Applying Next-Generation Sequencing to Track HIV-1 Drug Resistance Mutations Circulating in Portugal.

BACKGROUND: The global scale-up of antiretroviral treatment (ART) offers significant health benefits by suppressing HIV-1 replication and increasing CD4 cell counts. However, incomplete viral suppression poses a potential threat for the emergence of drug resistance mutations (DRMs), limiting ART options, and increasing HIV transmission. OBJECTIVE: We investigated the patterns of transmitted drug resistance (TDR) and acquired drug resistance (ADR) among HIV-1 patients in Portugal. METHODS: Data were obtained from 1050 HIV-1 patient samples submitted for HIV drug resistance (HIVDR) testing from January 2022 to June 2023. Evaluation of DRM affecting viral susceptibility to nucleoside/tide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase strand transfer inhibitors (INSTIs) was performed using an NGS technology, the Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay. RESULTS: About 71% of patients were ART naïve and 29% were experienced. Overall, 20% presented with any DRM. The prevalence of TDR and ADR was 12.6% and 41.1%, respectively. M184V, T215S, and M41L mutations for NRTI, K103N for NNRTI, and M46I/L for PIs were frequent in naïve and treated patients. E138K and R263K mutations against INSTIs were more frequent in naïve than treated patients. TDR and ADR to INSTIs were 0.3% and 7%, respectively. Patients aged 50 or over (OR: 1.81, p = 0.015), originating from Portuguese-speaking African countries (PALOPs) (OR: 1.55, p = 0.050), HIV-1 subtype G (OR: 1.78, p = 0.010), and with CD4 < 200 cells/mm3 (OR: 1.70, p = 0.043) were more likely to present with DRMs, while the males (OR: 0.63, p = 0.003) with a viral load between 4.1 to 5.0 Log10 (OR: 0.55, p = 0.003) or greater than 5.0 Log10 (OR: 0.52, p < 0.001), had lower chances of presenting with DRMs. CONCLUSIONS: We present the first evidence on TDR and ADR to INSTI regimens in followed up patients presenting for healthcare in Portugal. We observed low levels of TDR to INSTIs among ART-naïve and moderate levels in ART-exposed patients. Regimens containing PIs could be an alternative second line in patients with intermediate or high-level drug resistance, especially against second-generation INSTIs (dolutegravir, bictegravir, and cabotegravir).

Serological screening in a large-scale municipal survey in Cascais, Portugal, during the first waves of the COVID-19 pandemic: lessons for future pandemic preparedness efforts.

Background: Serological surveys for SARS-CoV-2 were used early in the COVID-19 pandemic to assess epidemiological scenarios. In the municipality of Cascais (Portugal), serological testing combined with a comprehensive socio-demographic, clinical and behavioral questionnaire was offered to residents between May 2020 and beginning of 2021. In this study, we analyze the factors associated with adherence to this municipal initiative, as well as the sociodemographic profile and chronic diseases clinical correlates associated to seropositivity. We aim to contribute with relevant information for future pandemic preparedness efforts. Methods: This was a cross-sectional study with non-probabilistic sampling. Citizens residing in Cascais Municipality went voluntarily to blood collection centers to participate in the serological survey. The proportion of participants, stratified by socio-demographic variables, was compared to the census proportions to identify the groups with lower levels of adherence to the survey. Univariate and multivariate logistic regression were used to identify socio-demographic, clinical and behavioral factors associated with seropositivity. Results: From May 2020 to February 2021, 19,608 participants (9.2% of the residents of Cascais) were included in the study. Based on the comparison to census data, groups with lower adherence to this survey were men, the youngest and the oldest age groups, individuals with lower levels of education and unemployed/inactive. Significant predictors of a reactive (positive) serological test were younger age, being employed or a student, and living in larger households. Individuals with chronic diseases generally showed lower seroprevalence. Conclusion: The groups with low adherence to this voluntary study, as well as the socio-economic contexts identified as more at risk of viral transmission, may be targeted in future pandemic situations. We also found that the individuals with chronic diseases, perceiving higher risk of serious illness, adopted protective behaviors that limited infection rates, revealing that health education on preventive measures was effective for these patients.

The Role of Late Presenters in HIV-1 Transmission Clusters in Europe.

BACKGROUND: Investigating the role of late presenters (LPs) in HIV-1 transmission is important, as they can contribute to the onward spread of HIV-1 virus before diagnosis, when they are not aware of their HIV status. OBJECTIVE: To characterize individuals living with HIV-1 followed up in Europe infected with subtypes A, B, and G and to compare transmission clusters (TC) in LP vs. non-late presenter (NLP) populations. METHODS: Information from a convenience sample of 2679 individuals living with HIV-1 was collected from the EuResist Integrated Database between 2008 and 2019. Maximum likelihood (ML) phylogenies were constructed using FastTree. Transmission clusters were identified using Cluster Picker. Statistical analyses were performed using R. RESULTS: 2437 (91.0%) sequences were from subtype B, 168 (6.3%) from subtype A, and 74 (2.8%) from subtype G. The median age was 39 y/o (IQR: 31.0-47.0) and 85.2% of individuals were males. The main transmission route was via homosexual (MSM) contact (60.1%) and 85.0% originated from Western Europe. In total, 54.7% of individuals were classified as LPs and 41.7% of individuals were inside TCs. In subtype A, individuals in TCs were more frequently males and natives with a recent infection. For subtype B, individuals in TCs were more frequently individuals with MSM transmission route and with a recent infection. For subtype G, individuals in TCs were those with a recent infection. When analyzing cluster size, we found that LPs more frequently belonged to small clusters (<8 individuals), particularly dual clusters (2 individuals). CONCLUSION: LP individuals are more present either outside or in small clusters, indicating a limited role of late presentation to HIV-1 transmission.

