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The genus Pelophylax has been considered a model organism for understanding hybridization. Despite being the most diverse within the Western Palearctic group of Pelophylax, the ridibundus/bedriagae lineage that includes six species, remains largely understudied, revealing many knowledge gaps in regards to their evolution and conservation. Using genetic data from populations among species inhabiting contact zones could prove vital in filling these gaps. We tested 17 microsatellite markers for cross-species amplification in mainland Pelophylax species distributed in southern Balkans and evaluated their power to successfully detect population/species structure. Importantly, we examined their potential for identifying hybrids and backcrosses between known hybridized species. We detected 12 highly polymorphic loci that cross-amplified all species that showed no significant Linkage Disequilibrium and were able to discriminate among species and between parental and hybrids. We suggest their future use in genetic studies for the genus Pelophylax in Greece, including the identification of contact zones.

The Mediterranean basin constitutes one of the largest global biodiversity hotspots, hosting more than 11,000 endemic plants, and it is recognised as an area with a high proportion of threatened taxa. Nevertheless, only a tiny fraction of the threatened Mediterranean endemics have their genetic diversity assessed, and we are unaware if and how climate change might impact their conservation status. This is even more pronounced in Eastern Mediterranean countries with a rich endemic flora, such as Greece, which hosts a large portion of the plant taxa assessed at the European level under the IUCN criteria. Using inter simple sequence repeats (ISSR) markers and species distribution models, we analysed the genetic diversity and investigated the impacts of climate change on four critically endangered and extremely narrow and rare Greek island endemic plants, namely Aethionema retsina, Allium iatrouinum, Convolvulus argyrothamnos, and Saponaria jagelii. All four species are facing intense anthropogenic threats and display moderate genetic diversity (uHe: 0.254–0.322), while climate change is expected to have a profound impact on their range size during the coming decades. A combination of in- and ex-situ measures, such as population reinforcement and seed bank conservation, are urgently needed in order to preserve these highly threatened and rare Greek endemics.

Aedes aegypti bears the common name “the yellow fever mosquito,” although, today, it is of more concern as the major vector of dengue, chikungunya, and, most recently, Zika viruses. In the present article, we review recent work on the population genetics of this mosquito in efforts to reconstruct its recent (approximately 600 years) history and relate these findings to epidemiological records of occurrences of diseases transmitted by this species. The two sources of information are remarkably congruent. Ae. aegypti was introduced to the New World 400–550 years ago from its ancestral home in West Africa via European slave trade. Ships from the New World returning to their European ports of origin introduced the species to the Mediterranean region around 1800, where it became established until about 1950. The Suez Canal opened in 1869 and Ae. aegypti was introduced into Asia by the 1870s, then on to Australia (1887) and the South Pacific (1904).

Aedes albopictus, the “Asian tiger mosquito,” is an aggressive biting mosquito native to Asia that has colonized all continents except Antarctica during the last ~30–40 years. The species is of great public health concern as it can transmit at least 26 arboviruses, including dengue, chikungunya, and Zika viruses. In this study, using double-digest Restriction site-Associated DNA (ddRAD) sequencing, we developed a panel of ~58,000 single nucleotide polymorphisms (SNPs) based on 20 worldwide Ae. albopictus populations representing both the invasive and the native range. We used this genomic-based approach to study the genetic structure and the differentiation of Ae. albopictus populations and to understand origin(s) and dynamics of the recent invasions. Our analyses indicated the existence of two major genetically differentiated population clusters, each one including both native and invasive populations. The detection of additional genetic structure within each major cluster supports that these SNPs can detect differentiation at a global and local scale, while the similar levels of genomic diversity between native and invasive range populations support the scenario of multiple invasions or colonization by a large number of propagules. Finally, our results revealed the possible source(s) of the recent invasion in Americas, Europe, and Africa, a finding with important implications for vector-control strategies.

