Diversity and divergence: evolution of secondary metabolism in the tropical tree genus Inga.

Plants are widely recognized as chemical factories, with each species producing dozens to hundreds of unique secondary metabolites. These compounds shape the interactions between plants and their natural enemies. We explore the evolutionary patterns and processes by which plants generate chemical diversity, from evolving novel compounds to unique chemical profiles. We characterized the chemical profile of one-third of the species of tropical rainforest trees in the genus Inga (c. 100, Fabaceae) using ultraperformance liquid chromatography-mass spectrometry-based metabolomics and applied phylogenetic comparative methods to understand the mode of chemical evolution. We show: each Inga species contain structurally unrelated compounds and high levels of phytochemical diversity; closely related species have divergent chemical profiles, with individual compounds, compound classes, and chemical profiles showing little-to-no phylogenetic signal; at the evolutionary time scale, a species' chemical profile shows a signature of divergent adaptation. At the ecological time scale, sympatric species were the most divergent, implying it is also advantageous to maintain a unique chemical profile from community members; finally, we integrate these patterns with a model for how chemical diversity evolves. Taken together, these results show that phytochemical diversity and divergence are fundamental to the ecology and evolution of plants.

Faecal metabarcoding reveals pervasive long-distance impacts of garden bird feeding.

Supplementary feeding of wildlife is widespread, being undertaken by more than half of households in many countries. However, the impact that these supplemental resources have is unclear, with impacts largely considered to be restricted to urban ecosystems. We reveal the pervasiveness of supplementary foodstuffs in the diet of a wild bird using metabarcoding of blue tit (Cyanistes caeruleus) faeces collected in early spring from a 220 km transect in Scotland with a large urbanization gradient. Supplementary foodstuffs were present in the majority of samples, with peanut (Arachis hypogaea) the single commonest (either natural or supplementary) dietary item. Consumption rates exhibited a distance decay from human habitation but remained high at several hundred metres from the nearest household and continued to our study limit of 1.4 km distant. Supplementary food consumption was associated with a near quadrupling of blue tit breeding density and a 5-day advancement of breeding phenology. We show that woodland bird species using supplementary food have increasing UK population trends, while species that do not, and/or are outcompeted by blue tits, are likely to be declining. We suggest that the impacts of supplementary feeding are larger and more spatially extensive than currently appreciated and could be disrupting population and ecosystem dynamics.

Phylogenomics reveals accelerated late Cretaceous diversification of bee flies (Diptera: Bombyliidae).

Bombyliidae is a very species-rich and widespread family of parasitoid flies with more than 250 genera classified into 17 extant subfamilies. However, little is known about their evolutionary history or how their present-day diversity was shaped. Transcriptomes of 15 species and anchored hybrid enrichment (AHE) sequence captures of 86 species, representing 94 bee fly species and 14 subfamilies, were used to reconstruct the phylogeny of Bombyliidae. We integrated data from transcriptomes across each of the main lineages in our AHE tree to build a data set with more genes (550 loci versus 216 loci) and higher support levels. Our overall results show strong congruence with the current classification of the family, with 11 out of 14 included subfamilies recovered as monophyletic. Heterotropinae and Mythicomyiinae are successive sister groups to the remainder of the family. We examined the evolution of key morphological characters through our phylogenetic hypotheses and show that neither the "sand chamber subfamilies" nor the "Tomophthalmae" are monophyletic in our phylogenomic analyses. Based on our results, we reinstate two tribes at the subfamily level (Phthiriinae stat. rev. and Ecliminae stat. rev.) and we include the genus Sericosoma Macquart (previously incertae sedis) in the subfamily Oniromyiinae, bringing the total number of bee fly subfamilies to 19. Our dating analyses indicate a Jurassic origin of the family (165-194 Ma), with the sand chamber evolving early in bee fly evolution, in the late Jurassic or mid-Cretaceous (100-165 Ma). We hypothesize that the angiosperm radiation and the hothouse climate established during the late Cretaceous accelerated the diversification of bee flies, by providing an expanded range of resources for the parasitoid larvae and nectarivorous adults.

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.

Hybrid capture of 964 nuclear genes resolves evolutionary relationships in the mimosoid legumes and reveals the polytomous origins of a large pantropical radiation.

