Phytophagous insects have specialized on virtually every plant lineage. Parasitic plants, however, are uncommon hosts. Among insects, only a single lineage of weevils, the Smicronychini, has successfully radiated on both parasitic and non-parasitic plants in a large panel of distantly related Asterid families. This unusual pattern suggests that major host plant shifts have occurred over the course of their diversification. Through the analysis of a phylogenomic dataset, we reconstruct for the first time their evolutionary history and ancestral host-plant repertoire. Our results show that independent host plant shifts occurred both from parasitic to non-parasitic hosts and between distinct parasitic lineages. These results suggest that host shift mechanisms can be driven by ecological opportunities provided by plant-plant interactions. This first evidence of extreme insect host plant shifts, apparently mediated by parasitic plant-plant interactions, emphasizes the core importance of ecological interactions as driving forces behind insect host plant shifts.

Representatives of the weevil tribe Ochyromerini (Coleoptera: Curculionidae: Curculioninae) have been increasingly reported as specialized pollinators of Annonaceae Juss., with some species in the genus Endaeus Schoenherr, 1826 engaging in brood-site pollination with their host. Recent investigations into the poorly documented life history of these pollinators have led to the discovery of new species in the genus Endaeus and provided details of their life histories. The following species are herein described: E. canangae Haran sp. nov. from Southeast Asia, associated with Cananga odorata (Lam.) Hook.f. & Thomson (Annonaceae); E. lenticulatus Haran sp. nov. from Central Africa, associated with Uvariodendron molundense (Diels) R.E.Fr. (Annonaceae); E. xylopiae Haran & Zelvelder sp. nov., E. jaculifer Haran & Zelvelder sp. nov. and E. convexiculus Haran & Zelvelder sp. nov. from Central Africa, associated with Xylopia aethiopica (Dunal) A.Rich. (Annonaceae) and E. staminicola Haran sp. nov. also from Central Africa, associated with Allanblackia floribunda Oliv. (Clusiaceae Lindl.). Pictures of habitus and terminalia of adults, molecular and morphological diagnostic tools along with life history data are provided for each species. These new associations are discussed in light of the general patterns of specialized brood-site pollination mutualism between plants and weevils.

The presence of field margins in agricultural landscapes increases plant and animal diversity, providing food resources and shelter. Margins also increase ecosystem services’ delivery by hosting a diverse insect fauna. Beetles, in particular, are important contributors, acting as auxiliaries, decomposers and pollinators. However, these habitats are threatened by agricultural intensification, representing potential traps for biodiversity due to pesticide and fertilizer drift. In this work we aim at understanding the unintended effects of agricultural practices on beetle diversity, abundance and composition in agricultural field margins, at a national scale. More precisely, we investigated which agricultural practices directly impact beetles and which ones indirectly impact them through their effects on plants. Here we used data from a national standardized monitoring network in metropolitan France where both plants and beetles were identified to the species level in more than 300 different field margins, representing the main crops (cereals, vineyards and market gardening) and a gradient of agricultural intensification practices at the national level. We ran structural equation modeling (SEM) to disentangle the direct and indirect effects of management on beetles. We showed that intensive agricultural practices such as high pesticide pressure and fertilization are mostly associated with a decrease in beetle diversity and abundance, as well as in the proportion of floral visitors, while increasing the proportion of pests in each community. We also showed that many of these impacts are mediated by plant diversity and traits. These results suggest that managing composition and structure of field margin vegetation, as well as reducing agricultural intensification both at the regional and at the field-margin level, are key to guarantee high beetle diversity and optimal ecosystem services such as pollination and pest regulation in the field margins.

