Patterns of sexual dimorphism in the armoured tardigrades.

Sexual dimorphism is widespread among animals, with diverse patterns and proposed explanations observed across the Tree of Life. Here we present the first formal analysis of the patterns of sexual dimorphism in body size and cephalic sensory appendages across 40 species (from 10 genera) of armoured tardigrades (Echiniscidae). Phylogenetic signal was found for body size traits and the cephalic papilla relative size, indicating that the association between these traits between the sexes has high evolutionary persistence. The Echiniscidae body size dimorphism is generally female-biased, which would be in accordance with the fecundity hypothesis. No strong evidence of allometric patterns of body size sexual dimorphism was found. In contrast, some of the cephalic appendages show male-biased sexual dimorphism, particularly those that, by being more innervated, are thought to function as chemodetection organs used by males during mate search. The latter is consistent with the sexual selection hypothesis. As the first systematic quantification and analysis of the patterns of sexual dimorphism in the phylum Tardigrada, this study provides important insights into their ecology and evolution, such as corroborating the suggestion that cephalic appendages evolved for mate searching.

Expanding Acutuncus: Phylogenetics and morphological analyses reveal a considerably wider distribution for this tardigrade genus.

The tardigrade genus Acutuncus has been long thought to be an Antarctic endemism, well adapted to this harsh environment. The Antarctic endemicity of Acutuncus was recently dispelled with the description of Acutuncus mariae Zawierucha, 2020 found in the Svalbard archipelago. The integrated analyses on two newly found Acutuncus populations from UK and Italy, and a population of Acutuncus antarcticus found close to its type locality allowed us to expand the climatic and geographic range of the genus Acutuncus. These findings also allowed us to re-evaluate the morphological diagnoses of Acutuncus and accommodate it in the newly proposed monotypic family Acutuncidae fam. nov. Two new Acutuncus species morpho-groups are instituted based on eggs morphology: one (Acutuncus antarcticus morphogroup) including the Antarctic Acutuncus taxa characterized by eggs with long pillars within the chorion and eggs laid freely to the environment, the other (Acutuncus mariae morphogroup) including the European species, characterized by eggs with short pillars within the chorion and eggs laid in the exuvium. Finally, we describe two new Acutuncus species from Europe: Acutuncus mecnuffisp. nov. and Acutuncus giovanniniaesp. nov.

First evidence of sex-specific responses to chemical cues in tardigrade mate searching behaviour.

Chemical cues are widely used in intraspecific and interspecific communication, either as substances deposited in the substrate or as molecules diffused in water or air. In tardigrades, an emerging microscopic study system, chemical communication and its role in reproduction are poorly known. Here, we assessed sex differences in the detection of (a) short-range diffusing signals and (b) deposited cue trails during the mate-searching behaviour of freely moving virgin male and female Macrobiotus polonicus. We tracked individual behaviour (a) in simultaneous double-choice chambers, where live conspecifics of each sex were presented in water and (b) of freely moving pairs on agar without water. We found that males, but not females, preferentially associated with opposite-sex individuals in trials conducted in water. In contrast, neither sex detected nor followed cues deposited on agar. In conclusion, our study suggests that mate discrimination and approach are male-specific traits and are limited to waterborne chemical cues. These results support the existence of Darwinian sex roles in pre-mating behaviour in an animal group with virtually non-existing sex differences in morphology or ecology.

New multilocus phylogeny reorganises the family Macrobiotidae (Eutardigrada) and unveils complex morphological evolution of the Macrobiotus hufelandi group.

