Bt maize is a genetically modified crop that produces insecticidal proteins derived from the bacterium Bacillus thuringiensis . The proteins act against susceptible herbivorous pests, including lepidopteran insects. Wind-dispersed maize pollen can settle on larval host plants, where caterpillars may ingest Bt proteins and experience additional mortality or sublethal adverse effects.

European Union rules require environmental risk assessment of genetically modified organisms before market placement, and controlled laboratory tests form a central part of assessing potential hazards to non-target organisms. Because thousands of European butterfly and moth species exist, however, it is not feasible to test every species that could be exposed. The researchers began with the LEPIDAT database of the German Federal Agency for Nature Conservation, which contained 8,670 entries for lepidopteran taxonomic units, including genera, species and subspecies.

A preliminary screen retained 1,478 entries for which information was available on altitude, habitat, larval feeding period and feeding type. Most “Micro-Lepidoptera” were excluded before the formal selection because their biological information was insufficient, although the diamondback moth, Plutella xylostella , was retained as an exception. The researchers regarded it as an abundant farmland moth, sensitive to Bt, and easy to breed in the laboratory.

The first formal step excluded 350 listed species in the family Noctuidae, some of which are now classified in Nolidae, because members of this group had been reported to be comparatively insensitive to Bt toxins. The next steps removed species that did not occur in Germany and those unlikely to be exposed to Bt maize pollen. The latter exclusions covered species restricted to elevations above 700 metres, where maize is not cultivated in Germany; species whose larval periods did not overlap with the approximate German maize pollen-shedding period from late June to late August; and species whose larvae feed within host plants or below ground on roots, where deposited pollen would not normally reach them.

These filters reduced the candidate pool to 652 potentially exposed species. The remaining species were classified into broad habitat types: farmland, dryland, wetland, woodland and settlements. A species could qualify for more than one habitat category.

The researchers selected candidates found concurrently in at least four habitat types, including farmland, to favor widespread species capable of representing a range of landscapes surrounding maize cultivation. This stage retained 54 species. Laboratory practicality then provided the next filter.

Candidate species needed evidence that females could lay eggs under laboratory conditions and that their larvae could be reared indoors on host-plant leaves or an artificial diet. Twenty-two species without such evidence were removed, leaving 32 species considered suitable for laboratory experiments. Conservation status and expert judgment shaped the final choice.

Protected species were ranked highly because risk assessment must consider potential harm to populations that are conservation targets under European legislation. The researchers then assessed taxonomic variety, the geographic and ecological range represented by the candidates, and differences in larval body size. These criteria produced a tentative panel of 15 focal species from four lepidopteran superfamilies.

Breeding feasibility was supported by a separate literature review covering nine superfamilies and 30 families of European Lepidoptera. The search identified 548 publications, including handbooks, reviews, traditional breeding guides, modern rearing studies and artificial-diet protocols. Some records dated from more than a century ago, while others described controlled methods and detailed environmental requirements.

Although the review found evidence for laboratory culture across a broad taxonomic range, it also showed that an individual species may require time and adaptation before a reliable method can produce enough healthy larvae for testing. Natural host plants most closely reproduce the circumstances in which caterpillars would encounter deposited maize pollen, and the source study notes that they are the recommended approach for Bt maize effect tests. Artificial diets can nevertheless aid standardization, reproducibility or rapid screening.

Common formulations contain flour from legumes or cereals, protein, yeast, cholesterol, vitamins, minerals and agar as a binder; some also include dried plant material or sucrose to stimulate feeding. Such diets are not universally accepted, especially by newly hatched larvae, and larvae that have already fed on natural food may not transfer successfully. Artificial food must be replaced before it dries or develops mould, and the cited guidance advises against allowing larvae to pupate within it.

Laboratory culture also requires control of several biological and physical factors, including host-plant identity and availability, temperature, humidity, photoperiod, diapause, mating conditions, oviposition substrates and population density. Regular replacement of food, adequate ventilation and routine disinfection of containers and handling equipment help limit condensation, mould and disease. Colonies maintained over several generations may require measures to avoid inbreeding and, in some cases, supplementation with field-collected individuals.

Eggs or pupae may be disinfected before mass rearing, while field-collected food plants can introduce pathogens or parasitoid eggs. These practical requirements are why the researchers treated successful breeding as a central selection criterion rather than a detail to be addressed only after choosing test organisms. The procedure is presented as a selection protocol, not as evidence that particular butterflies or moths are hazardous or protected from Bt exposure.

Susceptibility to Bt maize pollen remains poorly known for most non-target lepidopterans, which is why it was not used as the main screening factor. The final two stages also relied on expert assessment, although that judgment was applied within the documented sequence. The German list cannot be transferred unchanged to other regions: researchers would need to adapt altitude limits, pollen-shedding periods, habitat information and the species present in the relevant receiving environment.

Some excluded species may also deserve testing because information was missing rather than because they were unsuitable. Even with those limitations, the framework offers a systematic way to turn a large regional fauna into a diverse, ecologically informed and practically testable set of candidate non-target insects.

Subject of Research: Selection of non-target butterflies and moths for laboratory assessment of Bt maize pollen effects.

Article Title: Selection of non-target Lepidoptera species to test Bt maize effects in the laboratory: which species and how to breed them?

Article References: Lang, A., Dolek, M., Lee, M. S., Freese-Hager, A., & Otto, M. (2020). Selection of non-target Lepidoptera species to test Bt maize effects in the laboratory: which species and how to breed them?. BioRisk, 15, 45-65. https://doi.org/10.3897/biorisk.15.59823

Image Credits: AI Generated

DOI: 10.3897/biorisk.15.59823

Keywords: Bt maize, non-target Lepidoptera, environmental risk assessment, laboratory breeding, maize pollen, butterflies, moths, species selection, insect conservation, artificial diets, Selection, non-target

The study, published in the journal Nota Lepidopterologica, focused on six butterfly species classified as mire specialists, meaning tyrphobiontic species that are essentially restricted to peat bogs or tyrphophilous species that preferentially occur there. The selected species were the Moorland Clouded Yellow (Colias palaeno), the Cranberry Fritillary (Boloria aquilonaris), the Bog Fritillary (Boloria eunomia), the Cranberry Blue (Plebejus optilete), the Large Heath (Coenonympha tullia) and the Jutta Arctic (Oeneis jutta). The researchers compiled presence and absence records from raised bogs in northwest and northeast Germany, northern Poland, Lithuania and northern Belarus, drawing on two decades of expert field surveys conducted along transect lines between roughly 1998 and 2018.

Every site was visited during three to five different years, typically in the June to August flight period, and transect lengths of one to three kilometres covered the most important habitats at each locality. By relying on presence and absence data rather than abundance counts, the authors ensured comparability across regions that differed in survey intensity. Because the underlying records were collected over many seasons by experienced specialists, the authors argue that the broad picture of which species occur where should be highly reliable, even without standardised quantitative sampling.

The headline result is a clear gradient. Mean species number per site dropped from about four species in northern Belarus and just over three in Lithuania, to roughly 2.3 in northeastern Poland, to around 1.1 in both northwest and northeast Germany. Individual species showed equally revealing patterns. Colias palaeno and Boloria eunomia were entirely absent from northwest German sites and nearly absent from the northeast, while their occurrence soared eastwards: Colias palaeno was found at more than 90 percent of sampled bogs in Lithuania and northern Belarus, and Boloria eunomia reached 100 percent occupancy in northern Belarus. The boreal Oeneis jutta followed a similar eastward-increasing trajectory, reaching two-thirds of Belarusian bogs but never occurring in Germany.

One species broke the mould. The Cranberry Fritillary, Boloria aquilonaris, was continuously distributed across all sampled regions, present at 50 percent of northwest German sites and between roughly 32 and 75 percent elsewhere. Unlike the more sensitive specialists, it appears to be the most tolerant of the tyrphobiontic butterflies in the face of human impact. The authors note that it persists in smaller bogs with partial forest cover and recolonises open patches of former peat-cutting holes rapidly after hydrological restoration. Its sensitivity is nonetheless real: about half of the historically occupied peat bog sites in northwestern Germany have suffered local extinctions of this species.

