Phylogenetic relationships of the subgenus AvaritiaFox, 1955 including Culicoides obsoletus (Diptera, Ceratopogonidae) in Italy based on internal transcribed spacer 2 ribosomal DNA sequences

Abstract. Biting midges of the genus Culicoides include vector species for orbiviral diseases, such as bluetongue and African horse sickness. Although the Afro-Asiatic species C. imicola is the major vector of bluetongue in the Mediterranean basin, recent outbreaks in regions where C. imicola is absent has incriminated other Culicoides, including those belonging to the Obsoletus Complex of the subgenus Avaritia Fox, 1955. The classical taxonomy of this species complex is unclear and this stimulated the molecular analysis of twenty Culicoides populations sampled from eighteen localities across Italy. Ribosomal internal transcribed spacer 2 sequences were used to characterize the intra- and interspecific variation between Italian members of the Obsoletus Complex and related species, by means of an analysis of molecular variance and phylogenetic analyses. Although morphological differentiation is often extremely difficult, the molecular analysis clearly demonstrated a high degree of divergence between most species. The study showed that at least seven species of the subgenus Avaritia occur in Italy; these are C. obsoletus, C. scoticus, C. montanus, C. dewulfi, C. imicola and two species that could not be identified with certainty, but one of which is similar to C. chiopterus. Finally, a simple polymerase chain reaction assay was developed that rapidly discriminates between four members of the Obsoletus Complex in Italy, a prerequisite for vector identification.     

Climate change and the spread of vector‐borne diseases: using approximate Bayesian computation to compare invasion scenarios for the bluetongue virus vector Culicoides imicola in Italy

Bluetongue (BT) is a commonly cited example of a disease with a distribution believed to have recently expanded in response to global warming. The BT virus is transmitted to ruminants by biting midges of the genus Culicoides, and it has been hypothesized that the emergence of BT in Mediterranean Europe during the last two decades is a consequence of the recent colonization of the region by Culicoides imicola and linked to climate change. To better understand the mechanism responsible for the northward spread of BT, we tested the hypothesis of a recent colonization of Italy by C. imicola, by obtaining samples from more than 60 localities across Italy, Corsica, Southern France, and Northern Africa (the hypothesized source point for the recent invasion of C. imicola), and by genotyping them with 10 newly identified microsatellite loci. The patterns of genetic variation within and among the sampled populations were characterized and used in a rigorous approximate Bayesian computation framework to compare three competing historical hypotheses related to the arrival and establishment of C. imicola in Italy. The hypothesis of an ancient presence of the insect vector was strongly favoured by this analysis, with an associated P ≥ 99%, suggesting that causes other than the northward range expansion of C. imicola may have supported the emergence of BT in southern Europe. Overall, this study illustrates the potential of molecular genetic markers for exploring the assumed link between climate change and the spread of diseases.     

First molecular identification of the vertebrate hosts of Culicoides imicola in Europe and a review of its blood‐feeding patterns worldwide: implications for the transmission of bluetongue disease and African horse sickness

Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife, livestock and, occasionally, humans. Culicoides imicola (Kieffer, 1913) is considered to be the main vector of the pathogens that cause bluetongue disease (BT) and African horse sickness (AHS) in southern Europe. The study of blood‐feeding patterns in Culicoides is an essential step towards understanding the epidemiology of these pathogens. Molecular tools that increase the accuracy and sensitivity of traditional methods have been developed to identify the hosts of potential insect vectors. However, to the present group's knowledge, molecular studies that identify the hosts of C. imicola in Europe are lacking. The present study genetically characterizes the barcoding region of C. imicola trapped on farms in southern Spain and identifies its vertebrate hosts in the area. The report also reviews available information on the blood‐feeding patterns of C. imicola worldwide. Culicoides imicola from Spain feed on blood of six mammals that include species known to be hosts of the BT and AHS viruses. This study provides evidence of the importance of livestock as sources of bloodmeals for C. imicola and the relevance of this species in the transmission of BT and AHS viruses in Europe.     