The family Macrobiotidae is one of the most speciose and diverse groups among tardigrades. Although there have been attempts to reconstruct the phylogeny of this family, the evolutionary relationships within Macrobiotidae are only superficially determined as available genetic data cover only a small fraction of this vast group. Here, we present the first extensive molecular phylogeny of the family based on four molecular markers (18S rRNA, 28Sr RNA, ITS-2 and COI) associated with detailed morphological data for the majority of taxa. The phylogenetic analysis includes nearly two hundred sequences representing more than sixty species, including sixteen taxa that have never been sequenced and/or analysed phylogenetically before. Our results recovered a new monophyletic group, comprising Macrobiotus spectabilis Thulin, 1928 and Macrobiotus grandis Richters, 1911, for which we erect a new genus, Sisubiotusgen. nov., to accommodate its evolutionary distinctiveness. The largest, so far, dataset for the family Macrobiotidae showed that the genus Xerobiotus is nested within the clade representing the genus Macrobiotus deeper than it was earlier assumed, therefore we propose to suppress Xerobiotus and transfer its species to Macrobiotus. Moreover, mapping key morphological traits onto macrobiotid phylogeny exposed complex evolution of phenotypes within the Macrobiotus hufelandi group, i.e. Macrobiotus s.s. Finally, our findings enabled a detailed revision and discussion on species compositions of the most ubiquitous tardigrade genera, species groups and species complexes, which resulted in changes of taxonomic statuses of a number of macrobiotid species. All this contributes to the reconstruction of the morphological evolution within Macrobiotidae.

Nosy neighbours: large broods attract more visitors. A field experiment in the pied flycatcher, Ficedula hypoleuca.

Life is uncertain. To reduce uncertainty and make adaptive decisions, individuals need to collect information. Individuals often visit the breeding sites of their conspecifics (i.e., "prospect"), likely to assess conspecifics' reproductive success and to use such information to identify high-quality spots for future breeding. We investigated whether visitation rate by prospectors and success of visited sites are causally linked. We manipulated the reproductive success (enlarged, reduced, and control broods) in a nest-box population of migratory pied flycatchers, Ficedula hypoleuca, in Finland. We measured the visitation rates of prospectors at 87 nest-boxes continuously from manipulation (day 3 after hatching) to fledging. 302 adult pied flycatchers prospected 9194 times on these manipulated nests (at least 78% of detected prospectors were successful breeders). While the number of visitors and visits was not influenced by the relative change in brood size we induced, the resulting absolute brood size predicted the prospecting behaviour: the larger the brood size after manipulation, the more visitors and visits a nest had. The parental provisioning rate at a nest and brood size pre-manipulation did not predict the number of visitors or visits post-manipulation. More visitors, however, inspected early than late nests and broods in good condition. Our study suggests that individuals collect social information when visiting conspecific nests during breeding and provides evidence that large broods attract more visitors than small broods. We discuss the results in light of individual decision-making by animals in their natural environments.

Predator encounters have spatially extensive impacts on parental behaviour in a breeding bird community.

Predation risk has negative indirect effects on prey fitness, partly mediated through changes in behaviour. Evidence that individuals gather social information from other members of the population suggests that events in a community may impact the behaviour of distant individuals. However, spatially wide-ranging impacts on individual behaviour caused by a predator encounter elsewhere in a community have not been documented before. We investigated the effect of a predator encounter (hawk model presented at a focal nest) on the parental behaviour of pied flycatchers (Ficedula hypoleuca), both at the focal nest and at nearby nests different distances from the predator encounter. We show that nest visitation of both focal pairs and nearby pairs were affected, up to 3 h and 1 h, respectively. Parents also appeared to compensate initial disrupted feeding by later increasing nest visitation rates. This is the first evidence showing that the behaviour of nearby pairs was affected away from an immediate source of risk. Our results indicate that the impacts of short-term predator encounters may immediately extend spatially to the broader community, affecting the behaviour of distant individuals. Information about predators is probably quickly spread by cues such as intra- and heterospecific alarm calls, in communities of different taxa.

Resolving variation in the reproductive tradeoff between sperm size and number.

Spermatozoa are amongst the most variable cells, and three factors are thought to account for this variation in design: fertilization mode, phylogeny, and postcopulatory sexual selection. In addition, it has long been assumed that a tradeoff exists between sperm size and number, and although postcopulatory sexual selection affects both traits, empirical evidence for a tradeoff has so far been elusive. Our recent theoretical model predicts that the nature of a direct tradeoff between sperm size and number varies with sperm competition mechanism and sperm competition risk. We test these predictions using a comparative approach in two very different taxa with different sperm competition mechanisms: passerine birds (mechanism: simple raffle) and Drosophila fruit flies (sperm displacement). We show that in both groups, males increase their total ejaculate investment with increasing sperm competition risk, but whereas passerine birds allocate disproportionately to sperm number, drosophilids allocate disproportionately to sperm size. This striking difference between the two groups can be at least partly explained by sperm competition mechanisms depending on sperm size relative to the size of the female reproductive tract: in large animals (passerines), sperm numbers are advantageous in sperm competition owing to dilution inside the female tract, whereas in small animals (drosophilids), large sperm are advantageous for physical competition (sperm displacement). Our study provides two important results. First, we provide convincing evidence for the existence of a sperm size-number tradeoff. Second, we show that by considering both sperm competition mechanism and dilution, can we account for variation in sperm size between different taxa.

