Costas Stamatis

Mitochondrial DNA variation in Northeast Atlantic and Mediterranean populations of Norway lobster, Nephrops norvegicus

Analysis of the genetic structure of the Norway lobster (Nephrops norvegicus), a marine crustacean with high commercial value, was undertaken to gain information regarding the differentiation of Atlantic from Mediterranean populations of marine invertebrates. Restriction fragment length polymorphism analysis of two mitochondrial DNA segments, 3.6 kilobases in total, was performed. Twelve populations from the North Sea, Irish Sea, Portuguese coast and Aegean Sea were analysed. Low levels of differentiation were found among them (FST = 0.018, P < 0.001) and there were no signs of an Atlantic–Mediterranean divide or of an isolation‐by‐distance scheme of differentiation. Possible reasons for these low levels of differentiation can be found in the recent expansion of N. norvegicus populations. This is supported by the mismatch distribution of pairwise haplotype differences, as well as by the high mean haplotype diversity (h = 0.93) combined with medium nucleotide diversity (π = 0.0057) (in comparison to values for marine crustaceans or teleosts) found in this study. This combination of high levels of haplotype diversity with moderate to low levels of nucleotide diversity has also been frequently attributed to a recent time of divergence for various marine species. No evidence was found for a Mediterranean refugium during glaciation periods, separate from the Atlantic, as has been reported for some marine species. The Irish Sea population was the most differentiated as a result of reduced levels of diversity. Results are also discussed in the light of future management of N. norvegicus stocks.

Genetic structure of Greek brown hare (Lepus europaeus) populations as revealed by mtDBNA RFLP-PCR analysis : implications for conserving genetic diversity

The genetic differentiation and the phylogenetic status of brown hare (Lepus europaeus) populations from central Greece as well as the impact of the releases of reared individuals on the native populations genetic structure was assessed, using mtDNA RFLP-RCR analysis. Data analysis revealed extensive haplotype diversity (42 out of 56 haplotypes were unique) within and among wild populations. Haplotype diversity was equally distributed within and between geographical regions, while significant genetic structuring was evident from heterogeneity of haplotype frequencies among sampling sites. Specific mtDNA profiles clearly differentiated reared from wild individuals and proved highly indicative for reared hares from past releases caught within wild populations. MtDNA analysis suggests the introgression of allochthonous gene pools into the native populations. To conserve indigenous genotypes and to prevent loss of genetic diversity, restocking operations should be stopped and an appropriate management adjusted to the local population dynamics should be developed.

Mitochondrial tRNA Mutations: Clinical and Functional Perturbations

During the last decade, there has been a progressive accumulation of reports that connect the identification of specific mitochondrial tRNA gene mutations to severe disorders in human. As a result, mitochondrial tRNA genes and their products have emerged as novel and essential molecular markers for wide biochemical and genetic screenings among different human populations. So far, 139 pathogenic and 243 polymorphic mt tRNA mutations have been described and they have become the foreground of numerous case reports. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogeneous. They range from lesions of single tissues or structures to more severe impairements including myopathies, encephalomyopathies, cardiomyopathies, or complex multisystem syndromes. Moreover, the exact mechanisms by which biochemical cascades can be dramatically affected by mitochondrial tRNA mutations still remain uncharacterized. However and regardless of the vast amount of information that daily emerges, only few efforts have been carried out to systematically record all the mitochondrial tRNA-associated pathogenic mutations or polymorphisms. In this report, we summarize all the clinical phenotypes associated with mitochondrial tRNA pathogenic mutations that have been reported so far. In a next step we describe in detail all the pathogenic and polymorphic mutations that have been recorded so far and we categorize them per tRNA species and per associated disease. Finally, we discuss the impact of the frequency of mitochondrial tRNA mutations in general population surveys and we preview any relevant implications on the essential functional integrity of mitochondrial biochemical pathways.

Major histocompatibility complex variation at class II DQA locus in the brown hare (Lepus europaeus)

