Integrative analysis reveals the divergence and speciation between sister Sooty Copper butterflies Lycaena bleusei and L. tityrus.

The comparison of closely related taxa is cornerstone in biology, as understanding mechanisms leading up to differentiation in relation to extant shared characters are powerful tools in interpreting the evolutionary process. Hotspots of biodiversity such as the west-Mediterranean, where many lineages meet are ideal grounds to study these processes. We set to explore the interesting example of Sooty Copper butterflies: widespread Eurasian Lycaena tityrus (Poda, 1761) comes into contact in Iberia with closely related and local endemic, L. bleusei (Oberthür, 1884), which hasn't always been considered a distinct species. An integrative analysis was designed, combining the use of extensive molecular data (five genes), geometric morphometrics analyses, verified and up-to-date distribution data, and environmental niche modelling, aimed at deciphering their true relationship, their placement within European Lycaena and trace their evolutionary history. We revealed several levels of differentiation: L. bleusei and L. tityrus appear to be reciprocally monophyletic independent gene-pools, distinct in all genes analysed, having mutually diverged 4.8 Ma ago. L. tityrus but not L. bleusei, further displays a genetic structure compatible with several glacial refugia, where populations assignable to infraspecific taxa surface. Conversely, L. bleusei shows a loss in mtDNA diversity in relation to nuDNA. Morphological analyses differentiate both species according to size and shape but also discriminate strong seasonal and sexual traits and a geographical phenotype segregation in L. tityrus. Finally, updated distribution and its modelling for current and glacial timeframes reveal both species respond differently to environmental variables, defining a mostly parapatric distribution and an overlapping belt where sympatry was recovered. During the last glacial maximum, a wider expansion in L. bleusei distribution explains current isolated populations. Our study highlights the importance of gathering several lines of evidence when deciphering the relationships between closely related populations in the fringe of cryptic species realm.

Testing drivers of acoustic divergence in cicadas (Cicadidae: Tettigettalna).

Divergence in acoustic signals may have a crucial role in the speciation process of animals that rely on sound for intra-specific recognition and mate attraction. The acoustic adaptation hypothesis (AAH) postulates that signals should diverge according to the physical properties of the signalling environment. To be efficient, signals should maximize transmission and decrease degradation. To test which drivers of divergence exert the most influence in a speciose group of insects, we used a phylogenetic approach to the evolution of acoustic signals in the cicada genus Tettigettalna, investigating the relationship between acoustic traits (and their mode of evolution) and body size, climate and micro-/macro-habitat usage. Different traits showed different evolutionary paths. While acoustic divergence was generally independent of phylogenetic history, some temporal variables' divergence was associated with genetic drift. We found support for ecological adaptation at the temporal but not the spectral level. Temporal patterns are correlated with micro- and macro-habitat usage and temperature stochasticity in ways that run against the AAH predictions, degrading signals more easily. These traits are likely to have evolved as an anti-predator strategy in conspicuous environments and low-density populations. Our results support a role of ecological selection, not excluding a likely role of sexual selection in the evolution of Tettigettalna calling songs, which should be further investigated in an integrative approach.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data.

Understanding patterns of population differentiation and gene flow in insect vectors of plant diseases is crucial for the implementation of management programs of disease. We investigated morphological and genome-wide variation across the distribution range of the spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Auchenorrhyncha, Aphrophoridae), presently the most important vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. We found genome-wide divergence between P. spumarius and a very closely related species, P. tesselatus Melichar, 1899, at RAD sequencing markers. The two species may be identified by the morphology of male genitalia but are not differentiated at mitochondrial COI, making DNA barcoding with this gene ineffective. This highlights the importance of using integrative approaches in taxonomy. We detected admixture between P. tesselatus from Morocco and P. spumarius from the Iberian Peninsula, suggesting gene-flow between them. Within P. spumarius, we found a pattern of isolation-by-distance in European populations, likely acting alongside other factors restricting gene flow. Varying levels of co-occurrence of different lineages, showing heterogeneous levels of admixture, suggest other isolation mechanisms. The transatlantic populations of North America and Azores were genetically closer to the British population analyzed here, suggesting an origin from North-Western Europe, as already detected with mitochondrial DNA. Nevertheless, these may have been produced through different colonization events. We detected SNPs with signatures of positive selection associated with environmental variables, especially related to extremes and range variation in temperature and precipitation. The population genomics approach provided new insights into the patterns of divergence, gene flow and adaptation in these spittlebugs and led to several hypotheses that require further local investigation.

