Characterization of Two Transposable Elements and an Ultra-Conserved Element Isolated in the Genome of Zootoca vivipara (Squamata, Lacertidae).

Transposable elements (TEs) constitute a considerable fraction of eukaryote genomes representing a major source of genetic variability. We describe two DNA sequences isolated in the lizard Zootoca vivipara, here named Zv516 and Zv817. Both sequences are single-copy nuclear sequences, including a truncation of two transposable elements (TEs), SINE Squam1 in Zv516 and a Tc1/Mariner-like DNA transposon in Zv817. FISH analyses with Zv516 showed the occurrence of interspersed signals of the SINE Squam1 sequence on all chromosomes of Z. vivipara and quantitative dot blot indicated that this TE is present with about 4700 copies in the Z. vivipara genome. FISH and dot blot with Zv817 did not produce clear hybridization signals. Bioinformatic analysis showed the presence of active SINE Squam 1 copies in the genome of different lacertids, in different mRNAs, and intronic and coding regions of various genes. The Tc1/Mariner-like DNA transposon occurs in all reptiles, excluding Sphenodon and Archosauria. Zv817 includes a trait of 284 bp, representing an amniote ultra-conserved element (UCE). Using amniote UCE homologous sequences from available whole genome sequences of major amniote taxonomic groups, we performed a phylogenetic analysis which retrieved Prototheria as the sister group of Metatheria and Eutheria. Within diapsids, Testudines are the sister group to Aves + Crocodylia (Archosauria), and Sphenodon is the sister group to Squamata. Furthermore, large trait regions flanking the UCE are conserved at family level.

Transposable Elements and Stress in Vertebrates: An Overview.

Since their identification as genomic regulatory elements, Transposable Elements (TEs) were considered, at first, molecular parasites and later as an important source of genetic diversity and regulatory innovations. In vertebrates in particular, TEs have been recognized as playing an important role in major evolutionary transitions and biodiversity. Moreover, in the last decade, a significant number of papers has been published highlighting a correlation between TE activity and exposition to environmental stresses and dietary factors. In this review we present an overview of the impact of TEs in vertebrate genomes, report the silencing mechanisms adopted by host genomes to regulate TE activity, and finally we explore the effects of environmental and dietary factor exposures on TE activity in mammals, which is the most studied group among vertebrates. The studies here reported evidence that several factors can induce changes in the epigenetic status of TEs and silencing mechanisms leading to their activation with consequent effects on the host genome. The study of TE can represent a future challenge for research for developing effective markers able to detect precocious epigenetic changes and prevent human diseases.

Dynamic Characterization of the Biomechanical Behaviour of Bovine Ovarian Cortical Tissue and Its Short-Term Effect on Ovarian Tissue and Follicles.

The ovary is a dynamic mechanoresponsive organ. In vitro, tissue biomechanics was reported to affect follicle activation mainly through the Hippo pathway. Only recently, ovary responsiveness to mechanical signals was exploited for reproductive purposes. Unfortunately, poor characterization of ovarian cortex biomechanics and of the mechanical challenge hampers reproducible and effective treatments, and prevention of tissue damages. In this study the biomechanical response of ovarian cortical tissue from abattoir bovines was characterized for the first time. Ovarian cortical tissue fragments were subjected to uniaxial dynamic testing at frequencies up to 30 Hz, and at increasing average stresses. Tissue structure prior to and after testing was characterized by histology, with established fixation and staining protocols, to assess follicle quality and stage. Tissue properties largely varied with the donor. Bovine ovarian cortical tissue consistently exhibited a nonlinear viscoelastic behavior, with dominant elastic characteristics, in the low range of other reproductive tissues, and significant creep. Strain rate was independent of the applied stress. Histological analysis prior to and after mechanical tests showed that the short-term dynamic mechanical test used for the study did not cause significant tissue tear, nor follicle expulsion or cell damage.

From Fish Eggs to Fish Name: Caviar Species Discrimination by COIBar-RFLP, an Efficient Molecular Approach to Detect Fraud in the Caviar Trade.

