Historical climatic oscillations and geographic barriers have profoundly shaped genetic diversity and speciation across Europe, particularly within glacial refugia. The widespread butterfly Cyaniris semiargus (Mazarine Blue) is a striking example of such divergence, especially in its southernmost populations, which are fragmented across montane and coastal habitats. Using genome-wide ddRADseq data and environmental analyses, we investigated the genetic structure, gene flow and ecological differentiation of C. semiargus across the western Palearctic, with a focus on its isolated southern populations. Our results reveal four deeply diverged genetic lineages within the Iberian Peninsula and one in North Africa. These lineages exhibit strong genetic structure, ecological specialization and minimal gene flow, consistent with allopatric divergence strengthened by local adaptation. Coastal populations show host plant specialization and adaptation to milder and more stable climates, while montane lineages persist in climatically extreme refugia. Interestingly, another lineage in the Peloponnese is following similar high-altitude climatic selective pressures. Together, these findings point to parallel processes of ecological speciation across both montane and coastal environments. Adopting a conservative taxonomic approach, albeit reflecting their evolutionary and conservation significance, we recognize these lineages as subspecies. Our study highlights the role of glacial refugia, habitat specialization and ecological divergence in shaping patterns of biodiversity, and underscores the urgent need for lineage-specific conservation measures.

Aim Comparison between islands and equivalent mainland areas to dissect the effect of area, isolation and species traits in determining island genetic endemicity and genetic differentiation.Location The Western Mediterranean region.Time Period Current.Major Taxa Studied Butterflies (Lepidoptera, Papilionoidea).Methods We analysed 10,367 COI sequences from 105 species, 34 islands and 47 sea straits, along with four functional species traits. We compared determinants of genetic diversity (nucleotide diversity, NucDiv), endemic mutations (EM) and the Dst fixation index between island populations and similarly sized mainland populations (Continental Area Equivalents, CAEs). Generalised linear mixed models tested fixed effects and interactions of island characteristics and species traits. We also evaluated whether population-level effects (individual species increasing differentiation with island size and isolation) or community-level effects (larger, less isolated islands hosting more genetically divergent species) better explain observed island patterns.Results CAEs and islands exhibited highly distinct genetic signatures. NucDiv was higher in CAEs while Dst was higher across sea straits than between land areas separating CAEs, particularly for larger islands and non-migratory species. EM increased with island area, especially in non-migratory and small species. Island communities were significantly nested. Widespread species also showed lower genetic variation, thus producing a strong community-level effect. The population-level effects showed a weaker effect. Counterintuitively, increasing isolation did not increase divergence, as both processes tended to reduce genetic differentiation in more isolated islands.Main Conclusions Comparing islands with CAEs reveals that even narrow sea barriers of a few kilometres greatly reduce gene flow in non-migratory butterflies, increasing genetic differentiation to a greater extent than similar mainland distances. After controlling for the presence of sea straits, island size predicts genetic divergence while increasing isolation does not. Overall, endemicity remains low, indicating that mainland-driven genetic turnover, rather than in situ evolution, dominates insular genetic patterns.

Based on a dataset of 585 samples originating from across the Palearctic region and four genes (COI, CAD, Ca-ATPase, and 28S) for a subset of these samples, we studied the phylogenetic relationships within and between the Old World Swallowtail (Papilio machaon Linnaeus, 1758) and its closest relatives in the Palearctic region. We used Machine Learning (ML) to investigate the concordance of morphological characteristics with the molecular data. Our phylogenetic analyses showed that the Palearctic taxa everesti, archias and hippocrates formed strongly supported sister-group relationships with P. machaon. Within the Palearctic machaon a few distinct mitochondrial linages were observed, including one from the Himalayas and Central Asia, and another one that included all of the north African samples (taxa mauretanica, saharae and neosaharae) without any distinction. ML clustering was supportive of the haplotype and geographic analyses and a positive correlation was measured between average genetic phylogenetic and machine learnt specimen-image distances. Screening for Wolbachia revealed infection only in females of P. archias. We present a biogeographic scenario for the evolution of the P. machaon species group in the Palearctic region.