Genomic signatures of introgression between commercial and native bumblebees, Bombus terrestris, in western Iberian Peninsula-Implications for conservation and trade regulation.

Human-mediated introductions of species may have profound impacts on native ecosystems. One potential impact with largely unforeseen consequences is the potential admixture of introduced with autochthonous species through hybridization. Throughout the world, bumblebees have been deliberately introduced for crop pollination with known negative impacts on native pollinators. Given the likely allochthonous origin of commercial bumblebees used in Portugal (subspecies Bombus terrestris terrestris and B. t. dalmatinus), our aim was to assess their putative introgression with the native Iberian subspecies B. terrestris lusitanicus. We analysed one mitochondrial gene, cytochrome c oxidase subunit I (COX1) and genomic data involving thousands of genome-wide restriction-site-associated DNA markers (RAD-seq). In the mitochondrial COX1 analyses, we detected one relatively common haplotype in commercial bumblebees, also present in wild samples collected nearby the greenhouses where the commercial hives are used. In the RAD-seq analysis, we found a clear genetic differentiation between native and commercial lineages. Furthermore, we detected candidate hybrids in the wild, as well as putatively escaped commercial bumblebees, some of which being potentially fertile males. Although we cannot assess directly the fitness effects of introgressed alleles, there is a risk of maladaptive allele introgression to the local bumblebee subspecies, which can negatively impact autochthon populations. One immediate recommendation to farmers is for the proper disposal of hive boxes, after their use in greenhouses, so as to minimize the risk of escapees contaminating native populations. On the other hand, the feasibility of using local subspecies B. t. lusitanicus, preferably with local production, should be evaluated.

The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), a vector of dengue, chikungunya and zika viruses, reaches Portugal (Diptera: Culicidae).

The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), is a tropical species originally from south-eastern Asia (Hawley, 1988). It has experienced a rampant human-mediated range expansion since the 1970s to now occupy almost the whole of the tropical and subtropical areas of the world. As an eclectic haematophagous species, it attacks humans and is able to use a number of man-made and natural structures where stagnant water is present (Estrada-Franco Craig, 1995). Eggs are able to survive for extended periods of time in complete dryness and diapause over unsuitable cold season, making it an especially resilient species (Hawley, 1988).

Morphology, songs and genetics identify two new cicada species from Morocco: Tettigettalna afroamissa sp. nov. and Berberigetta dimelodica gen. nov. & sp. nov. (Hemiptera: Cicadettini).

Morocco has been the subject of very few expeditions on the last century with the objective of studying small cicadas. In the summer of 2014 an expedition was carried out to Morocco to update our knowledge with acoustic recordings and genetic data of these poorly known species. We describe here two new small-sized cicadas that could not be directly assigned to any species of North African cicadas: Tettigettalna afroamissa sp. nov. and Berberigetta dimelodica gen. nov. & sp. nov. In respect to T. afroamissa it is the first species of the genus to be found outside Europe and we frame this taxon within the evolutionary history of the genus. Acoustic analysis of this species allows us to confidently separate T. afroamissa from its congeners. With B. dimelodica, a small species showing a remarkable calling song characterized by an abrupt frequency modulation, a new genus had to be erected. Bayesian inference and maximum likelihood phylogenetic analyses with DNA-barcode sequences of Cytochrome C Oxidase 1 support the monophyly of both species, their distinctness and revealed genetic structure within B. dimelodica. Alongside the descriptions we also provide GPS coordinates of collection points, distributions and habitat preferences.

The Afrotropical Miomantiscaffra Saussure 1871 and Miomantispaykullii Stal 1871: first records of alien mantid species in Portugal and Europe, with an updated checklist of Mantodea in Portugal (Insecta: Mantodea).

The recent growing interest on the Mantodea fauna of southern Europe and Portugal in particular, has enabled the discovery of two geographically separated populations of hitherto unknown species in Europe. Analysis of specimens shows that they belong to two Afrotropical mantids: Miomantiscaffra Saussure, 1871 and Miomantispaykullii Stal, 1871, thus raising the number of known species in Europe to 39 and in Portugal to 11. While these are remarkable findings, they also represent the first alien mantis species recorded from this continent. As yet, these species appear to be confined to artificial humanised gardened areas but call for more attention to the problem of biological invasions and the need for better bio-security measures for the conservation of natural ecosystems. In the absence of recent revisionary work on the Mantodea of Portugal and given the need to provide an accessible identification tool, both a checklist and a key to species are provided for all species in the country.

