Understanding the impact of ecological factors on biodiversity is central in the context of accelerating climate change and biodiversity loss. Urban areas, as landscapes under particularly strong anthropogenic influence, are undergoing rapid ecological change, yet the consequences for urban biodiversity and ecosystem functioning remain poorly understood. In this study, we focused on fruit flies of the genus Drosophila-a diverse group of dipterans with variable ecological niches and degrees of synanthropy, i.e., the adaptation to human-modified habitats. We investigated species composition and community ecology in the metropolitan area of Vienna, Austria. With the help of numerous citizen scientists, we have collected approximately 18,000 specimens through dense spatio-temporal sampling both indoors and outdoors of human dwellings. A total of 13 Drosophila species were identified, with communities dominated by widespread cosmopolitan synanthropic species. Among these, D. mercatorum and D. virilis represent novel records for Austria. Comparisons to a previous study from more than 30 years ago revealed that the species richness in Vienna was more than 50% lower than before and showed that formerly common species were potentially replaced by non-indigenous drosophilids (neobiota). We further assessed ecological niches by intersecting species abundance data with high-dimensional, high-resolution earth observation datasets, which revealed distinct ecological preferences among species. In particular, the neozoan D. mercatorum emerged as a highly synanthropic species, tightly confined to urban areas with high levels of imperviousness. In summary, our study underpins the versatility of the Drosophila system as an indicator of biodiversity loss in a rapidly changing world.

Natural variation in circadian clock genes provides a powerful framework for understanding how organisms respond to environmental heterogeneity. The Clock ( Clk ) gene encodes a core transcriptional regulator of circadian rhythms and contains a polymorphic polyglutamine (polyQ) tract whose evolutionary significance remains unclear. Here, we integrate population genomic, behavioral, and molecular analyses to investigate the functional and geographic patterns of Clk polyQ variation in Drosophila melanogaster . Using data from 127 European populations, we identify 11 Clk polyQ alleles whose frequencies show significant associations with latitude, longitude, and principal components derived from bioclimatic variables, indicating strong geographic structure. Behavioral assays of near-isogenic lines revealed that polyQ length modulates circadian function under thermal challenge: most alleles maintained stable free-running periods across temperatures, whereas the intermediate-length Q25 allele exhibited reduced temperature compensation. Circadian phase showed pronounced allele-specific sensitivity to elevated temperature in laboratory assays, although phase variation did not display a consistent relationship with geographic variables. At the molecular level, luciferase reporter assays showed that longer polyQ alleles exhibited higher transcriptional activity, linking polyQ length to CLK-mediated gene expression. Together, these results demonstrate that natural variation in Clk polyQ length has measurable functional consequences for circadian regulation and exhibits strong geographic structuring, highlighting the potential for low-complexity regions to modulate clock function in a context-dependent manner across environmental gradients. ### Competing Interest Statement The authors have declared no competing interest. Israel Science Foundation, 2121/23

Abstract Animal microbiomes are shaped by both environmental exposure and host-associated filtering, but the relative importance of these processes remains poorly understood. Dung-associated insects provide an ideal model because they develop and feed in highly dynamic microbial environments. We investigated the gut microbiomes of six sympatric dung fly species of the genus Sepsis (Diptera: Sepsidae) and compared them with microbial communities in cow dung throughout a growing season in Switzerland. Using full-length 16S rRNA gene sequencing (PacBio), we characterized bacterial communities from 74 fly and 15 dung samples. Seasonal variation was the strongest predictor of microbiome composition, whereas host species exerted weaker effects that persisted after removing dung-associated taxa, indicating that gut communities are not merely passive reflections of environmental exposure. Only few gut microbiome reads were attributable to dung-associated taxa, and environmental overlap differed among fly species rather than season. A highly non-random core microbiome persisted across all six species: 36 bacterial genera (of 469) were shared by all hosts at ∼119-fold enrichment above random expectation and remained after removing dung-associated taxa. These findings support a two-layer model of microbiome assembly, in which seasonal environmental variation determines microbial availability while host-specific processes selectively retain a subset of taxa.

