What Goes in Must Come Out? The Metabolic Profile of Plants and Caterpillars, Frass, And Adults of Asota (Erebidae: Aganainae) Feeding on Ficus (Moraceae) in New Guinea.

Insect herbivores have evolved a broad spectrum of adaptations in response to the diversity of chemical defences employed by plants. Here we focus on two species of New Guinean Asota and determine how these specialist moths deal with the leaf alkaloids of their fig (Ficus) hosts. As each focal Asota species is restricted to one of three chemically distinct species of Ficus, we also test whether these specialized interactions lead to similar alkaloid profiles in both Asota species. We reared Asota caterpillars on their respective Ficus hosts in natural conditions and analyzed the alkaloid profiles of leaf, frass, caterpillar, and adult moth samples using UHPLC-MS/MS analyses. We identified 43 alkaloids in our samples. Leaf alkaloids showed various fates. Some were excreted in frass or found in caterpillars and adult moths. We also found two apparently novel indole alkaloids-likely synthesized de novo by the moths or their microbiota-in both caterpillar and adult tissue but not in leaves or frass. Overall, alkaloids unique or largely restricted to insect tissue were shared across moth species despite feeding on different hosts. This indicates that a limited number of plant compounds have a direct ecological function that is conserved among the studied species. Our results provide evidence for the importance of phytochemistry and metabolic strategies in the formation of plant-insect interactions and food webs in general. Furthermore, we provide a new potential example of insects acquiring chemicals for their benefit in an ecologically relevant insect genus.

Spatial covariance of herbivorous and predatory guilds of forest canopy arthropods along a latitudinal gradient.

In arthropod community ecology, species richness studies tend to be prioritised over those investigating patterns of abundance. Consequently, the biotic and abiotic drivers of arboreal arthropod abundance are still relatively poorly known. In this cross-continental study, we employ a theoretical framework in order to examine patterns of covariance among herbivorous and predatory arthropod guilds. Leaf-chewing and leaf-mining herbivores, and predatory ants and spiders, were censused on > 1000 trees in nine 0.1 ha forest plots. After controlling for tree size and season, we found no negative pairwise correlations between guild abundances per plot, suggestive of weak signals of both inter-guild competition and top-down regulation of herbivores by predators. Inter-guild interaction strengths did not vary with mean annual temperature, thus opposing the hypothesis that biotic interactions intensify towards the equator. We find evidence for the bottom-up limitation of arthropod abundances via resources and abiotic factors, rather than for competition and predation.

Vertical stratification of a temperate forest caterpillar community in eastern North America.

Vertical niche partitioning might be one of the main driving forces explaining the high diversity of forest ecosystems. However, the forest's vertical dimension has received limited investigation, especially in temperate forests. Thus, our knowledge about how communities are vertically structured remains limited for temperate forest ecosystems. In this study, we investigated the vertical structuring of an arboreal caterpillar community in a temperate deciduous forest of eastern North America. Within a 0.2-ha forest stand, all deciduous trees ≥ 5 cm diameter at breast height (DBH) were felled and systematically searched for caterpillars. Sampled caterpillars were assigned to a specific stratum (i.e. understory, midstory, or canopy) depending on their vertical position and classified into feeding guild as either exposed feeders or shelter builders (i.e. leaf rollers, leaf tiers, webbers). In total, 3892 caterpillars representing 215 species of butterflies and moths were collected and identified. While stratum had no effect on caterpillar density, feeding guild composition changed significantly with shelter-building caterpillars becoming the dominant guild in the canopy. Species richness and diversity were found to be highest in the understory and midstory and declined strongly in the canopy. Family and species composition changed significantly among the strata; understory and canopy showed the lowest similarity. Food web analyses further revealed an increasing network specialization towards the canopy, caused by an increase in specialization of the caterpillar community. In summary, our study revealed a pronounced stratification of a temperate forest caterpillar community, unveiling a distinctly different assemblage of caterpillars dwelling in the canopy stratum.

Compound Specific Trends of Chemical Defences in Ficus Along an Elevational Gradient Reflect a Complex Selective Landscape.