Euscorpius tauricus (C. L. Koch, 1837) was previously known only from the Crimea Peninsula, Ukraine. We report an unexpected presence of this species in the Cyclades Islands (Greece) and northwestern Anatolia (Turkey). In addition we designate a neotype for this species. We synonymize Euscorpius carpathicus aegaeus Di Caporiacco, 1950 syn. n. , from Antiparos Island and Euscorpius rahsenae Yagmur et Tropea, 2013 syn. n. , from Anatolia, with E. tauricus . In addition, we describe two new species related to E. tauricus , from the Cyclades Islands: E. curcici sp. n. , from Ios and Sikinos Islands, and E. amorgensis sp. n. , from Amorgos Island. Identity and level of divergence of these taxa is confirmed by multiple DNA markers.

In an attempt to control the mosquito-borne diseases yellow fever, dengue, chikungunya, and Zika fevers, a strain of transgenically modified Aedes aegypti mosquitoes containing a dominant lethal gene has been developed by a commercial company, Oxitec Ltd. If lethality is complete, releasing this strain should only reduce population size and not affect the genetics of the target populations. Approximately 450 thousand males of this strain were released each week for 27 months in Jacobina, Bahia, Brazil. We genotyped the release strain and the target Jacobina population before releases began for21,000 single nucleotide polymorphisms (SNPs). Genetic sampling from the target population six, 12, and 27-30 months after releases commenced provides clear evidence that portions of the transgenic strain genome have been incorporated into the target population. Evidently, rare viable hybrid offspring between the release strain and the Jacobina population are sufficiently robust to be able to reproduce in nature. The release strain was developed using a strain originally from Cuba, then outcrossed to a Mexican population. Thus, Jacobina Ae. aegypti are now a mix of three populations. It is unclear how this may affect disease transmission or affect other efforts to control these dangerous vectors. These results highlight the importance of having in place a genetic monitoring program during such releases to detect un-anticipated outcomes.

Background In the last four decades, the Asian tiger mosquito, Aedes albopictus, vector of several human arboviruses, has spread from its native range in South-East Asia to all over the world, largely through the transportation of its eggs via the international trade in used tires. Albania was the first country invaded in Europe in 1979, followed by Italy in 1990 and other Mediterranean countries after 2000. Methods/Principal findings We here inferred the invasion history and migration patterns of Ae. albopictus in Italy (today the most heavily-infested country in Europe), Greece and Albania, by analyzing a panel of >100,000 single nucleotide polymorphisms (SNPs) obtained by sequencing of double-digest Restriction site-Associated DNA (ddRADseq). The obtained dataset was combined with samples previously analyzed from both the native and invasive range worldwide to interpret the results using a broader spatial and historical context. The emerging evolutionary scenario complements the results of other studies in showing that the extraordinary worldwide expansion of Ae. albopictus has occurred thanks to multiple independent invasions by large numbers of colonists from multiple geographic locations in both native and previously invaded areas, consistently with the role of used tires shipments to move large numbers of eggs worldwide. By analyzing mosquitoes from nine sites across ~1,000-km transect in Italy, we were able to detect a complex interplay of drift, isolation by distance mediated divergence, and gene flow in shaping the species very recent invasion and range expansion, suggesting overall high connectivity, likely due to passive transportation of adults via ground transportation, as well as specific adaptations to local conditions. Conclusions/Significance Results contribute to characterize one of the most successful histories of animal invasion, and could be used as a baseline for future studies to track epidemiologically relevant characters (e.g. insecticide resistance)., Author summary Studying the genomes of invasive populations allows for the tracing of invasion processes. In this study, the authors have focused on the invasion of Albania, Italy and Greece by the Asian tiger mosquito, Aedes albopictus, one of the most successful invasive animal species worldwide, which in the last 40 years has invaded all continents resulting from the transportation of its eggs via human induced dispersion, especially the international used tire trades. Results showed that the invasion process was triggered by multiple independent (and most likely ongoing) importations of large number of eggs from different continents. Afterward, ground transportation of adult mosquitoes (e.g. by cars and trucks) and the capacity of the species to adapt to novel environmental conditions have allowed the colonization of whole countries, as clearly shown to have occurred in Italy. Overall, the results are consistent with a major role of human activities in the extraordinary expansion of the species, which today can be considered a permanent pest in all Mediterranean European countries, where it has already caused the infections of Europeans with exotic viruses (such as chikungunya) once confined to tropical regions and today representing a major concern also in temperate regions.

