Karl M. Kjer

Advances in Insect Phylogeny at the Dawn of the Postgenomic Era

Most species on Earth are insects and thus, understanding their evolutionary relationships is key to understanding the evolution of life. Insect relationships are increasingly well supported, due largely to technological advances in molecular sequencing and phylogenetic computational analysis. In this postgenomic era, insect systematics will be furthered best by integrative methods aimed at hypothesis corroboration from molecular, morphological, and paleontological evidence. This review of the current consensus of insect relationships provides a foundation for comparative study and offers a framework to evaluate incoming genomic evidence. Notable recent phylogenetic successes include the resolution of Holometabola, including the identification of the enigmatic Strepsiptera as a beetle relative and the early divergence of Hymenoptera; the recognition of hexapods as a crustacean lineage within Pancrustacea; and the elucidation of Dictyoptera orders, with termites placed as social cockroaches. Regions of the tree that require further investigation include the earliest winged insects (Palaeoptera) and Polyneoptera (orthopteroid lineages).

Phylogeny of Trichoptera (Caddisflies): Characterization of Signal and Noise Within Multiple Datasets

Trichoptera are holometabolous insects with aquatic larvae that, together with the Lepidoptera, make up the Amphiesmenoptera. Despite extensive previous morphological work, little phylogenetic agreement has been reached about the relationship among the three suborders--Annulipalpia, Spicipalpia, and Integripalpia--or about the monophyly of Spicipalpia. In an effort to resolve this conflict, we sequenced fragments of the large and small subunit nuclear ribosomal RNAs (1078 nt; D1, D3, V4-5), the nuclear elongation factor 1 alpha gene (EF-1 alpha; 1098 nt), and a fragment of mitochondrial cytochrome oxidase I (COI; 411 nt). Seventy adult and larval morphological characters were reanalyzed and added to molecular data in a combined analysis. We evaluated signal and homoplasy in each of the molecular datasets and attempted to rank the particular datasets according to how appropriate they were for inferring relationships among suborders. This evaluation included testing for conflict among datasets, comparing tree lengths among alternative hypotheses, measuring the left-skew of tree-length distributions from maximally divergent sets of taxa, evaluating the recovery of expected clades, visualizing whether or not substitutions were accumulating with time, and estimating nucleotide compositional bias. Although all these measures cast doubt on the reliability of the deep-level signal coming from the nucleotides of the COI and EF-1 alpha genes, these data could still be included in combined analyses without overturning the results from the most conservative marker, the rRNA. The different datasets were found to be evolving under extremely different rates. A site-specific likelihood method for dealing with combined data with nonoverlapping parameters was proposed, and a similar weighting scheme under parsimony was evaluated. Among our phylogenetic conclusions, we found Annulipalpia to be the most basal of the three suborders, with Spicipalpia and Integripalpia forming a clade. Monophyly of Annulipalpia and Integripalpia was confirmed, but the relationships among spicipalpians remain equivocal.

Site specific rates of mitochondrial genomes and the phylogeny of eutheria

BackgroundTraditionally, most studies employing data from whole mitochondrial genomes to diagnose relationships among the major lineages of mammals have attempted to exclude regions that potentially complicate phylogenetic analysis. Components generally excluded are 3rd codon positions of protein-encoding genes, the control region, rRNAs, tRNAs, and the ND6 gene (encoded on the opposite strand). We present an approach that includes all the data, with the exception of the control region. This approach is based on a site-specific rate model that accommodates excessive homoplasy and that utilizes secondary structure as a reference for proper alignment of rRNAs and tRNAs.ResultsMitochondrial genomic data for 78 eutherian mammals, 8 metatherians, and 3 monotremes were analyzed with a Bayesian analysis and our site specific rate model. The resultant phylogeny revealed strong support for most nodes and was highly congruent with more recent phylogenies based on nuclear DNA sequences. In addition, many of the conflicting relationships observed by earlier mitochondrial-based analyses were resolved without need for the exclusion of large subsets of the data.ConclusionRather than exclusion of data to minimize presumed noise associated with non-protein encoding genes in the mitochondrial genome, our results indicate that selection of an appropriate model that accommodates rate heterogeneity across data partitions and proper treatment of RNA genes can result in a mitochondrial genome-based phylogeny of eutherian mammals that is reasonably congruent with recent phylogenies derived from nuclear genes.

Opinions on Multiple Sequence Alignment, and an Empirical Comparison of Repeatability and Accuracy between POY and Structural Alignment

An extensive literature, special symposia, and even a formal organization (International Society for Phylogenetic Nomenclature) have been devoted to various other taxonomic proposals some of which seem far more radical and/or less utilitarian than the timeclip proposal advanced here. Thus, we now invite open discussion on the temporal-banding strategy and on timeclip-like tactics that could greatly enrich existing taxonomies while still maintaining established Linnaean traditions and nomenclatures.

