Karyotype Diversification and Chromosome Rearrangements in Squamate Reptiles.

Karyotype diversification represents an important, yet poorly understood, driver of evolution. Squamate reptiles are characterized by a high taxonomic diversity which is reflected at the karyotype level in terms of general structure, chromosome number and morphology, and insurgence of differentiated simple or multiple-sex-chromosome systems with either male or female heterogamety. The potential of squamate reptiles as unique model organisms in evolutionary cytogenetics has been recognised in recent years in several studies, which have provided novel insights into the chromosome evolutionary dynamics of different taxonomic groups. Here, we review and summarize the resulting complex, but promising, general picture from a systematic perspective, mapping some of the main squamate karyological characteristics onto their phylogenetic relationships. We highlight how all the major categories of balanced chromosome rearrangements contributed to the karyotype evolution in different taxonomic groups. We show that distinct karyotype evolutionary trends may occur, and coexist, with different frequencies in different clades. Finally, in light of the known squamate chromosome diversity and recent research advances, we discuss traditional and novel hypotheses on karyotype evolution and propose a scenario of circular karyotype evolution.

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.

Life History Traits and Longevity of the Invasive Asian Common Toad Duttaphrynus melanostictus (Schneider, 1799) in Madagascar.

We analyzed the body length, age structure, and age at sexual maturity of the invasive Asian common toad Duttaphrynus melanostictus from different sites in Toamasina, east Madagascar. We used skeletochronology as a proxy for age estimation, while gonads were histologically analyzed to determine the age of sexual maturity. The analysis of pooled age data from three sites investigated in 2016 showed that both sexes were larger, although not older, than those of native populations. For the individuals from Madagascar, the males were significantly smaller and younger (mean ± SD, SVL: 71.4 ± 1.6 mm; age: 1.8 ± 0.7 years) than the females (SVL: 78.42 ± 1.9 mm; age: 2.7 ± 1.3 years), when the data were pooled, but when the data were analyzed separately for each of the three sites, similar results were obtained only for one site. The oldest recorded male and female were 3 and 6 years old, respectively. Gonadal histology showed that the males and females reach sexual maturity after the first and second years of age, respectively. Further studies are needed to understand if the larger size and faster growth rates observed in the invasive population of D. melanostictus in Madagascar are a consequence of more favorable environmental conditions with respect to the native range (e.g., the availability of larger trophic niches, a lack of competitors, and lower predatory pressure), and we suggest to extend the monitoring of these life history traits to understand how they might influence the invasion.

Microchromosome fusions underpin convergent evolution of chameleon karyotypes.

Evolutionary shifts in chromosome compositions (karyotypes) are major drivers of lineage and genomic diversification. Fusion of ancestral chromosomes is one hypothesized mechanism for the evolutionary reduction of the total chromosome number, a frequently implied karyotypic shift. Empirical tests of this hypothesis require model systems with variable karyotypes, known chromosome features, and a robust phylogeny. Here we used chameleons, diverse lizards with exceptionally variable karyotypes ($2n=20\text{-}62$), to test whether chromosomal fusions explain the repeated evolution of karyotypes with fewer chromosomes than ancestral karyotypes. Using a multidisciplinary approach including cytogenetic analyses and phylogenetic comparative methods, we found that a model of constant loss through time best explained chromosome evolution across the chameleon phylogeny. Next, we tested whether fusions of microchromosomes into macrochromosomes explained these evolutionary losses using generalized linear models. Multiple comparisons supported microchromosome fusions as the predominant agent of evolutionary loss. We further compared our results to various natural history traits and found no correlations. As such, we infer that the tendency of microchromosomes to fuse was a quality of the ancestral chameleon genome and that the genomic predisposition of ancestors is a more substantive predictor of chromosome change than the ecological, physiological, and biogeographical factors involved in their diversification.

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.

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.

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.

Comparative cytogenetics on eight Malagasy Mantellinae (Anura, Mantellidae) and a synthesis of the karyological data on the subfamily.