Evolutionary history of the European free-tailed bat,a tropical affinity species spanning across the Mediterranean Basin

The Mediterranean Basin is a global biodiversity hotspot, hosting a number of native species belonging to families that are found almost exclusively in tropical climates. Yet, whether or not these taxa were able to survive in the Mediterranean region during the Quaternary climatic oscillations remains unknown. Focusing on the European free-tailed bat (Tadarida teniotis), we aimed to (a) identify potential ancient populations and glacial refugia; (b) determine the post-glacial colonization routes across the Mediterranean; and (c) evaluate current population structure and demography. Mitochondrial and nuclear markers were used to understand T. teniotis evolutionary and demographic history. We show that T. teniotis is likely restricted to the Western Palearctic, with mitochondrial phylogeny suggesting a split between an Anatolian/Middle East clade and a European clade. Nuclear data pointed to three genetic populations, one of which is an isolated and highly differentiated group in the Canary Islands, another distributed across Iberia, Morocco, and France, and a third stretching from Italy to the east, with admixture following a pattern of isolation by distance. Evolutionary and demographic reconstruction supports a pre-Last Glacial Maximum (LGM) colonization of Italy and the Anatolian/Middle East, while the remaining populations were colonized from Italy after the Younger Dryas. We also found support for demographic expansion following the Iberian colonization. The results show that during the LGM T. teniotis persisted in Mediterranean refugia and has subsequently expanded to its current circum-Mediterranean range. Our findings raise questions regarding the physiological and ecological traits that enabled species with tropical affinities to survive in colder climates.     

Protection of livestock against bluetongue virus vector Culicoides imicola using insecticide‐treated netting in open areas

The protection of livestock against Culicoides species (Diptera: Ceratopogonidae) using physical barriers or chemically treated barriers is difficult owing to the small size of these biting midges and animal welfare concerns associated with the reduction of air flow. Culicoides imicola Kieffer is the main bluetongue virus vector in the Mediterranean basin, including the southern Iberian peninsula, where livestock is mainly housed in open pens or sheds which offer no physical protection against C. imicola. In this study we assessed the efficacy of surrounding yearling ewe pens with a canvas barrier or a cypermethrin‐treated canvas barrier in reducing the entry of Culicoides spp. and C. imicola. Analyses were based on comparisons of Culicoides catches in traps in pens with and without barriers, and in traps located outside pens. Although there was no clear reduction in the abundance of Culicoides other than C. imicola in pens with either barrier, the C. imicola presence was markedly reduced by the insecticide‐treated barrier compared with the untreated barrier; the latter did not reduce the abundance of this species in pens. Estimates of the protection conferred against C. imicola by the treated barrier differed depending on whether catch comparisons were based on outside traps or on traps located inside no‐barrier pens. The results suggest that the use of insecticide‐treated barriers may reduce contact between livestock and C. imicola in open areas or sheds. More research is necessary to assess the degree of protection as a function of barrier height, C. imicola abundance, and the size of the area to be protected.     

Worldwide Niche and Future Potential Distribution of Culicoides imicola,a Major Vector of Bluetongue and African Horse Sickness Viruses

We modelled the ecoclimatic niche of Culicoides imicola, a major arthropod vector of midge-borne viral pathogens affecting ruminants and equids, at fine scale and on a global extent, so as to provide insight into current and future risks of disease epizootics, and increase current knowledge of the species'' ecology. Based on the known distribution and ecology of C. imicola, the species'' response to monthly climatic conditions was characterised using CLIMEX with 10′ spatial resolution climatic datasets. The species'' climatic niche was projected worldwide and under future climatic scenarios. The validated model highlights the role of irrigation in supporting the occurrence of C. imicola in arid regions. In Europe, the modelled potential distribution of C. imicola extended further West than its reported distribution, raising questions regarding ongoing process of colonization and non-climatic habitat factors. The CLIMEX model highlighted similar ecological niches for C. imicola and the Australasian C. brevitarsis raising questions on biogeography and biosecurity. Under the climate change scenarios considered, its'' modelled potential distribution could expand northward in the Northern hemisphere, whereas in Africa its range may contract in the future. The biosecurity risks from bluetongue and African horse sickness viruses need to be re-evaluated in regions where the vector''s niche is suitable. Under a warmer climate, the risk of vector-borne epizootic pathogens such as bluetongue and African horse sickness viruses are likely to increase as the climate suitability for C. imicola shifts poleward, especially in Western Europe.     