Species with disjunct distributions have long puzzled evolutionary biologists and biogeographers. Long-distance dispersal can play a pivotal role in generating intra-specific disjunct distributions, initiating early stages of allopatric speciation and leading to eventual interspecific disjunctions. Vanessa butterflies exhibit diverse movement behaviours, from low-dispersal species with restricted distributions to others that engage in annual extensive migratory cycles. The biogeographic history of Vanessa presents intriguing cases of both intra- and interspecific disjunctions. Vanessa atalanta is present in the Nearctic and Western Palearctic but is absent in Asia, while its sister species V. tameamea is endemic to Hawaii. Vanessa indica occurs only in Asia, and its sister species, V. vulcania, is endemic to Macaronesia. Here, we investigate this conundrum through population genomics and demographic analyses of Vanessa atalanta using ddRAD data from 70 samples across its entire distributional range, identifying two genetically differentiated populations separated by the Atlantic Ocean. Demographic simulations and phylogenetic analyses suggest that these originated via long-distance dispersal from the Nearctic to Europe around the Last Glacial Maximum. Hybridisation tests revealed introgression between the Palearctic population of V. atalanta and V. indica, indicating that their distributions overlapped during V. atalanta's colonisation of Europe. We hypothesise that V. atalanta caused a species displacement of V. indica from Europe to Asia, explaining their current allopatric distributions-a scenario that is supported by ecological niche modelling. Together, our results illustrate the role of long-distance dispersal and species interactions in shaping complex biogeographic patterns.

Background: Zinc oxide nanobiocomposites were successfully synthesized using a green synthesis approach. The process involves the utilization of the isoflavone puerarin, resulting in the formation of PUE-ZnO NPs. Methods: Physico-chemical and biological characterization techniques including X-ray dif-fraction (XRD), UV-vis spectroscopy, Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), atomic force microscopy (AFM), and in ovo methods were employed to study the main characteristics of this novel hybrid material. Results: The PUE-ZnO NPs were confirmed to have been successfully synthesized with a UV absorption peak at 340 nm, the XRD analysis demonstrating their high purity and crystallinity. The energy band-gap value of 3.30 eV suggests possible photocatalytic properties. Both SEM and AFM images revealed the nanoparticle`s quasi-spherical shape, roughness, and size. Good tolerability and anti-irritative effects were recorded in ovo on the chorioallantoic membrane (CAM). Conclusions: According to these results, the synthesis of green PUE-ZnO NPs may be a promising future approach for biomedical and personal care applications.

ABSTRACT Human induced environmental changes are accelerating at an unprecedented pace, forcing organisms to rapidly adjust their behaviours. There is broad evidence that the main driver of the ongoing biodiversity crisis is land-use change, that reduces and fragments natural habitats. However, the consequence of habitat fragmentation on behavioural responses of fitness-related traits such as oviposition site selection in insects, which represent about 50% of \ Earth’s species diversity, have been so far understudied. In herbivorous insects, oviposition-related behaviours determine larval food access, and thus the fate of the next generation. We present a pilot study to assess differences in oviposition-related behaviours in Limenitis camilla butterflies from Wallonia (Belgium), one of the most fragmented regions in Europe. We first quantified variation in functional habitat connectivity for L. camilla across Wallonia and found that fragmented habitats had more abundant, but less evenly distributed Lonicera periclymenum , the host plant of L. camilla . Secondly, we compared in a semi-natural experimental setting the behaviours of field-caught L. camilla females originating from habitats with contrasted landscape connectivity. We found differences in behaviours related to flight investment: butterflies from fragmented woodlands spent more time in non-compass orientation flight, which we associated with dispersal, than butterflies from homogenous woodlands, where L. periclymenum was less abundant and more evenly distributed. Although results from this study should be interpreted with caution given the limited sample size, they provide valuable insights for the advancement of behavioral research that aims to assess the effects of global changes on insects.