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.

A Tale of Three Recent Pandemics: Influenza, HIV and SARS-CoV-2.

Emerging infectious diseases are one of the main threats to public health, with the potential to cause a pandemic when the infectious agent manages to spread globally. The first major pandemic to appear in the 20th century was the influenza pandemic of 1918, caused by the influenza A H1N1 strain that is characterized by a high fatality rate. Another major pandemic was caused by the human immunodeficiency virus (HIV), that started early in the 20th century and remained undetected until 1981. The ongoing HIV pandemic demonstrated a high mortality and morbidity rate, with discrepant impacts in different regions around the globe. The most recent major pandemic event, is the ongoing pandemic of COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused over 5.7 million deaths since its emergence, 2 years ago. The aim of this work is to highlight the main determinants of the emergence, epidemic response and available countermeasures of these three pandemics, as we argue that such knowledge is paramount to prepare for the next pandemic. We analyse these pandemics' historical and epidemiological contexts and the determinants of their emergence. Furthermore, we compare pharmaceutical and non-pharmaceutical interventions that have been used to slow down these three pandemics and zoom in on the technological advances that were made in the progress. Finally, we discuss the evolution of epidemiological modelling, that has become an essential tool to support public health policy making and discuss it in the context of these three pandemics. While these pandemics are caused by distinct viruses, that ignited in different time periods and in different regions of the globe, our work shows that many of the determinants of their emergence and countermeasures used to halt transmission were common. Therefore, it is important to further improve and optimize such approaches and adapt it to future threatening emerging infectious diseases.

Genome-wide diversity of Zika virus: Exploring spatio-temporal dynamics to guide a new nomenclature proposal.

The Zika virus (ZIKV) disease caused a public health emergency of international concern that started in February 2016. The overall number of ZIKV-related cases increased until November 2016, after which it declined sharply. While the evaluation of the potential risk and impact of future arbovirus epidemics remains challenging, intensified surveillance efforts along with a scale-up of ZIKV whole-genome sequencing provide an opportunity to understand the patterns of genetic diversity, evolution, and spread of ZIKV. However, a classification system that reflects the true extent of ZIKV genetic variation is lacking. Our objective was to characterize ZIKV genetic diversity and phylodynamics, identify genomic footprints of differentiation patterns, and propose a dynamic classification system that reflects its divergence levels. We analysed a curated dataset of 762 publicly available sequences spanning the full-length coding region of ZIKV from across its geographical span and collected between 1947 and 2021. The definition of genetic groups was based on comprehensive evolutionary dynamics analyses, which included recombination and phylogenetic analyses, within- and between-group pairwise genetic distances comparison, detection of selective pressure, and clustering analyses. Evidence for potential recombination events was detected in a few sequences. However, we argue that these events are likely due to sequencing errors as proposed in previous studies. There was evidence of strong purifying selection, widespread across the genome, as also detected for other arboviruses. A total of 50 sites showed evidence of positive selection, and for a few of these sites, there was amino acid (AA) differentiation between genetic clusters. Two main genetic clusters were defined, ZA and ZB, which correspond to the already characterized 'African' and 'Asian' genotypes, respectively. Within ZB, two subgroups, ZB.1 and ZB.2, represent the Asiatic and the American (and Oceania) lineages, respectively. ZB.1 is further subdivided into ZB.1.0 (a basal Malaysia sequence sampled in the 1960s and a recent Indian sequence), ZB.1.1 (South-Eastern Asia, Southern Asia, and Micronesia sequences), and ZB.1.2 (very similar sequences from the outbreak in Singapore). ZB.2 is subdivided into ZB.2.0 (basal American sequences and the sequences from French Polynesia, the putative origin of South America introduction), ZB.2.1 (Central America), and ZB.2.2 (Caribbean and North America). This classification system does not use geographical references and is flexible to accommodate potential future lineages. It will be a helpful tool for studies that involve analyses of ZIKV genomic variation and its association with pathogenicity and serve as a starting point for the public health surveillance and response to on-going and future epidemics and to outbreaks that lead to the emergence of new variants.

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.

Different Genomic Changes Underlie Adaptive Evolution in Populations of Contrasting History.

