Zoological Journal of the Linnean Society

The diversity of body shapes is one of the most prominent features of phenotypic variation in mammals. Yet, mammalian body shapes are poorly quantified and the underlying components contributing to its diversity as well as its relationship to other components of the skeleton are rarely tested. Here, we use lagomorphs (hares, rabbits and pikas) as a model system to (1) investigate which components of the skeleton contributed the most to body shape diversity, (2) examine the relationships between body shape and relative limb lengths, and (3) test how body size, ecotype, burrowing behavior, and locomotor mode influenced variation in lagomorph body shape and appendicular morphology. We quantified the body shape and functional proxies of the appendicular skeleton in 40 lagomorph species from osteological specimens held at museum collections. Using phylogenetic comparative methods, we found the relative length of the ribs and elongation or shortening of the thoracic and lumbar regions contributed the most to body shape evolution across lagomorphs. Second, we found that only leporids (hares and rabbits) exhibited a significant relationship between limb length and body shape, where more elongate species exhibit relatively shorter forelimbs and hindlimbs. Lastly, we found that models incorporating body size were the best predictors of lagomorph body shape and the majority of the appendicular traits, whereas models incorporating burrowing behavior and locomotor mode were largely poor fits. Broadly, these results indicate that larger lagomorphs tend to exhibit more robust body shapes with longer, more gracile forelimbs, whereas smaller lagomorphs tend to exhibit more elongate body shapes with shorter, more robust forelimbs. Overall, this work contributes to the growing understanding of mammalian body shape evolution and demonstrates the importance of not omitting body size in ecomorphological analyses.

The genus Trichiurus is the most economically valuable fish in the family Trichiuridae, currently recognized to include 10 valid species. However, historically numerous morphologically similar congeners have been erroneously assigned as synonyms or subspecies of T. lepturus. In this study, we examined 16 hairtail specimens collected from the southern waters of Java Island, Indonesia. Integrated morphological and mitochondrial phylogenetic analyses (COX1 and 16S rRNA), compared against global Trichiurus sequences, revealed that these specimens form an independent lineage that diverged early from other congeners. Consequently, we describe this lineage as a previously undescribed cryptic species. Diagnostic characters include: first anal-fin spine below 36th-37th dorsal-fin rays; anus below 35th-36th dorsal-fin rays; anteriormost tip of supraoccipital well posterior to posterior distal margin of eye; anterior margin of the pectoral-fin spine non-serrated; fangs on both jaws with barb-like processes; upper jaw long, mean 16.6% (15.5-17.6%) of preanal length; snout short, 12.0% (10.9-13.1%) of preanal length; eye small, diameter 5.3% (4.3-5.7%) of preanal length; and absence of hyperostosis on dorsal cranium. We herein propose the name Trichiurus javaensis sp. nov., and provide a formal morphological description and diagnostic characterization of this species.

Silk glands have been found in two groups of amphipods: the Corophiida and the Ampeliscidae. The silk glands in Ampeliscidae, however, have yet to be examined in detail. Here we report, for the first time, the morphology and distribution of pereopodal glands in the Ampeliscidae, in non-thread producing Synopiidae, and in the Paragammaropsidae. In the Ampeliscidae we found two gland types distributed throughout all pereopods which have the ability to create threads. Pereopods three and four have additional silk extrusion morphology at the tip of the dactylus in which silk is transformed into semi-cylindrical threads used for building domiciles. Synopiid outgroup species have one of the gland types but lack silk extrusion morphology. Using ancestral state reconstruction analysis, we find that glands in the Synopiidae are likely ancestral and hypothesize that silk glands in Ampeliscidae are derived from these ancestral glands. Silk-spinning pereopods in the Paragammaropsidae had similarities with both Corophiida and Ampeliscidae but had distinctions. Ampeliscidae silk-spinning systems bear surprising resemblance to the Corophiida which presents one to reconsider the taxonomic placement of Ampeliscidae and the origins of silk-spinning in amphipods. This is the first comprehensive study on the glandular systems of Ampeliscidae, Synopiidae, and Paragammaropsidae using advanced microscopy, providing pertinent morphological data to the study of arthropod silk gland evolution and complex traits.

