A chromosome-level genome assembly of the Rhus gall aphid Schlechtendalia chinensis provides insight into the endogenization of Parvovirus-like DNA sequences.

The Rhus gall aphid, Schlechtendalia chinensis, feeds on its primary host plant Rhus chinensis to induce galls, which have economic importance in medicines and the food industry. Rhus gall aphids have a unique life cycle and are economically beneficial but there is huge gap in genomic information about this group of aphids. Schlechtendalia chinensis induces rich-tannin galls on its host plant and is emerging as a model organism for both commercial applications and applied research in the context of gall production by insects. Here, we generated a high-quality chromosome-level assembly for the S. chinensis genome, enabling the comparison between S. chinensis and non-galling aphids. The final genome assembly is 344.59 Mb with 91.71% of the assembled sequences anchored into 13 chromosomes. We predicted 15,013 genes, of which 14,582 (97.13%) coding genes were annotated, and 99% of the predicted genes were anchored to the 13 chromosomes. This assembly reveals the endogenization of parvovirus-related DNA sequences (PRDs) in the S. chinensis genome, which could play a role in environmental adaptations. We demonstrated the characterization and classification of cytochrome P450s in the genome assembly, which are functionally crucial for sap-feeding insects and have roles in detoxification and insecticide resistance. This genome assembly also revealed the whole genome duplication events in S. chinensis, which can be considered in comparative evolutionary analysis. Our work represents a reference genome for gall-forming aphids that could be used for comparative genomic studies between galling and non-galling aphids and provides the first insight into the endogenization of PRDs in the genome of galling aphids. It also provides novel genetic information for future research on gall-formation and insect-plant interactions.

Whole-genome sequence of the Cooley spruce gall adelgid, Adelges cooleyi (Hemiptera: Sternorrhyncha: Adelgidae).

The adelgids (Adelgidae) are a small family of sap-feeding insects, which, together with true aphids (Aphididae) and phylloxerans (Phylloxeridae), make up the infraorder Aphidomorpha. Some adelgid species are highly destructive to forest ecosystems such as Adelges tsugae, Adelges piceae, Adelges laricis, Pineus pini, and Pineus boerneri. Despite this, there are no high-quality genomic resources for adelgids, hindering advanced genomic analyses within Adelgidae and among Aphidomorpha. Here, we used PacBio continuous long-read and Illumina RNA-sequencing to construct a high-quality draft genome assembly for the Cooley spruce gall adelgid, Adelges cooleyi (Gillette), a gall-forming species endemic to North America. The assembled genome is 270.2 Mb in total size and has scaffold and contig N50 statistics of 14.87 and 7.18 Mb, respectively. There are 24,967 predicted coding sequences, and the assembly completeness is estimated at 98.1 and 99.6% with core BUSCO gene sets of Arthropoda and Hemiptera, respectively. Phylogenomic analysis using the A. cooleyi genome, 3 publicly available adelgid transcriptomes, 4 phylloxera transcriptomes, the Daktulosphaira vitifoliae (grape phylloxera) genome, 4 aphid genomes, and 2 outgroup coccoid genomes fully resolves adelgids and phylloxerans as sister taxa. The mitochondrial genome is 24 kb, among the largest in insects sampled to date, with 39.4% composed of noncoding regions. This genome assembly is currently the only genome-scale, annotated assembly for adelgids and will be a valuable resource for understanding the ecology and evolution of Aphidomorpha.

Description of a new species of Hamamelistes forming galls on Fothergilla spp. (Hamamelidaceae) and the generic limits of Hormaphidini (Sternorrhyncha: Aphididae: Hormaphidinae).