There is broad evidence that the main driver of the ongoing biodiversity crisis is land-use change, which reduces and fragments habitats. The consequence of habitat fragmentation on behavioural responses of fitness-related traits in insects have been so far understudied. In herbivorous insects, oviposition-related behaviours determine access to larval food, and the fate of the next generation. We present a pilot study to assess differences in behaviours related to movement and oviposition in Limenitis camilla butterflies from Wallonia (Belgium), one of the most fragmented regions in Europe. We first quantified variation in functional habitat connectivity across Wallonia and found that fragmented habitats had more abundant, but less evenly distributed host plants of L. camilla. Secondly, we quantified the behaviours of field-caught L. camilla females originating from habitats with contrasted landscape connectivity in an outdoor experimental setting. We found differences in behaviours related to flight investment: butterflies from fragmented woodlands spent more time in departing flight, which we associated with dispersal, than butterflies from homogenous woodlands. Although results from this study should be interpreted with caution given the limited sample size, they provide valuable insights for the advancement of behavioural research that aims to assess the effects of global changes on insects.

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.

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 degrees of longitude and 27.5-71.2 degrees 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), analysed from individual to species level. Software format Data and functions to manage the dataset are provided in the iodatabase R package () and in Dryad ().

Ruthenium, as an industrial by-product or from natural sources, represents an important economical resource due to its specific applications. A complex problem is represented by ruthenium separation during reprocessing operations, therefore, different materials and methods have been proposed. The present study aims to develop a new material with good adsorbent properties able to be used for ruthenium recovery by adsorption from aqueous solutions. Absorbent material was obtained using chitosan (Ch) surface modification with dibenzo-30-crown-10 ether (DB30C10). Chitosan represents a well-known biopolymer with applicability in different adsorptive processes due to the presence of hydroxyl-, carboxyl-, and nitrogen-containing groups in the structure. Additionally, crown ethers are macromolecules with a good complexation capacity for metallic ions. It is expected that the adsorptive efficiency of newly prepared material will be superior to that of the individual components. New synthesized material was characterized using scanning electron microscopy coupled with energy dispersive X-ray (SEM–EDX), Fourier transform infrared spectroscopy (FT-IR), Brunauer–Emmett–Teller surface area analysis (BET), and determination of point of zero charge (pZc). Results obtained from the performed kinetic, thermodynamic, and equilibrium studies confirmed the good adsorptive capacity of the prepared material, Ch-DB30C10, obtaining a maximum adsorption capacity of 52 mg Ru(III) per gram. This adsorption capacity was obtained using a solution with an initial concentration of 275 mg L−1, at pH 2, and 298 K. Ru(III) adsorption kinetics were studied by modeling the obtained experimental data with pseudo-first order and pseudo-second order models. Desorption studies established that the optimum eluent was represented by the 5M HNO3 solution. Based on the performed studies, a mechanism for recovery of ruthenium by adsorption was proposed.

The aim of this study was to develop an electrochemical procedure for the advanced detection of tetracycline (TC) in water using CoAl2O4 dispersed onto carbon nanofiber-epoxy composite electrode substrate, which was selected from a series of tested carbonbased electrode substrates, i.e., commercial glassy-carbon and boron-doped diamond electrodes and home-made carbon nanotubeepoxy and carbon nanofiber-epoxy composites. Carbon nanofiber-epoxy composite electrode was found the best host for CoAl2O4 modifier based on the electrocatalytic effect towards TC oxidation and detection using cyclic voltammetry (CV) technique. The electrochemical techniques applied for electrochemical detection applications were CV, differential-pulse voltammetry (DPV), square-wave voltammetry (SWV) and chronoamperometry (CA). All the tested electrochemical techniques allowed TC determination and the electroanalytical parameters varied related to the technique type. DPV operated at the step potential of 50 mV and the modulation amplitude of 200 mV allowed the lowest limit of detection of 9 nM for TC determination. The recovery test applied for real surface water spiked with known TC concentrations among the reproducibility and the repeatability showed the great potential for the advanced quantitative determination of TC in real water or in pharmaceutical formulations.

