Massimo Giovannotti

Mitochondrial DNA sequence analysis reveals multiple Pleistocene glacial refugia for Podarcis muralis (Laurenti, 1768) in the Italian Peninsula

Abstract The genetic variability of the wall lizard, Podarcis muralis, was analysed in that part of its range regarded as its area of origin and also its expansion centre. The analysis was performed on partial cytochrome b sequences (405 bp) of 117 specimens representing 31 sampling locations from southern, central and northern Italy. The results of this study revealed 30 haplotypes in the Italian sample belonging to three main genetic lineages. The genetic divergence among haplotypes ranged from 0.2 to 6.6%, whereas the estimation of divergence times among mtDNA lineages highlighted a complex pattern of genetic differentiation with both Pleistocene and Pliocene divergences (3.2–0.7 Ma). These results put in evidence the role played by the pre-Pleistocene and Pleistocene palaeogeographic and palaeoclimatic events in shaping the genetic diversity of the wall lizard in Italy and confirm the role of this peninsula as glacial refuge. In addition, AMOVA analysis revealed the presence of geographically structured clades indicating the occurrence of multiple glacial refugia in Italy, thus supporting a refugia-within-refugia scenario for the wall lizard in Italy. Finally, the results of this study seem to indicate a lack of congruence between the four morphological subspecies currently recognised in the Italian Peninsula and the main genetic lineages detected.

Evolution of digit identity in the three-toed Italian skink Chalcides chalcides: a new case of digit identity frame shift

SUMMARY Digit identity in the avian wing is a classical example of conflicting anatomical and embryological evidence regarding digit homology. Anatomical in conjunction with phylogenetic evidence supports the hypothesis that the three remaining digits in the bird wing are digits 1, 2, and 3. At the same time, various lines of embryological evidence support the notion that these digits develop in positions that normally produce digits 2, 3, and 4. In recent years, gene expression as well as experimental evidence was published that supports the hypothesis that this discrepancy arose from a digit identity shift in the evolution of the bird wing. A similar but less well‐known controversy has been ongoing since the late 19th century regarding the identity of the digits of the three‐toed Italian skink, Chalcides chalcides. Comparative anatomy identifies these digits as 1, 2, and 3, while embryological evidence suggests their derivation from embryological positions 2, 3, and 4. Here we re‐examine this evidence and add gene expression data to determine the identity of the three digits of C. chalcides. The data confirm that the adult and the embryological evidence for digit identity are in conflict, and the expression of Hoxd11 suggests that digits 1, 2, and 3 develop in positions 2, 3, and 4. We conclude that in C. chalcides, and likely in its close relatives, a digit identity frame shift has occurred, similar to the one in avian evolution. This result suggests that changes in of digit identity might be a more frequent consequence of digit reduction than previously assumed.

Phylogeographic inferences on the native brown trout mtDNA variation in central Italy

Genetic diversity was analysed in Salmo trutta populations living in an area of central Italy by sequencing 310 bp of the 5′ end of the mtDNA control region (D‐loop) and by Restriction Fragment Length Polymorphism (RFLP) analysis of three mtDNA segments. Data show a genetic structure profoundly altered by stockings with allochthonous material of Atlantic origin. In fact, 15 of the RFLP haplotypes detected are linked to an Atlantic sequence. The remaining 9 were instead coupled with sequences representing the three major phylogenetic lineages previously identified in the Mediterranean basin (Adriatic, marmoratus, and Mediterranean), representing the native genetic diversity of brown trout in that area. The close genetic affinity observed between these latter haplotypes and those found in the Balkan peninsula by other authors seems to be in accordance with a recent, natural, history of dispersion between the two borders of the Adriatic Sea. These results appear significant from a conservation point of view as, in spite of the massive stocking with hatchery‐reared specimens, they highlight the persistence of Salmo trutta native genetic diversity in central Italy.

Chromosomal evolution in Gekkonidae. I. Chromosome painting between Gekko and Hemidactylus species reveals phylogenetic relationships within the group.