Most land snails in order to ensure tlieir survival under adverse environmental conditions interrupt tlieir activity entering a state of dormancy. The transition from activity to dormancy is accompanied by several changes in the animais’ physiology. To identify these changes we examined the seasonal patterns in the biochemical composition and the LDH activity of three different tissues in four endangered land snail species. Additionally, within a phylogenetic framework we investigated the correlation of the measured parameters with the spatial and climatic variables of the sampling sites. Our results indicate that there are both differences and similarities in the pattern exhibited by the four species, depending on the physiological parameter investigated and the climatic conditions of the sampling sites, which sometimes have a significant effect on the seasonal pattern exhibited. Snails can successfully deal with the winter dormancy by maintaining high metabolite concentrations and stable water content whereas there is no indication of anaerobio pathways’ activation. At the same time, they deal successfully with the low humidity and high temperatures during the summer, but they are forced to maintain low metabolite concentrations and seem to activate anaerobio pathways to meet their energy demands. Therefore, from a biochemical perspective, it seems that winter is a less stressful period for snails compared to summer. According to the prevailing climate change scenarios, the Mediterranean region is going to exhibit a sudden transition towards a dryer and longer summer. This transition will exert a very high adaptation pressure on the already vulnerable Codringtonia species. Thus, it could be that this endemic Greek genus is truly on the verge of extinction.

Kotschy's Gecko, Mediodactylus kotschyi, is a small gecko native to southeastern Europe and the Levant. It displays great morphological variation with a large number of morphologically recognized subspecies. However, it has been suggested that it constitutes a species complex of several yet unrecognized species. In this study, we used multilocus sequence data (three mitochondrial and three nuclear gene fragments) to estimate the phylogenetic relationships of 174 specimens from 129 sampling localities, covering a substantial part of the distribution range of the species. Our results revealed high genetic diversity of M. kotschyi populations and contributed to our knowledge about the phylogenetic relationships and the estimation of the divergence times between them. Diversification within M. kotschyi began approximately 15 million years ago (Mya) in the Middle Miocene, whereas the diversification within most of the major clades have been occurred in the last 5 Mya. Species delimitation analysis suggests there exists five species within the complex, and we propose to tentatively recognize the following taxa as full species: M. kotschyi (mainland Balkans, most of Aegean islands, and Italy), M. orientalis (Levant, Cyprus, southern Anatolia, and south-eastern Aegean islands), M. danilewskii (Black Sea region and south-western Anatolia), M. bartoni (Crete), and M. oertzeni (southern Dodecanese Islands). This newly recognized diversity underlines the complex biogeographical history of the Eastern Mediterranean region.

International audience; Aedes aegypti, the major vector of dengue, yellow fever, chikungunya, and Zika viruses , remains of great medical and public health concern. There is little doubt that the ancestral home of the species is Africa. This mosquito invaded the New World 400-500 years ago and later, Asia. However, little is known about the genetic structure and history of Ae. aegypti across Africa, as well as the possible origin(s) of the New World invasion. Here, we use ~17,000 genome-wide single nucleotide polymor-phisms (SNPs) to characterize a heretofore undocumented complex picture of this mosquito across its ancestral range in Africa. We find signatures of human-assisted migrations, connectivity across long distances in sylvan populations, and of local ad-mixture between domestic and sylvan populations. Finally, through a phylogenetic analysis combined with the genetic structure analyses, we suggest West Africa and especially Angola as the source of the New World's invasion, a scenario that fits well with the historic record of 16th-century slave trade between Africa and Americas.

A new scorpion species, Euscorpius drenskii sp. nov., is described from the Western Rhodope Mts. in southern Bulgaria. It is characterized by an oligotrichous trichobothrial pattern, which shows a conspicuous loss of one trichobothrium in the external median patellar series (em = 3), also observed in E. carpathicus (Linnaeus, 1767) and the subgenus Alpiscorpius Gantenbein, Fet, Largiadèr & Scholl, 1999. Phylogenetic analysis of 16S rDNA marker sequences does not show any close relationship between these three groups, suggesting that the observed loss of a trichobothrium is an independent event.

