Population genetics of the spittlebug genus Philaenus (Homoptera: Gercopidae) in Greece

The electrophoretically detectable variability of 18 enzyme loci was studied in three species of the genus Philaenus: P. loukasi, P. spumarius and P. signatus . Four natural populations of P. loukasi , three of P. spumarius and two of P. signatus were analysed. Two loci, aldehyde oxidase-1 and aldehyde oxidase-2 were found to be in strong linkage disequilibrium in both populations of P. signatus . The enzyme loci acid phosphatase, peptidase-2, mannose-phosphate isomerase and aldehyde oxidase-2 proved to be powerful tools for the identification of the species in question. The colour polymorphism is discussed as well as the low average heterozygosities maintained by the three species (0.099 for P. loukasi , 0.027 for P. spumarius and 0.066 for P. signatus) . On the basis of electrophoretic data a phylogenetic tree is constructed.     

Differential population structuring of two closely related fish species, the mackerel (Scomber scombrus) and the chub mackerel (Scomber japonicus), in the Mediterranean Sea

Population genetic structures of the mackerel (Scomber scombrus) and chub mackerel (Scomber japonicus) were studied in the Mediterranean Sea. Fragments of 272 bp (S. scomber) and 387 bp (S. japonicus) of the 5'-end of the mitochondrial control region were sequenced from spawning individuals collected off the coasts of Greece, Italy, Spain, and Portugal. High levels of mitochondrial control region haplotypic diversity (> 0.98) were found for both Scomber species. Nucleotide diversity was higher in the mackerel (0.022) than in the chub mackerel (0.017). Global F(ST) values were also higher and significant in the mackerel (0.024, P < 0.0001) as opposed to the chub mackerel (0.003, P > 0.05). Molecular variance analyses showed differential genetic structuring for these two closely related species. There is extensive gene flow between Mediterranean Sea and Atlantic Ocean populations of chub mackerel, which are organized into a larger panmictic unit. In contrast, Mediterranean Sea populations of mackerel show some degree of genetic differentiation and are structured along an east-west axis. The analysed eastern Mediterranean Sea mackerel populations (Greece, Italy) are clearly separated from that of the western Mediterranean Sea (Barcelona), which forms a panmictic unit with eastern Atlantic Ocean populations. The genetic structures of both species showed asymmetric migration patterns and indicated population expansion.     

Genetic divergence in Greek populations of the genus Leuciscus and its evolutionary and biogeographical implications

In 23 populations of Greek Leuciscus ( Squalius ), the percentage of polymorphic allozyme loci ranged between 0·034 and 0·379 (P=0·19) and expected heterozygosity was 0·011–0·166 (H_e=0·067). Current taxonomy is confusing and does not correspond to genetic data that support the presence in Greece of at least seven different species: L. cephalus , L. peloponnensis , L. prespensis , L. moreoticus , L. borysthenicus , L. keadicus and Leuciscus sp. from Euboea Island. The maximum Nei modified genetic distance was found among L. keadicus and the rest of subgenus Squalius populations D * Nei=0·446–0·705). Accepting the molecular clock hypothesis, speciation for the genus Leuciscus in Greece must have occurred during the Cenozoic period (between the Middle Miocene and the Later Pliocene). The two main biogeographical events causing speciation on mainland Greece were the Uplift of the Pindic cordillera and the isolation of the southern part of Peloponnesus. The faunistic composition of the lakes studied, in which new taxa are reconsidered, suggests the same faunistic origin in the Early Pliocene for Lakes Prespa and Stymphalia and a younger one in the Late Pliocene for Lake Trichonis. Euboea Island was not a zoogeographical unit during the Cenozoic. The isolation of all the freshwater fish fauna of Euboea has occurred since the Pliocene. The biogeographical model proposed here differs from classical hypotheses in considering of lesser importance the dispersion of L. cephalus on the Greek mainland during the Neogene and Quaternary.     

High genetic diversity in southwest Asian populations of Philaenus spumarius (Hemiptera: Auchenorrhyncha)

The genus Philaenus is one of the best investigated among Auchenorrhyncha, and several morphological, ecological, karyological, and molecular data have led to a designation of up to 10 species, distributed mainly in the Mediterranean and south-western Asia. The only widespread Palaearctic species, P. spumarius, is known to be structured phylogeographically as it consists of two highly divergent mitochondrial clades (northeast, NE and southwest, SW), with several subclades. This study contributes to the species phylogeography through the study of the genetic diversity and affinity of P. spumarius populations from southwestern Asia. Mitochondrial DNA (cytochrome B) show a high level of genetic diversity within Turkish and Iranian populations, the majority of which belong to the SW clade, and only single populations from northeastern Turkey are found to be substantially highly divergent lineages within the NE clade. One of the NE populations also showed significant differences in the distribution and amount of heterochromatin compared to other populations. According to the results of this study and previous phylogenetic and phylogeographic works on this species, we conclude that Southwestern Asia is probably the place of origin of the Philaenus spumarius.     

