Metabarcoding and applied ecology with hyperdiverse organisms: Recommendations for biological control research.

Metabarcoding is revolutionizing fundamental research in ecology by enabling large-scale detection of species and producing data that are rich with community context. However, the benefits of metabarcoding have yet to be fully realized in fields of applied ecology, especially those such as classical biological control (CBC) research that involve hyperdiverse taxa. Here, we discuss some of the opportunities that metabarcoding provides CBC and solutions to the main methodological challenges that have limited the integration of metabarcoding in existing CBC workflows. We focus on insect parasitoids, which are popular and effective biological control agents (BCAs) of invasive species and agricultural pests. Accurately identifying native, invasive and BCA species is paramount, since misidentification can undermine control efforts and lead to large negative socio-economic impacts. Unfortunately, most existing publicly accessible genetic databases cannot be used to reliably identify parasitoid species, thereby limiting the accuracy of metabarcoding in CBC research. To address this issue, we argue for the establishment of authoritative genetic databases that link metabarcoding data to taxonomically identified specimens. We further suggest using multiple genetic markers to reduce primer bias and increase taxonomic resolution. We also provide suggestions for biological control-specific metabarcoding workflows intended to track the long-term effectiveness of introduced BCAs. Finally, we use the example of an invasive pest, Drosophila suzukii, in a reflective "what if" thought experiment to explore the potential power of community metabarcoding in CBC.

Deployment of Aggregation-Sex Pheromones of Longhorned Beetles (Coleoptera: Cerambycidae) Facilitates the Discovery and Identification of their Parasitoids.

Longhorned beetles (Coleoptera: Cerambycidae) include many species that are among the most damaging pests of managed and natural forest ecosystems worldwide. Many species of cerambycids use volatile chemical signals (i.e., pheromones) to locate mates. Pheromones are often used by natural enemies, including parasitoids, to locate hosts and therefore can be useful tools for identifying host-parasitoid relationships. In two field experiments, we baited linear transects of sticky traps with pheromones of cerambycid beetles in the subfamily Cerambycinae. Enantiomeric mixtures of four linear alkanes or four linear alkanes and a ketol were tested separately to evaluate their attractiveness to hymenopteran parasitoids. We hypothesized that parasitoids would be attracted to these pheromones. Significant treatment effects were found for 10 species of parasitoids. Notably, Wroughtonia ligator (Say) (Hymenoptera: Braconidae) was attracted to syn-hexanediols, the pheromone constituents of its host, Neoclytus acuminatus acuminatus (F.) (Coleoptera: Cerambycidae). Location and time of sampling also significantly affected responses for multiple species of parasitoids. These findings contribute to the basic understanding of cues that parasitoids use to locate hosts and suggest that pheromones can be used to hypothesize host relationships between some species of cerambycids and their parasitoids. Future work should evaluate response by known species of parasitoids to the complete blends of pheromones used by the cerambycids they attack, as well as other odors that are associated with host trees of cerambycids.

Comprehensive phylogenomic analyses re-write the evolution of parasitism within cynipoid wasps.

BACKGROUND: Parasitoidism, a specialized life strategy in which a parasite eventually kills its host, is frequently found within the insect order Hymenoptera (wasps, ants and bees). A parasitoid lifestyle is one of two dominant life strategies within the hymenopteran superfamily Cynipoidea, with the other being an unusual plant-feeding behavior known as galling. Less commonly, cynipoid wasps exhibit inquilinism, a strategy where some species have adapted to usurp other species' galls instead of inducing their own. Using a phylogenomic data set of ultraconserved elements from nearly all lineages of Cynipoidea, we here generate a robust phylogenetic framework and timescale to understand cynipoid systematics and the evolution of these life histories. RESULTS: Our reconstructed evolutionary history for Cynipoidea differs considerably from previous hypotheses. Rooting our analyses with non-cynipoid outgroups, the Paraulacini, a group of inquilines, emerged as sister-group to the rest of Cynipoidea, rendering the gall wasp family Cynipidae paraphyletic. The families Ibaliidae and Liopteridae, long considered archaic and early-branching parasitoid lineages, were found nested well within the Cynipoidea as sister-group to the parasitoid Figitidae. Cynipoidea originated in the early Jurassic around 190 Ma. Either inquilinism or parasitoidism is suggested as the ancestral and dominant strategy throughout the early evolution of cynipoids, depending on whether a simple (three states: parasitoidism, inquilinism and galling) or more complex (seven states: parasitoidism, inquilinism and galling split by host use) model is employed. CONCLUSIONS: Our study has significant impact on understanding cynipoid evolution and highlights the importance of adequate outgroup sampling. We discuss the evolutionary timescale of the superfamily in relation to their insect hosts and host plants, and outline how phytophagous galling behavior may have evolved from entomophagous, parasitoid cynipoids. Our study has established the framework for further physiological and comparative genomic work between gall-making, inquiline and parasitoid lineages, which could also have significant implications for the evolution of diverse life histories in other Hymenoptera.

