Wolbachia are the most pervasive bacterial endosymbionts yet described, infecting half of all arthropod species. These bacteria trigger outstanding phenotypes in their hosts including cytoplasmic incompatibility (CI), which has also been used as a mechanism to control pest species. Here, we analyzed peer-reviewed articles published in the 30 years from 1995 to 2024. Our results show that most studies continue to use traditional methods such as PCR and Sanger sequencing, approaches that remain sufficient and appropriate in many cases, particularly when working with well-characterized strains or applied systems. Only a smaller proportion of studies have employed newer genome-based techniques, which are increasingly important for exploring Wolbachia diversity and uncovering novel mechanisms. Research has focused mainly on a small number of insect groups and a limited set of Wolbachia strains. In addition, most work focuses on CI, especially since the discovery of cif genes related to this trait. Although the use of Wolbachia in pest and disease control is expanding, other possible effects and interactions with different microbes remain less explored. Our analysis shows how the field has advanced through some key discoveries, while many studies continue to use established approaches and concentrate on a limited set of hosts and strains. To make further progress, we suggest using a wider range of methods and sampling a broader set of hosts, alongside integrating genetic tools with studies of natural populations. This work outlines clear areas where more research is needed and points to ways the field can develop a fuller understanding of Wolbachia and its roles in nature.

The growing global population increases the demand for protein, while organic waste management has become more challenging. Alternative protein sources are essential to mitigate the environmental impact of food production. The black soldier fly (BSF; Hermetia illucens) has emerged as an alternative to traditional protein sources (e.g., soybean meal, fishmeal) due to its ability to convert diverse organic waste, addressing both issues simultaneously. This makes the BSF a promising candidate for industrial rearing, with its successful development closely tied to microbial influences on growth and behaviour, particularly bacterial influences on oviposition. In this study, we focus on the microbiota throughout insect development with a special focus on egg surface microbiota and their origin. We analysed the microbiota in the haemolymph and gut of larvae raised on sterilized and non-sterilized feed, pupal cell pulp, the wash of the ovipositor, eggs directly collected after oviposition, ovarian eggs, the empty female abdomen, eggs exposed to adult BSF, and surface-sterilized eggs. Our analysis revealed distinct bacterial community profiles across life stages, indicating a transition from larval dominance of Enterobacteriaceae to Burkholderiaceae on all analysed eggs. At the genus level, larval stages were characterized by Morganella, Escherichia, and Proteus, transitioning to less diverse communities in egg samples predominated by Burkholderia-Caballeronia-Paraburkholderia. Our study reveals that while predominant microbiota persist throughout all life stages, microbial community composition transforms progressively during maturation, particularly before oviposition. Understanding egg surface microbiota and the cues guiding oviposition has the potential to boost egg production and simplify mass harvesting of BSF larvae.

Lasius castor n.sp. and L. pollux n.sp., two new species of the ant subgenus Chthonolasius , are described and separated by high-resolution stereomicroscopy, Numeric Morphology-Based Alpha-Taxonomy (NUMOBAT), analysis of nest sample compositions and zoogeography. With a total of 922 nest samples of 11 Chthonolasius species, the study attempted to investigate all available nest samples found in the geographic range of the new species. The subgenus-specific mode of temporary socially parasitic colony foundation in Lasius s. str. species was observed in only about 6% of the L. castor n.sp. and L. pollux n.sp. nest populations and is considered a behavioral relict. In contrast, 94% formed mixed nest populations with the Chthonolasius species L. umbratus , L. distinguendus and L. sabularum. As much as 15% of the nests of the latter species contained workers of L. castor n.sp. or L. pollux n.sp. The new species differ in pilosity characters, zoogeography and selection of habitat. The mixed populations are long-term stable and contain on average 40% of fully functional workers of L. castor n.sp. or L. pollux n.sp. The new species actively search for association with a Chthonolasius host. The observations indicate an ongoing evolutionary transition from temporary social parasitism in Lasius s.str. to a form of association with closely related Chthonolasius species. We discuss the unresolved question of whether this transition represents (i) a switch to permanent social parasitism, (ii) a switch to parabiosis or (iii) a transition by establishment of male-transmitted genetic lineages. Deviating from the normal Chthonolasius morphology, the stouter body shape, appendage shortening and change of petiole shape in both new species, could signalize a beginning development to a degenerate parasite morphology.