Direct measurement of sodium and potassium in the transpiration stream of salt-excluding and non-excluding varieties of wheat

The xylem-feeding insect Philaenus spumarius was used to analyse sodium and potassium fluxes in the xylem of intact, transpiring wheat plants. Two cultivars were compared: the salt-excluding (Chinese Spring) and the non-excluding (Langdon). Chinese Spring accumulated much less sodium in its leaves than the salt-sensitive Langdon. After 7 d in 150 mol m(-3) NaCl, the sodium concentration in the leaf sap of Langdon reached over 600 mol m(-3). This was some three-fold greater than that in Chinese Spring. Similar findings have previously been reported from these cultivars. The reduced ion accumulation was specific to sodium; accumulation of K(+) was unaffected by NaCl in Chinese Spring, such that it developed a much lower leaf Na(+)/K(+) ratio than Langdon. The spittlebug, P. spumarius was used to sample xylem sap from both cultivars. This approach showed that the leaf xylem sap of Chinese Spring had much lower levels of sodium than that of Langdon. In the 150 mol m(-3) NaCl treatment, sodium levels in the leaf xylem reached only 2-3 mol m(-3) in Chinese Spring, compared with 8-10 mol m(-3) in Langdon. Transpiration rates were found to be similar in the two varieties. The lower leaf xylem content alone was thus sufficient to account for the reduced accumulation of sodium in leaves of Chinese Spring. The mechanisms by which xylem sodium might be lowered are discussed and it is concluded that sodium is probably excluded from the xylem in the root of Chinese Spring.     

Relics of the Europe's warm past: phylogeography of the Aesculapian snake

Understanding how species responded to past climate change can provide information about how they may respond to the current global warming. Here we show how a European reptile species responded to the last natural global warming event at the Pleistocene-Holocene transition that led to the Holocene climatic optimum approximately 5000-8000 years ago. The Aesculapian snake, Zamenis longissimus, is a thermophilous species whose present-day distribution in the southern half of Europe is a remnant of much wider range during the Holocene climatic optimum when populations occurred as far north as Denmark. These northern populations went extinct as the climate cooled, and presently the species is extinct from all central Europe, except few relic populations in locally suitable microhabitats in Germany and the Czech Republic. Our phylogenetic and demographic analyses identified two major clades that expanded from their respective western and eastern refugia after the last glacial maximum (18,000-23,000 years ago) and contributed approximately equally to the present range. Snakes from the relic northern populations carried the Eastern clade, showing that it was primarily the snakes from the eastern, probably Balkan, refugium that occupied the central and northern Europe during the Holocene climatic optimum. Two small, deep-branching clades were identified in near the Black Sea and in Greece. These clades provide evidence for two additional refugia, which did not successfully contribute to the colonization of Europe. If, as our results suggest, some populations responded to the mid-Holocene global warming by shifting their ranges further north than other populations of the same species, knowing what populations were able to expand in different species may provide information about what populations will be important for the species' ability to cope with the current global warming.     

Enemy at the gates: introduction potential of non-indigenous freshwater crayfish in Greece via the aquarium trade

Indigenous freshwater crayfish species (ICS) are important biodiversity components and desirable fishery targets. However, ICS populations are increasingly threatened by various anthropogenic stressors. Moreover, established populations of non-indigenous freshwater crayfish species (NICS) and new ‘waves’ of NICS introductions exert significant pressure on ICS populations at a pan-European level. These effects include direct competition for space and resources as well as crayfish ‘plague’ transmission from introduced North American species. Given low public knowledge of this problematic, considerable risk of future introductions exist as a result of conventional and internet-based aquarium trade, which often lead to deliberate and/or accidental releases of NICS into the wild. In 2011, we conducted a survey of freshwater crayfish species in eleven large-size pet shops located in three major cities and in three large internet-based aquarium companies in Greece. Overall, eight species belonging to three genera (Procambarus, Cherax and Cambarellus) were recorded, originating from the USA, Australia, New Guinea and Mexico. The invasion potential of the three most popular species was assessed using the Freshwater Invertebrate Invasiveness Scoring Kit (FI-ISK). Two species were determined to constitute a ‘very high risk’ of invasion. As such, regulatory measures need to be implemented to monitor the ornamental trade of NICS in Greece and a national framework developed for protecting ICS.     

