Stable pollination service in a generalist high Arctic community despite the warming climate.

Insects provide key pollination services in most terrestrial biomes, but this service depends on a multistep interaction between insect and plant. An insect needs to visit a flower, receive pollen from the anthers, move to another conspecific flower, and finally deposit the pollen on a receptive stigma. Each of these steps may be affected by climate change, and focusing on only one of them (e.g., flower visitation) may miss important signals of change in service provision. In this study, we combine data on visitation, pollen transport, and single-visit pollen deposition to estimate functional outcomes in the high Arctic plant-pollinator network of Zackenberg, Northeast Greenland, a model system for global warming-associated impacts in pollination services. Over two decades of rapid climate warming, we sampled the network repeatedly: in 1996, 1997, 2010, 2011, and 2016. Although the flowering plant and insect communities and their interactions varied substantially between years, as expected based on highly variable Arctic weather, there was no detectable directional change in either the structure of flower-visitor networks or estimated pollen deposition. For flower-visitor networks compiled over a single week, species phenologies caused major within-year variation in network structure despite consistency across years. Weekly networks for the middle of the flowering season emerged as especially important because most pollination service can be expected to be provided by these large, highly nested networks. Our findings suggest that pollination ecosystem service in the high Arctic is remarkably resilient. This resilience may reflect the plasticity of Arctic biota as an adaptation to extreme and unpredictable weather. However, most pollination service was contributed by relatively few fly taxa (Diptera: Spilogona sanctipauli and Drymeia segnis [Muscidae] and species of Rhamphomyia [Empididae]). If these key pollinators are negatively affected by climate change, network structure and the pollination service that depends on it would be seriously compromised.

Correction: Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

[This corrects the article DOI: 10.1371/journal.pgen.1007533.].

A tale of two tissues: Probing gene expression in a complex insect-induced gall.

Plant galls are novel and sometimes dramatic plant organs whose development is initiated and controlled by parasitic microbes, nematodes, insects and mites. For arthropods, galls provide relative safety from enemies and abiotic stresses while providing nutrition. Galls are formed entirely by the plant, whose transcriptional pathways are modified and coopted to produce a structure specific to the galler species; they comprise a classic example of Dawkins' "extended phenotype". Arthropod-elicited galls are unique in that they are often anatomically complex (Figure 1a), with multiple differentiated tissue types (Figure 1b). A growing number of investigators have studied changes in hostplant gene expression to understand arthropod gall development. In this issue of Molecular Ecology, Martinson et al. (2021) report using RNA sequencing to explore tissue-specific gene expression associated with anatomical and functional gall complexity, demonstrating for the first time that gall tissues are as different transcriptionally as they are anatomically.

Delimiting the cryptic diversity and host preferences of Sycophila parasitoid wasps associated with oak galls using phylogenomic data.

Cryptic species diversity is a major challenge regarding the species-rich community of parasitoids attacking oak gall wasps due to a high degree of sexual dimorphism, morphological plasticity, small size and poorly known biology. As such, we know very little about the number of species present, nor the evolutionary forces responsible for generating this diversity. One hypothesis is that trait diversity in the gall wasps, including the morphology of the galls they induce, has evolved in response to selection imposed by the parasitoid community, with reciprocal selection driving diversification of the parasitoids. Using a rare, continental-scale data set of Sycophila parasitoid wasps reared from 44 species of cynipid galls from 18 species of oak across the USA, we combined mitochondrial DNA barcodes, ultraconserved elements (UCEs), morphological and natural history data to delimit putative species. Using these results, we generate the first large-scale assessment of ecological specialization and host association in this species-rich group, with implications for evolutionary ecology and biocontrol. We find most Sycophila target specific subsets of available cynipid host galls with similar morphologies, and generally attack larger galls. Our results suggest that parasitoid wasps such as Sycophila have adaptations allowing them to exploit particular host trait combinations, while hosts with contrasting traits are resistant to attack. These findings support the tritrophic niche concept for the structuring of plant-herbivore-parasitoid communities.

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Discordant Pleistocene population size histories in a guild of hymenopteran parasitoids.