The Dinaric karst of Croatia encompasses a network of over 10,000 caves and represents one of the worlds most important subterranean biodiversity hotspots. It is inhabited by remarkably diverse and often endemic species, including planarian flatworms, which are among the rarest macroinvertebrates encountered in cave habitats. Although the presence of cave planarians has long been known, no integrative research on this group has been conducted to date, and the evolutionary relationships between these animals and their surface water counterparts are currently unresolved. To address these gaps, we combined field sampling, phylogenetic analysis based on COI and 18S genes, and phenotypic characterization. Our results show that cave planariids in Croatia belong to at least three genera and are more widespread and diverse across both Croatia, and the broader Dinaric karst, than previously assumed. We increased the number of cave records in the Dinaric karst from 26 to 37 and documented cf. Atrioplanaria and Phagocata in Croatian caves for the first time. Phylogenetic reconstructions suggest numerous independent cave colonization events, including multiple instances within the genera Crenobia and cf. Atrioplanaria. Variation in pigmentation and eye reduction, both within and between populations, further reveal heterogeneous evolutionary trajectories of cave-associated phenotypes. The biogeographical patterns and high genetic diversity we report here point to a complex evolutionary history of planariids in the Dinarides. Our newly generated molecular phylogenies and systematic documentation of trait variability establish Planariidae as a valuable model for studying mechanisms underlying convergent evolution of pigment loss and eye reduction in cave environments.

The ichneumonid subfamily Eucerotinae has been thought to be almost absent from the tropics, with the only known Afrotropical species found in Madagascar. We report the subfamily to be present in the mainland Afrotropics, and describe a new species, Euceros species 1 from Uganda and Cameroon (name not yet shown in preprint). The subfamily had likely not been observed in the mainland Afrotropics before due to low abundances and insufficient sampling. More Eucerotinae likely remain to be discovered in tropical Africa and Asia, although tropical America may genuinely have few eucerotine species. Much more extensive sampling will be needed before it is possible to make confident estimates of how eucerotine diversity is distributed globally.

For over four decades, the bivalve Anomalocardia flexuosa has been recorded in Hong Kong coastal waters. However, the known native distribution of this heavily exploited commercial species is restricted to the Atlantic coast of South America, raising questions about the biogeographical validity of the Hong Kong populations. By employing an integrative taxonomic approach combining morphological re-evaluations and molecular phylogenetic analysis of the COI gene, we confirm that the species in Shui Hau, Hong Kong, China, has been historically misidentified. The population belongs to Cryptonema producta (syn. Anomalocardia producta).

Animal weapons are ecologically important traits that mediate contests over limiting resources and can strongly influence survival and reproduction. Weapon traits often exhibit substantial intraspecific morphological diversity, raising questions about the ecological drivers of this variation. Acrorhagi are weapons produced by sea anemones that are used in intraspecific territorial encounters. Although acrorhagial morphology varies widely within species, patterns of intraspecific variation remain poorly characterized, and the extent to which such variation reflects differences in local intraspecific competition is unclear. Here, we conduct morphometric analyses to characterize within-population variation and allometry in acrorhagial traits of the solitary anemone Anthopleura sola. We show that these traits covary with habitats differing in conspecific density. The number of acrorhagi scaled positively with body size, and individuals occupying a high-density habitat tended to possess more acrorhagi than did similar sized individuals from a low-density habitat. In addition, anemones from high-density habitats exhibited longer acrorhagial cnidae, a pattern that was not explained by differences in body size or acrorhagial density. Together, these results suggest that competitive context influences weapon-related traits at multiple levels of biological organization, potentially via phenotypic plasticity or selective processes. More broadly, our findings highlight how fine-scale ecological variation may contribute to the maintenance of trait diversity within and across species.