Allozyme diversity in Pinus virginiana (Pinaceae): intraspecific and interspecific comparisons

Two of the four members of subsection Contortae of the genus Pinus occur in the southeastern United States: Pinus virginiana, which ranges throughout the southern and central Appalachian Mountains, and P. clausa, which is restricted to Florida and southern Alabama. We examined allozyme variation within P. virginiana and genetic relationships between this species and the two varieties of P. clausa (var. clausa and var. immuginata). P. virginiana maintains more genetic diversity at both the species (Hes = 0.139) and population (Hep = 0.128) levels than the other three species in the subsection, which may reflect the combination of its widespread distribution and the absence of cone serotiny. Genetic differentiation among populations in P. virginiana was relatively low (GST = 0.053), but significant contrasts in allozyme frequencies and genetic diversity were apparent between populations to the northwest vs. outheast of the Appalachian Mountains. These regional differences likely resulted initially from historical processes that occurred during the Pleistocene and early Holocene, and have been reinforced by modern selective pressures and barriers to gene flow. The mean genetic distance between populations of P. virginiana and P. clausa (D = 0.071) was greater than that between populations of the two varieties of P. clausa (D = 0.012), which suggests that the two varieties diverged at some point after the separation of the two species.     

Population genetic structure and secondary endosymbionts of Q Bemisia tabaci (Hemiptera: Aleyrodidae) from Greece

We investigated the molecular diversity of the major agricultural pest Bemisia tabaci and of its associated secondary endosymbionts in Greece. Analyzing mitochondrial DNA, we found that the Q1 (=Q west) is predominant. We used eight microsatellite polymorphic markers to study the genetic structure of 37 populations from mainland and insular Greece, collected on different host species from outdoor and protected crops as well as from non-cultivated plants. In some cases, gene flow was found to be low even between populations separated by just a few kilometres. Bayesian analysis identified two main genetic groups, the first encompassing populations from south Crete and the second composed of populations from north Crete, two other Aegean islands and mainland Greece. Genetic differentiation was not correlated with different host plant species or habitat, or greenhouse versus open environment populations. Gene flow significantly decreased with geographic distance, but no isolation by distance existed when only the samples from mainland Greece or only the samples from Crete were considered. The secondary symbionts Wolbachia and Hamiltonella were present at high frequencies while Arsenophonus, Cardinium and Rickettsia were absent from Greek populations. Multilocus sequence typing of Wolbachia identified two Wolbachia strains. These two strains were found together in most of the populations studied but never in the same host individual. Their role on the observed population structure is discussed.     

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.     

Molecular phylogeny and biogeography of the wall-lizard Podarcis erhardii (Squamata: Lacertidae)

Erhard's wall lizard, Podarcis erhardii (Sauria: Lacertidae), is highly diversified in Greece and especially in the southern Aegean region. Out of the 28 recognized subspecies, 27 are found in Greece from the North Sporades island-complex in the North Aegean (grossly south of the 39th parallel) to the island of Crete in the South. The species exhibits great morphological and ecological plasticity and inhabits many different habitats from rocky islets and sandy shores to mountaintops as high as 2000m. By examining intraspecific variability at a segment of the mitochondrial gene cytochrome b we have found that that extant populations of P. erhardii are paraphyletic. Furthermore, we have found that subspecies previously defined on the basis of morphological characteristics do not correspond to different molecular phylogenetic clades, so that their status should be reconsidered. The DNA based biogeographical and phylogenetic history of Podarcis in Southern Greece is congruent with available paleogeographic data of the region, which supports the view that DNA sequences may be a useful tool for the study of palaeogeography.     

The cytogeographical distribution pattern ofAllium (Alliaceae) in the Greek Peninsula and Islands

Greece is considered as a secondary centre of evolution for the genusAllium since it possesses about 50% of the species known from the whole Flora Europaea area. In the present investigation 44 GreekAllium spp. have been studied and new chromosome counts are reported from 40 populations and 17 species. The distribution of the different cytotypes (x = 7, x = 8, x = 11 and 2n = 2x, 3x, 4x, 5x, 6x, 7x) in Greece is discussed. From the four phytogeographical subdivisions recognized, South continental Greece shows the greatest species and karyotype diversity. This phenomenon is probably due to the geographical position and to the geological history of this area which has received species and populations from different directions. Subsequently, hybridization apparently has been of evolutionary importance.The genusAllium in Greece I.     

