Genetic differentiation and hybridization in two naturally occurring sympatric trout Salmo spp. forms from a small karstic lake.

In this study, multiple molecular markers [genotyping of 12 nuclear microsatellite loci and the protein-coding gene ldh-c1* plus sequencing of the mitochondrial DNA (mtDNA) control region] were employed to investigate the genetic structure of the two trout forms, Salmo cettii and Salmo fibreni, inhabiting Lake Posta Fibreno, central Italy. The two forms were found to share a unique mtDNA haplotype, belonging to a widespread Mediterranean haplogroup (AD). Bayesian clustering analyses showed that these two forms correspond to well-defined autochthonous gene pools. Genetic introgression between the two gene pools, however, was observed, whose frequency appears to correlate with the environmental features of the spawning sites. The interplay of selection for the spawning sites, philopatry and natural selection can be argued to maintain genetic differentiation despite the lack of complete reproductive isolation.

Pleistocene evolutionary history of the Clouded Apollo (Parnassius mnemosyne): genetic signatures of climate cycles and a 'time-dependent' mitochondrial substitution rate.

Genetic data are currently providing a large amount of new information on past distribution of species and are contributing to a new vision of Pleistocene ice ages. Nonetheless, an increasing number of studies on the 'time dependency' of mutation rates suggest that date assessments for evolutionary events of the Pleistocene might be overestimated. We analysed mitochondrial (mt) DNA (COI) sequence variation in 225 Parnassius mnemosyne individuals sampled across central and eastern Europe in order to assess (i) the existence of genetic signatures of Pleistocene climate shifts; and (ii) the timescale of demographic and evolutionary events. Our analyses reveal a phylogeographical pattern markedly influenced by the Pleistocene/Holocene climate shifts. Eastern Alpine and Balkan populations display comparatively high mtDNA diversity, suggesting multiple glacial refugia. On the other hand, three widely distributed and spatially segregated lineages occupy most of northern and eastern Europe, indicating postglacial recolonization from different refugial areas. We show that a conventional 'phylogenetic' substitution rate cannot account for the present distribution of genetic variation in this species, and we combine phylogeographical pattern and palaeoecological information in order to determine a suitable intraspecific rate through a Bayesian coalescent approach. We argue that our calibrated 'time-dependent' rate (0.096 substitutions/ million years), offers the most convincing time frame for the evolutionary events inferred from sequence data. When scaled by the new rate, estimates of divergence between Balkan and Alpine lineages point to c. 19 000 years before present (last glacial maximum), and parameters of demographic expansion for northern lineages are consistent with postglacial warming (5-11 000 years before present).

Development of primers to amplify mitochondrial DNA control region of Old World porcupines (subgenus Hystrix).

Eight primers were developed for the amplification of mitochondrial DNA control region of Old world porcupines (subgenus Hystrix). Successful amplifications of low-quality DNA extracted from old (12 years old) and recent quills were performed, thus facilitating field sampling. Successful cross-species amplifications were obtained for Hystrix africaeaustralis, H. cristata and H. indica. Length and structure of mitochondrial DNA control region were analysed and its usefulness as genetic marker for interspecific and population investigation was discussed.

Molecular biogeography of cave life: a study using mitochondrial DNA from bathysciine beetles.

This study focuses on phylogenetic relationships in two distinct species assemblages of cave-dwelling beetles with similar disjunct distributions in the Pyrenees and Sardinia. One assemblage contains six species in the genera Ovobathysciola (four species) and Patriziella (two species) on Sardinia and one species of Anillochlamys in the Pyrenees. Species within the two Sardinian genera co-occur in the same karst area. Although, they are believed to be each others closest relative, they have very different body types (globular body with short appendages in Ovobathysciola; elongated body with long appendages in Patriziella), which are believed to reflect different degrees of adaptation to cave life. The other assemblage of Bathysciine beetles includes three species in the genus Speonomus in the Pyrenees and one on Sardinia. All the species are rare and many are endangered. One issue of particular interest was whether Ovobathysciola and Patriziella are reciprocally monophyletic or whether each of the Patriziella species evolved independently from the co-occurring Ovobathysciola species, as the similar morphology of the Patriziella species might be due to convergence rather than common descent. Based on DNA sequences of the cytochrome oxidase I (COI) region of the mtDNA, neither scenario was supported. Rather, the two Patriziella species are sister taxa embedded within the Ovobathysciola radiation. In addition, the well-dated geological history of this region allowed us to calibrate absolute rates of COI evolution, the first such estimates for any insect. Finally this study suggests that the evolutionary acquisition of typical cave adaptations (e.g., elongated body and appendages) may occur at about the same rate as loss of traits (e.g., eyes and pigmentation) associated with cave life.

