Butterfly-parasitoid-hostplant interactions in Western Palaearctic Hesperiidae: a DNA barcoding reference library

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.Support for this research was provided by the Spanish National Research Council (CSIC) with a JAE-Intro fellowship for the introduction to research to ETD (reference numbers JAEINT_20_00248 and JAEINT20_EX_0638) and by projects PID2019-107078GB-I00/MCIN/AEI/10.13039/501100011033 and 2017-SGR-991 (Generalitat de Catalunya) to RV, and PID2020-117739GA-I00/MCIN/AEI/10.13039/501100011033 to GT. We thank the Rachadaphiseksomphot Fund, Graduate School, Chulalongkorn University, for the award of a Senior Postdoctoral Fellowship to DLJQ. Further support for this research was provided by the Academy of Finland (Academy Research Fellow, decision no. 328895) to VD. PDNH acknowledges support from Genome Canada through Ontario Genomics. BV has been funded by the CERCA Programme of the Generalitat de Catalunya and by the Grant RYC-22243-2017, whose PI is Josep Sardanyés. SV was supported by the Spanish Ministry of Economy and Competitiveness, grant PID2020-117822GB-I00 MINEICO/AEI/ FEDER and the European Union.INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSION SUPPORTING INFORMATION ACKNOWLEDGEMENTS DATA AVAILABILITY REFERENCES Supplementary dat

Genetics and extreme confinement of three overlooked butterfly species in Romania call for immediate conservation actions

Abstract A good knowledge of species distributions and their genetic structure is essential for numerous types of research such as population genetics, phylogeography, or conservation genetics. We document the presence of extremely local populations of three butterfly species (Iolana iolas, Satyrus ferula and Melanargia larissa) in the Romanian fauna. Satyrus ferula and M. larissa are reported for the first time in the country, while I. iolas is rediscovered following presumed extinction. Based on mitochondrial DNA (cytochrome c oxidase subunit 1—COI sequences), we assessed the genetic structure of these populations and placed them into a broader context through comparisons with other populations from across the range of these species. Each of the three species had a single haplotype in Romania, suggesting low female effective population size possibly under genetic erosion. Two of the populations (S. ferula and M. larissa) are genetically unique, displaying endemic haplotypes in south-western Romania. The Romanian populations of the three species likely remained unnoticed due to their extremely limited extent of occurrence. Their restricted range, close to the northern limits of distribution in the Balkans, their apparent low female effective population size, the presence of endemic haplotypes, and habitat vulnerability (especially for I. iolas) highlight the need for monitoring and conservation measures for the safeguarding of these populations

Integrative analyses on Western Palearctic Lasiommata reveal a mosaic of nascent butterfly species

Abstract Satyrinae butterflies occurring in the Mediterranean apparently have reduced gene flow over sea straits, and for several species, recent wide-scale biodiversity surveys indicate the existence of divergent mitochondrial lineages. Here, we apply an integrative approach and examine the phylogeography of the genus Lasiommata in the Western Palearctic. Our research comprised molecular analyses (mitochondrial and nuclear DNA) and geometric morphometrics (wings and genitalia) for two main species groups, and a comparative GMYC analysis, based on COI, of all the tribes within Satyrinae from this region. The GMYC approach revealed a particularly fast coalescence rate in the Parargina subtribe. The Lasiommata group was divided into 12 evolutionary significant units: six clades for the L. maera species group, five for the L. megera species group, and one for L. petropolitana, with divergences of about 1%. The patterns of COI were mirrored by ITS2 in L. maera, but the two markers were generally inconsistent in L. megera. On the contrary, morphological differences were coherent with the results of COI for L. megera, but less clearly so for L. maera. L. paramegaera and L. meadewaldoi were considerably differentiated for all the analyzed markers and likely proceeded faster in the process of speciation because of geographic isolation and reduced effective population size, rendering the rest paraphyletic. Our study illustrates the continuous nature of speciation and the difficulties of delimiting species. In Lasiommata, the recognition of taxa as diverging lineages or distinct, possibly paraphyletic species, mostly depends on the criteria adopted by different species concepts

Molecular phylogeny of the Palaearctic butterfly genus Pseudophilotes (Lepidoptera: Lycaenidae) with focus on the Sardinian endemic P. barbagiae

