BACKGROUND:Tandemly repeated satellite DNA sequences are an important part of animal genomes. They are involved in chromosome interactions and the maintenance of the integral structure of the nucleus, regulation of chromatin conformation and gene expression, and chromosome condensation and movement during cell division. Satellite DNAs located in the centromeric heterochromatin evolve rapidly and likely affect hybrid fertility and fitness. However, their studies are taxonomically highly biased. In lacertid lizards, satDNA has been extensively studied in the subfamily Lacertinae, but the subfamily Eremiadinae has been largely overlooked. RESULTS:In this work, we describe a novel 177-bp-long centromeric satDNA family EremSat177, which is present in all studied species of the genus Eremias, but not in related genera. EremSat177 is not homologous to any previously identified centromeric satellites. Using fluorescence in situ hybridization, we demonstrate its centromeric localization in E. velox and E. arguta. We also show its tandem organization and intra-genomic homogenization by in silico analysis in the genome of E. argus. The phylogenetic analysis of consensus EremSat177 sequences from 12 Eremias species demonstrates that the same monomer subfamily is the most abundant in all these species, and its evolution mainly follows the species phylogeny as revealed by the mtDNA sequences. CONCLUSION:The EremSat177 represents a novel, lineage-specific centromeric satellite DNA, and its role in centromere functioning should be revealed in further research.

Lacertid and amphisbaenian lizards are two squamate reptile lineages very divergent morphologically. In fact, adaptation to burrowing deeply modified amphisbaenian worm-like body. Lacertids instead have a typical reptile morphology with a long tail and four well-developed limbs. Despite so different in appearance, they are evolutionarily very close as evidenced by molecular phylogenetic approaches. Consistently, a slow-evolving satellite DNA (IMO-TaqI) described in lacertid lizards was isolated from the genome of the amphisbaenian Trogonophis wiegmanni too. Comparison with lacertid repeats evidenced great similarity, highlighting that molecular characters appeared very suitable especially when morphology is subjected to strong selective pressures.

Onopordum tauricum Willd., a species distributed in Eastern Europe, has been the subject of various research endeavors aimed at assessing its suitability for extracting vegetable rennet for use in the production of local cheeses as a substitute for animal-derived rennet. In Italy, the species has an extremely fragmented and localized distribution in six locations scattered across the central-northern Apennines and some areas of southern Italy. In this study, both the morphology and genetic diversity of the six known Italian populations were investigated to detect putative ecotypes. To this end, 33 morphological traits were considered for morphometric measurements, while genetic analysis was conducted on the entire genome using the ddRAD-Seq method. Both analyses revealed significant differences among the Apennine populations (SOL, COL, and VIS) and those from southern Italy (ROT, PES, and LEC). Specifically, the southern Italian populations appear to deviate significantly in some characteristics from the typical form of the species. Therefore, its attribution to O. tauricum is currently uncertain, and further genetic and morphological analyses are underway to ascertain its systematic placement within the genus Onopordum.

The veiled chameleon (Chamaeleo calyptratus) is a typical member of the family Chamaeleonidae and a promising object for comparative cytogenetics and genomics. The karyotype of C. calyptratus differs from the putative ancestral chameleon karyotype (2n = 36) due to a smaller chromosome number (2n = 24) resulting from multiple chromosome fusions. The homomorphic sex chromosomes of an XX/XY system were described recently using male-specific RADseq markers. However, the chromosomal pair carrying these markers was not identified. Here we obtained chromosome-specific DNA libraries of C. calyptratus by chromosome flow sorting that were assigned by FISH and sequenced. Sequence comparison with three squamate reptiles reference genomes revealed the ancestral syntenic regions in the C. calyptratus chromosomes. We demonstrated that reducing the chromosome number in the C. calyptratus karyotype occurred through two fusions between microchromosomes and four fusions between micro-and macrochromosomes. PCR-assisted mapping of a previously described Y-specific marker indicates that chromosome 5 may be the sex chromosome pair. One of the chromosome 5 conserved synteny blocks shares homology with the ancestral pleurodont X chromosome, assuming parallelism in the evolution of sex chromosomes from two basal Iguania clades (pleurodonts and acrodonts). The comparative chromosome map produced here can serve as the foundation for future genome assembly of chameleons and vertebrate-wide comparative genomic studies.