This investigation aimed at compiling all phylogenetic lineages within and around genus Cyanoboletus. The evolutionary inference obtained from the nuclear ribosomal genes internal transcribed spacer region (ITS) suggests that part of the species currently classified in Cyanoboletus belong in lineages separate from the genus, thus suggesting a narrower boundary that includes only the species that develop a strong staining reaction to touch and to air exposure of the context. The separate lineages are the monotypic Cupreoboletus genus and a few species that do not develop such reaction, which are part of a clade together with genera Cacaoporus and Acyanoboletus, thus broadening the concept of Cacaoporus to encompass all of them. The emerging 3C perspective of Cupreoboletus, Cacaoporus and Cyanoboletus offers a remarkably consistent morphological diagnosis, overcoming the problems of a too broad concept for Cyanoboletus. This work reveals that Boletus neotropicus, B. novae-zelandiae and B. sensibilis belong respectively in Cyanoboletus, Cacaoporus and Lanmaoa, and by studying multigene alignment concatenates it identifies lineages that probably represent undescribed species: at least four in Cacaoporus and at least five in Cyanoboletus. Diagnostic tables and dichotomic keys are presented by geographic region. The present work also includes a study of the phylogenetic position of Neoboletus flavosanguineus, a species once classified in Cyanoboletus. The complexity of assigning species epithets in some lineages is addressed, namely for the boundaries between Cacaoporus instabilis and Ca. fagaceophilus as well as the diversity under the names Cyanoboletus sinopulverulentus and Cy. pulverulentus. The overall picture of evolutionary lineages sets a framework for the choice of reference data that can provide, in future phylogenetic studies that involve the 3C, a balanced and efficient coverage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/724631v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@7f618corg.highwire.dtl.DTLVardef@dd6a14org.highwire.dtl.DTLVardef@5f7399org.highwire.dtl.DTLVardef@9e7443_HPS_FORMAT_FIGEXP M_FIG C_FIG

AO_SCPLOWBSTRACTC_SCPLOWEnvironmental heterogeneity across freshwater systems often promotes phenotypic variation, yet disentangling environmentally induced variation from heritable differentiation remains a central goal in evolutionary ecology. We investigated the geographic distribution and morphological differentiation, and heritability of shell traits among populations of the freshwater lymnaeid snail Pectinidens diaphanus in Patagonia. Extensive field surveys across 196 freshwater sites revealed that the species occupies a broad range of lentic and lotic habitats and constitutes the only lymnaeid inhabiting southern Patagonia. While reproductive anatomical structures were conserved across populations, shell shape differed markedly among populations from contrasting habitat types, with population identity explaining nearly 50% of total shape variation. Populations from hydrologically unstable habitats (ponds and streams) exhibited more elongated shells and relatively smaller apertures, a pattern consistent with functional responses to hydroperiod variability and desiccation risk. To assess the heritability of this differentiation, we conducted a common-garden experiment across two generations. Shell shape differences between permanent- (lagoon) and temporary- (pond) habitat-derived populations persisted into the G2 generation reared under standardized laboratory conditions, indicating that the observed variation is not solely a response to local environmental conditions but includes a heritable component. Together, our findings demonstrate that P. diaphanus constitutes the sole lymnaeid across southern Patagonia, occupying a broader range than previously documented, and that populations show heritable shell differentiation potentially associated with contrasting freshwater habitats. By integrating large-scale biogeographic surveys with morphometric and experimental approaches, this study provides new insight into how habitat variation may contribute to ecological and evolutionary differentiation in freshwater gastropods.

AimHome range size is a fundamental aspect of animal spatial ecology, and understanding the factors that shape it is important for conservation purposes. Several hypotheses, based on energy needs or competition, assume that home range size negatively correlates with population density. However, this pattern has been little investigated on a global scale, and it remains unclear whether it would stand at both intra- and interspecific levels. To fill this gap, we conducted a global exploration of this relationship at the level of an animal family. Location: Global. Time period: Contemporary. Major taxa studied: Wild Felidae. MethodsIndividual home range size records (n = 1022) and population density estimates (n = 1061) were retrieved from the literature for 23 felid species across the world. We first investigated the interspecific relationship by modelling the median home range size of a species as a function of its median population density. To study the intraspecific relationship, we spatially merged data points based on their spatial or temporal proximity. We then applied a mixed-effects linear model using species as a random factor. ResultsWe found that home range size was negatively associated with population density, at both interspecific (-1.323 {+/-} 0.180, p < 0.001) and intraspecific levels (-0.569 {+/-} 0.201 to - 0.537 {+/-} 0.201 depending on the merging approach, p < 0.01). Landscape features were also predictors of home range size, without confounding the effect of population density. Main conclusionsSeveral processes likely govern the relationship between home range size and population density: differences in body mass between species may drive the interspecific relationship, whereas the intraspecific pattern is probably explained by conspecific competition. Although more research is needed to quantify their relative contribution, our study highlights a worldwide ecological pattern that exists at multiple biological levels in the wild.