Testes size seen through the glass of amphibian care.

Despite the great diversity of parental care types found in amphibians, studies linking them to post-copulatory sexually selected traits are scarce, presumably due to a lack of data. Valencia-Aguilar et al. used fieldwork and museum collections to show that paternal care appears to trade-off with testes size in glass frogs.

A first look into moss living tardigrades in boreal peatlands.

Tardigrades (Tardigrada) are a phylum of micrometazoans found in all biomes on Earth, but their ecology and habitat preferences remain vastly understudied. Boreal peatlands include a diversity of habitat types and high structural heterogeneity that represents an interesting system to study some of the poorly known habitat preferences of tardigrades. Here, we investigate for the first time tardigrade communities in peatland mosses and the latter's potential associations with key environmental variables. We collected 116 moss samples from 13 sites representing different peatland types and management histories. We found that tardigrades are common and diverse in boreal peatlands, as tardigrades were present in 72% of the collected samples and we identified 14 tardigrade genera. Tardigrade abundance seemed to increase alongside the increasing tree basal area and the density was higher in the microtopographic level further from the water table level, that is, hummocks (mean 117/moss gram) than in lawns/hollows (mean 84/moss gram). Furthermore, the highest tardigrade density was found in the moss taxa that are associated with forested peatland types (i.e., feather mosses) (321 mean/moss gram). Finally, we found interesting patterns regarding tardigrade functional diversity, as carnivorous tardigrades were found only in peatlands with tree basal area > 20 m2 and mostly in hummocks. Our study demonstrates that the habitat heterogeneity of peatlands (e.g., variation in moisture and vegetation cover) represents an interesting system to study tardigrade ecology and habitat preferences. However, since we found variation in tardigrade abundance and communities across peatland types and microhabitats within peatlands, our results highlight that such studies should be conducted with numerous replicate samples and a systematic study design that properly addresses the habitat heterogeneity between and within different peatland types.

Glutaraldehyde-enhanced autofluorescence as a general tool for 3D morphological imaging.

Routine histochemical techniques are capable of producing vast amount of information from diverse samples types, but these techniques are limited in their ability to generate 3D information. Autofluorescence imaging can be used to analyse samples in 3D but it suffers from weak/low signal intensities. Here, we describe a simple chemical treatment with glutaraldehyde to enhance autofluorescence for 3D fluorescence imaging and to generate detailed morphological images on whole-mount samples. This methodology is straightforward and cost-effective to implement, suitable for a wide range of organisms and sample types. Furthermore, it can be readily integrated with standard confocal and fluorescence microscopes for analysis. This approach has the potential to facilitate the analysis of biological 3D structures and research in developmental biology, including studies on model and non-model organisms.

The impact of life stage and pigment source on the evolution of novel warning signal traits.

Our understanding of how novel warning color traits evolve in natural populations is largely based on studies of reproductive stages and organisms with endogenously produced pigmentation. In these systems, genetic drift is often required for novel alleles to overcome strong purifying selection stemming from frequency-dependent predation and positive assortative mating. Here, we integrate data from field surveys, predation experiments, population genomics, and phenotypic correlations to explain the origin and maintenance of geographic variation in a diet-based larval pigmentation trait in the redheaded pine sawfly (Neodiprion lecontei), a pine-feeding hymenopteran. Although our experiments confirm that N. lecontei larvae are indeed aposematic-and therefore likely to experience frequency-dependent predation-our genomic data do not support a historical demographic scenario that would have facilitated the spread of an initially deleterious allele via drift. Additionally, significantly elevated differentiation at a known color locus suggests that geographic variation in larval color is currently maintained by selection. Together, these data suggest that the novel white morph likely spread via selection. However, white body color does not enhance aposematic displays, nor is it correlated with enhanced chemical defense or immune function. Instead, the derived white-bodied morph is disproportionately abundant on a pine species with a reduced carotenoid content relative to other pine hosts, suggesting that bottom-up selection via host plants may have driven divergence among populations. Overall, our results suggest that life stage and pigment source can have a substantial impact on the evolution of novel warning signals, highlighting the need to investigate diverse aposematic taxa to develop a comprehensive understanding of color variation in nature.

