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The accuracy of the DNA barcoding tool depends on the existence of a comprehensive archived library of sequences reliably determined at species level by expert taxonomists. However, misidentifications are not infrequent, especially following large-scale DNA barcoding campaigns on diverse and taxonomically complex groups. In this study we used the species-rich flea beetle genus Longitarsus, that requires a high level of expertise for morphological species identification, as a case study to assess the accuracy of the DNA barcoding tool following several optimization procedures. We built a cox1 reference database of 1502 sequences representing 78 Longitarsus species, among which 117 sequences (32 species) were newly generated using a non-invasive DNA extraction method that allows keeping reference voucher specimens. Within this dataset we identified 69 taxonomic inconsistencies using barcoding gap analysis and tree topology methods. Threshold optimisation and a posteriori taxonomic revision based on newly generated reference sequences and metadata allowed resolving 44 sequences with ambiguous and incorrect identification and provided a significant improvement of the DNA barcoding accuracy and identification efficacy. Unresolved taxonomic uncertainties, due to overlapping intra- and inter-specific levels of divergences, mainly regards the Longitarsus pratensis species complex and polyphyletic groups L. melanocephalus, L. nigrofasciatus and L. erro. Such type of errors indicates either poorly established taxonomy or any biological processes that make mtDNA groups poorly predictive of species boundaries (e.g. recent speciation or interspecific hybridisation), thus providing directions for further integrative taxonomic and evolutionary studies. Overall, this study underlines the importance of reference vouchers and high-quality metadata associated to sequences in reference databases and corroborates, once again, the key role of taxonomists in any step of the DNA barcoding pipeline in order to generate and maintain a correct and functional reference library.

Cave crickets of the genus Dolichopoda (Orthoptera; Rhaphidophoridae) represent a key component of cave ecosystems. In Italy, nine species are currently known, distributed from the northwestern regions to the southernmost Apennines, with occurrences also along various Tyrrhenian coastal areas and islands, including Sardinia. In this study, we focus on the Apennine region, where we sampled 18 populations of Dolichopoda spp. and sequenced mitochondrial markers (cox1 and 16S) from newly collected individuals to investigate their distribution and genetic diversity. Our analyses identified two previously unrecognized lineages within D. geniculata. Moreover, the sampled caves in the northern Apennines allowed us to refine the distributional ranges of D. geniculata, D. letitiae, and D. schiavazzii. Finally, we provide comments to support a future taxonomic revision of the group.

The recently published mitochondrial genome of the fingerprint oyster Alectryonella plicatula (Gmelin, 1791) with GenBank accession number MW143047 was resolved in an unexpected phylogenetic position, as sister to the Pacific cupped oyster Magallana gigas (Thunberg, 1793) and share with this species three typical gene duplications that represent robust synapomorphies of the Magallana clade. In this study, we verified the identity of MW143047 using direct comparisons of single gene sequences, DNA barcoding and phylogenetic analyses. BLAST searches using as query each of the 12 protein coding genes (PCGs) and rRNA genes extracted from MW143047 retrieved M. gigas as best hit with 100% sequence identity for all genes. MW143047 is nested within the clade formed by M. gigas sequences, with virtually zero-length terminal branch, both in the cox1 gene tree (based on 3639 sequences) and in the 16S gene tree (based on 1839 sequences), as well as in the Maximum Likelihood mitogenomic tree based on concatenated sequence of 12 PCGs. Our findings suggest that the original specimen used for mitogenome sequencing was misidentified and represents an individual of M. gigas . This study reinforces the notion that morphological shell analysis alone is not sufficient for oyster identification, not even at high taxonomic ranks such as subfamilies. While it is well established that morphological identification of oysters should be validated by molecular data, this study emphasizes that also molecular data should be taxonomically verified by means of DNA barcoding and phylogenetic analyses. The implications of the publication of taxonomically misidentified sequences and mitogenomes are discussed.

Here we provide a Code-compliant description of Ostrea oleomargarita Oliver, Salvi, and Al-Kandari, sp. nov as reported in Salvi et al. (2022) and complemented with the ZooBank registration numbers of the publication and of the new species name. Results of phylogenetic, species delimitation, and morphological analyses on which the systematic assessment of this new species is based can be found in Salvi et al. (2022).

Since the opening of the Suez Canal in 1869, hundreds of Indo-Pacific species have rapidly colonised the Mediterranean. Understanding the spatial and temporal patterns of this biological invasion is crucial for assessing its ecological impact. A notable example is the non-indigenous oyster Dendostrea sp., first discovered in Türkiye in 1998 and later found throughout the easternmost Mediterranean, though its identity remained uncertain. This study clarifies the taxonomic identity and the introduction pathways of Dendostrea sp. using molecular analyses. Over 100 specimens from 25 sites in the eastern Mediterranean, as well as Île d’Ambre and Rodrigues in the Mauritius Archipelago (the native range), were sequenced for mitochondrial DNA (COI) and compared to 422 sequences from GenBank. Phylogenetic and species delimitation analyses identified the Mediterranean oysters as D. cf. crenulifera, conspecific with oysters from Rodrigues. The Mediterranean populations exhibited high genetic diversity, lack of phylogeographic structure and showed no evidence of a founder effect. These findings suggest that D. cf. crenulifera entered the Mediterranean over two decades ago through multiple shipping-mediated introductions from its native range and successfully established, likely aided by the decline of native biodiversity. The observed genetic diversity pattern across the Mediterranean indicates high propagule pressure driving the species’ invasion history, which likely underpins its establishment success by reducing the deleterious consequences of population bottlenecks and overcoming the so-called genetic paradox. This study underscores the value of molecular surveys in identifying taxonomically challenging non-indigenous species and uncovering their invasion histories.

