Natural oil seeps remain poorly explored in terms of their biodiversity. Most studies on these habitats have focused on prokaryotic communities involved in hydrocarbon metabolism, whereas the microscopic invertebrates inhabiting these environments have received little attention, and existing observations rarely include species-level identifications. Here, we collected samples from a natural oil seep and characterized the meiofaunal community using DNA barcoding to support species identification. We detected representatives of four animal phyla known for their tolerance of extreme conditions: Annelida, Nematoda, Rotifera and Tardigrada. Across these groups, we identified at least seven putative species, including several that may represent previously unrecognized taxa. The meiofaunal assemblage comprised both widespread species and rarer taxa occasionally found in other habitats. Although hydrocarbon-polluted environments likely restrict many species, they also appear to provide ecological niches for extremophile animals and may serve as important reservoirs of specialized biodiversity. Nevertheless, the association between meiofaunal taxa and seep habitats requires further validation and investigations. Overall, meiofauna offer a promising model for eco-evolutionary studies of hydrocarbon-impacted environments and for applications in biomonitoring and bioremediation.

Whole genome amplification (WGA), and in particular multiple displacement amplification (MDA), has become a key technique for genomic sequencing of microscopic organisms, yet it introduces artefacts such as palindromic (inverted chimeric) reads that may compromise downstream analyses. We assessed how pervasive palindromic reads generated by MDA impact the assembly of tardigrade (Acutuncus giovanniniae and A. mecnuffi) mitogenomes sequenced with Oxford Nanopore technology. We show that the MDA produces a high proportion of palindromic reads, often exceeding one-third of mitochondrial reads and frequently exhibiting complex multi-inversion structures. These artefacts severely impair long-read assembly, leading to low success rates and inconsistent genome reconstruction. To solve this issue, a strategy based on in silico fragmentation of long reads into short, high-quality fragments, followed by short-read assembly, consistently produced complete and accurate circularised mitochondrial genomes. Our results demonstrate that palindromic read formation can be, in some cases, a limitation of MDA coupled with long-read sequencing, but this issue can be mitigated through read fragmentation. This approach provides a simple, robust and scalable solution for mitogenome assembly from data heavily affected by amplification artefacts, particularly in microscopic taxa where whole genome amplification is often unavoidable.

Microscopic invertebrates often exhibit variation in eye presence within closely related taxa, yet the ecological correlates of this variation remain poorly understood. In tardigrades, eyes may occur or be absent even among species inhabiting similar environments. Because structures that enable vision are frequently linked to environmental conditions, we investigated which factors influence the occurrence of eyes in bryophyte-dwelling tardigrade communities. We tested whether eye presence is primarily associated with broad-scale light conditions or with fine-scale microhabitat characteristics related to bryophyte structure. Bryophyte samples were collected in alpine sites, and the presence of eyes in tardigrades found in those samples was analyzed at the individual level using mixed-effects models that incorporated bryophyte light preference and its morphological complexity. Eyeless tardigrades were relatively more frequent in morphologically complex bryophytes and, more weakly, in bryophytes associated with high-light environments. Together, these patterns suggest that eye reduction may be associated with low light availability within bryophyte interiors rather than with external habitat exposure. Therefore, our study indicates the role of fine-scale environmental filtering in structuring sensory trait variation in microscopic animals.

Abstract Three Sicilian populations belonging to the enigmatic genus Xerobiotus were analyzed using integrative taxonomic methods combining detailed morphological and genetic data. A new population of X. inermis was found at its locus typicus in the coastal dunes of Gela, Sicily, while X. euxinus was recorded for the first time in Sicily at Viagrande (Catania) and Serra La Nave (Etna). Both species were examined using phase contrast microscopy (PCM) and scanning electron microscopy (SEM), and sequenced for four molecular markers: 18S rDNA, 28S rDNA, ITS-2, and COI. For X. inermis, the complete mitochondrial genome was also obtained. These data enabled us to redescribe X. inermis and to re-evaluate and amend the description of the widespread and morphologically variable X. euxinus. To explore phenotypic and quantitative variation, we conducted a principal component analysis (PCA) of morphometric traits across Xerobiotus populations and selected Macrobiotus taxa. The analysis revealed extensive character overlap among species, limiting diagnostic resolution and complicating genus-level delimitation. Phylogenetic reconstructions and species delimitation analyses were partially congruent with earlier studies but also challenged the current boundaries of Xerobiotus, especially with Macrobiotus, suggesting potential synonymy or misclassification among genus- and species-level taxa. In this paper, we integrate multiple lines of evidence from our analyses and comparisons in order to synonymize two genera and two species and identify two additional taxa as species in need of urgent revision to test for further potential synonymies.