The White-bellied Heron (Ardea insignis) is one of the world's rarest birds, with fewer than 60 known individuals remaining in the wild. Whether this extreme rarity reflects a recent anthropogenic collapse or a long history of persistently small population size has remained unknown, limiting our understanding of the species' evolutionary resilience and conservation needs. Here, we present the first high-quality reference genome for A. insignis, generated using Oxford Nanopore long-read sequencing and complemented with Illumina whole-genome data. Comparative mitochondrial and nuclear phylogenomic analyses consistently recover A. insignis as the sister species of Purple Heron (A. purpurea), while revealing moderate mitonuclear discordance among deeper ardeid lineages. Genome-wide analyses demonstrate exceptionally low heterozygosity and extensive runs of homozygosity relative to the widespread and closely related Great Blue Heron (A. herodias), indicating pronounced genomic erosion and long-term inbreeding. However, the predominance of short and intermediate-length homozygous tracts, together with robust Pairwise Sequentially Markovian Coalescent (PSMC) reconstructions across alternative parameterizations, indicates that A. insignis has persisted with comparatively small effective population sizes over much of its evolutionary history rather than experiencing only a recent demographic collapse. The two sampled individuals nevertheless differ in the abundance of longer homozygous tracts, indicating that inbreeding accumulated over the past few generations has not been uniform among the surviving birds, despite their shared history of chronic rarity. Our results indicate that the White-bellied Heron represents a lineage that has survived prolonged demographic adversity and that its greatest genetic challenge may be limited adaptive potential rather than recent genomic deterioration alone. Beyond providing the first genomic resource for this critically endangered species, our study establishes an evolutionary baseline for future monitoring and highlights the importance of integrating genomic and ecological data to guide conservation strategies for species persisting at the edge of extinction.

A particularly well-studied evolutionary model is the vinegar fly Drosophila melanogaster, a cosmopolitan insect of ancestral southern-central African origin. Recent work suggests that it expanded out of Africa ∼9,000 years ago, and spread from the Middle East into Europe ∼1,800 years ago. During its global expansion, this human commensal adapted to novel climate zones and habitats. Despite much work on phenotypic differentiation and adaptation on several continents (especially North America and Australia), typically in the context of latitudinal clines, little is known about phenotypic divergence among European populations. Here, we sought to provide a continent-wide study of phenotypic differentiation among European populations of D. melanogaster. In a consortium-wide phenomics effort, we assayed 16 fitness-related traits on a panel of 173 isofemale lines from 9 European populations, with the majority of traits measured by several groups using semi-standardized protocols. For most fitness-related traits, we found significant differentiation among populations on a continental scale. Despite inevitable differences in assay conditions among labs, the reproducibility and hence robustness of our measurements were overall remarkably good. Several fitness components (e.g., viability, development time) exhibited significant latitudinal or longitudinal clines, and populations differed markedly in multivariate trait structure. Notably, populations experiencing higher humidity/rainfall and lower maximum temperature showed higher viability, fertility, starvation resistance, and lifespan at the expense of lower heat-shock survival, suggesting a pattern of local adaptation. Our results indicate that derived populations of this tropical fly have been shaped by pervasive spatially varying multivariate selection and adaptation to different climates on the European continent.

Many similar sepsid dung fly species coexist on European pastures, contradicting conventional wisdom of niche theory and competitive exclusion. We hypothesized that closely-related sepsid species on the same pasture in Switzerland avoid each other by having different spatio-temporal microhabitat niche preferences, thus enabling coexistence. A thermal racetrack experiment in the laboratory tested the thermal preferences of males and females of 9 coexisting temperate Sepsis dung fly species from Switzerland at two acclimation temperatures. The sepsid species investigated here showed no strong differences in thermal preferences. Flies of all species preferred to settle at cooler temperatures, and otherwise utilized the entire range (from 12°C to 30°C) offered for their activities. This was the case for both sexes, and also for both acclimation temperatures (18°C, 24°C). Our findings suggest that physiological thermal adaptation or acclimation is not an important mechanism by which adult sepsid flies avoid interspecific competition. Our experiment supports previous findings of widespread sepsid flies lacking local adaptation but high phenotypic plasticity, again highlighting the necessity of experimentally assessing putative biological mechanisms facilitating coexistence.

