range in arm ratio in chromosome length 3.5-8 m 2.5-8 m 2-8 m 2-8 m 1-10.4 1- 1- 1- 1- 1- Range in CI 0-50 0-50 0-50 0-50 0-50 Total length 268 m 228.8 m 157.5 m 138 m 267.3 m FN 154 150 142 138 140

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1966; OHNO et al. 1968; OHNO 1970; FItZSIMONS 1972; LEGRANDE 1975) and the karyotypes with biarmed chromosomes are regarded to represent a derived condition (OHNO et al. 1968; OHNO 1970; DENtON 1973; GOLD 1979). Keeping this into consideration, S. plagiostomus and S. labiatus seem to be primitive shes when compared to S. niger, S. esocinus and S. curvifrons which possess a more derived karyotype. The trend of gradual increase in the FN from 138 in S. plagiostomus to 154 in S. niger supports the above assertion. The overall dissimilarity in the 2n, karyotypic conguration and FN points to the role of almost all types of chromosomal rearrangements in the karyological evolution of Schizothoracinae, however deviation in the chromosome number can be possibly according to NAVASHINS (1932) dislocation hypothesis of evolution of chromosomes. This hypothesis explains that each chromosome is monocentric and an evolutionary change in the chromosome number must involve duplication of centromere together with a region within it while a decrease in the number must mean a permanent loss from the karyotype of the region containing centromere. Family cyprinidae points to the role of almost all types of chromosomal rearrangements for their karyological evolution (RISHI et al. 1998). It is interesting that some of the Kashmir Valley species of Schizothorax show diploid number similar to that recorded for other species inhabiting different geographical locations e.g. Schizothorax richardsonii, 2n=98 (SHARMA et al. 1992; LAkARA et al. 1997), Schizothorichthys progsastus, 2n=98 (RISHI et al. 1983) and S.

kumaonensis, 2n=98 (RISHI et al. 1998; LAkARA et al. 1997), suggesting origin from the same primitive ancestor. The overall similarity in the chromosome number and morphology implies that Schizothorax species are very closely related in that they have not been isolated as evolving entities long enough for random chromosome changes to have taken place and become xed, and that a particular karyotype would be selected implies an adaptive advantage for that particular conguration. 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 different animals have been reported by WILSON et al. (1974) and WHItE (1978). Cyprinid karyotypes have not been without systematic implications (JOSWIAk et al. 1980) because comparative karyology has become a useful tool in sh systematic studies (ARAI 1982; BUtH et al. 1991) as chromosome number and morphology shows changes that modied 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 also undertaken with the same objective to generate the information regarding the species status of the Schizothorax in Kashmir Valley. The

Fig. 5 Karyotype of S. curvifrons.

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study revealed that despite overlap in the general morphological features, the ve species of Schizothorax investigated are genetically different and hence denite species as the chromosomal differentiation in animal species usually precedes strong morphological differentiation (HOWELL AND VILLA 1976). Most morphologic features of shes have been shown to have the potential of being modied by the environmental conditions (SVARDSON 1965; FOWLER 1970). Therefore, a morphologically based classication should be tested by the features not likely to be environmentally plastic and Chromosome structure is best suited for this purpose as it reects genetic divergence and is least affected by environmental distortion (CAMpOS 1972). The present study conclusively conrms the specic status of the ve species of Schizothorax on the basis of their genetic material. The study negates the proposition of SILAS (1960) regarding the taxonomic status of Schizothorax niger and Schizothorax curvifrons, who had combined these two species into a single species Schizothorax niger treating curvifrons and niger as two varieties or subspecies. The chromosome study has clearly shown that these two species of sh be treated as distinct species and not varieties or subspecies of the same species. The present study has also cleared the taxonomic position of Schizothorax plagiostomus vis-a-vis S. richardsonii as for a long time the two species were regarded as synonyms. A comparison of the two species clearly indicates that the two species are distinct from each other as was proposed by KULLANDER et al. (1999).
Acknowledgement Authors wish to thank Director of centre for providing the research facilities and cSIR New Delhi for providing nancial assistance to FAROOQ AHMAD. We are grateful to DR. RUbENS PAZZA for positive and critical evaluation of the manuscript.

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Received March 16th 2011; accepted November 20th 2011

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