Aim: Freshwater rock pools are ephemeral and fragile habitats that support specialised animal taxa. While distributed worldwide, these habitats are usually neglected and overlooked. We used DNA metabarcoding and metaphylogeographic approaches to study inter and intraspecific tardigrade biodiversity to identify their biogeographic patterns to inform conservation measures. Location: Europe. Period: Present. Major Taxa Studied: Tardigrades. Methods: We conducted DNA metabarcoding targeting a fragment of the cytochrome oxidase subunit 1 gene of 163 freshwater rock pools from four sites in Sweden, Poland and Italy to analyse the effect of both short and long distance on their alpha diversity and genetic differentiation. Results: A total of 85 tardigrades OTUs (species-level clusters) were detected, with the most prevalent taxa across all samples being species from the genus Ramazzottius. OTUs richness was mostly driven by a negative relationship with rock pool depth and site-specific differences. For seven OTUs a phylogeographic analyses revealed a strong founder effect and a variable effect of geographic distance and climate on their population structures. Main Conclusions: The high biodiversity and presence of potential specialised taxa highlight the conservation value of freshwater rock pools. While some tardigrade species may be able to easily disperse and have a large geographic range, other species show isolation by distance patterns and their genetic diversity may be negatively affected by habitat loss. Our results call for a bigger effort in identifying, mapping, characterising and protecting freshwater rock pools as underlooked but important habitats.

Species are fundamental units of biodiversity, yet their delimitation remains challenging in many organismal groups. The increasing use of DNA data has revealed widespread cryptic diversity, in which genetically distinct lineages are morphologically indistinguishable. Consequently, many morphology-based biogeographical inferences have likely overestimated species ranges, particularly in microscopic animals traditionally considered ubiquitous. We investigated bdelloid rotifer diversity and biogeographical patterns in small aquatic, moss-associated habitats across South-Western Greenland, a poorly explored and geographically isolated region. Using morphological identification combined with mitochondrial COI sequencing and molecular species delimitation, we analysed Greenlandic populations in a global context. Seven morphologically defined species were recorded, all new for Greenland, and each represented a complex of cryptic lineages. Using DNA taxonomy, we identified 24 putative species, most lacking close matches in public databases. Species richness estimates exceeded observed values, indicating extensive hidden diversity. Most putative species exhibited restricted distributions, whereas only a minority were shared with other Holarctic regions. Our results suggest that apparent ubiquity in bdelloid rotifers largely reflects unresolved cryptic diversity and limited sampling. Additional barcoding of tardigrades, collected opportunistically from the same samples together with rotifers, revealed similar gaps in reference libraries for aquatic meiofauna. Overall, this study highlights the importance of DNA-based taxonomy for robust biogeographical inference in microscopic animals.

Soil-dwelling invertebrates shape belowground microbial communities through feeding, movement and organic inputs, yet the microbiota of many taxa remain poorly characterized. Antlions (Neuroptera: Myrmeleontidae), sedentary predators of sandy habitats, are ecologically important but microbiologically understudied. A comprehensive analysis of larval-associated bacterial communities in two widespread species, Myrmeleon bore (Tjeder, 1941) and Euroleon nostras (Fourcroy & Geoffroy, 1785) across developmental stages was performed using 16S rRNA amplicon sequencing. M. bore, which inhabits more thermally variable, open habitats, exhibited slightly higher microbial diversity and more pronounced ontogenetic shifts than E. nostras. This suggests that environmental input also has some impact on the microbial composition of antlion larvae. Some of the detected taxa, including Serratia, Lactococcus or Enterococcus, are common in antlions or other soil invertebrates and may contribute to nutrient cycling, though their functional roles in antlions remain speculative. We propose that antlion larvae represent a promising model for investigating microbiota assembly in arid, low-nutrient habitats and emphasize the need for longitudinal and functional studies to resolve microbial roles in these insects.