Reptiles show a remarkable diversity of sex determination mechanisms and sex chromosome systems, derived from different autosomal pairs. The origin of the ZW sex chromosomes ofLacerta agilis, a widespread Eurasian lizard species, is a matter of discussion: is it a small macrochromosome from the 11-18 group common to all lacertids, or does this species have a unique ZW pair derived from the large chromosome 5? Using independent molecular cytogenetic methods, we investigated the karyotype ofL. agilis exiguafrom Siberia, Russia, to identify the sex chromosomes. FISH with a flow-sorted chromosome painting probe derived fromL. strigataand specific to chromosomes 13, 14, and Z confirmed that the Z chromosome ofL. agilisis a small macrochromosome, the same as inL. strigata. FISH with the telomeric probe showed an extensive accumulation of the telomere-like repeat in the W chromosome in agreement with previous studies, excluding the possibility that the lineages ofL.agilisstudied in different works could have different sex chromosome systems due to a putative intra-species polymorphism. Our results reinforce the idea of the stability of the sex chromosomes and lack of evidence for sex-chromosome turnovers in known species of Lacertidae.

The sand tiger shark (Carcharias taurus) is a coastal species distributed in temperate and sub-tropical waters, classified as 'Vulnerable' at global level and 'Critically endangered' in eastern Australia, south-western Atlantic Ocean, and Mediterranean Sea. Six populations (north-western Atlantic, Brazil, South Africa, Japan, eastern Australia, and western Australia) with low genetic diversity and limited gene flow were identified worldwide, but genetic information for many other geographic areas are still missing. Specifically, this species is listed in several reports as part of the Mediterranean fauna, even if there has been a lack of catches and sightings in recent years in this basin. To clarify the origin of C. taurus individuals caught in the past in the Mediterranean Sea, historical samples were genetically analysed. Nine samples with certain Mediterranean origin were collected from different European museums. DNA was extracted and 600 bp of the mitochondrial DNA control region were amplified using eight overlapping species-specific primer pairs. Sequences obtained were aligned with all the haplotypes globally known so far. Genetic analysis revealed the misidentification of one museum specimen. Among the remaining Mediterranean historical samples, three different haplotypes were recovered. Two of them previously observed only in South Africa and one described in both South African and Brazilian populations. Results suggest a genetic relationship between Mediterranean sand tiger sharks and those from the western Indian Ocean. According to previous studies, we hypothesized that, during the Pleistocene, the cold Benguela upwelling barrier was temporarily reduced allowing the passage of C. taurus individuals from the Indian to Atlantic Ocean. After the restoration of this phylogeographic barrier some individuals were trapped in the Atlantic Ocean and probably migrated northward colonizing the western African coasts and the Mediterranean Sea.

The brown trout Salmo trutta is characterised by both anadromous (sea trout) and resident populations, naturally occurring in Atlantic and Ponto-Caspian rivers. Sea trout are currently considered absent from rivers of the Mediterranean area, probably because of the non-optimal chemical-physical characteristics of the Mediterranean Sea. However, the occasional bycatch of smoltified S. trutta in the Adriatic Sea is well known among fishermen and the biological explanation of this phenomenon is still controversial. The aim of this study was to compare the genetic diversity of freshwater and marine brown trout to try to understand the factors underlying the presence of putative anadromous brown trout in the Adriatic Sea. In this study, we analysed the genetic diversity of: (1) wild brown trout collected from the Esino River (central Italy); (2) a domestic strain of brown trout used for stocking the study area; and (3) a sample of Adriatic sea trout collected near the outlet of the Esino River. Together with genetic analysis, we carried out scale analysis in order to track the freshwater/marine stages of the life cycle in the sea trout samples. The genetic characterisation was carried out by polymerase chain reaction-restriction fragment length polymorphism analysis of the mtDNA fragment ND-5/6 and the nuclear locus LDH-C1* and by genotyping 15 microsatellite loci. The genetic polymorphism obtained was used to investigate intra- and inter-population genetic diversity, rates of genetic introgression between wild and domestic samples and the origin of sea trout specimens by using assignment tests. Our genetic analyses demonstrated that the sea trout analysed in this study are from the domestic strain of Atlantic origin used in central Italy for stocking activities. The level of genetic introgression between native and domestic samples is high in the Esino River. The populations more resilient to introgressive hybridisation appeared to be those living in the portion of the river network dominated by carbonate rocks. Assignment tests (GeneClass) suggest the existence of a link between stocking efforts and the freshwater origin of the sea trout. In addition, data obtained from the analysis of scales, size measurement, and sex determination showed a pattern of smolt age, size, and sex ratio very similar to those observed in other anadromous populations. In conclusion, the present study highlighted that sea trout from the central Adriatic Sea originated from brown trout of Atlantic origin inhabiting the Esino River. Their seaward migratory behaviour could represent a consequence of an active migration instead of a passive displacement by water flow. Our results also showed that traditional stocking practices represent a negative activity for the conservation of the last Mediterranean native S. trutta populations.