We performed a molecular and cytogenetic analysis on different Mantellinae species and revised the available chromosomal data on this group to provide an updated assessment of its karyological diversity and evolution. Using a fragment of the mitochondrial 16S rRNA, we performed a molecular taxonomic identification of the samples that were used for cytogenetic analyses. A comparative cytogenetic analysis, with Giemsa's staining, Ag-NOR staining and sequential C-banding + Giemsa + CMA + DAPI was performed on eight species: Gephyromantis sp. Ca19, G.striatus (Vences, Glaw, Andreone, Jesu et Schimmenti, 2002), Mantidactylus (Chonomantis) sp. Ca11, M. (Brygoomantis) alutus (Peracca, 1893), M. (Hylobatrachus) cowanii (Boulenger, 1882), Spinomantispropeaglavei "North" (Methuen et Hewitt, 1913), S.phantasticus (Glaw et Vences, 1997) and S. sp. Ca3. Gephyromantisstriatus, M. (Brygoomantis) alutus and Spinomantispropeaglavei "North" have a karyotype of 2n = 24 chromosomes while the other species show 2n = 26 chromosomes. Among the analysed species we detected differences in the number and position of telocentric elements, location of NOR loci (alternatively on the 6th, 7th or 10th pair) and in the distribution of heterochromatin, which shows species-specific patterns. Merging our data with those previously available, we propose a karyotype of 2n = 26 with all biarmed elements and loci of NORs on the 6th chromosome pair as the ancestral state in the whole family Mantellidae. From this putative ancestral condition, a reduction of chromosome number through similar tandem fusions (from 2n = 26 to 2n = 24) occurred independently in Mantidactylus Boulenger, 1895 (subgenus Brygoomantis Dubois, 1992), Spinomantis Dubois, 1992 and Gephyromantis Methuen, 1920. Similarly, a relocation of NORs, from the putative primitive configuration on the 6th chromosome, occurred independently in Gephyromantis, Blommersia Dubois, 1992, Guibemantis Dubois, 1992, Mantella Boulenger, 1882 and Spinomantis. Chromosome inversions of primitive biarmed elements likely generated a variable number of telocentric elements in Mantellanigricans Guibé, 1978 and a different number of taxa of Gephyromantis (subgenera Duboimantis Glaw et Vences, 2006 and Laurentomantis Dubois, 1980) and Mantidactylus (subgenera Brygoomantis, Chonomantis Glaw et Vences, 1994, Hylobatrachus Laurent, 1943 and Ochthomantis Glaw et Vences, 1994).

Lizards as Model Organisms of Sex Chromosome Evolution: What We Really Know from a Systematic Distribution of Available Data?

Lizards represent unique model organisms in the study of sex determination and sex chromosome evolution. Among tetrapods, they are characterized by an unparalleled diversity of sex determination systems, including temperature-dependent sex determination (TSD) and genetic sex determination (GSD) under either male or female heterogamety. Sex chromosome systems are also extremely variable in lizards. They include simple (XY and ZW) and multiple (X1X2Y and Z1Z2W) sex chromosome systems and encompass all the different hypothesized stages of diversification of heterogametic chromosomes, from homomorphic to heteromorphic and completely heterochromatic sex chromosomes. The co-occurrence of TSD, GSD and different sex chromosome systems also characterizes different lizard taxa, which represent ideal models to study the emergence and the evolutionary drivers of sex reversal and sex chromosome turnover. In this review, we present a synthesis of general genome and karyotype features of non-snakes squamates and discuss the main theories and evidences on the evolution and diversification of their different sex determination and sex chromosome systems. We here provide a systematic assessment of the available data on lizard sex chromosome systems and an overview of the main cytogenetic and molecular methods used for their identification, using a qualitative and quantitative approach.

Chromosome Diversity and Evolution in Helicoide a (Gastropoda: Stylommatophora): A Synthesis from Original and Literature Data.