The major histocompatability complex (MHC) is a multigene family of receptors that bind and present antigenic peptides to T‐cells. Genes of the MHC are characterized by an outstanding genetic polymorphism, which is considered to be maintained by positive selection. Sites involved in peptide binding form binding pockets (P) that are collectively termed the peptide‐binding region (PBR). In this study, we examined the level of MHC genetic diversity within and among natural populations of brown hare (Lepus europaeus) from Europe and Anatolia choosing for analysis of the second exon of the DQA locus, one of the most polymorphic class II loci. We aimed at an integrated population genetic analysis of L. europeaus by (i) correlating MHC polymorphism to genetic variability and phylogenetic status estimated previously from maternally (mtDNA) and biparentally (allozymes, microsatellites) inherited loci; and (ii) comparing full‐length exon amino acid polymorphism with functional polymorphism in the PBR and the binding pockets P1, P6 and P9. A substantial level of DQA exon 2 polymorphism was detected with two completely different set of alleles between the Anatolian and European populations. However, the phylogeny of full‐length exon 2 Leeu‐DQA alleles did not show a strong phylogeographic signal. The presence of balancing selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous in the PBR and a trans‐species pattern of evolution detected after phylogenetic reconstruction. The differentiating patterns detected between genetic and functional polymorphism, i.e. the number and the distribution of pocket variants within and among populations, indicated a hierarchical action of selection pressures.

Discrimination of the closely related biocontrol agents Macrolophus melanotoma (Hemiptera: Miridae) and M. pygmaeus using mitochondrial DNA analysis

The separation of the closely related predatory species Macrolophus melanotoma Costa (= M. caliginosus Wagner) and Macrolophus pygmaeus (Rambur) based exclusively on the different colour pattern of the first antennal segment (white central band in M. melanotoma and entirely black in M. pygmaeus) is rather precarious and their taxonomic status is still in doubt. In the present study their taxonomic status was evaluated by DNA confirmatory analysis and hybridization experiments between M. pygmaeus and a Macrolophus taxon, resembling M. melanotoma, with a first antennal segment entirely black or with a white central band collected from Dittrichia viscosa (L.) W. Greuter (Asteraceae) in southern Greece. Adult females from Dittrichia plants hybridized with males of M. pygmaeus and vice versa did not produce viable eggs. The Macrolophus species from Dittrichia irrespective of the first antennal segment coloration differed from M. pygmaeusin digestive patterns generated by AseI, XbaI, and MseI on 16S rRNA and in RAPD profiles produced by the primers OPA-18 and OPA-20. These results demonstrate that on Dittrichia plants there is a distinct dimorphic taxon, M. melanotoma, as it is the only species of the genus Macrolophus bearing a first antennal segment with a central white band. Given the limitation of the coloration pattern, the mtDNA genetic markers are the appropriate method for the identification of M. melanotomaand M. pygmaeus.

Biochemical Genetic Variability in Brown Hares (Lepus europaeus) From Greece

Allozyme variability of 91 brown hares (Lepus europaeus) from seven regions in Greece was compared to existing data of Bulgarian populations to test the hypothesis of the occurrence of specific alleles in Greece, likely stemming from an isolated Late Pleistocene refugial population in the southern Balkans. This hypothesis is particularly suggested by some subfossil Late Pleistocene hare remains in Greece and the reported high mtDNA diversity in Greek hares. Allozymic diversity could be higher in Greek hares than in hares from neighboring regions as a result of the accumulation of variants in a long-lasting Pleistocene refugium. Conversely, Greek hares could exhibit reduced genetic diversity because of long-lasting low effective population sizes during the Late Glacial Maximum and a lower chance of postglacial gene flow from other populations into this rather marginal part in the southern Balkans. Horizontal starch gel electrophoresis of proteins from 35~loci revealed three alleles (Es-1−162, Pep-2114, Mpi88) at low frequencies, which were not found in Bulgarian or any other brown hare population. In contrast, some alleles from the populations from Bulgaria and other regions of Europe were absent in the Greek samples. Population genetic statistics indicated only a slight tendency of increased gene pool diversity in Greek hares, little substructuring in Greek and Bulgarian populations, respectively, as well as an only slightly lower level of gene flow between the two neighboring regions, as compared to the gene flow within each region. The results conform to the hypothesis of a Late Pleistocene refugial population in the southern Balkans, with some few specific nuclear gene pool characteristics, but little effect on the overall genetic differentiation between Greek and Bulgarian hares.

RFLP analysis of mitochondrial DNA to evaluate genetic variation in striped red mullet (Mullus surmuletus L.) and red mullet (Mullus barbatus L.) populations.