Two satellite DNAs (satDNAs) have been isolated and characterized from three populations of Atlantolacerta andreanskyi. One satDNA (AAN-TaqI) has been isolated here from the first time. It is characterized by a tendency to AT enrichment (AT = 54.2%) and monomer length ranging from 187 to 199 bp. FISH experiments showed that this element occurs in subterminal position on the short arms of all chromosomes of the complement. The analyses of genetic variability of AAN-TaqI showed that the concerted evolution is acting effectively on these repeats that form separate clusters consistent with the geographic origin in the phylogenetic tree, thus supporting the hypothesis that A. andreanskyi would be a species complex. In addition, in the population from Jbel Aoulime this satDNA is already differentiated into two subfamilies. The other satDNA belongs to the family of IMO-TaqI already isolated in other lacertids. Differently from AAN-TaqI, concerted evolution does not seem to act effectively on this element that is not differentiated between populations. These results confirm that IMO-TaqI (AT = 53.4%) is conserved in both chromosomal position and most of its sequence in the lacertids from which it has been characterized so far. Its remarkable evolutionary conservation for about 45 million years could indicate that this satDNA may have a functional role that future investigations could unveil. Once again, this study shows how satDNAs coexisting in the same genome may differ in their evolutionary pattern, even though the reasons underlying this phenomenon in the species here studied have still to be fully understood.

The Apennine chamois (Rupicapra cf. pyrenaica) is a very endangered mountain mammal. At the beginning of the 20th century, only a small population survived in the Abruzzo, Lazio and Molise National Park (Central Italy) and, despite its reintroduction in different Apennine massifs and an increased census size, its genetic variability is the lowest among bottlenecked mammals. The ancient DNA analysis of a skull dated back to approximate to 3000 cal yr BP allowed us to describe a new haplotype belonging to the mitochondrial Central Clade (including Chartreuse and Apennine populations) but never found in extant chamois. This result underlines that the demographic collapse of Apennine populations, which probably started in the Pleistocene, was combined with an ever-increasing genetic erosion in gradually smaller and isolated populations.

Pleurodont lizards are characterized by an ancient system of sex chromosomes. Along with stability of the central component of the system (homologous to the X chromosome of Anolis carolinensis [Dactyloidae], ACAX), in some genera the ancestral sex chromosomes are fused with microautosomes, forming neo-sex chromosomes. The genus Ctenonotus (Dactyloidae) is characterized by multiple X1X1X2X2/X1X2Y sex chromosomes. According to cytogenetic data, the large neo-Y chromosome is formed by fusion of the ancestral Y chromosome with 2 microautosomes (homologous to ACA10 or ACA11 and ACA12), the X1 chromosome is formed by fusion of the ancestral X chromosome with the autosome homologous to ACA10 or ACA11, and the X2 chromosome is homologous to autosome ACA12. To determine more precisely the content and evolution of the Ctenonotus sex chromosomes, we sequenced flow-sorted chromosomes (both sex chromosomes and microautosomes as control) of 2 species with a similar system: C. pogus and C. sabanus. Our results indicate that the translocated part of the X1 is homologous to ACA11, X2 is homologous to ACA12, and the Y contains segments homologous to both ACA11 and ACA12. Molecular divergence estimates suggest that the ancestral X-derived part has completely degenerated in the Y of Ctenonotus, similar to the degeneration of the Norops sagrei Y chromosome (Dactyloidae). The newly added regions show loss of DNA content, but without degeneration of the conserved regions. We hypothesize that the translocation of autosomal blocks onto sex chromosomes facilitated rapid degeneration of the pseudoautosomal region on the ancestral Y.

Mediterranean brown trout is subject to several serious threats such as pollution, water abstraction, habitat alteration and especially genetic introgression with domestic strains used for stocking activities. Despite this latter issue has largely been debated by scientists, local managers and stakeholders for decades, official stocking practices with domestic trout still persists in several countries (Italy included), even if there are laws explicitly prohibiting introduction of organisms of non-local origin. Probably, the last opportunity to conserve native brown trout populations is represented by protected areas. Therefore, in the present study, we aimed to verify the role of the Nature 2000 network and a national park as valid tools to guarantee the survival of native brown trout in the Apennines. Partial mitochondrial DNA control region sequence analysis and genotypingof the locus LDH-C1* and 11 microsatellites were used to investigate the genetic diversity of three rivers from central Italy. For all rivers investigated a temporal analysis of introgression was also carried out. The genetic diversity of three domestic stocks was included in the sampling design for comparison. The main results of this study indicated that: (i) the genetic diversity of brown trout in central Italy is very complex and (ii) its conservation is seriously threatened by genetic introgression phenomena still ongoing. The only samples showing no introgression or a decrease in genetic introgression were those isolated by the presence of natural and/or artificial barriers to fish movements rather than protected by inhabiting rivers withinthe Natura 2000 network. This observation prompts an important reflection on issues concerning fluvial continuity restoration and suggests that barrier removal should be undertaken with caution in order to avoid the concrete risk of domestic trout spreading that could promote additional loss of native brown trout biodiversity.