We performed a molecular and a comparative cytogenetic analysis on different Helicoidea species and a review of all the available chromosome data on the superfamily to provide an updated assessment of its karyological diversity. Standard karyotyping, banding techniques, and Fluorescence in situ hybridization of Nucleolus Organizer Region loci (NOR-FISH) were performed on fifteen species of three families: two Geomitridae, four Hygromiidae and nine Helicidae. The karyotypes of the studied species varied from 2n = 44 to 2n = 60, highlighting a high karyological diversity. NORs were on a single chromosome pair in Cernuella virgata and on multiple pairs in four Helicidae, representing ancestral and derived conditions, respectively. Heterochromatic C-bands were found on pericentromeric regions of few chromosomes, being Q- and 4',6-diamidino-2-phenylindole (DAPI) negative. NOR-associated heterochromatin was C-banding and chromomycin A3 (CMA3) positive. Considering the available karyological evidence on Helicoidea and superimposing the chromosome data gathered from different sources on available phylogenetic inferences, we describe a karyotype of 2n = 60 with all biarmed elements as the ancestral state in the superfamily. From this condition, an accumulation of chromosome translocations led to karyotypes with a lower chromosome number (2n = 50-44). This process occurred independently in different lineages, while an augment of the chromosome number was detectable in Polygyridae. Chromosome inversions were also relevant chromosome rearrangements in Helicoidea, leading to the formation of telocentric elements in karyotypes with a relatively low chromosome count.

Karyological Diversification in the Genus Lyciasalamandra (Urodela: Salamandridae).

We performed the first cytogenetic analysis on five out of the seven species of the genus Lyciasalamandra, including seven subspecies, and representatives of its sister genus Salamandra. All the studied species have a similar karyotype of 2n = 24, mostly composed of biarmed elements. C-bands were observed on all chromosomes, at centromeric, telomeric and interstitial position. We found a peculiar taxon-specific NOR configuration, including either heteromorphic and homomorphic NORs on distinct regions of different chromosomes. Lyciasalamandra a.antalyana and L. helverseni showed two homomorphic NORs (pairs 8 and 2, respectively), while heteromorphic NORs were found in L. billae (pairs 6, 12), L. flavimembris (pairs 2, 12), L. l. luschani (pairs 2, 12), L. l. basoglui (pairs 6, 12), L. l. finikensis (pairs 2, 6) and S. lanzai (pairs 8, 10). Homomorphic NORs with an additional supernumerary site were shown by S. s. salamandra (pairs 2, 8) and S. s. gigliolii (pairs 2, 10). This unexpected highly variable NOR configuration is probably derived from multiple independent NOR translocations and paracentric inversions and correlated to lineage divergence in Lyciasalamandra. These results support the taxonomic validity of the studied taxa and are consistent with a hypothesized scenario of synchronous evolution in the genus.

Karyological and bioinformatic data on the common chameleon Chamaeleo chamaeleon.

The data presented in this paper stand as supplementary information of the associated article "Karyological characterization of the common chameleon (Chamaeleo chamaeleon) provides insights on the evolution and diversification of sex chromosomes in Chamaeleonidae" [1]. This work provides (i) raw experimental data on the karyology of the common chameleon Chamaeleo chamaeleon and (ii) the results of bioinformatic analysis on sex-specific and repeated DNA sequences found in the same species. The karyological information here presented includes traditional staining method (Giemsa staining) and sequential C-banding + fluorochromes performed on Tunisian samples of the species. The sequence data include the alignments of the isolated DNA sequences with homologous sequences found in squamate Short Read Archives (SRAs) and the results of searches in public nucleic acid databases.

Karyological characterization of the common chameleon (Chamaeleo chamaeleon) provides insights on the evolution and diversification of sex chromosomes in Chamaeleonidae.

