Amphibia-Reptilia 27 (2006) 113-119

The karyology of Vipera aspis V atra V hugyi and Cerastes vipera

Gennaro Aprea1 Augusto Gentilli2 Marco AL 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 Viperaaspis V atra and V 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 pairand absence of cytologically evident sex chromosomes at least with the methods used The three taxa of Vipera studiedshowed chromosomal characters either derived or primitive or at an initial stage of differentiation All three species showeda karyotype (derived) of 2n = 42 chromosomes with 22 macro- and 20 micro-chromosomes they all showed NORs on onemicro-chromosome pair and presented Z and W chromosomes at an initial stage of differentiation Sexchromosomes Z andW were in fact homomorphic but the former was near all euchromatic while the W chromosome was almost completelyheterochromatic All the three taxa of Vipera resulted however karyologically diversified mainly due to the number ofmacro-chromosomes pairs with a centromeric CMA3 positive heterochromatin almost all the pairs in V aspis two pairs inV atra and absent in V hugyi

Introduction

Vipera aspis is among the most widely studiedsnakes of the European herpetofauna but its dis-tribution and taxonomic status is still highly dis-cussed (Zuffi and Bonnet 1999 Conelli 2002Zuffi 2002 Ursenbacher et al 2003) (fig 1)Four subspecies belonging to the Vipera aspisgroup have been recently recognized as goodspecies Vipera aspis (France except for thePyrenean pre-Pyrenean and south eastern areasItaly central northern Italy east from the virtualline Ticino river-Staffora valley-Gulf of Genoanorth of line Gargano-Vesuvio volcano) Viperaatra (northern Italy west from the virtual line

1 - Dipartimento di Biologia Evolutiva e Comparata Uni-versitagrave di Napoli Federico II Dipartimento di BiologiaStrutturale e Funzionale Via Cintia Napoli ItalyCorresponding authorrsquos e-mail gaetanoodiernauninait

2 - Dipartimento di Biologia Animale Universitagrave di Paviapza Botta 9 27100 Pavia Italye-mail gentilliunipvit

3 - Museo di Storia Naturale e del Territorio Universitagrave diPisa via Roma 79 56011 Calci (Pisa) Italye-mail marcozmuseounipiit

Ticino river-Staffora valley-Gulf of Genoa cen-tral and western Switzerland and south east-ern France to the Rhone valley) Vipera hugyi(southern Italy south of the Gargano-Vesuviovolcano-Sicily) Vipera zinnikeri (Pyrenees andsouth-western France) Italian populations ofVipera aspis are still under investigation andshould be considered as Italian Vipera aspispopulations Distinction between the differentspecies was made on a morphological basisusing meristic characteristics such as ventraland subcaudal scales number of dorsal barsand hemipenial anatomy (Zuffi 2002) Theanalysis of skull morphology using geomet-ric morphometry led to results similar to thosefound using the previous morphometric ap-proach (Gentilli et al unpublished data) Onthe contrary recent analyses on mtDNA are incontrast to what has just been described V as-pis has been synonymised with V atra and theother Italian taxa have been highly differenti-ated (Conelli 2002 Ursenbacher et al 2003)Research into any further differences within thefield of karyology starting from what has been

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114 G Aprea et al

briefly reported is therefore particularly inter-esting

Chromosomal studies particularly when thebanding technique was used have given rele-vant information for systematic and phyloge-netic purposes in several snake groups as inelapids and boids (Mengden and Stock 1980Mengden 1982) and in species of the genderVipera (V seoanei V latastei and V ammodytesV zinnikeri Saint Girons 1977) In all the re-maining taxa of the Vipera aspis group chro-mosomal analyses have been performed up tonow on male specimens of the nominal sub-species only (Kobel 1967 Saint Girons 1977)for which a karyotype of 2n = 42 chromosomeshas been described In our paper we presentthe results of a chromosomal analysis on speci-mens of Italian V atra V aspis and V hugyi inwhich in addition to standard coloration meth-ods we also performed banding staining tech-niques (Ag-NOR- CMA3- C- digestions withAlu I endonucleasis and sequential stainingwith C- and Alu I banding + fluorochromes)The results obtained are compared to those ob-tained on specimens of Cerastes vipera becausefor this species also no one chromosomal in-formation is known Karyological data on thisspecies thus contributes to the information forthe whole subfamily