First insights into female sperm storage duration in tardigrades.

Female sperm storage is ubiquitous in the animal kingdom and it has been shown to be linked to several evolutionary processes, from postcopulatory sexual selection to dispersal. Here we report, for the first time, long-term sperm storage in females of the tardigrade Macrobiotus polonicus. Females, isolated after a short contact with a male, were able to use the stored sperm for up to 5 weeks (mean of 2 weeks), which translates to a considerable proportion of female post-mating longevity under controlled laboratory conditions (60% on average). Our study provides the first insights into the duration of sperm storage, an underexplored feature of the reproductive biology of tardigrades. Additionally, we discuss important considerations for reproductive studies on these non-model animals.

Macrobiotus naginae sp. nov., a New Xerophilous Tardigrade Species from Rokua Sand Dunes (Finland).

Animals that colonize soil show specific adaptations to soil. Compared to closely related species living on the surface, the limbs of soil-dwelling animals are often shortened, reduced, or absent to allow a less restricted passage through cavities between soil particles. This pattern of limb reduction has also been observed in tardigrades, where multiple lineages that colonized the below-ground habitat show independent reduction and/or loss of legs and claws. In the tardigrade superfamily Macrobiotoidea, leg and claw reductions are a common trait found in the Macrobiotus pseudohufelandi complex. This rarely found species complex currently contains four nominal taxa. Here we describe, with the use of integrative taxonomy, Macrobiotus naginae sp. nov., a new species in the Macrobiotus pseudohufelandi complex from inland sand dunes in Finland. We also provide a dichotomous key to the Macrobiotus pseudohufelandi complex to assist with their identification in future studies.

Integrative taxonomy resolves species identities within the Macrobiotus pallarii complex (Eutardigrada: Macrobiotidae).

The taxonomy of many groups of meiofauna is challenging due to their low number of diagnostic morphological characters and their small body size. Therefore, with the advent of molecular techniques that provide a new source of traits, many cryptic species have started to be discovered. Tardigrades are not an exception, and many once thought to be cosmopolitan taxa are being found to be complexes of phenotypically similar species. Macrobiotus pallarii Maucci, 1954 was originally described in South Italy and has been subsequently recorded in Europe, America, and Asia. This allegedly wide geographic range suggests that multiple species may be hidden under this name. Moreover, recently, genetic evidence to support this was put forward, and the Macrobiotus pallarii complex has been proposed to accommodate putative species related to M. pallarii. Here, we describe three new pseudocryptic species based on populations that would have been all classified as Macrobiotus pallarii if molecular methods were not employed. Using an integrative taxonomy approach, we analyzed animals and eggs from the topotypic population of Macrobiotus pallarii, together with four other populations of the complex. We recovered four distinct phylogenetic lineages that, despite the overlap of morphometric traits, can be separated phenotypically by subtle but discrete morphological characters. One lineage corresponds to Macrobiotus pallarii, whereas the other three are newly described as Macrobiotus margoae Stec, Vecchi & Bartels, sp. nov. from the USA, Macrobiotus ripperi Stec, Vecchi & Michalczyk, sp. nov. from Poland and Finland, and Macrobiotus pseudopallarii Stec, Vecchi & Michalczyk, sp. nov. from Montenegro. To facilitate species identification, we provide a dichotomous key for species of the M. pallarii complex. Delimitation of these pseudocryptic taxa highlights the need for an integrative approach to uncover the phylum's diversity in full.

Tardigrades of Finland: new records and an annotated checklist.

