Elena Klossa-Kilia

Genetic Divergence and Phylogenetic Relationships in Grey Mullets (Teleostei: Mugilidae) Based on PCR–RFLP Analysis of mtDNA Segments

The genetic differentiation and phylogenetic relationships among five species of the Mugilidae family (Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens) were investigated at the mtDNA level, on samples taken from Messolongi lagoon-Greece. RFLP analysis of three PCR-amplified mtDNA gene segments (12s rRNA, 16s rRNA, and CO I) was used. Ten, eight, and nine restriction enzymes were found to have at least one recognition site at 12s rRNA, 16s rRNA, and CO I genes, respectively. Several fragment patterns were revealed to be species-specific, and thus they could be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. Seven different haplotypes were detected. The greatest amount of genetic differentiation was observed at the interspecific level, while little variation was revealed at the intraspecific level. The highest values of nucleotide sequence divergence were observed between M. cephalus and all the other species, while the lowest was found between C. labrosus and L. saliens. Dendrograms obtained by the three different methods (UPGMA, Neighbor-Joining, and Dollo parsimony), were found to exhibit in all cases the same topology. According to this, the most distinct species is M. cephalus, while the other species are clustered in two separate groups, the first one containing L. aurata and L. ramada, the other L. saliens and C. labrosus. This last clustering makes the monophyletic origin of the genus Liza questionable.

Genetic Divergence and Phylogenetic Relationships in Grey Mullets (Teleostei: Mugilidae) Using Allozyme Data

Genetic divergence and phylogenetic relationships among five species of the Mugilidae family (Liza saliens, Liza aurata, Liza ramada, Chelon labrosus, and Mugil cephalus) were investigated, in the present study, on samples taken from Messolongi lagoon in Greece, using allozyme electrophoresis. Ten enzymic systems corresponding to 22 genetic loci were assayed, among which, four were found to be polymorphic in Liza saliens, Liza aurata, and Chelon labrosus, seven in Liza ramada, while only two were polymorphic in Mugil cephalus. Several loci showed different electrophoretic patterns among the species and thus, they can be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. It must be underlined that among them the aGPD-2* locus proved to be species-specific, while the other ones can be also used in various combinations for the same purpose. The observed heterozygosity was found to range from 0.020 to 0.051. Allele frequencies of all loci were used to estimate Neis (1972) genetic distance, which was found to range between 0.249 and 1.171 among the five species studied. UPGMA and NJ trees, obtained by genetic distance matrix methods, as well as, a tree based on the discrete character parsimony analysis were found to exhibit the same topology. Our result show that the three species of the genus Liza are clustered together, Chelon labrosus being closer to the previous clade, while Mugil cephalus being more distinct.

Mitochondrial DNA diversity in Atherina boyeri populations as determined by RFLP analysis of three mtDNA segments.

The genetic differentiation and the phylogenetic relationships of eight Atherina boyeri Greek populations have been investigated at the mtDNA level. The populations studied are from two different lakes, a lagoon, the interface zone between the lagoon and the sea, and four marine sites. RFLP analysis of three mtDNA segments (12s rRNA, 16s rRNA and D-loop) amplified by PCR was used. Six, seven and eight restriction enzymes were found to have at least one recognition site at 12s rRNA, 16s rRNA and D-loop respectively. Twenty-one different haplotypes were detected among the populations studied. Several restriction patterns were revealed. These patterns can be used for the discrimination of the populations living in the sea (‘marine’ type populations) from the others inhabiting the lagoon and the lakes (‘lagoon’ type populations). The estimated net nucleotide sequence divergence between the populations examined ranged from 0 to 10.385%, while the Nst value of 0.92 indicates the existence of high interpopulation genetic differentiation. This high degree of differentiation detected between the ‘lagoon’ and ‘marine’ type populations makes the classification of these two types of populations as a single taxon questionable.

Molecular Phylogeny of Grey Mullets (Teleostei: Mugilidae) in Greece: Evidence from Sequence Analysis of mtDNA Segments

Mitochondrial DNA sequence analysis has been used to explore genetic differentiation and phylogenetic relationships among five species of the Mugilidae family, Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens. DNA was isolated from samples originating from the Messolongi Lagoon in Greece. Three mtDNA segments (12s rRNA, 16s rRNA, and CO I) were PCR amplified and sequenced. Sequencing analysis revealed that the greatest genetic differentiation was observed between M. cephalus and all the other species studied, while C. labrosus and L. aurata were the closest taxa. Dendrograms obtained by the neighbor-joining method and Bayesian inference analysis exhibited the same topology. According to this topology, M. cephalus is the most distinct species and the remaining taxa are clustered together, with C. labrosus and L. aurata forming a single group. The latter result brings into question the monophyletic origin of the genus Liza.

Molecular phylogeny of the Greek populations of the genus Ligidium (Isopoda, Oniscidea) using three mtDNA gene segments

The phylogeny of Greek populations of the terrestrial isopod genus Ligidium is reconstructed based on three mtDNA gene segments: 12S rRNA, 16S rRNA and COI. Two widely distributed European species, as well as three outgroups belonging to different isopod genera, were also included in the analyses. The samples used represent almost all Ligidium species known to occur in Greece, as well as several populations of unknown specific status plus some new records. Phylogenetic analyses of the combined data set were performed using Bayesian inference and maximum parsimony. The two main sister clades with good support indicate the sympatric differentiation of two lineages in southern continental Greece (Peloponnisos), where Ligidium populations exhibit a mosaic distribution of sibling species. The insular populations of the Aegean Islands show increased genetic divergence and form separate clades. The presence of a third lineage of Asiatic origin is strongly suggested by both the molecular phylogeny and morphology. The only presumably valid diagnostic morphological character exhibits only partial correspondence to well supported clades of the molecular phylogeny. Genetic differentiation between populations is very high, a fact that can be attributed to the strict ecological specialization of these animals that leads to increased levels of isolation even between populations that are in close proximity. As a consequence, Greek Ligidium populations, especially those present on islands, are unique genetic pools and extremely vulnerable to extinction.