Leafhoppers (Cicadellidae) are major pests of key crops, including okra, eggplant, and cotton, in tropical Africa. Several closely related species, which are often challenging to differentiate, infest these crops, yet little is known about their relative roles in causing crop damage. The study aims to clarify the taxonomy of the species and provide a preliminary assessment of their impact on eggplant, okra, and cotton production. A biomolecular analysis was conducted on 180 leafhopper individuals collected from 13 locations across the three crops during the 2021 and 2022 cropping seasons. The findings revealed that the same species were present on cotton, okra, and eggplant. Three primary species were identified: Empoasca papayae, Jacobiasca lybica, and Amrasca biguttula. The abundance of these species varied between the two years. In 2021, the predominant species found on okra, eggplant, and cotton were J. lybica and E. papayae; J. lybica was the most abundant, proportions on okra, eggplant, and cotton were 90, 95.8, and 91.7%, respectively. In contrast, by 2022, A. biguttula emerged as the most abundant species with proportions of 100, 100, and 85% on okra, eggplant, and cotton, respectively. Although J. lybica was present on cotton, it was less abundant. The introduction of A. biguttula, a species new to West Africa, marked a shift in the leafhopper community, replacing the local species on African eggplant, okra, and cotton. These results enhance our understanding of leafhopper species composition on these important crops and will inform the development of targeted pest management.

The species of the genus Derelomus Schoenherr, 1825 (Curculionidae: Curculioninae: Derelomini) from the Afrotropical (excluding Madagascar) and Mediterranean regions are here revised. In total, 32 valid species are recognized, including 14 described as new: D. acuminatus Haran sp. nov.; D. baka Haran sp. nov.; D. brevis Haran sp. nov.; D. brunneus Haran sp. nov.; D. caldarai Haran sp. nov.; D. crypticus Haran sp. nov.; D. discus Haran sp. nov.; D. karooensis Haran sp. nov.; D. peglerae Haran sp. nov.; D. prochesi Haran sp. nov.; D. setifer Haran sp. nov.; D. strangulatus Haran sp. nov.; D. strelitziae Haran sp. nov. and D. trinotatus Haran sp. nov. The following new synonymies are proposed: D. pallidus Hartmann, 1904 and D. hartmanni Klima, 1934 = D. pallidus Fåhraeus, 1844 syn. nov.; D. rectirostris Hustache, 1937 = D. languidus Fåhraeus, 1844 syn. nov.; D. subcostatus Boheman, 1844 = D. chamaeropis (Fabricius, 1798) syn. nov.; D. kocheri Hoffmann, 1957 = D. piriformis (Hoffamnn, 1938) syn. nov.; D. auberti Hustache, 1932 = D. ephippiger Gyllenhal, 1836 syn. nov and Psilocaulus elatus Richard, 1958 = Derelomus costiger Marshall, 1958 syn. nov. Morphological examination of specimens and molecular analyses enabled recognising four main species groups: the D. signatus Gyllenhal, 1836 group (eight species) associated with the inflorescences of species of Vachellia Wight & Arn. (Fabaceae), the D. pallidus Fåhraeus, 1844 group (three species) associated with Achariaceae and Ebenaceae, the D. nigrovariegatus Hustache, 1936 group (six species) associated with Strelitzia (Strelitziaceae) and Phoenix reclinata Jacq. (Arecaceae) and the D. ephippiger Gyllenhal, 1836 group (12 species) associated with male inflorescences of various palms (Chamaerops L., Cocos L., Phoenix L.; Arecaceae). A key and a description or redescription are provided for each species, with illustrations of habitus of adults and male terminalia.