The Large Heath told an unexpected story in the opposite direction. Although long considered a bog specialist, Coenonympha tullia was absent from all raised bogs surveyed east of the river Vistula in Poland and in Lithuania and Belarus, despite its Palaearctic range covering the entire study area. Previous research offers a likely explanation: the species can reach twentyfold higher densities in managed but undrained fens dominated by broad-leaved bog-cotton, and its larval food plants, sedges of the genus Carex, are far more abundant in fens than in raised bogs. Where pristine, traditionally mown fens survive in Eastern Europe, the raised bogs nearby may simply be suboptimal refuges. In Western Europe, where fenland has largely vanished, peat bogs increasingly serve that fallback role.

From a biogeographical perspective, the peat bogs of the European Lowlands function as palaeorefugia, isolated cold and nutrient-poor habitats in which boreo-montane and arcto-alpine insects established populations during the Holocene. Raised bogs began forming in the young moraine landscapes of northern Europe during the Early Holocene and developed through the Mid and Late Holocene, remaining cold islands within a warming temperate biome. The authors favour the term palaeorefugia over the familiar label glacial relicts, because these species do not have an obligatory connection to Pleniglacial or Late Glacial environments; rather, they colonised bogs over past millennia as the mires themselves developed.

The west-to-east decline in butterfly species richness, the study suggests, is mainly a consequence of human pressure rather than natural biogeography. Western European countries have lost far more than 90 percent of their intact peatland ecosystems, while several Eastern European countries have retained between 15 and 50 percent of their natural peatland heritage. The figures cited for remaining natural peatland are stark: roughly 1 percent in Germany, 15 percent in Poland, 40 percent in Lithuania and about 45 percent in Belarus. The geographic pattern of species depletion documented in the 105 bogs roughly coincides with this gradient in remaining habitat, and the almost complete gap in occurrences of Colias palaeno and Boloria eunomia in northern Germany, where Colias palaeno went extinct in the first half of the twentieth century, fits the same picture.

Ecological flexibility also varies regionally, which complicates simple categories of bog dependence. Colias palaeno is strongly tyrphobiontic in most of Central Europe but uses dwarf shrub communities above the treeline in the Alps. Boloria eunomia exists as two distinct ecotypes in the eastern part of its range, one feeding on Bistorta major on wet meadows and the other on Vaccinium oxycoccus on raised bogs, with recent findings suggesting the meadow form is ancestral. In Finland, some of the six species fall into the highest class of mire dependence, with over 95 percent of populations in mires, while others are less tightly bound. The lesson is that a species’ degree of association with peat bogs is not fixed across its range but shaped by the surrounding landscape and the availability of alternative wetlands.

Beyond diagnosis, the study offers practical guidance for restoration. The authors propose Boloria aquilonaris and Plebejus optilete as target species whose presence can signal success in peat bog restoration across the western part of the northern lowlands. Both are tyrphobiontic in the strict sense, threatened by drainage, degradation and afforestation, and yet ecologically flexible enough to respond positively to rewetting, deforestation and the return of vegetation heterogeneity. Boloria aquilonaris colonises open peat-cutting holes quickly after hydrological restoration, while Plebejus optilete tolerates a wider spectrum of larval host plants and restricted flowering resources. Because both are among the most widely distributed mire specialists across the entire region, their re-establishment in the few remaining German bogs, where only five sites currently host both together, could provide a living measure of whether Europe’s efforts to bring its cold islands back from the brink are working.

Subject of Research: Distribution patterns of mire specialist butterflies in raised bogs of the Central European lowlands

Article Title: The distribution pattern of mire specialist butterflies in raised bogs of the northern lowlands of Central Europe

Article References: Sommer, R. S., Thiele, V., Sushko, G., Sielezniew, M., Kolligs, D., & Dapkus, D. (2022). The distribution pattern of mire specialist butterflies in raised bogs of the northern lowlands of Central Europe. Nota Lepidopterologica, 45, 41-52. https://doi.org/10.3897/nl.45.75182

Image Credits: AI Generated

DOI: 10.3897/nl.45.75182

Keywords: raised bogs, butterflies, tyrphobiontic species, peatland restoration, Boloria aquilonaris, Colias palaeno, Plebejus optilete, biogeography, palaeorefugia, species richness gradient, Central Europe, biodiversity

The anomalous blues belong to the subgenus Agrodiaetus within the large genus Polyommatus, a lineage of more than 120 species that spread explosively across the Western Palaearctic and Central Asia in the last three million years. Males of this group often display striking dorsal wing colours, from blue and silver to violet and brown, while females are usually brown. The species studied here are part of the brown complex, in which males are coffee-brown rather than blue, and they are notoriously difficult to tell apart by appearance alone. The state of a white streak on the underside of the hindwing, long used as an identification trait, turned out to be unreliable, as shown by past misidentifications of the Greek species Polyommatus aroaniensis in Croatia, where specimens lacking the streak were initially assigned to the wrong species until mitochondrial COI barcoding corrected the record.

Until now, the Polyommatus aroaniensis species complex was thought to contain three closely related allopatric species distributed across the southern Balkan Peninsula. Polyommatus aroaniensis sensu stricto lives in southern Greece and the Peloponnese and carries 47 to 48 chromosomes per haploid set, together with mitochondrial haplogroup aroa1. Polyommatus timfristos, described in 2016 from the mountains of central Greece, has only 38 chromosomes and haplogroup aroa2. Polyommatus orphicus, found in north-eastern Greece, southern Bulgaria and North Macedonia, has 41 to 42 chromosomes and haplogroup orph1. Each species is differentiated primarily by its chromosome number and mitochondrial DNA, because external morphology varies so much geographically and individually that it rarely provides unambiguous separation.

Suspicion that a fourth taxon existed arose when the first author collected butterflies across 94 sites in Albania, from the southern province of Korçë to the north-eastern province of Kukës. Only seven sites yielded members of the aroaniensis complex, and one region stood out dramatically: the Lurë region in Dibër province, where many individuals showed a remarkably dark habitus and mostly lacked the white hindwing streak entirely. Even more striking was their habitat. These butterflies lived on dark-coloured ophiolitic soils, substrates derived from fragments of former oceanic crust pushed up during mountain building, while related populations in the nearby Valikardhë region occupied visibly paler karst, or chalky limestone, substrates. In the ophiolitic habitats, plants of Onobrychis alba, a known foodplant for various Agrodiaetus species, were found growing.

To resolve the identity of these butterflies, the team analysed DNA from 41 Albanian specimens and mined 19 additional COI sequences from GenBank for comparison. The full 658-base-pair DNA barcode fragment was amplified with the primers LepF1 and LepR1, and a 411 to 440-base-pair fragment of the nuclear ITS2 region was also sequenced to strengthen the phylogenetic signal. Phylogenies were reconstructed with Bayesian inference, maximum likelihood and maximum parsimony methods on both the COI alignment and a concatenated COI plus ITS2 matrix of 1039 base pairs. In every analysis, the Albanian specimens carrying the mitochondrial haplogroup aroa3 formed a monophyletic, well-differentiated clade with strong support. The haplotype network, built from 54 specimens collapsed into 28 haplotypes, confirmed that aroa3 is the most differentiated mitochondrial lineage within the entire aroaniensis species complex, clearly separated from aroa1, aroa2 and orph1 by multiple nucleotide substitutions.

Karyotyping provided the second decisive line of evidence. Testes were removed from freshly collected males within an hour of capture, stored in fixative, and stained with acetic orcein for chromosome analysis under high-magnification light microscopy. The Albanian populations of Polyommatus orphicus showed a haploid chromosome number of n=42, fitting the previously described variability of the species. The dark Lurë butterflies, however, carried n=42 to 43, with variation caused by polymorphism for a single chromosome fusion or fission. Crucially, the difference between the two taxa lies in a different chromosome pair, meaning they differ by at least one fixed chromosome fission. Chromosome rearrangements have been shown to be important drivers of speciation in Polyommatus butterflies, and even a slight reduction in fertility of heterozygotes for such rearrangements can create a genetic boundary between chromosomally diverged populations.