Hamamelistes and Hormaphis aphids of the tribe Hormaphidini are distributed disjunctly in eastern North America and Eurasia. Host-alternating species have life cycles encompassing generations in a gall on witch-hazel (Hamamelis spp.) and generations on leaves of birch (Betula spp.). In Hamamelistes, generations on witch-hazel induce globular pouch galls on flower or leaf buds. Herbarium specimens of a related Hamamelidaceae genus, Fothergilla, contain large galls in place of the seed head. We obtained a fresh sample of these elongate pouch galls collected from F. milleri in Alabama, USA. The galls were formed in place of fruiting structures and contained numerous aphids. Examination of morphology and the cytochrome oxidase subunit I barcode DNA sequence confirmed that the aphids are an undescribed species of Hamamelistes. Here, we describe the new species, Hamamelistes blackmani Dederich von Dohlen sp. n., from the morphology of foundresses, immatures, and winged forms in the gall. The life cycle is presumed monoecious. In addition, we review the evidence for including other genera in Hormaphidini and recommend that this tribe be restricted to Hamamelistes and Hormaphis.

Comparative mitochondrial genomes of the Rhus gall aphid Kaburagia rhusicola subspecies with variable gall shapes.

Rhus gall aphids (Hemiptera: Aphididae: Eriosomatinae) stimulate the formation of galls on their primary host plants (sumacs: Rhus spp., Anacardiaceae). The shapes of galls are often used as an extended phenotype to identify the aphid species and subspecies. We collected four Rhus galls with conspicuously different shapes formed by Kaburagia rhusicola aphids, whose sequences of the complete mitochondrial genomes (mitogenomes) were obtained by high-throughput sequencing. Each mitogenome was assembled into a circular molecule containing 13 protein-coding genes, two rRNAs, 22 tRNAs and one control region. All the protein-coding genes had a typical ATN initiation codon and TAA termination codon except for cox1 and nad4, which had a single T as stop codon. All the tRNAs could be folded as a typical clover-leaf secondary structure, except for trnS1 lacking a dihydrouridine (DHU) arm. The relative synonymous codon usage and ratio of nonsynonymous to synonymous substitution rates showed that the four K. rhusicola samples were highly similar to the subspecies K. r. ovogallis. The phylogenetic analyses grouped these samples with K. r. ovogallis in a clade sister to K. r. rhusicola. All these molecular analyses demonstrated that our current samples represented one subspecies of Kaburagia rhusicola, i.e., K. r. ovogallis, and the gall shape was variable even at the subspecies level in Kaburagia gall aphids.

Transitional genomes and nutritional role reversals identified for dual symbionts of adelgids (Aphidoidea: Adelgidae).

Many plant-sap-feeding insects have maintained a single, obligate, nutritional symbiont over the long history of their lineage. This senior symbiont may be joined by one or more junior symbionts that compensate for gaps in function incurred through genome-degradative forces. Adelgids are sap-sucking insects that feed solely on conifer trees and follow complex life cycles in which the diet fluctuates in nutrient levels. Adelgids are unusual in that both senior and junior symbionts appear to have been replaced repeatedly over their evolutionary history. Genomes can provide clues to understanding symbiont replacements, but only the dual symbionts of hemlock adelgids have been examined thus far. Here, we sequence and compare genomes of four additional dual-symbiont pairs in adelgids. We show that these symbionts are nutritional partners originating from diverse bacterial lineages and exhibiting wide variation in general genome characteristics. Although dual symbionts cooperate to produce nutrients, the balance of contributions varies widely across pairs, and total genome contents reflect a range of ages and degrees of degradation. Most symbionts appear to be in transitional states of genome reduction. Our findings support a hypothesis of periodic symbiont turnover driven by fluctuating selection for nutritional provisioning related to gains and losses of complex life cycles in their hosts.

A further contribution to the knowledge of Uroleucon species (Hemiptera, Aphididae) living on Adesmia (Fabaceae) in southern South America, with description of a new species from Chile.