Animals frequently display aggressive behavior, for example, when competing for food. Aggression is influenced by various extrinsic and intrinsic factors such as the microbiome and genetics. However, we currently lack understanding what factors cause animals to start aggression. Here, we use an ant species to test if chemical, microbiome, genomic, and/or transcriptomic traits correlate with the start of aggression and the reactions to it, namely reacting aggressively or peacefully. We found nine bacterial operational taxonomic units, mutations in two genes, and eight differentially expressed genes, which were positively or negatively associated with the start of aggression or reactions to it. The microbiome and genetic factors are mainly linked to hormone signaling and neurological and synaptic functions, respectively. The results indicate that multiple traits, possibly acting in concert, may affect the start of aggression and reactions to it. We speculate that such traits could promote aggression and play important evolutionary roles.

Amphibians are threatened worldwide by various environmental and anthropogenic factors, making non-invasive conservation studies particularly valuable. Newts are one example of a thus challenged amphibian group. In Austria, local population declines of newts have been observed, with the smooth newt ( Lissotriton vulgaris ) being strongly affected. In this study, skin swabs were used as a non-invasive method to gather DNA, combined with established microsatellite markers. We sampled 139 L. vulgaris individuals at ten sites in North Tyrol, Austria, and, for comparison, 22 L. vulgaris meridionalis individuals in Brixen, Italy. We genotyped all individuals and analysed their population structure. We demonstrate the presence of three distinct L. vulgaris population clusters and find differences in population structure between supposedly introduced allochthonous L. vulgaris individuals and autochthonous populations, as evidenced by differences in Bayesian clustering and elevated values of the fixation index FST. A captive population in a zoological garden, with origins in the Kramsacher Loar in the Tyrolean Unterland (eastern part of Tyrol), performed poorly in terms of conservation genetics, with low genetic diversity (number of alleles) and clear genetic differentiation from populations in the wild (high pairwise FST values with wild individuals, clear separation in cluster analysis). Habitat restoration programs are a crucial aspect of amphibian conservation, as they restore ecosystems that are critical to the animals′ survival. While breeding programs can play an additional role in the future, they must carefully consider genetic diversity to ensure resilient and viable populations, especially in the face of climate change and chytrid fungus infection. This study emphasizes the significance of considering the geographic origin and genetic diversity of newts in conservation efforts. It also serves as a foundation for future population genetic studies of newts in Austria. ### Competing Interest Statement The authors have declared no competing interest.

Communication allows organisms to quickly convey information vital for survival or fitness. Chemical communication and speed-accuracy trade-offs are ubiquitous in animal decision making. Most studies have used species which forage mainly above-ground species, tested in an epigean setting, but it remains unclear whether below-ground species behave similarly. Here, we use the below-ground ant Tetramorium alpestre to assess the efficacy of above- vs. below-ground mazes, the accuracy of decisions when using natural vs. artificial pheromones, the presence of a speed-accuracy trade-off, and the pheromones’ effect on aggression. Ants decided more quickly under below-ground than above-ground conditions, indicating they may be distracted by above-ground stimuli. Ants followed both natural and artificial trails but in direct competition preferred artificial trails, likely due to a higher pheromone concentration. Surprisingly, no speed-accuracy trade off was observed during path decision-making. Lastly, population origin but not pheromones affected if and how aggression occurred in presence of trail and home-range marking pheromones. We argue that the design of behavioural tests should match the lifestyle of the focal organism. We further speculate that speed-accuracy trade-offs likely are highly species- and context-specific and other factors besides chemicals seem important to trigger aggression, at least in this species.