UCE data reveal multiple origins of rose gallers in North America: Global phylogeny of Diplolepis Geoffroy (Hymenoptera: Cynipidae).

Gall wasps in the genus Diplolepis Geoffroy are specialized herbivores that induce galls exclusively on roses. Despite their wide distribution across the Holarctic, little is known about their evolutionary history. Here we present the first phylogenomic tree of global Diplolepis reconstructed using Ultraconserved Elements (UCEs), resulting in a robust phylogeny based on 757 genes. Results support the existence of two principal clades: a Nearctic stem-galler clade, and a Holarctic leaf-galler clade that further splits into two Palearctic groups and one Nearctic group. This topology is congruent with a previous study based on the mitochondrial gene COI, an unexpected result given the common occurrence of mitonuclear discordance in closely related oak gall wasp lineages. Most Diplolepis species were recovered as reciprocally monophyletic, with some notable exceptions such as the D. polita and the D. ignota complex, for which species boundaries remain unresolved. Historical biogeographic reconstruction was unable to pinpoint the origin of Diplolepis, but confirms two independent incursions into the Nearctic. Ancestral state reconstruction analysis highlights the conservatism of gall location on the host plants, as shifts to different host organs are relatively rare. We suggest that Diplolepis were originally leaf gallers, with a Nearctic stem-galler clade undergoing a major plant organ switch onto rose stems. Host organ switch or reversal is uncommon, which suggests a level of conservatism. Our study showcases the resolving power of UCEs at the species level while also suggesting improvements to advance future Cynipoidea phylogenomics. Our results also highlight the additional sampling needed to clarify taxonomic relationships in the Nearctic and eastern Palearctic regions.

Key to the North American tribes and genera of herb, rose, bramble, and inquiline gall wasps (Hymenoptera, Cynipoidea, Cynipidaesensu lato).

Robust keys exist for the family-level groups of Cynipoidea. However, for most regions of the world, keys to genera are not available. To address this gap as it applies to North America, a fully illustrated key is provided to facilitate identification of the tribes and genera of rose gall, herb gall, and inquiline gall wasps known from the region. For each taxon covered, a preliminary diagnosis and an updated overview of taxonomy, biology, distribution, and natural history are provided.

Genome divergence and reproductive incompatibility among populations of Ganaspis near brasiliensis.

During the last decade, the spotted wing drosophila, Drosophila suzukii, has spread from eastern Asia to the Americas, Europe, and Africa. This fly attacks many species of cultivated and wild fruits with soft, thin skins, where its serrated ovipositor allows it to lay eggs in undamaged fruit. Parasitoids from the native range of D. suzukii may provide sustainable management of this polyphagous pest. Among these parasitoids, host-specificity testing has revealed a lineage of Ganaspis near brasiliensis, referred to in this paper as G1, that appears to be a cryptic species more host-specific to D. suzukii than other parasitoids. Differentiation among cryptic species is critical for introduction and subsequent evaluation of their impact on D. suzukii. Here, we present results on divergence in genomic sequences and architecture and reproductive isolation between lineages of Ganaspis near brasiliensis that appear to be cryptic species. We studied five populations, two from China, two from Japan, and one from Canada, identified as the G1 vs G3 lineages based on differences in cytochrome oxidase l sequences. We assembled and annotated the genomes of these populations and analyzed divergences in sequence and genome architecture between them. We also report results from crosses to test reproductive compatibility between the G3 lineage from China and the G1 lineage from Japan. The combined results on sequence divergence, differences in genome architectures, ortholog divergence, reproductive incompatibility, differences in host ranges and microhabitat preferences, and differences in morphology show that these lineages are different species. Thus, the decision to evaluate the lineages separately and only import and introduce the more host-specific lineage to North America and Europe was appropriate.

