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Introduced species provide the opportunity to study evolutionary change on short time scales¿a key first step to understand and manage the ecosystem-level impact of invasions. This study examined mitochondrial DNA sequence variation in cytochrome c oxidase subunit 1 (COI) for 26 insect species - Coleoptera (9), Hymenoptera (9), and Lepidoptera (8) - introduced to the Nearctic from the Palearctic. A total of 6,302 barcode records were retrieved from BOLD (boldsystems.org) to compare sequence diversity between the native and introduced range. As expected, genetic variation averaged nearly an order of magnitude lower in introduced populations (2.19 × 10−5 substitutions per nucleotide) than in the native range (1.48 ×10-4 substitutions per nucleotide). Nonsynonymous and synonymous changes had a similar incidence in the introduced populations (p-value = 0.83, averaging respectively 1.08 × 10-5 and 1.11 × 10-5 substitutions). By contrast, nonsynonymous changes were ten-fold less frequent than synonymous changes in the native populations (p-value < 0.001, averaging 1.74 × 10-5 and 1.3 × 10-4 substitutions, respectively). Patterns of sequence variation in the introduced range were largely congruent across the three insect orders which suggests that they are produced by general processes. This study explores the molecular evolution of introduced species, a fundamental aspect to improve understanding of their biology and manage their impact on ecosystems.

A new subspecies of Leptidea sinapis from Northern Iran, discovered by means of DNA barcoding, is described as Leptidea sinapis tabarestana ssp. nov. The new subspecies is allopatric with respect to other populations of L. sinapis and is genetically distinct, appearing as a well-supported sister clade to all other populations in COI-based phylogenetic reconstructions. Details on karyotype, genitalia, ecology and behaviour for the new subspecies are given and a biogeographical speciation scenario is proposed.

Ecological Niche Modelling output and input.

Parapatrically distributed taxa pose a challenge for species delimitation due to the presence of gene flow and inherent arbitrariness of exactly defining the species boundaries in such systems. We tackled the problem of species delimitation in a parapatric species pair of Melitaea butterflies using two popular genomic methods—double digest restriction-site associated DNA sequencing (ddRAD) and target enrichment. We compared newly generated target enrichment dataset with 1733 loci to the already available ddRAD data from a previous study on the same set of specimens using a suite of phylogenetic, population genetic, and species delimitation methods. We recovered consistent phylogenetic relationships across the datasets, both demonstrating the presence of a genetically distinct Balkan lineage and paraphyly of Melitaea athalia with respect to Melitaea celadussa. Population genetic STRUCTURE analyses supported the presence of two species when using ddRAD data, but three species when using target enrichment, while a Bayes factor delimitation analysis found both two and three species scenarios equally decisive in both datasets. As the results obtained from both methods were largely congruent, we discuss some practical considerations and benefits of target enrichment over RAD sequencing. We conclude that the choice of method of genomic data collection does not influence the results of phylogenetic analyses at alpha taxonomic level, given a sufficient number of loci. Finally, we recommend a solution for delineating species in parapatric scenarios by proposing that parapatric taxa be consistently classified as subspecies or complete species, but not both, to promote taxonomic stability.

Climate change represents a threat to narrow-ranged mountain species living in low-altitude massifs. We studied the disjunct Apennine population of Lasiommata petropolitana (Lepidoptera, Nymphalidae) in the Gran Sasso and Monti della Laga National Park. We quantified the altitudinal shifts undergone in the last decades (1964–2021) in the Alps and Apennines and estimated the local extinction risk due to climate change. We also sequenced the COI mitochondrial marker of seven Apennine specimens, comparing them with those available across the Palearctic. We projected the probability of presence for the species under a future climatic scenario using an ensemble forecasting approach. We found that, despite geographical isolation, the Apennine population of L. petropolitana displays a single widespread COI haplotype also occurring in most European populations. In the Alps and Apennines, this species has shifted uphill an average of 6.3 m per year since 1964. Accordingly, our model predicted a likely extinction in the Apennines by about 2060, due to a reduction of the climatic suitability in this region. Implications for insect conservation Implications for insect conservation Despite its potential loss in the Apennines would not erode mitochondrial diversity, L. petropolitana characterises the butterfly community of the Gran Sasso massif as an alpine enclave. The loss of the Apennine population, together with those of other orophilous butterflies, could trigger a homogenization of alpha and beta diversity and induce a loss of functional diversity in the impoverished high-altitude biotas. As habitat heterogeneity is a key aspect for populations to endure climate change, the maintenance of varied microhabitats, mainly through grazing management, could address the decline of this population.

