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Except for polyploidy, parthenogenesis and hybridogenesis, the role of chromosome rearrangements in speciation is still highly debated. This paper highlights an unusual karyotype diversification and a putative case of chromosomal speciation in the endemic Malagasy tomato frogs. The tomato frogs include Dyscophus insularis as well as the sister species D. antongilii and D. guineti, which are characterized by a disjunct distribution and a low genetic divergence, probably due to a recent hybridization. Our results show that the three Dyscophus species present 2n = 26 biarmed chromosomes, differing in chromatin pattern. Interestingly, both sexes of D. guineti show a heteromorphic chromosome pair 11, with one element larger (11a) than the other (11b) due to the presence of a large terminal tract on the short arm with an euchromatic band enclosed between two heterochromatic bands. This fixed condition reminds that of some newts of the genus Triturus, but in Dyscophus it probably characterizes the beginning of a cladogenetic process. We hypothesize that in D. guineti the raising of the heteromorphic 11th chromosome pair followed the hybridization with D. antongilii, originating a post-zygotic barrier and representing a case of sympatric or parapatric chromosomal speciation. This speciation mechanism may also have had important biogeographic consequences, shaping the current disjunct distribution of D. antongilii and D. guineti, so far not sufficiently explained.

We performed a phylogenetic analysis using nuclear (RAG-1, RAG-2) and mitochondrial (16S) markers, a statistical Bayesian reconstruction of ancestral distribution areas and a karyological analysis on most Malagasy species of the gekkonid genus Lygodactylus. The phylogenetic analysis largely confirms major basal branching pattern of previous molecular studies, but highlights significant differences concerning both the relationships between different species groups as well as those within groups. The biogeographic analysis supports a Malagasy origin of Lygodactylus, an oversea dispersal to continental Africa and a return to Madagascar. The L. madagascariensis group (also including a new candidate species identified herein) is the most basal clade in Lygodactylus, and the sister group of a clade with all the remaining species. The second most basal clade is the L. verticillatus group, placed as the sister group of a clade comprising African and Malagasy species. The sister lineage of the L. verticillatus group originated the African radiation through an oversea dispersal out of Madagascar. Eventually, the sister lineage of the L. capensis group originated secondary dispersals from Africa to Madagascar. In Madagascar, lineage diversification in different species groups mainly occurred from southern to northern and eastern regions. Dispersal, vicariance and paleoclimatic refugia probably played a relevant role in the evolutionary history of closely related taxa and in speciation mechanisms. The cytogenetic analysis evidenced a high karyotypic variability in Lygodactylus (from 2n = 34 to 2n = 40), which is at least partly consistent with the phylogenetic relationships and the composition of the various species group. Chromosome evolution occurred independently in different lineages, mainly through a reduction in the chromosome number and starting from a putative primitive karyotype of 2n = 40 with all telocentric elements.

In this first cytogenetic survey on the lamprophiid snake subfamily Pseudoxyrhophiinae, we studied the karyology of ten snake species belonging to seven genera from Madagascar (Compsophis, Leioheterodon, Liophidium, Lycodryas, Madagascarophis, Phisalixella and Thamnosophis) using standard and banding methods. Our results show a wide range of different karyotypes ranging from 2n = 34 to 2n = 46 elements (FN from 40 to 48), with nucleolus organizer regions (NORs) on one (plesiomorphic) or two (derived/apomorphic) microchromosome pairs, and W chromosome at early or advanced states of diversification from the Z chromosome. The observed W chromosome variations further support the most accepted hypothesis that W differentiation from the Z chromosome occurred by progressive steps. We also propose an evolutionary scenario for the observed high karyotype diversity in this group of snakes, suggesting that it is derived from a putative primitive pseudoxyrhophiine karyotype with 2n = 46, similar to that of Leioheterodon geayi, via a series of centric fusions and inversions among macrochromosomes and translocations of micro- either to micro- or to macrochromosomes. This primitive Pseudoxyrhophiinae karyotype might have derived from a putative Lamprophiidae ancestor with 2n = 48, by means of a translocation of a micro- to a macrochromosome. In turn, the karyotype of this lamprophiid common ancestor may have derived from the assumed primitive snake karyotype (2n = 36 chromosomes, with 16 biarmed macro- and 20 microchromosomes) by a series of centric fissions and one inversion. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 450–460.