Authentication of Messolongi (Greece) fish roe using PCR-RFLP analysis of 16s rRNA mtDNA segment

Abstract The fish roe of Messolongi is a famous product from Greece with designation of origin. This processed sea food product originates from the whole ovaries of the fish M. cephalus which is captured in Messolongi lagoon – Greece. A PCR–RFLP based method has been developed in our laboratory for the authentication of fish roe of Messolongi. The polymerase chain reaction (PCR) was used for amplification of mitochondrial 16s rRNA gene segment. Digestion of the PCR products with the restriction enzymes BstN I, Taq I and Hinf I followed by agarose gel electrophoresis, yielded species specific restriction patterns, that enabled clear discrimination of the fish roe of Messolongi from the fish roe originating from the other Mugilidae species coexisting in the same area.

Phylogenetic relationships of Greek Apis mellifera subspecies based on sequencing of mtDNA segments (COI and ND5)

Summary Honey bee Apis mellifera L. populations from various areas of Albania, Bulgaria, Cyprus, Greece, Italy, Slovenia and Turkey, together with a genetically improved commercial strain exported all over the world were studied using sequencing analysis of two mitochondrial regions, the ND5 and the COI gene segments. The aim of this study was to examine the phylogenetic relationships among these honey bee populations, to investigate the existence of gene flow as a result of migratory beekeeping and commercial breeding, and to find out if these subspecies can be discriminated. Seven and eight different haplotypes were revealed for the COI and ND5 gene segments respectively, while the combined data set consisted of twelve different haplotypes. Among the two DNA segments studied, the highest genetic divergence values were observed in COI. In both genes the highest divergence value was among A. m. ligustica colonies and all others. All the phylogenetic trees constructed by Maximum Parsimony, Neighbour-Joining and Bayesian Inference analyses exhibited exactly the same topology for COI and ND5 separately, as well as for the concatenated data set; A. m. ligustica forms the most distinct clade. Our study presents the first comprehensive sequencing analysis of A. mellifera subspecies occurring in Greece and it is the first time that sequencing data from ND5 mtDNA gene segment have been obtained at the population level.

Genetic Divergence Among Marine and Lagoon Atherina boyeri Populations in Greece Using mtDNA Analysis

Genetic differentiation and phylogenetic relationships among 15 Atherina boyeri populations from several marine and lagoon or lake sites in Greece were investigated using mtDNA analysis. PCR-RFLP analysis of 12s, 16s rRNA genes and D-loop revealed 23 haplotypes. All the lake or lagoon populations, as well as the Kymi and Kalymnos populations that originated from sites with lagoonlike environmental conditions, showed haplotypes 1–6, clearly distinguishable from the marine populations, which exhibited types 7–23. The genetic divergence values estimated between the lagoon and the marine populations ranged from 5.55 to 10.45%. The high genetic differentiation observed between these two types of populations is also highlighted by the dendrograms obtained using UPGMA and maximum parsimony methods.

Old Aegean palaeoevents driving the diversification of an endemic isopod species (Oniscidea, Trachelipodidae)

The Greek endemic isopod species Trachelipus aegaeus is distributed in Aegean islands and the adjacent coastal parts of the Greek mainland. Major palaeogeographic events of the Aegean archipelago, such as the formation of the mid‐Aegean trench and the Messinian Salinity Crisis, have been often employed as major causal factors of evolutionary events and phylogeographic patterns exhibited by several taxa. Herein, we infer phylogenetic relationships among T. aegaeus populations using partial cytochrome oxidase subunit I (COI) and 16S rRNA sequences. Due to the poor preservation of the specimens, we propose a modified DNA extraction protocol, which returned highly positive results in terms of the quality of the total extracted DNA. We implement a calibrated molecular clock and path sampling analysis, using alternative palaeogeographic events and rates of substitution, to evaluate the biogeographic history of the species and to estimate the chronology of diversification events among its populations. Our results are clearly in favour of the scenario of the MAT triggering vicariance among most T. aegaus populations. Moreover, the large intraspecific genetic divergence (0–19% for COI and 0–20.3% for the 16S rRNA) and the overall phylogeographic patterns depicted herein seem not to have been obscured by more recent palaeogeological events. A role of dispersal, probably human‐aided, is assumed for certain ‘deviant’ cases.

Genetic diversity of Atherina hepsetus (Osteichthyes: Atherinidae) populations as determined by RFLP analysis of three mtDNA regions

The genetic differentiation of 11 Greek Atherina hepsetus populations have been investigated at the mtDNA level. Restriction fragment length polymorphism analysis of three mtDNA regions (12S rRNA, 16S rRNA and D‐loop) amplified by PCR was used. Eighteen restriction endonucleases were found to have at least one recognition site at the three mtDNA regions studied. Fifteen different composite haplotypes were detected among the populations studied while 80.6% of the individuals were grouped in two common haplotypes. The estimated net nucleotide sequence divergence values between the populations ranged from 0.003 to 0.379%, while the Nst value of 0.092 indicates the existence of low interpopulation genetic differentiation. The findings clearly support the existence of a single panmictic population in the sampling area.