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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 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.

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 genotyping of 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 within the 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.

Acanthodactylus lineomaculatus is now regarded as an ecotype of A. erythrurus with which it has been recently synonymized. Despite the wide range of A. erythrurus, karyological data for this species are scarce and limited to classical cytogenetic studies carried out in individuals from only 2 locations (central Spain and Spanish enclave of Melilla on the northwestern Mediterranean Moroccan coast). Here, for the first time, we cytogenetically characterized individuals of A. lineomaculatus from the southwestern Moroccan Atlantic coast with the aim to increase the karyological knowledge of this wide-ranging species and to assess if any chromosomal changes can be found in this ecotype in comparison to other populations of this species. The diploid number of the individuals investigated is 2n = 38 which is typical of most lacertids. Active NORs were located telomerically in a medium-small pair of chromosomes, and no inactive NORs were detected. C-banding revealed an intensely heterochromatic W chromosome composed of AT-rich (centromere and long arm telomeric region) and GC-rich (most of the long arm) regions, with extended interstitial telomeric sequences. These telomere-like repeats occupy the GC-rich heterochromatin of the W. The DNA composition of the W represents a trait distinguishing A. lineomaculatus (southwestern Morocco) from A. erythrurus from Spain that possess a DAPI-positive (AT-rich) W chromosome. In conclusion, these results add further evidence to the remarkable karyotype conservation in lacertid lizards, although differences in NOR location and in W chromosome structure among populations could suggest an incipient speciation mediated by chromosome changes in this wide-ranging lizard species.

Acanthodactylus lineomaculatus is now regarded as an ecotype of A. erythrurus with which it has been recently synonymized. Despite the wide range of A. erythrurus, karyological data for this species are scarce and limited to classical cytogenetic studies carried out in individuals from only 2 locations (central Spain and Spanish enclave of Melilla on the northwestern Mediterranean Moroccan coast). Here, for the first time, we cytogenetically characterized individuals of A. lineomaculatus from the southwestern Moroccan Atlantic coast with the aim to increase the karyological knowledge of this wide-ranging species and to assess if any chromosomal changes can be found in this ecotype in comparison to other populations of this species. The diploid number of the individuals investigated is 2n = 38 which is typical of most lacertids. Active NORs were located telomerically in a medium-small pair of chromosomes, and no inactive NORs were detected. C-banding revealed an intensely heterochromatic W chromosome composed of AT-rich (centromere and long arm telomeric region) and GC-rich (most of the long arm) regions, with extended interstitial telomeric sequences. These telomere-like repeats occupy the GC-rich heterochromatin of the W. The DNA composition of the W represents a trait distinguishing A. lineomaculatus (southwestern Morocco) from A. erythrurus from Spain that possess a DAPI-positive (AT-rich) W chromosome. In conclusion, these results add further evidence to the remarkable karyotype conservation in lacertid lizards, although differences in NOR location and in W chromosome structure among populations could suggest an incipient speciation mediated by chromosome changes in this wide-ranging lizard species.

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.

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. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 13–26, 2014. © 2013 Wiley Periodicals, Inc.

It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (N-e) are much smaller than those expected in a fishes with large population census sizes (N-c). Estimates of N-e indicate low effective population sizes, even before the population collapse. The ratio N-e/N-e ranged between 10(-6) and 10(-8), indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.

The transparent goby (Aphia minuta) is a small progenetic goby that displays a short life cycle with early reproductive maturity and represents a valuable fishery resource harvested by artisanal fleets in most part of its distributional range. This study aimed to define the genetic variability in A. minuta at five sampling sites within the Mediterranean Sea and one in the Atlantic Ocean through the analysis of 11 nuclear microsatellite loci. The results revealed that several genetic diversity estimators (expected and observed heterozygosities, mean number of alleles and allelic richness) were lower in the Atlantic Ocean than in the Mediterranean Sea, suggesting the role of past or current demographic events in shaping this pattern. The genetic structure was investigated using both classical genetic differentiation descriptors and Bayesian approaches, and by defining the current and past migration rates. The results obtained revealed a pronounced genetic structure within the Mediterranean Sea and suggest a very low current migration rate. The pattern of historical migration suggests the possible role of hydrographic boundaries in shaping the genetic structure detected in this species. In addition, the identification of loci under selection suggests the possible implication of selective pressures that are acting on genes connected with the peculiar life cycle of this gobiid fish.

