The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA‐based monitoring relies on validated reference data sets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658‐bp 5′ end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the data set to 1,294 DNA barcodes. A multi‐approach species delimitation analysis involving two distance (OT and ABGD) and four tree‐based (PTP, MPTP, GMYC and bGMYC) methods was used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct molecular operational units, whereas the remaining ones were classified as ‘warnings’ (i.e. showing a mismatch between morphospecies assignment and DNA‐based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped into three categories depending on if they showed low, high or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA‐based monitoring studies.

Because of the high risk of going unnoticed, cryptic species represent a major challenge to biodiversity assessments, and this is particularly true for taxa that include many such species, for example, bats. Long‐eared bats from the genus Plecotus comprise numerous cryptic species occurring in the Mediterranean Region and present complex phylogenetic relationships and often unclear distributions, particularly at the edge of their known ranges and on islands. Here, we combine Species Distribution Models (SDMs), field surveys and molecular analyses to shed light on the presence of a cryptic long‐eared bat species from North Africa, Plecotus gaisleri, on the islands of the Sicily Channel, providing strong evidence that this species also occurs in Europe, at least on the islands of the Western Mediterranean Sea that act as a crossroad between the Old Continent and Africa. Species Distribution Models built using African records of P. gaisleri and projected to the Sicily Channel Islands showed that all these islands are potentially suitable for the species. Molecular identification of Plecotus captured on Pantelleria, and recent data from Malta and Gozo, confirmed the species' presence on two of the islands in question. Besides confirming that P. gaisleri occurs on Pantelleria, haplotype network reconstructions highlighted moderate structuring between insular and continental populations of this species. Our results remark the role of Italy as a bat diversity hotspot in the Mediterranean and also highlight the need to include P. gaisleri in European faunal checklists and conservation directives, confirming the usefulness of combining different approaches to explore the presence of cryptic species outside their known ranges—a fundamental step to informing conservation.

37Freshwater macroinvertebrates, and specifically Odonata, are considered among the most 38 endangered faunal groups. Their biomonitoring has been progressively supported by DNA-39 based tools whose performance and accuracy rely on validated reference datasets that, in some 40 cases, are lacking or do not cover important biogeographical centres of diversification. 41This study reports the results of a DNA barcoding campaign on Odonata, involving the 42 collection of 812 specimens (448 of which barcoded) from Italy and its main islands (328 43 localities), belonging to the 88 species (31 Zygoptera and 57 Anisoptera) inhabiting the country. 44 Additional BOLD and GenBank data from Holarctic samples of the same taxa expanded the 45 dataset to 1294 DNA barcodes. An integrative species delimitation analysis involving two 46 distance (OT and ABGD) and four tree-based (PTP, MPTP, GMYC, bGMYC) approaches 47 identified warnings of possible taxonomic relevance. Of the 88 investigated species, 85% could 48 be successfully identified by their DNA barcodes, with damselflies showing a percentage of 49 warnings (29%) higher than dragonflies (7%), contrasting with the other available DNA 50 barcoding studies on Odonata (showing up to 95% of identification success). The species 51causing warnings were grouped in three categories depending on if they showed low, high or 52 mixed genetic divergence patterns. Moreover, for the second class of warnings, the analysis of 53 haplotypes revealed unexpected structure at the Italian, Palearctic and Holarctic scale. Overall, 54 the DNA barcoding inventory assembled in this study will provide valuable insights into the 55 systematics and conservation of many odonate species with implications for future DNA and 56 eDNA monitoring-based studies. 57 58

Molecular-based approaches for species identification and delimitation strongly rely, in terms of universality and efficiency, on the selected markers. Conventionally, when adopting a DNA barcoding approach to discriminate (or identify) metazoans species, the marker choice falls on the 658 base pair region at the 5ʹ end of the mitochondrial COI gene. However, a growing number of studies suggest to use alternative and more variable genetic regions, even from the same gene, such as the 3ʹ end of the COI. In this work, we compared the identification performance of the 5ʹ and 3ʹ end COI regions on a large sequence dataset of odonate species, an order of arthropods among the most studied in terms of conservation importance for aquatic ecosystems. The genetic datasets comprised a total of 236 specimens, 113 species, 51 genera and 12 families spanning the two odonate suborders Zygoptera and Anisoptera, and were analysed under an integrative multiple approach including descriptive statistics and variability of the sequences, phylogenetic reconstructions, DNA-based species delimitations, genetic distances, identification of diagnostic characters and saturation plots. All analyses were congruent in recovering the COI-3ʹ region to be slightly more variable than the COI-5ʹ one, and both regions showed a saturation of transversion at the third codon position. However, phylogenetic reconstructions, genetic distances, and diagnostic characters identification resulted in a similar discrimination power for the two COI regions. Therefore, the COI-3ʹ region does not add much information to the standard COI-5ʹ barcode region, which has in turn largely been demonstrated to successfully delineate invertebrate communities through DNA and eDNA metabarcoding, and to have a much more extensive taxonomic coverage in public databases. Overall, the DNA barcoding inventory assembled in this study will provide valuable insights into the systematics and conservation of many odonate species with implications for future DNA and eDNA monitoring-based studies.

The genus Crocidura (Eulipotyphla, Soricidae) is the most speciose genus amongst mammals, i.e., it includes the highest number of species. Different species are distinguished by skull morphology, which often prevents the identification of individuals in the field and limits research on these species’ ecology and biology. We combined species distribution models and molecular analyses to assess the distribution of cryptic Crocidura shrews in Italy, confirming the occurrence of the greater white-toothed shrew Crocidura russula in the northwest of the country. The molecular identification ascertained the species’ presence in two distinct Italian regions. Accordingly, species distribution modelling highlighted the occurrence of areas suitable for C. russula in the westernmost part of northern Italy. Our results confirm the role of Italy as a mammal hotspot in the Mediterranean; additionally, they also show the need to include C. russula in Italian faunal checklists. To conclude, we highlight the usefulness of combining different approaches to explore the presence of cryptic species outside their known ranges. Since the similar, smaller C. suaveolens may be displaced by the larger C. russula through competitive exclusion, the latter might be the species actually present where C. suaveolens had been reported previously. A comprehensive and detailed survey is therefore required to assess the current distribution of these species.