Tropea, Gioele, Fet, Victor, Parmakelis, Aristeidis, Kotsakiozi, Panayiota, Stathi, Iasmi (2015): A new species of Euscorpius from Bulgaria and Greece (Scorpiones: Euscorpiidae). Euscorpius 207 (207): 1-15, DOI: 10.18590/euscorpius.2015.vol2015.iss207.1, URL: https://mds.marshall.edu/euscorpius/vol2015/iss207/1/

The Balkan Peninsula constitutes a biodiversity hotspot with high levels of species richness and endemism. The complex geological history of the Balkans in conjunction with the climate evolution are hypothesized as the main drivers generating this biodiversity. We investigated the phylogeography, historical demography, and population structure of closely related wall-lizard species from the Balkan Peninsula and southeastern Europe to better understand diversification processes of species with limited dispersal ability, from Late Miocene to the Holocene. We used several analytical methods integrating genome-wide SNPs (ddRADseq), microsatellites, mitochondrial and nuclear DNA data, as well as species distribution modelling. Phylogenomic analysis resulted in a completely resolved species level phylogeny, population level analyses confirmed the existence of at least two cryptic evolutionary lineages and extensive within species genetic structuring. Divergence time estimations indicated that the Messinian Salinity Crisis played a key role in shaping patterns of species divergence, whereas intraspecific genetic structuring was mainly driven by Pliocene tectonic events and Quaternary climatic oscillations. The present work highlights the effectiveness of utilizing multiple methods and data types coupled with extensive geographic sampling to uncover the evolutionary processes that shaped the species over space and time.

Background Cornu aspersum is a quite intriguing species from the point of view of ecology and evolution and its potential use in medical and environmental applications. It is a species of economic importance since it is farmed and used for culinary purposes. However, the genomic tools that would allow a thorough insight into the ecology, evolution, nutritional and medical properties of this highly adaptable organism, are missing. In this work, using next-generation sequencing (NGS) techniques we assessed a significant portion of the transcriptome of this non-model organism. Results Out of the 9445 de novo assembled contigs, 2886 (30.6%) returned significant hits and for 2261 (24%) of them Gene Ontology (GO) terms associated to the hits were retrieved. A high percentage of the contigs (69.4%) produced no BLASTx hits. The GO terms were grouped to reflect biological processes, molecular functions and cellular components. Certain GO terms were dominant in all groups. After scanning the assembled transcriptome for microsatellites (simple sequence repeats, SSRs), a total of 563 SSRs were recovered. Among the identified SSRs, trinucleotide repeats were the predominant followed by tetranucleotide and dinucleotide repeats. Conclusion The annotation success of the transcriptome of C. aspersum was relatively low. This is probably due to the very limited number of annotated reference genomes existing for mollusc species, especially terrestrial ones. Several biological processes being active in the aestivating species were revealed through the association of the transcripts to enzymes relating to the pathways. The genomic tools provided herein will eventually aid in the study of the global genomic diversity of the species and the investigation of aspects of the ecology, evolution, behavior, nutritional and medical properties of this highly adaptable organism.

Seasonal differences in the water content of four Codringtonia species were investigated using specimens collected from the field. In addition, rate of water loss and expression of heat-shock protein 70 (Hsp70) were measured in a laboratory setting with six Codringtonia species subjected to a short-term heat shock. Using a phylogenetic framework, both the Hsp70 expression levels and the rate of water loss were investigated for their correlation with spatial and climatic variables. As indicated by the field-collected samples, during summer aestivation only C. helenae exhibited a tendency for water loss. During the short-term heat shock the rate of water loss in C. helenae was also significantly greater. No interspecific differences could be detected in the levels of Hsp70 in the species subjected to short-term heat shock. A single Codringtonia species seemed to maintain increased Hsp70 protein levels. In the species subjected to short-term heat shock, a positive relationship was found between Hsp70 levels and rate of water loss. On the other hand, the Hsp70 levels under normal conditions showed a negative correlation with altitude and mean summer precipitation of the sampling localities. Thus, species seem to adapt to harsher environmental conditions by maintaining higher levels of Hsp70.