Wild bees are decreasing in species diversity and populations due to human impact. The abundance of the western honey bee (Apis mellifera L.) experiences an inverse trend, enhancing competition with wild bees and the probability of microbiome exchange. Addressing this exchange, we studied the gut microbiome composition of wild and honey bees, focusing on patterns indicating honey bee influence. Three solitary wild bee species (large scabious mining bee [Andrena hattorfiana F.], grey-backed mining bee (Andrena vaga Panzer), and European orchard bee [Osmia cornuta Latreille]) as well as bumble bees as representatives of eusocial wild bees (Bombus spp. Latreille) and honey bees were sampled in the Austrian Alps. Subsequent 16S ribosomal DNA sequencing revealed the composition of the bacterial communities. The bee groups differed concerning their bacterial composition, with honey bees having the least variation among individuals and a low number of exclusive bacterial taxa and bumble bees the highest bacterial diversity. High honey bee densities corresponded with lower bacterial diversity in wild bees and a higher bacterial similarity between wild and honey bees. Some bacterial taxa were found for the first time in the studied bee groups. Furthermore, the composition of bacterial communities differed between solitary and social bees. We found the first hints that high honey bee density negatively impacts wild bees through alterations of wild bee microbiomes. Future studies should focus on understanding microbiome transmission mechanisms and their consequences for wild bees. Suggestions on how to consider wild bee fitness are indispensable in halting the biodiversity crisis.

Urban and agricultural areas are covering ever more land, deteriorating animal habitats. So far, there exist a limited number of studies on urbanisation and even fewer studies on the impact of agriculture. To find out how this overall human pressure, that is, hemeroby, affects arthropods, we chose an interdisciplinary approach by studying genetic diversity and trophic position of the spiders Araneus diadematus and Nuctenea umbratica and the ant Formica fuscocinerea. We collected five specimens per species from fifty 500 × 500 m square plots in a medium-sized Central-European city with green surroundings. Firstly, genetic analyses using newly developed microsatellites revealed a significant effect of hemeroby on Araneus diadematus, that is, reduced genetic diversity. Secondly, likewise in Araneus diadematus, the stable isotope δ15N increased with hemeroby, possibly due to a major food-spectrum change. Thus, we found that hemeroby can negatively impact arthropods. We believe our findings are relevant to urban planning, particularly given that even stronger effects can be expected for larger and more urbanised cities and in less vagile organisms.

Social interactions among conspecifics can have significant fitness implications, but how social behaviours develop in wild animals remains poorly understood. Here, we examine the intrinsic drivers of a social behaviour, communal winter roosting, in red kites Milvus milvus. In this species, communal roosting is a facultative behaviour, and the mechanisms underlying its emergence at the individual and population levels are unclear. Through longitudinal GPS tracking of 635 bird-winters from 216 red kites, we derived multi-year roosting histories to investigate (i) which individual characteristics associate with the decision to join communal roosts, (ii) how these patterns change across life stages and (iii) whether roost composition reflects assortative associations among breeding pairs or kin. Based on 33,930 nights across six consecutive winters on the breeding grounds, we identified red kites from our tagged sample joining communal roosts on average 38% of the time. Males occurred more at communal roosts than females, but in both sexes this probability drastically decreased with age and additionally decreased once they started breeding. These ontogenetic changes in communal roosting behaviour were driven by behavioural plasticity at the individual level rather than selective mortality. Red kites displayed assortative behaviour both in communal and non-communal roosting contexts. Breeding pairs showed the strongest affiliation, roosting more often together than expected by chance in non-communal roosting sites, when in proximity to their breeding territory. In contrast, sibling and parent-offspring dyads were rare, and roosting less frequently together than expected by chance within communal roosts. Overall, our results show that the structure of communal roosts in the red kite is shaped by the age, sex and social relationships of individuals. The influence of these factors may stem from trade-offs across various life history stages, driven by changes in the net benefits associated with foraging, territory and mate prospecting, as well as territory maintenance throughout an individual's life.

Biodiversity loss is increasing worldwide due to climate change, land-use change, and other anthropogenic impacts, increasing the importance of conservation measures. In Europe, government-led monitoring programs under directives regularly monitor certain species to resolve conservation plans. However, the focus is primarily on population size estimation, while genetic monitoring is sparse. Birds are among the most frequently monitored groups, including two grouse species, Tetrao urogallus (western capercaillie) and Lyrurus tetrix (black grouse), for which non-invasive samples (feces/feathers) are well established for genetic analysis. In this study, we analyzed non-invasive genetic samples from T. urogallus and L. tetrix collected in Tyrol, Austria, between 2011 and 2019, within the framework of a governmental monitoring program, for extensive population genetics and parentage and sibship analysis. Overall, 526 T. urogallus genotypes and 960 L. tetrix genotypes were identified. In both species, we found high heterozygosity and allelic richness, as well as good connectivity, across populations. Furthermore, parentage and sibship analysis suggested a female-biased dispersal in both species. We showed that the populations of T. urogallus and L. tetrix are overall genetically diverse and connected, which adds important information to the estimated population sizes for further conservation measures. Furthermore, the results provide valuable insights for the early detection of connectivity loss and the potential onset of inbreeding, essential for proactive management strategies. This study emphasizes the importance of integrating genetic monitoring into conservation efforts and demonstrates the importance of collaboration between academia and conservation to improve both species conservation and ecological understanding.

