Federica Venanzetti

Characterization of a species-specific satellite DNA family of Dolichopoda schiavazzii (Orthoptera, Rhaphidophoridae) cave crickets.

The satellite DNA family pDoP102 is species specific for the cave cricket Dolichopoda schiavazzii, an endemic species of mainland and insular Tuscany. It consists of numerous tandemly arranged repeats, 102 bp in length, and evolved most probably after cladogenesis of D. schiavazzii from the D. baccettii-aegilion group within the last 2.3 ± 0.8 million years. A sequence comparison of 31 clones (53 repetition units) from three isolated populations reveals a very high degree of sequence homogeneity within the species with no evidence for any specific population features. This appears to be in contrast to the results of allozyme analyses which account for a relatively old evolutionary divergence of the Elba island population from the mainland ones. Since the assumption of actual gene flow and recent colonization is rejected, the observed sequence homogeneity is hypothesized to be maintained by recombination processes preventing fixation of newly introduced mutations on pDoP102 sequence clusters.

Tandemly repeated satellite DNA of Dolichopoda schiavazzii: a test for models on the evolution of highly repetitive DNA

Three specific satellite DNA families can be detected in the genome of the cave cricketDolichopoda schiavazzii. ThepDoP102 and thepDsPv400 families are species specific forD. schiavazzii; thepDoP500 family is probably present in allDolichopoda species. The three satellite DNA families were characterized from individuals of three isolated populations ofD. schiavazzii with respect to nucleotide sequence, sequence complexity, sequence variability, and copy number. This unique data set on satellite DNAs of D. schiavazzii seems to allow one to test the significance of theoretical approaches to the mode of evolution of noncoding, tandemly arranged satellite DNA. At least for satellite DNAs ofD. schiavazzii two clear trends were observed: (1) sequence variability increases with copy number and (2) the repeat length decreases with copy number. The first trend is in good agreement with the theory but the second is not. Thus, a revision of the models is proposed.

Phylogeography and mitochondrial DNA divergence in Dolichopoda cave crickets (Orthoptera, Rhahidophoridae)

Dolichopoda cave crickets are found in caves in the northern Mediterranean region from the Pyrenees in the west to the Caucasus in the east. In this study we analysed the phylogeny within the genus Dolichopoda using parts of the mitochondrial cytochrome oxidase I and 16S ribosomal genes, and explored biogeographic patterns through a dispersal-vicariance analysis (DIVA). Phylogenetic analyses grouped the 15 species into the four geographically restricted main lineages corresponding to the Caucasus, Greece, the Pyrenees and Italy, respectively. The species occur largely in allopatry. The Caucasian and Greek species were basal in the phylogeny, as was the clade including the nine Italian species, which grouped into two major lineages, one mainly including species from western Italian coastal regions and islands, and the other including species with a predominantly inland distribution. Thus it seems likely that there have been two main immigrations into Italy followed by multiple consecutive speciation events. The DIVA analysis supported the assumption of an east-west migration route, and indicated that there have been four major dispersal events. Since the statistical support for the basal node connecting D. remyi and D. hussoni with the west Mediterranean species is low, alternative interpretations for the colonization of the Mediterranean, namely parallel colonization of the main areas of the current Dolichopoda distribution, i.e. the Caucasus, Greece, Italy, and the Pyrenees is also possible. Particular emphasis was put on the D. geniculata-laetitiae species complex. D. geniculata included several recently diverged lineages and constitutes a paraphyletic species complex, also embracing the closely related D. laetitiae.

Molecular evolution of the pDo500 satellite DNA family in Dolichopoda cave crickets (Rhaphidophoridae)

BackgroundNon-coding satellite DNA (satDNA) usually has a high turn-over rate frequently leading to species specific patterns. However, some satDNA families evolve more slowly and can be found in several related species. Here, we analyzed the mode of evolution of the pDo500 satDNA family of Dolichopoda cave crickets. In addition, we discuss the potential of slowly evolving satDNAs as phylogenetic markers.ResultsWe sequenced 199 genomic or PCR amplified satDNA repeats of the pDo500 family from 12 Dolichopoda species. For the 38 populations under study, 39 pDo500 consensus sequences were deduced. Phylogenetic analyses using Bayesian, Maximum Parsimony, and Maximum Likelihood approaches yielded largely congruent tree topologies. The vast majority of pDo500 sequences grouped according to species designation. Scatter plots and statistical tests revealed a significant correlation between genetic distances for satDNA and mitochondrial DNA. Sliding window analyses showed species specific patterns of variable and conserved regions. The evolutionary rate of the pDo500 satDNA was estimated to be 1.63-1.78% per lineage per million years.ConclusionsThe pDo500 satDNA evolves gradually at a rate that is only slightly faster than previously published rates of insect mitochondrial COI sequences. The pDo500 phylogeny was basically congruent with the previously published mtDNA phylogenies. Accordingly, the slowly evolving pDo500 satDNA family is indeed informative as a phylogenetic marker.

