Understanding how life is adapting to urban environments represents an important challenge in evolutionary biology. In this work, we investigate a widely cited example of urban adaptation, Culex pipiens form molestus, also known as the London Underground mosquito. Population genomic analysis of ~350 contemporary and historical samples counters the popular hypothesis that molestus originated belowground in London <200 years ago. Instead, we show that molestus first adapted to human environments aboveground in the Mediterranean or Middle East over the course of more than 1000 years, possibly in association with ancient agricultural civilizations of the Middle East. Our results highlight the role of early human society in priming taxa for contemporary urban evolution. They also provide insight into whether and how molestus contributes to West Nile virus transmission in modern cities.

The emergence of infectious diseases, particularly those caused by fungal pathogens, poses serious threats to public health, wildlife and ecosystem stability1. Host-fungus interactions and environmental factors have been extensively examined2-4. However, the role of genetic variability in pathogens is often less well-studied, even for diseases such as white-nose in bats, which has caused one of the highest disease-driven death tolls documented in nonhuman mammals5. Previous research on white-nose disease has primarily focused on variations in disease outcomes attributed to host traits or environmental conditions6-8, but has neglected pathogen variability. Here we leverage an extensive reference collection of 5,479 fungal isolates from 27 countries to reveal that the widespread causative agent is not a single species but two sympatric cryptic species, each exhibiting host specialization. Our findings provide evidence of recombination in each species, but significant genetic differentiation across their genomes, including differences in genome organization. Both species contain geographically differentiated populations, which enabled us to identify the species introduced to North America and trace its source population to a region in Ukraine. In light of our discovery of the existence of two cryptic species of the causative agent of white-nose disease, our research underscores the need to integrate the study of pathogen variability into comprehensive disease surveillance, management and prevention strategies. This holistic approach is crucial for enhancing our understanding of diseases and implementing effective measures to prevent their spread.

The Myotis nattereri species complex has been the focus of several recent morphological and molecular surveys to assess the species status of various named forms, including three informally referred to as Myotis sp. A, M. sp. B and M. sp. C. The first two forms have now been formally described as distinct biological species, and named M. crypticus and M. zenatius, respectively, both distinct from the nominotypical M. nattereri s. str. and M. escalerai. The latter form, Myotis sp. C is known only from Corsica. Here we demonstrate that this form has not only unique mitochondrial haplotypes but also several nuclear alleles that are divergent and not found anywhere else, which emphasizes its long independent evolution. We therefore confirm its specific status and describe it as a new species. Its ecology and rupicolous roosting habits resemble those of the Iberian M. escalerai, but it is otherwise morphologically most similar to M. crypticus. This new species is endemic to Corsica and is apparently very rare and essentially localised to mountain forests. Owing to its restricted distribution, its small population size, and limited population connectivity, it seems highly vulnerable to climate change and thus should be classified as endangered.

A recent publication in Ecosphere claimed to report the first case of a predation event by a theridiid spider on a bat globally and the first case of a member of the genus Steatoda preying on a mammal globally, and the paper concludes with possible implications for public health. Predation is typically understood to mean “capture, kill, and eat”. However, none of these aspects had been observed. Moreover, key aspects of bat biology were not taken into account, including that the bat in question was a flightless newborn, the capture of which would require some explanation. We revisit this interesting observation considering both bat and spider ecology.

Recently diverged or diverging populations can offer unobstructed insights into early barriers to gene flow during the initial stages of speciation. The current study utilised a novel insect system (order Mantophasmatodea) to shed light on the early drivers of speciation. The members of this group have limited dispersal abilities, small allopatric distributions and strong habitat associations in the Cape Floristic Region biodiversity hotspot in South Africa. Sister taxa from the diverse family Austrophasmatidae were chosen as focal species (Karoophasma biedouwense, K. botterkloofense). Population genetics and Generalized Dissimilarity Modelling (GDM) were used to characterise spatial patterns of genetic variation and evaluate the contribution of environmental factors to population divergence and speciation. Extensive sampling confirmed the suspected allopatry of these taxa. However, hybrids were identified in a narrow region occurring between the species' distributions. Strong population structure was found over short geographic distances; particularly in K. biedouwense in which geographic distance accounted for 32% of genetic variation over a scale of 50 km (r = .56, p < .001). GDM explained 42%-78% of the deviance in observed genetic dissimilarities. Geographic distance was consistently indicated to be important for between species and within population differentiation, suggesting that limited dispersal ability may be an important neutral driver of divergence. Temperature, altitude, precipitation and vegetation were also indicated as important factors, suggesting the possible role of adaptation to local environmental conditions for species divergence. The discovery of the hybrid-zone, and the multiple allopatric species pairs in Austrophasmatidae support the idea that this could be a promising group to further our understanding of speciation modes.