The importance of genital morphology for alpha-and supraspecific taxonomy of pulmonate land snails has been known since the 19th century. Despite that, the genital anatomy of the family Clausiliidae has been considered of low discriminatory importance at the species level, although it has great potential for discriminating genera. No statistical analyses of fundamental aspects of genital morphology of clausiliids, such as measurements, size ranges, mean values, standard deviation, and trait categorization, are yet found in the literature. Moreover, no data on statistical correlations among the various morphological parts (genitalia and shell) have been reported to date. In the present study we investigate the clausiliid genus Montenegrina as a model for such morphometric analyses. Measurements show a high degree of variability and large overlaps among the Montenegrina taxa. Correlation tests show positive correlations among some shell chracters, while correlations among genital characters prove to be generally weaker. Additionally, we compare quantitative (morphometric measurements) and qualitative (categorization of traits) data with principal component analyses (PCA) to test if genitalia and shell morphological features correspond to the phylogeny based on published previously mitochondrial DNA sequences. The morphometric analyses of shell and genital characters show that, although specimens of the same taxon mostly cluster together, many clades overlap in the PCA scatter plots and/or form intermingled clusters in the neighbour-joining (NJ) trees. Finally, we ask if the taxa studied show character displacement. Among the 4 syntopic pairs of taxa that exist in the genus Montenegrina, 3 show a morphological Euclidean distance well above the average for the genus, although the discriminant power is notably weak. Assumptions on how ecological factors should result in differences of shell and/or genital morphology-thus, ecological character displacement (ECD)-remain speculative. Our findings are compatible with both the assumption of reproductive character displacement (RCD) as well as the non-adaptive radiation hypothesis raised by earlier work on this group.

Large-scale genomic resources can place genetic variation into an ecologically informed context. To advance our understanding of the population genetics of the fruit fly Drosophila melanogaster, we present an expanded release of the community-generated population genomics resource Drosophila Evolution over Space and Time (DEST 2.0; https://dest.bio/). This release includes 530 high-quality pooled libraries from flies collected across six continents over more than a decade (2009 to 2021), most at multiple time points per year; 211 of these libraries are sequenced and shared here for the first time. We used this enhanced resource to elucidate several aspects of the species' demographic history and identify novel signs of adaptation across spatial and temporal dimensions. For example, we showed that the spatial genetic structure of populations is stable over time, but that drift due to seasonal contractions of population size causes populations to diverge over time. We identified signals of adaptation that vary between continents in genomic regions associated with xenobiotic resistance, consistent with independent adaptation to common pesticides. Moreover, by analyzing samples collected during spring and fall across Europe, we provide new evidence for seasonal adaptation related to loci associated with pathogen response. Furthermore, we have also released an updated version of the DEST genome browser. This is a useful tool for studying spatiotemporal patterns of genetic variation in this classic model system.

The Tasmanian mountain shrimps (Malacostraca: Anaspides) are enigmatic freshwater crustaceans that entered the subterranean habitat multiple times independently during Pleistocene times. Some lineages are exclusively stygomorphic species, such as Anaspides eberhardi (Ahyong, 2016), whereas others, such as Anaspides richardsoni (Ahyong, 2016), have surface and cave forms. Two different models for speciation have been suggested for recent colonization events of caves: the climatic relict hypothesis and the adaptive shift hypothesis. The major difference is the absence (climatic relict hypothesis) or presence (adaptive shift hypothesis) of gene flow during divergence, corresponding to allopatric and parapatric speciation. Herein, we present a phylogenetic analysis of the obligate cave-dwelling species A. eberhardi and two closely related species using double digest restriction-site associated DNA (ddRAD) and COI datasets. Despite the detected mito-nuclear discordance, the extensive ddRAD datasets clearly support the monophyly of each species. However, we detected one instance of localized introgression into A. eberhardi from a surface population of A. richardsoni, whereas syntopically occurring populations with an undescribed species showed no evidence of interbreeding. Our data support a single origin of A. eberhardi during the Pleistocene, followed by underground dispersal and extinction of surface populations, all in correlation with glacial events. This clearly favours the climatic relict hypothesis as the mode of speciation.