Signatures of past changes in population size have been detected in genome-wide variation in many species. However, the causes of such demographic changes and the extent to which they are shared across co-distributed species remain poorly understood. During Pleistocene glacial maxima, many temperate European species were confined to southern refugia. While vicariance and range expansion processes associated with glacial cycles have been widely documented, it is unclear whether refugial populations of co-distributed species have experienced shared histories of population size change. We analyse whole-genome sequence data to reconstruct and compare demographic histories during the Quaternary for Iberian refuge populations in a single ecological guild (seven species of chalcid parasitoid wasps associated with oak cynipid galls). For four of these species, we find support for large changes in effective population size (Ne ) through the Pleistocene that coincide with major climate events. However, there is little evidence that the timing, direction and magnitude of demographic change are shared across species, suggesting that demographic histories in this guild are largely idiosyncratic, even at the scale of a single glacial refugium.

Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient, and probably shared by most animal (metazoan) phyla. Indeed, while vertebrates had been thought an exception, it has recently been argued that mammals also possess an antiviral RNAi pathway, although its immunological relevance is currently uncertain and the viral small RNAs (viRNAs) are not easily detectable. Here we use a metagenomic approach to test for the presence of viRNAs in five species from divergent animal phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga-which represents an independent origin of multicellularity from plants, fungi, and animals. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify viRNAs derived from those viruses. We identified 21U small RNAs derived from an RNA virus in the brown alga, reminiscent of plant and fungal viRNAs, despite the deep divergence between these lineages. However, contrary to our expectations, we were unable to identify canonical (i.e. Drosophila- or nematode-like) viRNAs in any of the animals, despite the widespread presence of abundant micro-RNAs, and somatic transposon-derived piwi-interacting RNAs. We did identify a distinctive group of small RNAs derived from RNA viruses in the mollusc. However, unlike ecdysozoan viRNAs, these had a piRNA-like length distribution but lacked key signatures of piRNA biogenesis. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an endogenous RNA virus in the mollusc. The absence of canonical virus-derived small RNAs from our samples may suggest that the majority of animal phyla lack an antiviral RNAi response. Alternatively, these phyla could possess an antiviral RNAi response resembling that reported for vertebrates, with cryptic viRNAs not detectable through simple metagenomic sequencing of wild-type individuals. In either case, our findings show that the antiviral RNAi responses of arthropods and nematodes, which are highly divergent from each other and from that of plants and fungi, are also highly diverged from the most likely ancestral metazoan state.

Whole-genome data reveal the complex history of a diverse ecological community.

How widespread ecological communities assemble remains a key question in ecology. Trophic interactions between widespread species may reflect a shared population history or ecological fitting of local pools of species with very different population histories. Which scenario applies is central to the stability of trophic associations and the potential for coevolution between species. Here we show how alternative community assembly hypotheses can be discriminated using whole-genome data for component species and provide a likelihood framework that overcomes current limitations in formal comparison of multispecies histories. We illustrate our approach by inferring the assembly history of a Western Palearctic community of insect herbivores and parasitoid natural enemies, trophic groups that together comprise 50% of terrestrial species. We reject models of codispersal from a shared origin and of delayed enemy pursuit of their herbivore hosts, arguing against herbivore attainment of "enemy-free space." The community-wide distribution of species expansion times is also incompatible with a random, neutral model of assembly. Instead, we reveal a complex assembly history of single- and multispecies range expansions through the Pleistocene from different directions and over a range of timescales. Our results suggest substantial turnover in species associations and argue against tight coevolution in this system. The approach we illustrate is widely applicable to natural communities of nonmodel species and makes it possible to reveal the historical backdrop against which natural selection acts.

Low-coverage genomic data resolve the population divergence and gene flow history of an Australian rain forest fig wasp.

Population divergence and gene flow are key processes in evolution and ecology. Model-based analysis of genome-wide data sets allows discrimination between alternative scenarios for these processes even in nonmodel taxa. We used two complementary approaches (one based on the blockwise site frequency spectrum [bSFS], the second on the pairwise sequentially Markovian coalescent [PSMC]) to infer the divergence history of a fig wasp, Pleistodontes nigriventris. Pleistodontes nigriventris and its fig tree mutualist Ficus watkinsiana are restricted to rain forest patches along the eastern coast of Australia and are separated into The Northern population is to the north of the Southern populations by two dry forest corridors (the Burdekin and St. Lawrence Gaps). We generated whole genome sequence data for two haploid males per population and used the bSFS approach to infer the timing of divergence between northern and southern populations of P. nigriventris, and to discriminate between alternative isolation with migration (IM) and instantaneous admixture (ADM) models of postdivergence gene flow. Pleistodontes nigriventris has low genetic diversity (π = 0.0008), to our knowledge one of the lowest estimates reported for a sexually reproducing arthropod. We find strongest support for an ADM model in which the two populations diverged ca. 196 kya in the late Pleistocene, with almost 25% of northern lineages introduced from the south during an admixture event ca. 57 kya. This divergence history is highly concordant with individual population demographies inferred from each pair of haploid males using PSMC. Our analysis illustrates the inferences possible with genome-level data for small population samples of tiny, nonmodel organisms and adds to a growing body of knowledge on the population structure of Australian rain forest taxa.

