Comparative cytogenetics of two sympatric species (Gymnocypris, Schizothoracinae) from the Lake Langcuo, Qinghai-Tibet Plateau

Abstract

Schizothoracinae is a subfamily of naked carp which is widespread throughout the Qinghai-Tibet Plateau region. Cytogenetic analyses were performed on two sympatric species of naked carp, Gymnocypris chui and G. scleracanthus from the Lake Langcuo, Qinghai-Tibet Plateau. The results showed G. chui and G. scleracanthus had the same chromosome number (2n = 92), but different karyotypes:, (36m + 16sm + 12st + 28a) for G. chui and (26m + 12sm + 18st + 36a) for G. scleracanthus, respectively. The six loci of 45S ribosomal genes (rDNA) were detected through fluorescent in situ hybridization, and their locations were highly conserved between two species. This study depicted the chromosomal characteristics of the Schizothoracinae species and improved our understanding about the relationships and the evolution of two sympatric species.

Keywords:

• Karyotypic divergence
• Schizothoracinae
• sympatry
• Qinghai-Tibet Plateau
• FISH

Introduction

Karyological studies provide basic information on the number, size and morphology of chromosomes that is important to study evolution and diversification in fish (Almeida-Toledo et al. 2000; Borba et al. 2012; Poletto et al. 2010). The karyotype is unique for each species, therefore is widely used for distinguishing different organisms. Traditionally, fish karyotypes have been described using C-banding and N-banding. In the last few decades, fluorescence in situ hybridization (FISH) was introduced into cytogenetic studies of fish, which enables the chromosomal localization of specific genes or repetitive sequence. The different numbers and positions of the rRNA genes provided useful information for understanding the evolutionary relationships among closely related species (Rubert et al. 2011; Cardoso et al. 2013).

The fish of the subfamily Schizothoracinae (Family: Cyprinidae) are important members of the ichthyofauna in the Qinghai-Tibet Plateau and its surrounding areas, where they are widely distributed in a variety of rivers and lakes (Wu and Wu 1992; Chen and Cao 2000). Currently, this subfamily includes 15 genera and over a hundred species distributed all over the world (Mirza 1991; Wu and Wu 1992; Chen and Cao 2000). The karyotypes of four Schizothoracinae fish were first reported by Zan et al (1985). Available cytogenetic data for this subfamily showed a wide variation with tetraploid, hexaploid and octoploid numbers, ranging from 2n = 66 in Gymnocypris dobula to 2n = 417–470 in Ptychobarbus dipogon (Table 1). However, cytogenetic research on Schizothoracinae lags behind that for other fishes because of the similar chromosomal organization, large number of chromosomes, and autotetraploidy, with four nearly identical genomes (Zan et al.1985; Yu et al.1987; Wu et al. 1998 ; Qi 2004; Li et al.2010; Kong et al.2011 ).

Table 1. A summary of the cytogenetic data available for the Schizothoracinae.