Chameleons display high karyological diversity in chromosome number (from 2n = 20 to 62), morphology, heterochromatin distribution and location of specific chromosomal markers, making them unique study models in evolutionary cytogenetics. However, most available cytogenetic data are limited to the description of the chromosome number and morphology. Concerning sex chromosomes, our knowledge is limited to ZZ/ZW and Z1Z1Z2Z2/Z1Z2W systems in the genus Furcifer and the isolation of sex-linked, male-specific, sequences in Chamaeleo calyptratus, but the putative XY chromosomes have still to be identified in Chamaeleo and the conservation of male heterogamety in the genus needs confirmation from other species. In this study we performed a molecular and a cytogenetic analysis on C. chamaeleon, using standard, banding methods and molecular cytogenetics to provide a throughout karyological characterization of the species and to identify and locate the putative XY chromosomes. We confirm that the chromosome formula of the species is 2n = 24, with 12 metacentric macrochromosomes, 12 microchromosomes and NORs on the second chromosome pair. Heterochromatin was detected as weak C-bands on centromeric regions, differently from what was previously reported for C. calyptratus. Fluorescence in situ hybridization (FISH) showed the occurrence of interspersed telomeric signals on most macrochromosomes, suggesting that ancient chromosome fusions may have led to a reduction of the chromosome number. Using a combination of molecular and FISH analyses, we proved that male specific Restriction site-Associated DNA sequences (RADseq) isolated in C. calyptratus are conserved in C. chamaeleon and located the putative XY chromosomes on the second chromosome pair. We also identified different transposable elements in the focal taxa, which are highly interspersed on most chromosome pairs.

Isolation and Characterization of Interspersed Repeated Sequences in the Common Lizard, Zootoca vivipara, and Their Conservation in Squamata.

The common lizard (Zootoca vivipara) displays characteristic cytogenetic, reproductive, molecular, and biogeographic variability. This species comprises oviparous and viviparous populations with disjunct distribution and sex chromosome polymorphisms, from simple ZZ/ZW to complex Z1Z1Z2Z2/Z1Z2W systems with different morphologies of the W chromosome. In this study, we used the primers SINE A and SINE B and a newly designed primer pair to (1) obtain information on the presence and distribution of transposable elements (TEs) in 8 squamate families and (2) assess the chromosomal location of SINE Squam elements in Z. vivipara. PCR amplification with SINE A and SINE B produced single or multiple products in different Z. vivipara populations, subsequently used to design the SINE-Zv primers. Using the newly designed SINE-Zv primers, we identified 2 sequences of about 700 and 300 bp (SINE-Zv 700 and SINE-Zv 300) in all the investigated populations of Z. vivipara. Fluorescence in situ hybridizations showed a preferential localization of SINE-Zv sequences in the peritelomeric regions of almost all chromosomes, with the exception of the W. Both sequences contained a distinct segment of SINE Squam2. SINE-Zv 700 appeared to be restricted to Z. vivipara, while SINE-Zv 300 contained a partial Gypsy sequence that is highly conserved among Squamata and showed high identity values (72-93%) with several transcripts from different species. Using the same primers, we also highlighted the presence of another highly conserved Gypsy-like fragment in snakes which displayed significant similarity with the stomatin-like protein 2 of colubrids. Our results suggest that SINEs and the Gypsy-like elements are widely distributed among squamates and may have played an active role in their genomic evolution and differentiation.

Cold-blooded in the Ice Age: "refugia within refugia", inter-and intraspecific biogeographic diversification of European whipsnakes (Squamata, Colubridae, Hierophis).

In this work, we performed a biogeographic analysis with Bayesian binary MCMC (BBM) statistical dispersal-vicariance analysis (S-DIVA) and species distribution models (SDM) on three phylogenetically closely related Mediterranean whipsnakes (Hierophis gemonensis, H. carbonarius, H. viridiflavus), to investigate the pathways of their geographical diversification and locate putative refugial areas in the last glacial maximum (LGM). Our analysis suggests that the diversification processes between the studied species overall followed an east-west route, from eastern Greece to the Iberian Peninsula and continental France, highlighting a significant role of dispersal and vicariance processes at both inter- and intraspecific levels. In particular, the main lineage-splitting events between H. gemonensis, H. carbonarius and H. viridiflavus coincide with two events of vicariance, involving respectively eastern Greece and eastern Italy, and eastern Italy and western Italy, Iberian Peninsula and continental France. SDM analyses highlight the occurrence of multiple putative glacial refugia in the Balkans, Italy and southern France, which represent well the occurrence of distinct "refugia within refugia" in the main Mediterranean peninsulas. Furthermore, our results suggest how these refugia may have played an important role during the Quaternary climatic oscillations in shaping the current haplotype distribution of European whipsnakes.