The demand for caviar is growing as is its price on the market. Due to the decline of true caviar production from sturgeons, eggs from other fish species and other animals have been used as substitutes for caviar. The labels on these products should indicate the species from which the eggs were derived, but the label can be misleading in some cases. In this context, species identification using DNA analysis is crucial for traceability and authentication of caviar products. In this work, we applied the COIBar-RFLP procedure to obtain species-specific endonuclease restriction patterns useful to discriminate "caviar" species. The tested caviar products were identified as originating from eight species: Acipenser transmontanus, A. gueldenstaedtii, A. stellatus, A. baerii, Mallotus villosus, Huso huso, Cyclopterus lumpus and Eumicrotremus orbis. The results demonstrated that 14% of the caviar products examined have a label that does not indicate the species from which the eggs were originated. The MboI restriction enzyme produced specific profiles discriminating the eight species, confirming that the COIBar-RFLP is a useful approach for routine screening of seafood products due to its ease and rapid execution, as the results of screening can be obtained within 7 h, by-passing the need for sequencing.

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

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

In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development.

Several studies have demonstrated that overexposure to pesticides can reduce mammalian sperm quality, impairing male fertility. Chlorpyrifos (CPF), a widely used organophosphate pesticide, was shown to impair spermatogenesis by inducing the formation of highly reactive toxic intermediates. To gain further insight into the mechanisms underlying the cytotoxicity and genotoxicity of CPF, bovine spermatozoa were exposed in vitro to environmental CPF concentrations and the motility, in vitro fertilization rates, DNA fragmentation, chromatin alterations, and methylation patterns were assessed. Motility and in vitro fertilization rates were significantly reduced in spermatozoa exposed to CPF, while DNA fragmentation and putative chromatin deconstruction appeared to increase at higher pesticide concentrations. In situ hybridization was carried out with X and Y probes on sperm samples exposed to different CPF concentrations, and subsequent analysis highlighted a significant percentage of spermatozoa with a peculiar morphological malformation, in which a narrowing occurred at the level of the hybridization. Analysis of potential abnormalities in the methylation pattern of NESP55-GNAS and XIST promoters displayed no differentially methylated regions in GNAS promoter relative to the control, whereas spermatozoa exposed to 10 µg/mL CPF had increased methylation variance in one region of imprinted XIST promoter. Our results provide support that CPF can induce a genotoxic effect on spermatozoa, impairig their ability to fertilize and support preimplantation embryo development in vitro. These observations are worrying since altered levels of sporadic methylation in genes of male gametes may affect the success of reproduction and contribute to infertility. Environ. Mol. Mutagen. 60:85-95, 2019. © 2018 Wiley Periodicals, Inc.

Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture.

OBJECTIVE: To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended in vitro culture. DESIGN: Randomized experimental study. SETTING: University research laboratory. PATIENT(S): Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9 days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time-PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture. RESULT(S): The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription-PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10-35) and a markedly lower up-regulation of only 5 genes (range, 3.6-7.8) in SN samples. Long-term in vitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue. CONCLUSION(S): Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.

Specific Effects of Chronic Dietary Exposure to Chlorpyrifos on Brain Gene Expression-A Mouse Study.

chlorpyrifos (CPF) is an organophosphate insecticide used to control pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce cerebral cortex thinning, alteration of long-term brain cognitive function, and Parkinson-like symptoms, but the mechanisms of these processes are not fully understood. In this study, we aimed to gain a deeper understanding of the alterations induced in the brains of mice chronically exposed to CPF by dietary intake. For our purpose, we analysed F1 offspring (sacrificed at 3 and 8 months) of Mus musculus, treated in utero and postnatally with 3 different doses of CPF (0.1-1-10 mg/kg/day). Using RT² Profiler PCR Arrays, we evaluated the alterations in the expression of 84 genes associated with neurodegenerative diseases. In the brains of exposed mice, we evidenced a clear dose-response relationship for AChE inhibition and alterations of gene expression. Some of the genes that were steadily down-regulated, such as Pink1, Park 2, Sv2b, Gabbr2, Sept5 and Atxn2, were directly related to Parkinson's onset. Our experimental results shed light on the possibility that long-term CPF exposure may exert membrane signalling alterations which make brain cells more susceptible to develop neurodegenerative diseases.