Motivation Butterflies represent a model in biology and a flagship group for invertebrate conservation. We provide four new resources for the Western Palaearctic butterflies: (1) an updated checklist comprising 552 species; (2) a curated dataset of 32,126 mitochondrial cytochrome c oxidase subunit I (COI) sequences for 532 species, including a de novo reference library for the Maghreb (Morocco, northern Algeria and Tunisia) and Macaronesia (Azores, Madeira and Canary Islands); (3) seven indexes of intraspecific genetic variation (IGV): observed and expected number of haplotypes, haplotype and nucleotide diversity, two fixation indexes and maximum p-distance; and (4) species-level maps illustrating the distribution of COI variability and haplotype networks. The updated checklist will be fundamental for any application dealing with butterfly diversity in the Western Palaearctic. The IGV indexes provide measures for genetic polymorphism and spatial structure and represent proxies for dispersal capacity. These resources will facilitate comparative studies of macrogenetics, foster integrative taxonomy and aid conservation strategies. Main types of variables contained A complete species checklist in table format, 32,126 mitochondrial DNA barcodes provided with metadata (species membership, WGS84 coordinates and sequence length) and a book in PDF format, including the IGV atlas and indexes, are provided. Spatial location and grain The checklist encompasses Europe up to the Urals in the east, north Macaronesia (the Azores, Madeira and the Canary Islands) and the Maghreb (Morocco, northern Algeria and Tunisia). COI sequences have been retained in the geographical interval of -31.3 to 67.5° of longitude and 27.5 - 71.2° of latitude. Time period and grain COI sequences originate from studies published between 1998 and 2022 and from de novo sequencing of 2541 specimens done between 2007 and 2022. Major taxa and level of measurement Butterflies (Lepidoptera: Papilionoidea), anal

One of the main consequences of globalization is the intensification of biological introductions. Because of their negative impact on environments, the early detection and monitoring of introduced species through molecular approaches is gaining increased uptake. This study assembles 2,278 DNA barcode records to examine contemporary patterns of sequence variation in mitochondrial cytochrome c oxidase I (COI) in five butterfly species introduced to the Nearctic, with a focus on Pieris rapae Linnaeus (Lepidoptera: Pieridae) and Thymelicus lineola Ochsenheimer (Lepidoptera: Hesperiidae). Parameters of genetic diversity were low (i.e., h < 0.606, ¿ < 0.0039) for Nearctic populations of all analyzed species. Those of P. rapae and T. lineola showed marked genetic differentiation from their source populations in the Palearctic. Haplotype distributions in their Nearctic populations exposed a starburst pattern with a few common haplotypes known from Palearctic, and infrequent haplotypes diverging from them at only one or two nucleotide sites. Some uncommon haplotypes were only found in the Nearctic suggesting they originated after invasion, while others also occur in the Palearctic. This study provides an example of genetic paradox of invasion, where species often rapidly expand their distribution and become dominant in the new habitat despite their depleted levels of sequence variation.

[EN] Although both positive (expansion of thermophilous species) and negative effects (retraction of cold-adapted species) have been attributed to global warming, range shifts may be constrained by the ecological traits of species. This can be especially true for highly specialised organisms like the Spanish Greenish Black-tip (Euchloe bazae), an Iberian endemic specialist of semiarid steppes with a debated taxonomic and conservation status. Here, we first seek to clarify the taxonomic status of this butterfly and its populations by using multilocus phylogenetic inference. Then, we update its distribution range and employ ecological niche modelling, combined with other sources of data, to re-evaluate its conservation status. Our results confirm E. bazae as a well-differentiated species with one of the most restricted distribution ranges among the European butterflies. We demonstrate that its two disjunct populations, regarded as subspecies, are genetically differentiated and should be treated as independent management units. Climate models under two future emission scenarios suggest an increase of the area climatically suitable. However, the suitability of the areas currently occupied is estimated to decrease, meaning that rapid range shifts could be required for the survival of the species. Given the presumably low dispersal capabilities of E. bazae, its current restricted distribution, high degree of population fragmentation (divided in two distant populations), low intrapopulation genetic variability, decline in extension and abundance, low densities, high yearly fluctuations, and numerous threats to its habitat, we consider that their populations are extremely fragile and we propose to consider it `Endangered¿ [EN B1ab(i,ii,iii,iv); B2ab(i,ii,iii,iv)]., [CA] Tot i que s’han atribuït tant efectes positius (expansió d’espècies termòfiles) com negatius (contracció d’espècies adaptades al fred) a l’escalfament global, canvis en les distribucions poden estar condicionats pels trets ecològics de les espècies. Això pot ser especialment cert per a organismes altament especialitzats com la grogueta del desert (Euchloe bazae), un endemisme ibèric especialista d’estepes semiàrides amb un estatus taxonòmic i de conservació debatut. En aquest estudi, primer cerquem esclarir l’estatus taxonòmic d’aquesta papallona ide les seves poblacions mitjançant la inferència filogenètica multilocus. També actualitzem la seva distribució i fem servir la modelització de nínxols ecològics com-binada amb altres dades per reavaluar el seu estatus de conservació. Els nostres resultats confirmen que E. bazae és una espècie ben diferenciada i amb una de les distribucions més restringides entre les papallones europees. Demostrem que els seus dos nuclis de població, cadascun considerat una sub-espècie, són diferents genèticament i s’han de tractar com a unitats de gestió independents. Els models climàtics elaborats amb dos escenaris d’emissions futurs suggereixen un augment de l’àrea climàticament adient. No obstant això, s’estima que la idoneïtat de l’àrea actualment ocupada disminuirà, el que podria requerir canvis ràpids en la seva distribució per garantir-ne la supervivència. Tenint en compte que E. bazae disposaria d’una capacitat de dispersió baixa, la distribució actual reduïda, l’alt grau de fragmentació (dividida en dues poblacions llunyanes), la baixa variabilitat genètica intra poblacional, la disminució de l’extensió i l’abundància, les baixes densitats, les elevades fluctuacions anuals i les nombroses amenaces al seu hàbitat, considerem que les poblacions d’aquesta papallona són extremament fràgils i proposem considerar-la “En perill”