Materials and methods

Number sex and locality of each examined specimen are re-

ported in table 1 Chromosomes were obtained from all the

specimens using the air-drying method starting either from

in vivo blood cultures according to the method of Backer et

al (1972) or in vitro blood cultures by taking 05 ml blood

from tail and keeping this sample for 72 hours in 5 ml of

MEM+15 calf serum+3 PHA+antibiotic (see Odierna

et al 1999 for further details) In addition to standard stain-

ing the following banding techniques were carried out Ag-

NOR banding (Howell and Black 1980) CMA3methylic

green (Sahar and Latt 1980) C-banding (Sumner 1972)

sequential staining of C-banding+CMA3+DAPI and diges-

tion with the Alu I+CMA3+DAPI (Odierna et al 1999

2001)

Results

Cerastes vipera

Both sexes showed 2n = 36 chromosomes16 were macrochromosomes and 20 microchro-mosomes Among the macrochromosomes thepairs 1 3 4 5 and 8 were metacentric pairs2 and 7 were submetacentric and the sixth wasacrocentric (fig 2A B) After the C band-ing staining centromeric regions of all themacrochromosomes showed a scarce presenceof heterochromatin which was CMA3 negativeand DAPI positive (fig 2D E) Also the telom-eric regions of these elements were positive tothe DAPI only but were more weakly stainedwith respect to the centromeric regions (fig 2DE) Among the microchromosomes only onepair was marked differently and only by theCMA3 (fig 2D E) probably corresponding tothat carrying NORs in fact the specific stain-ing that highlight NORs the Ag-NOR- (fig 2B)and CMA3-banding (fig 2D) mark selectivelyone pair of microchromosomes

Vipera

All the studied specimens of the three con-sidered taxa independently of sex and originshowed a karyotype 2n = 42 chromosomes22 of which were macro and 20 microchro-mosomes (fig 3A B) In macrochromosomesthe first three pairs were markedly larger thanthe other pairs the fourth and sixth pairs werealso metacentric the tenth and eleventh weresubmetacentric and the others were acrocentric(fig 3A B) Staining with Ag-NOR- and CMA3

banding found NORs on a pair of microchro-mosomes (fig 3C D) The CMA3 combinedboth with green methyl and after the C-bandingcoloured differently all the macrochromosomesof the three Vipera taxa studied in V atraand V hugyi the centromeric regions of all themacrochromosomes were respectively positive(fig 3D H) and negative to CMA3 (fig 3F)in V aspis only the centromeric regions oftwo pairs of macrochromosomes were positive(fig 3G) C-banding staining evidenced how-

The karyology of Vipera aspis V atra V hugyi and Cerastes 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

ViperaV 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 onthe centromeric regions of all the macrochro-mosomes while among the microchromosomesonly one pair was marked probably the pairthat shows the NORs (fig 3F) This bandingstaining however highlights in all the exam-ined females namely of V atra and V aspisthe existence of a pair of sex chromosomes Zand W This pair of chromosomes was homo-morphic both being sub telocentric and corre-sponding in size to the eleventh pair The Wchromosome differed from the Z chromosomein that it was almost entirely heterochromatic(fig 3E) containing a heterochromatin posi-tive both to CMA3 and to DAPI (fig 3G H JK) Digestions with Alu I and those sequentialof Alu I+CMA3+DAPI produced results per-fectly overlapping with those of C-banding andC-banding+CMA3+DAPI (evidence not shown)

Discussion

Cerastes vipera

The chromosomal formula of Cerastes vipera(2n = 36 chromosomes with 16 macro and 20microchromosomes) is found in all other con-generic species both in the subfamily Viperi-nae (except for Vipera aspis group and of V am-modytes) and in the subfamily Crotalinae (Gor-man 1973 Saint Girons 1977 Olmo 1986)Furthermore the presence of homomorphic sex-ual chromosomes cytologically unidentifiedleads us to consider C vipera a basal specieswithin the Viperidae family all the other inves-tigated species show in fact the sexual chromo-somes Z and W as being heteromorphic (Gor-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)