Abstract Background: The Palaearctic butterfly genus Pseudophilotes Beuret, 1958 (Lycaenidae: Polyommatinae), that today occurs in North Africa and in Eurasia, includes ten described species with various distribution ranges, including endemics such as the Sardinian P. barbagiae. Phylogenetic relationships among these species are largely unresolved. In the present study, we analysed 158 specimens, representing seven species out of ten described in the genus, from widely distributed sites throughout the western Palaearctic region, using nuclear markers (28S rRNA, wingless, Internal Transcribed Spacer 2 and Elongation Factor 1α) and the barcoding region of the mitochondrial cytochrome c oxidase subunit 1 gene to investigate if the current taxonomic entities match the phylogenetic pattern. Further, we attempt to infer the geographic origin of the genus Pseudophilotes and estimate the timing of its radiations, including the split of the Sardinian endemic P. barbagiae. Results: Maximum Likelihood and Bayesian inference analyses confirmed the monophyly of the genus Pseudophilotes and clearly supported the closer affinity of P. barbagiae to the species assemblage of P. baton, P. vicrama and P. panoptes as opposed to P. abencerragus. The currently accepted species P. baton, P. vicrama and P. panoptes turned out to be weakly differentiated from each other, while P. bavius and P. fatma emerged as highly distinct and formed a well supported clade. The split between the lineage comprising bavius and fatma (sometimes treated as a distinct genus, Rubrapterus) with Salvia species as larval host plants, and the remaining Pseudophilotes that utilize Thymus and other Lamiaceae (but not Salvia), dates back to about 4.9 million years ago (Mya). Conclusions: Our results show that the last common ancestor of the genus probably lived in the Messinian period (5.33–7.25 Mya). At species level, they support the current taxonomy of the genus, although P. panoptes, P. baton and P. vicrama display complex patterns based on phylogeographic relationships inferred from mtDNA. The Sardinian endemic P. barbagiae turned out to be a young endemic, but clearly with European instead of North African origin and evolved through allopatric isolation on the island of Sardinia only about 0.74 Mya

How long is 3 km for a butterfly?:ecological constraints and functional traits explain high mitochondrial genetic diversity between Sicily and the Italian Peninsula

Abstract 1.Populations inhabiting Mediterranean islands often show contrasting genetic lineages, even on islands that were connected to the mainland during glacial maxima. This pattern is generated by forces acting in historical and contemporary times. Understanding these phenomena requires comparative studies integrating genetic structure, functional traits and dispersal constraints. 2.Using as a model the butterfly species living across the Messina strait (3 km wide) separating Sicily from the Italian Peninsula, we aimed to unravel the mechanisms limiting the dispersal of matrilines and generating genetic differentiation across a narrow sea strait. 3.We analysed the mitochondrial COI gene of 84 butterfly species out of 90 documented in Sicily and compared them with populations from the neighbouring southern Italian Peninsula (1,398 sequences) and from the entire Palaearctic region (8,093 sequences). For each species, we regressed 13 functional traits and 2 ecological constraints to dispersal (winds experienced at the strait and climatic suitability) against genetic differentiation between Sicily and Italian Peninsula to understand the factors limiting dispersal. 4.More than a third of the species showed different haplogroups across the strait and most of them also represented endemic haplogroups for this island. One fifth of Sicilian populations (and 32.3% of endemic lineages) had their closest relatives in distant areas, instead of the neighbouring Italian Peninsula, which suggests high relictuality. Haplotype diversity was significantly explained by the length of the flight period, an intrinsic phenology trait, while genetic differentiation was explained by both intrinsic traits (wingspan and degree of generalism) and contemporary local constraints (winds experienced at the strait and climatic suitability). 5.A relatively narrow sea strait can produce considerable differentiation among butterfly matrilines and this phenomenon showed a largely deterministic fingerprint. Because of unfavourable winds, populations of the less dispersive Sicilian butterflies tended to differentiate into endemic variants or to maintain relict populations. Understanding these phenomena required the integration of DNA sequences, species traits and physical constraints for a large taxon at continental scale. Future studies may reveal if the patterns here shown for mitochondrial DNA are also reflected in the nuclear genome or, alternatively, are the product of limited female dispersal

Butterfly–parasitoid–hostplant interactions in Western Palaearctic Hesperiidae:a DNA barcoding reference library

Abstract 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