Key innovations and the diversification of Hymenoptera.

The order Hymenoptera (wasps, ants, sawflies, and bees) represents one of the most diverse animal lineages, but whether specific key innovations have contributed to its diversification is still unknown. We assembled the largest time-calibrated phylogeny of Hymenoptera to date and investigated the origin and possible correlation of particular morphological and behavioral innovations with diversification in the order: the wasp waist of Apocrita; the stinger of Aculeata; parasitoidism, a specialized form of carnivory; and secondary phytophagy, a reversal to plant-feeding. Here, we show that parasitoidism has been the dominant strategy since the Late Triassic in Hymenoptera, but was not an immediate driver of diversification. Instead, transitions to secondary phytophagy (from parasitoidism) had a major influence on diversification rate in Hymenoptera. Support for the stinger and the wasp waist as key innovations remains equivocal, but these traits may have laid the anatomical and behavioral foundations for adaptations more directly associated with diversification.

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.

Hymenopteran Parasitoids of Leucotaraxis argenticollis (Diptera: Chamaemyiidae) and Leucotaraxis piniperda: Implications for Biological Control of Hemlock Woolly Adelgid (Hemiptera: Adelgidae).

The hemlock woolly adelgid (Adelges tsugae (Annand)) is a serious invasive pest of hemlock trees in eastern North America. Multiple biological control agents have been the focus of research aimed at pest management and conserving hemlock communities. Three promising A. tsugae specialist predators are the beetle Laricobius nigrinus (Fender) (Coleoptera: Derodontidae) and flies in the genus Leucotaraxis (Diptera: Chamaemyiidae), Leucotaraxis argenticollis (Zetterstedt), and Leucotaraxis piniperda (Malloch). However, these flies are vulnerable to parasitism by wasps in the genera Pachyneuron (Walker) (Hymenoptera: Pteromalidae) and Melanips (Walker) (Hymenoptera: Figitidae). This study explores parasitoid wasp interactions with these Leucotaraxis species in their native western North American range and potential impacts on the biological control program in the East. Leucotaraxis, La. nigrinus, and parasitoid emergences were observed from adelgid-infested foliage collected from Washington State and British Columbia in 2018, 2019, and 2020. Undescribed species of Pachyneuron and Melanips emerged from puparia as solitary parasitoids. Parasitoid emergence was positively correlated with Leucotaraxis emergence. Percent parasitism increased between February and July, with the months of June and July experiencing higher parasitoid emergence than Leucotaraxis. Differences in emergence patterns suggest that Pachyneuron may be more closely associated with Le. argenticollis as a host, and that Melanips may be associated with Le. piniperda. High parasitism in Leucotaraxis had no effect on La. nigrinus larval abundance, whereas the combined emergence of parasitoids and Leucotaraxis was positively correlated with La. nigrinus. This suggests that there is limited competition among these predators.

First Record and Distribution of Ganaspis brasiliensis (Hymenoptera: Figitidae: Eucoilinae), a Parasitoid of Drosophila suzukii (Diptera: Drosophilidae) in Argentina.

