Evolutionary history of an Alpine Archaeognath (Machilis pallida): Insights from different variant.

Reconstruction of species histories is a central aspect of evolutionary biology. Patterns of genetic variation within and among populations can be leveraged to elucidate evolutionary processes and demographic histories. However, interpreting genetic signatures and unraveling the contributing processes can be challenging, in particular for non-model organisms with complex reproductive modes and genome organization. One way forward is the combined consideration of patterns revealed by different molecular markers (nuclear vs. mitochondrial) and types of variants (common vs. rare) that differ in their age, mode, and rate of evolution. Here, we applied this approach to RNAseq data generated for Machilis pallida (Archaeognatha), an Alpine jumping bristletail considered parthenogenetic and triploid. We generated de novo transcriptome and mitochondrial assemblies to obtain high-density data to investigate patterns of mitochondrial and common and rare nuclear variation in 17 M. pallida individuals sampled from all known populations. We find that the different variant types capture distinct aspects of the evolutionary history and discuss the observed patterns in the context of parthenogenesis, polyploidy, and survival during glaciation. This study highlights the potential of different variant types to gain insights into evolutionary scenarios even from challenging but often available data and the suitability of M. pallida and the genus Machilis as a study system for the evolution of sexual strategies and polyploidization during environmental change. We also emphasize the need for further research which will be stimulated and facilitated by these newly generated resources and insights.

Global change may make hostile - Higher ambient temperature and nitrogen availability increase ant aggression.

Behaviour is a response of organisms to internal and external stimuli and comprises various activities such as searching for food. Aggression is important in such activities, for example, improving the chances of winning competition for food, but animals differ in their level of aggression. This behavioural plasticity allows individuals to respond to environmental changes and is important for the survival of animals. It may be an important asset in facing global changes, which affect all organisms, for example, via rising temperature and eutrophication. The latter have steadily increased since 1900, especially in high elevations. Their effects may first become visible in stationary organisms such as ants because their nests are strictly associated with the conditions on site. Here, we analysed eight populations of the high-elevation ant Tetramorium alpestre along several elevations spanning the European Alps. We conducted a correlative approach and analysed several genetic and environmental proxies, namely within- and across-colony genetic relatedness, cuticular hydrocarbons, body size, across-colony geographic distance, air temperature, and worker nitrogen values additionally to within-population aggressive behaviour. We hypothesised that a) these proxies and aggressive behaviour differ among populations and that b) one or more of these proxies influence aggression. We found that a) some environmental proxies and aggression differed among populations but not the genetic proxies and that b) air temperature and worker nitrogen-isotope values correlated positively with worker aggression. The results indicate an environmental but not social-structural influence on this ant's aggressive behaviour, even though social structure varied among populations (single- and multiple-queened colonies). We infer that global change affects aggression in our study system and propose five mutually non-exclusive scenarios to explain the behavioural change mechanistically. Using the space-for-time principle, we speculate that aggression may increase due to future increases in temperature and nitrogen availability in this ant and other species living in high elevations.

Differential gene expression in Drosophila melanogaster and D. nigrosparsa infected with the same Wolbachia strain.

Wolbachia are maternally inherited endosymbionts that infect nearly half of all arthropod species. Wolbachia manipulate their hosts to maximize their transmission, but they can also provide benefits such as nutrients and resistance against viruses to their hosts. The Wolbachia strain wMel was recently found to increase locomotor activities and possibly trigger cytoplasmic incompatibility in the transinfected fly Drosophila nigrosparsa. Here, we investigated, in females of both D. melanogaster and D. nigrosparsa, the gene expression between animals uninfected and infected with wMel, using RNA sequencing to see if the two Drosophila species respond to the infection in the same or different ways. A total of 2164 orthologous genes were used. The two fly species responded to the infection in different ways. Significant changes shared by the fly species belong to the expression of genes involved in processes such as oxidation-reduction process, iron-ion binding, and voltage-gated potassium-channel activity. We discuss our findings also in the light of how Wolbachia survive within both the native and the novel host.

Contribution to the molecular phylogeny of Sciaridae (Diptera) with special attention to the genera emBradysia/em and emCorynoptera/em.

