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Amphibia-Reptilia 27 (2006): 113-119 The karyology of Vipera aspis, V. atra, V. hugyi,andCerastes vipera Gennaro Aprea1, Augusto Gentilli2, Marco A.L. Zuffi3, Gaetano Odierna1 Abstract. In the current paper we show the results obtained using standard and banding staining methods (Ag-NOR-, CMA3-, C-banding and sequential colorations (or Alu I digestions) + CMA3 + DAPI) in specimens of Cerastes vipera, Vipera aspis, V. atra,andV. hugyi. Cerastes vipera presented chromosomal characters, primitive in snakes, as a karyotype of 2n = 36 chromosomes, with 16 biarmed macrochromosomes and 20 microchromosomes, NORs on one microchromosome pair and absence of cytologically evident sex chromosomes, at least with the methods used. The three taxa of Vipera studied showed chromosomal characters either derived, or primitive or at an initial stage of differentiation. All three species showed a karyotype (derived) of 2n = 42 chromosomes with 22 macro- and 20 micro-chromosomes; they all showed NORs on one micro-chromosome pair and presented Z and W chromosomes at an initial stage of differentiation. Sexchromosomes Z and W, were in fact homomorphic, but the former was near all euchromatic, while the W chromosome was almost completely heterochromatic. All the three taxa of Vipera resulted, however, karyologically diversified, mainly due to the number of macro-chromosomes pairs with a centromeric, CMA3 positive heterochromatin: almost all the pairs in V. aspis,twopairsin V. atra and absent in V. hugyi. Introduction Ticino river-Staffora valley-Gulf of Genoa, cen- tral and western Switzerland and south east- Vipera aspis is among the most widely studied ern France to the Rhone valley); Vipera hugyi snakes of the European herpetofauna, but its dis- (southern Italy south of the Gargano-Vesuvio tribution and taxonomic status is still highly dis- volcano-Sicily); Vipera zinnikeri (Pyrenees and cussed (Zuffi and Bonnet, 1999; Conelli, 2002; Zuffi, 2002; Ursenbacher et al., 2003) (fig. 1). south-western France). Italian populations of Four subspecies belonging to the Vipera aspis Vipera aspis are still under investigation and group have been recently recognized as good should be considered as Italian Vipera aspis species: Vipera aspis (France: except for the populations. Distinction between the different Pyrenean, pre-Pyrenean and south eastern areas; species was made on a morphological basis, Italy: central northern Italy east from the virtual using meristic characteristics, such as ventral line Ticino river-Staffora valley-Gulf of Genoa; and subcaudal scales, number of dorsal bars north of line Gargano-Vesuvio volcano); Vipera and hemipenial anatomy (Zuffi, 2002). The atra (northern Italy: west from the virtual line analysis of skull morphology, using geomet- ric morphometry, led to results similar to those found using the previous morphometric ap- 1 - Dipartimento di Biologia Evolutiva e Comparata, Uni- versità di Napoli Federico II, Dipartimento di Biologia proach (Gentilli et al., unpublished data). On Strutturale e Funzionale, Via Cintia, Napoli, Italy. the contrary, recent analyses on mtDNA are in Corresponding author’s e-mail: gaetano.odierna@ contrast to what has just been described: V. as- unina.it 2 - Dipartimento di Biologia Animale, Università di Pavia, pis has been synonymised with V. atra and the p.za Botta 9, 27100 Pavia, Italy. other Italian taxa have been highly differenti- e-mail: [email protected] ated (Conelli, 2002; Ursenbacher et al., 2003). 3 - Museo di Storia Naturale e del Territorio, Università di Pisa, via Roma 79, 56011 Calci (Pisa), Italy. Research into any further differences within the e-mail: [email protected] field of karyology, starting from what has been © Koninklijke Brill NV, Leiden, 2006. Also available online - www.brill.nl 114 G. Aprea et al. briefly reported, is therefore particularly inter- Results esting. Cerastes vipera Chromosomal studies, particularly when the = banding technique was used, have given rele- Both sexes showed 2n 36 chromosomes, vant information for systematic and phyloge- 16 were macrochromosomes and 20 microchro- netic purposes in several snake groups, as in mosomes. Among the macrochromosomes the elapids and boids (Mengden and Stock, 1980; pairs 1, 3, 4, 5 and 8 were metacentric, pairs Mengden, 1982) and in species of the gender 2 and 7 were submetacentric and the sixth was acrocentric (fig. 2A, B). After the C band- Vipera (V. seoanei, V. latastei and V. ammodytes, ing staining, centromeric regions of all the V. zinnikeri; Saint Girons, 1977). In all the re- macrochromosomes showed a scarce presence maining taxa of the Vipera aspis group, chro- of heterochromatin, which was CMA negative mosomal analyses have been performed up to 3 and DAPI positive (fig. 2D, E). Also the telom- now on male specimens of the nominal sub- eric regions of these elements were positive to species only (Kobel, 1967; Saint Girons, 1977), the DAPI