The third line of evidence came from an innovative morphometric technique based on wing colour. The researchers photographed set male specimens under a standardized triangular arrangement of light sources in a darkened room, positioning the wings at a 20-degree angle to measure maximum light reflectance from the wing scale structures. Reflectance measurements were taken from the intervein space of the inner postdiscal zone, and exact RGB and hue values were extracted for statistical analysis. Non-metric multidimensional scaling plots of 27 specimens showed that wing reflectance matched the phylogenetic clades significantly. Polyommatus orphicus generally showed a measurable greenish reflectance on the postdiscal band of the forewing, a trait completely absent in the new species. Statistical analysis also revealed a significant link between each species and its typical soil substrate, dark ophiolitic versus light karst soils.

At the contact zone between the two species, where mixed ophiolite and karst substrates occur, the team detected specimens with unusual combinations of mitochondrial haplogroup and karyotype, including individuals with the orph2 haplogroup and n=43 chromosomes, together with intermediate morphology and intermediate wing reflectance. The researchers interpret these as hybrids between the new species and Polyommatus orphicus. Notably, the karyotypes expected for first-generation hybrids, which should all show 41 visible elements in the first meiotic metaphase, were not observed, suggesting that there is no complete reproductive isolation between the two species and that the observed patterns result from repeated hybridization and backcrosses. Alternatively, the Albanian orphicus lineage may itself carry n=42 to 43, though the restriction of these specimens to the contact zone and their intermediate morphology favour the hybridization hypothesis.

Based on this consensus of molecular, cytogenetic and morphometric evidence, the team formally described the Albanian taxon as Polyommatus lurae sp. nov., named after the Lurë region where the type locality lies. The species name also alludes to a local legend in which the Lura tribe, during the Ottoman wars, sent armed men disguised in bridal veils to surprise the enemy camp, a fitting reference for a species that has survived periods of close contact with more widely distributed congeners without complete admixture. The new species is distinguished by its dark chocolate-brown upperside, the absence of a white postdiscal streak on the forewing underside, a minimal or invisible hindwing streak, lack of wing reflectance, and its karyotype. Worn individuals may be externally indistinguishable from related taxa, but can still be identified by chromosome number and COI barcode.

The discovery carries an urgent conservation message. The Lurë region has been a National Park since 1966, yet illegal logging and forest fires are estimated to have destroyed as much as half of the original park, and overgrazing by sheep threatens the Onobrychis foodplants on which the butterfly depends. Because Polyommatus lurae occupies a very restricted area linked to a unique ophiolitic biotope found mostly in Albania, the researchers argue it should be considered a candidate for protected species lists in Albania and Europe. As Albania moves toward European Union membership, adequate protection legislation for its biodiversity heritage, including control of illegal logging, burning and uncontrolled grazing, will be essential to secure the future of this newly recognized species and the unique ecosystems that harbour it.

Subject of Research: Cryptic butterfly speciation in Albania

Article Title: Integrative analysis reveals cryptic speciation linked to habitat differentiation within Albanian populations of the anomalous blues ( Lepidoptera , Lycaenidae , Polyommatus Latreille, 1804)

Article References: Parmentier, L., Vila, R., & Lukhtanov, V. (2022). Integrative analysis reveals cryptic speciation linked to habitat differentiation within Albanian populations of the anomalous blues (Lepidoptera, Lycaenidae, Polyommatus Latreille, 1804). Comparative Cytogenetics, 16(4), 211-242. https://doi.org/10.3897/compcytogen.v16.i4.90558

Image Credits: AI Generated

DOI: 10.3897/CompCytogen.v16.i4.90558

Keywords: butterflies, Albania, cryptic species, Agrodiaetus, karyotype, DNA barcoding, speciation, biodiversity, Integrative, analysis, reveals, cryptic

The genus Erebia, whose members are among the most intensively studied alpine insects in the Holarctic, most likely originated in Asia and colonised Europe roughly 17 to 23 million years ago. It diversified into dozens of species during the Miocene and Pliocene, and then underwent a rapid burst of intraspecific differentiation driven by the Pleistocene glacial cycles. The large ringlet is one of the most variable species in the genus, with sixteen morphological subspecies described, five of which are endemic to the Alps. Their territories are separated by intergradation zones so narrow that gene flow between neighbouring forms is evidently extremely restricted, a hallmark of long isolation.

Previous molecular work had painted a confusing picture. Allozyme studies identified two lineages in the Alps, matching the subspecies adyte and isarica, plus one lineage in the Pyrenees and another in the Carpathians and Balkans. But a mitochondrial study suggested that the subspecies ocellaris and isarica were closer to the Carpathian syrmia than to each other, and two poorly known southern Alpine subspecies, pseudoadyte and kunzi, had never been sampled at all. The number of genetic lineages in the Alps therefore ranged anywhere from two to five depending on which data one trusted. To resolve the confusion, the team sequenced 1,496 base pairs of the mitochondrial COI gene, covering both the standard DNA barcode region and the less commonly used 3-prime section of the gene, in sixteen Alpine and Jurassian populations, and combined the new sequences with published data from the Pyrenees, Cantabria, the Carpathians and the Sudetes.

The resulting dataset of 158 individuals contained fifty-three haplotypes defined by fifty-one variable sites. The haplotype network resolved into five clusters, labelled A through E, and these clusters turned out to be largely congruent with the morphological delineation of subspecies. Remarkably, ninety-eight percent of the haplotypes were private to a single subspecies and eighty-eight percent to a single population, evidence of profound genetic isolation despite the proximity of some ranges. The 3-prime section of COI proved far more discriminating than the barcode region alone, revealing three clades that the standard barcode fragment had left hidden. The analysis confirmed that pseudoadyte and kunzi are genuine, distinct lineages rather than mere look-alikes of adyte and ocellaris, and that the morphologically intermediate western form of kunzi is genetically inseparable from the eastern one, vindicating its original assignment.

The study also settled the contested status of isarica and ocellaris. Earlier work had declared them genetically identical, but the new data showed that these two subspecies share only a single common haplotype, from which each has evolved its own private mutations. The shared haplotype becomes more frequent as one approaches the wide hybrid zone where the two forms meet, a pattern consistent with secondary contact after independent differentiation. Intriguingly, isarica itself split into two genetically distinct sub-lineages, one corresponding to western and northern Alpine populations and the other to eastern Alpine ones, meaning the Alps actually harbour six mitochondrial lineages, four of which had gone unrecognised until now.

To reconstruct where these lineages survived the last ice age, the researchers overlaid the current distributions onto the six broadly recognised categories of potential refugia during the Würm glaciation. When the great glaciers filled the main valleys, habitable conditions persisted in unglaciated lower mountains at the western and eastern edges of the Alps, on the sun-exposed, wind-sheltered slopes along the southern Alpine border, and in pockets along the lobed northern ice front. Each subspecies mapped neatly onto one of these compartments. Pseudoadyte, for example, occupies a territory sharply bounded by the Adda and Adige valleys, exactly matching a single refugial compartment, while kunzi clings to the relict foothills between Val d’Adige and Valcellina. The valleys that dissect the southern Alpine fringe, carved ever deeper by repeated glacial erosion, acted as barriers that fragmented the butterfly into the three lineages adyte, pseudoadyte and kunzi, which now show pairwise genetic divergences of up to 1.15 percent, consistent with long-term isolation within their current borders.

The deepest story, however, lies in the broader geography of the lineages. The clusters sort into three groups: a southern Alpine group, a circum-Pannonian group uniting Carpathian butterflies with those of the northern and eastern Alps, and a western extra-Alpine group. This structure implies an ancient Alpine-Carpathian disjunction, with the common ancestor most plausibly resident in the Carpathians, a hint reinforced by the close genetic affinity between the Carpathian samples and the outgroup species Erebia ligea. During severe cold phases the ancestors retreated to the Pannonian plain, where pollen evidence indicates taiga-like forests and even patches of temperate woodland persisted even at the glacial maximum. When the climate ameliorated, they colonised both the Alps and the Carpathians. A second such retreat and re-expansion then split the circum-Pannonian group into the Carpathian subspecies euryale and syrmia on one side and the Alpine ocellaris, eastern isarica and western isarica on the other.