The karyology of Vipera aspis V atra V hugyi and Cerastes vipera 117

Figure 3 Karyotypes and metaphase plates of V aspis group Giemsa stained karyotypes of a male of V hugyi (A) and afemale of V atra from Novara (B) Ag-NOR- (C) CMA3- (D) C-banded (E) and C-banding + CMA3 (F G H) + DAPI(I J K) stained metaphase plates of a male V hugyi (F I) female from Mantova of V aspis (C G J) and a female fromNovara of V atra (F H K) Solid and empty arrows point to the W sex chromosome and NOR-bearing microchromosomesrespectively

118 G Aprea et al

Vipera

Specimens of Italian V aspis and V hugyi showa karyotype 2n = 42 chromosomes which doesnot differ from that described for specimens ofthe nominal subspecies (Matthey 1931 Kobel1967 Saint Girons 1977) Also the specimensof V atra show the same chromosomal formulawith 2n = 42 chromosomes This formula alsoshown by V ammodytes and V zinnikeri dif-fered markedly from the 2n = 36 chromosomesexhibited by all the other species of viperidsnakes studied so far (Gorman 1973 Olmo1986) including Cerastes vipera (present pa-per) It has been hypothesised that the karyotypeof 2n = 42 chromosomes in the V aspis grouphas been derived from that of 2n = 36 chro-mosomes after a series of fissions and inver-sions (Kobel 1967 Gorman 1973) From ouranalyses we highlight that NORs were not in-volved in this process since both Vipera andC vipera present NORs on microchromosomesLocalisation of NORs is known for one othersnake species only Crotalus viridis belongingto the subfamily Crotalinae which also displaysNORs on michrochromosomes (Porter et al1991 1994) This result in addition to thoseobtained in this study suggests that the NORsposition has remained unaltered during the spe-cific differentiation within the two phyletic lin-eages of the family Viperidae the Crotalinaeand Viperinae

Our results evidence that the number ofmacrochromosomes possessing centromericCMA3 positive heterochromatin discriminatesthe three examined taxa of Vipera Intraspecificdifferences in amount and distribution of het-erochromatin have been found in taxa (John1988) and considered as polymorphisms thentaxonomically irrelevant Therefore the differ-entiation observed at a morphological level be-tween the three taxa of Vipera (Zuffi 2002) hasnot been accomplished with any substantial dif-ferences at a karyological level On the otherhand the role of inter- and intra-specific vari-ations of heterochromatin and highly repeatedDNA families associated with this genomic ma-

terial in speciation processes is still contro-versial Variation of this genomic material isfrequent and already recorded in Squamates(Capriglione et al 1991 1998 Yonenaga-Yassuda et al 1996) Furthermore in severaleukaryotes cases are reported in which the hete-rochromatin could influence genetic variabilityit being able to activate or to inhibit some geneexpressions (King 1993 Redi et al 2001)Nevertheless there is no convincing evidenceof a reduced fertility of the hybrid betweencytotypes with a structural chromosomal het-erozigosity derived from a different amount ordistribution of heterochromatine (John 1988)Variations in heterochromatin and in highly re-peated DNA families associated to this genomicmaterial could play an important role in allow-ing the incoming or fission of other mutations(Charlesworth et al 1994 Capriglione et al1998 Redi et al 2001) The variability ob-served in the heterochromatin between the threetaxa of Vipera therefore could be an indicationof a their active tendency to chromosomal vari-ability This variability could tentatively be hy-pothesised as one of the relevant mechanismsacting to reinforce the barrier against the hy-bridisation between incoming species On theother hand the Vipera aspis group with the ex-ception of V ammodytes among Viperidae arethe only species that have a modified karyotype(2n = 42 chromosomes) with respect to that ofthe 2n = 36 chromosomes found in all the otherstudied viperid species It should be stressedhowever that the specimens of V aspis groupcan however in nature hybridise with those ofV ammodytes (Saint Girons 1977 and refer-ences therein) even if the reproductive abilityof the resulting hybrids is unknown

References

Backer RJ Mengden GA Bull JJ (1972) Karyotipicstudies of thirtyeight species of North American snakesCopeia 1972 257-265

Capriglione T Cardone A Odierna G Olmo E (1991)Evolution of a centromeric satellite DNA and phylogeny

The karyology of Vipera aspis V atra V hugyi and Cerastes 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