Lasius fuliginosus, a fungus-growing ant species distributed across Europe, hosts various fungi inside its carton nests in trees, including the nest fungus SP1 of the order Chaetothyriales, as well as the nest fungi SP5 and SP4 of the order Venturiales. The goal of this study was to gain a better understanding of the fungal interactions inside the L. fuliginosus nests as well as of potential interactions around the nests, including the effects of Armillaria mellea-a root-rot fungus infecting potential host trees. We performed two types of confrontation experiments on Petri dishes between the isolated nest fungi and A. mellea. Firstly, using de Wit experiments, we tested the fungal species in pairwise combinations at three different initial confrontation concentrations. Secondly, a linear confrontation setup focused on differences in directional growth of the fungal species in pairwise combinations as well as on the development of A. mellea rhizomorphs. For the fungi SP1, SP5, and SP4, we found positive influences on each other (SP1 on SP5, SP4 on SP1 and SP5, and SP5 on SP1) alongside no influence (SP1 on SP4, SP5 on SP4). SP1 had a significantly negative impact on the surface growth and directional growth of A. mellea, and SP5 triggered the strongest rhizomorph development of A. mellea, possibly a stress reaction of the root-rot fungus. Armillaria mellea did not negatively impact any of the nest fungi and even promoted the surface growth of SP1. The de Wit setup and the linear setup turned out to be complementary and together facilitated first insights into potential roles of the nest fungi in this association of ants and fungi in trees. Follow-up studies will need to assess how these findings under Petri dish conditions transfer to conditions in natural habitat, in the presence of both the ant and the tree host.

Industrial insect farming has potential for converting low-value organic waste into nutrient-rich insect biomass, producing valuable by-products like organic fertilizers. However, a better understanding of density-related thermogenesis and microbial dynamics is needed to enhance standardization and bridge gaps between laboratory and industry needs. This lab-scale study focuses on the black soldier fly (Hermetia illucens), which exhibits thermogenesis that intensifies with larval population size and its natural crowding behavior. Using high-resolution temperature monitoring and biomolecular methods, we found that doubling larval density (0, 1.25, 2.5, and 5 larvae/cm2) increased temperatures by 0.6°C-2.4°C, depending on the treatment. Adding potassium sorbate altered microbial profiles, increasing Enterobacter and decreasing Providencia, while promoting lactic acid bacteria. Density also impacted pH, water content, dry matter, volatile solids, and ash in the substrate. Our findings provide essential insights into managing microbial and thermal dynamics, offering valuable information for optimizing and standardizing conditions in rearing trials.

Habitat fragmentation threatens wildlife populations and may cause reduced genetic variation, fitness, and even local extinction, but effects differ across species. Here, we addressed effects of habitat fragmentation using the harvester ant Messor structor, a habitat specialist in natural xerothermic grassland, in 54 habitat islands of a fragmented 200 km2 area in Austria analysing microsatellites, mitochondrial DNA, infection with the endocellular bacterium Wolbachia, and morphology. We found (a) pronounced genetic diversity and two mitochondrial lineages that we infer to originate from separate glacial refugia. (b) Wolbachia infection rates were high, but the two strains detected, correlating with the mitochondrial lineages, do not seem to have induced a reproductive barrier. (c) Habitat islands with fewer ant nests had less allelic richness, and the ones with less allelic richness had reduced heterozygosity, as measured by mean locus heterozygosity; this indicated inbreeding. (d) Fluctuating asymmetry of workers, known to be low in fit individuals, positively correlated with heterozygosity, as measured by the interallelic distance of heterozygous loci; high levels of this distance are probably due to the mixing of lineages, and facing such a potential outbreeding depression, vulnerability for inbreeding may increase. (e) Gyne mesosoma size in less connected habitat was smaller than that of gynes in better connected habitat, indicating reduced flight ability. Our work highlights the use of multiple approaches to evaluate species responses to habitat fragmentation, showcasing the importance of historical colonisation and current habitat connectivity to the maintenance of genetic and phenotypic diversity.