Uroleucon (Hemiptera, Aphididae, Macrosiphini) is a highly diverse and widely distributed genus. Most of its species are hosted by plants of the Asteraceae or Campanulaceae, but three species from the southern end of South America have specialized to live on plants of the genus Adesmia (Fabaceae). New morphological and chorological data are provided for U. adesmiae Mier Durante and Ortego and U. naheulhuapense Nieto Nafría von Dohlen, with a description of the alate viviparous female of the latter species. U. australe Nieto Nafría Mier Durante, sp. n. is described from apterous viviparous females, oviparous females and males from four localities in the Chilean regions of Aysén and Magallanes; it is very close to U. nahuelhuapense, both morphologically and genetically. Contrary to what is typical for aphids, the males of U. australe have a greater number of distinguishing characters than are found in viviparous females. A modification of the identification key to the apterous viviparous females of the known Uroleucon species in South America by Nieto Nafría et al. (2019) is presented.

Congruent phylogenetic relationships of Melaphidina aphids (Aphididae: Eriosomatinae: Fordini) according to nuclear and mitochondrial DNA data with taxonomic implications on generic limits.

Melaphidina aphids (Rhus-gall aphids; Eriosomatinae: Fordini) comprise five genera from eastern Asia and one monotypic genus from eastern North America. Melaphidina are unique in feeding on plant species of Rhus subgenus Rhus (Anacardiaceae), on which they form galls during the summer. The phylogenetic relationships among some species of Melaphidina aphids remain controversial. In this study, we sought to resolve the backbone phylogeny of Melaphidina aphids by sampling 15 accessions representing all six genera, all species, and all subspecies except Meitanaphis microgallis using 20 gene regions: five nuclear genes as well as 13 protein-coding genes and two rRNA genes of the mitochondrial genome. Phylogenetic analyses included Bayesian and maximum likelihood methods. Independent analyses of nuclear and mitochondrial genes returned congruent topologies, and analyses of all gene regions combined showed well-supported relationships among Melaphidina species. In particular, these were: (1) Nurudea (excluding N. ibofushi) is sister to a clade composed of the five remaining genera; (2) the monotypic North American genus Melaphis is sister to a clade comprising the four remaining genera; and (3) (Schlechtendalia + N. ibofushi) is sister to the clade (Floraphis (Meitanaphis + Kaburagia). Our results support the transfer of Meitanaphis flavogallis to Kaburagia as an additional subspecies or species, and the recognition of Floraphis as a distinct genus. This study provides important molecular resources for subsequent evolutionary studies using more nuclear genes on the Melaphidina aphids and their close allies.

The species of Uroleucon (Hemiptera: Aphididae) living on Adesmia (Fabaceae) in Argentina, with the description of a new species.

Uroleucon Mordvilko is the largest genus in the Macrosiphini (Hemiptera, Aphididae) and most of its species live on species of Asteraceae. Uroleucon nahuelhuapense Nieto Nafría von Dohlen sp. n. is described from apterous viviparous females collected on Adesmia boronioides (Fabaceae) in a locality of Neuquén province (Argentina). The morphological characters and the sequence of the tRNAleu-COII mitochondrial locus support the inclusion of the new species in the genus Uroleucon. Apterous viviparous females of Uroleucon adesmiae Mier Durante Ortego collected in two Argentinean localities have also been studied and ranges of several quantitative features have been modified. The identification key for the Uroleucon species known in South America by Nieto Nafría et al. (2007) has been modified to include the new species.

Ecological factors influencing the beneficial endosymbionts of the hemlock woolly adelgid (Hemiptera: Adelgidae).