The shaping of mitochondrial DNA phylogeographic patterns of the brown hare (Lepus europaeus) under the combined influence of Late Pleistocene climatic fluctuations and anthropogenic translocations

The phylogeographic structure of the brown hare (Lepus europaeus) was studied by analysing mtDNA control region sequences of 98 individuals from continental and insular Greece, Bulgaria, Cyprus and northern Israel, together with 44 published sequences from Italy and central Europe. We found two distinct clades separated by an average nucleotide divergence of 6.6%, which may correspond to a Balkan and to an Asia Minor refugium. The estimated time of separation of the two clades was dated back to 105,000- 490,000 years ago. These two clades coexist in the area of northeastern Greece and Bulgaria, most likely as a result of a post-glacial northward expansion. Within the southern Balkan refugium, network analyses showed geographical structuring, which supports the hypothesis of several isolated Late Pleistocene populations. The central European and Italian populations appear to have originated from a non-detected northern Balkan population that was genetically closely related to some northern Greek populations, as a result of postglacial expansion, translocations or a combination of both. Moreover, several cases of ancient and recent translocations by humans were detected, especially for some island populations, while the eastern Aegean islands off the Asia Minor coast were most likely colonized naturally through Late Pleistocene land bridge connection. The genetic analysis presented here provides a framework for designing proper conservation and management guidelines for this species.     

Phenetic analysis of populations of Porcellionides pruinosus (Brandt, 1833) (Crustacea, Isopoda, Oniscidea)

The cosmopolitan species, Porcellionides pruinosus (Brandt, 1833), exhibited a geographical variation of its morphological features and its reproduction pattern. In fact, some Tunisian populations had a seasonal reproductive period and other ones showed a reproductive activity. A phenetic analysis has been performed to compare populations belonging to P. pruinosus from different geographical localities. For this, nineteen quantitative characters in 800 specimens of P. pruinosus issued from nine populations located in the North, the Centre, and the South of Tunisia, and one population from Athens (Greece) were studied. These populations were characterized by different reproductive behaviours. The populations of Tabarka, Korba, and Tamerza showed reproductive activity, whereas those of Garat Naam, Raccada, Sahline, Gafsa, Rdayef, Gabès, and Athens exhibited a seasonal reproductive period. The variability of the first three axes of the principal components analysis (PCA) and their significance showed an intraspecific variability structure. The graphical representation of the population's dispersion generated by the two first axes (63.55% of the total variability) revealed a great heterogeneity among females of the different populations of P. pruinosus. The Garat Naam population was set apart from the other ones by its cephalic width and length and also by its apophysis length. Furthermore, cephalic and uropod length separated the Garat Naam and Tabarka populations. The dendrogram, based on Euclidean coordinates, confirms an isolation of the populations of Tabarka and Garat Naam, exhibiting respectively reproductive activity and seasonal reproduction. However, the other populations do not show any relationship with the reproduction behaviour. They were clustered in two groups. The first one is represented by the populations of the Southeast and the West of Tunisia. The second pooled the populations of the Northeast and the Centre of Tunisia with the population of Athens (Greece). Phenetic analysis of Tunisian populations exhibits an important heterogeneity, which is worth noting. Thus, we need to complete and deepen this study by genetic analysis and crossbreeding tests in order to define the taxonomic status of these populations.     

Adaptation for oviposition into concealed cycad ovules in the cycad weevils Antliarhinus zamiae and A. signatus (Goleoptera: Gurculionoidea)

Antliarhinus zamiae (Thunberg) and A. signatus Gyllenhal are the only insects known in which the larvae feed exclusively on the cycad gametophyte. In their host plants, species of the cycad genus Encephalartos (Zamiaceae), the gametophyte is protected by a three-layered integument and substantial sporophyll tissues. Adaptation for oviposition into the gametophyte may, therefore, be crucial to this feeding specialization. Ovipositional behaviour and ovipositor structure in four species of Antliarhinus suggest that ovipositional traits suited to ovule predation preceded the evolution of this habit and that ovipositional traits in ancestral species provided the right environment for the evolution of ovule predation. Current ovipositional traits in A. zamiae and A. signatus probably represent responses to changes in cone structure after the evolution of ovule predation.     

Genetic variation of Marchalina hellenica (Hemiptera: Margarodidae) sampled from different hosts and localities in Greece

Random amplified polymorphic DNA (RAPD) analysis was applied to 120 individuals of Marchalina hellenica (Gennadius) representing six populations collected in northern, central and southern mainland Greece. One population was sampled on one species of fir tree and the others on two species of pine trees. Four random decamer primers were used to evaluate genetic variation among the populations examined. The results revealed intra- and interpopulation polymorphism both related to host type and region of origin. Phylogenetic analysis based on genetic distances estimated by the RAPD frequencies revealed an important genetic differentiation in samples collected on fir trees in southern Greece and to a lesser extent in samples from pine trees in central and northern Greece. Furthermore, considerable subdivision and restricted gene flow among the populations examined were observed. The results are discussed in relation to the biology and geographical distribution of M. hellenica in Greece.     