Dating these events proved subtle. Bayesian molecular dating, calibrated against the roughly 3.5-million-year split between E. euryale and its nearest relative, placed the diversification of the present subspecies between about 0.75 and 2 million years ago. But the geological constraints tell a tighter story: only the most severe glacial phases, beginning with marine isotope stage 22 around 870,000 to 880,000 years ago, drove eastern Alpine glaciers down into the main valleys and expelled subalpine organisms into refugial isolation. The first Alpine-Carpathian split therefore cannot be much older than a million years, and the southern Alpine differentiation of adyte, pseudoadyte and kunzi must predate the Alpine-Pyrenean disjunction, placing it at least as far back as marine isotope stage 10. The star-like haplotype network of the Pyrenean and Jurassian butterflies, typical of a recent expansion after a bottleneck, marks them as a comparatively young divergence, separated from their Alpine relatives no later than the Riss glaciation.

Not every question is closed. Allozyme data place the Pyrenean lineage closer to the northern than to the southern Alps, conflicting with the mitochondrial signal that ties it to the southern Alpine adyte group, a mismatch that only genome-wide nuclear data will be able to arbitrate. The authors also note that Wolbachia infections, known to occur in E. euryale, could in principle have influenced the mitochondrial patterns, and that nuclear genomic data would address this possibility directly. A striking morphological detail offers independent support for the deep splits: the three southern Alpine subspecies share white-pupilled eyespots on the forewing upperside, a trait that appears to be a shared derived character marking the basal division of the species’ history. For a butterfly that most hikers barely notice, Erebia euryale has turned out to be a remarkable archive of Quaternary climate history, one whose pages were written by advancing and retreating ice.

Subject of Research: Phylogeography and Pleistocene diversification of the large ringlet butterfly Erebia euryale based on mitochondrial COI sequences

Article Title: Mitochondrial DNA-based phylogeography of the large ringlet Erebia euryale (Esper, 1805) suggests recurrent Alpine-Carpathian disjunctions during Pleistocene ( Nymphalidae , Satyrinae )

Article References: Cupedo, F., & Doorenweerd, C. (2022). Mitochondrial DNA-based phylogeography of the large ringlet Erebia euryale (Esper, 1805) suggests recurrent Alpine-Carpathian disjunctions during Pleistocene (Nymphalidae, Satyrinae). Nota Lepidopterologica, 45, 65-86. https://doi.org/10.3897/nl.45.68138

Image Credits: AI Generated

DOI: 10.3897/nl.45.68138

Keywords: Erebia euryale, phylogeography, mitochondrial DNA, COI, Alps, Carpathians, Pleistocene, glacial refugia, butterflies, Nymphalidae, subspecies, haplotype network

Before 1999, when the species was recorded for the first time in Portugal, the Iberian distribution of Thecla betulae was considered to be limited to the temperate Euro-Siberian northern third of Spain. The first Portuguese record was itself a deceased adult, found at Serra da Lousã, a location far from the nearest known localities in Galicia at the time, and a live adult was not observed in the country until 2011. This late addition to the national species list is a direct reflection of the butterfly’s arboreal lifestyle. Because adults are so seldom seen, the species is overwhelmingly monitored through the detection of its bright white overwintering eggs on the dark bark of blackthorn and related food plants during winter, when the deciduous hosts have shed their leaves and the eggs contrast sharply with the bare branches.

The caterpillars of Thecla betulae are moderately polyphagous and feed on a variety of rosaceous shrubs and trees in the genus Prunus, especially blackthorn, Prunus spinosa, but also plum, Prunus domestica, its wild relative Prunus insititia, and other allied species in the subfamily Amygdaloideae. The species is univoltine, with adults on the wing from July to October in one extended generation. Eggs are laid most often at the base of lateral shoots of the food plant, positioned between half a metre and two metres above ground, and overwintering happens in the egg stage. In early spring, right after the mass flowering of the host plant, young caterpillars emerge synchronously with the first leaves and feed on their underside until pupation by May or June, accomplished either at ground level in the leaf litter or on nearby branches.

The research team reviewed the literature from 2002 to 2021, since the publication of the first comprehensive Iberian butterfly atlas, which had mapped the species in 122 landscape-scale quadrats of 10 by 10 kilometres. They complemented this with opportunistic field surveys in Portugal from 2004 to 2021, searching for adults in summer and eggs later in the year, and with records from online databases such as GBIF and iNaturalist, which added a further 16 quadrats not covered by any other source. All point coordinates and area data were standardised to 10 by 10 kilometre UTM quadrats, producing an up-to-date distribution map built from a total of 442 occurrence points for the peninsula.

The resulting map reveals an almost continuous distribution across the northern third of Iberia, from the pre-Pyrenean area and coastal ranges of Catalonia in the east, through the Pyrenees and Cantabrian ranges, westwards to the Galician and Leonese ranges, and widespread in Galicia up to the coast and into northern Portugal. Further south, populations are mostly mountain-bound, clustering in the Central Iberian Mountain System from Serra da Lousã to Serra da Estrela, in Serra da Malcata, Sierra de Gata, Candelario and Sierra de Gredos. Notably, no records yet exist eastwards in the Sierra de Guadarrama or Segovia province that would connect these southern pockets to the main northern distribution area through Spain, a gap that popular generalist European sources have long failed to bridge, often not extending the species beyond the Pyrenees and the Cantabrian belt at all.

In Portugal alone, the fieldwork confirmed the species at 19 new sites, an increase of about 428 percent in known Portuguese localities, bringing the total to roughly 31 sites of occurrence. These records cluster in three to four areas: the Portuguese part of the Central Mountain System in the ranges of Serra da Lousã, Serra do Açor, Serra da Estrela and Serra da Malcata; the environs of Bragança around Serra de Montesinho and Serra da Nogueira; and a newly discovered, more fragmented cluster from the northern border with Ourense near Boticas and Montalegre southwards to Serra do Alvão near Vila Real. The species was also found in Serra da Peneda, although whether these populations are in contact with the others remains to be confirmed.

To help target future searches, the team built a species-distribution model using maximum entropy analysis implemented in MAXENT, contrasting the 442 occurrence points against 19 current bioclimatic variables from the WORLDCLIM database. The model achieved an AUC of 0.977, well above the 0.7 usually desired in such studies, meaning the known occupation of the territory is very well explained by the variables used. Four variables framed the distribution especially well: temperature seasonality, the mean temperature of the driest quarter, the temperature annual range and annual precipitation. In essence, Thecla betulae is positively affected by relatively stable temperatures over the year, summers that are not too hot, and relatively high annual precipitation for Iberian conditions, confirming that a temperate climate is essential for the species’ presence in a peninsula with a predominantly Mediterranean climate.

The model highlights several areas as highly climatically suitable but still under-sampled or poorly known, including the extreme north of Aragon, the northern half of Burgos and Palencia provinces, most of Cantabria, western Zamora, the north-east of Trás-os-Montes in Portugal, and the northern slopes of Sierra de Guadarrama, from which no records are yet known. The mountain ranges of all northern and central Portugal appear suitable, possibly extending southwards to the Gardunha range or even the Serra de Aire area. The authors expect that these projections will help naturalists and researchers plan future fieldwork in both Spain and Portugal to complete the Iberian distribution map of the species.

One of the most intriguing outcomes of the fieldwork was the detection of eggs on almond trees, Prunus dulcis, a food plant hitherto unknown for Thecla betulae anywhere in its range. The observation comes from the south-western part of Serra da Estrela, under a thermophilic situation that allows young almond trees to grow, a condition not usually met at the butterfly’s other potential sites. This discovery broadens the ecological spectrum of the species towards a more Mediterranean environment, and the finding that the flight period in Portugal can start as early as late May, earlier than expected elsewhere in Europe, further supports this broadening of the niche and shows how novel life-history traits may still turn up in a relatively well-known butterfly.