Bacterial endosymbionts of sap-sucking insects provide their host with a number of beneficial qualities, including the supply of nutrition, defense against parasitoids, and protection from heat stress. Damage to these bacterial associates can therefore have a negative impact on the fitness of their insect host. We evaluated observational and experimental factors regarding the nonnative hemlock woolly adelgid (Adelges tsugae Annand) (Hemiptera: Adelgidae) to help understand the roles of its three recently identified symbionts, including under heat stress conditions. The prevalence of A. tsugae's facultative symbiont (Serratia symbiotica) was examined at different spatial scales to determine how variable infection rates are for this symbiont. There was no significant difference found in infection rates between adelgids on a tree, within a plot, or within a state. However, significantly more adelgids in Georgia (95%) had S. symbiotica compared to those in New York (68%). Microsatellite genotyping of the adelgids found that this difference was most likely not the result of a second introduction of A. tsugae into eastern North America. Comparison of S. symbiotica proportions between first and fourth instars showed that symbiont absence did not affect the ability of A. tsugae to survive aestivation. Evaluations of symbiont densities within each adelgid found that when S. symbiotica was absent, the density of obligate symbionts was significantly higher. Exposure to heat stress (32.5 °C) was not consistently correlated with changes in symbiont densities over a 4-d period. Overall, we have shown that symbiont prevalence and densities vary within the broad population of A. tsugae in eastern North America, with potentially significant effects upon the ecology of this important pest.

Partnering With a Pest: Genomes of Hemlock Woolly Adelgid Symbionts Reveal Atypical Nutritional Provisioning Patterns in Dual-Obligate Bacteria.

Nutritional bacterial symbionts enhance the diets of sap-feeding insects with amino acids and vitamins missing from their diets. In many lineages, an ancestral senior symbiont is joined by a younger junior symbiont. To date, an emergent pattern is that senior symbionts supply a majority of amino acids, and junior symbionts supply a minority. Similar to other hemipterans, adelgids harbor obligate symbionts, but have higher diversity of bacterial associates, suggesting a history of symbiont turnover. The metabolic roles of dual symbionts in adelgids and their contributions to the consortium are largely unexplored. Here, we investigate the symbionts of Adelges tsugae, the hemlock woolly adelgid (HWA), an invasive species introduced from Japan to the eastern United States, where it kills hemlock trees. The response of hemlocks to HWA feeding has aspects of a defensive reaction against pathogens, and some have speculated that symbionts may be involved. We sequenced the genomes of "Ca. Annandia adelgestsuga" and "Ca. Pseudomonas adelgestsugas" symbionts to detail their metabolic capabilities, infer ages of relationship, and search for effectors of plant defenses. We also tested the relationship of "Ca. Annandia" to symbionts of other insects. We find that both symbionts provide nutrients, but in more balanced proportions than dual symbionts of other hemipterans. The lesser contributions of the senior "Ca. Annandia" support our hypothesis for symbiont replacements in adelgids. Phylogenomic results were ambiguous regarding the position of "Ca. Annandia". We found no obvious effectors of plant defenses related to insect virulence, but hypothetical proteins in symbionts are unknown players.

Taxonomic contributions to Ageniella Banks, 1912 (Hymenoptera: Pompilidae) from Brazil.

Ageniella Banks is the second most diverse genus in the tribe Ageniellini (Pompilidae: Pepsinae) with about 200 valid names. Ageniella is known to be paraphyletic; yet, no revision has been made. Lack of a taxonomic catalogue and of identification keys has further delayed taxonomic studies. Additionally, many of the currently valid names should be treated as synonyms. This present study is the first attempt to organize the species of Ageniella, focusing on the species occurring in Brazil. A total of six subgenera and 40 species of Ageniella occur in Brazil. Of these, 25 species are endemic to Brazil. The new synonyms are proposed for (valid names are first): Alasagenia Banks, 1944 =Lissagenia Banks, 1946, syn. nov.; Ageniella (Alasagenia) erichsoni Banks, 1944 =Priophanes major Banks, 1945, syn. nov.; Ageniella (Ameragenia) zeteki (Banks, 1925) =Priophanes marcida Banks, 1946, syn. nov.; Ageniella (Ameragenia) agitata (Smith, 1873), comb. nov. =Salius (Priocnemis) setaceicornis Fox, 1897, syn. nov.; Ageniella (Ameragenia) sanguinolenta (Smith, 1864) =Agenia ruficeps Smith, 1864, syn. nov.; =Ageniella alternata Banks, 1946, syn. nov.; Ageniella (Priophanes) dolorosa (Banks, 1946), comb. nov. =Priophanes plagosa Banks, 1946, syn. nov.; Ageniella (Ameragenia) fabricii (Banks, 1944) =Ameragenia notabilis Banks, 1946, syn. nov. A new combination is proposed for: Ageniella (Alasagenia) cursor (Smith, 1873), comb. nov. (from Agenia Schiødte); Ageniella (Ameragenia) clypeata (Fox, 1897), comb. nov. (from Salius Fabricius), Ageniella (Ameragenia) rutila (Fox, 1897), comb. nov. (from Salius), Ageniella (Priophanes) tegularis (Fox, 1897), comb. nov. (from Salius), Ageniella (Priophanes) rufitarsis (Fox, 1897), comb. nov. (from Salius), Ageniella (Ameragenia) citricornis (Fox, 1897), comb. nov. (from Salius), Ageniella (Ameragenia) serrula (Fox, 1897), comb. nov. (from Salius), Ageniella (Priophanes) cingulata (Fox, 1897), comb. nov. (from Agenia). A new name Ageniella (Ameragenia) banksii Waichert, nom. nov. is proposed for Ameragenia festina Banks, 1946, secondary homonym of Ageniella festina Banks, 1917, and Ageniella (Priophanes) otiosa is reinstate, stat. resurr.