PREMISE: Targeted enrichment methods facilitate sequencing of hundreds of nuclear loci to enhance phylogenetic resolution and elucidate why some parts of the "tree of life" are difficult (if not impossible) to resolve. The mimosoid legumes are a prominent pantropical clade of ~3300 species of woody angiosperms for which previous phylogenies have shown extensive lack of resolution, especially among the species-rich and taxonomically challenging ingoids. METHODS: We generated transcriptomes to select low-copy nuclear genes, enrich these via hybrid capture for representative species of most mimosoid genera, and analyze the resulting data using de novo assembly and various phylogenomic tools for species tree inference. We also evaluate gene tree support and conflict for key internodes and use phylogenetic network analysis to investigate phylogenetic signal across the ingoids. RESULTS: Our selection of 964 nuclear genes greatly improves phylogenetic resolution across the mimosoid phylogeny and shows that the ingoid clade can be resolved into several well-supported clades. However, nearly all loci show lack of phylogenetic signal for some of the deeper internodes within the ingoids. CONCLUSIONS: Lack of resolution in the ingoid clade is most likely the result of hyperfast diversification, potentially causing a hard polytomy of six or seven lineages. The gene set for targeted sequencing presented here offers great potential to further enhance the phylogeny of mimosoids and the wider Caesalpinioideae with denser taxon sampling, to provide a framework for taxonomic reclassification, and to study the ingoid radiation.

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.

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.

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.

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.

Likelihood-based inference of population history from low-coverage de novo genome assemblies.

Short-read sequencing technologies have in principle made it feasible to draw detailed inferences about the recent history of any organism. In practice, however, this remains challenging due to the difficulty of genome assembly in most organisms and the lack of statistical methods powerful enough to discriminate between recent, nonequilibrium histories. We address both the assembly and inference challenges. We develop a bioinformatic pipeline for generating outgroup-rooted alignments of orthologous sequence blocks from de novo low-coverage short-read data for a small number of genomes, and show how such sequence blocks can be used to fit explicit models of population divergence and admixture in a likelihood framework. To illustrate our approach, we reconstruct the Pleistocene history of an oak-feeding insect (the oak gallwasp Biorhiza pallida), which, in common with many other taxa, was restricted during Pleistocene ice ages to a longitudinal series of southern refugia spanning the Western Palaearctic. Our analysis of sequence blocks sampled from a single genome from each of three major glacial refugia reveals support for an unexpected history dominated by recent admixture. Despite the fact that 80% of the genome is affected by admixture during the last glacial cycle, we are able to infer the deeper divergence history of these populations. These inferences are robust to variation in block length, mutation model and the sampling location of individual genomes within refugia. This combination of de novo assembly and numerical likelihood calculation provides a powerful framework for estimating recent population history that can be applied to any organism without the need for prior genetic resources.

Life cycle closure of Cerroneuroterus minutulus (Giraud, 1859) with C. aggregatus (Wachtl, 1880) (Hymenoptera: Cynipidae: Cynipini): experimental, taxonomic and molecular approaches.

The life cycle of Cerroneuroterus minutulus (Giraud, 1859), a species previously known only from its asexual generation, is closed. Our study demonstrates that C. minutulus exhibits the heterogony typical of Cynipini species, with alternating sexual and asexual generations. The identity of the sexual generation is demonstrated by laboratory experiments and confirmed by molecular data, with Cerroneuroterus aggregatus (Wachtl, 1880) determined to be the sexual generation, and junior synonym, of Cerroneuroterus minutulus (Giraud, 1859). Illustrations and diagnoses for adults and galls, observations on biology and distribution information are provided as is a detailed redescription of the sexual generation of C. minutulus.

Plastid phylogenomics reveals evolutionary relationships in the mycoheterotrophic orchid genus Dipodium and provides insights into plastid gene degeneration.

The orchid genus Dipodium R.Br. (Epidendroideae) comprises leafy autotrophic and leafless mycoheterotrophic species, with the latter confined to sect. Dipodium. This study examined plastome degeneration in Dipodium in a phylogenomic and temporal context. Whole plastomes were reconstructed and annotated for 24 Dipodium samples representing 14 species and two putatively new species, encompassing over 80% of species diversity in sect. Dipodium. Phylogenomic analysis based on 68 plastid loci including a broad outgroup sampling across Orchidaceae found that sect. Leopardanthus is the sister lineage to sect. Dipodium. Dipodium ensifolium, the only leafy autotrophic species in sect. Dipodium, was found to be a sister to all leafless, mycoheterotrophic species, supporting a single evolutionary origin of mycoheterotrophy in the genus. Divergence-time estimations found that Dipodium arose ca. 33.3 Ma near the lower boundary of the Oligocene and that crown diversification commenced in the late Miocene, ca. 11.3 Ma. Mycoheterotrophy in the genus was estimated to have evolved in the late Miocene, ca. 7.3 Ma, in sect. Dipodium. The comparative assessment of plastome structure and gene degradation in Dipodium revealed that plastid ndh genes were pseudogenised or physically lost in all Dipodium species, including in leafy autotrophic species of both Dipodium sections. Levels of plastid ndh gene degradation were found to vary among species as well as within species, providing evidence of relaxed selection for retention of the NADH dehydrogenase complex within the genus. Dipodium exhibits an early stage of plastid genome degradation, as all species were found to have retained a full set of functional photosynthesis-related genes and housekeeping genes. This study provides important insights into plastid genome degradation along the transition from autotrophy to mycoheterotrophy in a phylogenomic and temporal context.