The sustained exploitation of marine populations requires an understanding of a species' adaptive seascape so that populations can track environmental changes from short- and long-term climate cycles and from human development. The analysis of the distributions of genetic markers among populations, together with correlates of life-history and environmental variability, can provide insights into the extent of adaptive variation. Here, we examined genetic variability among populations of mature European anchovies (n = 531) in the Adriatic (13 samples) and Tyrrhenian seas (2 samples) with neutral and putative non-neutral microsatellite loci. These genetic markers failed to confirm the occurrence of two anchovy species in the Adriatic Sea, as previously postulated. However, we found fine-scale population structure in the Adriatic, especially in northern areas, that was associated with four of the 13 environmental variables tested. Geographic gradients in sea temperature, salinity and dissolved oxygen appear to drive adaptive differences in spawning time and early larval development among populations. Resolving adaptive seascapes in Adriatic anchovies provides a means to understand mechanisms underpinning local adaptation and a basis for optimizing exploitation strategies for sustainable harvests.

In this pilot study for the first time, ancient DNA has been extracted from bone remains of Salmo trutta. These samples were from a stratigraphic succession located in a coastal cave of Calabria (southern Italy) inhabited by humans from upper Palaeolithic to historical times. Seven pairs of primers were used to PCR-amplify and sequence from 128 to 410 bp of the mtDNA control region of eleven samples. Three haplotypes were observed: two (ADcs-1 and MEcs-1) already described in rivers from the Italian peninsula; one (ATcs-33) belonging to the southern Atlantic clade of the AT Salmo trutta mtDNA lineage (sensu Bernatchez). The prehistoric occurrence of this latter haplotype in the water courses of the Italian peninsula has been detected for the first time in this study. Finally, we observed a correspondence between frequency of trout remains and variation in haplotype diversity that we related with ecological and demographic changes resulting from a period of rapid cooling known as the Younger Dryas.

Karyotype and other chromosomal characteristics in the Adriatic brook lamprey Lampetra zanandreai, representative of one of the most ancestral group of vertebrates, were examined using conventional (Ag-staining, C-banding as well as CMA(3) and DAPI fluorescence) and molecular (FISH with 18/28S rDNA and EcoRI satDNA as probes) protocols with metaphase chromosomes derived from whole blood cultures. The chromosome complement had a modal diploid chromosome number of 2n = 164, as in other petromyzontid lamprey species. Ag-staining and CMA(3) fluorescence, as well as FISH with 18/28S rDNA probes, detected nucleolar organizer regions (NORs) close to the centromeres of the biarmed chromosomes of pairs 1 and 2, the largest chromosome pairs of the complement. In addition to NORs, CMA(3) fluorescence revealed positive signals in approximately 40 other chromosomes. DAPI stained mostly centromeric regions of many chromosomes as well as conspicuously massive blocks overlapping NOR sites. C-banding evidenced a large amount of constitutive heterochromatin in somatic chromosomes, with approximately 40 C-positive acrocentric elements completely heterochromatic, corresponding with the 40 CMA(3)+ chromosomes and positive heterochromatic blocks in pericentromeric regions of chromosome pairs 1 and 2. Polymerase chain reaction (PCR)-based cloning of satellite DNA with primers derived from Petromyzon marinus centromeric sequences was successful for L. zanandreai genomic DNA. The sequence was AT-rich (59%) and characterized by short consensus motifs similar to other centromeric satellite motifs. FISH using satDNA clones as a probe produced a fluorescent signal on a single pair of small chromosomes. This sequence was PCR-amplified also in L. planeri and P. marinus genomic DNA, and the evolution of this repetitive element in the above species was analysed.

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.

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.

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 ...

The karyotypes of five species of Scorpaenidae (genera Scorpenopsis, Dendrochirus and Pterois) from the Indian Ocean were analysed using various banding methods and in situ hybridisation with a ribosomal probe. All the species investigated are characterised by a diploid set of 48 chromosomes (mainly acrocentric and/or subtelocentric) and by a NOR location on the small arm of a medium‐sized pair. All the chromosomes stained uniformly with DAPI, whereas C‐banding evidenced a small amount of hete‐rochromatin. Despite the marked morphological differences among these species, the low degree of diversification of the chromosome sets with respect to the ancestral set of teleosts (2n = 48 acrocentric chromosomes) suggests that chromosome morphology has not undergone profound rearrangements during the evolution of these taxa.