Swollen Shoot is a viral disease affecting cocoa trees, transmitted by several species of mealybugs (Insecta, Hemiptera, Sternorrhyncha, Pseudococcidae). These insects maintain trophobiotic relationships with a complex and species-rich assemblage of ants protecting them and natural enemies controlling their populations. Here, we provide a curated DNA barcode database to characterise this insect community. Systematic observation of 7,500 cocoa trees was conducted, coupled with the collection of mealybug colonies and associated insect communities (parasitoids, predators and ants). Natural enemies were reared from mealybug colonies collected from 1,430 cocoa trees. Specimens were identified morphologically and sequenced for fragments of the standard DNA barcode region of the COI. We recovered 17 species of mealybugs from the family Pseudococcidae. Amongst these species, eight are new to the Ivorian cocoa orchard: Dysmicoccusneobrevipes Beardsley, Ferrisiadasylirii (Cockerell), Maconellicoccusugandae (Laing), Paracoccusmarginatus Williams & Granara de Willink, Phenacoccussolenopsis Tinsley, Planococcusminor (Maskell), Pseudococcusconcavocerarii James and Pseudococcusocciduus De Lotto. Three of these species were identified for the first time in cocoa orchards in Africa: D.neobrevipes, Fe.dasylirii and Ph.solenopsis. A total of 54 ant species were identified and represented the first record of these species associated with mealybug colonies in cocoa in Côte d'Ivoire. Amongst the species associated with the mealybugs, 22 primary parasitoids, eight hyperparasitoids, 11 ladybirds beetles (Coccinellidae), seven gall midges (Cecidomyidae), one predatory lepidopteran species and four spider species were identified. Nine species of mealybugs parasitoids are newly recorded in the African cocoa orchards: Acerophagusaff.dysmicocci, Aloencyrtus sp., Anagyruskamali, Anagyrusaff.pseudococci, Aenasiusadvena, Clauseniaaff.corrugata, Gyranusoideaaff.tebygi, Zaplatycerusaff.natalensis (Encyrtidae) and Coccophaguspulvinariae (Aphelinidae) and one hyperparasitoid, Pachyneuronmuscarum (Pteromalidae). For Côte d'Ivoire in particular, besides the previously mentioned nine parasitoids and one hyperparasitoid, five additional species are recorded for the first time, including four primary parasitoids, Blepyrusinsularis (Encyrtidae), Clauseniacorrugata (Encyrtidae), Clausenia sp. (Encyrtidae), and Coccidoctonuspseudococci (Encyrtidae) and one hyperparasitoid, Cheiloneuruscyanonotus (Encyrtidae). These results significantly enhance the knowledge of the diversity of the entomofauna associated with Swollen Shoot disease and pave the way for developing control methods based on the natural regulation of its mealybug (Pseudococcidae) vectors.

In a context of unprecedented insect decline, it is critical to have reliable monitoring tools to measure species diversity and their dynamic at large-scales. High-throughput DNA-based identification methods, and particularly metabarcoding, were proposed as an effective way to reach this aim. However, these identification methods are subject to multiple technical limitations, resulting in unavoidable false-positive and false-negative species detection. Moreover, metabarcoding does not allow a reliable estimation of species abundance in a given sample, which is key to document and detect population declines or range shifts at large scales. To overcome these obstacles, we propose here a human-assisted molecular identification (HAMI) approach, a framework based on a combination of metabarcoding and image-based parataxonomic validation of outputs and recording of abundance. We assessed the advantages of using HAMI over the exclusive use of a metabarcoding approach by examining 492 mixed beetle samples from a biodiversity monitoring initiative conducted throughout France. On average, 23% of the species are missed when relying exclusively on metabarcoding, this percent being consistently higher in species-rich samples. Importantly, on average, 20% of the species identified by molecular-only approaches correspond to false positives linked to cross-sample contaminations or mis-identified barcode sequences in databases. The combination of molecular methodologies and parataxonomic validation in HAMI significantly reduces the intrinsic biases of metabarcoding and recovers reliable abundance data. This approach also enables users to engage in a virtuous circle of database improvement through the identification of specimens associated with missing or incorrectly assigned barcodes. As such, HAMI fills an important gap in the toolbox available for fast and reliable biodiversity monitoring at large scales.

The bestRAD technique is a reduced genome representation approach with high-capacity sample multiplexing and physical isolation of biotin-labelled target DNA fragments using streptavidin beads, which should reduce total cost and genotyping errors. While we here formalise the relevance of this approach within the HTS landscape, our foremost aim was to improve its replicability, validity, and transparency. We first optimised the molecular laboratory protocol and shared the associated protocols (e.g., final detailed methodologies, quality control, best practices) under the FAIR principles. Using 84 worldwide individual samples of the Oriental fruit fly, Bactrocera dorsalis, a major invasive pest, we revealed a low rate of PCR duplicates, robustness to DNA quality and quantity, high genotype call rate, insignificant genotyping error rate, high nuclear and mitochondrial genome representativeness, and a high level of genetic information. This in-depth data quality assessment, along with total cost and handling time reduced by an estimated one-third relative to the parent RAD-Seq version, demonstrates that bestRAD is an excellent compromise between cost and quality. While we generated high-quality genomic resources for B. dorsalis, we also share details and recommendations for the bestRAD technique that can be readily used in any laboratory and applied to all organisms, even without published genome sequence.