ViperaV 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 onthe centromeric regions of all the macrochro-mosomes while among the microchromosomesonly one pair was marked probably the pairthat shows the NORs (fig 3F) This bandingstaining however highlights in all the exam-ined females namely of V atra and V aspisthe existence of a pair of sex chromosomes Zand W This pair of chromosomes was homo-morphic both being sub telocentric and corre-sponding in size to the eleventh pair The Wchromosome differed from the Z chromosomein that it was almost entirely heterochromatic(fig 3E) containing a heterochromatin posi-tive both to CMA3 and to DAPI (fig 3G H JK) Digestions with Alu I and those sequentialof Alu I+CMA3+DAPI produced results per-fectly overlapping with those of C-banding andC-banding+CMA3+DAPI (evidence not shown)

Discussion

Cerastes vipera

The chromosomal formula of Cerastes vipera(2n = 36 chromosomes with 16 macro and 20microchromosomes) is found in all other con-generic species both in the subfamily Viperi-nae (except for Vipera aspis group and of V am-modytes) and in the subfamily Crotalinae (Gor-man 1973 Saint Girons 1977 Olmo 1986)Furthermore the presence of homomorphic sex-ual chromosomes cytologically unidentifiedleads us to consider C vipera a basal specieswithin the Viperidae family all the other inves-tigated species show in fact the sexual chromo-somes Z and W as being heteromorphic (Gor-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)

The karyology of Vipera aspis V atra V hugyi and Cerastes vipera 117

Figure 3 Karyotypes and metaphase plates of V aspis group Giemsa stained karyotypes of a male of V hugyi (A) and afemale of V atra from Novara (B) Ag-NOR- (C) CMA3- (D) C-banded (E) and C-banding + CMA3 (F G H) + DAPI(I J K) stained metaphase plates of a male V hugyi (F I) female from Mantova of V aspis (C G J) and a female fromNovara of V atra (F H K) Solid and empty arrows point to the W sex chromosome and NOR-bearing microchromosomesrespectively

118 G Aprea et al

Vipera

Specimens of Italian V aspis and V hugyi showa karyotype 2n = 42 chromosomes which doesnot differ from that described for specimens ofthe nominal subspecies (Matthey 1931 Kobel1967 Saint Girons 1977) Also the specimensof V atra show the same chromosomal formulawith 2n = 42 chromosomes This formula alsoshown by V ammodytes and V zinnikeri dif-fered markedly from the 2n = 36 chromosomesexhibited by all the other species of viperidsnakes studied so far (Gorman 1973 Olmo1986) including Cerastes vipera (present pa-per) It has been hypothesised that the karyotypeof 2n = 42 chromosomes in the V aspis grouphas been derived from that of 2n = 36 chro-mosomes after a series of fissions and inver-sions (Kobel 1967 Gorman 1973) From ouranalyses we highlight that NORs were not in-volved in this process since both Vipera andC vipera present NORs on microchromosomesLocalisation of NORs is known for one othersnake species only Crotalus viridis belongingto the subfamily Crotalinae which also displaysNORs on michrochromosomes (Porter et al1991 1994) This result in addition to thoseobtained in this study suggests that the NORsposition has remained unaltered during the spe-cific differentiation within the two phyletic lin-eages of the family Viperidae the Crotalinaeand Viperinae

Our results evidence that the number ofmacrochromosomes possessing centromericCMA3 positive heterochromatin discriminatesthe three examined taxa of Vipera Intraspecificdifferences in amount and distribution of het-erochromatin have been found in taxa (John1988) and considered as polymorphisms thentaxonomically irrelevant Therefore the differ-entiation observed at a morphological level be-tween the three taxa of Vipera (Zuffi 2002) hasnot been accomplished with any substantial dif-ferences at a karyological level On the otherhand the role of inter- and intra-specific vari-ations of heterochromatin and highly repeatedDNA families associated with this genomic ma-