The Tc1 family of transposable elements is composed of DNA‐mediated transposons with inverted terminal repeats and an open reading frame encoding a putative transposase. These elements have been identified in a wide range of organisms, among which several fish species. Here we report on the presence of a Tc1‐like element in the ice‐fish Chionodraco hamatus. This element, only 800 bp long, is shorter than those observed so far and contains a degenerate open reading frame of approximately 440 bp bearing several deletions. Corresponding sequences were found in the genomes of other notothenioid species after Southern‐blot hybridization and polymerase chain reaction amplification. Although the efficiency of this widespread Tel element as a phylogenetic marker is still debated, it can be hypothesized that the Tc1‐like family isolated in C. hamatus appeared early in the genome of notothenioids, and its divergence probably accompanied or occurred in parallel to the evolutionary history of this taxon.

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.

Glutathione and glyoxalase II levels were evaluated in cytosolic and mitochondrial compartments of rat liver after 7 and 24 hr of cold storage in University of Wisconsin (UW) and Euro-Collins solutions. 1-4 A similar time-dependent depletion of cytosolic glutathione up to about 60% of control values was observed in both Euro-Collins and UW solutions. Cytosolic glyoxalase II showed activity oscillations in livers stored in Euro-Collins but not in UW. Mitochondrial glutathione and glyoxalase II were severely depleted soon after 7 hr of cold storage in Euro-Collins, whereas the same parameters did not change in liver stored in UW after 24 hr. UW is confirmed to be the most suitable solution for liver cold storage and we conclude that mitochondrial glutathione and glyoxalase II can be important parameters in assessing mitochondrial and cell function.

Pelagic gobies are considered of particular zoological interest because the acquisition of a pelagic lifestyle is achieved through the persistence of larval anatomical features. The Mediterranean Sea is inhabited by three goby species (Aphia minuta, Crystallogobius linearis and Pseudaphya ferreri) characterized by paedomorphic traits. Owing to the shared larval morphological features, these species have generally been considered as a monophyletic group. This study aimed at establishing the phylogenetic relationships of these paedomorphic species within the family Gobiidae to ascertain whether the pelagic lifestyle achieved through paedomorphosis is due to an event that took place in a common ancestor or not. For this purpose, we amplified by polymerase chain reaction and sequenced the mitochondrial 12S rDNA (complete sequence) and 16S rDNA (partial sequence) of 15 Mediterranean gobies. The phylogenetic analysis strongly supported the polyphyletic origin of the three paedomorphic gobies, indicating that the heterochronic change leading to the retention of larval features seems to have occurred independently in the ancestors of these species. Thus, the sharing of morphological traits can be considered homoplasious and the classification of these species in the same taxonomic group is rejected on the basis of molecular data.

Centromere and telomere composition and organization were studied in various gobiid species exhibiting and not exhibiting chromosome polymorphisms involving Robertsonian rearrangements. In Gobius cobitis, we isolated an AT-rich centromeric DNA satellite, designated pCOB, and found that several sequences contain adenine stretches, various CA/TG dinucleotide steps, and a sequence 76% homologous to the yeast CDE III centromeric sequence. All of these traits are generally considered important for centromeric function, and the hypothesis has been advanced that some are involved in the control of DNA curvature and thus in the degree of centromeric chromatin compactness. Based on these features, and on the fact that they are found only in the species not exhibiting Robertsonian biarmed chromosomes, a role for pCOB in preventing centric fusions has been hypothesized. Our data also suggest that, as in other species, the formation of Robertsonian biarmed chromosomes is accompanied by the loss of telomeric sequences.

The DNA of the Antarctic scallop Adamussium colbecki was found to contain a highly repeated sequence identifiable upon restriction with endonuclease BglII. The monomeric unit - denominated pACS (about 170bp long) - was cloned. Southern blot hybridization yielded a ladder-like banding pattern, indicating that the repeated elements are tandemly arranged in the genome and therefore represent a sequence of satellite DNA. Sequence analysis of five different clones revealed the presence of various subfamilies, some of which showed a high degree of divergence. In each clone, regions homologous to the mammalian CENP-B box were observed. A region homologous to the CDEIII centromeric sequence of yeast was also found in one of the clones. These observations suggest a relationship of the pACS family to the centromeric area in A. colbecki.