Elevational gradients affect the production of plant secondary metabolites through changes in both biotic and abiotic conditions. Previous studies have suggested both elevational increases and decreases in host-plant chemical defences. We analysed the correlation of alkaloids and polyphenols with elevation in a community of nine Ficus species along a continuously forested elevational gradient in Papua New Guinea. We sampled 204 insect species feeding on the leaves of these hosts and correlated their community structure to the focal compounds. Additionally, we explored species richness of folivorous mammals along the gradient. When we accounted for Ficus species identity, we found a general elevational increase in flavonoids and alkaloids. Elevational trends in non-flavonol polyphenols were less pronounced or showed non-linear correlations with elevation. Polyphenols responded more strongly to changes in temperature and humidity than alkaloids. The abundance of insect herbivores decreased with elevation, while the species richness of folivorous mammals showed an elevational increase. Insect community structure was affected mainly by alkaloid concentration and diversity. Although our results show an elevational increase in several groups of metabolites, the drivers behind these trends likely differ. Flavonoids may provide figs with protection against abiotic stressors. In contrast, alkaloids affect insect herbivores and may provide protection against mammalian herbivores and pathogens. Concurrent analysis of multiple compound groups alongside ecological data is an important approach for understanding the selective landscape that shapes plant defences.

A highly resolved food web for insect seed predators in a species-rich tropical forest.

The top-down and indirect effects of insects on plant communities depend on patterns of host use, which are often poorly documented, particularly in species-rich tropical forests. At Barro Colorado Island, Panama, we compiled the first food web quantifying trophic interactions between the majority of co-occurring woody plant species and their internally feeding insect seed predators. Our study is based on more than 200 000 fruits representing 478 plant species, associated with 369 insect species. Insect host-specificity was remarkably high: only 20% of seed predator species were associated with more than one plant species, while each tree species experienced seed predation from a median of two insect species. Phylogeny, but not plant traits, explained patterns of seed predator attack. These data suggest that seed predators are unlikely to mediate indirect interactions such as apparent competition between plant species, but are consistent with their proposed contribution to maintaining plant diversity via the Janzen-Connell mechanism.

Community structure of insect herbivores is driven by conservatism, escalation and divergence of defensive traits in Ficus.

Escalation (macroevolutionary increase) or divergence (disparity between relatives) in trait values are two frequent outcomes of the plant-herbivore arms race. We studied the defences and caterpillars associated with 21 sympatric New Guinean figs. Herbivore generalists were concentrated on hosts with low protease and oxidative activity. The distribution of specialists correlated with phylogeny, protease and trichomes. Additionally, highly specialised Asota moths used alkaloid rich plants. The evolution of proteases was conserved, alkaloid diversity has escalated across the studied species, oxidative activity has escalated within one clade, and trichomes have diverged across the phylogeny. Herbivore specificity correlated with their response to host defences: escalating traits largely affected generalists and divergent traits specialists; but the effect of escalating traits on extreme specialists was positive. In turn, the evolution of defences in Ficus can be driven towards both escalation and divergence in individual traits, in combination providing protection against a broad spectrum of herbivores.

The global distribution of diet breadth in insect herbivores.

Understanding variation in resource specialization is important for progress on issues that include coevolution, community assembly, ecosystem processes, and the latitudinal gradient of species richness. Herbivorous insects are useful models for studying resource specialization, and the interaction between plants and herbivorous insects is one of the most common and consequential ecological associations on the planet. However, uncertainty persists regarding fundamental features of herbivore diet breadth, including its relationship to latitude and plant species richness. Here, we use a global dataset to investigate host range for over 7,500 insect herbivore species covering a wide taxonomic breadth and interacting with more than 2,000 species of plants in 165 families. We ask whether relatively specialized and generalized herbivores represent a dichotomy rather than a continuum from few to many host families and species attacked and whether diet breadth changes with increasing plant species richness toward the tropics. Across geographic regions and taxonomic subsets of the data, we find that the distribution of diet breadth is fit well by a discrete, truncated Pareto power law characterized by the predominance of specialized herbivores and a long, thin tail of more generalized species. Both the taxonomic and phylogenetic distributions of diet breadth shift globally with latitude, consistent with a higher frequency of specialized insects in tropical regions. We also find that more diverse lineages of plants support assemblages of relatively more specialized herbivores and that the global distribution of plant diversity contributes to but does not fully explain the latitudinal gradient in insect herbivore specialization.

Variably hungry caterpillars: predictive models and foliar chemistry suggest how to eat a rainforest.

