Sex chromosome diversification in the smooth snake Coronella austriaca (Reptilia, Serpentes)

The smooth snake Coronella austriaca is a widespread Palearctic colubrid species. The species has been the subject of several molecular and phylogeographic studies which highlighted the occurrence of distinct genetic lineages in different areas of the species distribution, but scarce cytogenetic data are currently available on the species. In this paper we present a molecular and karyological study performed with several banding, staining methods and NOR-FISH on samples of C. austriaca from different geographical areas (Italy and Greece) of the species distribution. The molecular and phylogenetic analysis unambiguously placed the studied samples in different clades with a clear geographical pattern. The karyotype of the two female samples studied was composed of 2n = 36 chromosomes with 16 macro- and 20 microchromosomes and a mix of plesiomorphic and derivate chromosome features. All macrochromosomes were biarmed with the exception of pair 5 that was telocentric. NORs were detected on a microchromosome pair. In both females, the pair 4 was heteromorphic (and completely heterochromatic after C-banding in the Italian female), representing the first report of a ZZ/ZW sex chromosome system with female heterogamety in C. austriaca. In addition, the W chromosome showed a different morphology between the two female studied (submetacentric and subtelocentric), highlighting the occurrence of a chromosomal diversification among distinct geographical areas of the species distribution and further supporting that the species contains different diverging evolutionary clades

First Insights on the Karyotype Diversification of the Endemic Malagasy Leaf-Toed Geckos (Squamata: Gekkonidae: Uroplatus)

We provide here the first karyotype description of eight Uroplatus species and a characterization of their chromosomal diversity. We performed a molecular taxonomic assessment of several Uroplatus samples using the mitochondrial 12S marker and a comparative cytogenetic analysis with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, +Chromomycin A3 (CMA3), +4',6-diamidino-2-phenylindole (DAPI). We found chromosomal variability in terms of chromosome number (2n = 34-38), heterochromatin composition and number and localization of loci or Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair). Chromosome morphology is almost constant, with karyotypes composed of acrocentric chromosomes, gradually decreasing in length. C-banding evidenced a general low content of heterochromatin, mostly localized on pericentromeric and telomeric regions. Centromeric bands varied among the species studied, resulting in CMA3 positive and DAPI negative or positive to both fluorochromes. We also provide evidence of a first putative heteromorphic sex chromosome system in the genus. In fact, in U. alluaudi the 10th pair was highly heteromorphic, with a metacentric, largely heterochromatic W chromosome, which was much bigger than the Z. We propose an evolutionary scenario of chromosome reduction from 2n = 38 to 2n = 34, by means of translocations of microchromosomes on larger chromosomes (often involving the NOR-bearing microchromosomes). Adding our data to those available from the literature, we show that similar processes characterized the evolutionary radiation of a larger gecko clade. Finally, we hypothesize that sex chromosome diversification occurred independently in different genera

Evolutionary and Genomic Diversity of True Polyploidy in Tetrapods

: True polyploid organisms have more than two chromosome sets in their somatic and germline cells. Polyploidy is a major evolutionary force and has played a significant role in the early genomic evolution of plants, different invertebrate taxa, chordates, and teleosts. However, the contribution of polyploidy to the generation of new genomic, ecological, and species diversity in tetrapods has traditionally been underestimated. Indeed, polyploidy represents an important pathway of genomic evolution, occurring in most higher-taxa tetrapods and displaying a variety of different forms, genomic configurations, and biological implications. Herein, we report and discuss the available information on the different origins and evolutionary and ecological significance of true polyploidy in tetrapods. Among the main tetrapod lineages, modern amphibians have an unparalleled diversity of polyploids and, until recently, they were considered to be the only vertebrates with closely related diploid and polyploid bisexual species or populations. In reptiles, polyploidy was thought to be restricted to squamates and associated with parthenogenesis. In birds and mammals, true polyploidy has generally been considered absent (non-tolerated). These views are being changed due to an accumulation of new data, and the impact as well as the different evolutionary and ecological implications of polyploidy in tetrapods, deserve a broader evaluation

Chromosome Diversity and Evolution of the Endemic Malagasy Velvet Geckos of the Genus Blaesodactylus (Reptilia, Gekkonidae)