The study also sounds a note of conservation concern. As more records surface, Portuguese populations seem to face an increasing risk from the spread of Eucalyptus globulus monocultures and the invasion of semi-natural habitats by alien Acacia trees, which is rampant in central Portugal. These changes bring an elevated fire risk, and the massive wildfires of 2017 burnt a large part of central Portugal, affecting known sites for the butterfly. Following the fires, new management rules oblige landowners to clear vegetation around houses and along roads, removing the marginal vegetation such as blackthorn where the species survives. For a butterfly already near its southern bioclimatic threshold in Europe, these pressures should be accounted for in any conservation status evaluation. The new baseline of knowledge set by this review should foster further studies and a better understanding of other elusive, underrecorded species that share its temperate woodland refuges.

Subject of Research: Ecology

Article Title: A review of the distribution and ecology of the elusive Brown Hairstreak butterfly Thecla betulae ( Lepidoptera , Lycaenidae ) in the Iberian Peninsula

Article References: Marabuto, E., Pires, P., Romão, F., Lemos, P., & Merckx, T. (2022). A review of the distribution and ecology of the elusive Brown Hairstreak butterfly Thecla betulae (Lepidoptera, Lycaenidae) in the Iberian Peninsula. Nota Lepidopterologica, 45, 101-118. https://doi.org/10.3897/nl.45.76222

Image Credits: AI Generated

DOI: 10.3897/nl.45.76222

Keywords: Brown Hairstreak, Thecla betulae, butterflies, Iberian Peninsula, species distribution modelling, Portugal, Spain, conservation, review, distribution, ecology, elusive

The discovery was, by the authors’ own account, accidental. The team had set out to answer a different question: whether any of the Iranian populations of the Wood White actually belonged to Leptidea juvernica, a cryptic sister species that was itself only recognized as distinct in recent decades. Fourteen museum and field specimens from disjunct populations across northwestern and northern Iran had their legs submitted for DNA barcoding, processed at the Center for Biodiversity Genomics in Guelph, Canada, using standard LepF and LepR primers supplemented by mini-primers for degraded samples. Eleven additional sequences from the Javaherdeh locality, generated in 2012, were later added, and thirty-six new barcode sequences were deposited in GenBank. None of the barcoded Iranian specimens turned out to be L. juvernica, but something unexpected emerged instead.

The mitochondrial COI barcodes, mostly full-length sequences of 658 base pairs, told a striking story. Samples from East Azerbaijan province in the northwest, around Arasbaran, carried haplotypes identical to those of the widespread Eurasian nominate subspecies. But specimens collected across the Alborz mountains, from Talesh in the west to the forests of Golestan in the northeast, formed a unique and well-supported clade. In both Maximum Likelihood and Bayesian phylogenetic reconstructions, this lineage appeared as the sister group to all other populations of L. sinapis, meaning it branched off earliest in the species’ genealogy. The average uncorrected genetic distance between the new lineage and other L. sinapis populations was 0.74 percent, with a range from 0.42 to 1.76 percent, clearly smaller than the distances separating L. sinapis from its recognized sibling species L. reali at 0.92 percent and L. juvernica at 1.97 percent, but far from trivial.

What makes the finding remarkable is that the new butterfly is morphologically indistinguishable from the nominate subspecies. The researchers dissected and measured the male genitalia of eight specimens, focusing on three characters known to be the most informative for separating Leptidea species: phallus length, saccus length and vinculum width. The measurements, 1.47, 0.60 and 0.71 millimeters respectively, overlapped broadly with those of typical L. sinapis, and no single external character or combination of characters could reliably separate the two forms. Individual variation within the new taxon mirrors that seen in nominate populations, where butterflies on cold, high-altitude slopes tend to be smaller and darker than those in warmer lowland forests. Only the DNA barcodes betray the difference, placing the new subspecies within the Barcode Identification Number of L. sinapis while forming a distinct cluster of its own.

The chromosomes added another layer of evidence. Chromosome preparations from ten adult males caught at Javaherdeh yielded usable metaphase plates in only two specimens, a common difficulty in this genus. In mitotic cells of one sample, the diploid chromosome number was estimated at approximately 58, though exact counting was hampered by overlapping chromosomes. More intriguingly, the second metaphase plates of another sample showed traces of a phenomenon the researchers call inverted meiosis, in which heterozygosity for chromosomal fusions or fissions produces trivalent-like structures at the second meiotic division. Seven such structures were observed among 29 chromosome entities, allowing the haploid number to be estimated at between 29 and 33. This relatively low chromosome count matches the eastern end of a remarkable cline documented in L. sinapis, in which diploid numbers decline from 106 in Spain to 56 in Sweden and eastern Kazakhstan, marking the Iranian population as carrying an oriental karyotype variant.

The known distribution of the new subspecies, confirmed by DNA evidence, spans the northern Iranian provinces of Ardabil, Gilan, Mazandaran, Tehran and Golestan. It is strictly allopatric with respect to the nominate subspecies, whose nearest Iranian population lies about 100 kilometers away in the Arasbaran region. Intriguingly, specimens from the Talysh mountains across the border in the Republic of Azerbaijan show the same karyotype and may also belong to the new subspecies, though DNA confirmation is still pending, and unconfirmed reports from the Kopet Dagh mountains of Turkmenistan likely refer to it as well. Unlike the nominate form, which in Iran is restricted to humid and damp forest clearings, tabarestana occupies semi-humid and even semi-dry mountainous habitats, flying in humid meadows, along forest river banks and in clearings between 1,000 and 2,000 meters elevation, usually in undisturbed or lightly grazed sites with lush green vegetation.

The butterfly’s ecology follows a familiar seasonal rhythm. Adults appear in two, possibly three, generations each year, from April at lower altitudes to the end of September at higher elevations, flying alongside species such as Ochlodes hyrcana, Pieris napi mazandarana, Lasiommata adrastiodes and Maniola jurtina. Adults are often seen near Lathyrus plants, and the larval host plant is likely among the herbaceous legumes of genera such as Lathyrus, Vicia and Lotus, though it remains unrecorded, leaving open the question of whether the new subspecies shows host preferences different from other populations. The early stages are entirely unknown.

How did such a distinct lineage arise without any visible divergence in body form? The authors propose a biogeographical scenario rooted in the Pleistocene history of the region. Dense forests once covered all of northern Iran, from Azerbaijan province across the Alborz mountains and into the Kopet Dagh in the northeast. Since the Last Glacial Maximum, roughly 21,000 years ago, the Alborz range has been nearly entirely isolated from surrounding regions, and declining forest cover left isolated refugia in the southern Caucasus and northern Iran. As intervening populations died out, the range of the ancestral Wood White fragmented, geographically and genetically isolating the Alborz lineage. Recent estimates place the most recent common ancestor of L. sinapis at about 1.5 million years ago, and of sinapis plus juvernica at about 3 million years, with the eastward dispersal of the species occurring much later, before or after the Last Glacial Maximum.

The Wood White complex holds a special place in the history of cryptic species research. Differences in genital morphology and allozyme markers between L. sinapis and L. reali were among the first documented cases of cryptic species in Europe, and the subsequent discovery of the widespread hidden taxon L. juvernica turned the group into a model system for studying speciation. Yet modern taxonomic treatments had come to regard all populations from Europe to northwestern Mongolia as a single subspecies, and the idea of subspecies within L. sinapis had largely faded from view. The new discovery shows how much diversity can still be concealed within even well-studied butterflies, and how much of Eurasia remains poorly sampled.

The authors caution that the presence of Wolbachia endosymbionts, known to affect mitochondrial DNA in Leptidea, cannot be ruled out as a factor in their results, and further studies are needed to confirm the subspecies’ presence in Azerbaijan and Turkmenistan. If the two taxa are eventually found to co-occur in the intervening areas of northwestern Iran without gene flow, or if additional evidence from nuclear DNA, karyotype or morphology signals full species-level separation, tabarestana may yet be elevated to a bona species. For now, the mountains south of the Caspian have yielded one more reminder that the map of life is still being drawn, one barcode at a time.

Subject of Research: Discovery and description of a new cryptic subspecies of the Wood White butterfly Leptidea sinapis from northern Iran using DNA barcoding, genital morphology and karyotype analysis.