Hammerhead-mediated processing of satellite pDo500 family transcripts from Dolichopoda cave crickets.

This work reports the discovery and functional characterization of catalytically active hammerhead motifs within satellite DNA of the pDo500 family from several DOLICHOPODA: cave cricket species. We show that in vitro transcribed RNA of some members of this satellite DNA family do self-cleave in vitro. This self-cleavage activity is correlated with the efficient in vivo processing of long primary transcripts into monomer-sized RNA. The high sequence conservation of the satellite pDo500 DNA family among genetically isolated DOLICHOPODA: schiavazzii populations, as well as other DOLICHOPODA: species, along with the fact that satellite members are actively transcribed in vivo suggests that the hammerhead-encoding satellite transcripts are under selective pressure, perhaps because they fulfil an important physiological role or function. Remarkably, this is the third example of hammerhead ribozyme structures associated with transcribed repetitive DNA sequences from animals. The possibility that such an association may not be purely coincidental is discussed.

Mitochondrial DNA rates and biogeography in European newts (genus Euproctus).

Sequence divergence for segments of three mitochondrial DNA (mtDNA) genes encoding the 12S and 16S ribosomal RNA and cytochrome b was examined in newts belonging to the genus Euproctus (E. asper, E. montanus, E. platycephalus) and in three other species belonging to the same family (Salamandridae), Triturus carnifex, T. vulgaris, and Pleurodeles waltl. The three Euproctus species occur (one species each) in Corsica, Sardinia, and the Pyrenees. This vicariant distribution is believed to have been determined by the disjunction and rotation of the Sardinia-Corsica microplate from the Pyreneean region. Because time estimates are available for the tectonic events that led to the separation of the three landmasses, we used sequence data to estimate rates of evolution for the three gene fragments and investigated whether they conform to the rate-constancy hypothesis. By the Tajima (1993, Genetics 135:599-607) test, we could not detect rate heterogeneities for the ribosomal genes and for transversions in the cytochrome b gene. Assuming that these sites are evolving linearly over time and that cessation of gene flow occurred simultaneously with vicariant events, we compared the time of divergence estimated by molecular distances with the divergence times based on the geological estimates. Because we have two estimates of divergence time from the geological record, the split of Corsica/Sardinia from the Pyrenees and the split of Corsica from Sardinia, we could compare ratios of molecular divergence with the ratio of geological time divergence. The ratios are very similar, indicating that the molecular clock hypothesis cannot be rejected. These geological events also allowed us to calculate absolute rates of evolution for ribosomal and cytochrome b genes and compare them to rates for the same regions in other salamandrids and other vertebrates. Ribosomal mtDNA rates are comparable to those reported for other vertebrates, but cytochrome b rates are 3-7 times lower in salamanders than in other ectotherms. From a phylogenetic perspective, our data suggest that the cladogenic events leading to species formation in Euproctus and Triturus occurred very closely in time, indicating that the two genera may not be monophyletic. A duplication of the cytochrome b gene in T. carnifex was found, and the implications of this finding for mtDNA phylogenetic studies are discussed.

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 cricket Dolichopoda schiavazzii. The pDoP102 and the pDsPv400 families are species specific for D. schiavazzii; the pDoP500 family is probably present in all Dolichopoda species. The three satellite DNA families were characterized from individuals of three isolated populations of D. 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 of D. 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.

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.

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.

Molecular phylogenies in Dolichopoda cave crickets and mtDNA rate calibration.

Five species of Dolichopoda cave crickets have been studied by means of mtDNA RFLPs. The phylogenetic relationships among them were previously inferred from two different molecular measures: allozyme polymorphisms and DNA-DNA hybridization. mtDNA data generate a phylogeny exactly matching those obtained from the other two genetic markers. This is not always the case for other organisms studied so far. This result is discussed in respect of the performance of the three molecular approaches and the population biology of these cave dwelling insects. A tentative calibration of mtDNA rate for Dolichopoda produces an estimate of about 2%/lineage/Myr. The comparison between mtDNA and scnDNA divergence estimates suggests that in these crickets changes accumulate approximately 2-3 times faster in mitochondrial than single copy nuclear sequences.