The present paper describes a new way of obtaining an efficient adsorbent material by functionalization of Mg3Al layered double hydroxides (LDH) with methyl trialkyl ammonium chloride-ionic liquid (IL) using two methods: ultrasound and cosynthesis. Layered double hydroxides are good solid support for the functionalization with ionic liquids due to their well-ordered structure. The immobilization of the ILs in suitable solid supports combine the advantages of the ILs with the properties of the solid supports bringing more benefits such as use of lower quantity of ILs and avoiding of ILs loss in the aqua phase which overall decrease the treatment costs. In case of ultrasound method of functionalization is assured a uniform distribution of IL on the solid surface, but through immobilization by cosynthesis due to the tunable properties of LDH, is assured an intercalation of the ILs between the LDH layers. This fact was highlighted by the X-ray diffraction (RXD), scanning electron microscopy (SEM) analyses and Fourier-transform infrared (FTIR) spectroscopy of the obtained adsorbent. The added value brought by the functionalization of Mg3Al with the studied IL was underlined by the adsorption studies conducted in the treatment process of water with diclofenac content. Kinetic, thermodynamic, and equilibrium studies were performed. DCF adsorption onto the studied materials correspond to a chemisorption, the pseudo-second-order kinetic model describing the most accurately the experimental data. DCF adsorption onto the studied materials occurs as a heterogeneous process, with the experimental data fitting best with the SIPS isotherm. The sample obtained through cosynthesis developed a maximum adsorption capacity of 648 mg/g.

Research on social learning has centered around vertebrates, but evidence is accumulating that small-brained, non-social arthropods also learn from others. Social learning can lead to social inheritance when socially acquired behaviors are transmitted to subsequent generations. Using oviposition site selection, a critical behavior for most arthropods, as an example, we first highlight the complementarities between social and classical genetic inheritance. We then discuss the relevance of studying social learning and transmission in non-social arthropods and document known cases in the literature, including examples of social learning from con- and hetero-specifics. We further highlight under which conditions social learning can be adaptive or not. We conclude that non-social arthropods and the study of oviposition behavior offer unparalleled opportunities to unravel the importance of social learning and inheritance for animal evolution.

Due to the increased demand for palladium, as well due to its reduced availability in nature, its recovery from diluted waste solutions becomes a necessity, and perhaps an emergency. As a result of economic and technological development, new materials with improved adsorbent properties that are more efficient for metallic ions’ recovery were synthesized and introduced to market. The goal of this study was to obtain a new adsorbent material by functionalizing through impregnation a commercial polymeric support that was both inexpensive and environmentally friendly (Amberlite XAD7) with crown ether (di-benzo-18-crown-6—DB18C6). Crown ethers are known for their ability to form complexes within metallic ions, by including them inside of the ring, regardless of its atomic size. Adsorbent material was prepared by impregnation using the solvent-impregnated resin method (SIR). To highlight the presence of crown ether on the resin surface, a new synthesized material was characterized by scanning electron microscopy (SEM), elemental analysis X-ray energy dispersive spectroscopy (EDX) and Fourier transform infrared spectroscopy (FT-IR). The specific surface of the adsorbent material was also determined by the Brunauer–Emmett–Teller (BET) method. Adsorbent performances of the prepared material were highlighted by kinetic, thermodynamic and equilibrium studies and a possible mechanism was also proposed. The influence of specific parameters for the adsorption process (contact time, temperature, Pd(II) initial concentration) on the maximum adsorption capacity was pursued.

In this study we apply a new monitoring technique for butterflies in order to get more insights into the habitat use patterns of Erebia christi Rätzer 1890 (Nymphalidae: Satyrinae). This European endemic is a rare and very localized butterfly species, found alongside steep slopes in a restricted area in the Alps, at the border between Italy and Switzerland. Even though it was discovered almost 140 years ago, captivating the interest of many lepidopterists, there are still several aspects about the biology of E. christi that are not fully known. We selected two monitoring sites in Veglia–Devero Natural Park, Italy and tested whether E. christi was more likely to use rocky, almost vertical slopes than other types of habitat. Since this habitat was almost inaccessible to humans, we used the single rope access technique, in which field operators rappelled down the slopes to explore the rock faces. We demonstrate that the main characteristics of the typical habitat of E. christi are indeed very steep rock faces with grassy ledges where Festuca sp. plants are present. We also show that, E. christi is more abundant than it was previously thought, with populations that could probably be structured in a metapopulation system. Long-term monitoring with the rope access technique could represent an optimal method to provide key insights into the biology and ecology not only of E. christi but of other butterflies that use similar habitats. Finally, we propose the upgrading of its IUCN category from Vulnerable to Endangered at European level. Our findings demonstrate how appropriate, long-term monitoring can contribute to improve the scarce knowledge of the ecology of an elusive species, and to devise informed proposals regarding the management and protection of populations, especially for species of conservation concern.

