Nuclear and mitochondrial phylogenetic analysis of big-scale sand smelt Atherina boyeri complex in Greece.

Genetic differentiation and phylogenetic relationships of big-scale sand smelt Atherina boyeri specimens from 23 sampling sites in Greece and one from a lake in Turkey were investigated. A total of 2180 base pairs (bp) corresponding to the partial sequence of the mitochondrial gene cytochrome oxidase I (coI), cytochrome b (cytb) and control region, as well as of the nuclear protein-coding gene rhodopsin (rh), were determined for the 143 specimens studied. Phylogenetic analysis of each gene segment separately and of the combined dataset revealed the existence of three different and well divergent lineages in Greece. The first corresponds to the lagoon form, in which a clear distinction between the Aegean and Ionian Sea was observed. The other two correspond to the punctuated and non-punctuated marine forms that have been previously reported. The fact that in the present study a population without black spots on the flanks is clustered with the punctuated form and vice versa, however, suggests that differences in colour pattern do not seem to be a sufficient marker to discriminate the two marine forms. In contrast, the presence of a different length insertion between transfer RNA (tRNA) glutamic acid (tRNA(glu)) and cytb genes in the lagoon and in one of the two forms of marine populations, and its absence from the rest marine-form specimens confirms that this character is conserved and capable to be used for distinguishing the different forms. Even though the divergence values among the different forms were high, their phylogenetic relationships were not able to be resolved.

Allozyme variability and phylogenetic relationships in honey bee (Hymenoptera: Apidae: Apis mellifera) populations from Greece and Cyprus.

Ten gene enzymic systems (alpha-GPDH, AO, MDH, ADH, LAP, SOD, ALP, ACPH, ME, and EST), corresponding to 12 genetic loci, were assayed from five Greek populations representing three subspecies of Apis mellifera, A. m. cecropia (Pthiotida, Kythira), A. m. macedonica (Macedonia), and the "Aegean race" of A. mellifera, which is supposed to be very similar to A. m. adami (Ikaria, Kasos), as well as a population from Cypus (A. m. cypria). ADH( *)-1, ADH( *)-2, and LAP( *) electrophoretic patterns discriminate the Cyprus population from the Greek populations. MDH( *)-1, EST( *)-3, SOD( *), ALP( *), and ME( *) loci were found to be polymorphic in almost all populations. The observed heterozygosity was found to range from 0.066 to 0.251. Allele frequencies of all loci were used to estimate Nei's genetic distance, which was found to range between 0.011 and 0.413 among the populations studied. UPGMA and neighbor-joining phylogenetic trees obtained by genetic distance matrix methods, as well as a Wagner tree based on the discrete character parsimony method, support the hypothesis that the most distant population is that from Cyprus. Our allozymic data support A. m. cypria as a distinct subspecies, but there was no allozymic support for the distinction of the other subspecies existing in Greece.

Indirect thermal selection in Drosophila melanogaster and adaptive consequences.

Short-term indirect selection in Drosophila melanogaster for heat-sensitivity and heat resistance resulted in two strains, one heat sensitive and another heat resistant, and correlated responses were found for the rate of heat shock protein synthesis, behavioral patterns (asymmetrical sexual isolation) and fitness components (fecundity, fertility, viability, developmental time), as well as for several enzyme activities (MDH, G-6-PDH, ADH, ACHE). These responses associated with temperature selection may reflect the effects of differential inbreeding depression caused by homozygosity of temperature sensitive mutations with different pleiotropic effects. Selection even of a very short duration can induce significant adaptive and evolutionary changes.

The effect of metals and alcohol on sexual isolation in Drosophila melanogaster.

Strong sexual isolation established between D. melanogaster long-term cage populations (originated from common parents and being under selection pressure since 1972) is maintained (with a tendency to increase) for twelve years after the origin of the populations. The sexual isolation is also maintained when the populations are kept in common conditions for about two years, while it dramatically decreases when the populations live on a food medium supplemented with strong chemical selective factors, such as various metals or ethanol. Seasonal or geographical studies of sexual isolation between natural and our cage populations did not reveal significant deviation from random matings. The genetic nature of sexual isolation is discussed.

Mating propensities and variations in enzyme activities in long-term cage populations of Drosophila melanogaster.

Environment-dependent reproductive isolation was established between cage populations (Bs) of Drosophila melanogaster originated from a Greek natural population (summer 1973) and maintained for about five years under different diets (poor-rich). The detected deviation from random mating involved no homogametic or heterogametic preference but rather, a significantly increased activity of males from populations maintained on the rich food medium. This observation indicates that the male parental investment is not negligible and under certain conditions sexual isolation can be a function not only of female behavior but also of male behavior. Differences also were found in various enzyme activities on the inter- and intra-population levels. Given those observations as well as the observed different behavioral patterns of Bs and Cs-Ds populations 19, a preliminary attempt was made to associate adaptive evolution with differences in enzyme activities. The differences in enzyme activities between populations reared on different media are not due to allozymic differences. It also was shown that in some populations environmental effects do not always elicit differences in enzyme activity. It was concluded, therefore, that the observed variations were the result of environmental effects interacting with modifier genes.

Adaptation of Drosophila enzymes to temperature--V. Heat shock effect on the malate dehydrogenase of Drosophila melanogaster.

Drosophila melanogaster cMdh allozymic variants (MdhF MdhF/S, MdhS) were subjected to heat shock (33 degrees C/30 min----40 degrees C/30 min). This stress increases differentially MDH specific activity, with the cMdhF strain showing greater response as compared with the cMdhS one; heterozygotes exhibited generally intermediate values. Correlative differences were also revealed for some catalytic properties (Vmax, Vmax/Km ratio, thermostability) of the cMDH; this is not true for the mMDH. The catalytic behavior of the enzyme is correlated with the differential survival of the cMdh variants, with the cMdhF showing again higher survival than the cMdhS one, a fact which seems to contribute to temperature adaptation of D. melanogaster.

Drosophila lactate dehydrogenase. Functional and evolutionary aspects.

The functional characteristics of homogeneously purified LDH were studied in the eight D. melanogaster species subgroup at two different growing temperatures (14 degrees C, 25 degrees C). The Vmax, kcat, Vmax/KeNAD.KmLac and Kcat/KsNAD KmLac values detected at the permissive growing temperature of 25 degrees C, were found to converge with the consensus phylogeny of these species which consists of two (melanogaster, yakuba) complexes. This scheme, also verified by the Micro-Complement Fixation (MC'F) method in another study in our Laboratory, substantiates the connection between the enzyme function and the phylogeny of these species. We propose that the major variation of the Ldh gene in this subgroup has arisen prior to the first species divergence, the final result of which is the eight sibling species. On the other hand, the variable catalytic differentiation observed at the restrictive temperature of 14 degrees C may enrich the species with hidden adaptive possibilities.

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 wasfound to range from 0.020 to 0.051. Allele frequencies of all loci were used to estimate Nei's (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.

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, thefirst 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.

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.