Three new species of the genus Euscorpius Thorell, 1876 are described from Greece: E. stahlavskyi sp.n. from Epirus in the northwestern Greece; E. kinzelbachi sp.n. from Mt. Olympus at the eastern border between Thessaly and Central Macedonia, and E. vignai sp.n. from Karpathos and Kasos Islands (eastern Aegean Sea). Species-level divergence of these taxa is also confirmed by multiple DNA markers in Parmakelis et al. (2013).

Phylogenetic analysis of the genus Euscorpius (Scorpiones: Euscorpiidae) across the Mediterranean region (86 specimens, 77 localities, four DNA markers: 16S rDNA, COI, COII, and ITS1), focusing on Greek fauna, revealed high variation, deep clade divergences, many cryptic lineages, paraphyly at subgenus level, and sympatry of several new and formerly known lineages. Numerous specimens from mainland and insular Greece, undoubtedly the least studied region of the genus' distribution, have been included. The reconstructed phylogeny covers representative taxa and populations across the entire genus of Euscorpius. The deepest clades detected within Euscorpius correspond (partially) to its current subgeneric division, outlining subgenera Tetratrichobothrius and Alpiscorpius. The rest of the genus falls into several clades, including subgenus Polytrichobothrius and a paraphyletic subgenus Euscorpius s.s. Several cryptic lineages are recovered, especially on the islands. The inadequacy of the morphological characters used in the taxonomy of the genus to delineate species is discussed. Finally, the time frame of differentiation of Euscorpius in the study region is estimated and the distributional patterns of the lineages are contrasted with those of other highly diversified invertebrate genera occurring in the study region. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110, 728–748.

Extensive variation in land snail shell morphology has been widely documented, although few studies have attempted to investigate the ecological and evolutionary drivers of this variation. Within a comparative phylogenetic framework, we investigated the temporal and spatial evolution of the shell morphology of the Greek endemic land snail genus Codringtonia. The contribution of both inter- and intraspecies shell differentiation in the overall shell variability is assessed. The effect of climate, space, and evolutionary history on the shell variability was inferred using a variance partitioning framework. For Codringtonia species, intraspecies divergence of shell traits contributes substantially to the overall shell variability. By decomposing this variability, it was shown that the overall shell size of Codringtonia clades is phylogenetically constrained, related to early speciation events, and strongly affected by large-scale spatial variability (latitudinal gradient). The effect of climate on shell size cannot be disentangled from phylogeny and space. Shell and, to a larger extent, aperture shape are not phylogenetically constrained, and appear to be mostly related to conspecific populations divergence events. Shell shape is substantially explained by both climate and space that greatly overlap. Aperture shape is mainly interpreted by medium to small-scale spatial variables. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110, 796–813.

Euscorpius avcii Tropea et al., 2012 has been recently described from Dilek Peninsula in western Anatolia (Turkey, Aydin Province). The population from Samos Island in eastern part of the Aegean Sea is found to match closely the Anatolian E. avcii , making it a new, rare species for the Greek fauna, confirmed by two DNA markers as well as morphology. Samos also shares with western Anatolia two other local recently described scorpion species, Iurus kinzelbachi and Neocalchas gruberi (family Iuridae).

Land snails usually exhibit cycles of activity and dormancy (aestivation or hibernation). The transition between these two states is accompanied by a range of behavioural and physiological responses to ensure their survival under adverse environmental conditions. Furthermore, aestivation plays an important role in shaping species’ distribution patterns. We examined the seasonal patterns in biochemical tissue composition in relation to aspects of behavioural ecology in three land snail populations: one mainland and one insular population of the widespread Helix aspersa and a population (sympatric with the latter) of Helix figulina, a congeneric species with a narrow and declining distribution. Helix figulina aestivates in underground borrows, while H. aspersa spends the summer under stones and may interrupt aestivation when conditions become favourable. Prior to aestivation H. figulina accumulates metabolic fuels, which it consumes later during summer, and at the same time loses substantial body water and increases lactate dehydrogenase (LDH) activity. The insular H. aspersa population follows a similar pattern (regarding metabolites and LDH activity), with the difference that water loss is limited. However, the mainland population of H. aspersa deviates from this model with energy metabolites and water levels showing little variation throughout the year, while LDH activity is reduced. These differences probably reflect the particular behavioural and physiological patterns adopted by each species. The specialist and range-restricted H. figulina shows a constant and more predictable seasonal pattern, which may be effective for surviving in its historical biogeographic range, but it seems to be more vulnerable to possibly changing environmental conditions. On the other hand the generalist and cosmopolitan H. aspersa adopts a more flexible pattern that compensates for the effects of adverse conditions during aestivation and permits a more effective exploitation of energy resources.