The evolutionary history of holometabolous insects inferred from transcriptome-based phylogeny and comprehensive morphological data

BackgroundDespite considerable progress in systematics, a comprehensive scenario of the evolution of phenotypic characters in the mega-diverse Holometabola based on a solid phylogenetic hypothesis was still missing. We addressed this issue by de novo sequencing transcriptome libraries of representatives of all orders of holometabolan insects (13 species in total) and by using a previously published extensive morphological dataset. We tested competing phylogenetic hypotheses by analyzing various specifically designed sets of amino acid sequence data, using maximum likelihood (ML) based tree inference and Four-cluster Likelihood Mapping (FcLM). By maximum parsimony-based mapping of the morphological data on the phylogenetic relationships we traced evolutionary transformations at the phenotypic level and reconstructed the groundplan of Holometabola and of selected subgroups.ResultsIn our analysis of the amino acid sequence data of 1,343 single-copy orthologous genes, Hymenoptera are placed as sister group to all remaining holometabolan orders, i.e., to a clade Aparaglossata, comprising two monophyletic subunits Mecopterida (Amphiesmenoptera + Antliophora) and Neuropteroidea (Neuropterida + Coleopterida). The monophyly of Coleopterida (Coleoptera and Strepsiptera) remains ambiguous in the analyses of the transcriptome data, but appears likely based on the morphological data. Highly supported relationships within Neuropterida and Antliophora are Raphidioptera + (Neuroptera + monophyletic Megaloptera), and Diptera + (Siphonaptera + Mecoptera). ML tree inference and FcLM yielded largely congruent results. However, FcLM, which was applied here for the first time to large phylogenomic supermatrices, displayed additional signal in the datasets that was not identified in the ML trees.ConclusionsOur phylogenetic results imply that an orthognathous larva belongs to the groundplan of Holometabola, with compound eyes and well-developed thoracic legs, externally feeding on plants or fungi. Ancestral larvae of Aparaglossata were prognathous, equipped with single larval eyes (stemmata), and possibly agile and predacious. Ancestral holometabolan adults likely resembled in their morphology the groundplan of adult neopteran insects. Within Aparaglossata, the adult’s flight apparatus and ovipositor underwent strong modifications. We show that the combination of well-resolved phylogenies obtained by phylogenomic analyses and well-documented extensive morphological datasets is an appropriate basis for reconstructing complex morphological transformations and for the inference of evolutionary histories.

Phylogeny of caddisflies (Insecta, Trichoptera)

Trichoptera are holometabolous insects with aquatic larvae that, together with the Lepidoptera, comprise the Amphiesmenoptera. Previous phylogenetic hypotheses and progress on our ongoing data collection are summarized. Fragments of the large and small subunit nuclear ribosomal RNAs (D1, D3, V4–5), the nuclear elongation factor 1 alpha gene and a fragment of mitochondrial cytochrome oxidase 1 (COI) were sequenced, and molecular data were combined with previously published morphological data. Equally and differentially weighted parsimony analyses were conducted in order to present a phylogeny of Trichoptera, including 43 of 45 families. Our phylogeny closely resembles that proposed by Herbert Ross with respect to the relationships among suborders, with a monophyletic Annulipalpia at the base of the tree, and a clade consisting of Spicipalpia plus a monophyletic Integripalpia. The monophyly of Spicipalpia is weakly supported in the combined equally weighted analysis, and Spicipalpia is paraphyletic in the differentially weighted analysis. Within Integripalpia, our phylogeny recovered monophyletic Plenitentoria, Brevitentoria and Sericostomatoidea. Leptoceroidea was unresolved in the equally weighted analysis and monophyletic in the differentially weighted analysis. Within Annulipalpia, we recovered a basal but paraphyletic Philopotamoidea and a monophyletic Hydropsychoidea.

Associating larvae and adults of Chinese Hydropsychidae caddisflies (Insecta:Trichoptera) using DNA sequences

Abstract The utility of hydropsychid (Trichoptera:Hydropsychidae) caddisfly larvae for freshwater biomonitoring has been demonstrated, but the major impediment to its implementation has been the lack of species-level larval descriptions and illustrations. A rapid and reliable molecular protocol that also uses morphology is proposed because conventional approaches to associating undescribed larvae with adults have been slow and problematic. Male adults were identified before DNA sequence analyses were used. These identifications established morphospecies boundaries that were mapped on phylograms constructed from 2 independent gene fragments: mitochondrial cytochrome c oxidase subunit I (COI) and large subunit (28S) nuclear ribosomal DNA expansion fragment D2 (D2). Species boundaries were confirmed if they were monophyletic on both molecular phylograms. Larval associations were made with reference to the phylogenetic analyses under 2 criteria: sequence identity across both genes or nested placement within a reference species boundary. A total of 133 individuals belonging to Chinese Hydropsyche sensu lato group (including Hydropsyche [Hydropsyche], Hydropsyche [Occutanspsyche], Ceratopsyche, Mexipsyche, Hydatomanicus, and Herbertorossia) were included in our study to test the new protocol. D2 sequences (all individuals) and COI sequences (101 individuals) were obtained, and 2 independent phylograms were constructed using neighbor joining. Both fragments provided enough nucleotide changes to differentiate independently most Hydropsyche sensu lato species, with ambiguity in only a few species that eventually could be resolved with additional sequences and specimens. COI diverges significantly within some species, suggesting a need for caution when applying typical genetic divergence thresholds in species diagnoses. The study enabled us to establish a procedure for delimiting species boundaries and associating larvae and adults using DNA sequences and morphological evidence. Ideal sampling strategies for larval–adult association are suggested. Associating larvae and adults of hydropsychids using DNA sequences appears to be promising in terms of both reliability and speed.