AimBiological invasions result from the combination of (i) population dispersal opportunities and (ii) adaptations to the recipient environment. Identifying complex migration histories, made of long-distance dispersal from the native range and secondary introductions, or genetic patterns indicative of adaptation is crucial to build coherent management efforts of problematic invasive species. We here aimed at determining the routes of introduction and describing the genetic peculiarities of recent introductions of Wasmannia auropunctata, a destructive invasive ant species with a polymorphic clonal/sexual reproduction system.LocationWe focused on three recently established European populations (Cyprus, France and Spain) and their relationship with earlier worldwide introductions and the native South American range of W. auropunctata.MethodsWe used a combination of cytochrome c oxidase subunit I (COI) sequencing and genotyping of a total of 686 European individuals at 12 microsatellite loci (from 76 nests and seven localities), together with previous worldwide datasets totalling 503 COI sequences and 6963 genotyped individuals. Phylogenetic reconstruction and genetic differentiation analyses were used to infer the origin, reproduction systems and genetic diversity of the three European populations.ResultsWe show that the history of the invasion of Europe is a mix of secondary introductions from a Mediterranean bridgehead population (Israel), and a novel long-distance introduction from a climatically similar area of northeastern Argentina. All newly introduced populations reproduce clonally and display an outbred genotypic pattern, consistent with all prior introductions and with anthropised areas of the native range.Main ConclusionsThis study confirms that preventing novel introductions is complex and requires adequate surveillance tools to simultaneously monitor areas of the native range with potential prior adaptation and previous introductions that could act as bridgeheads. Because of their ecological and genetic similarities, the fate of more ancient introductions could help us foresee what the future holds for European introductions.

ABSTRACTLinnaean and Wallacean shortfalls (Uncertainties on species taxonomy and distribution, respectively) are major factors hampering efficient conservation planning in the current context of biodiversity erosion. These shortfalls concern even widespread and abundant species in relatively well-studied regions such as the Mediterranean biodiversity hotspot which still hosts a large fraction of unrecognised biodiversity, notably in small vertebrates. Species delimitations have long been based on phylogenetic analyses of a small number of standard markers, but accurate lineage identification in this context can be obscured by incomplete lineage sorting, introgression or isolation by distance. Recently, integrative approaches coupling various sets of characters or analyses of contact zones aiming at estimating reproductive isolation (RI) have been advocated instead. Analyses of introgression patterns in contact zone with genomic data represent a powerful way to confirm the existence of independent lineages and estimate the strength of their RI at the same time. The Spiny-footed Lizard Acanthodactylus erythrurus (Schinz, 1833) is widespread in the Iberian Peninsula and the Maghreb and exhibits a large amount of genetic diversity, although the precise number and distribution of its genetic lineages remain poorly understood. We applied a RADseq approach to obtain a genome wide SNPs dataset on a contact zone in central Morocco between the previously described Rif and Middle-Atlas lineages. We show that these two lineages exhibit strong RI across this contact zone, as shown by the limited amount and restricted spatial extant of gene flow. We interpret these results as evidence for species-level divergence of these two lineages. Our study confirms the usefulness of RADseq approaches applied on contact zones for cryptic diversity studies and therefore to resolve Linnaean and Wallacean shortfalls.