Rhodnius prolixus is a primary insect vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected parasitosis endemic in Latin American countries. It has been estimated that Chagas disease affects 7-8 million people worldwide and is responsible for approximately 1000 deaths per year. Genetic and molecular studies in this species remain challenging due to its life cycle and feeding habits, thus hindering the development of new strategies to control their populations and reduce the diffusion of Chagas disease. Recently, two stable cell lines - RPE/LULS53 and RPE/LULS57 - were derived from Rhodnius embryos, which represent promising new tools to investigate the genetics of this insect vector. Here, we describe their gene expression landscapes through transcriptomic approaches. We show that 8,968 expressed genes are shared between the two cell lines, whereas 391 and 1,088 genes are uniquely expressed in RPE/LULS53 and RPE/LULS57, respectively. Although key components of primary developmental, immune and redox signaling pathways are expressed in both cell lines, some genes such as Frizzled-10-a-like and catalase show marked differences in expression. Our results strongly suggest that RPE/LULS53 and RPE/LULS57 likely represent two different cell phenotypes. Consistent with this, gene ontology analysis reveals that RPE/LULS53 is enriched for animal organ morphogenesis and stress response, while RPE/LULS57 for DNA-directed RNA polymerase activity, among others. Despite these differences, both cell lines express comparable levels of transcripts from resident transposable elements, including the highly abundant Mariner and LINE/I elements, as well as horizontally transferred transposons. Our findings shed light on the nature of the RPE/LULS53 and RPE/LULS57 embryo-derived cell lines and provide valuable transcriptomic resources for future genetic and functional studies in Rhodnius and other triatomine insect vectors. Author summaryRhodnius prolixus is a blood-feeding insect and a major vector of Chagas disease, a parasitosis endemic in Latin America and affecting millions of people worldwide. In the absence of effective drugs and vaccines, the control of the insect population represents a promising strategy to reduce the diffusion of the disease. Yet, genetic and functional studies in Rhodnius are extremely challenging due to its feeding habit and life cycle. To overcome these limitations, researchers have previously developed two stable cell lines derived from Rhodnius embryos. In this study, we provide the first characterization of the genes expressed in these cell lines. We found that, while the two cell lines share many expressed genes, each of them also has distinct gene expression patterns pointing to two different cell types with specialized functions. These differences likely affect the way they respond to stress and regulate biological processes. Our findings provide an important resource for researchers studying Rhodnius prolixus and other insect vectors, helping advance our understanding of the genetic and molecular mechanisms that control the insect development and mediate the interactions between insect vectors and the parasites they transmit

Fasciolosis caused by Fasciola hepatica is an economically important disease in sheep and cattle. Knowledge of the population genetic structure of F. hepatica is important for understanding gene flow and informing disease control. In the present study, we designed, developed, and validated a multilocus sequence typing (MLST) scheme based on six markers. These markers were selected by aligning newly sequenced whole-genome sequence (WGS) data with available reference genomes and selecting variable regions with five or more single-nucleotide polymorphisms SNPs from different scaffolds of the F. hepatica reference genome Fasciola 10x pilon (GCA_900302435.1). Twenty markers were initially identified, of which 12 were multiplexed for deep amplicon sequencing after validation on worm and faecal eggs DNA; six markers were ultimately retained for downstream population genetics analysis. These markers were used to investigate population genetic structure in 15 cattle- and 27 sheep-derived F. hepatica populations in UK. A total of 53 unique alleles from six MLST markers were identified from 30 faecal (cattle = 13, sheep = 17) and 12 adult worm (cattle = 2, sheep = 10) populations. Shared alleles were observed in sheep- and cattle-derived populations. The highest allelic variation was observed in the Scottish Borders, Southern Scotland, and South-West England, and the lowest in North-West England. Minimal genetic differentiation was observed between cattle- and sheep-derived populations, with most genetic structuring within rather than between populations. Five markers showed high allelic polymorphism, whereas one marker showed low levels of allelic polymorphism, highlighting the importance of multilocus approaches. Overall, this six MLST-marker panel provides a tool for population genetic studies, revealing high gene flow and clonal expansion of F. hepatica across hosts and regions in the UK.