Geckos are a large group of lizards characterized by a rich variety of species, different modes of sex determination and diverse karyotypes. In spite of many unresolved questions on lizards’ phylogeny and taxonomy, the karyotypes of most geckos have been studied by conventional cytogenetic methods only. We used flow-sorted chromosome-specific painting probes of Japanese gecko (Gekko japonicus), Mediterranean house gecko (Hemidactylus turcicus) and flat-tailed house gecko (Hemidactylus platyurus) to reveal homologous regions and to study karyotype evolution in seven gecko species (Gekko gecko, G. japonicus, G. ulikovskii, G. vittatus, Hemidactylus frenatus, H. platyurus and H. turcicus). Generally, the karyotypes of geckos were found to be conserved, but we revealed some characteristic rearrangements including both fissions and fusions in Hemidactylus. The karyotype of H. platyurus contained a heteromorphic pair in all female individuals, where one of the homologues had a terminal DAPI-negative and C-positive heterochromatic block that might indicate a putative sex chromosome. Among two male individuals studied, only one carried such a polymorphism, and the second one had none, suggesting a possible ZZ/ZW sex determination in some populations of this species. We found that all Gekko species have retained the putative ancestral karyotype, whilst the fission of the largest ancestral chromosome occurred in the ancestor of modern Hemidactylus species. Three common fissions occurred in the ancestor of Mediterranean house and flat-tailed house geckos, suggesting their sister group relationships. PCR-assisted mapping on flow-sorted chromosome libraries with conserved DMRT1 gene primers in G. japonicus indicates the localization of DMRT1 gene on chromosome 6.

Genetic characterization of loggerhead turtle ( Caretta caretta ) individuals stranded and caught as bycatch from the North-Central Adriatic Sea

The North Adriatic Sea is considered a critical feeding and developmental area for Mediterranean loggerhead turtles. In this study, a comparative analysis of mitochondrial DNA control region sequences was carried out on sixty-five loggerhead individuals stranded and caught as bycatch in the Adriatic Sea from 1999 to 2002. We demonstrated the existence of genetic relationships between the North-Central Adriatic (NCA) aggregate and the Central-Eastern Mediterranean rookeries. Short-range and long-range migrations towards the Adriatic Sea were hypothesized by the finding of individuals bearing haplotypes endemic to the nesting populations of Greece and Turkey. The occurrence of individuals belonging to dimensional classes from juveniles to mature adults strongly support both the feeding and developmental role of the NCA.

Chromosomal study of native and hatchery trouts from Italy (Salmo trutta complex, Salmonidae): conventional and FISH analysis

A cytogenetic analysis was carried out using conventional staining, banding techniques and fluorescence in situ hybridization (FISH) in Italian populations of brown trout (Salmo truttacomplex). All individuals analysed, belonging to the Atlantic (At), Marmoratus (Ma), Adriatic (Ad) and Mediterranean (Me) lineages, showed remarkable karyotype uniformity, with diploid complement of 2n = 80 chromosomes, arm number (NF) of 102 and invariable karyotype composition. Such uniformity was also observed with respect to the location of 5S rDNA and the active, i.e. silver-positive NOR sites. On the contrary, FISH with 28S ribosomal probe and fluorescent staining with CMA3 revealed that inactive NOR sites are more numerous in Ad and Me than in At and Ma lineages. A centromeric sequence was successfully isolated from Salmo trutta individuals by polymerase chain reaction (PCR)-based cloning, using primers designed from published Atlantic salmon (Salmo salar) satellite DNA sequences. This sequence had high AT content (65.3%) and short consensus motif (A/T)(G/C)AAA(T/C) similar to other centromeric satellite repeats. The isolated satellite DNA clones were localized with FISH in the centromeric regions of the brown trout chromosomes, showing lineage-specific patterns. Because it is well known that AT-rich sequences can induce a pronounced DNA curvature, which in turn would promote faster and higher chromatin spiralization, it may be hypothesised that the wide distribution of this satellite in the S. trutta genome may have played a role in its karyotype stability. The presence of this sequence in other salmonid species was also tested by Southern blot hybridization and used to analyze its evolution within salmonids.

Evolutionary dynamics of Anolis sex chromosomes revealed by sequencing of flow sorting-derived microchromosome-specific DNA.

Squamate reptiles show a striking diversity in modes of sex determination, including both genetic (XY or ZW) and temperature-dependent sex determination systems. The genomes of only a handful of species have been sequenced, analyzed and assembled including the genome of Anolis carolinensis. Despite a high genome coverage, only macrochromosomes of A. carolinensis were assembled whereas the content of most microchromosomes remained unclear. Most of the Anolis species have homomorphic XY sex chromosome system. However, some species have large heteromorphic XY chromosomes (e.g., A. sagrei) and even multiple sex chromosomes systems (e.g. A. pogus), that were shown to be derived from fusions of the ancestral XY with microautosomes. We applied next generation sequencing of flow sorting-derived chromosome-specific DNA pools to characterize the content and composition of microchromosomes in A. carolinensis and A. sagrei. Comparative analysis of sequenced chromosome-specific DNA pools revealed that the A. sagrei XY sex chromosomes contain regions homologous to several microautosomes of A. carolinensis. We suggest that the sex chromosomes of A. sagrei are derived by fusions of the ancestral sex chromosome with three microautosomes and subsequent loss of some genetic content on the Y chromosome.