The systematics of the dipteran family Sciaridae is based mainly on morphological characters and has remained quite controversial. In this study, we used two mitochondrial DNA markers (CO1, 16S) and a nuclear one (28S) to take a glimpse into phylogenetic relationships of part of the North and Central European Sciaridae. A total of 91 species from 19 genera were analysed using Maximum Likelihood based phylogenetics (depending on the availability of valid sequences, 50-70 per gene). We strengthen the suggestion of the Chaetosciara group as an independent subfamily. Within the subfamily Megalosphyinae, two separate Bradysia clades were identified, suggesting a close relation between the genera Zygoneura, Austrosciara, and Scatopsciara. The genus Alpinosciara gen. n. is established to place the species of the former Corynoptera crassistylata group inside the subfamily Megalosphyinae. This new genus now includes 22 species.

Effect of social structure and introduction history on genetic diversity and differentiation.

Invasive species are a global threat to biodiversity, and understanding their history and biology is a major goal of invasion biology. Population-genetic approaches allow insights into these features, as population structure is shaped by factors such as invasion history (number, origin and age of introductions) and life-history traits (e.g., mating system, dispersal capability). We compared the relative importance of these factors by investigating two closely related ants, Tetramorium immigrans and Tetramorium tsushimae, that differ in their social structure and invasion history in North America. We used mitochondrial DNA sequences and microsatellite alleles to estimate the source and number of introduction events of the two species, and compared genetic structure among native and introduced populations. Genetic diversity of both species was strongly reduced in introduced populations, which also differed genetically from native populations. Genetic differentiation between ranges and the reduction in microsatellite diversity were more severe in the more recently introduced and supercolonial T. tsushimae. However, the loss of mitochondrial haplotype diversity was more pronounced in T. immigrans, which has single-queen colonies and was introduced earlier. Tetramorium immigrans was introduced at least twice from Western Europe to North America and once independently to South America. Its monogyny might have limited genetic diversity per introduction, but new mutations and successive introductions over a long time may have added to the gene pool in the introduced range. Polygyny in T. tsushimae probably facilitated the simultaneous introduction of several queens from a Japanese population to St. Louis, USA. In addition to identifying introduction pathways, our results reveal how social structure can influence the population-genetic consequences of founder events.

Performance comparison of two reduced-representation based genome-wide marker-discovery strategies in a multi-taxon phylogeographic framework.

Multi-locus genetic data are pivotal in phylogenetics. Today, high-throughput sequencing (HTS) allows scientists to generate an unprecedented amount of such data from any organism. However, HTS is resource intense and may not be accessible to wide parts of the scientific community. In phylogeography, the use of HTS has concentrated on a few taxonomic groups, and the amount of data used to resolve a phylogeographic pattern often seems arbitrary. We explore the performance of two genetic marker sampling strategies and the effect of marker quantity in a comparative phylogeographic framework focusing on six species (arthropods and plants). The same analyses were applied to data inferred from amplified fragment length polymorphism fingerprinting (AFLP), a cheap, non-HTS based technique that is able to straightforwardly produce several hundred markers, and from restriction site associated DNA sequencing (RADseq), a more expensive, HTS-based technique that produces thousands of single nucleotide polymorphisms. We show that in four of six study species, AFLP leads to results comparable with those of RADseq. While we do not aim to contest the advantages of HTS techniques, we also show that AFLP is a robust technique to delimit evolutionary entities in both plants and animals. The demonstrated similarity of results from the two techniques also strengthens biological conclusions that were based on AFLP data in the past, an important finding given the wide utilization of AFLP over the last decades. We emphasize that whenever the delimitation of evolutionary entities is the central goal, as it is in many fields of biodiversity research, AFLP is still an adequate technique.

The Core Gut Microbiome of Black Soldier Fly (Hermetia illucens) Larvae Raised on Low-Bioburden Diets.

An organism's gut microbiome handles most of the metabolic processes associated with food intake and digestion but can also strongly affect health and behavior. A stable microbial core community in the gut provides general metabolic competences for substrate degradation and is robust against extrinsic disturbances like changing diets or pathogens. Black Soldier Fly larvae (BSFL; Hermetia illucens) are well known for their ability to efficiently degrade a wide spectrum of organic materials. The ingested substrates build up the high fat and protein content in their bodies that make the larvae interesting for the animal feedstuff industry. In this study, we subjected BSFL to three distinct types of diets carrying a low bioburden and assessed the diets' impact on larval development and on the composition of the bacterial and archaeal gut community. No significant impact on the gut microbiome across treatments pointed us to the presence of a predominant core community backed by a diverse spectrum of low-abundance taxa. Actinomyces spp., Dysgonomonas spp., and Enterococcus spp. as main members of this community provide various functional and metabolic skills that could be crucial for the thriving of BSFL in various environments. This indicates that the type of diet could play a lesser role in guts of BSFL than previously assumed and that instead a stable autochthonous collection of bacteria provides the tools for degrading of a broad range of substrates. Characterizing the interplay between the core gut microbiome and BSFL helps to understand the involved degradation processes and could contribute to further improving large-scale BSFL rearing.