Sex ratio distorters (SRDs) are heritable elements that bias offspring sex ratios to enhance their transmission. In the terrestrial isopod Armadillidium vulgare, feminization of genetic males can occur through vertical transmission of the sex ratio distorter known as the f-element, as well as through infection by Wolbachia, a maternally inherited bacterial endosymbiont that can alter host reproduction. Previous studies have focused on the distribution of SRDs and their associations with mitochondrial haplotypes in native European populations, but these patterns are poorly understood in the United States. In this study, we sampled A. vulgare in 12 states, screening individuals for Wolbachia infection, the presence of the f-element, and mitochondrial haplotypes. We found that Wolbachia shows a heterogeneous distribution across populations and haplotypes, in contrast with stronger associations in Europe. The f-element occurred in lower overall frequencies but showed a strong association with mitochondrial haplotype VI. These results indicate that patterns associated with SRD differ from those observed in Europe and suggest that multiple introductions and population mixing have shaped these distributions.

The nuclear, internal transcribed spacer (ITS) and mitochondrial cox1 markers are widely used to differentiate Schistosoma haematobium from its livestock counterparts, S. bovis and S. curassoni. Schistosoma isolated from humans that have ITS and cox1 markers from livestock schistosomes are typically inferred as zoonotic infections, those with mixed species, heterozygous ITS are classified as F1s or recent hybrids, while those with discordant ITS and cox1 markers are considered to reflect older hybridization events. We evaluated the reliability of this classification scheme by genotyping ITS and cox1 from 132 parasites isolated from human urine, and from 37 adult schistosomes collected from cattle at 14 Nigerian locations. We also genome sequenced each sample to empirically determine livestock schistosome ancestry. ITS/cox1 genotyping suggested extensive recent hybridization and zoonotic infection. Among parasites from humans, 10.1% carried both S. curassoni and S. haematobium ITS, consistent with F1 or early generation hybrids, 21% had livestock schistosome markers at both cox1 and ITS suggesting zoonotic infection, while 13.7% carried S. bovis cox1 alongside mixed S. curassoni and S. haematobium ITS, suggesting more complex ancestry. Genome sequencing revealed a very different picture. All parasites from humans formed a tight cluster regardless of ITS or cox1 genotype, while all worms from cattle were well differentiated. We found no schistosomes containing 50% livestock parasite ancestry consistent with F1s. Instead, we observed regionally varying levels of S. bovis introgression, with modest levels in southern Nigeria (mean = 4.9%) and low levels in northern Nigeria (mean = 0.06%). These results demonstrate that: (i) two-locus genotyping is uninformative for detecting zoonotic infection or recent hybridization between S. haematobium and livestock schistosomes and (ii) previous data generated using this approach requires reinterpretation. These findings reveal the limitations of widely-used approaches for documenting zoonotic infection and hybridization between S. haematobium and livestock schistosome species.

Cities are characterized by elevated temperatures, increased pollution, and high-density human populations which often are accompanied by changes in available resources, like food. These shifts have the potential to drive phenotypic divergence in urban wildlife. Functional morphological traits, like body size, can mediate interactions between wildlife and habitat and are closely tied to life history and fitness. While examples of functional morphological variation associated with urbanization are increasing, variation in such traits as a response to urbanization remains unexplored for most taxa. Here, we investigated morphological divergence between urban and rural populations of house mice (Mus musculus domesticus). House mice are globally distributed in diverse habitats and are a model system with a wealth of phenotypic data, making them useful for the study of the impacts of urbanization on morphology. Using a paired replicate design, we sampled urban and rural populations in three distinct metropolitan regions in the eastern United States. We found that body size was smaller in urban populations. Using 3D geometric morphometrics, we also analyzed variation in cranial shape across habitats. Differences in cranial shape were largely allometric, that is, driven by differences in body size. However, we also uncovered evidence of cranial shape variation between habitats not explained by size. In contrast, we did not find evidence for habitat-driven differences in cranial capacity independent of size. Overall, our results suggest a key role for body size in mediating morphological responses to urbanization and highlight the potential of house mice as a globally-distributed model for urbanization.