Species	2n	Karyotypic formula	Locality	Reference
Chuanchia labiosa	92	32m+26sm+18st+16t	Yellow River, China	Yu et al. 1990
Diptychus sp.	98	28m+32sm+38st-t	Lake Fuxian, China	Zan et al. 1985
Diptychus maculatus	100	70m/sm+30st/a	Kirghiz Tan	Mazik et al. 1989
Diptychus sewerzowi	98	80m/sm +18st/a	Kirghiz Tan	Mazik et al. 1989
Gymnodiptychus dybowskii	98	28m+30sm+12st+28t	Manasi River, China	Kong et al. 2011
Gymnodiptychus pachycheilus	90–98	—	—	Collares-Pereira, 1994
Gymnocypris dobula	66	32m+10sm+4st+20t	Tingri County, China	Wu et al. 1999
Gymnocypris eckloni	94	26m+28sm+22st+18t	Yellow River, China	Yu et al. 1990
Gymnocypris przewalskii	92	18m+18sm+16st+40t	Lake Qinghai, China	Qi 2004
	92	32m+22sm+24st+14t	Lake Qinghai, China	Yan et al. 2007
Gymnocypris waddelli	94	24m+14sm+22st+34t	Pumqu River, China	Wu et al. 1999
Oxygymnocypris stewartii	92	26m+30sm+22st+14t	Lasha River, China	Yu et al. 1990
	86	24m+12sm+12st+18t	Nianchu River, China	Wu et al. 1999
Platypharodon extremus	90	24m+30sm+20st+16t	Yellow River, China	Yu et al. 1990
Ptychobarbus dipogon	424–432	—	Lasha River, China	Wu et al. 1999
	417–470	—	—	Yu and Yu 1990
Schizopygopsis pylzovi	92	32m+26sm+20st+14t	Yellow River, China	Yu et al. 1990
	92	24m+24sm+22st+22t	Yellow River, China	Chen et al. 2006
Schizopygopsis younghusbandi younghusbandi	90	26m+28sm+20st+16t	Lasha River, China	Yu et al. 1990
Schizopygopsis younghusbandi himalayensis	88	40m+16sm+12st+20t	—	Wu et al. 1999
Schizothorax sp.	148	50m+28sm+70st-t	ErYuan County, China	Zan et al. 1985
Schizothorax curvifrons	94	26m+20sm+20st+28t	Sindh stream, India	Ganai et al. 2011, 2012, 2014
Schizothorax daliensis	148	48m+30sm+70st-t	Erhai, China	Zan et al. 1985
Schizothorax davidi	98	20m+34sm+24st+20t	Qingyi River, China	Li et al. 1987
Schizothorax esocinus	98	30m+22sm+10st+36t	Lake Dal, India	Ganai et al. 2011, 2014
Schizothorax grahami	148	52m+30sm+66st-t	Luquan County, China	Zan et al. 1985
Schizothorax kozlovi	96	36m+16sm+10st+34t	Wujiang River, China	Chen 2013
	128	26m+22sm+24st+56t	Wujiang River, China	Zou 2009
Schizothorax kumaonensis	98	24m+6sm+68st-a	—	Lakara et al. 1997
Schizothorax labiatus	98	24m+20sm+2st+52t	Lake Dal, India	Ganai et al. 2011, 2014
Schizothorax lissolabiatus	148	38m+32sm+78st/a	Yunan, China	Zan et al. 1986
Schizothorax macropogon	90–98	—	—	Collares-Pereira, 1994
	102	20m+28sm+22st+16t	Lasha River, China	Wu et al. 1999
Schizothorax niger	98	24m+32sm+22st+20t	Lake Dal, India	Ganai et al. 2011, 2012, 2014
	98	22m+26sm+50st-t	India	Khuda-Bukhsh and Nayak 1982
Schizothorax oconnori	92	30m+26sm+20st+16t	Lasha River, China	Yu et al. 1990
	106	24m+26sm+30st+25t	Nianchu River, China	Wu et al. 1999
Schizothorax plagiostomus	96	24m+18sm+54t	Jhelum River, India	Ganai et al. 2011, 2014
Schizothorax prenanti	148	28m+40sm+36st+44t	Qingyi River, China	Li et al. 1987
Schizothorax progastus	98	16m+20sm+16st+40t	Haryana, India	Rishi et al. 1983
Schizothorax richardsonii	98	24m+28sm+24st+22a	—	Lakara et al. 1997
Schizothorax taliensis	148	48m+28sm+72st/a	Lake Erhai, China	Zan et al. 1985
Schizothorax waltoni	92	30m+26sm+20st+16t	Lasha River, China	Yu et al. 1990
	112	26m+24sm+28st+34t	Nianchu River, China	Wu et al. 1999
Schizothorax yunnanensis	148	—	—	Collares-Pereira, 1994
Schizothorax zarudnyi	96	9m + 14sm + 25st	Hamoon Lake, Iran	Kalbassi et al. 2008

G. chui Tchang, Yueh et Hwang and G. scleracanthus Tsao et al (Cyprinidae: Schizothoracinae) are endemic fish that are sympatric distributed in Lake Langcuo in Tibet, China (Wu and Wu 1992). Morphological difference was investigated (Wu and Wu 1992), and more evidence was required to explain the relationship between these two sympatric fish. In the present work, we obtained G. chui and G. scleracanthus from the Lake Langcuo, Qinghai-Tibet Plateau and illustrated their different karyotypes, which helps to explore their karyotypic evolution and contributes to cytogenetic information for members of the subfamily Schizothoracinae.

Material and methods

Samples of G. chui (two males and six females) and G. scleracanthus (four males and three females) from the Lake Langcuo, Qinghai-Tibet Plateau, were analyzed. All necessary permits for collection and experimentation were acquired for the described field study from the Agriculture Department of Tibet Autonomous Region, China.

Chromosomal preparations followed the methods described by Lin (1982) with some modifications. In brief, a live fish was administered an intraperitoneal injection of a 10 μg/1 g solution of phytohemagglutinin (PHA) to stimulate mitosis and kept alive for 20 h in a fully aerated aquarium. Subsequently the fish was injected with PHA again. Two hours later, a 0.025% colchicine solution was injected at the proportion of 0.5 ml/100 g of body weight. After 120 min the fish was euthanized and the visceral cavity was opened to collect the kidneys. The kidneys were macerated in a hypotonic solution of 0.04 M KCl and incubated at 37°C for 60 min. The cell solution was then suspended in a fixative (3:1 methanol: acetic acid) and centrifuged twice. The resulting pellet was then suspended in fresh fixative and dropped onto warmed slides. The slides were analyzed after conventional Giemsa staining with 6-diamidino-2-phenylindole (DAPI) (Pieczarka et al. 2006) and FISH with 45S ribosomal DNA (45S rDNA) probes. The 45S probes were labeled with tetramethyl-rhodamine-5-dUTP (Roche Diagnostics, Mannheim, Germany) by a nick-translation method. Images were acquired with a cooled CCD camera (Photometrics CoolSNAP, USA) under a fluorescence microscope (Leica, Wetzlar, Germany) and analyzed with the MetaVue imaging system (Universal Imaging Corporation, USA). Finally, images were adjusted with Adobe Photoshop (version 6.0; Adobe Inc. USA) for contrast and background optimization.