Gradients in richness and turnover of a forest passerine's diet prior to breeding: A mixed model approach applied to faecal metabarcoding data.

Rather little is known about the dietary richness and variation of generalist insectivorous species, including birds, due primarily to difficulties in prey identification. Using faecal metabarcoding, we provide the most comprehensive analysis of a passerine's diet to date, identifying the relative magnitudes of biogeographic, habitat and temporal trends in the richness and turnover in diet of Cyanistes caeruleus (blue tit) along a 39 site and 2° latitudinal transect in Scotland. Faecal samples were collected in 2014-2015 from adult birds roosting in nestboxes prior to nest building. DNA was extracted from 793 samples and we amplified COI and 16S minibarcodes. We identified 432 molecular operational taxonomic units that correspond to putative dietary items. Most dietary items were rare, with Lepidoptera being the most abundant and taxon-rich prey order. Here, we present a statistical approach for estimation of gradients and intersample variation in taxonomic richness and turnover using a generalised linear mixed model. We discuss the merits of this approach over existing tools and present methods for model-based estimation of repeatability, taxon richness and Jaccard indices. We found that dietary richness increases significantly as spring advances, but changes little with elevation, latitude or local tree composition. In comparison, dietary composition exhibits significant turnover along temporal and spatial gradients and among sites. Our study shows the promise of faecal metabarcoding for inferring the macroecology of food webs, but we also highlight the challenge posed by contamination and make recommendations of laboratory and statistical practices to minimise its impact on inference.

Coevolutionary arms race versus host defense chase in a tropical herbivore-plant system.

Coevolutionary models suggest that herbivores drive diversification and community composition in plants. For herbivores, many questions remain regarding how plant defenses shape host choice and community structure. We addressed these questions using the tree genus Inga and its lepidopteran herbivores in the Amazon. We constructed phylogenies for both plants and insects and quantified host associations and plant defenses. We found that similarity in herbivore assemblages between Inga species was correlated with similarity in defenses. There was no correlation with phylogeny, a result consistent with our observations that the expression of defenses in Inga is independent of phylogeny. Furthermore, host defensive traits explained 40% of herbivore community similarity. Analyses at finer taxonomic scales showed that different lepidopteran clades select hosts based on different defenses, suggesting taxon-specific histories of herbivore-host plant interactions. Finally, we compared the phylogeny and defenses of Inga to phylogenies for the major lepidopteran clades. We found that closely related herbivores fed on Inga with similar defenses rather than on closely related plants. Together, these results suggest that plant defenses might be more evolutionarily labile than the herbivore traits related to host association. Hence, there is an apparent asymmetry in the evolutionary interactions between Inga and its herbivores. Although plants may evolve under selection by herbivores, we hypothesize that herbivores may not show coevolutionary adaptations, but instead "chase" hosts based on the herbivore's own traits at the time that they encounter a new host, a pattern more consistent with resource tracking than with the arms race model of coevolution.

Seeing-good-gene-based mate choice: From genes to behavioural preferences.

Although vertebrates have been reported to gain higher reproductive outputs by choosing mates, few studies have been conducted on threatened species. However, species recovery should benefit if natural mate choice could improve reproductive output (i.e. pair performance related to offspring number, such as increased clutch size, numbers of fertilized egg and fledglings). We assessed the evidence for major histocompatibility complex (MHC)-based mate preference in the endangered crested ibis (Nipponia nippon) and quantified the impacts of such choice on reproductive output. We tested the hypothesis that crested ibis advertise "good genes" through external traits, by testing whether nuptial plumage characteristics and body morphology mediate mate choice for underlying genetic MHC variation. We found differences between males and females in preferred MHC genotypes, external traits used in mate choice and contributions to reproductive outputs. Females preferred MHC-heterozygous males, which had darker [i.e. lower total reflectance and ultraviolet (UV) reflectance] nuptial plumage. Males preferred females lacking the DAB*d allele at the MHC class II DAB locus, which had higher average body mass. DAB*d-free females yielded heavier eggs and more fledglings, while MHC-heterozygous males contributed to more fertilized eggs and fledglings. Fledging rate was highest when both parents had the preferred MHC genotypes (i.e. MHC-heterozygous father and DAB*d-free mother). Comparisons showed that free-mating wild and semi-natural pairs yielded more fertilized eggs and more fledglings, with a higher fledging rate, than captive pairs matched artificially based on pedigree. Conservation programmes seldom apply modern research results to population management, which could hinder recovery of threatened species. Our results show that mate choice can play an important role in improving reproductive output, with an example in which an endangered bird selects mates using UV visual capability. Despite the undoubted importance of pedigree-based matching of mates in conservation programmes, we show that free mating can be a better alternative strategy.