terial in speciation processes is still contro-versial Variation of this genomic material isfrequent and already recorded in Squamates(Capriglione et al 1991 1998 Yonenaga-Yassuda et al 1996) Furthermore in severaleukaryotes cases are reported in which the hete-rochromatin could influence genetic variabilityit being able to activate or to inhibit some geneexpressions (King 1993 Redi et al 2001)Nevertheless there is no convincing evidenceof a reduced fertility of the hybrid betweencytotypes with a structural chromosomal het-erozigosity derived from a different amount ordistribution of heterochromatine (John 1988)Variations in heterochromatin and in highly re-peated DNA families associated to this genomicmaterial could play an important role in allow-ing the incoming or fission of other mutations(Charlesworth et al 1994 Capriglione et al1998 Redi et al 2001) The variability ob-served in the heterochromatin between the threetaxa of Vipera therefore could be an indicationof a their active tendency to chromosomal vari-ability This variability could tentatively be hy-pothesised as one of the relevant mechanismsacting to reinforce the barrier against the hy-bridisation between incoming species On theother hand the Vipera aspis group with the ex-ception of V ammodytes among Viperidae arethe only species that have a modified karyotype(2n = 42 chromosomes) with respect to that ofthe 2n = 36 chromosomes found in all the otherstudied viperid species It should be stressedhowever that the specimens of V aspis groupcan however in nature hybridise with those ofV ammodytes (Saint Girons 1977 and refer-ences therein) even if the reproductive abilityof the resulting hybrids is unknown

References

Backer RJ Mengden GA Bull JJ (1972) Karyotipicstudies of thirtyeight species of North American snakesCopeia 1972 257-265

Capriglione T Cardone A Odierna G Olmo E (1991)Evolution of a centromeric satellite DNA and phylogeny

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)

The karyology of Vipera aspis V atra V hugyi and Cerastes vipera 117

Figure 3 Karyotypes and metaphase plates of V aspis group Giemsa stained karyotypes of a male of V hugyi (A) and afemale of V atra from Novara (B) Ag-NOR- (C) CMA3- (D) C-banded (E) and C-banding + CMA3 (F G H) + DAPI(I J K) stained metaphase plates of a male V hugyi (F I) female from Mantova of V aspis (C G J) and a female fromNovara of V atra (F H K) Solid and empty arrows point to the W sex chromosome and NOR-bearing microchromosomesrespectively

118 G Aprea et al

Vipera

Specimens of Italian V aspis and V hugyi showa karyotype 2n = 42 chromosomes which doesnot differ from that described for specimens ofthe nominal subspecies (Matthey 1931 Kobel1967 Saint Girons 1977) Also the specimensof V atra show the same chromosomal formulawith 2n = 42 chromosomes This formula alsoshown by V ammodytes and V zinnikeri dif-fered markedly from the 2n = 36 chromosomesexhibited by all the other species of viperidsnakes studied so far (Gorman 1973 Olmo1986) including Cerastes vipera (present pa-per) It has been hypothesised that the karyotypeof 2n = 42 chromosomes in the V aspis grouphas been derived from that of 2n = 36 chro-mosomes after a series of fissions and inver-sions (Kobel 1967 Gorman 1973) From ouranalyses we highlight that NORs were not in-volved in this process since both Vipera andC vipera present NORs on microchromosomesLocalisation of NORs is known for one othersnake species only Crotalus viridis belongingto the subfamily Crotalinae which also displaysNORs on michrochromosomes (Porter et al1991 1994) This result in addition to thoseobtained in this study suggests that the NORsposition has remained unaltered during the spe-cific differentiation within the two phyletic lin-eages of the family Viperidae the Crotalinaeand Viperinae

Our results evidence that the number ofmacrochromosomes possessing centromericCMA3 positive heterochromatin discriminatesthe three examined taxa of Vipera Intraspecificdifferences in amount and distribution of het-erochromatin have been found in taxa (John1988) and considered as polymorphisms thentaxonomically irrelevant Therefore the differ-entiation observed at a morphological level be-tween the three taxa of Vipera (Zuffi 2002) hasnot been accomplished with any substantial dif-ferences at a karyological level On the otherhand the role of inter- and intra-specific vari-ations of heterochromatin and highly repeatedDNA families associated with this genomic ma-

terial in speciation processes is still contro-versial Variation of this genomic material isfrequent and already recorded in Squamates(Capriglione et al 1991 1998 Yonenaga-Yassuda et al 1996) Furthermore in severaleukaryotes cases are reported in which the hete-rochromatin could influence genetic variabilityit being able to activate or to inhibit some geneexpressions (King 1993 Redi et al 2001)Nevertheless there is no convincing evidenceof a reduced fertility of the hybrid betweencytotypes with a structural chromosomal het-erozigosity derived from a different amount ordistribution of heterochromatine (John 1988)Variations in heterochromatin and in highly re-peated DNA families associated to this genomicmaterial could play an important role in allow-ing the incoming or fission of other mutations(Charlesworth et al 1994 Capriglione et al1998 Redi et al 2001) The variability ob-served in the heterochromatin between the threetaxa of Vipera therefore could be an indicationof a their active tendency to chromosomal vari-ability This variability could tentatively be hy-pothesised as one of the relevant mechanismsacting to reinforce the barrier against the hy-bridisation between incoming species On theother hand the Vipera aspis group with the ex-ception of V ammodytes among Viperidae arethe only species that have a modified karyotype(2n = 42 chromosomes) with respect to that ofthe 2n = 36 chromosomes found in all the otherstudied viperid species It should be stressedhowever that the specimens of V aspis groupcan however in nature hybridise with those ofV ammodytes (Saint Girons 1977 and refer-ences therein) even if the reproductive abilityof the resulting hybrids is unknown