The order Atheriniformes (silversides, rainbowfishes, and blue-eyes) is a globally distributed group of fishes with frequent evolutionary transitions between marine and freshwater ecosystems. However, understanding the tempo and mode of these transitions has been hampered by poor phylogenetic resolution and limited taxonomic sampling, particularly within the suborder Atherinoidei. We generated a phylogenomic dataset of 1,100 exon loci for 150 species to resolve interfamilial relationships and reconstruct the groups biogeographic history. We were also able to incorporate a large number of existing GenBank sequences, producing a phylogeny with 265 species sampled for at least some genetic data (67% of known species diversity). While the New World suborder Atherinopsidae is well-resolved, we found that the family Atherinidae is polyphyletic across all analyses. We propose a revised classification that restricts Atherinidae to the genus Atherina and recognizes Atherinomoridae and Craterocephalidae as separate families. Our biogeographic inferences using explicit geographic areas suggests more frequent marine-to-freshwater transitions than previously inferred with simplified binary (marine vs. freshwater) coding, and uncover habitat transitions where marine ancestors may have gone extinct. These results highlight how explicit geographic modeling can uncover marine ancestry erased by extinction, providing a robust phylogenetic framework for future evolutionary studies of Atheriniformes.

Intraspecific morphological variability presents a complex challenge for biological systematics and biomonitoring, particularly for organisms with high phenotypic plasticity, such as zooplankton. Morphological differences between individuals of the water flea species Bosmina longirostris (Crustacea: Cladocera) are difficult to distinguish visually, parthenogenetic females look morphologically uniform within the species; nevertheless, they demonstrate differences attributable to their geographic origin and developmental stage. A reference dataset of microscopic images was created for the study, including populations from two geographically separated regions (seven ones from European Russia and seven ones from Sakhalin Island in the Pacific Ocean (Far East of Russia) and two age groups, demonstrating the ability of a neural network classify to successfully the intraspecific morphological variation. This study demonstrates that deep learning methods are prospective for the detection and understanding of fine morphological intraspecific differences in the cladocerans.

Gastropods are a highly diverse and often overlooked taxonomic group of significant ecological and economic importance. Some terrestrial gastropods are critical pests of commercial agriculture and home gardens worldwide. Malacopathogenic nematodes offer an effective biological control method of managing pest slugs and snails as a natural enemy. Pellioditis (syn. Phasmarhabditis) hermaphrodita and Pellioditis (syn. Phasmarhabditis) californica are two species of biocontrol nematodes that have been commercialized, sold as Nemaslug(R) and Nemaslug(R) 2.0 respectively on three continents. Although there is interest in bringing Nemaslug(R) products to the US, they are currently not permitted due to limited knowledge on their North American distribution and effects on non-target and native species. In this study, we investigated the impact of P. hermaphrodita and P. californica on Ariolimax columbianus across two slug-host life stages, in laboratory infectivity assays. The objectives were to 1. determine whether P. hermaphrodita and P. californica nematodes impact survival of A. columbianus, and 2. evaluate whether there are differential effects on survival in juvenile and adult life stages of A. columbianus, in laboratory infectivity trials. We found that P. hermaphrodita caused significant mortality in A. columbianus with 100% mortality observed in both juvenile and adult slug hosts. The P. californica treatment had significant effects on the juvenile A. columbianus group only, with 80% mortality. By contrast, only 16% of unexposed control juveniles and 4% of control adult slugs died during the experiment. These results indicate that P. hermaphrodita and P. californica are lethal to the native, non-target Pacific banana slug (A. columbianus) under laboratory conditions, with mortality differing between juvenile and adult host life stages. Given the ecological importance of A. columbianus, these findings raise concerns for potential non-target effects of P. californica and P. hermaphrodita on terrestrial gastropod communities and emphasize the need for testing biocontrol agents against multiple life stages.