A long-term goal in evolutionary ecology is to explain the incredible diversity of insect herbivores and patterns of host plant use in speciose groups like tropical Lepidoptera. Here, we used standardized food-web data, multigene phylogenies of both trophic levels and plant chemistry data to model interactions between Lepidoptera larvae (caterpillars) from two lineages (Geometridae and Pyraloidea) and plants in a species-rich lowland rainforest in New Guinea. Model parameters were used to make and test blind predictions for two hectares of an exhaustively sampled forest. For pyraloids, we relied on phylogeny alone and predicted 54% of species-level interactions, translating to 79% of all trophic links for individual insects, by sampling insects from only 15% of local woody plant diversity. The phylogenetic distribution of host-plant associations in polyphagous geometrids was less conserved, reducing accuracy. In a truly quantitative food web, only 40% of pair-wise interactions were described correctly in geometrids. Polyphenol oxidative activity (but not protein precipitation capacity) was important for understanding the occurrence of geometrids (but not pyraloids) across their hosts. When both foliar chemistry and plant phylogeny were included, we predicted geometrid-plant occurrence with 89% concordance. Such models help to test macroevolutionary hypotheses at the community level.

Phylogenetic trophic specialization: a robust comparison of herbivorous guilds.

Resource specialization is a key concept in ecology, but it is unexpectedly difficult to parameterize. Differences in resource availability, sampling effort and abundances preclude comparisons of incompletely sampled biotic interaction webs. Here, we extend the distance-based specialization index (DSI) that measures trophic specialization by taking resource phylogenetic relatedness and availability into account into a rescaled version, DSI*. It is a versatile metric of specialization that expands considerably the scope and applicability, hence the usefulness, of DSI. The new metric also accounts for differences in abundance and sampling effort of consumers, which enables robust comparisons among distinct guilds of consumers. It also provides an abundance threshold for the reliability of the metric for rare species, a very desirable property given the difficulty of assessing any aspect of rare species accurately. We apply DSI* to an extensive dataset on interactions between insect herbivores from four folivorous guilds and their host plants in Papua New Guinean rainforests. We demonstrate that DSI*, contrary to the original DSI, is largely independent of sample size and weakly and non-linearly related with several host specificity measures that do not adjust for plant phylogeny. Thus, DSI* provides further insights into host specificity patterns; moreover, it is robust to the number and phylogenetic diversity of plant species selected to be sampled for herbivores. DSI* can be used for a broad range of comparisons of distinct feeding guilds, geographical locations and ecological conditions. This is a key advance in elucidating the interaction structure and evolution of highly diversified systems.

Calibrating the taxonomy of a megadiverse insect family: 3000 DNA barcodes from geometrid type specimens (Lepidoptera, Geometridae).

It is essential that any DNA barcode reference library be based upon correctly identified specimens. The Barcode of Life Data Systems (BOLD) requires information such as images, geo-referencing, and details on the museum holding the voucher specimen for each barcode record to aid recognition of potential misidentifications. Nevertheless, there are misidentifications and incomplete identifications (e.g., to a genus or family) on BOLD, mainly for species from tropical regions. Unfortunately, experts are often unavailable to correct taxonomic assignments due to time constraints and the lack of specialists for many groups and regions. However, considerable progress could be made if barcode records were available for all type specimens. As a result of recent improvements in analytical protocols, it is now possible to recover barcode sequences from museum specimens that date to the start of taxonomic work in the 18th century. The present study discusses success in the recovery of DNA barcode sequences from 2805 type specimens of geometrid moths which represent 1965 species, corresponding to about 9% of the 23 000 described species in this family worldwide and including 1875 taxa represented by name-bearing types. Sequencing success was high (73% of specimens), even for specimens that were more than a century old. Several case studies are discussed to show the efficiency, reliability, and sustainability of this approach.

Global arthropod beta-diversity is spatially and temporally structured by latitude.

Global biodiversity gradients are generally expected to reflect greater species replacement closer to the equator. However, empirical validation of global biodiversity gradients largely relies on vertebrates, plants, and other less diverse taxa. Here we assess the temporal and spatial dynamics of global arthropod biodiversity dynamics using a beta-diversity framework. Sampling includes 129 sampling sites whereby malaise traps are deployed to monitor temporal changes in arthropod communities. Overall, we encountered more than 150,000 unique barcode index numbers (BINs) (i.e. species proxies). We assess between site differences in community diversity using beta-diversity and the partitioned components of species replacement and richness difference. Global total beta-diversity (dissimilarity) increases with decreasing latitude, greater spatial distance and greater temporal distance. Species replacement and richness difference patterns vary across biogeographic regions. Our findings support long-standing, general expectations of global biodiversity patterns. However, we also show that the underlying processes driving patterns may be regionally linked.