Noninvasive genetic sampling is often used in conservation biology to assess population genetic parameters and population size of rare and elusive species. However, this sampling method poses some challenges, such as identifying an appropriate approach to sampling in the field, the optimal number of samples to be analyzed, and the number of sampling rounds. In this study, we compare the influence of 1 (OneRound) vs. 2 (TwoRounds) noninvasive sampling rounds on drawing population genetic inferences on Tetrao urogallus (Western Capercaillie) to create a framework for future development of grouse-related genetic studies. We identified unique genotypes using microsatellite markers and compared population genetic parameters and population size estimations for OneRound vs. TwoRounds. Under the premise that fieldwork is conducted at the same sampling sites during lekking season, we found that the decreased fieldwork effort of OneRound results in overall similar conclusions as TwoRounds for population genetic indices. In our study area, both approaches indicated a well-connected T. urogallus population with no signs of inbreeding or isolation. Population size estimations were slightly higher when based on OneRound of sampling, with high overlaps (86%) for males with TwoRounds. The largest discrepancy between the 2 datasets OneRound and TwoRounds became evident when comparing the data on females, indicating a sex bias. We conclude that 1 sampling round is more cost-, time- and personnel-efficient sampling approach, as highly similar results for various important population parameters can be obtained with half the fieldwork effort compared with 2 sampling rounds. When deciding about the number of sampling rounds, the weather dynamics of the target area as well as the likelihood to obtain sufficient numbers of samples should be considered. center dot The monitoring of wildlife using noninvasive samples, such as feces or feathers, poses several challenges (e.g., the number of sampling rounds to ensure a sufficient sample size).center dot In this study, we compared the outcome of population genetic parameters and population size estimates from 1 vs. 2 sampling rounds, using noninvasive sampling to monitor the Tetrao urogallus (Western Capercaillie) population in Tyrol, Austria.center dot We had similar population genetics results for both sampling approaches, revealing a well-mixed population with no signs of inbreeding.center dot Population size estimates for both sampling approaches were similar for males but differed for females.center dot We found that overall one sampling round was sufficient for genetic grouse monitoring; however, for population size estimation, a sex bias due to sex-specific behavior must be considered.center dot These results are relevant for future genetic monitoring programs, as savings cost can help facilitate longer-term monitoring, which in turn improves species conservation. In der Naturschutzbiologie wird h & auml;ufig nicht-invasiv beprobt, um genetische Parameter und Populationsgr & ouml;ss en zur Evaluierung der Populationen von seltenen oder schwer erfassbaren Arten zu ermitteln. Derartige Probennahme bringt jedoch einige Herausforderungen mit sich, wie beispielsweise eine geeignete Kartierungsmethode, die Entscheidung & uuml;ber die optimale Anzahl der zu analysierenden Proben und auch die Anzahl der Beprobungsrunden. In dieser Studie vergleichen wir den Einfluss von einer (OneRound) versus zwei (TwoRounds) nicht-invasiven Beprobungsrunden auf populationsgenetische R & uuml;ckschl & uuml;sse auf Tetrao urogallus, um einen Rahmen f & uuml;r k & uuml;nftige genetische Studien zu Raufu ss h & uuml;hnern zu schaffen. Mit Hilfe von Mikrosatellitenmarkern wurden Individuen identifiziert und populationsgenetische Parameter sowie Sch & auml;tzungen der Populationsgr & ouml;ss en zwischen den beiden Ans & auml;tzen verglichen. Unter der Voraussetzung, dass die Beprobung w & auml;hrend der Balzzeit an denselben Probenahmestellen durchgef & uuml;hrt werden, ergibt sich, dass der geringere Aufwand f & uuml;r die Feldarbeit bei OneRound insgesamt zu & auml;hnlichen Schlussfolgerungen f & uuml;r die genetischen Indizes der Population f & uuml;hrt wie der gr & ouml;ss ere Aufwand bei TwoRounds. In unserem Untersuchungsgebiet ergaben beide Ans & auml;tze eine gut vernetzte Population ohne Anzeichen von Inzucht oder Isolation. Die Sch & auml;tzungen der Populationsgr & ouml;ss e waren etwas h & ouml;her, wenn sie auf der Grundlage von OneRound-Probennahmen erfolgten, wobei es bei den m & auml;nnlichen Daten zu gro ss en & Uuml;berschneidungen (86%) mit den TwoRound Ergebnissen kam. Die gr & ouml;ss te Diskrepanz zwischen den beiden Datens & auml;tzen OneRound und TwoRounds wurde beim Vergleich der weiblichen Daten deutlich, was auf die schlechtere Auffindbarkeit der Weibchen und die damit verbundene gr & ouml;ss ere Schwankungsbreite der Beprobung zur & uuml;ckgef & uuml;hrt werden kann. Durch diese Ergebnisse kommen wir zu dem Schluss, dass OneRound der kosten-, zeit- und personeneffizientere Beprobungsansatz ist, da sehr & auml;hnliche Ergebnisse f & uuml;r verschiedene wichtige Populationsparameter mit der H & auml;lfte des Feldarbeitsaufwands erzielt werden k & ouml;nnen; bei der Entscheidung & uuml;ber die Anzahl der Beprobungsrunden sollten die Wetterdynamik des Zielgebiets sowie die Wahrscheinlichkeit, eine ausreichende Anzahl von Proben zu erhalten, jedoch ber & uuml;cksichtigt werden.