多叶重楼遗传多样性的ISSR分析

采用ISSR分子标记对多叶重楼种下3个变种的8个居群共208份样品的遗传多样性进行了分析。l4条引物共检测到251个清晰的扩增位点,其中多态位点235个。在物种水平上,多态位点百分率(PPB)达93.63%,Nei′s基因多样性指数(HE)为0.2204、Shannon′s信息指数(HO)为0.3532。在居群水平上,多态位点百分率(PPB)为50.45%,Nei′s基因多样性指数(HE)为0.1405,Shannon′s信息指数(HO)为0.2194,这些均表明多叶重楼的遗传多样性水平较高。此外,还用NTSYS软件对样品进行了UPGMA聚类分析,结果显示滇重楼的6个居群聚为一支,滇重楼与狭叶重楼有较近的亲缘关系,而与长药隔重楼之间的遗传分化较大。本研究提出了合理保护重楼植物资源的若干措施,为进一步开展滇重楼优质种质引种驯化,实现种质资源多样性的合理保护和可持续利用提供参考。     

Reproductive attributes of <Emphasis Type="Italic">Campanula</Emphasis> populations from Mt Olympos,Greece

We studied five reproductive attributes (fruit production and volume, seed content and weight, and seed germination) of different populations of Campanula species occurring along the elevation gradient of Mt Olympos, the highest mountain of Greece (2917 m), in order to examine whether and how they change with elevation, and if there are trade-offs among them. We also studied flower and fruit (reproductive) losses and population features in order to estimate whether there might be a threat to the survival of Campanula species on Mt Olympos. The genus is represented by nine species, ranging from cosmopolitan to steno-endemic. All species were found non-autogamous, and hence they depend on pollinators for their sexual reproduction. At the genus level, there is a trade-off between seed content per fruit and seed weight. At the species level, this is manifested only for C. glomerata, which produces the heaviest seeds, far exceeding all other species, and for C. spatulata, represented on Mt Olympos by two elevation distinct subspecies. Changes of the reproductive attributes are not continuous all along the elevation gradient at the genus level nor are they monotonic at the species level. Nevertheless, upland Campanula populations (≥1500 m) produce fewer fruits per individual, containing fewer but heavier seeds than lowland populations. This indicates a transition from ‘r’ to ‘K’ strategies, from low to high elevations, respectively. The patterns of change of reproductive attributes indicate prevalence of phylogenetic over environmental constraints. Laboratory tests showed that for many populations germinability is high at 15 °C, but there are both inter- and intraspecific differences regarding temperature optima. Ecotypic variation among populations of the same species at different elevations was evident; populations from higher elevations had lower optima and vice versa. Reproductive losses, ranging from zero to over 90%, occurred mainly at the stage of fruit maturation; grazing by sheep and goats was the usual cause. Though such losses might affect the short-lived species (the annual C. sparsa and the biennial C. lingulata), which depend entirely on fruit maturation and seed dispersal for their reproduction, there does not seem to be a serious threat to the survival of Campanula species on the mountain.     

Nested clade and phylogeographic analyses of the chub, Leuciscus cephalus (Teleostei, cyprinidae), in Greece: implications for Balkan Peninsula biogeography

Phylogenetic relationships among Greek populations of the chub, Leuciscus cephalus, were investigated using 600 bp of the cytochrome b gene. The aim of this study was to test the assumption that the main difference in ichthyological composition between both sides of the Balkan Peninsula is directly linked to differences in the dispersion mechanisms used by fish in order to extend their distribution range. Phylogenetic and nested clade analyses clearly showed that populations in Greece are significantly differentiated. Greek populations were found to descend from three lineages in three geographical provinces: Western, Central, and Eastern Greece. The chub reached Western Greece at the beginning of the Pleistocene and Eastern Greece during the mid-Pleistocene. Chub dispersion occurred mainly by river confluence due to sea level lowering and river capture in Western Greece and sea dispersal with low-salinity conditions within the Aegean Sea in Eastern Greece. However, in Central Greece, the original mtDNA lineage has presumably been lost owing to a genetic introgression following a second invasion from the Danube during the final stage of the last glaciation. This study provides new elements for a better understanding of the composition of the contemporary ichthyofauna in Greece and highlights possible evolutionary mechanisms responsible for the high endemism rate in the Western Greek biogeographic province.