Chromosome morphology was determined on the basis of arm ratio, as proposed by Levan et al. (1964) and chromosomes were classified as metacentrics (M), submetacentrics (SM), subtelocentrics (ST) and acrocentrics (A). All analyzed specimens were preserved in 95% ethyl alcohol, and deposited in Northwest Institute of Plateau Biology, Chinese Academy of Sciences.

Results

Conventional cytogenetic analysis

A total of 250 metaphasic cells were analyzed for the two species, 100 for G. chui and 150 for G. scleracanthus. G. chui showed 2n = 92 chromosomes, a 36m + 16sm + 12st + 28a karyotypic formula and a fundamental number (FN) of 144 (Figure 1). The chromosome number of G. scleracanthus was 2n = 92, with the karyotype formula of 26m + 12sm + 18st + 36a and FN of 130 (Figure 2). Both males and females were analyzed for G. chui and G. scleracanthus, but no sex chromosomes were identified.

Figure 1. Giemsa stained karyotypes of Gymnocypris chui. m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric. The bar represents 10 μm.

Figure 2. Giemsa stained karyotypes of Gymnocypris scleracanthus. m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric. The bar represents 10 μm.

Chromosomal localization of 45S rDNA by fluorescence in situ hybridization

45S rDNA signals were physically mapped on the chromosomes of G. chui and G. scleracanthus (Figure 3(a, d)). Six 45S rDNA signals were detected at the NOR region of chromosomes in G. chui (Figure 3(a, c)). Similarly, the same number of 45s rRNA genes were found in three pairs of chromosomes in the G. scleracanthus (Figure 3(d, f)). No signals were detected outside these regions. Differences of hybridization signals were not observed between G. chui and G. scleracanthus. The chromosomes of these two fish appeared painted with shades of bands after DAPI staining (Figure 3(b, e)). The pericentromere of most chromosomes showed bright bands. No obvious difference was observed between the two fish.

Figure 3. Physical localization of ribosomal genes on metaphase chromosomes in (a–c) Gymnocypris chui: (a) fluorescent in situ hybridization with 45S rDNA probe; (b) metaphase showing DAPI staining; and (c) sites of 45S rDNA; and (d–f) G. scleracanthus: (d) fluorescent in situ hybridization with 45S rDNA probe; (e) metaphase showing DAPI staining; (f) sites of 45S rDNA.

Discussion

This is the first report of the karyotypes of G. chui and G. scleracanthus from the Lake Langcuo. The chromosome number of G. chui and G. scleracanthus is consistent with some species of the Schizothoracinae (Table 1). Nearly 40 Schizothoracinae species have already been submitted to some cytogenetic study, and show considerable interspecific chromosomal diversity (Table 1). A karyotype comprising 50 chromosomes could be considered as the ancestral state for the cyprinid fish; it is frequently found and widespread across all of the family (Arai 1982). Consequently, we speculate that G. chui and G. scleracanthus may be derived from another non-polyploid cyprinid with 2n = 50 chromosomes, probably with some merged or lost chromosomes after polyploidization. Polyploidization is the process by which Schizothoracinae fishes respond to the complicated environment during the uplift of the Qinghai-Tibet Plateau.

Differences in karyotypic formula were found between G. chui and G. scleracanthus, showing a variation in their karyotypic macrostructure. These species have the same chromosome number but different karyotype formula probably because of structural chromosomal rearrangements. The karyotypic differences may play a role in post-zygotic reproductive isolation during the speciation process (Cardoso et al. 2013). Therefore, hybridizations between G. chui and G. scleracanthus are unlikely to occur because of the identified karyotypic differences. However, a deeper insight into the reproductive isolation will probably be achieved by studies of reproductive biology and ethology.

Karyotypic variability can be detected by the chromosomal localization of specific genes, such as 45S rDNA and 5S rDNA. In fish, the location of the 45S rDNA (18S + 5.8S + 28S) is an important cytogenetic marker. This technique has not been applied previously for chromosome studies in Schizothoracinae, and the present study is the first to report the number of nuclear ribosomal loci in Schizothoracinae using FISH. The location of 45S rDNA loci indicated G. chui and G. scleracanthus both had multiple NORs. Little difference was observed between the two species, presumably for the following reasons. First, it is difficult to study the karyotype of Schizothoracinae because of the small chromosome size, high chromosome number, and autotetraploid nature of its chromosomes. Moreover, G. chui and G. scleracanthus, as closely related Schizothoracinae species, have similar chromosome karyotypes. Therefore, some new FISH chromosome markers would be needed in future to identify chromosomes in Schizothoracinae.

In conclusion, accurate nuclear DNA measurements and further information about chromosome banding in these two sympatric species are needed to clarify their karyotypic evolution.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding information

This work was supported by the National Natural Science Foundation of China [grant numbers 31172070, 31572258].

Acknowledgments

We thank Dr Feng Yu for the assistance with the laboratory work. We sincerely thank Dr Fei Tian for her insightful comments and suggestions. We are also very grateful to Prof. Quanwen Dou for providing 45S rDNA.