Life history, natural enemies, and management of Disholcaspis quercusvirens (Hymenoptera: Cynipidae) on live oak trees.

Live oak (Quercus virginiana Mill.) trees are hosts to a complex of gall making arthropods. However, the bullet galls produced by the asexual generation of the cynipid Disholcaspis quercuscirens (Ashmead) can esthetically and physically damage nursery and street trees, and thus reduce tree value. We sought to describe the unknown sexual generation of D. quercusvirens, describe the development of galls from both generations, record adult cynipid and parasitoid activity periods, and evaluate the efficacy of several insecticides to suppress the gall makers and prevent additional gall formation. The oviposition period for asexual females occurred from late November to January in both years of the caging study. Eggs laid into dormant buds resulted in small bud galls in which the sexual generation developed for 4-5 mo. Sexual adults emerged and laid eggs in young elongating shoots in April. Bullet galls began protruding from branches in June, and asexual wasps emerged 5-7 mo later. Cynipids that emerged from the bullet (asexual generation) and bud (sexual generation) galls were genetically identical. Both generations were heavily parasitized. Targeting asexual females with an early December treatment of bifenthrin or acephate significantly reduced the number of bud galls, but control did not extend to the next generation of bullet galls, possibly because of reinvasion from neighboring infested trees.

A new genus of oak gallwasp, Cyclocynips Melika, Tang & Sinclair (Hymenoptera: Cynipidae: Cynipini), with descriptions of two new species from Taiwan.

A new genus of cynipid oak gallwasp-Cyclocynips Melika, Tang, & Sinclair (Hymenoptera: Cynipidae: Cynipini), with two new species--C. uberis and C. tumorvirgae--reared from galls on oaks of the Quercus subgenus Cyclobalanopsis is described from Taiwan. Descriptions of asexual generation adults and their diagnostic characters are presented. The likelihood of yet undiscovered sexual generations and the evolution of host-plant associations in these species are discussed.

Holocynips illinoiensis, sp. nov., a new species of oak gall wasp (Hymenoptera: Cynipidae) from the USA.

A new species, Holocynips illinoiensis Melika & Nicholls, sp. nov. is described from the Nearctic (America north of Mexico). Description, diagnoses, information on biology and host association is given for the new species, as well as preliminary discussion on the coherence of the genus Holocynips.

Re-establishment of the Nearctic oak cynipid gall wasp genus Feron Kinsey, 1937 (Hymenoptera: Cynipidae: Cynipini), including the description of six new species.