Structural alignment of 18S and 28S rDNA sequences provides insights into phylogeny of Phytophaga (Coleoptera: Curculionoidea and Chrysomeloidea)

We performed a comparative study of partial rDNA sequences from a variety of Coleoptera taxa to construct an annotated alignment based on secondary structure information, which in turn, provides improved rRNA structure models useful for phylogenetic reconstruction. Subsequent phylogenetic analysis was performed to test monophyly and interfamilial relationships of the megadiverse plant feeding beetle group known as ‘Phytophaga’ (Curculionoidea and Chrysomeloidea), as well as to discover their closest relatives among the Cucujiformia. Parsimony and Bayesian analyses were performed based on the structural alignment of segments of 18S rRNA (variable regions V4‐V5, V7‐V9) and 28S rRNA (expansion segment D2). A total of 104 terminal taxa of Coleoptera were included: 96 species of Cucujiformia beetles, representing the families and most ‘subfamilies’ of weevils and chrysomeloids (Phytophaga), as well as several families of Cleroidea, Tenebrionoidea and Cucujoidea, and eight outgroups from three other polyphagan series: Scarabaeiformia, Elateriformia and Bostrichiformia. The results from the different methods of analysis agree — recovering the monophyly of the ‘Phytophaga’, including Curculionoidea and Chrysomeloidea as sister groups. The curculionoid and chrysomeloid phylogeny recovered from the aligned 18S and 28S rDNA segments, which is independent of morphological data, is in agreement with recent hypotheses or concepts based on morphological evidence, particularly with respect to familial relationships. Our results provide clues about the evolutionary origin of the phytophagan beetles within the megaclade Cucujiformia, suggesting that the sister group of ‘Curculionoidea + Chrysomeloidea’ is a clade of the ‘Cucujoidea’, represented in this study by species in Boganiidae, Erotylidae, Nitidulidae, Cucujidae and Silvanidae. The Coccinellidae and Endomychidae are not grouped with the latter, and the remaining terminal taxa are nested in Tenebrionoidea and Cleroidea. We propose that the combination of structurally aligned ribosomal RNA gene regions 18S (V4‐V5, V7‐V9) and 28S (D2) are useful in testing monophyly and resolving relationships among beetle superfamilies and families.

MITOCHONDRIAL DNA SEQUENCE-BASED PHYLOGENY AND THE EVOLUTION OF VIVIPARITY IN THE SCELOPORUS SCALARIS GROUP (REPTILIA, SQUAMATA)

The lizard genus Sceloporus contains both oviparous and viviparous species. The scalaris complex is the only monophyletic group within the genus that includes both reproductive modes, thus it is particularly well suited for studies of the evolution of viviparity. Approximately 874 nucleotides of mtDNA sequence data, collected from 38 specimens, comprising 25 populations of all five recognized species within the group, were used in a phylogenetic analysis of the origin of viviparity. Viviparity appears to have evolved twice in this group: once in S. goldmani, included in a clade formed by a northern group consisting of S. scalaris, S. chaneyi, and S. goldmani, and one more time in S. bicanthalis, included in the southern group formed by S. bicanthalis and S. aeneus. An oviparous population of S. bicanthalis nested within that viviparous clade, indicates that reversal from viviparity to oviparity may be possible. Degree of sequence divergence among several S. bicanthalis individuals pertaining to a population in which both parity modes occur, was no larger between oviparous and viviparous lizards than among viviparous lizards. This suggests that this population is a single species, and it may represent a transition from oviparity to viviparity or vice‐versa.

Molecular dating and biogeography of fig-pollinating wasps

Figs and fig-pollinating wasps are obligate mutualists that have coevolved for over 60 million years. But when and where did pollinating fig wasps (Agaonidae) originate? Some studies suggest that agaonids arose in the Late Cretaceous and the current distribution of fig-wasp faunas can be explained by the break-up of the Gondwanan landmass. However, recent molecular-dating studies suggest divergence time estimates that are inconsistent with the Gondwanan vicariance hypothesis and imply that long distance oceanic dispersal could have been an important process for explaining the current distribution of both figs and fig wasps. Here, we use a combination of phylogenetic and biogeographical data to infer the age, the major period of diversification, and the geographic origin of pollinating fig wasps. Age estimates ranged widely depending on the molecular-dating method used and even when using the same method but with slightly different constraints, making it difficult to assess with certainty a Gondwanan origin of agaonids. The reconstruction of ancestral areas suggests that the most recent common ancestor of all extant fig-pollinating wasps was most likely Asian, although a southern Gondwana origin cannot be rejected. Our analysis also suggests that dispersal has played a more important role in the development of the fig-wasp biota than previously assumed.