References

Backer RJ Mengden GA Bull JJ (1972) Karyotipicstudies of thirtyeight species of North American snakesCopeia 1972 257-265

Capriglione T Cardone A Odierna G Olmo E (1991)Evolution of a centromeric satellite DNA and phylogeny

The karyology of Vipera aspis V atra V hugyi and Cerastes vipera 117

Figure 3 Karyotypes and metaphase plates of V aspis group Giemsa stained karyotypes of a male of V hugyi (A) and afemale of V atra from Novara (B) Ag-NOR- (C) CMA3- (D) C-banded (E) and C-banding + CMA3 (F G H) + DAPI(I J K) stained metaphase plates of a male V hugyi (F I) female from Mantova of V aspis (C G J) and a female fromNovara of V atra (F H K) Solid and empty arrows point to the W sex chromosome and NOR-bearing microchromosomesrespectively

118 G Aprea et al

Vipera

Specimens of Italian V aspis and V hugyi showa karyotype 2n = 42 chromosomes which doesnot differ from that described for specimens ofthe nominal subspecies (Matthey 1931 Kobel1967 Saint Girons 1977) Also the specimensof V atra show the same chromosomal formulawith 2n = 42 chromosomes This formula alsoshown by V ammodytes and V zinnikeri dif-fered markedly from the 2n = 36 chromosomesexhibited by all the other species of viperidsnakes studied so far (Gorman 1973 Olmo1986) including Cerastes vipera (present pa-per) It has been hypothesised that the karyotypeof 2n = 42 chromosomes in the V aspis grouphas been derived from that of 2n = 36 chro-mosomes after a series of fissions and inver-sions (Kobel 1967 Gorman 1973) From ouranalyses we highlight that NORs were not in-volved in this process since both Vipera andC vipera present NORs on microchromosomesLocalisation of NORs is known for one othersnake species only Crotalus viridis belongingto the subfamily Crotalinae which also displaysNORs on michrochromosomes (Porter et al1991 1994) This result in addition to thoseobtained in this study suggests that the NORsposition has remained unaltered during the spe-cific differentiation within the two phyletic lin-eages of the family Viperidae the Crotalinaeand Viperinae

Our results evidence that the number ofmacrochromosomes possessing centromericCMA3 positive heterochromatin discriminatesthe three examined taxa of Vipera Intraspecificdifferences in amount and distribution of het-erochromatin have been found in taxa (John1988) and considered as polymorphisms thentaxonomically irrelevant Therefore the differ-entiation observed at a morphological level be-tween the three taxa of Vipera (Zuffi 2002) hasnot been accomplished with any substantial dif-ferences at a karyological level On the otherhand the role of inter- and intra-specific vari-ations of heterochromatin and highly repeatedDNA families associated with this genomic ma-

terial in speciation processes is still contro-versial Variation of this genomic material isfrequent and already recorded in Squamates(Capriglione et al 1991 1998 Yonenaga-Yassuda et al 1996) Furthermore in severaleukaryotes cases are reported in which the hete-rochromatin could influence genetic variabilityit being able to activate or to inhibit some geneexpressions (King 1993 Redi et al 2001)Nevertheless there is no convincing evidenceof a reduced fertility of the hybrid betweencytotypes with a structural chromosomal het-erozigosity derived from a different amount ordistribution of heterochromatine (John 1988)Variations in heterochromatin and in highly re-peated DNA families associated to this genomicmaterial could play an important role in allow-ing the incoming or fission of other mutations(Charlesworth et al 1994 Capriglione et al1998 Redi et al 2001) The variability ob-served in the heterochromatin between the threetaxa of Vipera therefore could be an indicationof a their active tendency to chromosomal vari-ability This variability could tentatively be hy-pothesised as one of the relevant mechanismsacting to reinforce the barrier against the hy-bridisation between incoming species On theother hand the Vipera aspis group with the ex-ception of V ammodytes among Viperidae arethe only species that have a modified karyotype(2n = 42 chromosomes) with respect to that ofthe 2n = 36 chromosomes found in all the otherstudied viperid species It should be stressedhowever that the specimens of V aspis groupcan however in nature hybridise with those ofV ammodytes (Saint Girons 1977 and refer-ences therein) even if the reproductive abilityof the resulting hybrids is unknown