Abstract: The genetic differentiation of striped red mullet (Mullus surmuletus) and red mullet (Mullus barbatus) was investigated in 6 Mediterranean populations of each species by means of restriction fragment length polymorphism analysis of mitochondrial DNA. Three segments amplified by polymerase chain reaction (control region, COI, and 12S–16S ribosomal RNA) were digested with 20 restriction endonucleases, revealing 71 haplotypes for M. surmuletus and 30 for M. barbatus. For the two species nucleotide diversity was equally distributed within and among populations, leading to NST values of 0.545 and 0.500 for M. surmuletus and M. barbatus, respectively. However, intrapopulation and interpopulation genetic structuring appeared to be much higher for M. surmuletus than for M. barbatus (1.88% vs. 0.46% of mean intrapopulation nucleotide diversity; 1.94% vs. 0.47% of mean interpopulation nucleotide diversity; 0.055% vs. 0.002% of net interpopulation divergence). Furthermore, 81.69% of the haplotypes observed for M. surmuletus were unique, whereas 70.29% of M. barbatus individuals were grouped in 3 common haplotypes. Given that fishing pressure and population sizes are similar for both species, this differentiation could be attributed to differences in biological parameters and life histories between the two species, coupled with oceanographic conditions prevailing in the studied area.

Domestication does not narrow MHC diversity in Sus scrofa

The Major Histocompatibility Complex (MHC) is a multigene family of outstanding polymorphism. MHC molecules bind antigenic peptides in the peptide-binding region (PBR) that consists of five binding pockets (P). In this study, we compared the genetic diversity of domestic pigs to that of the modern representatives of their wild ancestors, the wild boar, in two MHC loci, the oligomorphic DQA and the polymorphic DRB1. MHC nucleotide polymorphism was compared with the actual functional polymorphism in the PBR and the binding pockets P1, P4, P6, P7, and P9. The analysis of approximately 200 wild boars collected throughout Europe and 120 domestic pigs from four breeds (three pureblood, Pietrain, Leicoma, and Landrace, and one mixed Danbred) revealed that wild boars and domestic pigs share the same levels of nucleotide and amino acid polymorphism, allelic richness, and heterozygosity. Domestication did not appear to act as a bottleneck that would narrow MHC diversity. Although the pattern of polymorphism was uniform between the two loci, the magnitude of polymorphism was different. For both loci, most of the polymorphism was located in the PBR region and the presence of positive selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous substitutions in the PBR. P4 and P6 were the most polymorphic binding pockets. Functional polymorphism, i.e., the number and the distribution of pocket variants within and among populations, was significantly narrower than genetic polymorphism, indicative of a hierarchical action of selection pressures on MHC loci.

Evolution of European Cockchafers (Melolonthinae: Scarabaeidae: Coleoptera): a morphological, molecular and chromosomal study of intra- and inter-specific variations

In cockchafers of the genus Melolontha, there is a marked intraspecific polymorphism for morphological characters, making some specimens of one species resemble another. A cytogenetic and molecular (mitochondrial COI gene sequence) study of typical and atypical forms of M. melolontha and M. hippocastani, captured at the same period and area, was performed. Karyotypes and haplotypes clearly characterize each taxon, placing atypical specimens in one or the other species unambiguously. This formally discards the role of hybridization in phenotypic resemblance, as usually proposed. Karyotypes and haplotypes were compared to those of M. pectoralis and Phyllophaga pleei, a more distantly related Melolonthinae, and some Dynastinae species, to reconstruct their ancestral karyotype. The karyotype of M. melolontha is the most derivative and that of P. pleei the most conserved among the Melolonthinae studied, which fits with the phylogeny established by COI gene analysis. Both karyotypes and COI haplotypes demonstrate the proximity of M. pectoralis and M. melolontha. The karyotype of M. melolontha is polymorphic, without relationship with morphological variations. Finally, the existence of similar morphological variations in different Melolontha species and chromosomal polymorphism in M. melolontha is discussed in relation with a network (reticulated) mode of speciation.

Genetic structure of three marine fishes from the Gulf of Pagasitikos (Greece) based on allozymes, RAPD, and mtDNA RFLP markers

In the present work we used three molecular techniques (allozymes, RAPDs and mtDNA RFLPs) in order to study the genetic structure of three commercial marine species (Mullus surmuletus, Mullus barbatus, and Pagellus erythrinus). Each species was sampled from three locations within the Gulf of Pagasitikos, Greece and from two neighbouring locations outside the Gulf (Trikeri and Alonissos). Values of genetic heterozygosity and nucleotide diversity for all populations studied were similar or above the mean values observed in marine fishes. None of the three types of molecular markers used revealed diagnostic patterns, which could allow the allocation of individuals to one of the populations. The analyses revealed that the three populations within Pagasitikos were homogenous representing thus a panmictic stock. However, there were evidences of genetic population subdivision between localities from inside and outside of the Pagasitikos Gulf. The results provide essential information for the design of a sustainable management plan of the Gulf of Pagasitikos and its demersal fish resources.