Parasitism rate, parasitoid community composition and host specificity on exposed and semi-concealed caterpillars from a tropical rainforest.

The processes maintaining the enormous diversity of herbivore-parasitoid food webs depend on parasitism rate and parasitoid host specificity. The two parameters have to be evaluated in concert to make conclusions about the importance of parasitoids as natural enemies and guide biological control. We document parasitism rate and host specificity in a highly diverse caterpillar-parasitoid food web encompassing 266 species of lepidopteran hosts and 172 species of hymenopteran or dipteran parasitoids from a lowland tropical forest in Papua New Guinea. We found that semi-concealed hosts (leaf rollers and leaf tiers) represented 84% of all caterpillars, suffered a higher parasitism rate than exposed caterpillars (12 vs. 5%) and their parasitoids were also more host specific. Semi-concealed hosts may therefore be generally more amenable to biological control by parasitoids than exposed ones. Parasitoid host specificity was highest in Braconidae, lower in Diptera: Tachinidae, and, unexpectedly, the lowest in Ichneumonidae. This result challenges the long-standing view of low host specificity in caterpillar-attacking Tachinidae and suggests higher suitability of Braconidae and lower suitability of Ichneumonidae for biological control of caterpillars. Semi-concealed hosts and their parasitoids are the largest, yet understudied component of caterpillar-parasitoid food webs. However, they still remain much closer in parasitism patterns to exposed hosts than to what literature reports on fully concealed leaf miners. Specifically, semi-concealed hosts keep an equally low share of idiobionts (2%) as exposed caterpillars.

Estimating global arthropod species richness: refining probabilistic models using probability bounds analysis.

A key challenge in the estimation of tropical arthropod species richness is the appropriate management of the large uncertainties associated with any model. Such uncertainties had largely been ignored until recently, when we attempted to account for uncertainty associated with model variables, using Monte Carlo analysis. This model is restricted by various assumptions. Here, we use a technique known as probability bounds analysis to assess the influence of assumptions about (1) distributional form and (2) dependencies between variables, and to construct probability bounds around the original model prediction distribution. The original Monte Carlo model yielded a median estimate of 6.1 million species, with a 90 % confidence interval of [3.6, 11.4]. Here we found that the probability bounds (p-bounds) surrounding this cumulative distribution were very broad, owing to uncertainties in distributional form and dependencies between variables. Replacing the implicit assumption of pure statistical independence between variables in the model with no dependency assumptions resulted in lower and upper p-bounds at 0.5 cumulative probability (i.e., at the median estimate) of 2.9-12.7 million. From here, replacing probability distributions with probability boxes, which represent classes of distributions, led to even wider bounds (2.4-20.0 million at 0.5 cumulative probability). Even the 100th percentile of the uppermost bound produced (i.e., the absolutely most conservative scenario) did not encompass the well-known hyper-estimate of 30 million species of tropical arthropods. This supports the lower estimates made by several authors over the last two decades.

Low beta diversity of herbivorous insects in tropical forests.

Recent advances in understanding insect communities in tropical forests have contributed little to our knowledge of large-scale patterns of insect diversity, because incomplete taxonomic knowledge of many tropical species hinders the mapping of their distribution records. This impedes an understanding of global biodiversity patterns and explains why tropical insects are under-represented in conservation biology. Our study of approximately 500 species from three herbivorous guilds feeding on foliage (caterpillars, Lepidoptera), wood (ambrosia beetles, Coleoptera) and fruit (fruitflies, Diptera) found a low rate of change in species composition (beta diversity) across 75,000 square kilometres of contiguous lowland rainforest in Papua New Guinea, as most species were widely distributed. For caterpillars feeding on large plant genera, most species fed on multiple host species, so that even locally restricted plant species did not support endemic herbivores. Large plant genera represented a continuously distributed resource easily colonized by moths and butterflies over hundreds of kilometres. Low beta diversity was also documented in groups with differing host specificity (fruitflies and ambrosia beetles), suggesting that dispersal limitation does not have a substantial role in shaping the distribution of insect species in New Guinea lowland rainforests. Similar patterns of low beta diversity can be expected in other tropical lowland rainforests, as they are typically situated in the extensive low basins of major tropical rivers similar to the Sepik-Ramu region of New Guinea studied here.

Population genetics of ecological communities with DNA barcodes: an example from New Guinea Lepidoptera.