Phylogenetic footprinting of non-coding RNA: hammerhead ribozyme sequences in a satellite DNA family of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae)

BackgroundThe great variety in sequence, length, complexity, and abundance of satellite DNA has made it difficult to ascribe any function to this genome component. Recent studies have shown that satellite DNA can be transcribed and be involved in regulation of chromatin structure and gene expression. Some satellite DNAs, such as the pDo500 sequence family in Dolichopoda cave crickets, have a catalytic hammerhead (HH) ribozyme structure and activity embedded within each repeat.ResultsWe assessed the phylogenetic footprints of the HH ribozyme within the pDo500 sequences from 38 different populations representing 12 species of Dolichopoda. The HH region was significantly more conserved than the non-hammerhead (NHH) region of the pDo500 repeat. In addition, stems were more conserved than loops. In stems, several compensatory mutations were detected that maintain base pairing. The core region of the HH ribozyme was affected by very few nucleotide substitutions and the cleavage position was altered only once among 198 sequences. RNA folding of the HH sequences revealed that a potentially active HH ribozyme can be found in most of the Dolichopoda populations and species.ConclusionsThe phylogenetic footprints suggest that the HH region of the pDo500 sequence family is selected for function in Dolichopoda cave crickets. However, the functional role of HH ribozymes in eukaryotic organisms is unclear. The possible functions have been related to trans cleavage of an RNA target by a ribonucleoprotein and regulation of gene expression. Whether the HH ribozyme in Dolichopoda is involved in similar functions remains to be investigated. Future studies need to demonstrate how the observed nucleotide changes and evolutionary constraint have affected the catalytic efficiency of the hammerhead.

Cloning and characterization of the European seabass, Dicentrarchus labrax, mitochondrial genome

Mitochondrial DNA (mtDNA) from the European seabass, Dicentrarchus labrax, has been cloned and characterized. Its gene organization was deduced by a comparison of the sequenced termini of different subclones obtained from European seabass mtDNA to the completely-sequenced mtDNAs from carp and freshwater loach. The difference in genome size between the European seabass mtDNA (approximately 18 kb) and most of the other characterized fish mtDNAs (approximately 16.5 kb) is accounted for by the displacement-loop (D-loop). Comparisons have been performed between the derived amino-acid sequences of three sequenced genes, cytochrome c oxidase subunit 2 (COII), NADH dehydrogenase subunit 4L (ND4L) and ATP synthase subunit 8 (ATPase8), from D. labrax, and their counterparts in other fishes and Xenopus laevis.

Length variation in mtDNA control region in hatchery stocks of European sea bass subjected to acclimation experiments

Length polymorphism of the mtDNA control region was analysed in samples from the sea bass Dicentrarchus labrax before and after acclimation to freshwater. Acclimation trials were repeated twice for two samples originating from the same broodstock. DNA amplification of 221 individuals made it possible to detect 40 different D-loop length variants and a mean level of gene diversity (/c!) of 0.850. Out of 52 scored distinct genotypes, 18 were homoplasmic and the remaining ones were heteroplasmic for up to four variants. Shifts in frequencies between starting and acclimated samples in the same direction of the same genotypes in both years were not observed. Patterns of mtDNA control region variation were compared to previous allozymic and RAPD studies carried out on the same samples. While variation found in the allozyme alleles and the RAPD markers in replicate experiments revealed differential survival of the genotypes, stochastic processes, such as genetic drift, explained the observed variation of mtDNA control region. Very high levels of genotype diversity were observed within each sample, ranging from 0.77 to 0.97. This high diversity associated with the maternal inheritance of mtDNA makes this molecular marker especially suitable for studies in aquaculture genetics, when the allozyme studies fail to reveal genetic variation or when the identification of progeny based on the mothers contribution is relevant.

Restriction enzymes induced bands in the cave cricket Dolichopoda schiavazzii (Orthoptera, Rhaphidophoridae): Implications for heterochromatin characterization and satellite DNA distribution

Abstract In this study the activity of some class II restriction endonucleases, RE, on fixed metaphase chromosomes has been tested on two sample populations of the cave cricket Dolichopoda schiavazzii, a species endemic in Tuscany. In particular, the 6‐base cutter PstI was chosen to detect the occurrence and localization on chromosomes of a satellite DNA family (named pDoP102) whose repetition units, analyzed in a previous study, contain the cleavage site of this enzyme. A comparison of the PstI digestion pattern and the C‐banding one, suggests that this satDNA family is mainly localized in certain heterochromatic regions of chromosomes 1, 3, 8, 10 and in the Y chromosome. As a negative control in situ digestion was performed with RE AluI whose cleavage site is not contained in the pDoP102 satDNA sequence. The partial overlap between the PstI and Alul digested regions suggests the occurrence of at least another satDNA family detected by PstI and containing the Alul target. Alul restriction banding also sh...