Article Title: More hidden diversity in a cryptic species complex: a new subspecies of Leptidea sinapis ( Lepidoptera , Pieridae ) from Northern Iran

Article References: Nazari, V., Lukhtanov, V. A., Naderi, A., Fric, Z. F., Dincă, V., & Vila, R. (2023). More hidden diversity in a cryptic species complex: a new subspecies of Leptidea sinapis (Lepidoptera, Pieridae) from Northern Iran. Comparative Cytogenetics, 17, 113-128. https://doi.org/10.3897/compcytogen.17.102830

Image Credits: AI Generated

DOI: 10.3897/compcytogen.17.102830

Keywords: Leptidea sinapis, cryptic species, DNA barcoding, COI, karyotype, Alborz mountains, Iran, Pieridae, butterflies, speciation, biogeography, taxonomy

The controversy centers on the identity of the type species for the genus Hipparchia, which was established by Fabricius in 1807. In 1868, the entomologist Walter Rothschild’s contemporary, A. E. Butler, designated Papilio hermione as the nominal type species for this genus. Butler made this choice under the firm belief that hermione was a subjective synonym of the senior name, Papilio fagi. This designation was accepted by the scientific community because it aligned with Linnaeus’s own earlier synonymy. The situation remained stable until Kudrna, in his 1977 revision of the genus, designated a specific specimen of the Rock Grayling, Hipparchia alcyone, as the lectotype for Papilio hermione. This action effectively broke the synonymy between hermione and fagi, rendering the genus Hipparchia based on a misidentified species and causing significant confusion in the application of these names.

The validity of Kudrna’s lectotype designation has been challenged on several grounds, primarily concerning the survival of the original type material. Linnaeus based the name Papilio hermione on specimens from the collection of Queen Louisa Ulrika of Sweden. Historical records indicate that this collection fell into neglect after the Queen’s death in 1782. When the collection was eventually bequeathed to Uppsala University in 1803 and catalogued by Carl Peter Thunberg, no specimen of Papilio hermione was present. This absence suggests that any original syntypes had been lost to neglect or decay before the collection reached the university. The authors of the recent paper examined the remnants of the Queen’s collection in Uppsala and confirmed that no specimen of hermione extant, consistent with Thunberg’s earlier observations.

Further investigation into the historical context of Linnaeus’s naming reveals that he likely muddled together multiple species of graylings. Linnaeus cited illustrations by James Petiver and August Johann Rösel von Rosenhof in his 1764 publication. Modern analysis suggests that these illustrations represent different species, specifically Hipparchia fidia and Brintesia circe. This indicates that Linnaeus’s original concept of Papilio hermione was not based on a single, clearly defined taxon. The lack of surviving syntypes from the Queen’s collection, combined with the ambiguity of the original illustrations, means that there is no valid material from which a lectotype could be selected. Therefore, Kudrna’s 1977 designation, which relied on a specimen that was not a syntype, is technically invalid under the rules of the International Commission on Zoological Nomenclature.

The consequences of Kudrna’s invalid designation have been far-reaching, leading to a split in the scientific community regarding the correct name for the Rock Grayling. A majority of lepidopterists have ignored Kudrna’s action and continue to apply the name Hipparchia alcyone to the Rock Grayling, maintaining the traditional synonymy of hermione with fagi. However, a significant minority have accepted Kudrna’s lectotype designation, leading to the application of the name hermione to the Rock Grayling and the designation of alcyone as a junior synonym. This divergence has created nomenclatural instability, with different publications and databases using different names for the same species, complicating research and conservation efforts.

To resolve this issue, the authors propose the designation of a neotype for both Papilio hermione and Papilio fagi. A neotype is a specimen selected to serve as the single physical embodiment of the type status of a nominal taxon when the original type material is lost or destroyed. By designating a single specimen of the Woodland Grayling as the neotype for both names, the authors render the two names objectively synonymous. This action restores the synonymy that was accepted by Linnaeus and Butler, thereby stabilizing the application of the name Papilio hermione as the nominal type species of the genus Hipparchia. The neotype specimen is a fresh male from Kreuzberg in Styria, Austria, collected in 1979 and deposited in the Tiroler Landesmuseen in Innsbruck.

The choice of the neotype specimen was guided by historical and geographical considerations. Kreuzberg is located in a region that corresponds to the historical areas of Germania and Carniola, where the original material for Papilio hermione and Papilio fagi likely originated. In the 18th century, these regions were part of the broader German-speaking lands, including the Archduchy of Austria. The specimen exhibits the wing characters typical of the species universally known as Hipparchia fagi, ensuring that the neotype accurately represents the taxon intended by Linnaeus and Scopoli. This designation provides a clear and objective reference point for future taxonomic work, eliminating the ambiguity that has plagued the nomenclature of these butterflies for decades.

The restoration of the synonymy between Papilio hermione and Papilio fagi has significant implications for the stability of the genus Hipparchia. By ensuring that the nominal type species is correctly identified as the Woodland Grayling, the authors preserve the application of the genus name as originally intended by Fabricius and Butler. This action also re-establishes the senior name for the Rock Grayling as Hipparchia alcyone, a name that has been widely used by the majority of authors. The resolution of this taxonomic dispute not only clarifies the nomenclature of these specific species but also serves as a case study in the importance of adhering to the rules of zoological nomenclature and the need for careful examination of historical type material.

The recent publication in Nota Lepidopterologica represents a significant contribution to the field of lepidopterology, providing a clear and well-supported solution to a long-standing nomenclatural problem. By demonstrating the invalidity of Kudrna’s lectotype designation and proposing a valid neotype, the authors restore stability to the application of the names Papilio hermione and Papilio fagi. This work underscores the importance of historical context and rigorous taxonomic analysis in resolving disputes over scientific names. As the scientific community continues to refine its understanding of biodiversity, such efforts are essential for maintaining the accuracy and consistency of biological nomenclature.

Subject of Research: Taxonomic nomenclature and type species designation in Satyrinae butterflies

Article Title:  Papilio hermione Linnaeus, type species of Hipparchia Fabricius ( Lepidoptera , Satyrinae ): restoring stability to the application of these names

Article References: Russell, P. J. C., & Vane-Wright, R. I. (2022). Papilio hermione Linnaeus, type species of Hipparchia Fabricius (Lepidoptera, Satyrinae): restoring stability to the application of these names. Nota Lepidopterologica, 45, 279-294. https://doi.org/10.3897/nl.45.85341

Image Credits: AI Generated

DOI: 10.3897/nl.45.85341

Keywords: Lepidoptera, Taxonomy, Nomenclature, Hipparchia, Neotype, Lectotype, Butterflies, Zoological Nomenclature, Systematics, Entomology, Papilio, hermione

The encounter took place on 19 July 2016 at approximately 13:25 CEST near the village of Hynčice in the Protected Landscape Area of Broumovsko, in the Czech Republic, at an elevation of roughly 400 metres beside the river Stěnava. One of the authors of the study took random photographs of butterflies throughout the survey as part of routine documentation. Only during later analysis of the photo archive did the team notice that what had initially appeared to be an interesting shot of two species sharing a single inflorescence was in fact something far more unusual: the abdomen tips of a female P. nausithous and a male P. teleius were visibly connected, indicating an attempt to copulate. The researchers do not know how long the pair remained in copula, because the connection was discovered only in the photographs.

Both species belong to the genus Phengaris, a group whose members are famous for a parasitic relationship with Myrmica ants. The caterpillars of these large blues are carried into ant nests, where they are fed or feed on ant brood, a life history so complex that it has made the genus a model system for evolutionary ecology. Despite this intensive research attention, and despite the fact that P. teleius and P. nausithous frequently occur side by side at the same sites and share the same food plant across Europe, no previous record of interspecific mating or hybridization between members of this genus has ever been documented. The new observation therefore fills a surprising gap in the knowledge of one of the best-studied butterfly lineages in Europe.