Emerging infectious diseases pose a major threat to human, animal, and plant health. The risk of species-extinctions increases when pathogens can survive in the absence of the host, for example in environmental reservoirs. However, identifying such reservoirs and modes of infection is often highly challenging. In this study, we investigated the presence and nature of an environmental reservoir for the ascomycete fungus Pseudogymnoascus destructans , the causative agent of white-nose disease. We also characterised the modes and timing of transmission of the pathogen; key elements to better understand the disease dynamics. Using 18 microsatellite markers, we determined the genotypic and genic (based on allele frequencies) differentiation between 1,497 P. destructans isolates collected from nine closely situated hibernacula in North-Eastern Germany. One hibernaculum was the focus of intensive sampling in which both the bats and walls of the site were sampled at regular intervals over five consecutive winter seasons (1,062 isolates). We found significant genic differentiation between sites and few multi-locus genotypes shared across hibernacula (genotypic differentiation). This demonstrates that each hibernaculum has an essentially unique population of the fungus. This would be expected if bats purge viable P. destructans over the summer, preventing the mixing and exchange of the pathogen in maternity colonies, where bats from all of the studied hibernacula meet. Results from the intensively sampled site show higher measures of genotypic richness on walls compared to bats, the absence of genic differentiation between bats and walls, and stable relative abundance of multi-locus genotypes over multiple winter seasons. This clearly implicates hibernacula walls as the main environmental reservoir of the pathogen, from which bats become re-infected annually.

Emerging infectious diseases are a severe conservation threat for a variety of plants and animals. In North America, several bat species are threatened by white-nose disease, which has caused an unprecedented mass mortality of > 6 million bats since 2006. The fungus Pseudogymnoascus ( Geomyces ) destructans is the causative agent of the disease. Though asexual reproduction is the norm, sexual reproduction is possible as two mating types exist. Sexual reproduction has been implicated in the emergence of virulent strains of fungi but to date no rapid means of mating type characterisation was available for P. destructans . In this study, three mating type-specific primer sets were designed and tested on 80 isolates. The primers were multiplexed with microsatellite loci allowing for rapid and concurrent genotyping and mating type assignment. These markers will have great utility in better understanding and predicting the population dynamics and evolutionary potential of this fungus, including the emergence of virulent strains.

Emerging infectious diseases rank among the most important threats to human and wildlife health. A comprehensive understanding of the mode of infection and presence of potential reservoirs is critical for the development of effective counter strategies. Fungal pathogens can remain viable in environmental reservoirs for extended periods of time before infecting susceptible individuals. This may be the case forPseudogymnoascus destructans(Pd), the causative agent of bat white-nose disease. Owing to its cold-loving nature, this fungal pathogen only grows on bats during hibernation, when their body temperature is reduced. Bats only spend part of their life cycle in hibernation and do not typically show signs of infection in summer, raising the question of whetherPdremains viable in hibernacula during this period (roughly six months). If so, this could facilitate the re-infection of bats when they return to the sites the following winter. In a laboratory experiment, we determined the germination rate ofPdspores kept under constant conditions on a wall-like substrate, over the course of two years. Results showed that the seasonal pattern inPdgermination mirrored the life cycle of the bats, with an increased germination rate at times when hibernating bats would naturally be present and lower germination rates during their absence. We suggest thatPdis dependent on the presence of hibernating bats and has therefore coupled its germination rate to host availability. Furthermore, we demonstrate thatPdspores survive extended periods of host absence and can remain viable for at least two years. There is, however, a strong decrease in spore viability between the first and second years (98%).Pdviability for at least two years on a solid mineral-based substrate establishes the potential for environmental reservoirs in hibernacula walls and has strong implications for the efficacy of certain management strategies (e.g. bat culling).

To increase the applicability and success of physiological approaches in conservation plans, conservation physiology should be based on ecologically relevant relationships between physiological markers and environmental variation that can only be obtained from wild populations. Given their integrative and multifaceted aspects, markers of oxidative status have recently been considered in conservation physiology, but still need to be validated across environmental conditions and locations. Here, we examined whether inter-annual variation in two oxidative markers, plasma antioxidant capacity and plasma hydroperoxides, followed inter-annual variation in temperature anomalies and associated vegetation changes in four colonies of long-lived greater mouse-eared bats (Myotis myotis) monitored over five consecutive years. We found that the plasma antioxidant capacity of bats decreased while plasma hydroperoxide concentrations increased with increasing temperature anomalies occurring in the two weeks before blood sampling. Moreover, the antioxidant defences of these bats reflected vegetation indices, which themselves reflected the thermal conditions experienced by bats in their foraging habitat. Variation in oxidative markers therefore appears to be due to variation in thermoregulatory costs and to indirect changes in foraging costs. Overall, these results validate the use of markers of oxidative status in conservation physiology to monitor thermal perturbations recently experienced by animals in their natural habitat. However, even though oxidative markers varied in the same direction in all four bat colonies across years, the amplitude of their response differed. If these different physiological responses reflect different performances (e.g. productivity, survival rate) between colonies, this implies that, if necessary, conservation measures may need to be applied at the local scale.