Interspecific interactions influence contrasting spatial genetic structures in two closely related damselfly species

Spatial genetic structure (SGS) is largely determined by colonization history, landscape and ecological characteristics of the species. Therefore, sympatric and ecologically similar species are expected to exhibit similar SGSs, potentially enabling prediction of the SGS of one species from that of another. On the other hand, due to interspecific interactions, ecologically similar species could have different SGSs. We explored the SGSs of the closely related Calopteryx splendens and Calopteryx virgo within Finland and related the genetic patterns to characteristics of the sampling localities. We observed different SGSs for the two species. Genetic differentiation even within short distances in C. splendens suggests genetic drift as an important driver. However, we also observed indication of previous gene flow (revealed by a negative relationship between genetic differentiation and increasing potential connectivity of the landscape). Interestingly, genetic diversity of C. splendens was negatively related to density of C. virgo, suggesting that interspecific interactions influence the SGS of C. splendens. In contrast, genetic differentiation between C. virgo subpopulations was low and only exhibited relationships with latitude, pointing to high gene flow, colonization history and range margin effects as the drivers of SGS. The different SGSs of the two ecologically similar species caution indirect inferences of SGS based on ecologically similar surrogate species.     

High genomic diversity in the bank vole at the northern apex of a range expansion: The role of multiple colonizations and end‐glacial refugia

The history of repeated northern glacial cycling and southern climatic stability has long dominated explanations for how genetic diversity is distributed within temperate species in Eurasia and North America. However, growing evidence indicates the importance of cryptic refugia for northern colonization dynamics. An important geographic region to assess this is Fennoscandia, where recolonization at the end of the last glaciation was restricted to specific routes and temporal windows. We used genomic data to analyse genetic diversity and colonization history of the bank vole (Myodes glareolus) throughout Europe (>800 samples) with Fennoscandia as the northern apex. We inferred that bank voles colonized Fennoscandia multiple times by two different routes; with three separate colonizations via a southern land‐bridge route deriving from a “Carpathian” glacial refugium and one via a north‐eastern route from an “Eastern” glacial refugium near the Ural Mountains. Clustering of genome‐wide SNPs revealed high diversity in Fennoscandia, with eight genomic clusters: three of Carpathian origin and five Eastern. Time estimates revealed that the first of the Carpathian colonizations occurred before the Younger Dryas (YD), meaning that the first colonists survived the YD in Fennoscandia. Results also indicated that introgression between bank and northern red‐backed voles (Myodes rutilus) took place in Fennoscandia just after end‐glacial colonization. Therefore, multiple colonizations from the same and different cryptic refugia, temporal and spatial separations and interspecific introgression have shaped bank vole genetic variability in Fennoscandia. Together, these processes drive high genetic diversity at the apex of the northern expansion in this emerging model species.     

A pre‐Miocene Irano‐Turanian cradle: Origin and diversification of the species‐rich monocot genus Gagea (Liliaceae)

The Irano‐Turanian (IT) floristic region is considered an important center of origin for many taxa. However, there is a lack of studies dealing with typical IT genera that also occur in neighboring areas. The species‐rich monocot genus Gagea Salisb. shows a center of diversity in IT region and a distribution in adjacent regions, therefore representing a good study object to investigate spatial and temporal relationships among IT region and its neighboring areas (East Asia, Euro‐Siberia, Himalaya, and Mediterranean). We aimed at (a) testing the origin of the genus and of its major lineages in the IT region, (b) reconstructing divergence times, and (c) reconstructing colonization events. To address these problems, sequences of the ribosomal DNA internal transcribed spacer (ITS) region of 418 individuals and chloroplast intergenic spacers sequences (psbA‐trnH, trnL‐trnF) of 497 individuals, representing 116 species from all sections of the genus and nearly its entire distribution area were analyzed. Divergence times were estimated under a random molecular clock based on nrITS phylogeny, which was the most complete data set regarding the representation of species and distribution areas. Ancestral distribution ranges were estimated for the nrITS data set as well as for a combined data set, revealing that Gagea most likely originated in southwestern Asia. This genus first diversified there starting in the Early Miocene. In the Middle Miocene, Gagea migrated to the Mediterranean and to East Asia, while migration into Euro‐Siberia took place in the Late Miocene. During the Pleistocene, the Arctic was colonized and Gagea serotina, the most widespread species, reached North America. The Mediterranean basin was colonized multiple times from southwestern Asia or Euro‐Siberia. Most of the currently existing species originated during the last 3 Ma.     