In light of recent technological advances – in fields such as computer science, imaging technologies, and molecular analysis methods – the possibilities for studying biological museum specimens and linking information across disciplines have expanded exponentially in recent years. In particular, scientific disciplines operating on the scale of so-called -omics technologies, originally developed in molecular biology, have found their way into the museum realm. This development has led to a proliferation of neologisms aimed at describing comprehensive approaches for analyzing museum specimens and collections with the help of large-scale datasets. Among these terms, “museomics” has become a frequently used, albeit rather loosely defined, synonym for a wide range of large-scale, integrative scientific approaches that are centered on, or incorporate, museum specimens. In this article, we aim to provide an overview of the most commonly used key terms, examine their conceptual interrelations, and present a literature-based survey of their usage. We advocate for using three terms at different hierarchical levels: (1) museomics for analytical approaches using biomolecules on museum specimen; (2) the extended specimen concept linking voucher specimens with diverse physical, digital, and contextual information; and (3) collectomics linking the data of the extended specimen with quantitative datasets from other domains of research. A key prerequisite for this linkage is the unambiguous assignment of data to the corresponding specimen using a Persistent Identifier (PID). Finally, we conclude the paper by reflecting on the current approaches and initiatives adopted at the Natural History Museum Vienna, emphasizing the importance of adopting a museum-wide strategy for developing extended specimen datasets using standardized data formats in accordance with Open Science principles.

Chromosomal inversions are structural mutations resulting in the reversal of the gene order along the corresponding genomic region. Due to their influence on recombination patterns, they can have a major influence on genetic variation and the evolutionary process. Accordingly, inversions can act as supergenes that keep together co-adapted gene complexes that form the genetic basis of many complex phenotypes in diverse organisms. In this book chapter, I will present an analysis pipeline to investigate the influence of two common cosmopolitan inversion, In(2L)t and In(3R)Payne, on genome-wide genetic variation and differentiation in world-wide populations of the vinegar fly Drosophila melanogaster. We will use single-individual and pooled resequencing data in combination with population genomics analysis tools to explore the impact of these two inversions on genetic variation, population structure, and clinal variation in natural populations.

To efficiently mitigate biodiversity loss both robust ecological data and broader societal engagement are needed. Citizen science offers a pathway to address these dual challenges by combining data collection with public involvement. Here, we introduce the citizen science project Vienna City Fly, conducted at the Natural History Museum Vienna (NHMW), which used fruit flies ( Drosophila ) as a model organism group for urban biodiversity research and public engagement. Participants deployed standardized traps across Vienna, enabling systematic sampling and active participation in research, which resulted in hundreds of fly collections, thousands of sampled fly specimens and new insights into the biodiversity and ecology of the collected fly species, demonstrating the feasibility of large-scale ecological research through public collaboration. To assess motivational drivers and perceived impacts of participation, we conducted an online survey (N=59) using the validated psychometric MORFEN-CS scale. Survey results revealed that nature conservation values and the intention to contribute to biodiversity conservation emerged as the strongest drivers of engagement, complemented by sociopolitical responsibility and citizen science-based motivations. Reported outcomes include knowledge gain (61%), more positive attitude toward study organisms (50%), and increased awareness of biodiversity (30%). Satisfaction was very high, with 85% rating their experience at the top of the scale and all participants expressing willingness to join future citizen science projects. Recruitment occurred mainly via social networks, and the sample of participants was highly educated, indicating limits to inclusivity and reach. Our findings demonstrate that citizen science can contribute to robust ecological data collection while gaining knowledge and awareness. Natural history museums, as trusted institutions, play a key role in facilitating such initiatives. Overall, Drosophila research proves to be a suitable field for citizen science, combining accessibility with strong potential for advancing ecological and biodiversity research as well as public engagement in urban biodiversity research. ### Competing Interest Statement The authors have declared no competing interest. Horizon Europe, 101059238