Interkingdom communication through volatile organic compounds influences interactions among organisms at a level often imperceptible to humans. Artificial settings that depend on the biotechnical exploitation of biological processes, such as the rapidly expanding sector of insect farming, are strongly affected by this often-overlooked multiway communication. Here, we aim to portray the significance of interkingdom communication influencing insect behavior. We use the black soldier fly (Hermetia illucens) as a model system to introduce the necessary actions to improve our understanding of communication between insects and microbes. Successful exploration of this phenomenon could transform the bioeconomy by improving insect mass-rearing processes and enhancing insect welfare. By interlocking behavioral and molecular ecology, chemistry, microbiology, and bioinformatics, we can uncover the molecular mechanisms underlying these interactions and develop practical applications for improved industrial practices. Future work should focus on pursuing research avenues to untangle the interwoven nature of insect behavior and microbial communities.

Once widespread in Europe, the lesser horseshoe bat (Rhinolophus hipposideros) suffered massive population declines in the second half of the 20th century and became extinct in some countries. For future conservation programs, it is important to understand the major causes of extinction. Here, we compared extinct and extant populations in Bavaria (Germany) and Tyrol (Austria) concerning the availability of roof trusses as roosts, as well as concerning heavy metals found in feces (lead, Pb; cadmium, Cd), persistent organic pollutants in the air of the roosts (lindane, dichlorodiphenyldichloroethane, dichlorodiphenyldichloroethylene, dichlorodiphenyltrichloroethane (DDT), and pentachloroanisole), landscape parameters (broadleaf forest, cropland, urbanisation), and light-pollution proxies (minimum and maximum radiance). We did not detect any recolonisation in extinct populations. One third of the buildings concerned are currently inaccessible for R. hipposideros because of closed entrances. Most roosts contained residues of lindane and pentachloroanisole, some of DDT. Pb and Cd concentrations were significantly higher for extinct than for extant colonies; these contaminations may at least partly explain the lack of recolonisation for the two thirds of buildings with open entrances. None of the parameters analyzed correlated significantly with the size of extant colonies. For further insight in the conservation biology of the lesser horseshoe bat in the Greater Alpine Space, assessment of transitional and hibernation roosts, linear habitat elements around roosts, and food availability in the face of insect decline will be necessary.