Wolbachia affect behavior and possibly reproductive compatibility but not thermoresistance, fecundity, and morphology in a novel transinfected host, Drosophila nigrosparsa.

Wolbachia, intracellular endosymbionts, are estimated to infect about half of all arthropod species. These bacteria manipulate their hosts in various ways for their maximum benefits. The rising global temperature may accelerate species migration, and thus, horizontal transfer of Wolbachia may occur across species previously not in contact. We transinfected and then cured the alpine fly Drosophila nigrosparsa with Wolbachia strain wMel to study its effects on this species. We found low Wolbachia titer, possibly cytoplasmic incompatibility, and an increase in locomotion of both infected larvae and adults compared with cured ones. However, no change in fecundity, no impact on heat and cold tolerance, and no change in wing morphology were observed. Although Wolbachia increased locomotor activities in this species, we conclude that D. nigrosparsa may not benefit from the infection. Still, D. nigrosparsa can serve as a host for Wolbachia because vertical transmission is possible but may not be as high as in the native host of wMel, Drosophila melanogaster.

Long-term isolation of European steppe outposts boosts the biome's conservation value.

The European steppes and their biota have been hypothesized to be either young remnants of the Pleistocene steppe belt or, alternatively, to represent relicts of long-term persisting populations; both scenarios directly bear on nature conservation priorities. Here, we evaluate the conservation value of threatened disjunct steppic grassland habitats in Europe in the context of the Eurasian steppe biome. We use genomic data and ecological niche modelling to assess pre-defined, biome-specific criteria for three plant and three arthropod species. We show that the evolutionary history of Eurasian steppe biota is strikingly congruent across species. The biota of European steppe outposts were long-term isolated from the Asian steppes, and European steppes emerged as disproportionally conservation relevant, harbouring regionally endemic genetic lineages, large genetic diversity, and a mosaic of stable refugia. We emphasize that conserving what is left of Europe's steppes is crucial for conserving the biological diversity of the entire Eurasian steppe biome.

Towards an evolutionary history of European-Alpine Trechus ground beetles: Species groups and wing reduction.

The evolution of flight triggered the rise of pterygote insects, but secondary flightlessness has evolved numerous times and is often associated with reduced gene flow among populations and patterns of diversification. With 85 species most of which are wing reduced, the ground beetle genus Trechus in the European Alps may be one such example. Here, we reconstructed a molecular phylogeny using 72 of these species based on mitochondrial and nuclear DNA sequences as a basis for reconstructing their evolutionary history. We rearranged the species into 20 monophyletic species groups, of which five are novel and 15 were already established but with different species allocated. Wing measurements revealed a strong tendency for wing reduction but also variation within and among species, with the few fully-winged species distributed across multiple species groups containing also wing-reduced species. Using character mapping and phylogenetic independent contrasts, we found that neither distribution area, body size, pigmentation, elevational zone, nor hygrophily explained wing status in our sample. Assessing five completely sampled clades, we inferred that each of their ancestors had most likely already been wing reduced. We discuss putative scenarios explaining this pattern and the presence of wing polymorphism across the phylogeny. One plausible scenario would be an already wing-reduced last common ancestor of all Trechus species and multiple regains of full wing length via back mutation and/or hybridisation. Alternatively and possibly more likely, the ancestors were either fully winged, with subsequent rapid and repeated wing reduction explaining the current wing-status pattern, or polymorphic, with long-term polymorphism or reselection acting on standing genetic variation explaining the recent fully-winged species. Either way, Trechus ground beetles are a promising, taxonomically and ecologically diverse system for studying the evolution of flightlessness. Areas for future research include morphological assessment of flight muscles, functional analysis of flight capability, and exploration of the mechanistic and genetic bases of wing and flight evolution.

Correction: Influence of three artificial light sources on oviposition and half-life of the Black Soldier Fly, Hermetia illucens (Diptera: Stratiomyidae): Improving small-scale indoor rearing.

[This corrects the article DOI: 10.1371/journal.pone.0197896.].

Wolbachia megadiversity: 99% of these microorganismic manipulators unknown.