Peatlands and peat-like habitats are among the most vulnerable ecosystems to climate change. Owing to their specific microclimatic conditions, which maintain high and stable moisture levels, they host assemblages dominated by hydrophilous taxa, including several groups of tardigrades. Despite this, tardigrade diversity in peat-related habitats remains poorly known. Here, we conducted an extensive taxonomic survey of tardigrades based on 35 samples collected from alpine fens in northern Italy. We detected 30 species-level tardigrade taxa, of which 23 were successfully DNA barcoded. One species of the genus Crenubiotus is new to science and is formally described herein as Crenubiotus meg sp. nov. We also provide updated morphological and taxonomic information for two enigmatic taxa, Fontourion secchii and Microhypsibius minimus, which have long been hindered by data deficiency. Notably, the assemblage recovered comprises several taxa with boreo-alpine or cold-adapted affinities, highlighting alpine peatlands as potential refugial habitats for relict tardigrade lineages. In addition, we performed phylogenetic analyses to resolve the placement of the newly described Crenubiotus species and to clarify the relationships of four Murrayon lineages recovered in this study. This work represents the first integrative taxonomic inventory of tardigrades inhabiting peat-like habitats and contributes an extensive, curated set of reference DNA sequences for this understudied habitat. These data provide an essential foundation for high-throughput biodiversity assessments using mega- and metabarcoding approaches and offer new insights into tardigrade taxonomy and evolutionary relationships.

Abstract African tropical forests are highly underexplored regarding microinvertebrate diversity, including tardigrades. Here, we describe two new tardigrade species from the genera Parahypsibius Gąsiorek et al., 2024 and Echiniscus C.A.S. Schultze, 1840, collected in the tropical rainforest of the Kibale National Park in southwestern Uganda. Species delimitation was based on an integrative approach combining morphological, morphometric, and molecular data. Morphological analyses employed light and scanning electron microscopy, while molecular characterization used mitochondrial COI and nuclear 18S rRNA, 28S rRNA, ITS1, and ITS2 markers. Additionally, we report Echiniscus lineatus Pilato, Fontoura, Lisi and Beasley, 2008 from Uganda for the first time, providing new sequences and reconstructing haplotype networks using previously published genetic data of this species. Phylogenetic analyses of subfamily Hypsibiinae Pilato, 1969 and genus Echiniscus clarify the relationships of the new taxa and place them within the broader phylogenetic context. Our study highlights the underestimated diversity of African tardigrades and demonstrates the importance of integrative taxonomy for species delineation. The new records from Kibale National Park provide also baseline data for understanding the distribution and phylogeny of tardigrades of the Central Africa.

Cryptogam habitats support a wide range of limno-terrestrial meiofauna, but the factors that shape their communities are still not well understood. The physical substrate that cryptogams grow on (e.g., soil, the base of a tree, or its trunk) can influence local moisture, temperature, and nutrient conditions, yet its role in structuring meiofaunal assemblages has rarely been tested systematically. We examined this question in montane forests of the Ukrainian Carpathians, using DNA metabarcoding to compare the diversity and composition of tardigrade and rotifer communities across substrates. We expected that rotifers, with shorter life cycles and often higher population densities, would respond more strongly to environmental differences among substrates, whereas tardigrades would be less affected and more influenced by chance colonization. Our results supported these expectations. Rotifer richness and community composition varied across substrates, while tardigrade assemblages showed weaker and more variable responses. Analysis of phylogenetic diversity indicated phylogenetic clustering in rotifers, suggesting that closely related species have similar ecological preferences. Despite these contrasts, both groups showed signs of largely stochastic community assembly, with species turnover dominating differences among samples. Overall, our findings suggest that meiofaunal communities in cryptogam-associated habitats are shaped mainly by random colonization events, modulated by subtle and taxon-specific effects of microhabitat structure and vertical positioning.