References

Backer RJ Mengden GA Bull JJ (1972) Karyotipicstudies of thirtyeight species of North American snakesCopeia 1972 257-265

Capriglione T Cardone A Odierna G Olmo E (1991)Evolution of a centromeric satellite DNA and phylogeny

118 G Aprea et al

Vipera

Specimens of Italian V aspis and V hugyi showa karyotype 2n = 42 chromosomes which doesnot differ from that described for specimens ofthe nominal subspecies (Matthey 1931 Kobel1967 Saint Girons 1977) Also the specimensof V atra show the same chromosomal formulawith 2n = 42 chromosomes This formula alsoshown by V ammodytes and V zinnikeri dif-fered markedly from the 2n = 36 chromosomesexhibited by all the other species of viperidsnakes studied so far (Gorman 1973 Olmo1986) including Cerastes vipera (present pa-per) It has been hypothesised that the karyotypeof 2n = 42 chromosomes in the V aspis grouphas been derived from that of 2n = 36 chro-mosomes after a series of fissions and inver-sions (Kobel 1967 Gorman 1973) From ouranalyses we highlight that NORs were not in-volved in this process since both Vipera andC vipera present NORs on microchromosomesLocalisation of NORs is known for one othersnake species only Crotalus viridis belongingto the subfamily Crotalinae which also displaysNORs on michrochromosomes (Porter et al1991 1994) This result in addition to thoseobtained in this study suggests that the NORsposition has remained unaltered during the spe-cific differentiation within the two phyletic lin-eages of the family Viperidae the Crotalinaeand Viperinae

Our results evidence that the number ofmacrochromosomes possessing centromericCMA3 positive heterochromatin discriminatesthe three examined taxa of Vipera Intraspecificdifferences in amount and distribution of het-erochromatin have been found in taxa (John1988) and considered as polymorphisms thentaxonomically irrelevant Therefore the differ-entiation observed at a morphological level be-tween the three taxa of Vipera (Zuffi 2002) hasnot been accomplished with any substantial dif-ferences at a karyological level On the otherhand the role of inter- and intra-specific vari-ations of heterochromatin and highly repeatedDNA families associated with this genomic ma-

terial in speciation processes is still contro-versial Variation of this genomic material isfrequent and already recorded in Squamates(Capriglione et al 1991 1998 Yonenaga-Yassuda et al 1996) Furthermore in severaleukaryotes cases are reported in which the hete-rochromatin could influence genetic variabilityit being able to activate or to inhibit some geneexpressions (King 1993 Redi et al 2001)Nevertheless there is no convincing evidenceof a reduced fertility of the hybrid betweencytotypes with a structural chromosomal het-erozigosity derived from a different amount ordistribution of heterochromatine (John 1988)Variations in heterochromatin and in highly re-peated DNA families associated to this genomicmaterial could play an important role in allow-ing the incoming or fission of other mutations(Charlesworth et al 1994 Capriglione et al1998 Redi et al 2001) The variability ob-served in the heterochromatin between the threetaxa of Vipera therefore could be an indicationof a their active tendency to chromosomal vari-ability This variability could tentatively be hy-pothesised as one of the relevant mechanismsacting to reinforce the barrier against the hy-bridisation between incoming species On theother hand the Vipera aspis group with the ex-ception of V ammodytes among Viperidae arethe only species that have a modified karyotype(2n = 42 chromosomes) with respect to that ofthe 2n = 36 chromosomes found in all the otherstudied viperid species It should be stressedhowever that the specimens of V aspis groupcan however in nature hybridise with those ofV ammodytes (Saint Girons 1977 and refer-ences therein) even if the reproductive abilityof the resulting hybrids is unknown

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Received September 14 2004 Accepted December 282004