This Collective Article presents information about 30 species with records in eight countries (Greece, Israel, Italy, Montenegro, Slovenia, Spain, Syria and Türkiye) and six ecoregions extending from the Alboran to the Levantine Seas. The recorded species belong to eight Phyla (4 Chlorophyta, 1 Rhodophyta, 1 Porifera, 3 Cnidaria, 2 Platyhelminthes, 2 Arthropoda, 2 Mollusca and 15 Chordata) as follows: Chlorophyta: Didymosporangium repens, Ochlochaete hystrix and Phaeophila hirsuta are reported for the first time from the Aegean coasts of Türkiye and Penicillus capitatus is firstly recorded in Slovenian coastal waters; Rhodophyta: Ptilophora dentata is recorded for the first time in Turkish coasts, from the entrance of a marine cave; Porifera: Tethya meloni is reported from Montenegrin waters; Cnidaria: Savalia savaglia and Dendrophyllia ramea are firstly observed north of the Almeria-Oran front in the southeastern Iberian Peninsula, while Spinimuricea cf. atlantica is firstly recorded in the Gulf of Lion constituting the easternmost record of the species in the Mediterranean Sea; Platyhelminthes: the polyclad flatworms Thysanozoon brocchii and Planocera graffi are reported for the first time from Greek waters, observed inside marine caves; Mollusca: Ascobulla fragilis is firstly reported from the Eastern Levantine Sea while the blanket octopus Tremoctopus violaceus is recorded in Izmir Bay constituting its fifth sighting in the Aegean Sea after a quarter of a century; Arthropoda: the copepod Ditrychocorycaeus africanus is firstly recorded in the Ionian Sea while the tufted ghost crab Ocypode cursor is detected further north in the Tyrrhenian Sea; Chordata: the bothid flounder Arnoglossus grohmanni is firstly reported in Spain while specimens of the rare bythitid Bellottia apoda are presented for the Adriatic Sea; the chondrichthyans Chimaera monstrosa, Dalatias licha, Heptranchias perlo, Leucoraja circularis, Mustelus mustelus, Oxynotus centrina, Squatina aculeata and Torpedo marmorata are presented as collected within 13 continuous years in the bathyal zone of the Antalya Bay; the speleophilic fish Grammonus ater is firstly recorded in the Alboran Sea, observed in a marine cave; the critically endangered sandy ray Leucoraja circularis is reported from the eastern Ionian Sea; the crested oarfish Lophotus lacepede is reported for the first time from Sardinia, based on evidence dating back 20 years; the white trevally Pseudocaranx dentex is firstly recorded in Tremiti Islands (Adriatic Sea, Italy) while the phaeton dragonet Synchiropus phaeton and the gobid Zebrus pallaoroi are firstly reported from Syrian and Italian waters, respectively.

Uncertainties on species taxonomy and distribution are major factors hampering efficient conservation planning in the current context of biodiversity erosion, even concerning widespread and abundant species in relatively well-studied regions. Species delimitation have long been based on phylogenetic analyses of a small number of standard markers, but accurate lineage identification through this approach can be hampered by incomplete lineage sorting, introgression or isolation by distance. In that context, analyses of introgression patterns at secondary contact zones offer an interesting alternative by allowing a direct estimation of reproductive isolation, especially when using genome-wide markers. Here, we investigated a contact zone between two genetic groups of the Spiny-footed Lizard Acanthodactylus erythrurus (Schinz, 1833) in Morocco, whose status as separate lineages remained disputed in previous multilocus studies. Based on thousands of genome-wide markers obtained through a RADseq approach, we confirmed that they represent distinct evolutionary lineages. Furthermore, the transition at their contact zone was very steep, with spatially restricted gene flow, highlighting levels of reproductive isolation consistent with species-level lineages. Our study further illustrates the power of RADseq-based studies of contact zones to understand cryptic diversity in non-model organisms.

Phytophagous insects have specialized on virtually every plant lineage. Parasitic plants, however, are uncommon hosts. Among insects, only a single lineage of weevils, the Smicronychini, has successfully radiated on both parasitic and non-parasitic plants in a large panel of distantly related Asterid families. This unusual pattern suggests that major host plant shifts have occurred over the course of their diversification. Through the analysis of a phylogenomic dataset, we reconstruct for the first time their evolutionary history and ancestral host plant associations. Our results show that independent host plant shifts occurred both from parasitic to non-parasitic hosts and between distinct parasitic lineages. These results suggest that host shift mechanisms can be driven by ecological opportunities provided by plant-plant interactions. This first evidence of extreme insect host plant shifts mediated by parasitic plant-plant interactions emphasizes the core importance of ecological interactions as driving forces behind insect host plant shifts.### Competing Interest StatementThe authors have declared no competing interest.

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.

