A large-scale, higher-level, molecular phylogenetic study of the insect order Lepidoptera (moths and butterflies).

BACKGROUND: Higher-level relationships within the Lepidoptera, and particularly within the species-rich subclade Ditrysia, are generally not well understood, although recent studies have yielded progress. We present the most comprehensive molecular analysis of lepidopteran phylogeny to date, focusing on relationships among superfamilies. METHODOLOGY PRINCIPAL FINDINGS: 483 taxa spanning 115 of 124 families were sampled for 19 protein-coding nuclear genes, from which maximum likelihood tree estimates and bootstrap percentages were obtained using GARLI. Assessment of heuristic search effectiveness showed that better trees and higher bootstrap percentages probably remain to be discovered even after 1000 or more search replicates, but further search proved impractical even with grid computing. Other analyses explored the effects of sampling nonsynonymous change only versus partitioned and unpartitioned total nucleotide change; deletion of rogue taxa; and compositional heterogeneity. Relationships among the non-ditrysian lineages previously inferred from morphology were largely confirmed, plus some new ones, with strong support. Robust support was also found for divergences among non-apoditrysian lineages of Ditrysia, but only rarely so within Apoditrysia. Paraphyly for Tineoidea is strongly supported by analysis of nonsynonymous-only signal; conflicting, strong support for tineoid monophyly when synonymous signal was added back is shown to result from compositional heterogeneity. CONCLUSIONS SIGNIFICANCE: Support for among-superfamily relationships outside the Apoditrysia is now generally strong. Comparable support is mostly lacking within Apoditrysia, but dramatically increased bootstrap percentages for some nodes after rogue taxon removal, and concordance with other evidence, strongly suggest that our picture of apoditrysian phylogeny is approximately correct. This study highlights the challenge of finding optimal topologies when analyzing hundreds of taxa. It also shows that some nodes get strong support only when analysis is restricted to nonsynonymous change, while total change is necessary for strong support of others. Thus, multiple types of analyses will be necessary to fully resolve lepidopteran phylogeny.

A molecular phylogeny for yponomeutoidea (insecta, Lepidoptera, ditrysia) and its implications for classification, biogeography and the evolution of host plant use.

BACKGROUND: Yponomeutoidea, one of the early-diverging lineages of ditrysian Lepidoptera, comprise about 1,800 species worldwide, including notable pests and insect-plant interaction models. Yponomeutoids were one of the earliest lepidopteran clades to evolve external feeding and to extensively colonize herbaceous angiosperms. Despite the group's economic importance, and its value for tracing early lepidopteran evolution, the biodiversity and phylogeny of Yponomeutoidea have been relatively little studied. METHODOLOGY/PRINCIPAL FINDINGS: Eight nuclear genes (8 kb) were initially sequenced for 86 putative yponomeutoid species, spanning all previously recognized suprageneric groups, and 53 outgroups representing 22 families and 12 superfamilies. Eleven to 19 additional genes, yielding a total of 14.8 to 18.9 kb, were then sampled for a subset of taxa, including 28 yponomeutoids and 43 outgroups. Maximum likelihood analyses were conducted on data sets differing in numbers of genes, matrix completeness, inclusion/weighting of synonymous substitutions, and inclusion/exclusion of "rogue" taxa. Monophyly for Yponomeutoidea was supported very strongly when the 18 "rogue" taxa were excluded, and moderately otherwise. Results from different analyses are highly congruent and relationships within Yponomeutoidea are well supported overall. There is strong support overall for monophyly of families previously recognized on morphological grounds, including Yponomeutidae, Ypsolophidae, Plutellidae, Glyphipterigidae, Argyresthiidae, Attevidae, Praydidae, Heliodinidae, and Bedelliidae. We also assign family rank to Scythropiinae (Scythropiidae stat. rev.), which in our trees are strongly grouped with Bedelliidae, in contrast to all previous proposals. We present a working hypothesis of among-family relationships, and an informal higher classification. Host plant family associations of yponomeutoid subfamilies and families are non-random, but show no trends suggesting parallel phylogenesis. Our analyses suggest that previous characterizations of yponomeutoids as predominantly Holarctic were based on insufficient sampling. CONCLUSIONS/SIGNIFICANCE: We provide the first robust molecular phylogeny for Yponomeutoidea, together with a revised classification and new insights into their life history evolution and biogeography.