Comparative population genetics of ecological guilds can reveal generalities in patterns of differentiation bearing on hypotheses regarding the origin and maintenance of community diversity. Contradictory estimates of host specificity and beta diversity in tropical Lepidoptera (moths and butterflies) from New Guinea and the Americas have sparked debate on the role of host-associated divergence and geographic isolation in explaining latitudinal diversity gradients. We sampled haplotypes of mitochondrial cytochrome c oxidase I from 28 Lepidoptera species and 1,359 individuals across four host plant genera and eight sites in New Guinea to estimate population divergence in relation to host specificity and geography. Analyses of molecular variance and haplotype networks indicate varying patterns of genetic structure among ecologically similar sympatric species. One-quarter lacked evidence of isolation by distance or host-associated differentiation, whereas 21% exhibited both. Fourteen percent of the species exhibited host-associated differentiation without geographic isolation, 18% showed the opposite, and 21% were equivocal, insofar as analyses of molecular variance and haplotype networks yielded incongruent patterns. Variation in dietary breadth among community members suggests that speciation by specialization is an important, but not universal, mechanism for diversification of tropical Lepidoptera. Geographically widespread haplotypes challenge predictions of vicariance biogeography. Dispersal is important, and Lepidoptera communities appear to be highly dynamic according to the various phylogeographic histories of component species. Population genetic comparisons among herbivores of major tropical and temperate regions are needed to test predictions of ecological theory and evaluate global patterns of biodiversity.

A workflow for expanding DNA barcode reference libraries through 'museum harvesting' of natural history collections.

Natural history collections are the physical repositories of our knowledge on species, the entities of biodiversity. Making this knowledge accessible to society - through, for example, digitisation or the construction of a validated, global DNA barcode library - is of crucial importance. To this end, we developed and streamlined a workflow for 'museum harvesting' of authoritatively identified Diptera specimens from the Smithsonian Institution's National Museum of Natural History. Our detailed workflow includes both on-site and off-site processing through specimen selection, labelling, imaging, tissue sampling, databasing and DNA barcoding. This approach was tested by harvesting and DNA barcoding 941 voucher specimens, representing 32 families, 819 genera and 695 identified species collected from 100 countries. We recovered 867 sequences (> 0 base pairs) with a sequencing success of 88.8% (727 of 819 sequenced genera gained a barcode > 300 base pairs). While Sanger-based methods were more effective for recently-collected specimens, the methods employing next-generation sequencing recovered barcodes for specimens over a century old. The utility of the newly-generated reference barcodes is demonstrated by the subsequent taxonomic assignment of nearly 5000 specimen records in the Barcode of Life Data Systems.

Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History.

The use of DNA barcoding has revolutionised biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian's National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases. Our workflow used a mix of Sanger and Next-Generation Sequencing (NGS) approaches to maximise sequence recovery while ensuring affordable cost. In total, COI sequences were obtained for 5,686 specimens belonging to 3,737 determined species in 3,886 genera and 205 families distributed in 137 countries. Success rates varied widely according to collection data and focal taxon. NGS helped recover sequences of specimens that failed a previous run of Sanger sequencing. Success rates and the optimal balance between Sanger and NGS are the most important drivers to maximise output and minimise cost in future projects. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, the Global Genome Biodiversity Network Data Portal and the NMNH data portal.

Insects on plants: explaining the paradox of low diversity within specialist herbivore guilds.

Classical niche theory explains the coexistence of species through their exploitation of different resources. Assemblages of herbivores coexisting on a particular plant species are thus expected to be dominated by species from host-specific guilds with narrow, coexistence-facilitating niches rather than by species from generalist guilds. Exactly the opposite pattern is observed for folivores feeding on trees in New Guinea. The least specialized mobile chewers were the most species rich, followed by the moderately specialized semiconcealed and exposed chewers. The highly specialized miners and mesophyll suckers were the least species-rich guilds. The Poisson distribution of herbivore species richness among plant species in specialized guilds and the absence of a negative correlation between species richness in different guilds on the same plant species suggest that these guilds are not saturated with species. We show that herbivore assemblages are enriched with generalists because these are more completely sampled from regional species pools. Herbivore diversity increases as a power function of plant diversity, and the rate of increase is inversely related to host specificity. The relative species diversity among guilds is thus scale dependent, as the importance of specialized guilds increases with plant diversity. Specialized insect guilds may therefore comprise a larger component of overall diversity in the tropics (where they are also poorly known taxonomically) than in the temperate zone, which has lower plant diversity.

A DNA barcode library for the butterflies of North America.

Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.