Counts obtained for the 1056 plates.

Introduced pathogens causing emerging infectious diseases (EIDs) are serious contemporary threats to animal, plant, and ecosystem health. The invasive fungus, Pseudogymnoascus destructans, has established populations of European origin in North America, resulting in mass mortality of several hibernating bat species. Extensive monitoring for this pathogen exists in Europe and North America, but limited screening is taking place elsewhere. We report results from cave surveys on Hawai'i Island. Substrates in 10 lava-tube caves with elevations up to 3,045 m were swabbed providing samples for screening P. destructans. Interior cave air temperatures spanned temperatures suitable for the growth and survival of P. destructans. Using quantitative PCR, all 85 samples tested were negative for the presence of P. destructans. The biology of the Hawaiian hoary bat (Lasiurus cinereus semotus) in relation to its unusual use of high elevation caves is discussed because these bats could come into contact with P. destructans should it arrive in Hawai'i. Large numbers of cave enthusiasts visit Hawaiian caves from across the world after having been inside caves elsewhere including areas with P. destructans. Thus, resource managers in Hawai'i and other remote areas may want to consider the potential for P. destructans to arrive unintentionally via human activities. Biosecurity measures and periodic screening for P. destructans are especially important in Hawai'i given the presence of high elevation caves with suitable temperatures for its growth. If P. destructans was introduced to Hawaiian caves, it could affect the local fauna but also act as a source population for colonisations elsewhere.

The recently discovered insect order Mantophasmatodea currently comprises 19 Southern African species. These mainly occur in allopatry, have high levels of color polymorphism and communicate via species- and gender-specific vibratory signals. High levels of interspecific morphological conservatism mean that cryptic species are likely to be uncovered. These aspects of Mantophasmatodean biology make them an ideal group in which to investigate population divergence due to habitat-specific adaptation, sexual selection, and potentially sensory speciation. Lack of appropriate genetic markers has thus far rendered such studies unfeasible. To address this need, the first microsatellite loci for this order were developed. Fifty polymorphic loci were designed specifically for Karoophasma biedouwense (Austrophasmatidae), out of which 23 were labeled and tested for amplification across the order using 2-3 individuals from 10 species, representing all 4 currently known families. A Bayesian mitochondrially encoded cytochrome c oxidase I (COI) topology was reconstructed and divergence dates within the order were estimated for the first time. Amplification success and levels of polymorphism were compared with genetic divergence and time since divergence. In agreement with studies on vertebrate taxa, both amplification and variability were negatively correlated with distance (temporal and genetic). The high number of informative loci will offer sufficient resolution for both broad level population genetic analysis and individual based pedigree or parentage analyses for most species in Austrophasmatidae, with at least some loci available for the other families. This resource will facilitate research into the evolutionary biology of this understudied but fascinating group.

Multiple biotic and abiotic parameters influence the dynamics of individual fungal species and entire communities. Major drivers for tropical plant endophytes are undoubtedly seasonality, local habitat conditions and biogeography. However, host specialization and tissue preferences also contribute to the structuring of endophytic mycobiomes. To elucidate such specializations and preferences, we sampled two commercially important, unrelated plant species, Amorphophallus albispathus and Camellia sinensis (tea plant) simultaneously at close proximity. The mycobiomes of different tissue types were assessed with high-throughput amplicon sequencing of the internal transcribed spacer DNA region. Both plants hosted different fungal communities and varied in α- and β-diversity, despite their neighboring occurrence. However, the fungal assemblages of Amorphophallus leaflets shared taxa with the mycobiomes of tea leaves, thereby suggesting common driving forces for leaf-inhabiting fungi irrespective of host plant identity. The mycobiome composition and diversity of tea leaves was clearly driven by leaf age. We suggest that the very youngest tea leaves are colonized by stochastic processes, while mycobiomes of old leaves are rather similar as the result of progressive succession. The biodiversity of fungi associated with A. albispathus was characterized by a large number of unclassified OTUs (at genus and species level) and by tissue-specific composition.This study is the first cultivation-independent high-throughput assessment of fungal biodiversity of an Amorphophallus species, and additionally expands the knowledge base on fungi associated with tea plants.

Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8 % identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.