The geological record and phylogeny of spider wasps (Hymenoptera: Pompilidae): A revision of fossil species and their phylogenetic placement.

Accurate fossil identification has become increasingly relevant with the widespread use of phylogenetic divergence time estimation methods, which rely on fossil data to determine clade hard-minimum ages. Here we revise, diagnose and illustrate known spider wasp (Hymenoptera: Pompilidae) fossil species and place them within the latest Pompilidae phylogenetic hypothesis. Ceropalites infelix Cockerell, from the Florissant Fossil Beds (Priabonian), is no longer recognized as Pompilidae, but as Aulacidae. Agenioideus saxigenus (Cockerell) comb. nov., Deuteragenia wettweri (Statz) comb. nov., Caputelus scudderi (Cockerell, 1906) comb. nov., Pepsinites avitula (Cockerell, 1941) comb. nov., Pepsinites contentus (Theobald, 1937) comb. nov., Pepsinites florissantensis (Cockerell, 1906) comb. nov., Pepsinites laminarum (Rohwer, 1909) comb. nov., Pepsinites scelerosus (Meunier, 1919) comb. nov., Pepsinites cockerellae (Rohwer, 1909) comb. nov., Pompilinites coquandi (Theobald, 1937) comb. nov., Pompilinites depressus (Statz, 1936) comb. nov., Pompilites incertus (Theobald, 1937) comb. nov., Pompilites induratus (Heer, 1849) comb. nov., Pompilites fasciatus (Theobald, 1937) comb. nov., and Pompilites senex comb. nov. are new combinations. Twenty-three fossil species of spider wasps are now recognized in 13 genera. Four new genera are proposed: Caputelus Waichert & Pitts gen. nov., Pompilites Rodriguez gen. nov., Pompilinites Rodriguez & Waichert gen. nov., and Pepsinites Rodriguez & Waichert gen. nov., of which the three latter are collective-group names for fossils with taxonomic uncertainty. One species of fossil spider wasp is described: Deuteragenia catalunyia Rodriguez, Waichert & Pitts sp. nov., from the Bellver deposits in Catalonia, Spain. Five of the 23 known species can be used to determine hard-minimum age for calibrations of genera stem-groups (Agenioideus, Anoplius, Cryptocheilus, Deuteragenia, Priocnemis). The fossil belonging to the stem-group of the tribe Ageniellini (Chubutholites) is not recommended for calibration because of the high uncertainty in its age and taxonomy. The remaining taxa can be assigned to the lineage comprising Pompilinae + Pepsinae (12 species) or crown-group Pompilidae (four species).

Dynamic Acquisition and Loss of Dual-Obligate Symbionts in the Plant-Sap-Feeding Adelgidae (Hemiptera: Sternorrhyncha: Aphidoidea).