Rapid genetic restoration of a keystone species exhibiting delayed demographic response

Genetic founder effects are often expected when animals colonize restored habitat in fragmented landscapes, but empirical data on genetic responses to restoration are limited. We examined the genetic response of banner‐tailed kangaroo rats (Dipodomys spectabilis) to landscape‐scale grassland restoration in the Chihuahuan Desert of New Mexico, USA. Dipodomys spectabilis is a grassland specialist and keystone species. At sites treated with herbicide to remove shrubs, colonization by D. spectabilis is slow and populations persist at low density for ≥10 years (≥6 generations). Persistence at low density and low gene flow may cause strong founder effects. We compared genetic structure of D. spectabilis populations between treated sites and remnant grasslands, and we examined how the genetic response to restoration depended on treatment age, area, and connectivity to source populations. Allelic richness and heterozygosity were similar between treated sites and remnant grasslands. Allelic richness at treated sites was greatest early in the restoration trajectory, and genetic divergence did not differ between recently colonized and established populations. These results indicated that founder effects during colonization of treated sites were weak or absent. Moreover, our results suggested founder effects were not mitigated by treatment area or connectivity. Dispersal is negatively density‐dependent in D. spectabilis, and we hypothesize that high gene flow may occur early in the restoration trajectory when density is low. Our study shows genetic diversity can be recovered more rapidly than demographic components of populations after habitat restoration and that founder effects are not inevitable for animals colonizing restored habitat in fragmented landscapes.     

Phylogeography,historical demography and habitat suitability modelling of freshwater fishes inhabiting seasonally fluctuating Mediterranean river systems: a case study using the Iberian cyprinid Squalius valentinus

The Mediterranean freshwater fish fauna has evolved under constraints imposed by the seasonal weather/hydrological patterns that define the Mediterranean climate. These conditions have influenced the genetic and demographic structure of aquatic communities since their origins in the Mid‐Pliocene. Freshwater species in Mediterranean‐type climates will likely constitute genetically well‐differentiated populations, to varying extents depending on basin size, as a consequence of fragmentation resulting from drought/flood cycles. We developed an integrative framework to study the spatial patterns in genetic diversity, demographic trends, habitat suitability modelling and landscape genetics, to evaluate the evolutionary response of Mediterranean‐type freshwater fish to seasonal fluctuations in weather. To test this evolutionary response, the model species used was Squalius valentinus, an endemic cyprinid of the Spanish Levantine area, where seasonal weather fluctuations are extreme, although our findings may be extrapolated to other Mediterranean‐type species. Our results underscore the significant role of the Mediterranean climate, along with Pleistocene glaciations, in diversification of S. valentinus. We found higher nuclear diversity in larger drainage basins, but higher mitochondrial diversity correlated to habitat suitability rather than basin size. We also found strong correlation between genetic structure and climatic factors associated with Mediterranean seasonality. Demographic and migration analyses suggested population expansion during glacial periods that also contributed to the current genetic structure of S. valentinus populations. The inferred models support the significant contribution of precipitation and temperature to S. valentinus habitat suitability and allow recognizing areas of habitat stability. We highlight the importance of stable habitat conditions, fostered by typical karstic springs found on the Mediterranean littoral coasts, for the preservation of freshwater species inhabiting seasonally fluctuating river systems.     

Contrasted levels of genetic diversity in a benthic Mediterranean octocoral: Consequences of different demographic histories?