Species checklists are powerful and important tools of communication between taxonomists and applied environmental biologists, which in turn lead to well-planned and successful conservation strategies and ecological studies. Despite this, only recently the interest on compiling systematic checklists is growing among taxonomists who study tardigrades-micrometazoans that inhabit almost every habitat worldwide. As the Finnish records of tardigrades (a.k.a. water bears) species are incomplete, outdated and no checklist has ever been compiled for this country, an easy-to-consult checklist is here reported. This checklist covers all Finnish tardigrade taxa identified in the past and in the 13 samples collected for this study. A total of 68 tardigrade species are recorded from Finland, with 6 of them being new records presented in this contribution. Of these species, four have their loci tipici in Finland and we provide an English translation of their original German descriptions. A Generalised Linear Model was used to test the effect of sampling effort and area size on the number of species recorded in each biogeographical province of Finland. The results showed that geographical differences in species richness can be explained solely by sampling effort. The number of tardigrade species recorded in Finland corresponds to about 5% of all described species in the phylum, thus indicating a potential high richness for this country. However, the results of the Generalised Linear Model highlight that a reliable knowledge of the tardigrade diversity in Finland will be reached only with a more uniform and intensive sampling effort.

Intraspecific variation in testis asymmetry in birds: evidence for naturally occurring compensation.

In many taxa, the left and right testes often differ in size. The compensation hypothesis states that one testis of the pair serves as a 'back-up' for any reduced function in the other and provides a mechanism to explain intraspecific variation in degree and direction of gonad asymmetry. Although testis asymmetry is common in birds, evidence for natural testis compensation is unknown. Using a novel quantitative approach that can be applied to any bilateral organ or structure, we show that testis compensation occurs naturally in birds and can be complete when one testis fails to develop. Owing to a recurrent risk of testis impairment and an evolutionary trade-off between natural and sexual selections acting on the arrangement of internal organs in species with abdominal and/or seasonal testes, compensation adds an important, but neglected, dimension to measures of male reproductive investment.

Sperm morphology and sperm velocity in passerine birds.

Sperm velocity is one of the main determinants of the outcome of sperm competition. Since sperm vary considerably in their morphology between and within species, it seems likely that sperm morphology is associated with sperm velocity. Theory predicts that sperm velocity may be increased by enlarged midpiece (energetic component) or flagellum length (kinetic component), or by particular ratios between sperm components, such as between flagellum length and head size. However, such associations have rarely been found in empirical studies. In a comparative framework in passerine birds, we tested these theoretical predictions both across a wide range of species and within a single family, the New World blackbirds (Icteridae). In both study groups, sperm velocity was influenced by sperm morphology in the predicted direction. Consistent with theoretical models, these results show that selection on sperm morphology and velocity are likely to be concomitant evolutionary forces.

Increased postcopulatory sexual selection reduces the intramale variation in sperm design.

Sperm competition is an important force driving the evolution of sperm design and function. Inter- and intraspecific variation in sperm design are strongly influenced by the risk of sperm competition in many taxa. In contrast, the variation among sperm of one male (intramale variation) is less well understood. We investigated intramale variation in sperm design in passerine birds and found that risk of sperm competition is negatively associated with intramale variation. This result is the first clear evidence that variation among sperm within an individual male is influenced by postcopulatory sexual selection. Our finding has important implications for male traits under pre- and postcopulatory sexual selection.

Fungal spore diversity reflects substrate-specific deposition challenges.

Sexual spores are important for the dispersal and population dynamics of fungi. They show remarkable morphological diversity, but the underlying forces driving spore evolution are poorly known. We investigated whether trophic status and substrate associations are associated with morphology in 787 macrofungal genera. We show that both spore size and ornamentation are associated with trophic specialization, so that large and ornamented spores are more probable in ectomycorrhizal than in saprotrophic genera. This suggests that spore ornamentation facilitates attachment to arthropod vectors, which ectomycorrhizal species may need to reach lower soil layers. Elongated spore shapes are more common in saprotrophic taxa, and genera associated with above ground substrates are more likely to have allantoid (curved elongated) spores, probably to lower the risk of wash out by precipitation. Overall, our results suggest that safe arrival on specific substrates is a more important driver of evolution in spore morphology than dispersal per se.