The European leaf toed Gecko, Euleptes europaea , is a strictly nocturnal species endemic to the western Mediterranean and has long been considered a rock-specialist as it is associated with this habitat during its entire daily and life cycle. In this study, we report observations of arboreal behaviour in E. europaea , collected during field research over a 40-year period from across the entire species range. We provide a review of the available information on this topic that contributes to a refined view of the habitat uses and arboreal abilities of this species. Arboreal behaviour in E. europaea was observed throughout the year, across different macrohabitats, on a wide variety of tree, shrub, and bush species, on various parts of the plant (trunk, branches, fronds, twigs, leaves), and at different height from the ground. Remarkably, E. europaea shows an extraordinarily agile arboreal locomotion associated with striking morphological adaptations to an arboreal lifestyle, namely a prehensile tail bearing a terminal adhesive pad that supplements grasping force, an equilibrium asset, and scansor adhesion both in static condition and during escape. We conclude that E. europaea is a climbing gecko (opposed to ground dwelling), occupying both rocky and arboreal microhabitats. While the evolutionary origin and ecological drivers of the arboreal behaviour of E. europaea remains to be fully investigated, this realization has important implications for designing fieldwork research and management strategies for conservation.

Tuber spp. (Ascomycota) forms hypogeous fruiting bodies (truffles) that host many microbial species as well as invertebrates which feed on them. Despite the larvae and adults of Diptera and Coleoptera are commonly found to inhabit truffles, molecular investigations assessing their occurrence are still few and the number of species is probably underestimated. In this study, 52 larvae and adults of Diptera from 23 T . aestivum ascomata collected in two provinces of northern and central Italy were molecularly characterized. The sequences fell into four Diptera families, and four taxa were identified as Cheilosia soror , Phaonia cf. trimaculata , Drosophila subobscura , and Suillia gigantea . Morphology of adults belonging to these species confirmed their identity. Additional three taxa belonging to the Helomyzidae remained unclassified. The study highlighted the coexistence of different Diptera species in the same ascoma, suggesting potential lack of competitive exclusion. Geographical distribution analysis reveals non-site specificity for most species. This research contributes insights into the diversity of Dipteran species and their interactions with truffles and lays the groundwork for their monitoring, at a time where truffle resources are threatened by anthropic and environmental factors.

Abstract European mountain systems have played a crucial role in shaping the distribution of species and of their genetic diversity during the Quaternary climatic changes, with the establishment of allopatric patterns across main mountain ranges. Here we investigated the evolutionary history of flea beetles of the Longitarsus candidulus species-group showing an uncommon disjunct biogeographic pattern across the Apennine and the Pyrenees. We applied a multilocus molecular approach and multispecies coalescent models to establish a phylogenetic and systematic framework for this morphologically homogeneous species-group and to estimate the time of main cladogenetic events underlying the origin of the Apennine-Pyrenees pattern. We found strong support for the monophyly of the candidulus group with a sister relationship between Longitarsus laureolae and L. leonardii endemic to the Apennine and the Pyrenees mountains respectively. The timing of speciation events in the candidulus species-group coincides with 2 major climatic transitions during the Early and Middle Pleistocene which resulted in significant environmental changes in Europe and suggest a scenario of allopatric isolation and divergence on distinct mountain ranges. The split between the thermophilic species L. candidulus and the ancestor of the temperate species L. laureolae and L. leonardii is estimated at ~3 Ma during the transition from Pliocene to Pleistocene and was probably triggered by their segregation in xerophilous and temperate habitats. The speciation between L. laureolae and L. leonardii, estimated at ~1 Ma during the Mid-Pleistocene Transition, can be explained by the establishment of unfavorable conditions in West Alps and Central Massif underlying the onset of the Apennine-Pyrenees disjunct pattern. Finally, the strict association between members of the candidulus group and distinct Thymelaeaceae plants suggests further studies to address the hypothesis that speciation in these flea beetles might have been also associated with Pleistocene range changes of their host plants.

Abstract Longitarsus candidulus (Foudras) is a thermophilic flea beetle species widely distributed in the Mediterranean Basin and associated with Daphne gnidium L. and Thymelaea hirsuta (L.). Longitarsus laureolae Biondi and Longitarsus leonardii Doguet, phylogenetically closely related to L. candidulus, show together a peculiar and rare disjunct distribution along the central-southern Apennines and the Cantabrian-Pyrenean mountain system, respectively. Both are associated with Daphne laureola L. in mesophilic habitats. We used “ecological niche modeling” to infer the Pleistocene dynamics in the distribution of the three flea beetle species and their host plants. We interpreted their current distributions, paying particular attention to the presumed time of species divergence as inferred from recent studies. The differentiation of L. laureolae and L. leonardii from L. candidulus likely represents a response to the marked climatic changes during the Late Pliocene. Such a split was likely associated with a trophic niche shift of the laureolae/leonardii ancestor towards the typically mesophilic host plant D. laureola. The subsequent split between L. laureolae and L. leonardii, possibly due at first to the niche competition, was then boosted by an allopatric divergence during the Middle Pleistocene, likely caused by a large area of low environmental suitability for both species, mainly located between the northern Apennines and the south-western Alps.