In this study we analysed mitochondrial DNA variation in Penaeus kerathurus prawns collected from seven locations along a transect across the Siculo-Tunisian region in order to verify if any population structuring exists over a limited geographical scale and to delineate the putative transition zone with sufficient accuracy. Partial DNA sequences of COI and 16S genes were analysed. In contrast to the highly conservative 16S gene, the COI sequences exhibited sufficient diversity for population analysis. The COI gene revealed low levels of haplotype and nucleotide diversities. The size of the annual landings of this commercial species suggests large population sizes. Hence, the low genetic diversity detected in this study could indicate a possible reduction in effective population sizes in the past. We detected significant genetic differentiation between eastern and western populations likely due to restricted gene flow across the Siculo-Tunisian boundary. We discuss the different evolutionary forces that may have shaped the genetic variation and suggest that the genetic divide is probably maintained by present-day dispersal limitation.

A karyological analysis on six Italian populations the slow worm (Anguis veronensis Pollini, 1818) was performed and their genetic differentiation at the mitochondrial 16S rRNA gene fragment from a Spanish sample has been assessed. The Italian populations were karyologically uniform, all showing 2n= 44 elements, of which 20 were macrochromosomes and 24 microchromosomes. Comparison with literature data on Central European populations showed a difference on the morphology of the 10th chromosome pair: submetacentric in Italian populations and telocentric in the Central European ones. Our analysis showed the presence of a fragile site on chromosomes of this pair, suggesting its propensity for structural rearrangements. Analysis of the 16S rRNA gene fragment showed uniformity among Italian populations (uncorrected genetic distance of 0.4%), and their genetic distinctness from the Spanish individual (uncorrected genetic distance of 4.2%). Our results confirm the existence of two different Anguis fragilis Linnaeus, 1758 lineages, each one characterized by a different cytotype.

Genetic diversity was analyzed in Chalcides chalcides populations from peninsular Italy, Sardinia, Sicily and Tunisia by sequencing 400 bp at the 5' end of the mitochondrial gene encoding cytochrome b (cyt b) and by restriction fragment length polymorphism (RFLP) analysis of two mitochondrial DNA segments (ND-1/2 and ND-3/4). The results of the phylogenetic analysis highlighted the presence of three main clades corresponding with three of the four main geographical areas (Tunisia, Sicily and the Italian peninsula), while Sardinia proved to be closely related to Tunisian haplotypes suggesting a colonization of this island from North Africa by human agency in historical times. On the contrary, the splitting times estimated on the basis of cyt b sequence data seem to indicate a more ancient colonization of Sicily and the Italian Peninsula, as a consequence of tectonic and climatic events that affected the Mediterranean Basin during the Pleistocene. Finally, the analysis of the genetic variability of C. chalcides populations showed a remarkable genetic homogeneity in Italian populations when compared to the Tunisian ones. This condition could be explained by a rapid post-glacial expansion from refugial populations that implied serial bottlenecking with progressive loss of haplotypes, resulting in a low genetic diversity in the populations inhabiting the more recently colonized areas.

Karyotype, location of the nucleolar organiser region (NOR) and heterochromatin presence and composition were studied in the Antarctic scallop Adamussium colbecki Smith, 1902. The karyotype exhibits 2n = 38 chromosomes with 11 pairs of metacentrics, 5 of submetacentrics, one subtelocentric and two telocentrics. Ag-NOR, CMA(3), DA/MM and NOR-FISH evidenced paracentromeric NORs on the short arm of 2nd pair chromosomes. Digestion with three restriction endonucleases followed by sequential staining with Giemsa, CMA(3) and DAPI evidenced on all chromosomes centromeric heterochromatin positive for both DAPI and CMA(3). In situ hybridisation analysis showed the presence of an AT-rich satellite DNA in the centromeric heterochromatin of several chromosomes. A mosaicism was detected in the germinal cell lines of one specimen, as in six of the 20 plates examined the set had 37 chromosomes with a missing pair of telocentrics and an unpaired metacentric. Comparison of the chromosome sets of all the pectinids studied to date and comparison with a phyletic tree obtained from molecular mitochondrial genes studies yielded good agreement between karyotype morphology and taxonomic classification.