Chemocoding as an identification tool where morphological- and DNA-based methods fall short: Inga as a case study.

The need for species identification and taxonomic discovery has led to the development of innovative technologies for large-scale plant identification. DNA barcoding has been useful, but fails to distinguish among many species in species-rich plant genera, particularly in tropical regions. Here, we show that chemical fingerprinting, or 'chemocoding', has great potential for plant identification in challenging tropical biomes. Using untargeted metabolomics in combination with multivariate analysis, we constructed species-level fingerprints, which we define as chemocoding. We evaluated the utility of chemocoding with species that were defined morphologically and subject to next-generation DNA sequencing in the diverse and recently radiated neotropical genus Inga (Leguminosae), both at single study sites and across broad geographic scales. Our results show that chemocoding is a robust method for distinguishing morphologically similar species at a single site and for identifying widespread species across continental-scale ranges. Given that species are the fundamental unit of analysis for conservation and biodiversity research, the development of accurate identification methods is essential. We suggest that chemocoding will be a valuable additional source of data for a quick identification of plants, especially for groups where other methods fall short.

Transcriptome mining for phylogenetic markers in a recently radiated genus of tropical plants (Renealmia L.f., Zingiberaceae).

The reconstruction of relationships within species-rich groups that have recently evolved in biodiversity hotspots is hampered by a lack of phylogenetically informative markers. It is also made difficult by the lack of sampling necessary to reconstruct a species-level phylogeny. We use transcriptome mining to search for markers to reconstruct a phylogeny of the amphi-Atlantic genus Renealmia L. f. (Zingiberaceae). We recover seven introns from single copy genes and use them to reconstruct the phylogeny of the genus together with a commonly used phylogenetic marker, internal transcribed spacers of ribosomal DNA (ITS) that has previously been used to reconstruct the phylogeny of the genus. We targeted genes with low numbers of base pairs that improves sequencing success using highly degraded DNA from herbarium specimens. The use of herbarium specimens greatly increased the number of species in the study as these were readily available in historical collections. Data were obtained for 14 of the 17 African species and 54 of the 65 Neotropical species. The phylogeny was well-supported for a number of Renealmia subgroups although relationships among those clades remained poorly supported.

Sweet Tetra-Trophic Interactions: Multiple Evolution of Nectar Secretion, a Defensive Extended Phenotype in Cynipid Gall Wasps.

Many herbivores employ reward-based mutualisms with ants to gain protection from natural enemies. We examine the evolutionary dynamics of a tetra-trophic interaction in which gall wasp herbivores induce their host oaks to produce nectar-secreting galls, which attract ants that provide protection from parasitoids. We show that, consistent with other gall defensive traits, nectar secretion has evolved repeatedly across the oak gall wasp tribe and also within a single genus (Disholcaspis) that includes many nectar-inducing species. Once evolved, nectar secretion is never lost in Disholcaspis, consistent with high defensive value of this trait. We also show that evolution of nectar secretion is correlated with a transition from solitary to aggregated oviposition, resulting in clustered nectar-secreting galls, which produce a resource that ants can more easily monopolize. Such clustering is commonly seen in ant guard mutualisms. We suggest that correlated evolution between maternal oviposition and larval nectar induction traits has enhanced the effectiveness of this gall defense strategy.

Tournament ABC analysis of the western Palaearctic population history of an oak gall wasp, Synergus umbraculus.

