A subfamily-level classification of mealybugs (Hemiptera: Pseudococcidae) based on integrated molecular and morphological data

How should characters and taxa be sampled to resolve efficiently the phylogeny of ancient and highly speciose groups? We addressed this question empirically in the treefrog family Hylidae, which contains > 800 species and may be nonmonophyletic with respect to other anuran families. We sampled 81 species (54 hylids and 27 outgroups) for two mitochondrial genes (12S, ND1), two nuclear genes (POMC, c-myc), and morphology (144 characters) in an attempt to resolve higher-level relationships. We then added 117 taxa to the combined data set, many of which were sampled for only one gene (12S). Despite the relative incompleteness of the majority of taxa, the resulting trees placed all taxa in the expected higher-level clades with strong support, despite some taxa being > 90% incomplete. Furthermore, we found no relationship between the completeness of a taxon and the support (parsimony bootstrap or Bayesian posterior probabilities) for its localized placement on the tree. Separate analysis of the data set with the most taxa (12S) gives a somewhat problematic estimate of higher-level relationships, suggesting that data sets scored only for some taxa (ND1, nuclear genes, morphology) are important in determining the outcome of the combined analysis. The results show that hemiphractine hylids are not closely related to other hylids and should be recognized as a distinct family. They also show that the speciose genus Hyla is polyphyletic, but that its species can be arranged into three monophyletic genera. A new classification of hylid frogs is proposed. Several potentially misleading signals in the morphological data are discussed.

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.

Molecular phylogenetic studies reveal that many endosymbioses between bacteria and invertebrate hosts result from ancient infections followed by strict vertical transmission within host lineages. Endosymbionts display a distinctive constellation of genetic properties including AT-biased base composition, accelerated sequence evolution, and, at least sometimes, small genome size; these features suggest increased genetic drift. Molecular genetic characterization also has revealed adaptive, host-beneficial traits such as amplification of genes underlying nutrient provision.