The West Palearctic green toads, Bufo viridis , represent a species complex. Apart from tetraploid populations, which form at least one separate species, evidence exists for relevant differentiation among diploid populations. We present the results of a chromosomal (C-, Ag-NOR-, Replication pattern, DAPI and CMA3 banding) and molecular study (isolation and characterization of a satellite DNA family) carried out on a number of Central Asian, European and North African populations. For comparative purposes, our molecular analysis was also extended to specimens of three additional Bufo species (B. bufo, B. mauritanicus and B. cf. regularis ), as well as two rare African bufonids (Werneria mertensis and Wolterstoffina sp.). Our results demonstrate a remarkable karyological and molecular evolutionary stasis in the Bufo viridis complex. In fact, all chromatinic markers showed the same pattern and/or composition in all specimens, independently of their origin and ploidy levels. Even the NOR loci were invariably two and located on the telomeric regions of two chromosomes of the sixth pair, or quartet. Furthermore, very similar patterns of genomic hybridization of a monomeric unit of the Pst I satellite DNA family (named pBv) were observed in all diploid and tetraploid populations, as well as in B. bufo and B. mauritanicus . Finally, pBv hybridizes with monomeric units of Pst I satellite DNA in all species studied, including Werneria and Wolterstorffina, which are thought to have separated from Bufo as early as in the Mesozoic.

Karyotypes of three microhylid frog species of the Malagasy relict genus Scaphiophryne were studied: Scaphiophryne gottlebei, S. madagascariensis and S. spinosa. The latter two showed a plesiomorphic ranoid karyotype of 2n = 26. In contrast, tetraploidy was demonstrated in S. gottlebei, which constitutes an exceptional state among Malagasy amphibians. A combination of different banding techniques and of rDNA-FISH provided evidence for allopolyploidy in the species and for a completed subsequent functional and structural diploidization. Phylogenetic analysis of mitochondrial 16S rDNA sequences revealed a significant deceleration of nucleotide substitution rates in Scaphiophryne. The tetraploidy of S. gottlebei probably occurred early in their radiation. Ecological and behavioural patterns of Scaphiophryne probably favoured intraspecific gene flow and hybridization events, thereby leading to slow molecular substitution rates and to allopolyploid chromosome speciation in S. gottlebei.

Cytogenetic and molecular data on Alytes muletensis (Amphibia: Discoglossidae) are compared with other representatives of archaeobatrachian frogs: Bombina variegata pachypus, Pelobates cultripes, Pelodytes punctatus, Xenopus laevis, and Discoglossus. A. muletensis has the karyotype typical for the genus Alytes, 38 elements with either one or two arms, some of which can be considered as ‘microchromosomes’. The NORs are located on the telomeres of the tenth chromosome pair which agrees with the state in A. obstetricians but differs from A. cisternasii reflecting phylogenetic affinities. C-banding and staining with DAPI and chromomycin A3 revealed important blocks of telomeric CMA-positive heterochromatin on the smaller chromosomes of Alytes, similar to the state found in Discoglossus. Phylogenetic analysis of 750 bp of fragments of the mitochondrial 16S and 12S rRNA genes corroborated that Discoglossus and Alytes are sister taxa which together probably form the sister group of the Bombinatorinae. Centromeric heterochromatin in Alytes may be responsible for the retention of a plesiomorphic asymmetric karyotype which independently has evolved into a symmetric karyotype through centric fusions in Bombina and Discoglossus. The HindIII satellite DNA family was present in all archaeobatrachians studied but absent in hyloid and ranoid neobatrachians.

We conducted a cytogenetic and molecular analysis in six out of the eight Discoglossus taxa CD. pictus pictus, D. p. auritus, D. sardus, D. montalentii, D. galganoi galganoi and D. g. jean‐neae) All the taxa possessed 2n = 28 biarmed chromosomes, except D. pictus pictus where the 13th pair was acrocentric, and the NORs were located on the short arm of the 7th pair in all the taxa but D. galganoi, where they were on the short arm of the 13th pair. Heterochromatin was detected by banding methods (C‐band‐ing, base‐specific fluorochromes, replication patterns and digestions with restriction enzymes). The techniques used were able to discriminate among all the Discoglussus taxa studied, identifying several heterochromatin families with marked differences in chromosome location and/or in the behaviour of the banding techniques used. The differentiation of these heterochromatin families can have either a phylogenetic (as is the case with A+T rich cen‐tromeric heterochromatin) or an adaptive (G+C rich in...