: We performed a molecular and phylogenetic analysis and a comparative cytogenetic study with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, + fluorochromes on several Blaesodactylus samples. The phylogenetic inference retrieved two main clades, the first comprises B. victori, B. microtuberculatus and B. boivini, while the second includes B. sakalava, B. antongilensis and B. ambonihazo. The available samples of B. sakalava form two different clades (here named B. sakalava clade A and clade B), which probably deserve a taxonomic re-evaluation. We found a karyological variability in Blaesodactylus in terms of chromosome number (2n = 40-42), morphology, location of NORs, and heterochromatin distribution pattern. Blaesodactylus antongilensis and B. sakalava clade A and B showed a karyotype of 2n = 40 mostly telocentric chromosomes. Pairs 1 and 6 were metacentric in B. sakalava clade A and B, while pair 1 was composed of subtelocentric/submetacentric elements in B. antongilensis. In contrast, B. boivini displayed a karyotype with 2n = 42 only telocentric chromosomes. NORs were on the first chromosome pair in B. boivini, and on the second pair in B. antongilensis. Adding our data to those available from the literature on evolutionarily related species, we highlight that the chromosome diversification in the genus probably proceeded towards a progressive reduction in the chromosome number and the formation of metacentric elements

Karyotype, chromosome structure, reproductive modalities of three Southern Eurasian populations of the common lacertid lizard, Zootoca vivipara (Jacquin, 1787)

According to a hypothesis of the evolution of viviparity the lacertid lizard Zootoca vivipara, rare relict oviparous populations of the species might occur in southern-eastern part of its distribution area. Such a hypothesis has been verified by comparing the karyotype, chromosome structure, and reproductive modality of three populations of south-eastern part of Russia, including Altai and neighbouring regions, where small territories remained isolated during the Pleistocene cooling and where Pleistocenic fossils of Z. vivipara have been found. The chromosomal study was carried out by conventional staining method and banding methods, namely C-banding and sequential staining of C-banding+ fluorochromes, CMA3 and DAPI. All studied females displayed viviparous reproductive modality and showed a karyotype of 2N = 35 acrocentric chromosomes, with a Z1Z2W sex chromosome system. Chromosome W was subtelocentric. No inter-population variability on karyotype and heterochromatin distribution and composition was observed. From the obtained data the three studied south-eastern Russian viviparous populations belong to the Russian viviparous form of Z. v. vivipara

Comparative cytogenetics on Zamenis lineatus and Elaphe quatuorlineata (Serpentes: Colubridae)

Because of their peculiar genomic and chromosomal characteristics, reptiles are extraordinary model organisms to study karyotype and sex chromosome evolution, but despite the growing interest in their evolutionary cytogenetics, only a small fraction of species have a known karyotype. We performed a comparative cytogenetic analysis on Elaphe quatuorlineata and Zamenis lineatus, using classic and molecular techniques. We provide the karyotype of these two species and an assessment of their chromosomal features. Chromosome analysis was performed with standard karyotyping, C-banding, sequential C-banding + CMA3 + DAPI and Ag-NOR staining. On E. quatuorlineata, we also performed CMA3-methyl green staining and Fluorescence in situ Hybridization mapping NOR loci (NOR-FISH). Elaphe quatuorlineata and Z. lineatus show a very similar karyotype of 2n = 36, with 8 macro- and 10 microchromosome pairs, but differ in the morphology of the pair 8, which resulted submetacentric in the former and metacentric in the latter species. By comparing our data to those available from the literature on congeneric species, we analysed the occurrence of primitive and derivate chromosomal characters and provide cytotaxonomic insights, which further support the species status of Z. lineatus. In both species, the 4th pair was identified as the sex chromosome pair (ZZ/ZW) and NORs were localized on a microchromosome pair. We finally highlight in both genera Elaphe and Zamenis different stages of heterochromatinization of the W chromosome, in agreement with the progressive diversification model of sex chromosome as already shown in different reptile taxa

Chromosome analysis on Central and Southern Italy population of the common toad, Bufo bufo (Amphibia, Anura)