Understanding the factors explaining the observed patterns of genetic diversity is an important question in evolutionary biology. We provide the first data on the genetic structure of a Mediterranean octocoral, the yellow gorgonian Eunicella cavolini, along with insights into the demographic history of this species. We sampled populations in four areas of the Mediterranean Sea: continental France, Algeria, Turkey, and the Balearic and Corsica islands. Along French coasts, three sites were sampled at two depths (20 and 40 m). We demonstrated a high genetic structure in this species (overall F_ST = 0.13), and most pairwise differentiation tests were significant. We did not detect any difference between depths at the same site. Clustering analyses revealed four differentiated groups corresponding to the main geographical areas. The levels of allelic richness and heterozygosity were significantly different between regions, with highest diversity in Algeria and lowest levels in Turkey. The highest levels of private allelic richness were observed in Algeria followed by Turkey. Such contrasted patterns of genetic diversity were not observed in other Mediterranean octocorals and could be the result of different evolutionary histories. We also provide new empirical evidence of contrasting results between tests and model‐based studies of demographic history. Our results have important consequences for the management of this species.     

Large‐scale introduction of the Indo‐Pacific damselfish Abudefduf vaigiensis into Hawai'i promotes genetic swamping of the endemic congener A. abdominalis

Hybridization in the ocean was once considered rare, a process prohibited by the rapid evolution of intrinsic reproductive barriers in a high‐dispersal medium. However, recent genetic surveys have prompted a reappraisal of marine hybridization as an important demographic and evolutionary process. The Hawaiian Archipelago offers an unusual case history in this arena, due to the recent arrival of the widely distributed Indo‐Pacific sergeant (Abudefduf vaigiensis), which is hybridizing with the endemic congener, A. abdominalis. Surveys of mtDNA and three nuclear loci across Hawai'i (N = 396, Abudefduf abdominalis and N = 314, A. vaigiensis) reveal that hybridization is significantly higher in the human‐perturbed southeast archipelago (19.8%), tapering off to 5.9% in the pristine northwest archipelago. While densities of the two species varied throughout Hawai'i, hybridization was highest in regions with similar species densities, contradicting the generalization that the rarity of one species promotes interspecific mating. Our finding of later generation hybrids throughout the archipelago invokes the possibility of genetic swamping of the endemic species. Exaptation, an adaptation with unintended consequences, may explain these findings: the endemic species has transient yellow coloration during reproduction, whereas the introduced species has yellow coloration continuously as adults, in effect a permanent signal of reproductive receptivity. Haplotype diversity is higher in Hawaiian A. vaigiensis than in our samples from the native range, indicating large‐scale colonization almost certainly facilitated by the historically recent surge of marine debris. In this chain of events, marine debris promotes colonization, exaptation promotes hybridization, and introgression invokes the possible collapse of an endemic species.     

Genetic inference of epiphytic orchid colonization; it may only take one

Colonization of vacant habitat is a fundamental ecological process that affects the ability of species to persist and undergo range modifications in continually shifting landscapes. Thus, understanding factors that affect and limit colonization has important ecological and conservation implications. Epiphytic orchids are increasingly threatened by various factors, including anthropogenic habitat disturbance. As cleared areas (e.g. pastures) are recolonized by suitable host trees, the establishment and genetic composition of epiphytic orchid populations are likely a function of their colonization patterns. We used genetic analyses to infer the prevailing colonization pattern of the epiphytic orchid, Brassavola nodosa. Samples from three populations (i.e. individuals within a tree) from each of five pastures in the dry forest of Costa Rica were genotyped with neutral nuclear and chloroplast markers. Spatial autocorrelation and hierarchical genetic structure analyses were used to assess the relatedness of individuals within populations, among populations within pastures and among populations in different pastures. The results showed significant relatedness within populations (mean r = 0.166) and significant but lower relatedness among populations within a pasture (mean r = 0.058). Our data suggest that colonization of available habitats is by few individuals with subsequent population expansion resulting from in situ reproduction, and that individuals within a tree are not a random sample of the regional seed pool. Furthermore, populations within a pasture were likely colonized by seeds produced by founders of a neighbouring population within that pasture. These results have important ramifications for understanding conservation measures needed for this species and other epiphytic orchids.     