Wolbachia (Alphaproteobacteria) are the most widespread endosymbionts of arthropods, manipulating their hosts by various means to maximize the number of host individuals infected. Based on quantitative analyzes of the published literature from Web of Science® and of DNA sequences of arthropod-hosted Wolbachia from GenBank, we made plausible that less than 1% of the expected 100 000 strains of Wolbachia in arthropods is known. Our findings suggest that more and globally better coordinated efforts in screening arthropods are needed to explore the true Wolbachia diversity and to help us understand the ecology and evolution of these host-endosymbiont interactions.

Major range loss predicted from lack of heat adaptability in an alpine Drosophila species.

Climate warming is threatening biodiversity worldwide. Climate specialists such as alpine species are especially likely to be vulnerable. Adaptation by rapid evolution is the only long-term option for survival of many species, but the adaptive evolutionary potential of heat resistance has not been assessed in an alpine invertebrate. Here, we show that the alpine fly Drosophila nigrosparsa cannot readily adapt to heat stress. Heat-exposed flies from a regime with increased ambient temperature and a regime with increased temperature plus artificial selection for heat tolerance were less heat tolerant than the control group. Increased ambient temperature affected negatively both fitness and competitiveness. Ecological niche models predicted the loss of three quarters of the climatically habitable areas of this fly by the end of this century. Our findings suggest that, alongside with other climate specialists, species from mountainous regions are highly vulnerable to climate warming and unlikely to adapt through evolutionary genetic changes.

Comparing ant behaviour indices for fine-scale analyses.

Animal behaviour often is characterised by standardised assays. In social insects such as ants, behaviour assays are for example used to characterise aggressive and peaceful behaviour. Such assays differ in the number of individuals, the duration and place of assays, and the scoring scales. Also the behaviour indices used to summarise the results differ. Here, we compared five behaviour indices (Aggression Index, Mean Maximum Aggression Index; and the newly introduced Mean Maximum Peace Index, Mean Behaviour Index aggressive, and Mean Behaviour Index peaceful) using a scoring scale that comprises peaceful and aggressive behaviour. The indices were applied on eight simulations and three observed data sets. The five indices were correlated but frequently differed in their means. Multiple indices were needed to capture the complete behaviour range. Furthermore, subtle differences in workers' behaviour, that is, differences that go beyond the presence/absence of aggression, were only identified when considering multiple indices. We infer that the indices applied are differently suited for different analyses. Fine-scale analyses of behavioural variation profit from using more than one index. The particular choice of index or indices likely influences the interpretation of behaviour and should be carefully done in the light of study species and research question.

Evolution of morphological crypsis in the Tetramorium caespitum ant species complex (Hymenoptera: Formicidae).

Cryptic species are morphologically very similar to each other. To what extent stasis or convergence causes crypsis and whether ecology influences the evolution of crypsis has remained unclear. The Tetramorium caespitum complex is one of the most intricate examples of cryptic species in ants. Here, we test three hypotheses concerning the evolution of its crypsis: H1: The complex is monophyletic. H2: Morphology resulted from evolutionary stasis. H3: Ecology and morphology evolved concertedly. We confirmed (H1) monophyly of the complex; (H2) a positive relation between morphological and phylogenetic distances, which indicates a very slow loss of similarity over time and thus stasis; and (H3) a positive relation between only one morphological character and a proxy of the ecological niche, which indicates concerted evolution of these two characters, as well as a negative relation between p-values of correct species identification and altitude, which suggests that species occurring in higher altitudes are more cryptic. Our data suggest that species-specific morphological adaptations to the ecological niche are exceptions in the complex, and we consider the worker morphology in this complex as an adaptive solution for various environments.

Turning one into five: Integrative taxonomy uncovers complex evolution of cryptic species in the harvester ant Messor "structor".

Seed harvesting ants are ecosystem engineers that shape vegetation, nutrient cycles, and microclimate. Progress in ecological research is, however, slowed down by poor species delimitation. For example, it has not been resolved to date, how many species the European harvester ant Messor "structor" (Latreille, 1798) represents. Since its first description, splitting into additional taxa was often proposed but not accepted later on due to inconsistent support from morphology and ecology. Here, we took an iterative integrative-taxonomy approach - comparing multiple, independent data sets of the same sample - and used traditional morphometrics, Wolbachia symbionts, mitochondrial DNA, amplified fragment length polymorphism, and ecological niche modelling. Using the complementarity of the data sets applied, we resolved multiple, strong disagreements over the number of species, ranging from four to ten, and the allocation of individuals to species. We consider most plausible a five-species hypothesis and conclude the taxonomic odyssey by redescribing Messor structor, M. ibericus Santschi, 1925, and M. muticus (Nylander, 1849) stat.rev., and by describing two new species, M. ponticus sp.n. and M. mcarthuri sp.n. The evolutionary explanations invoked in resolving the various data conflicts include pronounced morphological crypsis, incomplete lineage-sorting or ongoing cospeciation of endosymbionts, and peripatric speciation - these ants' significance to evolutionary biology parallels that to ecology. The successful solution of this particular problem illustrates the usefulness of the integrative approach to other systematic problems of comparable complexity and the importance of understanding evolution to drawing correct conclusions on species' attributes, including their ecology and biogeography.