Sap-sucking insects typically engage in obligate relationships with symbiotic bacteria that play nutritional roles in synthesizing nutrients unavailable or in scarce supply from the plant-sap diets of their hosts. Adelgids are sap-sucking insects with complex life cycles that involve alternation between conifer tree species. While all adelgid species feed on spruce during the sexual phase of their life cycle, each adelgid species belongs to a major lineage that feeds on a distinct genus of conifers as their alternate host. Previous work on adelgid symbionts had discovered pairs of symbionts within each host species, and unusual diversity across the insect family, but left several open questions regarding the status of bacterial associates. Here, we explored the consistency of symbionts within and across adelgid lineages, and sought evidence for facultative vs. obligate symbiont status. Representative species were surveyed for symbionts using 16S ribosomal DNA gene sequencing, confirming that different symbiont pairs were consistently present within each major adelgid lineage. Several approaches were used to establish whether symbionts exhibited characteristics of long-term, obligate mutualists. Patterns of symbiont presence across adelgid species and diversification with host insects suggested obligate relationships. Fluorescent in situ hybridization and electron microscopy localized symbionts to bacteriocyte cells within the bacteriome of each species (with one previously known exception), and detection of symbionts in eggs indicated their vertical transmission. Common characteristics of long-term obligate symbionts, such as nucleotide compositional bias and pleomorphic symbiont cell shape were also observed. Superimposing microbial symbionts on the adelgid phylogeny revealed a dynamic pattern of symbiont gains and losses over a relatively short period of time compared to other symbionts associated with sap-sucking insects, with each adelgid species possessing an older, "senior" symbiont and a younger "junior" symbiont. A hypothesis relating adelgid life cycles to relaxed constraints on symbionts is proposed, with the degradation of senior symbionts and repeated acquisition of more junior symbionts creating opportunities for repeated colonization of new alternate-conifer hosts by adelgids.

Molecular phylogeny of Pompilinae (Hymenoptera: Pompilidae): Evidence for rapid diversification and host shifts in spider wasps.

Pompilinae is one of the largest subfamilies of spider wasps (Pompilidae). Most pompilines are generalist spider predators at the family level, but some taxa exhibit ecological specificity (i.e., to spider-host guild). Here we present the first molecular phylogenetic analysis of Pompilinae, toward the aim of evaluating the monophyly of tribes and genera. We further test whether changes in the rate of diversification are associated with host-guild shifts. Molecular data were collected from five nuclear loci (28S, EF1-F2, LWRh, Wg, Pol2) for 76 taxa in 39 genera. Data were analyzed using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic results were compared with previous hypotheses of subfamilial and tribal classification, as well as generic relationships in the subfamily. The classification of Pompilus and Agenioideus is also discussed. A Bayesian relaxed molecular clock analysis was used to examine divergence times. Diversification rate-shift tests accounted for taxon-sampling bias using ML and BI approaches. Ancestral host family and host guild were reconstructed using MP and ML methods. Ancestral host guild for all Pompilinae, for the ancestor at the node where a diversification rate-shift was detected, and two more nodes back in time was inferred using BI. In the resulting phylogenies, Aporini was the only previously proposed monophyletic tribe. Several genera (e.g., Pompilus, Microphadnus and Schistonyx) are also not monophyletic. Dating analyses produced a well-supported chronogram consistent with topologies from BI and ML results. The BI ancestral host-use reconstruction inferred the use of spiders belonging to the guild "other hunters" (frequenting the ground and vegetation) as the ancestral state for Pompilinae. This guild had the highest probability for the ML reconstruction and was equivocal for the MP reconstruction; various switching events to other guilds occurred throughout the evolution of the group. The diversification of Pompilinae shows one main rate-shift coinciding with a shift to ground-hunter spiders, as reconstructed by the BI ancestral character-state analysis.

The evolution of life cycle complexity in aphids: Ecological optimization or historical constraint?