Using genetic variation to infer associations with climate in the common frog,Rana temporaria

Recent and historical species' associations with climate can be inferred using molecular markers. This knowledge of population and species‐level responses to climatic variables can then be used to predict the potential consequences of ongoing climate change. The aim of this study was to predict responses of Rana temporaria to environmental change in Scotland by inferring historical and contemporary patterns of gene flow in relation to current variation in local thermal conditions. We first inferred colonization patterns within Europe following the last glacial maximum by combining new and previously published mitochondrial DNA sequences. We found that sequences from our Scottish samples were identical to (92%), or clustered with, the common haplotype previously identified from Western Europe. This clade showed very low mitochondrial variation, which did not allow inference of historical colonization routes but did allow interpretation of patterns of current fine‐scale population structure without consideration of confounding historical variation. Second, we assessed fine‐scale microsatellite‐based patterns of genetic variation in relation to current altitudinal temperature gradients. No population structure was found within altitudinal gradients (average F_ST = 0.02), despite a mean annual temperature difference of 4.5 °C between low‐ and high‐altitude sites. Levels of genetic diversity were considerable and did not vary between sites. The panmictic population structure observed, even along temperature gradients, is a potentially positive sign for R. temporaria persistence in Scotland in the face of a changing climate. This study demonstrates that within taxonomic groups, thought to be at high risk from environmental change, levels of vulnerability can vary, even within species.     

A phylogeographic investigation of the kelp genus Laminaria (Laminariales,Phaeophyceae), with emphasis on the South Atlantic Ocean

The genus Laminaria has a wide distribution range compared with other kelp genera because it is found in both the North and the South Atlantic, on both sides of the North Pacific, as well as in the Mediterranean. Hypotheses behind this biogeographical pattern have been discussed by several authors but have not yet been fully evaluated with time‐calibrated phylogenies. Based on the analysis of four molecular markers (ITS2, rbcL, atp8 and trnWI), our goal was to reassess the Laminaria species diversity in South Africa, assess its relationship with the other species distributed in the South Atlantic and reconstruct the historical biogeography of the genus. Our results confirm the occurrence of a single species, L. pallida, in southern Africa, and its sister relationship with the North Atlantic L. ochroleuca. Both species belonged to a clade containing the other South Atlantic species: L. abyssalis from Brazil, and the Mediterranean L. rodriguezii. Our time‐calibrated phylogenies suggest that Laminaria originated in the northern Pacific around 25 mya, followed by at least two migration events through the Bering Strait after its opening (~5.32 mya). Today, the first is represented by L. solidungula in the Arctic, while the second gave rise to the rest of the Atlantic species. The colonization of the North Atlantic was followed by a gradual colonization southward along the west coast of Europe, into the Mediterranean (~2.07 mya) and two recent, but disconnected, migrations (~1.34 and 0.87 mya) across the equator, giving rise to L. abyssalis in Brazil and L. pallida in southern Africa, respectively.     

Gut microbiota in the burying beetle,Nicrophorus vespilloides,provide colonization resistance against larval bacterial pathogens

Carrion beetles, Nicrophorus vespilloides, are reared on decomposing carrion where larvae are exposed to high populations of carcass‐derived bacteria. Larvae do not become colonized with these bacteria but instead are colonized with the gut microbiome of their parents, suggesting that bacteria in the beetle microbiome outcompete the carcass‐derived species for larval colonization. Here, we test this hypothesis and quantify the fitness consequences of colonization with different bacterial symbionts. First, we show that beetles colonized by their endogenous microbiome produce heavier broods than those colonized with carcass‐bacteria. Next, we show that bacteria from the endogenous microbiome, including Providencia rettgeri and Morganella morganii, are better colonizers of the beetle gut and can outcompete nonendogenous species, including Serratia marcescens and Escherichia coli, during in vivo competition. Finally, we find that Providencia and Morganella provide beetles with colonization resistance against Serratia and thereby reduce Serratia‐induced larval mortality. This effect is eliminated in larvae first colonized by Serratia, suggesting that while competition within the larval gut is determined by priority effects, these effects are less important for Serratia‐induced mortality. Our work suggests that an unappreciated benefit of parental care in N. vespilloides is the social transmission of the microbiome from parents to offspring.     