New mitochondrial and nuclear evidences support recent demographic expansion and an atypical phylogeographic pattern in the spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae).

Philaenus spumarius is a widespread insect species in the Holarctic region. Here, by focusing on the mtDNA gene COI but also using the COII and Cyt b genes and the nuclear gene EF-1α, we tried to explain how and when its current biogeographic pattern evolved by providing time estimates of the main demographic and evolutionary events and investigating its colonization patterns in and out of Eurasia. Evidence of recent divergence and expansion events at less than 0.5 Ma ago indicate that climate fluctuations in the Mid-Late Pleistocene were important in shaping the current phylogeographic pattern of the species. Data support a first split and differentiation of P. spumarius into two main mitochondrial lineages: the "western", in the Mediterranean region and the "eastern", in Anatolia/Caucasus. It also supports a following differentiation of the "western" lineage into two sub-lineages: the "western-Mediterranean", in Iberia and the "eastern-Mediterranean" in the Balkans. The recent pattern seems to result from postglacial range expansion from Iberia and Caucasus/Anatolia, thus not following one of the four common paradigms. Unexpected patterns of recent gene-flow events between Mediterranean peninsulas, a close relationship between Iberia and North Africa, as well as high levels of genetic diversity being maintained in northern Europe were found. The mitochondrial pattern does not exactly match to the nuclear pattern suggesting that the current biogeographic pattern of P. spumarius may be the result of both secondary admixture and incomplete lineage sorting. The hypothesis of recent colonization of North America from both western and northern Europe is corroborated by our data and probably resulted from accidental human translocations. A probable British origin for the populations of the Azores and New Zealand was revealed, however, for the Azores the distribution of populations in high altitude native forests is somewhat puzzling and may imply a natural colonization of the archipelago.

Sphodromantisviridis (Forskal, 1775): New for Portugal and new records of the rare and small mantids Apteromantisaptera (Fuente, 1894) and Perlamantisallibertii Guérin-Méneville, 1843 in the country (Mantodea: Mantidae and Amorphoscelidae).

Several new records are presented on some of the least known mantis species in the Iberian Peninsula. From data collected in Portugal, their analysis has proven to represent an important advancement in the knowledge of this group of insects for the country and the Western Mediterranean area. Twenty new citations provide a better understanding on the distribution of the crepuscular species Perlamantisallibertii, the IUCN red listed Apteromantisaptera and the first Portuguese records of Sphodromantisviridis, extending their western limits of occurrence in Europe. The data thus gathered emphasizes the need to invest in biodiversity assessment for increased knowledge on species distribution and phenology but also for monitoring over time, essential to better ascertaining ecosystem services, the effects of climate change and habitat conservation.

Conflicting patterns of DNA barcoding and taxonomy in the cicada genus Tettigettalna from Southern Europe (Hemiptera: Cicadidae).

DNA barcodes have great potential to assist in species identification, especially when high taxonomical expertise is required. We investigated the utility of the 5' mitochondrial cytochrome c oxidase I (COI) region to discriminate between 13 European cicada species. These included all nine species currently recognized under the genus Tettigettalna, from which seven are endemic to the southern Iberian Peninsula. These cicadas have species-specific male calling songs but are morphologically very similar. Mean COI divergence between congeners ranged from 0.4% to 10.6%, but this gene was proven insufficient to determine species limits within genus Tettigettalna because a barcoding gap was absent for several of its species, that is, the highest intraspecific distance exceeded the lowest interspecific distance. The genetic data conflicted with current taxonomic classification for T. argentata and T. mariae. Neighbour-joining and Bayesian analyses revealed that T. argentata is geographically structured (clades North and South) and might constitute a species complex together with T. aneabi and T. mariae. The latter diverges very little from the southern clade of T. argentata and shares with it its most common haplotype. T. mariae is often in sympatry with T. argentata but it remains unclear whether introgression or incomplete lineage sorting may be responsible for the sharing of haplotypes. T. helianthemi and T. defauti also show high intraspecific variation that might signal hidden cryptic diversity. These taxonomic conflicts must be re-evaluated with further studies using additional genes and extensive morphological and acoustic analyses.