Habitats created by cryptogamic vegetation support a wide range of limno-terrestrial microfauna, but the factors that shape their communities are still not well understood. The physical substrate that cryptogams grow on (e.g., soil, the base of a tree, or its trunk) can influence local moisture, temperature, and nutrient conditions, yet its role in structuring microfauna assemblages has rarely been tested systematically. We examined this question in montane forests of the Ukrainian Carpathians, using DNA metabarcoding to compare the diversity and composition of tardigrade and rotifer communities across three substrate categories. We expected that rotifers, with shorter life cycles and often higher population densities, would respond more strongly to environmental differences among substrates, whereas tardigrades would be less affected and more influenced by chance colonization. Our results supported these expectations. Rotifer richness and community composition varied across substrate categories, while tardigrade communities showed weaker and more variable responses. Analysis of phylogenetic diversity indicated phylogenetic clustering in rotifers, suggesting that closely related species have similar ecological preferences. Despite these contrasts, both groups showed signs of largely stochastic community assembly, with species turnover dominating differences among samples. Overall, our findings suggest that microfauna communities in cryptogam-associated habitats are shaped mainly by random colonization events, modulated by subtle and taxon-specific effects of microhabitat structure and vertical positioning.

Four new species of the genus Macrobiotus are described from Asia and Africa: Macrobiotus witalinskii sp. nov., M. dalaticus sp. nov., M. surmaczi sp. nov., and M. hoianicus sp. nov. Species delimitation is based on detailed morphological and morphometric analyses using phase-contrast light microscopy and scanning electron microscopy, complemented by molecular data from four commonly used genetic markers. Phylogenetic analyses consistently place all four species within Macrobiotus clade C, each forming a distinct, well-supported monophyletic lineage. The newly described taxa expand the known morphological and ecological diversity of clade C and show that egg and body coloration characters previously considered diagnostic for this lineage are not evolutionarily conserved. These results further emphasize the value of integrative taxonomy for documenting hidden diversity and for supporting future biodiversity assessments, including large-scale DNA barcoding and metabarcoding surveys of tardigrades.

Two freshwater tardigrade populations belonging to the genus Dactylobiotus were investigated using phase contrast microscopy, scanning electron microscopy, and molecular markers commonly employed in tardigrade phylogenetic studies (18S rRNA, 28S rRNA, ITS2, and COI). The population from Taiwan, discovered through social media, represents a new species, described here as Dactylobiotus taiwanensis sp. nov. This species is most similar to Dactylobiotus parthenogeneticus but differs in the presence of singular rings of pores surrounding the egg processes and specific morphometric traits. The second population, from Greenland, was provisionally identified as D. cf. octavi, and its morphological discrepancies are discussed in detail. A revision of the type material for Dactylobiotus caldarellai and Dactylobiotus lombardoi raises questions about their validity due to insufficient data. Finally, a phylogenetic analysis incorporating taxa from the family Murrayidae, along with the newly sequenced populations, is presented. An updated dichotomous key for the genus Dactylobiotus is also provided.