The eucoiline species Ganaspis brasiliensis (von Ihering) (Hymenoptera: Figitidae) is recorded for the first time in Argentina, with confirmation of parasitism of Drosophila suzukii (Matsumura) infesting healthy raspberry fruit (Rubus idaeus L. cv. "Heritage") still on the plant. Drosophila suzukii puparia were recovered from fruit collected in an organic farm in Tafí del Valle, Tucumán, Argentina. One G. brasiliensis specimen was obtained from an isolated D. suzukii puparium. An additional 83 specimens, deposited in the entomological collection of the Museo de la Plata, Argentina, were also identified as G. brasiliensis. This parasitoid species is distributed in four biogeographical provinces of Argentina. The specimens of G. brasiliensis collected in Argentina are assumed to belong to a worldwide distributed and generalist lineage, parasitizing several Drosophila species. A taxonomic key of known Ganaspis species from Argentina is provided.

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii.

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa over the last decade, becoming a devastating pest of various soft-skinned fruits in its invaded regions. Biological control, especially by means of self-perpetuating and specialized parasitoids, is expected to be a viable option for sustainable area-wide management of this highly mobile and polyphagous pest. Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae) is a larval parasitoid that is widely distributed in East Asia, and has been found to be one of the most effective parasitoids of D. suzukii. Following rigorous pre-introduction evaluations of its efficacy and potential non-target risks, one of the more host-specific genetic groups of this species (G1 G. brasiliensis) has been approved recently for introduction and field release in the United States and Italy. Another genetic group (G3 G. brasiliensis), which was also commonly found to attack D. suzukii in East Asia, may be considered for introduction in the near future. There is currently enormous interest in rearing G. brasiliensis for research or in mass-production for field release against D. suzukii. This protocol and associated video article describe effective rearing methods for this parasitoid, both on a small scale for research and a large scale for mass-production and field release. These methods may benefit further long-term research and use of this Asian-native parasitoid as a promising biological control agent for this global invasive pest.

Adventive Larval Parasitoids Reconstruct Their Close Association with Spotted-Wing Drosophila in the Invaded North American Range.

Two species of larval parasitoids of the globally invasive fruit pest, Drosophila suzukii (Diptera: Drosophilidae), Leptopilina japonica, and Ganaspis brasiliensis (both Hymenoptera: Figitidae), were detected in British Columbia, Canada in 2016 and 2019, respectively. Both are presumed to have been unintentionally introduced from Asia; however, the extent of their establishment across different habitats with diverse host plants used by D. suzukii was unclear. In addition, there was no knowledge of the temporal dynamics of parasitism of D. suzukii by these two parasitoids. To address these gaps, we repeatedly sampled the fruits of known host plants of D. suzukii over the entire 2020 growing season in British Columbia. We documented the presence of L. japonica and G. brasiliensis and estimated the apparent percentage of D. suzukii parasitized among host plant species. Across a large region of southwestern British Columbia, both L. japonica and G. brasiliensis were found to be very common across a variety of mostly unmanaged habitats over the entire course of the season (May-October) in the fruits of most host plants known to host D. suzukii larvae. Parasitism of D. suzukii was variable (0-66% percent parasitism) and appeared to be time-structured. Our study demonstrates that the close association between the two larval parasitoids and D. suzukii that exists in Asia has evidently been reconstructed in North America, resulting in the highest parasitism levels of D. suzukii yet recorded outside of its area of origin.

A Coordinated Sampling and Identification Methodology for Larval Parasitoids of Spotted-Wing Drosophila.

We provide recommendations for sampling and identification of introduced larval parasitoids of spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). These parasitoids are either under consideration for importation (aka classical) biological control introductions, or their adventive (presumed to have been accidentally introduced) populations have recently been discovered in North America and Europe. Within the context of the ecology of D. suzukii and its parasitoids, we discuss advantages and disadvantages of estimating larval parasitism levels using different methods, including naturally collected fruit samples and sentinel baits. For most situations, we recommend repeated sampling of naturally occurring fruit rather than using sentinel baits to monitor seasonal dynamics of host plant-Drosophila-parasitoid associations. We describe how to separate Drosophilidae puparia from host fruit material in order to accurately estimate parasitism levels and establish host-parasitoid associations. We provide instructions for identification of emerging parasitoids and include a key to the common families of parasitoids of D. suzukii. We anticipate that the guidelines for methodology and interpretation of results that we provide here will form the basis for a large, multi-research team sampling effort in the coming years to characterize the biological control and nontarget impacts of accidentally and intentionally introduced larval parasitoids of D. suzukii in several regions of the world.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Geographic and Seasonal Variation in Species Diversity and Community Composition of Frugivorous Drosophila (Diptera: Drosophilidae) and their Leptopilina (Hymenoptera: Figitidae) Parasitoids.