Experimental evolution is a powerful tool to understand the adaptive potential of populations under environmental change. Here, we study the importance of the historical genetic background in the outcome of evolution at the genome-wide level. Using the natural clinal variation of Drosophila subobscura, we sampled populations from two contrasting latitudes (Adraga, Portugal and Groningen, Netherlands) and introduced them in a new common environment in the laboratory. We characterized the genome-wide temporal changes underlying the evolutionary dynamics of these populations, which had previously shown fast convergence at the phenotypic level, but not at chromosomal inversion frequencies. We found that initially differentiated populations did not converge either at genome-wide level or at candidate SNPs with signs of selection. In contrast, populations from Portugal showed convergence to the control population that derived from the same geographical origin and had been long-established in the laboratory. Candidate SNPs showed a variety of different allele frequency change patterns across generations, indicative of an underlying polygenic basis. We did not detect strong linkage around candidate SNPs, but rather a small but long-ranging effect. In conclusion, we found that history played a major role in genomic variation and evolution, with initially differentiated populations reaching the same adaptive outcome through different genetic routes.

Predictable phenotypic, but not karyotypic, evolution of populations with contrasting initial history.

The relative impact of selection, chance and history will determine the predictability of evolution. There is a lack of empirical research on this subject, particularly in sexual organisms. Here we use experimental evolution to test the predictability of evolution. We analyse the real-time evolution of Drosophila subobscura populations derived from contrasting European latitudes placed in a novel laboratory environment. Each natural population was sampled twice within a three-year interval. We study evolutionary responses at both phenotypic (life-history, morphological and physiological traits) and karyotypic levels for around 30 generations of laboratory culture. Our results show (1) repeatable historical effects between years in the initial state, at both phenotypic and karyotypic levels; (2) predictable phenotypic evolution with general convergence except for body size; and (3) unpredictable karyotypic evolution. We conclude that the predictability of evolution is contingent on the trait and level of organization, highlighting the importance of studying multiple biological levels with respect to evolutionary patterns.

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.

4Pipe4--A 454 data analysis pipeline for SNP detection in datasets with no reference sequence or strain information.

BACKGROUND: Next-generation sequencing datasets are becoming more frequent, and their use in population studies is becoming widespread. For non-model species, without a reference genome, it is possible from a panel of individuals to identify a set of SNPs that can be used for further population genotyping. However the lack of a reference genome to which the sequenced data could be compared makes the finding of SNPs more troublesome. Additionally when the data sources (strains) are not identified (e.g. in datasets of pooled individuals), the problem of finding reliable variation in these datasets can become much more difficult due to the lack of specialized software for this specific task. RESULTS: Here we describe 4Pipe4, a 454 data analysis pipeline particularly focused on SNP detection when no reference or strain information is available. It uses a command line interface to automatically call other programs, parse their outputs and summarize the results. The variation detection routine is built-in in the program itself. Despite being optimized for SNP mining in 454 EST data, it is flexible enough to automate the analysis of genomic data or even data from other NGS technologies. 4Pipe4 will output several HTML formatted reports with metrics on many of the most common assembly values, as well as on all the variation found. There is also a module available for finding putative SSRs in the analysed datasets. CONCLUSIONS: This program can be especially useful for researchers that have 454 datasets of a panel of pooled individuals and want to discover and characterize SNPs for subsequent individual genotyping with customized genotyping arrays. In comparison with other SNP detection approaches, 4Pipe4 showed the best validation ratio, retrieving a smaller number of SNPs but with a considerably lower false positive rate than other methods. 4Pipe4's source code is available at https://github.com/StuntsPT/4Pipe4.

Keeping your options open: Maintenance of thermal plasticity during adaptation to a stable environment.

Phenotypic plasticity may allow species to cope with environmental variation. The study of thermal plasticity and its evolution helps understanding how populations respond to variation in temperature. In the context of climate change, it is essential to realize the impact of historical differences in the ability of populations to exhibit a plastic response to thermal variation and how it evolves during colonization of new environments. We have analyzed the real-time evolution of thermal reaction norms of adult and juvenile traits in Drosophila subobscura populations from three locations of Europe in the laboratory. These populations were kept at a constant temperature of 18ºC, and were periodically assayed at three experimental temperatures (13ºC, 18ºC, and 23ºC). We found initial differentiation between populations in thermal plasticity as well as evolutionary convergence in the shape of reaction norms for some adult traits, but not for any of the juvenile traits. Contrary to theoretical expectations, an overall better performance of high latitude populations across temperatures in early generations was observed. Our study shows that the evolution of thermal plasticity is trait specific, and that a new stable environment did not limit the ability of populations to cope with environmental challenges.

Tettigettalnajosei (Boulard, 1982) (Hemiptera: Cicadoidea): first record in Spain, with notes on the distribution, genetic variation and behaviour of the species.

The small cicada Tettigettalnajosei (Boulard, 1982) was until recently only known from southern Portugal and was considered endemic to this country. Fieldwork in 2013 led to the first record of the species in Spain, expanding its known eastern range to Andalusia. The northern limits remain poorly defined but it appears that the distribution of Tettigettalnajosei is restricted to the south Atlantic coastline in the Iberian Peninsula, with the highest densities found in Algarve. Some notes on behaviour and genetic variation of Tettigettalnajosei are also given.

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.

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.