Despite many studies illustrating the perils of utilising mitochondrial DNA in phylogenetic studies, it remains one of the most widely used genetic markers for this purpose. Over the last decade, nuclear introns have been proposed as alternative markers for phylogenetic reconstruction. However, the resolution capabilities of mtDNA and nuclear introns have rarely been quantified and compared. In the current study we generated a novel ∼5kb dataset comprising six nuclear introns and a mtDNA fragment. We assessed the relative resolution capabilities of the six intronic fragments with respect to each other, when used in various combinations together, and when compared to the traditionally used mtDNA. We focused on a major clade in the horseshoe bat family (Afro-Palaearctic clade; Rhinolophidae) as our case study. This old, widely distributed and speciose group contains a high level of conserved morphology. This morphological stasis renders the reconstruction of the phylogeny of this group with traditional morphological characters complex. We sampled multiple individuals per species to represent their geographic distributions as best as possible (122 individuals, 24 species, 68 localities). We reconstructed the species phylogeny using several complementary methods (partitioned Maximum Likelihood and Bayesian and Bayesian multispecies-coalescent) and made inferences based on consensus across these methods. We computed pairwise comparisons based on Robinson-Foulds tree distance metric between all Bayesian topologies generated (27,000) for every gene(s) and visualised the tree space using multidimensional scaling (MDS) plots. Using our supported species phylogeny we estimated the ancestral state of key traits of interest within this group, e.g. echolocation peak frequency which has been implicated in speciation. Our results revealed many potential cryptic species within this group, even in taxa where this was not suspected a priori and also found evidence for mtDNA introgression. We demonstrated that by using just two introns one can recover a better supported species tree than when using the mtDNA alone, despite the shorter overall length of the combined introns. Additionally, when combining any single intron with mtDNA, we showed that the result is highly similar to the mtDNA gene tree and far from the true species tree and therefore this approach should be avoided. We caution against the indiscriminate use of mtDNA in phylogenetic studies and advocate for pilot studies to select nuclear introns. The selection of marker type and number is a crucial step that is best based on critical examination of preliminary or previously published data. Based on our findings and previous publications, we recommend the following markers to recover phylogenetic relationships between recently diverged taxa (<20 My) in bats and other mammals: ACOX2, COPS7A, BGN, ROGDI and STAT5A.

Maintaining the genetic diversity of populations is important in conservation because it strongly influences the ability to adapt to changing environments. We characterised the genetic structure of the endemic and endangered New Zealand (NZ) long-tailed bat (Chalinolobus tuberculatus) in two valleys in Fiordland, NZ. Fiordland is one of the last strongholds of the species, which has drastically declined throughout NZ. C. tuberculatus has suffered from recent habitat fragmentation and episodic predation by exotic mammals over the last 150 years. Gene flow and structuring were measured using nine nuclear microsatellite loci. In addition, the hyper-variable domain HVI of the mitochondrial control region was sequenced to analyse population structure at the maternal level. Our results show that the nine colonies studied have retained high genetic diversity, with moderate signs of genetic bottlenecks. Furthermore, low F-ST and F-IS values indicated that all colonies are still connected by gene flow and do not show signs of inbreeding. Substantial gene flow among colonies was also demonstrated by Bayesian clustering and PCA analysis. At the mitochondrial level, substantial differentiation between colonies has resulted from strong natal philopatry in females. Overall, our results indicate that genetic diversity is maintained in the Fiordland population of C. tuberculatus despite regular population crashes and habitat fragmentation. Management should ensure that remaining habitat linkages are preserved and further predator-induced population bottlenecks are prevented so that current genetic diversity is maintained in the long-term.

Among the many anthropogenic modifications to earth's ecosystems, habitat loss and degradation pose the most immediate threat to many biota. The predicted consequences of fragmented habitats include lower species diversity, smaller population sizes, disrupted gene flow, increased drift and inbreeding and increased differentiation between neighbouring populations; all of which are thought to be further enhanced in species with low dispersal abilities. These factors, especially when occurring in tandem, can lead to an increased risk of extinction. To examine the genetic consequences of habitat fragmentation we selected an isolated population of a sedentary woodland specialist species (Rhinolophus hipposideros) to act as an indicator of disruptions to landscape level connectivity. Based on 491 individuals from 37 colonies our results revealed the presence of a broad North-Range/South-Range differentiation within this species in Ireland; a finding supported across datasets (mtDNA and nuclear microsatellites) and analyses. Analyses of echolocation data and microsatellites suggested further differentiation of the northern-most colonies. A landscape genetics framework to assess the impact of habitat versus geographic distance on population differentiation showed that habitat features (at a five km resolution) were equally likely to be correlated with differentiation as geographic distance considered alone. Further differentiation of the geographically disjunct groups is likely to occur in the future. The viability of either group alone is uncertain given their restricted distribution, small population sizes (based on census data and Ne estimates) and isolation. Roost provision and habitat restoration in the geographic region separating the differentiated groups will be fundamental to the recolonization of this area and the reestablishment of connectivity between the regional groups.