Many studies have investigated species diversity patterns across space and time, but few have explored patterns of coexistence of tightly interacting species. We documented species diversity patterns in a host-parasitoid system across broad geographic location and seasons. We calculated species diversity (H and eH   ') and compared the relationship between community similarity and geographic distances of frugivorous Drosophila host (Diptera: Drosophilidae) and Leptopilina parasitoid (Hymenoptera: Figitidae) communities across Eastern North America, from New Hampshire to Florida, at two time points during the breeding season. We also analyzed the influence of environmental factors on species assemblages via constrained correspondence analysis and lastly calculated cluster dendrograms to identify potential host-parasitoid interactions. We found that the composition of Drosophila-Leptopilina communities varied significantly with latitude. Interestingly, diversity increased with increasing latitude, a trend counter to latitudinal patterns of diversity observed in many other taxa. We also found seasonal effects of monthly temperature range and precipitation on host biodiversity patterns across geographic locations. Cluster dendrograms nominated potential parasitoid-hosts and competitive interactions to be validated in the future studies. The present study fills an important gap of knowledge in North American Drosophila-Leptopilina species diversity patterns and lays the groundwork for future ecological and evolutionary studies in this system.

Phylogenomic Insights into the Evolution of Stinging Wasps and the Origins of Ants and Bees.

The stinging wasps (Hymenoptera: Aculeata) are an extremely diverse lineage of hymenopteran insects, encompassing over 70,000 described species and a diversity of life history traits, including ectoparasitism, cleptoparasitism, predation, pollen feeding (bees [Anthophila] and Masarinae), and eusociality (social vespid wasps, ants, and some bees) [1]. The most well-studied lineages of Aculeata are the ants, which are ecologically dominant in most terrestrial ecosystems [2], and the bees, the most important lineage of angiosperm-pollinating insects [3]. Establishing the phylogenetic affinities of ants and bees helps us understand and reconstruct patterns of social evolution as well as fully appreciate the biological implications of the switch from carnivory to pollen feeding (pollenivory). Despite recent advancements in aculeate phylogeny [4-11], considerable uncertainty remains regarding higher-level relationships within Aculeata, including the phylogenetic affinities of ants and bees [5-7]. We used ultraconserved element (UCE) phylogenomics [7, 12] to resolve relationships among stinging-wasp families, gathering sequence data from >800 UCE loci and 187 samples, including 30 out of 31 aculeate families. We analyzed the 187-taxon dataset using multiple analytical approaches, and we evaluated several alternative taxon sets. We also tested alternative hypotheses for the phylogenetic positions of ants and bees. Our results present a highly supported phylogeny of the stinging wasps. Most importantly, we find unequivocal evidence that ants are the sister group to bees+apoid wasps (Apoidea) and that bees are nested within a paraphyletic Crabronidae. We also demonstrate that taxon choice can fundamentally impact tree topology and clade support in phylogenomic inference.

A Scalable and Modular Dome Illumination System for Scientific Microphotography on a Budget.

A scalable and modular LED illumination dome for microscopic scientific photography is described and illustrated, and methods for constructing such a dome are detailed. Dome illumination for insect specimens has become standard practice across the field of insect systematics, but many dome designs remain expensive and inflexible with respect to new LED technology. Further, a one-size-fits-all dome cannot accommodate the large breadth of insect size encountered in nature, forcing the photographer to adapt, in some cases, to a less than ideal dome design. The dome described here is scalable, as it is based on a isodecahedron, and the template for the dome is available as a downloaded file from the internet that can be printed on any printer, on the photographer's choice of media. As a result, a photographer can afford, using this design, to produce a series of domes of various sizes and materials, and LED ring lights of various sizes and color temperatures, depending on the need.