only, but were more weakly stained for which a karyotype of 2n = 42 chromosomes with respect to the centromeric regions (fig. 2D, has been described. In our paper we present E). Among the microchromosomes, only one the results of a chromosomal analysis on speci- pair was marked differently and only by the mens of Italian V. atra, V. aspis and V. hugyi,in CMA3 (fig. 2D, E), probably corresponding to which, in addition to standard coloration meth- that carrying NORs: in fact the specific stain- ods, we also performed banding staining tech- ing that highlight NORs, the Ag-NOR- (fig. 2B) niques (Ag-NOR-, CMA3-, C-, digestions with and CMA3-banding (fig. 2D) mark selectively Alu I endonucleasis, and sequential staining one pair of microchromosomes. with C- and Alu I banding + fluorochromes). The results obtained are compared to those ob- Vipera tained on specimens of Cerastes vipera, because for this species also no one chromosomal in- All the studied specimens of the three con- sidered taxa, independently of sex and origin, formation is known. Karyological data on this showed a karyotype 2n = 42 chromosomes, species thus contributes to the information for 22 of which were macro and 20 microchro- the whole subfamily. mosomes (fig. 3A, B). In macrochromosomes, the first three pairs were markedly larger than the other pairs; the fourth and sixth pairs were Materials and methods also metacentric, the tenth and eleventh were submetacentric and the others were acrocentric Number, sex and locality of each examined specimen are re- ported in table 1. Chromosomes were obtained from all the (fig. 3A, B). Staining with Ag-NOR- and CMA3 specimens using the air-drying method starting either from banding found NORs on a pair of microchro- in vivo blood cultures, according to the method of Backer et mosomes (fig. 3C, D). The CMA3, combined al. (1972), or in vitro blood cultures by taking 0.5 ml blood both with green methyl and after the C-banding, from tail and keeping this sample for 72 hours in 5 ml of coloured differently all the macrochromosomes MEM+15% calf serum+3% PHA+antibiotic (see Odierna of the three Vipera taxa studied: in V. atra et al., 1999 for further details). In addition to standard stain- and V. hugyi, the centromeric regions of all the ing, the following banding techniques were carried out: Ag- macrochromosomes were, respectively, positive NOR banding (Howell and Black, 1980); CMA3/methylic green (Sahar and Latt, 1980); C-banding (Sumner, 1972); (fig. 3D, H) and negative to CMA3 (fig. 3F); in V. aspis only the centromeric regions of sequential staining of C-banding+CMA3+DAPI and diges- tion with the Alu I+CMA3+DAPI (Odierna et al., 1999, two pairs of macrochromosomes were positive 2001). (fig. 3G). C-banding staining evidenced, how- The karyology of Vipera aspis, V. atra, V. hugyi,andCerastes vipera 115 Figure 1. European and Italian distribution of different taxa belonging to the Vipera aspis group (from Zuffi, 2002, modified). Table 1. Number, sex and origin of the studied specimens of snakes Species Number and sex Origin Vipera V. atra 1 ,1 Pavia (Italy) V. hugyi 1 Cosenza (Italy) Italian V. aspis 1 ,1 Mantova (Italy) 2 ,1 Novara (Italy) Cerastes C. vipera 3 ,2 Southern Ouarzarate (Morocco) 1 ,1, 1juv Northern Ouarzarate (Morocco) ever, very weak bands, which were present on Discussion the centromeric regions of all the macrochro- mosomes, while among the microchromosomes Cerastes vipera only one pair was marked, probably the pair that shows the NORs (fig. 3F). This banding The chromosomal formula of Cerastes vipera staining, however, highlights in all the exam- (2n = 36 chromosomes with 16 macro and 20 ined females, namely of V. atra and V. aspis, microchromosomes) is found in all other con- the existence of a pair of sex chromosomes Z generic species, both in the subfamily Viperi- and W. This pair of chromosomes was homo- nae (except for Vipera aspis group and of V. am- morphic, both being sub telocentric and corre- modytes), and in the subfamily Crotalinae (Gor- sponding in size to the eleventh pair. The W chromosome differed from the Z chromosome man, 1973; Saint Girons, 1977; Olmo, 1986). in that it was almost entirely heterochromatic Furthermore, the presence of homomorphic sex- (fig. 3E), containing a heterochromatin posi- ual chromosomes, cytologically unidentified, leads us to consider C. vipera a basal species tive both to CMA3 and to DAPI (fig. 3G, H, J, K). Digestions with Alu I and those sequential within the Viperidae family: all the other inves- of Alu I+CMA3+DAPI produced results per- tigated species show, in fact, the sexual chromo- fectly overlapping with those of C-banding and somes Z and W as being heteromorphic (Gor- C-banding+CMA3+DAPI (evidence not shown). man, 1973; Olmo, 1986). 116 G. Aprea et al. Figure 2. Male (A) and female (B, C, D, E) karyotypes of C. vipera, stained with Giemsa (A), Ag-NOR (B) and C-banding+ CMA3(D)+DAPI(E).

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