Background Aedes aegypti, commonly known as “the yellow fever mosquito”, is of great medical concern today primarily as the major vector of dengue, chikungunya and Zika viruses, although yellow fever remains a serious health concern in some regions. The history of Ae. aegypti in Brazil is of particular interest because the country was subjected to a well-documented eradication program during 1940s-1950s. After cessation of the campaign, the mosquito quickly re-established in the early 1970s with several dengue outbreaks reported during the last 30 years. Brazil can be considered the country suffering the most from the yellow fever mosquito, given the high number of dengue, chikungunya and Zika cases reported in the country, after having once been declared “free of Ae. aegypti”. Methodology/Principal findings We used 12 microsatellite markers to infer the genetic structure of Brazilian Ae. aegypti populations, genetic variability, genetic affinities with neighboring geographic areas, and the timing of their arrival and spread. This enabled us to reconstruct their recent history and evaluate whether the reappearance in Brazil was the result of re-invasion from neighboring non-eradicated areas or re-emergence from local refugia surviving the eradication program. Our results indicate a genetic break separating the northern and southern Brazilian Ae. aegypti populations, with further genetic differentiation within each cluster, especially in southern Brazil. Conclusions/Significance Based on our results, re-invasions from non-eradicated regions are the most likely scenario for the reappearance of Ae. aegypti in Brazil. While populations in the northern cluster are likely to have descended from Venezuela populations as early as the 1970s, southern populations seem to have derived more recently from northern Brazilian areas. Possible entry points are also revealed within both southern and northern clusters that could inform strategies to control and monitor this important arbovirus vector., Author summary Aedes aegypti (“yellow fever mosquito”) is of great medical concern because it is the primary vector of important viruses causing dengue fever, chikungunya and Zika, as well as increasingly transmitting yellow fever once again. Due to the Zika outbreak, which started in Brazil in 2015 and rapidly spread with million cases reported in Central/South America and the Caribbean, this mosquito has attained special notoriety. Brazil is by far the most affected country not only by Zika, but also by dengue and chikungunya. Interestingly, in 1950s Brazil had been declared “free of Ae. aegypti” after a well-documented eradication program. However, during the last 45 years Ae. aegypti reappeared in Brazil causing several dengue outbreaks, with millions of reported cases. Here, we used genetic data to study the reappearance of Ae. aegypti in Brazil after the putative eradication event. Our results support re-invasion; mosquitoes from the non-eradicated areas in Venezuela invaded North Brazil, later expanding their distribution southwards. We also identify specific locations in Brazil, as possible entry points. This knowledge can inform strategies to control the spread of the vector and of the diseases it transmits.

The aim of this work was to infer the phylogeny of the Greek endemic land-snail genus Codringtonia Kobelt 1898, estimate the time frame of the radiation of the genus, and propose a biogeographic scenario that could explain the contemporary distribution of Codringtonia lineages. The study took place in the districts of Peloponnese, Central Greece and Epirus of mainland Greece. Sequence data originating from three mtDNA genes (COI, COII, and 16S rDNA) were used to infer the phylogeny of the eight nominal Codringtonia species. Furthermore, the radiation time-frame of extant Codringtonia species was estimated using a relaxed molecular clock analysis and mtDNA substitution rates of land snails. The phylogenetic analysis supported the existence of six Codringtonia lineages in Greece and indicated that one nominal species ( Codringtonia neocrassa ) might belong to a separate genus distantly related to Codringtonia . The time frame of differentiation of Codringtonia species was placed in the Late Miocene–Pleistocene epoch. The dispersal–vicariance analysis performed indicated that most probably Codringtonia exhibited a north-to-south spread with the ancestral area being that of central Greek mainland, accompanied with duplication (speciation) and vicariance events.