Altitudinal gradients offer a unique opportunity to understand the drivers of species richness, as mountain regions cover vast areas and contribute disproportionately to global terrestrial biodiversity. However, such gradients are often studied without considering the role of microhabitats, which introduce fine-scale heterogeneity within coarse macroenvironmental conditions. This heterogeneity may be especially important for microscopic animals, whose distributions and interactions are largely confined to the microhabitat scale. In our study, we investigated altitudinal patterns of microscopic invertebrates (Tardigrada), testing the hypothesis that microhabitat characteristics modulate the effects of macroenvironmental gradients on diversity and community structure. We compiled an extensive inventory of tardigrade taxa inhabiting bryophytes across the Western Alps (Northern Italy) and analysed 546 bryophyte samples collected along a broad altitudinal gradient using DNA metabarcoding to characterise tardigrade communities. For each taxon, we gathered functional trait data to assess how species characteristics influence distribution. We then evaluated the effects of macroenvironmental variables (altitude, vegetation type, slope exposition) and microhabitat-level traits (bryophyte biological and structural features) using spatially explicit statistical modelling. We found that species richness decreased with altitude, whereas standardised phylogenetic and functional diversity increased, indicating higher redundancy at lower elevations. Our results reveal that tardigrade communities in bryophyte microhabitats are highly heterogeneous, with strong species turnover and prevalent phylogenetic and functional underdispersion. Despite the influence of stochastic processes in shaping their distributions, we show that macroenvironmental variables such as altitude and geographic location drive species turnover, while microhabitat traits govern trait-based community structure. These findings support the view that microhabitats act as fine-scale filters modulating the broader effects of altitude on diversity, highlighting the importance of incorporating microhabitat heterogeneity when studying organismal diversity along altitudinal gradients.

In this paper, we present an integrative description, utilizing morphological and genetic data, of a new eutardigrade species, Mesobiotus silesiacus sp. nov. The new species was discovered during research on Jasieniowa Mountain in Upper Silesia, Poland. While Mesobiotus silesiacus sp. nov. belongs to the harmsworthi group, it is distinguished from other group members i.e. Mesobiotus altitudinalis (Biserov, 1997/1998), Mesobiotus diffusus (Binda Pilato, 1987), Mesobiotus imperialis Stec, 2021, Mesobiotus krynauwi (Dastych Harris, 1995), Mesobiotus romani Roszkowska, Stec, Gawlak Kaczmarek. 2018, Mesobiotus mandalori Erdmann, Kosicki, Kayastha, Mioduchowska Kaczmarek, 2024 and Mesobiotus wuzhishanensis Yin, Wang Li, 2011 by several morphometric traits of the bucco-pharyngeal apparatus, claws, and/or eggs. The morphometric findings are further supported by a comparative genetic analysis utilizing four molecular markers: 28S rRNA, 18S rRNA, COI and ITS2. Based on the compiled data, we also conducted a phylogenetic analysis using available genetic sequences of Mesobiotus representatives. The presented analysis clearly illustrates the position of the new species in the phylogenetic tree enabling also the discussion on the taxonomic composition and relatedness of other taxa within the genus Mesobiotus.

Previous studies on various organisms have suggested that low doses of ethanol can have stimulatory effects, while higher doses may lead to toxicity, a response known as hormesis. Low ethanol concentrations occur naturally in the environment, particularly in fermenting fruits and flower nectar, where pollinators such as honey bees may encounter it. This study aimed to investigate the potential hormetic effects of low-level ethanol consumption on honey bees. Bees were divided into three groups: one provided with only sucrose solution, one both with sucrose and 0.5% ethanol in sucrose, and one with only 1% ethanol in sucrose. The bees were exposed to these diets for 14 days, and their performance was assessed through survivorship, flight endurance, body mass, lipid content, and trehalose and ethanol levels in the haemolymph. The results showed no significant differences in most parameters between the groups. However, bees constantly exposed to 1% ethanol had slightly higher trehalose levels compared to the control group, suggesting a possible adaptive response to ethanol exposure. Ethanol levels in the haemolymph differed significantly between groups, with bees exposed to ethanol showing detectable levels in their system. While no clear hormetic effects were observed in terms of improved performance, the elevated trehalose levels in bees constantly exposed to 1% ethanol may indicate adaptations protecting bees from ethanol-induced damage. The study provides insights into how honey bees tolerate low-level ethanol exposure and highlights the need for further research on the ecological implications of ethanol consumption in pollinators.