Romania represents one of the few European Union member states in which all four Old World vulture species historically maintained breeding populations: the Griffon Vulture (Gyps fulvus), Cinereous Vulture (Aegypius monachus), Egyptian Vulture (Neophron percnopterus) and Bearded Vulture (Gypaetus barbatus). Until the 2026 reintroduction efforts initiated by Foundation Conservation Carpathia and Rewilding Romania, Romania remained the last EU country whose former vulture guild had not been targeted for active recovery. Despite this exceptional significance in a European conservation context, no comprehensive synthesis of the historical and contemporary distribution of these species in Romania has been undertaken. We conducted a comprehensive review to gather all available vulture occurrence data and present a fully georeferenced database of 1,170 occurrence records spanning 1818-2025. We systematically searched museum collections, historical ornithological literature, modern field surveys and citizen-science platforms. The database documents substantial breeding populations across the Carpathian arc and Dobrogea until the early twentieth century, followed by near-total breeding collapse between the 1920s and 1960s driven by persecution, secondary poisoning and agrarian transformation. In total, 149 confirmed or probable breeding records have been documented for the four species combined, with the most recent confirmed breeding records dating to 1929 (Gyps fulvus), 1929 (Gypaetus barbatus), 1942 (Aegypius monachus) and 1966 (Neophron percnopterus). Non-breeding occurrences increase markedly after 2010, consistent with dispersal from expanding Balkan source populations. The F[a]g[a]ra {square} and Retezat Mountains emerge as the historically most important breeding strongholds for all four species. Our dataset constitutes the most detailed historical baseline currently available for vulture conservation in Romania and is intended to identify key historical sites with high potential for future reintroduction and recovery. Our results show that Romania historically supported the full guild of European obligate scavengers, and that its collapse occurred within barely four decades (1920s-1960s). The dataset highlights the value of reconstructing historical baselines in regions where functional extinction preceded the onset of modern monitoring, and provides an empirical foundation for reassembling a keystone scavenger guild at a continental scale.

Molecular data have revolutionised taxonomic and ecological research on the hyperdiverse communities of aquatic benthic microinvertebrates known as meiofauna. However, reference sequence databases remain highly incomplete, with variable barcode genes or fragments studied from taxon to taxon. Furthermore, there is a typical tradeoff between universality of primers and phylogenetic resolution, with rRNA markers being robustly recoverable but failing to resolve species-level divergences, and mitochondrial markers showing the reverse trend. Here, we introduce Oxford Nanopore rRNA and COI amplicon sequencing (OrCa-seq), a rapid, low-cost protocol for parallel long-range PCR amplification and multiplexed sequencing of four amplicons, spanning the nearly-complete rRNA cistron ([~]7-8 kb) and the widely studied Folmer region of COI (represented as overlapping 313 and 658 bp amplicons). This protocol, with its associated bioinformatic workflow, was designed for conducting biodiversity inventories of meiofauna and can be easily carried out in field research and educational contexts, with data available from 96-well plates of specimens within a day of lysis. To validate the method, we processed six plates of student-isolated freshwater and limno-terrestrial meiofauna, characterising the recovery of target genes and taxa with both automated and human-curated BLAST database comparisons. These data demonstrate the universal applicability of OrCa-seq across effectively all meiofauna, including the very smallest species. Nonetheless, recovery efficiency for each amplicon shows variation by taxon, with the full-length Folmer COI amplicon standing out as the most challenging. We present exemplar phylogenetic trees integrating reference sequences, demonstrating the utility of these data in confirming morphological determinations and in identifying anonymous specimens in a reverse taxonomy context. While developed in a specific educational context for use on meiofauna, the OrCa-seq approach should be readily scalable to larger research datasets, adaptable to many specimen types, and to any combination of taxon-or target-specific primers. As such, it represents a compelling multi-locus extension to the ever-growing repertoire of nanopore DNA barcoding protocols.