The Dinaric Karst, a biodiversity hotspot, features complex surface and subterranean hydrological networks that influence aquatic species distribution. This study investigates how karst hydrology shapes the genetic structure of the surface-dwelling minnow Phoxinus lumaireul, examining both large-scale and small-scale population patterns. Using mitochondrial DNA and genome-wide single nucleotide polymorphism (SNP) data of 827 specimens of P. lumaireul, three hypotheses were tested: (1) karst underground water connections facilitate genetic connectivity within and across river systems, whereas non-karst rivers exhibit genetic connectivity mostly within the same system; (2) historical and occasional hydrological connections have shaped present-day population structure, leaving genetic signatures of relatedness where no contemporary hydrological links exist; and (3) genomic approaches provide additional insights into biologically relevant connections that may not be captured by classical tracing tests. The large-scale analyses confirmed three main genetic groups (1a-c), whose structure was likely shaped by Pleistocene glaciations and associated microrefugia rather than by karst hydrology. Small-scale structure analyses revealed that while karst hydrology facilitated gene flow within specific areas, connectivity was uneven and influenced by local hydrological dynamics and historical admixture events. Furthermore, some underground pathways identified by classical tracing tests lacked evidence of genetic connectivity, underscoring the limitations of traditional methods and the added value of genomic data in indirectly detecting biologically relevant hydrological connections. These findings highlight the influence of both historical processes and contemporary karst hydrology on P. lumaireul populations, emphasizing their vulnerability in karst ecosystems and the need for targeted conservation efforts.

We herein present a phylogenetic and population genetic analysis of a Tasmanian Mountain Shrimp clade, based on ddRAD and cytochrome oxidase subunit‐1 data sets. Our data show that the morphologically well‐delineated and widespread Anaspides richardsoni Ahyong, 2016 is paraphyletic with respect to four other species ( A. eberhardi Ahyong, 2016, A. spinulae Williams, 1965 and two undescribed species). These four species all form discrete (monophyletic) lineages and exhibit clear morphological distinctions in relation to A. richardsoni and to one another. However, we detect signals of introgression between some populations of A. richardsoni , A. spinulae and an undescribed species. We also find two instances of syntopic occurrences without evidence for interbreeding. Also, A. richardsoni is split into several allopatric and comparably old lineages. Anaspides spinulae from Lake St. Clair, however, seems to be a young species that might have differentiated only after the last glacial maximum of central Tasmania (22 000–17 000 years ago). Moreover, we analyse the present population structure and recolonization of the Central Plateau and Western Mountain Ranges in regard to their glacial history. We distinguish several glacial refugia and show that the recolonization most likely occurred only from one or two of these.

Torpedo rays (Torpedinidae, Torpediniformes) are small to moderately large batoids that produce an electric discharge. They are distributed worldwide in temperate and tropical seas and are, as a result of their bottom-dwelling behaviour, susceptible to trawl fishing and often end up as victims of bycatch. The distribution ranges of most recognized species seem to be restricted; however, their species-level systematics is not adequately resolved. In the genus Torpedo, in which many species require revision, there are possibly several undescribed species, while numerous misidentifications add to the complexity of the issue. In the latest lists of living rays, 13 species are accepted in the genus Torpedo, including three of doubtful validity and several recently discovered undescribed species. Among the valid species is the critically endangered, possibly extinct, Torpedo suessii Steindachner, 1898, the Red Sea torpedo, of which only four specimens have been recorded in the literature until now, three of which still exist in the fish collection of the Natural History Museum of Vienna. Museum collections are the most important archive of biodiversity on Earth, and are increasingly being used for various studies, including phylogenetics, population genomics, and biogeography. Nevertheless, molecular analysis of old museum material remains challenging because the genetic material has degraded, is fragmented, and of low quantity. In molecular taxonomy, the necessity of including type specimens as name-bearing specimens is increasingly recognized. Here, the extended specimen approach was applied to re-describe the lectotype of T. suessii. The approach included research of historical information and whole genome sequencing, followed by genome assembly and phylogenetic analysis.

Little is known about the metabolic basis of life-history trade-offs but lipid stores seem to play a pivotal role. During reproduction, an energetically highly costly process, animals mobilize fat reserves. Conversely, reduced or curtailed reproduction promotes lipid storage in many animals. Systemic signals from the gonad seem to be involved: Caenorhabditis elegans lacking germline stem cells display endocrine changes, have increased fat stores and are long-lived. Similarly, germline-ablated Drosophila melanogaster exhibit major somatic physiological changes, but whether and how germline loss affects lipid metabolism remains largely unclear. Here we show that germline-ablated flies have profoundly altered energy metabolism at the transcriptional level and store excess fat as compared to fertile flies. Germline activity thus constrains or represses fat accumulation, and this effect is conserved between flies and worms. More broadly, our findings confirm that lipids represent a major energetic currency in which costs of reproduction are paid.