The sighting was made during a systematic survey conducted from 4 July to 5 August 2016 across 13 habitat patches near the villages of Hynčice, Ruprechtice and Vižňov, all of them containing flowering great burnet, the exclusive larval food plant of both species in Europe. The survey was carried out as part of conservation monitoring obligations, since both butterflies are listed for protection under the EU Habitats Directive. The researchers used the Mark-Release-Recapture method, analysed with the program MARK 8.1 and modified according to established procedures for Phengaris, to estimate the density of the butterflies. The colonies in the area form a single metapopulation, with individuals dispersing between patches.

The population estimates reveal that the mating attempt occurred in a community where both species were well represented. Using the Cormack-Jolly-Seber method, the team calculated that the metapopulation contained approximately 802 individuals of P. teleius and 1487 individuals of P. nausithous. These figures matter for interpreting the event: the researchers considered whether unusually high densities might have caused confusion between the species, but concluded that the density at the observation site was neither higher than average nor so low that finding a conspecific partner would have been difficult. The abundance of both species at the site was, in other words, entirely ordinary, which makes the unusual behaviour harder to explain.

Interspecific mating is a rare phenomenon in butterflies generally, and it is far more commonly documented in some families than in others. Records from the wild are relatively rich for swallowtails in the genera Ornithoptera, Papilio and Troides, for whites of the genus Colias, and for nymphalids including Danaus, Erebia, Heliconius and Limenitis. The family Lycaenidae, to which Phengaris belongs, has produced only a handful of such observations. Previous lycaenid examples include matings between species of Callophrys and between species of Lycaena, as well as reports from India of copulation between Castalius rosimon and Talicada nyseus and between Chilades parrhasius and Zizina otis. For the genus Phengaris itself, the authors of the new study found no comparable record anywhere in the literature.

The vast majority of documented interspecific copulations in butterflies occur between species of the same genus, as in Limenitis, Colias, Danaus, Heliconius and the classic examples in Papilio. Rarer still are matings between different genera, and exceptional are cases spanning entire families, such as an observed union between the burnet moth Zygaena filipendulae and the tiger moth relative Amata phegea, or even between a nymphalid butterfly, Melitaea athalia, and that same moth species. Interspecific mating is a prerequisite for hybridization, yet natural hybridization in Lepidoptera remains rare. One celebrated natural hybrid is Erebia serotina, produced by males of E. pronoe and females of E. epiphron. Most known hybrids, by contrast, have been created in captivity, particularly among Papilio species, where hybrids can remain fertile for several generations.

Why would two species that normally maintain separate identities attempt to mate at all? Reproductive barriers in butterflies typically operate on several levels, combining olfactory recognition with species-specific behaviour, pheromones and other signals. However, mating pheromones often share similar chemical compositions across related species, and their content varies not only between species but also within single species. The lock-and-key mechanism of butterfly genitalia, long assumed to prevent mechanical mating between different species, does not always function reliably. Molecular studies have repeatedly shown that reproductive barriers in Lepidoptera usually hold firm, but when they fail, the willingness to mate is often strongly asymmetric between the species involved, as demonstrated by observations of male Papilio canadensis showing strong preferences for females of Papilio glaucus.

The authors of the new report propose possible explanations for the Bohemian encounter. Because species of Phengaris differ in the number of androconia, the scent-producing scales on their wings, one possibility is that individuals became confused when pheromones of both species mixed in the air, particularly if many butterflies were concentrated at the spot. Alternatively, a suitable partner of the same species might have been harder to detect if the pheromones of the two species are chemically similar. Yet the researchers note that neither explanation seems particularly convincing, given that population densities at the site were unremarkable. The true cause of the behaviour therefore remains unresolved.

The consequences of such events can range from negligible to profound. Hybridization can threaten vulnerable species when a related species colonizes or is introduced into a landscape, but it can also trigger the emergence of new species, as documented in Heliconius butterflies. In the case of the Bohemian observation, the photographic evidence confirms that the butterflies achieved a physical connection, but it remains far from certain whether the copulation was successful or whether deeper isolation mechanisms prevented any possibility of hybrid offspring. What the images do establish is that even in a genus whose ecology has been scrutinized more intensively than almost any other butterfly group, the boundaries between species can still, on rare summer afternoons, briefly dissolve.

Subject of Research: First photographic record of an attempted interspecies mating between the butterflies Phengaris nausithous and Phengaris teleius in the Czech Republic

Article Title: An attempt of interspecies mating between Phengaris nausithous and P. teleius ( Lepidoptera , Lycaenidae )

Article References: Pilařová, A., Zachardová, S., Fric, Z. F., & Vrabec, V. (2024). An attempt of interspecies mating between Phengaris nausithous and P. teleius (Lepidoptera, Lycaenidae). Nota Lepidopterologica, 47, 105-111. https://doi.org/10.3897/nl.47.118947

Image Credits: AI Generated

DOI: 10.3897/nl.47.118947

Keywords: Phengaris, Phengaris nausithous, Phengaris teleius, Lycaenidae, interspecies mating, butterflies, hybridization, reproductive barriers, Czech Republic, Sanguisorba officinalis, lepidoptera, conservation

Hipparchia parisatis is not a species one could easily misidentify. On the upper side of the hindwing, both sexes carry a striking white marginal band, the feature that gives the species its vernacular name, and a narrower version of the same band appears along the forewing. Only one other member of the genus, Hipparchia alcyone, shares anything like this pattern, but it lacks the row of prominent eyespots on the underside of the hindwing that is diagnostic for parisatis. The combination of a white hindwing margin and a strongly spotted underside separates the Georgian specimens from every other satyrine known from the Caucasus, which is why the authors of the new report were able to confirm the identification from field photographs alone, without collecting or dissecting a single specimen.

The story of the discovery is as much about how modern biodiversity data are gathered as it is about the butterfly itself. During fieldwork in Vashlovani Nature Reserve, supported by a grant from the Rufford Foundation, researcher János Novák photographed a single individual in its rocky habitat in August 2023. He posted the image to the citizen science platform iNaturalist, where it was tentatively identified by another contributor. Only then did the team realise what they had: not a stray vagrant but a species apparently established in Georgia, with older records already sitting unnoticed in online databases. A targeted search of iNaturalist and the observation platform Observation.org turned up four additional photographic records made between 2018 and 2022 by observers including Noé Terorde, Siebe Roffel, and Guido Reijnen, several of them from the same reserve.

All five records come from an area of barely a few square kilometres in and around Vashlovani Nature Reserve, a strictly protected zone established in 1935 that forms the core of the wider Vashlovani Protected Areas. The reserve sits in the extreme southeast of Georgia, where the country meets Azerbaijan, and protects a mosaic of arid grassland, steppe, and open woodland at elevations between roughly 300 and 800 metres above sea level. The Georgian records fall between 370 and 800 metres, consistent with the altitudinal preferences the species shows elsewhere in its range. Four of the five observations were made in September, and one in August, a seasonal pattern that matches the species’ phenology in neighbouring Iran, where adults fly in a single generation with a summer aestivation pause.

That phenology is one of the more curious aspects of the species’ biology. Across West and Central Asia, Hipparchia parisatis emerges in May or June, but the adults then disappear for the hottest weeks of summer, a dormancy strategy known as aestivation, before reappearing in August and September to complete their flight period. The Georgian observations, clustered in late summer, fit this rhythm precisely and suggest the species is not merely present but following the same life cycle in the Caucasus that it exhibits in its Asian strongholds. The authors note that the butterfly’s food plants, grasses typical of arid habitats, are abundant in the reserve, providing the resources needed for a resident population.

The zoogeographic implications are considerable. Vashlovani is already celebrated as one of the richest butterfly localities in Georgia, and the new report adds parisatis to a growing list of species there that are otherwise associated with the subtropical and tropical faunas of the East Mediterranean and West Asia. The authors describe the reserve as a refugium where relict populations of thermophilic species survive at the northern edge of their ranges, and the presence of parisatis strengthens the case that Vashlovani preserves a fragment of a once more widespread arid-subtropical biotope. The finding also underscores how incomplete the faunistic knowledge of the South Caucasus remains, with a conspicuous species going unrecorded in a well-studied country until citizen science platforms made the evidence visible.