The Nearctic cynipid oak gall wasp genus Feron Kinsey, comb. rev., is re-established with 34 species: F. albicomus (Weld, 1952), comb. nov., F. amphorus (Weld, 1926), comb. nov., F. apiarium (Weld, 1944), comb. nov., F. atrimentum (Kinsey, 1922), comb. nov., F. bakkeri (Lyon, 1984), comb. nov., F. caepula (Weld, 1926), comb. nov., F. californicum (Beutenmueller, 1911), comb. nov., F. clarkei (Bassett, 1890), comb. nov., F. comatum (Weld, 1952), comb. nov., F. crystallinum (Bassett, 1900), comb. nov., F. cylindratum (Kinsey, 1937), comb. nov., F. discale (Weld, 1926), comb. nov., F. discularis (Weld, 1926), comb. nov., F. dumosae (Weld, 1957), comb. nov., F. gigas (Kinsey, 1922), comb. nov., F. izabellae Melika, Nicholls & Stone, sp. nov., F. kingi (Bassett, 1900), comb. nov., F. parmula (Bassett, 1900), comb. nov., F. pattersonae (Fullaway, 1911), comb. nov., F. roberti Melika, Nicholls & Stone, sp. nov., F. rucklei Melika, Nicholls & Stone, sp. nov., F. scutellum (Weld, 1930), comb. nov., F. serranoae Pujade-Villar & Cuesta-Porta, sp. nov., F. splendens (Weld, 1919), comb. nov., F. stellare (Weld, 1926), comb. nov., F. stellulum (Burnett, 1974), comb. nov., F. sulfureum (Weld, 1926), comb. nov., F. syndicorum Pujade-Villar & Cuesta-Porta, sp. nov., F. tecturnarum (Kinsey, 1920), comb. nov., F. tetyanae Melika, sp. nov., F. tibiale Kinsey, 1937, comb. rev., F. tubifaciens (Weld, 1926), comb. nov., F. verutum Kinsey, 1937, comb. rev., and F. vitreum Kinsey, 1937, comb. rev. Most species are known only from the asexual generation; F. clarkei, F. comatum, and F. dumosae are known only from the sexual generation, while both generations are recognised for F. atrimentum, F. crystallinum, F. gigas, F. kingi, and F. pattersonae. Matching of alternate sexual and asexual generations is established for the first time for F. kingi and F. pattersonae (= Andricus pedicellatus (Kinsey, 1922), syn. nov.) based on molecular data (both cytb and ITS2 sequences). Morphological descriptions, re-descriptions, diagnoses, and a key to species are given, as well as data on DNA sequences, biology, phenology, and distribution.

Revision of the Amphibolips niger group with the description of a new species (Hymenoptera: Cynipini).

Amphibolips is currently divided into two species-groups, clearly differentiated by adult and gall morphology. The niger group of Amphibolips species is revised. This complex includes eight species: A. gumia Kinsey, A. jubatus Kinsey, A. elatus Kinsey, A. maturus Kinsey, A. nebris Kinsey, A. niger Kinsey, A. pistrix Kinsey and A. ufo Cuesta-Porta & Pujade-Villar sp. nov. We provide descriptions of new species and re-descriptions of known species, diagnoses, keys to complexes and species. The possibility of the niger complex belonging to a new genus is also discussed.

Precipitation is the main axis of tropical plant phylogenetic turnover across space and time.

Early natural historians-Comte de Buffon, von Humboldt, and De Candolle-established environment and geography as two principal axes determining the distribution of groups of organisms, laying the foundations for biogeography over the subsequent 200 years, yet the relative importance of these two axes remains unresolved. Leveraging phylogenomic and global species distribution data for Mimosoid legumes, a pantropical plant clade of c. 3500 species, we show that the water availability gradient from deserts to rain forests dictates turnover of lineages within continents across the tropics. We demonstrate that 95% of speciation occurs within a precipitation niche, showing profound phylogenetic niche conservatism, and that lineage turnover boundaries coincide with isohyets of precipitation. We reveal similar patterns on different continents, implying that evolution and dispersal follow universal processes.

Mitochondrial barcodes are diagnostic of shared refugia but not species in hybridizing oak gallwasps.

Mitochondrial DNA barcodes provide a simple taxonomic tool for systematic and ecological research, with particular benefit for poorly studied or species-rich taxa. Barcoding assumes genetic diversity follows species boundaries; however, many processes disrupt species-level monophyly of barcodes leading to incorrect classifications. Spatial population structure, particularly when shared across closely related and potentially hybridizing taxa, can invalidate barcoding approaches yet few data exist to examine its impacts. We test how shared population structure across hybridizing species impacts upon mitochondrial barcodes by sequencing the cytochrome b gene for 518 individuals of four well-delimited Western Palaearctic gallwasp species within the Andricus quercuscalicis species group. Mitochondrial barcodes clustered individuals into mixed-species clades corresponding to refugia, with no difference in within- and between-species divergence. Four nuclear genes were also sequenced from 4 to 11 individuals per refugial population of each species. Multi-locus analyses of these data supported established species, with no support for the refugial clustering across species seen in mitochondrial barcodes. This pattern is consistent with mitochondrial introgression among populations of species sharing the same glacial refugium, such that mitochondrial barcodes identify a shared history of population structure rather than species. Many taxa show phylogeographic structure across glacial refugia, suggesting that mitochondrial barcoding may fail when applied to other sets of co-distributed, closely related species. Robust barcoding approaches must sample extensively across population structure to disentangle spatial from species-level variation. Methods incorporating multiple unlinked loci are also essential to accommodate coalescent variation among genes and provide power to resolve recently diverged species.