Aggression has multiple benefits and is often coupled with other behaviors ("behavioral syndromes"). The level of aggressiveness is influenced by an adaptive benefit-cost ratio suggesting that benefits should outweigh the costs of aggression. Here, we assess if several behaviors are coupled in two behaviorally different populations (aggressive, peaceful) of the high-elevation ant Tetramorium alpestre. For three weeks, we collected colony fragments and analyzed boldness, exploring, foraging, and risk-taking behaviors. We hypothesized that the aggressive population is bolder, more explorative and risk-prone, and forages more food than the peaceful population. To test whether (a) the combination of experiments and parameters used yields a good setup, (b) populations differ behaviorally, and (c) populations display behavioral syndromes, we assessed (a) the frequency of repeatable behaviors of each experiment, (b) the behavioral means among populations, and (c) the behavioral repeatability, respectively. We found that (a) boldness and exploring were most repeatable and represent a good experimental setup, (b) the aggressive population was bolder and more explorative and risk-prone than the peaceful population, (c) boldness and exploring behaviors were highly repeatable in both populations, thus corroborating our hypothesis. The results suggest that boldness, exploring, and risk-taking but not foraging are presumably coupled with aggression and indicate the presence of behavioral syndromes in this ant. Under specific ecological conditions, aggression may be coupled with other behaviors and important for finding food. Aggression is probably adaptive in T. alpestre, possibly indicating that selection favors aggression at least partially, which may counteract the complete loss of intraspecific aggression.

The origin and evolution in the Southern Hemisphere of the Austral teals, consisting of the grey-teal and brown-teal species complexes, remains poorly understood owing to limited molecular data. With the group containing multiple independent examples of flight loss, understanding the evolutionary history of the group is of significant interest for functional genomic studies into the evolution of flightlessness. Here, we present the first whole-mitogenome-based phylogeny of the Austral teals. We show that the group diverged from a common ancestor with mallards in the late Miocene and soon after radiated into the brown-teal and grey-teal lineages, in addition to the widely distributed pintails and green-winged teals. The brown-teal species complex, which includes the volant brown teal, the flight-impaired, sub-Antarctic Auckland Island teal, and the flightless, Chatham Island and sub-Antarctic Campbell Island teals, radiated within the past 0.9-1.8 Myr. The divergence of the extinct Chatham Island teal and the colonization of the Auckland and Campbell Islands occurred from mainland New Zealand. Morphological changes towards flightlessness are also present in the volant brown teal on mainland New Zealand, suggesting that this group was on the pathway to flightlessness, a trend that accelerated in some insular island lineages.

Population size is an important parameter to monitor for species conservation and management. This is especially important for rare and endangered species, as declines can give information about anthropogenic impacts and the need for new conservation measures. To estimate population size, various methods of analysis can be used, for which sample size is an important factor. Sample size is particularly important to consider when applying non‐invasive sampling strategies such as sampling faeces or feathers/hairs as a source of DNA, as a means to limit disturbance and stress for the species of concern. We investigated a Black Grouse Lyrurus tetrix population in the eastern part of the Alps, in East Tyrol (Austria), and estimated population size using two approaches: capture–recapture and rarefaction. With a set of 12 polymorphic microsatellite markers, we identified genotypes from faeces and feathers (backed up with 23 tissue samples) and checked for population substructure and gene flow among sampling sites. We estimated population size using four different models from the two approaches (molecular capture–recapture: TIRM, TIRMpart; rarefaction: hyperbolic function – Kohn, exponential function – Eggert). To evaluate the impact of sample size on the estimations, we used the full dataset of 500 samples (‘complete’ dataset) and half the dataset of 250 samples (‘half’ dataset). We also estimated the population size for each sex separately using complete and half datasets to check for sex‐specific differences in population size. We found similar results in three of four models (capture–recapture: capwire TIRM, capwire TIRMpart; rarefaction: rarefaction Kohn). Using just half of the data increased the uncertainties in the estimation of population size in all models used and deviations were particularly large in females, which indicated a sex bias. Only the complete dataset of males had an observation rate of more than two observations/individual, and this observation rate meets the recommendation for using the capwire models. This indicates that, for species with different sex‐specific detectability, larger sample sizes do not generally imply higher observation rates. We conclude that calculating the observation rates and population‐size estimations for each sex separately can improve overall population‐size estimation, especially in species with biased sex ratios and those that exhibit sex‐specific behaviour.

The specific model settings for the Bayesian model approach using brms. MBI agg = Mean Behaviour Index aggressive; Kue = Kühtai, a population collected in Austria; Pen = Penser Joch, a population collected in Italy. Iter = iterations used, thin = thinning interval, n chains = number of chains, WAIC = Watanabe-Akaike information criterion, LOO = leave-one-out cross-validation.