This study introduces deep learning (DL) methods for imputing missing allele-frequency information in large-scale genome-wide pooled re-sequencing (Pool-Seq) data, using the comprehensive DEST dataset based on over 270 global samples of the vinegar fly Drosophila melanogaster as a use case. The primary challenge addressed here is gap filling in DNA sequences, a critical issue in large-scale genomic studies. An empirical baseline for missing allele frequencies was established using an inverse-distance-weighting (IDW) method, leveraging geographical and temporal proximity among densely sampled populations. Additionally, a machine learning (ML) approach with k-means clustering grouped populations based on allele frequencies, independent of their spatiotemporal context. The core contribution of this research is the application of advanced DL models, specifically Masked Autoencoders (MAE), Variational Autoencoders (VAE) and Generative Adversarial Networks (GAN). These models excel in learning the data distribution and generating plausible imputations for missing sequences, outperforming the IDW and k-means based methods. Their effectiveness is due to their ability to handle high-dimensional genetic sequences and capture complex data correlations while maintaining sequential integrity. The study demonstrates the efficacy of DL in genomic data analysis, particularly for large-scale, complex datasets. VAE and GAN models offer a significant advancement over traditional ML methods, providing more accurate and efficient solutions for gap filling in genetic sequences. This research highlights the potential of DL in genomics, setting a precedent for future AI applications in biological data analysis and demonstrating a novel application area for deep learning techniques in handling complex long-sequence biological datasets.

The advent of third generation sequencing technology has revolutionized parallelized sequencing of DNA fragments of varying lengths, such as PCR amplicons, which provides unprecedented new opportunities for large-scale and diverse DNA barcoding projects that, for example, aim to quantify the accelerating biodiversity crisis. However, the broad-scale application of these new technologies for biodiversity research is often hindered by the demand for advanced bioinformatics skills to carry out quantitative analyses. To facilitate the application of multilocus amplicon sequencing (amplicon-seq) data for biodiversity and integrative taxonomic research questions, we present AmpliPiper, an automated and user-friendly software pipeline which carries out bioinformatics analyses of multilocus amplicon-seq data generated with Oxford Nanopore (ONT) sequencing. AmpliPiper combines analysis methods for DNA barcoding data that include demultiplexing of pooled amplicon-seq data, haplotype-specific consensus sequence reconstruction, species identification based on comparison to the BOLD and GenBank databases, phylogenetic analyses and species delimitation. We demonstrate the applicability and workflow of our approach based on a newly generated dataset of 14 hoverfly (Syrphidae) samples that were amplified and sequenced at four marker genes. We further benchmark our approach with Sanger sequencing and simulated amplicon-seq data which show that DNA barcoding with ONT is both accurate and sensitive to detect even subtle genetic variation. ### Competing Interest Statement The authors have declared no competing interest.

Temperature plays a fundamental role in the fitness of all organisms. In particular, it strongly affects metabolism and reproduction in ectotherms that have limited physiological capabilities to regulate their body temperature. The influence of temperature variation on the physiology and behaviour of ectotherms is well studied but we still know little about the influence of symbiotic interactions on thermal preference (T-p) of the host. A growing number of studies focusing on the Wolbachia-Drosophila host-symbiont system found that Wolbachia can influence T-p in Drosophila laboratory strains. Here, we investigated the effect of Wolbachia on T-p in wild-type D. melanogaster flies recently collected from nature. Consistent with previous data, we found reduced T-p compared to an uninfected control in one of two fly strains infected with the wMelCS Wolbachia type. Additionally, we, for the first time, found that Wolbachia titer variation influences the thermal preference of the host fly. These data indicate that the interaction of Wolbachia and Drosophila resulting in behavioural variation is strongly influenced by the genetic background of the host and symbiont. More studies are needed to better understand the evolutionary significance of T-p variation influenced by Wolbachia in natural Drosophila populations.