Amphibians constitute a very good model to explore the historical aspects of species distributions due to their low dispersal capacity and low individual vagility. Bufonidae are one of the most speciose family of Anura, including taxa, such as Bufo bufo, widespread in Eurasian regions. We performed a karyological study with standard and sequential C-banding + fluorochromes (Chromomycin A3 (CMA) and Diamidinophenylindole (DAPI) on several tadpoles from different populations of Central and Southern Italy. All the examined tadpoles exhibited the standard Bufokaryotype of 2n = 22 biarmed chromosomes, with the first six pairs larger than the other five (7 - 11) pairs and NOR associated heterochromatin distal on the long arms of the 6thchromosome pair, that was also the only chromosome CMA-positive region. C-banding evidenced centromeric heterochromatin, DAPI positive, on all the chromosomes in all the studied populations from Central Italy. The Southern Italy populations differed in additional paracentromeric C-bands on the short arms of chromosomes 1, 3 and 5. These results support the partition of Central populations of B. bufo from the Southern ones, as evidenced also from molecular phylogenetic studie

Characterization of Two Transposable Elements and an Ultra-Conserved Element Isolated in the Genome of Zootoca vivipara (Squamata, Lacertidae)

: Transposable elements (TEs) constitute a considerable fraction of eukaryote genomes representing a major source of genetic variability. We describe two DNA sequences isolated in the lizard Zootoca vivipara, here named Zv516 and Zv817. Both sequences are single-copy nuclear sequences, including a truncation of two transposable elements (TEs), SINE Squam1 in Zv516 and a Tc1/Mariner-like DNA transposon in Zv817. FISH analyses with Zv516 showed the occurrence of interspersed signals of the SINE Squam1 sequence on all chromosomes of Z. vivipara and quantitative dot blot indicated that this TE is present with about 4700 copies in the Z. vivipara genome. FISH and dot blot with Zv817 did not produce clear hybridization signals. Bioinformatic analysis showed the presence of active SINE Squam 1 copies in the genome of different lacertids, in different mRNAs, and intronic and coding regions of various genes. The Tc1/Mariner-like DNA transposon occurs in all reptiles, excluding Sphenodon and Archosauria. Zv817 includes a trait of 284 bp, representing an amniote ultra-conserved element (UCE). Using amniote UCE homologous sequences from available whole genome sequences of major amniote taxonomic groups, we performed a phylogenetic analysis which retrieved Prototheria as the sister group of Metatheria and Eutheria. Within diapsids, Testudines are the sister group to Aves + Crocodylia (Archosauria), and Sphenodon is the sister group to Squamata. Furthermore, large trait regions flanking the UCE are conserved at family level

Haplotype characterization of a stranded common minke whale calf (Balaenoptera acutorostrata lacépède, 1804): Is the mediterranean sea a potential calving or nursery ground for the species?

The stranding of a suckling calf of Common Minke Whale (Balaenoptera acutorostrata) on the coast near Salerno (Campania, Southern Italy) is reported. The molecular analysis of a partial sequence of the mitochondrial DNA control region shows that the animal bore a haplotype identical to haplotype Ba169 considered as typical of individuals from North Atlantic population. Historical data and our results suggest the possibility that the Mediterranean Sea might be a potential calving or nursery ground for this species

Helix straminea Briganti, 1825 in Italy (Gastropoda: Pulmonata): taxonomic history, morphology, biology, distribution and phylogeny

The land snail taxon Helix straminea Briganti, 1825 has been reintroduced as a valid species in 2014. We provide here a comprehensive account of its taxonomy, distribution, anatomy, phylogeny and karyology in Italy. An overview of the historical views on the validity of the species is presented and faunistic data are reviewed and implemented with new records from Campania and Basilicata. A lectotype is fixed for H. straminea from the syntypes stored in the Muséum d'Histoire Naturelle of Genève, as well as for three other taxa (Helix straminiformis Bourguignat, 1876, Helix yleobia Bourguignat, 1883 and Helix straminea ssp. elongata Bourguignat, 1860). Genital system, radula and karyotype are described for the first time. Molecular analysis of two mitochondrial genes combining GenBank data and the new sequences presented in this paper showed no differentiation between the northern and southern Italian populations. The conservation status of the species and its possible threats are discussed