Comparative Chromosome Painting and NOR Distribution Suggest a Complex Hybrid Origin of Triploid Lepidodactylus lugubris (Gekkonidae).

Parthenogenesis, unisexuality and triploidy are interesting but poorly studied phenomena occurring in some reptile species. The mourning gecko (Lepidodactylus lugubris) represents a complex of diploid and triploid parthenogenetic mostly all-female populations (males occur quite rarely) widely distributed in coastal areas of the Indian and Pacific Oceans. Here, we study karyotypes of a male and two female L. lugubris (LLU) triploid individuals (3n = 66) using comparative painting with Gekko japonicus, Hemidactylus turcicus and H. platyurus chromosome specific probes to visualize the homologous regions and to reveal genus specific rearrangements. Also, we applied a 28S ribosomal DNA probe and Ag-staining to detect nucleolus organizer regions (NORs). Our results suggest that the karyotype of L. lugubris underwent a chromosome fission and a fusion after its divergence from a common ancestor of the Gekko-Hemidactylus group. The NORs were found to be located on one out of three homologs on each of LLU8, LLU15 and LLU18, thus further confirming a hybrid origin of triploid individuals. It seems that three different bisexual populations might have contributed to the origin of this triploid parthenogenetic population. We postulate that the heterozygosity in NOR localization is maintained in the triploid clone studied by the absence of recombination as described in whiptail lizards. The pattern of NOR localizations and homologous regions in males and females, as well as the absence of other detectable karyotypic differences, suggest that males arise spontaneously in all female populations and do not arise from independent hybridizations with different species.

Isolation and Characterization of Two Satellite DNAs in some Iberian Rock Lizards (Squamata, Lacertidae)

Satellite DNAs represent a large portion of all high eukaryotic genomes. They consist of numerous very similar repeated sequences, tandemly arranged in large clusters up to 100 million base pairs in length, usually located in the heterochromatic parts of chromosomes. The biological significance of satDNAs is still under discussion, but most of their proposed functions are related to heterochromatin and/or centromere formation and function. Because information about the structure of reptilian satDNA is far from exhaustive, we present a molecular and cytogenetic characterization of two satDNA families in four lacertid species. Two families of tandemly repeated DNAs, namely TaqI and HindIII satDNAs, have been cloned and sequenced from four species belonging to the genus Iberolacerta. These satDNAs are characterized by a monomer length of 171-188 and 170-172 bp, and by an AT content of 60.5% and 58.1%, respectively. FISH experiments with TaqI satDNA probe produced bright signals in pericentromeric regions of a subset of chromosomes whereas all the centromeres were marked by HindIII probe. The results obtained in this study suggest that chromosome location and abundance of satDNAs influence the evolution of these elements, with centromeric families evolving tenfold faster than interstitial/pericentromeric ones. Such different rates render different satellites useful for phylogenetic investigation at different taxonomic ranks.

Evolutionary dynamics of two satellite DNA families in rock lizards of the genus Iberolacerta (Squamata, Lacertidae): different histories but common traits.

Satellite DNAs compose a large portion of all higher eukaryotic genomes. The turnover of these highly repetitive sequences is an important element in genome organization and evolution. However, information about the structure and dynamics of reptilian satellite DNA is still scarce. Two satellite DNA families, HindIII and TaqI, have been previously characterized in four species of the genus Iberolacerta. These families showed different chromosomal locations, abundances, and evolutionary rates. Here, we extend the study of both satellite DNAs (satDNAs) to the remaining Iberolacerta species, with the aim to investigate the patterns of variability and factors influencing the evolution of these repetitive sequences. Our results revealed disparate patterns but also common traits in the evolutionary histories of these satellite families: (i) each satellite DNA is made up of a library of monomer variants or subfamilies shared by related species; (ii) species-specific profiles of satellite repeats are shaped by expansions and/or contractions of different variants from the library; (iii) different turnover rates, even among closely related species, result in great differences in overall sequence homogeneity and in concerted or non-concerted evolution patterns, which may not reflect the phylogenetic relationships among taxa. Contrasting turnover rates are possibly related to genomic constraints such as karyotype architecture and the interspersed organization of diverging repeat variants in satellite arrays. Moreover, rapid changes in copy number, especially in the centromeric HindIII satDNA, may have been associated with chromosomal rearrangements and even contributed to speciation within Iberolacerta.