The introgressive hybridization with the Atlantic trout is one of the main threats to the Mediterranean trout. The LIFE+ TROTA – Trout population RecOvery in central iTAly (LIFE12 NAT/IT/000940) project, started in November 2013 and finished in January 2018, was aimed to detect the presence of residual Mediterranean trout populations in seven watersheds in the Apennine area. Within the project, a series of concrete conservation actions were undertaken in order to preserve these native populations. Based on the genetic, demographic, and ecological characterization, the suitable sites for the alien trout eradication programs and for the re-introduction of Mediterranean trout were selected. The eradication activities were carried out by electrofishing on four watercourses where highly introgressed trout populations occurred; the eradicated sites were isolated from the rest of the basin by the presence of natural and/or human-made weirs that are insurmountable from fish fauna. The removal activities, for a total of 13 eradication interventions for each site, were carried out between December 2014 and July 2017. A total of over 22,000 trout individuals accounting for over 700 kg were removed from the eradicated sites. The percentage of abundances reduction far exceeds the target of the project, whose target was a reduction of 60%. These results confirmed the effectiveness of electrofishing in the eradication programs, especially in watercourses of modest dimensions in terms of flow rates, width and depth, and for populations characterized by modest abundance values.

The native Mediterranean trout populations in central Italy are at risk of extinction. Hybridization with the alien Atlantic trout represents one of the major threats to the species. The main aim of the research was to test the efficiency of morphological characterization in revealing alien trout introduction, pure lineages and local adaptations, in five watersheds of central Apennines (Italy). Data analysis was carried out for a total of 258 specimens already genetically identified. The results of the multivariate analysis, performed using 10 qualitative and 47 quantitative variables, revealed two distinct phenotypic groups and confirmed, at a morphological level, the genetic separation between Atlantic and Mediterranean trout. The morphological variables that play a key role in the discrimination of the Mediterranean trout were: (i) number of parr marks with striped pattern, (ii) adipose fin mainly with white color and without black spots, and (iii) number of black opercular spots, compared to the Atlantic trout. The geometric analysis revealed the importance of the spatial arrangements of spots on the fish flank and the shape of caudal peduncle in discriminating different Mediterranean populations. The information collected confirmed the powerful of morphological surveys in the screening of conservation status of wild trout populations.

Given the lack of genetic information about Mediterranean sea lamprey, taking advantage of recent capture of two individuals in the western Adriatic Sea, molecular analysis of a partial sequence of the mitochondrial DNA control region has been performed on two Mediterranean specimens for the first time. A new haplotype is reported for this species. Furthermore, the finding of both juvenile and adult individuals suggests the presence of suitable rivers for reproduction in the Adriatic region.

Trifonov, VA, Lisachov, AP, Kichigin, IG, Makunin, AI, Pereira, JC, Druzhkova, AS, Ferguson-Smith, MA, & Giovannotti, M. (2018). Evolutionary sex chromosome translocations in amniotes. Comparative Cytogenetics, 12(3), 304-305 … Trifonov, Vladimir A. ; Lisachov, Artem P. ; Kichigin, Ilya G. ; Makunin, Alexey I. ; Pereira, Jorge C. ; Druzhkova, Anna S. ; Ferguson-Smith, Malcolm A. ; Giovannotti, Massimo. / Evolutionary sex chromosome translocations in amniotes. In: Comparative Cytogenetics. 2018 ; Vol. 12, No. 3. pp. 304-305 … Trifonov, VA, Lisachov, AP, Kichigin, IG, Makunin, AI, Pereira, JC, Druzhkova, AS, Ferguson-Smith, MA & Giovannotti, M 2018, 'Evolutionary sex chromosome translocations in amniotes', Comparative Cytogenetics, vol. 12, no. 3, pp. 304-305 … Evolutionary sex chromosome translocations in amniotes. / Trifonov, Vladimir A.; Lisachov, Artem P.; Kichigin, Ilya G.; Makunin, Alexey I.; …