The study of ecological interactions between plants, phytophagous insects and their natural enemies is an essential but challenging component for understanding ecosystem dynamics. Molecular methods such as DNA barcoding can help elucidate these interactions. In this study, we employed DNA barcoding to establish hostplant and parasitoid interactions with hesperiid butterflies, using a complete reference library for Hesperiidae of continental Europe and north-western Africa (53 species, 100% of those recorded) based on 2934 sequences from 38 countries. A total of 233 hostplant and parasitoid interactions are presented, some recovered by DNA barcoding larval remains or parasitoid cocoons. Combining DNA barcode results with other lines of evidence allowed 94% species-level identification for Hesperiidae, but success was lower for parasitoids, in part due to unresolved taxonomy. Potential cases of cryptic diversity, both in Hesperiidae and Microgastrinae, are discussed. We briefly analyse the resulting interaction networks. Future DNA barcoding initiatives in this region should focus attention on north-western Africa and on parasitoids, because in these cases barcode reference libraries and taxonomy are less well developed.

The lesser marbled fritillary, Brenthis ino (Rottemburg, 1775), is a species of Palearctic butterfly. Male Brenthis ino individuals have been reported to have between 12 and 14 pairs of chromosomes, a much-reduced chromosome number than is typical in butterflies. Here, we present a chromosome-level genome assembly for Brenthis ino, as well as gene and transposable element annotations. The assembly is 411.8 Mb in length with a contig N50 of 9.6 Mb and a scaffold N50 of 29.5 Mb. We also show evidence that the male individual from which we generated HiC data was heterozygous for a neo-Z chromosome, consistent with inheriting 14 chromosomes from one parent and 13 from the other. This genome assembly will be a valuable resource for studying chromosome evolution in Lepidoptera, as well as for comparative and population genomics more generally.

1. Climate change is causing shifts in the distribution of many species and populations inhabiting mountain tops are particularly vulnerable to these threats because they are constrained in altitudinal shifts. Apennines are a relatively narrow and low mountain chain located in Southern Europe, which hosts many isolated populations of mountain species. The butterfly Erebia pandrose was recorded for the last time in the Apennines in 1977, on the top of a single massif (Monti della Laga). 2. We confirmed the presence of a small, isolated population of E. pandrose in the Apennines, at a distance of more than 400¿km to any other known populations. Then, we examined the cytochrome c oxidase subunit 1 mitochondrial DNA marker of this species across the Palaearctic area and estimated the potential decline over the Alps and the Apennines due to future climatic changes. 3. The Apennine population represents an endemic lineage characterised by eight mutations over the 658¿bp analysed (1.2%). In the Alps and Apennines, this species has shifted uphill more than 3 m per year since the end of the 19th century and more than 22 m per year since 1995. Species distribution models suggested that these mountain populations will experience a generalised loss of climatic suitability, which, according to our projections, could lead to the extinction of the Apennine population in a few decades. 4. Erebia pandrose has the potential to become a flagship species for advertising the risk of losing unique fractions of genetic diversity for mountain species.