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

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

Chlorpyrifos exposure affects fgf8, sox9, and bmp4 expression required for cranial neural crest morphogenesis and chondrogenesis in Xenopus laevis embryos.

Chlorpyrifos (CPF) is an organophosphate insecticide used primarily to control foliage and soil-borne insect pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce dose-related abnormalities such as slower brain growth and cerebral cortex thinning. In lower vertebrates, for example, fish and amphibians, teratogenic activity of this compound is correlated with several anatomical alterations. Little is known about the effects of CPF on mRNA expression of genes involved in early development of the anatomical structures appearing abnormal in embryos. This study investigated the effects of exposure to different CPF concentrations (10, 15 and 20 mg/L) on Xenopus laevis embryos from stage 4/8 to stage 46. Some of the morphological changes we detected in CPF-exposed embryos included cranial neural crest cell (NCC)-derived structures. For this reason, we analyzed the expression of select genes involved in hindbrain patterning (egr2), cranial neural crest chondrogenesis, and craniofacial development (fgf8, bmp4, sox9, hoxa2 and hoxb2). We found that CPF exposure induced a reduction in transcription of all the genes involved in NCC-dependent chondrogenesis, with largest reductions in fgf8 and sox9; whereas, in hindbrain, we did not find any alterations in egr2 expression. Changes in the expression of fgf8, bmp4, and sox9, which are master regulators of several developmental pathways, have important implications. If these changes are confirmed to belong to a general pattern of alterations in vertebrates prenatally exposed to OP, they might be useful to assess damage during vertebrate embryo development. Environ. Mol. Mutagen. 57:589-604, 2016. © 2016 Wiley Periodicals, Inc.

Short interspersed DNA elements and miRNAs: a novel hidden gene regulation layer in zebrafish?

In this study, we investigated by in silico analysis the possible correlation between microRNAs (miRNAs) and Anamnia V-SINEs (a superfamily of short interspersed nuclear elements), which belong to those retroposon families that have been preserved in vertebrate genomes for millions of years and are actively transcribed because they are embedded in the 3' untranslated region (UTR) of several genes. We report the results of the analysis of the genomic distribution of these mobile elements in zebrafish (Danio rerio) and discuss their involvement in generating miRNA gene loci. The computational study showed that the genes predicted to bear V-SINEs can be targeted by miRNAs with a very high hybridization E-value. Gene ontology analysis indicates that these genes are mainly involved in metabolic, membrane, and cytoplasmic signaling pathways. Nearly all the miRNAs that were predicted to target the V-SINEs of these genes, i.e., miR-338, miR-9, miR-181, miR-724, miR-735, and miR-204, have been validated in similar regulatory roles in mammals. The large number of genes bearing a V-SINE involved in metabolic and cellular processes suggests that V-SINEs may play a role in modulating cell responses to different stimuli and in preserving the metabolic balance during cell proliferation and differentiation. Although they need experimental validation, these preliminary results suggest that in the genome of D. rerio, as in other TE families in vertebrates, the preservation of V-SINE retroposons may also have been favored by their putative role in gene network modulation.

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

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

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

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

Helinoto, a Helitron2 transposon from the icefish Chionodraco hamatus, contains a region with three deubiquitinase-like domains that exhibit transcriptional activity.

Transposable elements have accompanied the evolution of the eukaryotic genome for millions of years. The recently discovered Helitron order (class II, subclass 2 single-strand DNA transposons) is common in eukaryotes and seems to play a highly active role in genome reshuffling. This study provides novel insights into the characteristics of Helinoto, a helitron isolated in the genome of the Antarctic fish Chionodraco hamatus. In particular, investigation of the structure of its 5' and 3' ends, which are involved in the transposition process, enabled identification of the characteristic motifs of the Helitron2 group. Moreover, identification of a deubiquitinating protease domain in the region upstream two consecutive OTU domains extended and strengthened the "deubiquitinase" character of the N-terminal portion of Helinoto. Finally, Helinoto transcriptional activity was detected in several C. hamatus tissues. Taken together, these data are particularly intriguing because they document high transcription levels for genes involved in ubiquitination, which ensures protein homeostasis in the extreme Antarctic environment.