Rescue behavior aims to free a relative from danger. Ants are particularly known for such helpfulness and, perhaps not coincidentally, also show the highest level of social organization in the animal kingdom, i.e. eusociality. However, even among social species such as ants, there is a huge variation in rescue proneness, and little is understood about the underlying causes of this variation. In this study, we explore the relationship between helpfulness in the form of rescue and life expectancy, focusing on 14 ant species with diverse phylogenetic backgrounds. We posit that species with longer worker life expectancies are more prone to engaging in rescue actions. To test this, we assessed worker lifespan in each species and conducted behavioral tests simulating entrapment scenarios involving a nestmate ensnared by an artificial obstacle. Observed behaviors involved contact with the nestmate, digging around it, pulling at its body parts, and biting the entrapping obstacle. Our findings reveal that species with longer worker life expectancies exhibit higher proneness to rescue endangered nestmates, irrespective of phylogenetic relatedness. Furthermore, we found no trace of a phylogenetic signal in the life expectancies or helpfulness of workers belonging to different species. The results underscore the significance of life expectancy as a key factor influencing the likelihood of rescue behavior in ants. This phenomenon warrants further investigation, given the varied physiologies, life histories, and ecologies observed among species. Nevertheless, the impact of life expectancy on behavioral patterns in social insects suggests that this parameter is likely significant across diverse taxa. Rescue behavior in ants involves helping endangered nestmates. Our study examined several ant species, finding that those with longer worker lifespans were more likely to engage in rescue actions. This suggests life expectancy is a crucial factor influencing rescue behavior. These findings highlight the need for further research on the behavioral patterns of social insects.

We present the first record of Milnesium guanyinensis in the Palearctic region (S Poland), along with two other Milnesium species ( M. berladnicorum and M. inceptum ) that are reported as new for Poland. While recent studies have provided empirical evidence that tardigrades belonging to the genus Milnesium exhibit zoogeographical patterns, and only some taxa are recognised as truly cosmopolitan, all of the species re- ported in our study have broad distribution ranges. We contextualise our findings within the current under - standing of tardigrade phylogeography and discuss the potential for anthropogenic long-distance dispersal. The current study increases the number of known Milnesium species in Poland from four to seven and raises the total number of tardigrades in Poland to 118. All of the observations are based on an integrative taxonomy approach, which combines a morphological analysis with DNA barcoding.

DNA metabarcoding is revolutionizing biodiversity research by providing rapid and efficient ways of collecting species occurrence data. However, it has not yet been effectively applied to many taxonomic groups, mainly due to a significant lack of reference sequences and dedicated protocols. One such group is the tardigrades-a charismatic phylum of microinvertebrates known for their extremophilic and cryptobiotic capabilities. In this study, we provide the first curated database of 3194 tardigrade COI sequences sourced from public databases and supplemented with newly produced barcodes. We demonstrate tardigrade metabarcoding in action with optimized PCR primers and a sample processing protocol using 78 samples collected in Poland and Italy. The metabarcoding revealed the presence of more than a hundred operational taxonomic units classified as Tardigrada, representing 23 genera. We compared the metabarcoding results with a morphological survey, which revealed the presence of the same genera, but a lower number of species-level taxa identified morphologically. We observed congruent patterns of tardigrade species richness and taxonomic composition between metabarcoding and morphological surveys in both within-sample and regional fauna composition levels. The metabarcoding had a higher discriminatory power, revealing cryptic diversity, and distinguishing species belonging to taxonomically challenging species complexes. By combining metabarcoding with morphological study, we were able to find rare taxa, including novel biogeographic records and putative species new to science, showing also that this approach can be extremely powerful and effective in meiofauna research.

The tardigrade genus Richtersius Pilato & Binda, 1989 has been considered monotypic for more than 30 years since its establishment and is frequently used in experimental studies on physiological adaptations to stress. Only recently, integrative taxonomy has allowed us to disentangle and describe different but similar species. In this study, we provide a taxonomic reanalysis of the genus Richtersius with an integrative description of two new species based on light and scanning electron microscopy as well as DNA sequencing of four markers (18S rDNA, 28S rDNA, ITS-2, and COI). Richtersius nicolai sp. nov. and Richtersius ingemari sp. nov. are distinguished from congeneric species based on a combination of pore density in newborn’s dorsal cuticle, egg diameters, placoid sizes and reproductive modes. This reanalysis of the genus Richtersius will facilitate the future descriptions of new species and provides a solid taxonomic background for the identification of the species used in experimental research.