Morphological and Genomic Differences in the Italian Populations of Onopordum tauricum Willd.-A New Source of Vegetable Rennet.

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.

Identification of Iguania Ancestral Syntenic Blocks and Putative Sex Chromosomes in the Veiled Chameleon (Chamaeleo calyptratus, Chamaeleonidae, Iguania).

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.

Chromosome Painting Does Not Support a Sex Chromosome Turnover in Lacerta agilis Linnaeus, 1758.

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 of Lacerta 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 of L. agilis exigua from Siberia, Russia, to identify the sex chromosomes. FISH with a flow-sorted chromosome painting probe derived from L. strigata and specific to chromosomes 13, 14, and Z confirmed that the Z chromosome of L. agilis is a small macrochromosome, the same as in L. 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 of L. agilis studied 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.

Isolation and characterization of two satellite DNAs in Atlantolacerta andreanskyi (Werner, 1929) (Reptilia, Lacertidae).

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.

Genetic Content of the Neo-Sex Chromosomes in Ctenonotus and Norops (Squamata, Dactyloidae) and Degeneration of the Y Chromosome as Revealed by High-Throughput Sequencing of Individual Chromosomes.

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.

First report on B chromosome content in a reptilian species: the case of Anolis carolinensis.

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.

Characterization of a satellite DNA in the genera Lacerta and Timon (Reptilia, Lacertidae) and its role in the differentiation of the W chromosome.

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.

Cytogenetic Characterization of a Population of Acanthodactylus lineomaculatus Duméril and Bibron, 1839 (Reptilia, Lacertidae), from Southwestern Morocco and Insights into Sex Chromosome Evolution.

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.

Immunocytological analysis of meiotic recombination in two anole lizards (Squamata, Dactyloidae).

Although the evolutionary importance of meiotic recombination is not disputed, the significance of interspecies differences in the recombination rates and recombination landscapes remains under-appreciated. Recombination rates and distribution of chiasmata have been examined cytologically in many mammalian species, whereas data on other vertebrates are scarce. Immunolocalization of the protein of the synaptonemal complex (SYCP3), centromere proteins and the mismatch-repair protein MLH1 was used, which is associated with the most common type of recombination nodules, to analyze the pattern of meiotic recombination in the male of two species of iguanian lizards, Anolis carolinensis Voigt, 1832 and Deiroptyx coelestinus (Cope, 1862). These species are separated by a relatively long evolutionary history although they retain the ancestral iguanian karyotype. In both species similar and extremely uneven distributions of MLH1 foci along the macrochromosome bivalents were detected: approximately 90% of crossovers were located at the distal 20% of the chromosome arm length. Almost total suppression of recombination in the intermediate and proximal regions of the chromosome arms contradicts the hypothesis that "homogenous recombination" is responsible for the low variation in GC content across the anole genome. It also leads to strong linkage disequilibrium between the genes located in these regions, which may benefit conservation of co-adaptive gene arrays responsible for the ecological adaptations of the anoles.

Heteromorphism of "Homomorphic" Sex Chromosomes in Two Anole Species (Squamata, Dactyloidae) Revealed by Synaptonemal Complex Analysis.

Iguanians (Pleurodonta) are one of the reptile lineages that, like birds and mammals, have sex chromosomes of ancient origin. In most iguanians these are microchromosomes, making a distinction between the X and Y as well as between homeologous sex chromosomes in other species difficult. Meiotic chromosome analysis may be used to elucidate their differentiation, because meiotic prophase chromosomes are longer and less condensed than metaphase chromosomes, and the homologues are paired with each other, revealing minor heteromorphisms. Using electron and fluorescent microscopy of surface spread synaptonemal complexes (SCs) and immunolocalization of the proteins of the SC (SYCP3), the centromere, and recombination nodules (MLH1), we examined sex chromosome synapsis and recombination in 2 species of anoles (Dactyloidae), Anolis carolinensis and Deiroptyx coelestinus, in which the sex chromosomes represent the ancestral condition of iguanians. We detected clear differences in size between the anole X and Y microchromosomes and found an interspecies difference in the localization of the pseudoautosomal region. Our results show that the apparent homomorphy of certain reptile sex chromosome systems can hide a cryptic differentiation, which potentially may influence the evolution of sexual dimorphism and speciation.

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.

The Effects of Paleoclimatic Events on Mediterranean Trout: Preliminary Evidences from Ancient DNA.

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.

Biocomplexity in Populations of European Anchovy in the Adriatic Sea.

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.

Coupling Demographic and Genetic Variability from Archived Collections of European Anchovy (Engraulis encrasicolus).

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 (Ne) are much smaller than those expected in a fishes with large population census sizes (Nc). Estimates of Ne indicate low effective population sizes, even before the population collapse. The ratio Ne/Ne 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.

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.

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.

Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting.

In contrast to mammals, birds exhibit a slow rate of chromosomal evolution. It is not clear whether high chromosome conservation is an evolutionary novelty of birds or was inherited from an earlier avian ancestor. The evolutionary conservatism of macrochromosomes between birds and turtles supports the latter possibility; however, the rate of chromosomal evolution is largely unknown in other sauropsids. In squamates, we previously reported strong conservatism of the chromosomes syntenic with the avian Z, which could reflect a peculiarity of this part of the genome. The chromosome 1 of iguanians and snakes is largely syntenic with chromosomes 3, 5 and 7 of the avian ancestral karyotype. In this project, we used comparative chromosome painting to determine how widely this synteny is conserved across nine families covering most of the main lineages of Squamata. The results suggest that the association of the avian ancestral chromosomes 3, 5 and 7 can be dated back to at least the early Jurassic and could be an ancestral characteristic for Unidentata (Serpentes, Iguania, Anguimorpha, Laterata and Scinciformata). In Squamata chromosome conservatism therefore also holds for the parts of the genome which are homologous to bird autosomes, and following on from this, a slow rate of chromosomal evolution could be a common characteristic of all sauropsids. The large evolutionary stasis in chromosome organization in birds therefore seems to be inherited from their ancestors, and it is particularly striking in comparison with mammals, probably the only major tetrapod lineage with an increased rate of chromosomal rearrangements as a whole.

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.