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

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

Characterization of sex determination and sex differentiation genes in Latimeria.

Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique "living fossils", could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA.

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.

Identification of a novel helitron transposon in the genome of Antarctic fish.

Rolling-circle (RC) eukaryotic transposons, known as helitrons, are found in a wide range of organisms, from protist to mammals. Autonomous helitrons have a distinctive open reading frame (ORF) encoding a polypeptide that contains typical domains for RC replication (RCR): the Rep (RCR initiator) and the DNA helicase domains. These elements are believed to have an important role in the host genome evolution, owing to their frequent capture of host genes, some of which can evolve into novel genes or become essential for helitron transposition. We conducted a molecular analysis of the suborder Notothenioidei, a group of Perciformes that currently dominate the Antarctic waters by virtue of their remarkable cold-adaptation ability. A novel helitron from the genome of the icefish species Chionodraco hamatus, belonging to the Channichthyidae, the most derived Notothenioids family, was isolated, characterized and designated as HeliNoto (8.9 kb). Its ORF was compared to homologous sequences from different species in a comprehensive phylogenetic analysis. For the first time the putative functional domains of a helitron were subjected to a well accurate structural analysis including chromosomal localization. Finally, the distribution of HeliNoto among Notothenioids was investigated.

Polypteridae (Actinopterygii: Cladistia) and DANA-SINEs insertions.

SINE sequences are interspersed throughout virtually all eukaryotic genomes and greatly outnumber the other repetitive elements. These sequences are of increasing interest for phylogenetic studies because of their diagnostic power for establishing common ancestry among taxa, once properly characterized. We identified and characterized a peculiar family of composite tRNA-derived short interspersed SINEs, DANA-SINEs, associated with mutational activities in Danio rerio, in a group of species belonging to one of the most basal bony fish families, the Polypteridae, in order to investigate their own inner specific phylogenetic relationships. DANA sequences were identified, sequenced and then localized, by means of fluorescent in situ hybridization (FISH), in six Polypteridae species (Polypterus delhezi, P. ornatipinnis, P. palmas, P. buettikoferi P. senegalus and Erpetoichthys calabaricus) After cloning, the sequences obtained were aligned for phylogenetic analysis, comparing them with three Dipnoan lungfish species (Protopterus annectens, P. aethiopicus, Lepidosiren paradoxa), and Lethenteron reissneri (Petromyzontidae)was used as outgroup. The obtained overlapping MP, ML and NJ tree clustered together the species belonging to the two taxonomically different Osteichthyans groups: the Polypteridae, by one side, and the Protopteridae by the other, with the monotypic genus Erpetoichthys more distantly related to the Polypterus genus comprising three distinct groups: P. palmas and P. buettikoferi, P. delhezi and P. ornatipinnis and P. senegalus. In situ hybridization with DANA probes marked along the whole chromosome arms in the metaphases of all the Polypteridae species examined.

Mitochondrial DNA variation in the caramote prawn Penaeus (Melicertus) kerathurus across a transition zone in the Mediterranean Sea.

In this study we analysed mitochondrial DNA variation in Penaeus kerathurus prawns collected from seven locations along a transect across the Siculo-Tunisian region in order to verify if any population structuring exists over a limited geographical scale and to delineate the putative transition zone with sufficient accuracy. Partial DNA sequences of COI and 16S genes were analysed. In contrast to the highly conservative 16S gene, the COI sequences exhibited sufficient diversity for population analysis. The COI gene revealed low levels of haplotype and nucleotide diversities. The size of the annual landings of this commercial species suggests large population sizes. Hence, the low genetic diversity detected in this study could indicate a possible reduction in effective population sizes in the past. We detected significant genetic differentiation between eastern and western populations likely due to restricted gene flow across the Siculo-Tunisian boundary. We discuss the different evolutionary forces that may have shaped the genetic variation and suggest that the genetic divide is probably maintained by present-day dispersal limitation.