Impact of the Egg Parasitoid, Gryon pennsylvanicum (Hymenoptera: Scelionidae), on Sentinel and Wild Egg Masses of the Squash Bug (Hemiptera: Coreidae) in Maryland.

Seasonal changes in egg parasitism and predation rates on sentinel (laboratory-reared) and wild (naturally occurring) egg masses of the squash bug, Anasa tristis (De Geer), were evaluated in squash fields in Maryland from June through September in 2013 and 2014. Rates of egg parasitism were significantly higher on wild egg masses than on sentinel egg masses. Squash bug nymphal emergence was significantly higher on sentinel egg masses than on wild egg masses. Between the first week of July and the first week of September of both survey years, squash bug nymphs emerged from 24.2% of wild eggs compared with 46.2% of sentinel eggs and parasitoids emerged from 55.7% of wild eggs compared with only 21.8% of sentinel eggs. Sentinel egg masses significantly underestimated the rate of natural egg parasitism. The egg parasitoid, Gryon pennsylvanicum (Ashmead), was responsible for over 99% of parasitism of squash bug eggs. There was a significant negative correlation between parasitoid emergence and nymphal emergence, suggesting that parasitoids were able to suppress squash bug populations. The average rate of parasitoid emergence peaked on wild egg masses on the fifth week of July at 72.8%, whereas the average rate of nymphal emergence from wild egg masses was <20% from the fifth week of July until the first week of September. These results demonstrate that G. pennsylvanicum was able to efficiently track wild squash bug eggs throughout the season and that it has the potential to be an effective biological control agent of the squash bug in Maryland.

Afrotropical cynipoidea (hymenoptera).

The Afrotropical Cynipoidea are represented by 306 described species and 54 genera in four families: Cynipidae, Figitidae, Liopteridae and Ibaliidae, the latter represented by a single introduced species. Seven of these genera are only represented by undescribed species in the region. Seven new genus-level synonymies, one genus resurrected from synonymy, 54 new combinations, one combination reinstated, and one new replacement name are presented. We provide identification keys to the families, subfamilies and genera of cynipoid wasps occurring in the Afrotropical region (Africa south of the Sahara, including Madagascar and southern Arabian Peninsula). Online interactive Lucid Phoenix and Lucid matrix keys are available at: http://www.waspweb.org/Cynipoidea/Keys/index.htm. An overview of the biology and checklists of species for each genus are provided. This paper constitutes the first contributory chapter to the book on Afrotropical Hymenoptera.

Leaf beetles are ant-nest beetles: the curious life of the juvenile stages of case-bearers (Coleoptera, Chrysomelidae, Cryptocephalinae).

Although some species of Cryptocephalinae (Coleoptera: Chrysomelidae) have been documented with ants (Hymenoptera: Formicidae) for almost 200 years, information on this association is fragmentary. This contribution synthesizes extant literature and analysizes the data for biological patterns. Myrmecophily is more common in the tribe Clytrini than in Cryptocephalini, but not documented for Fulcidacini or the closely-related Lamprosomatinae. Myrmecophilous cryptocephalines (34 species in 14 genera) primarily live among formicine and myrmecines ants as hosts. These two ant lineages are putative sister-groups, with their root-node dated to between 77-90 mya. In the New World tropics, the relatively recent radiation of ants from moist forests to more xeric ecosystems might have propelled the association of cryptocephalines and ant nests. Literature records suggest that the defensive behavioral profile or chemical profile (or both) of these ants has been exploited by cryptocephalines. Another pattern appears to be that specialized natural enemies, especially parasitoid Hymenoptera, exploit cryptocephaline beetles inside the ant nests. With the extant data at hand, based on the minimum age of a fossil larva dated to 45 mya, we can infer that the origin of cryptocephaline myrmecophily could have arisen within the Upper Cretaceous or later. It remains unknown how many times myrmecophily has appeared, or how old is the behavior. This uncertainty is compounded by incongruent hypotheses about the origins of Chrysomelidae and angiosperm-associated lineages of cryptocephalines. Living with ants offers multiple advantages that might have aided the colonization of xeric environments by some cryptocephaline species.