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.

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.

Correction to: Assessing genotype-phenotype associations in three dorsal colour morphs in the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) using genomic and transcriptomic resources.

Following publication of the original article [1], it has been brought to the authors' attention that in their paper (Rodrigues et al. 2016) they reported the genome size based on 2C values (diploid genome) when it is more common to present it as 1C value.

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.

New data on polymorphism of the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) from the island of São Miguel (Azores).

The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera, Aphrophoridae) is a widespread insect species in the Holarctic region, exhibiting a dorsal colour balanced polymorphism. In the Azores the species is known from high elevations in Terceira and São Miguel islands. A sample of 235 individuals from Pico da Vara and Graminhais protected areas (São Miguel, Azores) (between 645 and 935 m a.s.l.), collected in 2000 and 2017, showed a remarkable high frequency of the melanic morphs flavicollis (FLA) and quadrimaculatus (QUA). In addition, a high frequency of melanics was observed in males. We explore the hypotheses for the origin of Azorean colonization and for the high proportion of melanism in the Azorean populations.

Assessing genotype-phenotype associations in three dorsal colour morphs in the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) using genomic and transcriptomic resources.

BACKGROUND: Colour polymorphisms are common among animal species. When combined with genetic and ecological data, these polymorphisms can be excellent systems in which to understand adaptation and the molecular changes underlying phenotypic evolution. The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera, Aphrophoridae), a widespread insect species in the Holarctic region, exhibits a striking dorsal colour/pattern balanced polymorphism. Although experimental crosses have revealed the Mendelian inheritance of this trait, its genetic basis remains unknown. In this study we aimed to identify candidate genomic regions associated with the colour balanced polymorphism in this species. RESULTS: By using restriction site-associated DNA (RAD) sequencing we were able to obtain a set of 1,837 markers across 33 individuals to test for associations with three dorsal colour phenotypes (typicus, marginellus, and trilineatus). Single and multi-association analyses identified a total of 60 SNPs associated with dorsal colour morphs. The genome size of P. spumarius was estimated by flow cytometry, revealing a 5.3 Gb genome, amongst the largest found in insects. A partial genome assembly, representing 24% of the total size, and an 81.4 Mb transcriptome, were also obtained. From the SNPs found to be associated with colour, 35% aligned to the genome and 10% to the transcriptome. Our data suggested that major loci, consisting of multi-genomic regions, may be involved in dorsal colour variation among the three dorsal colour morphs analysed. However, no homology was found between the associated loci and candidate genes known to be responsible for coloration pattern in other insect species. The associated markers showed stronger differentiation of the trilineatus colour phenotype, which has been shown previously to be more differentiated in several life-history and physiological characteristics as well. It is possible that colour variation and these traits are linked in a complex genetic architecture. CONCLUSIONS: The loci detected to have an association with colour and the genomic and transcriptomic resources developed here constitute a basis for further research on the genetic basis of colour pattern in the meadow spittlebug P. spumarius.

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.