The report is careful about what the photographs can and cannot establish. Because no specimens were collected, the authors cannot rule out entirely that the Georgian population belongs to a different subspecies from those found in Iran and Central Asia, and they note that the taxonomic status of parisatis populations in the Caucasus region, including those in Armenia and Azerbaijan, remains unresolved. What the images do establish, beyond reasonable doubt, is the presence of the species in Georgia, and the authors argue that the reserve’s protected status means the population is likely to persist. They recommend that future surveys combine targeted fieldwork with continued monitoring of citizen science platforms, an approach they say is proving essential for detecting new country records in a region where professional entomological coverage is thin.

For the authors, the discovery is a demonstration of the model they advocate: professional biologists and citizen observers working through shared online infrastructure. The initial photograph was taken by a professional researcher, but the identification was confirmed through iNaturalist’s community, and the additional records were found by searching the same platforms rather than by new fieldwork. The authors suggest that this workflow, in which observations are documented photographically, identified collaboratively, and then verified and published by specialists, is the most efficient way to advance faunistic knowledge in the South Caucasus, and they hope the Georgian record of Hipparchia parisatis will be the first of many such additions to the country’s butterfly fauna.

Subject of Research: On the presence of Hipparchia parisatis (Kollar, 1849) in the Republic of Georgia (Lepidoptera, Nymphalidae, Satyrinae)

Article Title: On the presence of Hipparchia parisatis (Kollar, 1849) in the Republic of Georgia ( Lepidoptera , Nymphalidae , Satyrinae )

Article References: Kolev, Z., Novák, J., & Seropian, A. (2024). On the presence of Hipparchia parisatis (Kollar, 1849) in the Republic of Georgia (Lepidoptera, Nymphalidae, Satyrinae). Caucasiana, 3, 263-271. https://doi.org/10.3897/caucasiana.3.140259

Image Credits: AI Generated

DOI: 10.3897/caucasiana.3.140259

Keywords: butterflies, citizen science, Georgia, Vashlovani, biodiversity, biogeography, presence, Hipparchia, parisatis, Kollar, Republic, Lepidoptera

A new study published in Nota Lepidopterologica by entomologist Enrique García-Barros of the Universidad Autónoma de Madrid tackles this mismatch head-on, and the results overturn a long-standing assumption. The best single predictors of dry body weight in adult male moths and butterflies turn out to be thorax length, total body length and head width — none of which is among the popular descriptors of size in this insect order. Forewing length, the metric most often published, is well correlated with body weight but is not the best linear predictor. The finding matters because researchers who want to estimate body mass from published descriptions have been working with suboptimal tools.

The scale of the new dataset is what gives the study its statistical muscle. García-Barros weighed and photographed 5,341 adult male specimens representing 2,645 species, a seven-fold increase in species sampling compared with his own earlier attempt published in 2015. Each insect was dried to constant weight at 70 degrees Celsius for 24 to 72 hours to obtain dry body weight in milligrams, then re-moistened, spread and digitized together with a metric scale. Thirteen linear measurements were taken from the digitized images using ImageJ, covering the head, thorax, abdomen, forewing, hindwing and two alternative estimates of wingspan. The largest part of the sample was deposited in the collections of the Madrid Natural History Museum, with vouchers of the Tortricidae held in Valencia.

The range of body masses covered is extraordinary. The lightest species, some Nepticulidae and Heliozelidae, weighed between 0.04 and 0.05 milligrams, and the study suggests that the tiniest known leaf-mining moths with forewings of just 1.2 to 1.3 millimeters would come in at roughly 0.017 to 0.020 milligrams. At the other extreme, some tropical Cossidae such as Xyleutes persona exceeded three grams — 3,000 milligrams — of dry weight. That means male dry body weight in known Lepidoptera spans five orders of magnitude, while forewing length spans only three. The allometric exponents recovered from the bivariate regressions fell between 1.92 and 2.96, exactly what length-to-volume scaling predicts, and every linear measurement correlated with dry body weight at R above 0.97 on logarithmically transformed values.

Eight multivariate regression models were fitted to the log-transformed species means, reflecting different combinations of measurements a researcher might realistically have available. The most complex models, based on five to seven body measurements, predicted dry body weight most accurately, with adjusted R-squared values of 0.980 and the lowest cross-validation errors. Models combining wingspan with body length performed reasonably but suboptimally, and simple models based on forewing length alone or wingspan alone were acceptable but clearly inferior. The author recommends two models in particular: one combining head width, thorax length and width, abdomen length and width, forewing length and forewing width, and another built on forewing length as the primary predictor with the residuals of the remaining variables added as supplementary terms — the latter having the advantage of near-complete linear independence among its predictors.

One of the study’s most technically interesting results concerns collinearity. Whenever more than one variable entered a regression model, the variance inflation factor climbed well above the threshold of 4 to 10 generally identified as excessive — with a single exception, the residual-based model 8, which kept minimum VIF at 1.00. High collinearity carries the risk of overprediction: species with unusual body plans not covered by the dataset could receive wildly inaccurate weight estimates. Variance partitioning of the regression residuals showed that most of the unexplained variation was attributable to morphology rather than phylogeny — the pure phylogenetic effect was virtually negligible, roughly a ten-fold smaller contribution than the shared morphology-phylogeny effect. This suggests the best models may be stable and reliable for prediction, provided the species being predicted falls within the morphological range the dataset covers.

The study also tested taxon-by-taxon approaches, fitting separate regressions for families and superfamilies, and ANCOVA-based methods that assign family-level coefficients to intercepts and slopes. The results were sobering. In the butterflies, an instructive example, three families — Lycaenidae, Riodinidae and Nymphalidae — proved homogeneous for slope and intercept, but all six families differed significantly from each other in intercept, slope, or both. That means a single family-level equation risks systematically overestimating the weight of some species and underestimating others. Because the phylogenetic structure in the residuals was so weak, García-Barros argues that clustering species on morphological grounds rather than taxonomic ones would be far preferable — a conclusion with implications for anyone building order-wide reference equations.

Two practical extensions round out the work. A limited sample of females from 362 species revealed a nearly isometric relationship between male and female dry body weight, described by the equation female DBw = 0.1574 + 0.9770(male DBw) with an adjusted R-squared of 0.953 — a clear-cut result, though the author cautions that evidence from other studies shows this relationship is not uniform across all moth and butterfly families. A subset of field-caught Spanish males, frozen shortly after capture and weighed within 24 hours, established that fresh body weight scales to dry weight with a slope close to isometry and that dry weight accounts for about 38 percent of live weight — a water content squarely within the typical range for adult insects. These conversions let field researchers translate predicted dry masses into approximate live weights, at some cost in accuracy.

The optimistic and pessimistic readings of the study are both on offer, and García-Barros presents them honestly. The pessimistic view notes that the sample represents barely more than 2 percent of described Lepidoptera species, most species contributed only one to three individuals, and female data are scarce. Yet the optimistic view is compelling: the seven-fold increase in species sampling raised the fit of the best model by merely 1 percent, suggesting that the broad morphological variation across the order is redundant enough for multivariate prediction to work well. The full dataset has been made openly available through the Dryad Data Repository, and the author argues that in the face of global change and documented insect biomass declines, publishing more standardized body size data on moths and butterflies should be actively encouraged. For a hyper-diverse group whose masses span five orders of magnitude, a ruler and the right equation may now be all that stands between a museum specimen and a usable number.

Subject of Research: A multivariate regression study of 2,645 Lepidoptera species refines estimates of adult dry body weight from thirteen linear measurements, showing that combinations of body and wing metrics outperform traditional size descriptors.

Article Title: Refining estimates of dry body weight from linear measurements in adult moths and butterflies ( Lepidoptera )

Article References: García-Barros, E. (2025). Refining estimates of dry body weight from linear measurements in adult moths and butterflies (Lepidoptera). Nota Lepidopterologica, 48, 165-184. https://doi.org/10.3897/nl.48.144747

Image Credits: AI Generated

DOI: 10.3897/nl.48.144747

Keywords: Lepidoptera, body size, allometry, dry body weight, insect ecology, moths, butterflies, morphometrics, Refining, estimates, body, weight