Weevils are an unusually species-rich group of phytophagous insects for which there is increasing evidence of frequent involvement in brood-site pollination. This study examines phylogenetic patterns in the emergence of brood-site pollination mutualism among one of the most speciose beetle groups, the flower weevils (subfamily Curculioninae). We analysed a novel phylogenomic dataset consisting of 214 nuclear loci for 202 weevil species, with a sampling that mainly includes flower weevils as well as representatives of all major lineages of true weevils (Curculionidae). Our phylogenomic analyses establish a uniquely comprehensive phylogenetic framework for Curculioninae and provide new insights into the relationships among lineages of true weevils. Based on this phylogeny, statistical reconstruction of ancestral character states revealed at least 10 independent origins of brood-site pollination in higher weevils through transitions from ancestral associations with reproductive structures in the larval stage. Broadly, our results illuminate the unexpected frequency with which true weevils-typically specialized phytophages and hence antagonists of plants-have evolved mutualistic interactions of ecological significance that are key to both weevil and plant evolutionary fitness and thus a component of their deeply intertwined macroevolutionary success.

The weevil genus Cryptolarynx Van Schalkwyk, 1966 is endemic to the Northern and Western Cape provinces of South Africa. The two previously known species of the genus, C. vitis (Marshall, 1957) and C. estriatus (Marshall, 1957), have an aberrant globular body and head shape, which has made it difficult to place the genus into the classification systems of the Curculionoidea. This paper presents the description of 21 new species of Cryptolarynx from South Africa (C. subglaber Haran sp. nov., C. squamulatus Haran sp. nov., C. muellerae Haran sp. nov., C. hirtulus Haran sp. nov., C. robustus Haran sp. nov., C. namaquanus Haran sp. nov., C. carinatus Haran sp. nov., C. variabilis Haran sp. nov., C. pyrophilus Haran sp. nov., C. pilipes Haran sp. nov., C. armatus Haran sp. nov., C. falciformis Haran sp. nov., C. oberprieleri Haran sp. nov., C. spinicornis Haran sp. nov., C. cederbergensis Haran sp. nov., C. homaroides Haran sp. nov., C. marshalli Haran sp. nov., C. endroedyi Haran sp. nov., C. oberlanderi Haran sp. nov., C. san Haran sp. nov., and C. luteipennis Haran sp. nov.) and of one new genus and species, Hadrocryptolarynx major Haran gen. et sp. nov., also from South Africa. A redescription of the genus Cryptolarynx is provided to incorporate the characters of the new species. The plant genus Oxalis (Oxalidaceae) is recorded as larval host for several species of Cryptolarynx and for Hadrocryptolarynx Haran gen. nov., as their larvae develop in the subterranean bulbs of members of the genus, and the egg, larva and pupa of C. variabilis are described. The characters of the Cryptolarynx larva confirm that Cryptolaryngini are an early-diverging group of Curculionidae, with a placement among taxa currently classified in the subfamily Brachycerinae sensu lato, and although their exact taxonomic position remains unresolved, some larval characters, and also pupal ones, suggest a close relationship between Cryptolaryngini and Stenopelmus Schoenherr. Potential use of species of Cryptolarynx in the biological control of weedy South African species of Oxalis is discussed.

The potential for population genomics to elucidate invasion pathways of a species is limited by taxonomic identification issues. The Oriental fruit fly pest, Bactrocera dorsalis (Hendel) belongs to a complex in which several sympatric species are attracted to the same lure used in trapping and are morphologically cryptic and/or reported to hybridize. In this study, we evaluated the taxonomic ambiguity between B. dorsalis and 2 major cryptic species, based on morphological expertise and 289 target specimens sampled across the whole distribution range. Specimens were then subjected to DNA sequence analyses of the COI mitochondrial barcode and the EIF3L nuclear marker to evaluate the potential for molecular identification, in particular for specimens for which morphological identification was inconclusive. To this aim, we produced reference datasets with DNA sequences from target specimens whose morphological identification was unambiguous, which we complemented with 56 new DNA sequences from closest relatives and 76 published and curated DNA sequences of different species in the complex. After the necessary morphological observation, about 3.5% of the target dataset and 47.6% of the specimens from Southeast Asian islands displayed ambiguous character states shared with B. carambolae and/or B. occipitalis. Critical interpretation of DNA sequence data solved morphological ambiguities only when combining both mitochondrial and nuclear markers. COI discriminated B. dorsalis from 5 species; EIF3L and ITS from another species. We recommend this procedure to ensure correct identification of B. dorsalis specimens in population genetics studies and surveillance programs.