Populations of Zootoca vivipara appear karyologically well differentiated by heterochromatin distribution both in autosomes and sex‐chromosomes; allopatric oviparous populations possess few elements with centromeric heterochromatin and W acrocen‐tric sex‐chromosomes with heterochromatin only on the centromere; Alpine, as well as central‐western European viviparous populations possess conspicuous centromeric bands of heterochromatin on all of the chromosome pairs and a metacentric W chromosome with paracentromeric and telomeric heterochromatin bands; viviparous populations, living east and possibly north of the Carpathian Mountains, have many elements with interstitial and telomeric heterochromatin and an acrocentric W chromosome with telomeric and subtelomeric heterochroma. These data suggest an origin and evolution of sex‐chromosomes in this species, that, interestingly, parallels the recent hypothesis (Heulin et al., 1993) on the rise and evolution of viviparity and speciation in Zootoca.

Chromosomes of various specimens belonging to four Italian and three African populations of Tarentola mauritanica were processed by Giemsa standard staining, by C‐, G‐, Ag‐NOR, DAPI‐banding methods and Alu I and Hae III endonuclease digestions. All specimens displayed a karyotype of 2n = 42 chromosomes, most of which were acrocentric and gradually decreasing in length. The karyotypes of the populations investigated proved different in the length of the short arms of acrocentric chromosomes, the number of dibrachial chromosomes and NOR localization. The presumptive chromosomal rearrangements are here indicated, and the implications of intraspecific diversification are discussed. Lastly, heterochromatin resistant to two common endonucleases may represent a relevant marker in the African Gekkoninae phylogenesis.

Well-defined G-bands were obtained on somatic metaphase chromosomes of Encarsia berlesei using trypsin and warm 2× SCC in sequence. The G-banded pattern allowed rapid identification of all five metacentric chromosomes, which appeared uniformly lighted when stained with DAPI fluorochrome dye. It is stressed that ageing affects G-banding in this insect species; in fact, good banded chromosomes were obtained on 1-month air-stored chromosomes. Evidence for asynchronous condensation on the chromosomes of this species is also provided.Key words: G-banding, chromosomes, insect, Encarsia.

We describe the karyotype, location of nucleolus-organizing regions (NORs) and heterochromatin distribution and composition in the Antarctic chiton Nuttallochiton mirandus. Specimens had a karyotype of 2n = 32 chromosomes, of which two were microchromosomes. Among macrochromosomes, the elements of the first and fourth pair were bi-armed, the others were telocentric. At least six NOR sites were detected with NOR-FISH, but only four were Ag-NOR-banding-positive. The two microchromosomes were essentially euchromatic, while all macrochromosomes exhibited clear pericentromeric C bands that were found to be AT-rich (being quinacrine- and DAPI-positive) and resistant to digestion with AluI and HaeIII. N. mirandus has the largest number of chromosomes (2n = 32) and telocentric elements (26) of all the chiton species studied to date. The karyological results of our study agree with previous molecular data indicating N. mirandus as a sister taxon of Acanthochitona crinita. The karyotypes of the two species could be related as a result of Robertsonian rearrangements. According to the more parsimonious hypothesis, the former would be the primitive karyotype, although other evolutionary events cannot be ruled out.

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, 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 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, two pairs in V. atra and absent in V. hugyi.

The 5S rRNA genes of Gasterosteus aculeatus were characterised. Genomic DNA was amplified by PCR using two primers constructed in different salmonid species in order both to extract the total 5S rDNA (coding region + nontranscribed spacer region) and to generate amplified DNA products only if 5S rDNAs were tandemly arrayed. Amplification revealed four bands (A, B, C, and D) of about 250, 370, 580, and 660 base pairs, respectively. The electrophoretic pattern yielded by PCR products appeared very similar in the various species examined, it was also present in a selachian, an amphibian and a reptile. However, differences were observed in the size of the bands. Band A was cloned and se‐quenced: it consisted of 225 base pairs (A+T = 48.6%; G+C = 51.4%) and showed a high sequence homology to 5S rDNA in many vertebrate species. Hybridization on nylon filters of genomic DNA digested with several restriction enzymes showed the typical tandem array of repeated DNAs.

A comparison of chromosome morphology of seventeen species of Boophis (Mantellidae, Boophinae) was carried out applying Giemsa, C‐, DAPI‐, CMA3and AgNOR‐ banding methods. All of the species studied showed a karyotype of 2n = 26 biarmed chromosomes. The nucleolar organizer regions (NORs) were interstitially located in a secondary constriction on the long arm of the 6th chromosome pair in all species, except B. cf. rufioculis and B. cf. madagascariensis in which the NORs were dislocated on the short arms of the 6th pair. Heterochromatin distribution and composition was very variable among the species, displaying C‐bands positive or negative both to DAPI and CMAj, or positive only at one of the two fluorochromes. Taxonomic and phylogenetic implications of these results are discussed.