Approximate Bayesian computation (ABC) is a powerful and widely used approach in inference of population history. However, the computational effort required to discriminate among alternative historical scenarios often limits the set that is compared to those considered more likely a priori. While often justifiable, this approach will fail to consider unexpected but well-supported population histories. We used a hierarchical tournament approach, in which subsets of scenarios are compared in a first round of ABC analyses and the winners are compared in a second analysis, to reconstruct the population history of an oak gall wasp, Synergus umbraculus (Hymenoptera, Cynipidae) across the Western Palaearctic. We used 4,233 bp of sequence data across seven loci to explore the relationships between four putative Pleistocene refuge populations in Iberia, Italy, the Balkans and Western Asia. We compared support for 148 alternative scenarios in eight pools, each pool comprising all possible rearrangements of four populations over a given topology of relationships, with or without founding of one population by admixture and with or without an unsampled "ghost" population. We found very little support for the directional "out of the east" scenario previously inferred for other gall wasp community members. Instead, the best-supported models identified Iberia as the first-regional population to diverge from the others in the late Pleistocene, followed by divergence between the Balkans and Western Asia, and founding of the Italian population through late Pleistocene admixture from Iberia and the Balkans. We compare these results with what is known for other members of the oak gall community, and consider the strengths and weaknesses of using a tournament approach to explore phylogeographic model space.

The city as a refuge for insect pollinators.

Research on urban insect pollinators is changing views on the biological value and ecological importance of cities. The abundance and diversity of native bee species in urban landscapes that are absent in nearby rural lands evidence the biological value and ecological importance of cities and have implications for biodiversity conservation. Lagging behind this revised image of the city are urban conservation programs that historically have invested in education and outreach rather than programs designed to achieve high-priority species conservation results. We synthesized research on urban bee species diversity and abundance to determine how urban conservation could be repositioned to better align with new views on the ecological importance of urban landscapes. Due to insect pollinators' relatively small functional requirements-habitat range, life cycle, and nesting behavior-relative to larger mammals, we argue that pollinators put high-priority and high-impact urban conservation within reach. In a rapidly urbanizing world, transforming how environmental managers view the city can improve citizen engagement and contribute to the development of more sustainable urbanization.

Molecular taxonomic analysis of the plant associations of adult pollen beetles (Nitidulidae: Meligethinae), and the population structure of Brassicogethes aeneus.

Pollen beetles (Nitidulidae: Meligethinae) are among the most abundant flower-visiting insects in Europe. While some species damage millions of hectares of crops annually, the biology of many species is little known. We assessed the utility of a 797 base pair fragment of the cytochrome oxidase 1 gene to resolve molecular operational taxonomic units (MOTUs) in 750 adult pollen beetles sampled from flowers of 63 plant species sampled across the UK and continental Europe. We used the same locus to analyse region-scale patterns in population structure and demography in an economically important pest, Brassicogethes aeneus. We identified 44 Meligethinae at ∼2% divergence, 35 of which contained published sequences. A few specimens could not be identified because the MOTUs containing them included published sequences for multiple Linnaean species, suggesting either retention of ancestral haplotype polymorphism or identification errors in published sequences. Over 90% of UK specimens were identifiable as B. aeneus. Plant associations of adult B. aeneus were found to be far wider taxonomically than for their larvae. UK B. aeneus populations showed contrasting affiliations between the north (most similar to Scandinavia and the Baltic) and south (most similar to western continental Europe), with strong signatures of population growth in the south.

Where is the UK's pollinator biodiversity? The importance of urban areas for flower-visiting insects.

Insect pollinators provide a crucial ecosystem service, but are under threat. Urban areas could be important for pollinators, though their value relative to other habitats is poorly known. We compared pollinator communities using quantified flower-visitation networks in 36 sites (each 1 km(2)) in three landscapes: urban, farmland and nature reserves. Overall, flower-visitor abundance and species richness did not differ significantly between the three landscape types. Bee abundance did not differ between landscapes, but bee species richness was higher in urban areas than farmland. Hoverfly abundance was higher in farmland and nature reserves than urban sites, but species richness did not differ significantly. While urban pollinator assemblages were more homogeneous across space than those in farmland or nature reserves, there was no significant difference in the numbers of rarer species between the three landscapes. Network-level specialization was higher in farmland than urban sites. Relative to other habitats, urban visitors foraged from a greater number of plant species (higher generality) but also visited a lower proportion of available plant species (higher specialization), both possibly driven by higher urban plant richness. Urban areas are growing, and improving their value for pollinators should be part of any national strategy to conserve and restore pollinators.