Genetic structure and population dynamics of the biting midges Culicoides obsoletus and Culicoides scoticus: implications for the transmission and maintenance of bluetongue

Culicoides species belonging to the Obsoletus complex (Diptera: Ceratopogonidae) have been indicated as primary bluetongue (BT) vectors in many European countries and their possible involvement in the maintenance and overwintering of BT viruses has been suggested, even in regions where Culicoides imicola Keiffer is the main vector. The Obsoletus complex includes two predominant taxa, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle. However, the role played by each species in the epidemiology of BT is still unknown. Taxonomic identification is mainly based on the morphology of male genitalia and the lack of other reliable diagnostic features makes the screening of trap‐collected vector populations, mainly females, particularly difficult. Although molecular markers have facilitated species identification, little information is yet available on the biology, abundance and population dynamics of the two taxa. The aim of this work was to investigate the genetic profile and temporal distribution of C. obsoletus and C. scoticus by using isozyme electrophoresis applied to adult midges, collected weekly at two selected farms in southern Sardinia. A total of nine enzyme loci were analysed and five of them provided diagnostic allozyme markers (Hk, Mdh, Pgi, Idh‐1 and Idh‐2). Nei's genetic distance between the two taxa was in the range of other well‐separated taxa (D = 1.792), supporting their status as true species. Culicoides scoticus represented almost 61% of the 562 specimens analysed; its genetic structure was characterized by a very low level of intra‐population variation (mean heterozygosity H_e = 0.019) and higher genetic divergence between populations (F_ST = 0.0016) than in C. obsoletus. The latter species had significantly more heterozygotes (H_e = 0.123), a higher percentage of polymorphic loci, and no inter‐population differentiation (F_ST?0). We suggest that different biological and ecological constraints, such as breeding habitat requirements, may contribute to shaping the genetic profiles of C. scoticus and C. obsoletus. However, enough gene flow was maintained between populations of each species as no spatial and temporal structuring was sustained by Fisher's exact probability test (P > 0.5). The seasonal distributions of C. scoticus and C. obsoletus only partially overlapped: both species were mainly found early in the year, when the main vector, C. imicola, was present in low numbers, and peaked in abundance in April and May. Culicoides scoticus was predominant until May, decreased rapidly in the following months and increased again in winter, whereas C. obsoletus decreased more slowly and was still present in early summer. Consequently, C. scoticus may be a good candidate for playing a role in the transmission and maintenance of BT virus in Sardinia, as well as in other Mediterranean countries, during the months of late winter and early spring when the seroconversion of sentinel animals is still occurring in the absence of the main vector.     

Diversity of bladed Bangiales (Rhodophyta) in western Mediterranean: recognition of the genus Themis and descriptions of T. ballesterosii sp. nov., T. iberica sp. nov., and Pyropia parva sp. nov.

The diversity of the bladed species of the red algal order Bangiales from the Iberian Mediterranean shores has been reassessed after a detailed study of this region. Prior to this study, 11 bladed species of Bangiales had been reported from Mediterranean waters: Porphyra atropurpurea, P. cordata, P. coriacea, P. dioica, P. linearis, P. purpurea, P. umbilicalis, Pyropia leucosticta, Pyropia koreana (as P. olivii), Py. elongata (as P. rosengurttii) and Py. suborbiculata. A combined analysis of the nuclear nSSU and the plastid rbcL genes together with detailed morphological studies has confirmed the presence of species within the genera Porphyra and Pyropia and also revealed a third, undescribed genus, Themis gen. nov. Porphyra linearis, Pyropia elongata and the introduced Pyropia koreana had been previously listed for the Mediterranean and were recorded in this study. An additional four species, including the introduced Pyropia suborbiculata and three new species: Pyropia parva sp. nov., Themis ballesterosii sp. nov., and Themis iberica sp. nov. were also observed. Hence, most of the Porphyra species traditionally reported along these shores were not reported in this survey. This new floristic Bangiales composition confirms the importance of the Mediterranean basin as a hotspot for biodiversity, possible endemics of ancient origin and high proportion of introductions. Our data also continue to confirm the extent of Bangiales diversity at regional and worldwide levels.