Influence of three artificial light sources on oviposition and half-life of the Black Soldier Fly, Hermetia illucens (Diptera: Stratiomyidae): Improving small-scale indoor rearing.

Hermetia illucens (L.), the Black Soldier Fly, has received increased scientific attention for its potential in circular waste management where larvae can serve as feedstuff for livestock and for biodiesel production. The flies occur naturally in (sub)-tropical and warm-temperate climates, and their mating depends on space and sunlight. Small-scale indoor rearing of Black Soldier Flies has been challenging because they react sensitive to artificial light sources and cage sizes, but recent studies have shown that small-scale rearing under artificial light is feasible. Here, we test the influence of three artificial light sources (light-emitting diodes, fluorescent lamps, and halogen lamps) on small-scale indoor rearing. Three experiments were conducted to compare oviposition traits (pre-oviposition period, total oviposition-period, and egg mass per female) and half-life among the three light sources. Oviposition did not differ among the three light sources, but male and female half-life did. Based on the performance of the light-emitting diodes and their outstanding energy efficiency, we recommend this light source for small-scale indoor rearing of Black Soldier Flies.

An Alpine ant's behavioural polymorphism: monogyny with and without internest aggression in Tetramorium alpestre.

Social structure influences animal societies on various levels (e.g., relatedness, behaviour). In ants, both the number of matings per queen and the number of queens per colony can vary strongly. While workers from both monogynous and polygynous colonies often fight fiercely, in supercolonies (an extreme form of polygyny comprising thousands of queens in spatially separated but interconnected nests), non-nestmates interact peacefully. Studies on social and behavioural polymorphism within ant species can help elucidate their influence on genetic diversity and behaviour and the factors triggering variation in social structure and behaviour. Here, we reveal a behavioural and social polymorphism comprising monogyny with and without internest aggression in Tetramorium alpestre sampled in Tyrol, Austria. The social polymorphism is based on genetic and behavioural evidence and contrasts with the supercolonial organisation known from another location in Austria (Carinthia), 150 km away. Microsatellite genotyping using eight polymorphic loci revealed monogyny-monandry and high intranest pairwise relatedness. Interestingly, various experimental one-on-one worker encounters revealed only occasional aggressive behaviour between monogynous colonies, and thus a behavioural polymorphism. Mantel tests revealed a significant negative correlation between spatial distance and relatedness, while worker behaviour was not correlated with relatedness or spatial distance. These results indicate that behaviour might be influenced by other factors - for example, the experience of workers, ecological, chemical, and/or genetic factors not characterised in this study. However, workers distinguished nestmates from non-nestmates also when aggression was lacking. We hypothesise an adaptive value of reduced aggression. We speculate that the non-aggressive and partly aggressive encounters observed represent different options in the social structure of T. alpestre, the non-aggressiveness possibly also promoting supercolony development. The social and behavioural polymorphisms observed offer opportunities to identify the factors triggering these changes and thus further explore the behavioural and social polymorphism of this ant species.

Life-history traits and physiological limits of the alpine fly Drosophila nigrosparsa (Diptera: Drosophilidae): A comparative study.

Interspecific variation in life-history traits and physiological limits can be linked to the environmental conditions species experience, including climatic conditions. As alpine environments are particularly vulnerable under climate change, we focus on the montane-alpine fly Drosophila nigrosparsa. Here, we characterized some of its life-history traits and physiological limits and compared these with those of other drosophilids, namely Drosophila hydei, Drosophila melanogaster, and Drosophila obscura. We assayed oviposition rate, longevity, productivity, development time, larval competitiveness, starvation resistance, and heat and cold tolerance. Compared with the other species assayed, D. nigrosparsa is less fecund, relatively long-living, starvation susceptible, cold adapted, and surprisingly well heat adapted. These life-history characteristics provide insights into invertebrate adaptations to alpine conditions which may evolve under ongoing climate change.