In this study, IMO-TaqI satDNA, previously isolated in several species of Lacertidae, was isolated and characterized from four species of the genus Lacerta and three of the genus Timon. The aim was to gain further insights into the evolutionary dynamics of this satDNA, its occurrence among lacertids and to understand if it plays any role in sex chromosome evolution in these seven species. The results here obtained highlighted the presence of this repetitive element in the genome of all the species investigated, thus indicating that IMO-TaqI satDNA is evolutionary conserved among a wide variety of lacertids. In addition, this element was found to be very abundant in the constitutive heterochromatin of the W-sex chromosome of the four Lacerta species investigated. The occurrence of IMO-TaqI satDNA on Lacerta heterochromosome suggests that it is involved in the differentiation of the W chromosome by heterochromatinization, and the fact that it is absent in the W of other lacertids investigated seems to confirm that repetitive DNA sequences would remain randomly trapped into the sex chromosomes, undergoing amplification as a consequence of the suppression of recombination.

In the Mediterranean area, one of the major threats to freshwater fish fauna is represented by the introduction of alien species. The bullhead, Cottus gobio, is a species of great conservation interest threatened by the massive introduction of hatchery-reared brown trout, Salmo trutta, for angling purposes. The aims of this research were: i) to present a case-study of interspecific and intraspecific competition in dwelling fish from two Mediterranean streams (central Italy); ii) to evaluate the bullhead population status before and after the alien brown trout removal, and iii) to investigate the influence of the environmental parameters on the abundance of both species. For the bullhead populations the abundances and the young-of-the-year percentage significantly increased after the removal activities. The growth analysis showed that, after the trout removal, bullhead were distinguished by greater performance and larger sizes in all age classes in comparison to the pre-removal period, while the condition factor (Kn) showed no differences, suggesting that the effects of trout population control acted mainly through predator-prey dynamics. An inverse correlation between the abundance of bullhead and its body condition suggested that the beneficial effects due to the reduction of predation could be compensated by the intraspecific competition due to the increase of the bullhead population density.

The Mediterranean brown trout (Salmo trutta complex) is one of the freshwater fish species complex at greater risk of extinction in the Mediterranean area. The introduction of alien invasive species and their interaction with the native fauna represent some of the major threats to the survival of this species. Currently, the genetic variability of the Mediterranean trout (Salmo trutta complex) is being compromised by the introgressive hybridization with the Atlantic trout (Salmo trutta Linnaeus, 1758). Therefore, it is necessary to gain further knowledge on genetic and demographic characteristics of Mediterranean trout populations, that, combined with environmental and angling data, will allow to undertake sound conservation strategies. The aims of the present study were to analyze: i) the demographic characteristics of the Mediterranean trout in seven central Apennine river basins where native populations are threatened by stocking with the domestic trout of north Atlantic origin; ii) the influence of both environmental parameters and different fishery management strategies on the status of native trout populations. The project focuses on 14 sites included in the Natura 2000 EU wide network of nature protection areas, established under the 1992 Habitat Directive. A total of 25 watercourses were investigated for a total of 32 sampling sites. A census of the fish fauna was carried out by electrofishing at each sampling location. Fish and environmental data were collected in spring and autumn 2014. The results obtained in the present research allowed us to detect the presence of three residual Mediterranean trout populations with a high degree of genetic integrity in the study area. The native trout populations were characterized by higher abundances and higher adult density values. Well-structured native populations with higher legal-size specimens density and poor body conditions in terms of relative weight were observed in the no-fishing areas, probably due to the presence of intraspecific competition phenomena. The environmental analysis confirmed the key role played by the geological characteristics of the central Apennines in shaping the introgression levels observed in the wild trout populations, while the recovery of the water quality appears decisive in the development of management strategies aimed at their conservation.

Supernumerary elements of the genome are often called B chromosomes. They usually consist of various autosomal sequences and, because of low selective pressure, are mostly pseudogenized and contain many repeats. There are numerous reports on B chromosomes in mammals, fish, invertebrates, plants, and fungi, but only a few of them have been studied using sequencing techniques. However, reptilian supernumerary chromosomes have been detected only cytogenetically and never sequenced or analyzed at the molecular level. One model squamate species with available genome sequence is Anolis carolinensis. The scope of the present article is to describe the genetic content of A. carolinensis supernumerary chromosomes. In this article, we confirm the presence of B chromosomes in this species by reverse painting and synaptonemal complex analysis. We applied low-pass high-throughput sequencing to analyze flow-sorted B chromosomes. Anole B chromosomes exhibit similar traits to other supernumerary chromosomes from different taxons: they contain two genes related to cell division control (INCENP and SPIRE2), are enriched in specific repeats, and show a high degree of pseudogenization. Therefore, the present study confirms that reptilian B chromosomes resemble supernumerary chromosomes of other taxons.