Brain Gene Expression is Influenced by Incubation Temperature During Leopard Gecko (Eublepharis macularius) Development.

Sexual differentiation (SD) during development results in anatomical, metabolic, and physiological differences that involve not only the gonads, but also a variety of other biological structures, such as the brain, determining differences in morphology, behavior, and response in the breeding season. In many reptiles, whose sex is determined by egg incubation temperature, such as the leopard gecko, Eublepharis macularius, embryos incubated at different temperatures clearly differ in the volume of brain nuclei that modulate behavior. Based on the premise that "the developmental decision of gender does not flow through a single gene", we performed an analysis on E. macularius using three approaches to gain insights into the genes that may be involved in brain SD during the thermosensitive period. Using quantitative RT-PCR, we studied the expression of genes known to be involved in gonadal SD such as WNT4, SOX9, DMRT1, Erα, Erß, GnRH, P450 aromatase, PRL, and PRL-R. Then, further genes putatively involved in sex dimorphic brain differentiation were sought by differential display (DDRT-PCR) and PCR array. Our findings indicate that embryo exposure to different sex determining temperatures induces differential expression of several genes that are involved not only in gonadal differentiation (PRL-R, Wnt4, Erα, Erß, p450 aromatase, and DMRT1), but also in neural differentiation (TN-R, Adora2A, and ASCL1) and metabolic pathways (GP1, RPS15, and NADH12). These data suggest that the brains of SDT reptiles might be dimorphic at birth, thus behavioral experiences in postnatal development would act on a structure already committed to male or female.

Laser microdissection-based analysis of the Y sex chromosome of the Antarctic fish Chionodracohamatus (Notothenioidei, Channichthyidae).

Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodracohamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Chionodracohamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Chionodracohamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action.

A novel satellite DNA isolated in Pecten jacobaeus shows high sequence similarity among molluscs.

The aim of this work is to investigate the sequence conservation and the evolution of repeated DNA in related species. Satellite DNA is a component of eukaryotic genomes and is made up of tandemly repeated sequences. These sequences are affected by high rates of mutation that lead to the occurrence of species-specific satellite DNAs, which are different in terms of both quantity and quality. In this work, a novel repetitive DNA family, named PjHhaI sat, is described in Pecten jacobaeus. The quantitative analyses revealed a different abundance of this element in the molluscan species investigated in agreement with the "library hypothesis" even if, in this case, at a high taxonomic level. In addition, the qualitative analysis demonstrated an astonishing sequence conservation not only among scallops but also in six other molluscan species belonging to three classes. These findings suggest that the PjHhaI sat may be considered as the most ancients of DNA described so far, which remained "frozen" during molluscan evolution. The widespread distribution of this sat DNA in molluscs as well as its long evolutionary preservation open up questions on the functional role of this element. A future challenge might be the identification of proteins or molecules which interact with the PjHhaI sat.

Novel repeated DNAs in the antarctic polyplacophoran Nuttallochiton mirandus (Thiele, 1906).

Within the scope of a project on the characterization of satellite DNAs in polar mollusks, the Antarctic chiton Nuttallochitonmirandus (Thiele, 1906) was analyzed. Two novel families of tandemly repeated DNAs, namely NmH and NmP, are described in their structure and chromosomal localization, and, furthermore, their presence was analyzed in related species. Data reported here display a particular variability in the structural organization of DNA satellites within this species. Processes driving satellite evolution, which are likely responsible for the intriguing variability of the identified satellite DNAs, are discussed.

Karyological evidence for diversification of Italian slow worm populations (Squamata, Anguidae).

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 10(th) 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.