For decades, biologists have debated why many parasites have obligate multihost life cycles. Here, we use comparative phylogenetic analyses of aphids to evaluate the roles of ecological optimization and historical constraint in the evolution of life cycle complexity. If life cycle complexity is adaptive, it should be evolutionarily labile, that is, change in response to selection. We provide evidence that this is true in some aphids (aphidines), but not others (nonaphidines)-groups that differ in the intensity of their relationships with primary hosts. Next, we test specific mechanisms by which life cycle complexity could be adaptive or a constraint. We find that among aphidines there is a strong association between complex life cycles and polyphagy but only a weak correlation between life cycle complexity and reproductive mode. In contrast, among nonaphidines the relationship between life cycle complexity and host breadth is weak but the association between complex life cycles and sexual reproduction is strong. Thus, although the adaptiveness of life cycle complexity appears to be lineage specific, across aphids, life cycle evolution appears to be tightly linked with the evolution of other important natural history traits.

Müllerian mimicry as a result of codivergence between velvet ants and spider wasps.

Recent studies have delineated a large Nearctic Müllerian mimicry complex in Dasymutilla velvet ants. Psorthaspis spider wasps live in areas where this mimicry complex is found and are phenotypically similar to Dasymutilla. We tested the idea that Psorthaspis spider wasps are participating in the Dasymutilla mimicry complex and that they codiverged with Dasymutilla. We performed morphometric analyses and human perception tests, and tabulated distributional records to determine the fit of Psorthaspis to the Dasymutilla mimicry complex. We inferred a dated phylogeny using nuclear molecular markers (28S, elongation factor 1-alpha, long-wavelength rhodopsin and wingless) for Psorthaspis species and compared it to a dated phylogeny of Dasymutilla. We tested for codivergence between the two groups using two statistical analyses. Our results show that Psorthaspis spider wasps are morphologically similar to the Dasymutilla mimicry rings. In addition, our tests indicate that Psorthaspis and Dasymutilla codiverged to produce similar color patterns. This study expands the breadth of the Dasymutilla Müllerian mimicry complex and provides insights about how codivergence influenced the evolution of mimicry in these groups.

Historical biogeography of Eastern Asian-Eastern North American disjunct Melaphidina aphids (Hemiptera: Aphididae: Eriosomatinae) on Rhus hosts (Anacardiaceae).

Intercontinental biotic disjunctions have been documented and analyzed in numerous Holarctic taxa. Patterns previously synthesized for animals compared to plants suggest that the timing of animal disjunctions are mostly Early Tertiary and were generated by migration and vicariance events occurring in the North Atlantic, while plant disjunctions are mostly Mid-Late Tertiary and imply migration and vicariance over Beringia. Melaphidina aphids (Hemiptera: Aphididae: Fordini) exhibit host-alternating life cycles comprising an obligate seasonal shift between Rhus subgenus Rhus species (Anacardiaceae) and mosses (Bryophyta). Similar to their Rhus hosts, melaphidines are distributed disjunctly between Eastern Asia and Eastern North America. We examined evolutionary relationships within Melaphidina to determine the position of the North American lineage, date its divergence from Asian relatives, and compare these results to a previous historical biogeographic study of Rhus. We sampled nine species and three subspecies representing all six genera of Melaphidina. Data included sequences of mitochondrial cytochrome c oxidase subunits I and II+leucine tRNA, cytochrome b, and nuclear elongation factor 1α genes. Phylogenetic analyses (Bayesian, maximum-likelihood, parsimony) of the combined data (3282 bp) supported the monophyly of all genera except Nurudea and Schlechtendalia, due to the position of N. ibofushi. While the exact position of the North American Melaphis was not well resolved, there was high support for a derived position within Asian taxa. The divergence of Melaphis from Asian relatives centered on the Eocene-Oligocene boundary (~33-35Ma), which coincides with closure of Beringian Land Bridge I. This also corresponded to the Asian-North American disjunction previously estimated for subgenus Rhus spp. We suggest the late-Eocene Bering Land Bridge as the most likely migration route for Melaphis ancestors, as was also hypothesized for North American Rhus ancestors. Results for the Melaphidina disjunction depart from the modal pattern in animal lineages, and present a case where insect and host-plant taxa apparently responded similarly to Tertiary climate change.

Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis.

The smallest reported bacterial genome belongs to Tremblaya princeps, a symbiont of Planococcus citri mealybugs (PCIT). Tremblaya PCIT not only has a 139 kb genome, but possesses its own bacterial endosymbiont, Moranella endobia. Genome and transcriptome sequencing, including genome sequencing from a Tremblaya lineage lacking intracellular bacteria, reveals that the extreme genomic degeneracy of Tremblaya PCIT likely resulted from acquiring Moranella as an endosymbiont. In addition, at least 22 expressed horizontally transferred genes from multiple diverse bacteria to the mealybug genome likely complement missing symbiont genes. However, none of these horizontally transferred genes are from Tremblaya, showing that genome reduction in this symbiont has not been enabled by gene transfer to the host nucleus. Our results thus indicate that the functioning of this three-way symbiosis is dependent on genes from at least six lineages of organisms and reveal a path to intimate endosymbiosis distinct from that followed by organelles.

Reconstructing the phylogeny of aphids (Hemiptera: Aphididae) using DNA of the obligate symbiont Buchnera aphidicola.

Reliable phylogenetic reconstruction, as a framework for evolutionary inference, may be difficult to achieve in some groups of organisms. Particularly for lineages that experienced rapid diversification, lack of sufficient information may lead to inconsistent and unstable results and a low degree of resolution. Coincidentally, such rapidly diversifying taxa are often among the biologically most interesting groups. Aphids provide such an example. Due to rapid adaptive diversification, they feature variability in many interesting biological traits, but consequently they are also a challenging group in which to resolve phylogeny. Particularly within the family Aphididae, many interesting evolutionary questions remain unanswered due to phylogenetic uncertainties. In this study, we show that molecular data derived from the symbiotic bacteria of the genus Buchnera can provide a more powerful tool than the aphid-derived sequences. We analyze 255 Buchnera gene sequences from 70 host aphid species and compare the resulting trees to the phylogenies previously retrieved from aphid sequences, only. We find that the host and symbiont data do not conflict for any major phylogenetic conclusions. Also, we demonstrate that the symbiont-derived phylogenies support some previously questionable relationships and provide new insights into aphid phylogeny and evolution.

Diversity of proteobacterial endosymbionts in hemlock woolly adelgid (Adelges tsugae) (Hemiptera: Adelgidae) from its native and introduced range.

Knowledge of intraspecific variation in symbioses may aid in understanding the ecology of widespread insects in different parts of their range. We investigated bacterial symbionts of Adelges tsugae, a pest of hemlocks in eastern North America introduced from Asia. Amplification, cloning, and sequencing of bacterial 16S rDNA, in situ hybridizations, and electron microscopy revealed that A. tsugae harbours up to five bacterial phylotypes, according to population. Three Gammaproteobacteria species are maternally transmitted. The first, designated 'Ca. Pseudomonas adelgestsugas' resides in the haemocoel, and was detected in all populations except Taiwan. The second phylotype, 'Ca. Serratia symbiotica', resides in bacteriocytes of populations on Tsuga sieboldii in Japan and in E. North America. The third phylotype, designated 'Ca. Annandia adelgestsuga', clustered within a lineage of several insect endosymbionts that included Buchnera aphidicola. It was detected in bacteriocytes in all populations, and in salivary glands of first instars. Two Betaproteobacteria phylotypes were detected in some Japanese T. sieboldii and eastern North America populations, and were observed only in salivary glands with no evidence of maternal transmission. Our results support the ideas that symbiont gain and loss has been volatile in adelgids, and that symbionts may help to trace the source of invasive species.