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.

Summary Highly reduced genomes of 144–416 kilobases have been described from nutrient-provisioning bacterial symbionts of several insect lineages [1–5]. Some host insects have formed stable associations with pairs of bacterial symbionts that live in specialized cells and provide them with essential nutrients; genomic data from these systems have revealed remarkable levels of metabolic complementary between the symbiont pairs [3, 4, 6, 7]. The mealybug, Planococcus citri (Hemiptera: Pseudococcidae), contains dual bacterial symbionts existing with an unprecedented organization: an unnamed Gammaproteobacteria, for which we propose the name Candidatus Moranella endobia, lives inside the Betaproteobacteria Candidatus Tremblaya princeps [8]. Here we describe the complete genomes and metabolic contributions of these unusual nested symbionts. We show that while there is little overlap in retained genes involved in nutrient production between symbionts, several essential amino acid pathways in the mealybug assemblage require a patchwork of interspersed gene products from Tremblaya, Moranella, and possibly P. citri. Furthermore, while Tremblaya has the smallest cellular genome yet described, it contains a genomic inversion present in both orientations in individual insects, starkly contrasting the extreme structural stability typical of highly reduced bacterial genomes [4, 9, 10].

A 658-bp fragment of mitochondrial DNA from the 5' region of the mitochondrial cytochrome c oxidase 1 (COI) gene has been adopted as the standard DNA barcode region for animal life. In this study, we test its effectiveness in the discrimination of over 300 species of aphids from more than 130 genera. Most (96%) species were well differentiated, and sequence variation within species was low, averaging just 0.2%. Despite the complex life cycles and parthenogenetic reproduction of aphids, DNA barcodes are an effective tool for identification.

Some insects have cultivated intimate relationships with mutualistic bacteria since their early evolutionary history. Most ancient 'primary' endosymbionts live within the cytoplasm of large, polyploid host cells of a specialized organ (bacteriome). Within their large, ovoid bacteriomes, mealybugs (Pseudococcidae) package the intracellular endosymbionts into 'mucus-filled' spheres, which surround the host cell nucleus and occupy most of the cytoplasm. The genesis of symbiotic spheres has not been determined, and they are structurally unlike eukaryotic cell vesicles. Recent molecular phylogenetic and fluorescent in situ hybridization (FISH) studies suggested that two unrelated bacterial species may share individual host cells, and that bacteria within spheres comprise these two species. Here we show that mealybug host cells do indeed harbour both beta- and gamma-subdivision Proteobacteria, but they are not co-inhabitants of the spheres. Rather, we show that the symbiotic spheres themselves are beta-proteobacterial cells. Thus, gamma-Proteobacteria live symbiotically inside beta-Proteobacteria. This is the first report, to our knowledge, of an intracellular symbiosis involving two species of bacteria.

Many aphids display a remarkably complex life cycle of host alternation, in which cyclical parthenogenesis is combined with the obligate use of two unrelated host plants. We used mitochondria1 ribosomal DNA @artial 12s and 16s) sequences to reconstruct the phylogeny of aphids, to determine how many origins of host alternation and correlated major hostplant shifts have occurred. Our results agreed with previous morphological studies in that species clustered with good support at the level of tribes. There was little well-supported phylogenetic structure at levels deeper than tribes, however, except for the monophyly of two subfamilies, Aphidinae and Lachninae. We argue that aphids experienced a rapid radiation at the tribal level, after host shifting from gymnosperms to angiosperms. A rapid radiation is consistent with aphid fossils, which record the presence of few subfamilies in the late Cretaceous, but most extant tribes by the early Tertiary. Plant fossils also record host plants of aphid tribes diversifying during this time. A hypothesized mechanism by which host alternation has evolved (fundatrix specialization), coupled with the rapid radiation, implies that this life cycle may have originated as often as in the ancestor of each tribe that displays it. We also consider, however, an alternative hypothesis of fewer origins. The basal radiation of Aphididae was dated from molecular sequences to have occurred at approximately 80-1 50 Mya.

Molecular phylogenetics of Vespoidea indicate paraphyly of the superfamily and novel relationships of its component families and subfamilies. -Zoologica Scripta, 37 , 539-560. The 24 000+ described species of Vespoidea include many well-known stinging wasps, such as paper wasps and hornets (Vespidae), velvet ants (Mutillidae), spider wasps (Pompilidae) and ants (Formicidae). The compelling behaviours of vespoids have been instrumental in developing theories of stepwise evolutionary transitions, which necessarily depend on an understanding of phylogeny, yet, existing morphological phylogenies for Vespoidea conflict. We collected molecular data from four nuclear genes (elongation factor-1 α F2 copy, long-wavelength rhodopsin, wingless and the D2-D3 regions of 28S ribosomal RNA (2700 bp in total)) to produce the first molecular phylogeny of Vespoidea. We analysed molecular data alone and in combination with published morphological data from Brothers and Carpenter. Parsimony analyses left many deeper nodes unsupported, but suggested paraphyly of three families. Total-evidence Bayesian inference produced a more resolved tree, in which the monophyly of Vespoidea was nevertheless ambiguous. Bayesian inference of molecular data alone returned a well-resolved consensus with posterior probabilities of over 95% for most nodes. We used this topology as the best estimate of phylogeny at the family and subfamily levels. Notable departures from previous estimates include: (i) paraphyly of Vespoidea resulting from the nesting of Apoidea within a lineage comprising Formicidae, Scoliidae and two subfamilies of Bradynobaenidae; (ii) paraphyly of Bradynobaenidae, Mutillidae and Tiphiidae; (iii) a sister relationship between Rhopalosomatidae and Vespidae; and (iv) Rhopalosomatidae + Vespidae as sister to all other vespoids/apoids. We discuss character evidence in light of the new phylogeny, and propose a new classification of Aculeata that recognizes eight superfamilies: Apoidea, Chrysidoidea,

Homoptera and Heteroptera comprise a large insect assemblage, the Hemiptera. Many of the plant sap-sucking Homoptera possess unusual and complex life histories and depend on maternally inherited, intracellular bacteria to supplement their nutritionally deficient diets. Presumably in connection with their diet and lifestyles, the morphology of many Homoptera has become greatly reduced, leading to major controversies regarding the phylogenetic affiliations of homopteran superfamilies. The most fundamental question concerns whether the Homoptera as a whole are monophyletic. Recent studies based on morphology have argued that the Homoptera Sternorrhyncha (Aphidoidea, Coccoidea, Psylloidea, Aleyrodoidea) is a sister group to a group comprising the Homoptera Auchenorrhyncha (Fulgoroidea, Cicadoidea, Cercopoidea, Cicadelloidea) and the Heteroptera, making the Homoptera paraphyletic. We sequenced the 5' 580-680 base pairs of small-subunit (18S) ribosomal DNA from a selection of Homoptera, Hemiptera, and their putative outgroups, the Thysanoptera and Psocoptera, to apply molecular characters to the problem of Homoptera phylogeny. Parsimony, distance, maximum-likelihood, and bootstrap methods were used to construct trees from sequence data and assess support for the topologies produced. Molecular data corroborate current views of relationships within the Sternorrhyncha and Auchenorrhyncha based on morphology and strongly support the hypothesis of homopteran paraphyly as stated above. In addition, it was found that Homoptera Sternorrhyncha have extra, GC-rich sequence concentrated in a variable region of the 18S rDNA, which indicates that some unique evolutionary processes are occurring in this lineage.

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