Platinum group metals (PGMs) palladium, platinum, and ruthenium represent the key materials for automotive exhaust gas treatment. Since there are no adequate alternatives, the importance of these metals for the automotive industry is steadily rising. The high value of PGMs in spent catalysts justifies their recycling. Therefore, it is really important to recovery platinum group metals from aqueous solutions. Of the many PGMs recovery procedures, adsorption is a process with a good efficiency, but an important role is played by the adsorbent material used into the process. In order to improve the adsorption properties of materials were developed new methods for chemical modification of the solid supports, through functionalization with different extractants. In present paper a new adsorbent material (Chitosan-DB18C6) was used for PGMs recovery. The new adsorbent material was produced by impregnating Chitosan with dibenzo-18-crown-6-ether using Solvent Impregnated Resin (SIR) method. The crown ethers were chosen as extractant due to their known ability to bind metallic ions, whether they are symmetrically or unsymmetrically substituted. In order to determine the PGMs recovery efficiency for new prepared adsorbent material the equilibrium and kinetic studies were performed. Also, to study the PGMs adsorption mechanism the experimental data were modelled using pseudo-first-order and pseudo-second order kinetic models. Experimental data were fitted with three equilibrium isotherm models: Langmuir, Freundlich and Sips. The results proved that new adsorbent material (Chitosan-DB18C6) is an efficient adsorbent for PGMs recovery from aqueous solutions.

The study of global biodiversity will greatly benefit from access to comprehensive DNA barcode libraries at continental scale, but such datasets are still very rare. Here, we assemble the first high-resolution reference library for European butterflies that provides 97% taxon coverage (459 species) and 22,306 COI sequences. We estimate that we captured 62% of the total haplotype diversity and show that most species possess a few very common haplotypes and many rare ones. Specimens in the dataset have an average 95.3% probability of being correctly identified. Mitochondrial diversity displayed elevated haplotype richness in southern European refugia, establishing the generality of this key biogeographic pattern for an entire taxonomic group. Fifteen percent of the species are involved in barcode sharing, but two thirds of these cases may reflect the need for further taxonomic research. This dataset provides a unique resource for conservation and for studying evolutionary processes, cryptic species, phylogeography, and ecology.

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. 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. 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. 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). 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.

The Black-veined White Aporia crataegi (Linnaeus, 1758), a common and widespread butterfly ranging from northwestern Africa to Europe and Asia, has been extinct in Britain since the 1920s and is on a steady decline in several other parts of its range. In order to investigate genetic diversity within A. crataegi and its correspondence with current subspecies-level taxonomy, we barcoded 173 specimens from across its range including, for the first time, extinct populations from Britain and Korea. Using next generation sequencing we also obtained a sequence for Aporia joubini , a peculiar taxon from China known only by its type specimen collected in the early twentieth century. Our phylogenetic analysis placed A. joubini sister to A. oberthuri , although further taxon sampling may reveal a different scheme. Within A. crataegi , we observed a shallow and weak mitogenomic structure with only a few distinct lineages in North Africa, Sicily, Iran, and Japan. Eurasian populations, including those extinct in Britain and Korea, clustered into a large set of closely allied lineages, consistent with a recent expansion during the Late Pleistocene glacial period. This study highlights the importance of museum collections and the unique opportunities they provide in documenting species diversity and helping conservation efforts.

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. We discover and study the genetics of previously unknown populations of three butterfly species (Iolana iolas, Satyrus ferula and Melanargia larissa) in Romania, which deserve conservation measures. This study exemplifies how biodiversity exploration and genetic information can contribute to informed decisions regarding the management and protection of threatened populations.