Resolving discrepancy between nucleotides and amino acids in deep-level arthropod phylogenomics: differentiating serine codons in 21-amino-acid models.

BACKGROUND: In a previous study of higher-level arthropod phylogeny, analyses of nucleotide sequences from 62 protein-coding nuclear genes for 80 panarthopod species yielded significantly higher bootstrap support for selected nodes than did amino acids. This study investigates the cause of that discrepancy. METHODOLOGY/PRINCIPAL FINDINGS: The hypothesis is tested that failure to distinguish the serine residues encoded by two disjunct clusters of codons (TCN, AGY) in amino acid analyses leads to this discrepancy. In one test, the two clusters of serine codons (Ser1, Ser2) are conceptually translated as separate amino acids. Analysis of the resulting 21-amino-acid data matrix shows striking increases in bootstrap support, in some cases matching that in nucleotide analyses. In a second approach, nucleotide and 20-amino-acid data sets are artificially altered through targeted deletions, modifications, and replacements, revealing the pivotal contributions of distinct Ser1 and Ser2 codons. We confirm that previous methods of coding nonsynonymous nucleotide change are robust and computationally efficient by introducing two new degeneracy coding methods. We demonstrate for degeneracy coding that neither compositional heterogeneity at the level of nucleotides nor codon usage bias between Ser1 and Ser2 clusters of codons (or their separately coded amino acids) is a major source of non-phylogenetic signal. CONCLUSIONS: The incongruity in support between amino-acid and nucleotide analyses of the forementioned arthropod data set is resolved by showing that "standard" 20-amino-acid analyses yield lower node support specifically when serine provides crucial signal. Separate coding of Ser1 and Ser2 residues yields support commensurate with that found by degenerated nucleotides, without introducing phylogenetic artifacts. While exclusion of all serine data leads to reduced support for serine-sensitive nodes, these nodes are still recovered in the ML topology, indicating that the enhanced signal from Ser1 and Ser2 is not qualitatively different from that of the other amino acids.

A molecular phylogeny for the leaf-roller moths (Lepidoptera: Tortricidae) and its implications for classification and life history evolution.

BACKGROUND: Tortricidae, one of the largest families of microlepidopterans, comprise about 10,000 described species worldwide, including important pests, biological control agents and experimental models. Understanding of tortricid phylogeny, the basis for a predictive classification, is currently provisional. We present the first detailed molecular estimate of relationships across the tribes and subfamilies of Tortricidae, assess its concordance with previous morphological evidence, and re-examine postulated evolutionary trends in host plant use and biogeography. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced up to five nuclear genes (6,633 bp) in each of 52 tortricids spanning all three subfamilies and 19 of the 22 tribes, plus up to 14 additional genes, for a total of 14,826 bp, in 29 of those taxa plus all 14 outgroup taxa. Maximum likelihood analyses yield trees that, within Tortricidae, differ little among data sets and character treatments and are nearly always strongly supported at all levels of divergence. Support for several nodes was greatly increased by the additional 14 genes sequenced in just 29 of 52 tortricids, with no evidence of phylogenetic artifacts from deliberately incomplete gene sampling. There is strong support for the monophyly of Tortricinae and of Olethreutinae, and for grouping of these to the exclusion of Chlidanotinae. Relationships among tribes are robustly resolved in Tortricinae and mostly so in Olethreutinae. Feeding habit (internal versus external) is strongly conserved on the phylogeny. Within Tortricinae, a clade characterized by eggs being deposited in large clusters, in contrast to singly or in small batches, has markedly elevated incidence of polyphagous species. The five earliest-branching tortricid lineages are all species-poor tribes with mainly southern/tropical distributions, consistent with a hypothesized Gondwanan origin for the family. CONCLUSIONS/SIGNIFICANCE: We present the first robustly supported phylogeny for Tortricidae, and a revised classification in which all of the sampled tribes are now monophyletic.

Can deliberately incomplete gene sample augmentation improve a phylogeny estimate for the advanced moths and butterflies (Hexapoda: Lepidoptera)?

This paper addresses the question of whether one can economically improve the robustness of a molecular phylogeny estimate by increasing gene sampling in only a subset of taxa, without having the analysis invalidated by artifacts arising from large blocks of missing data. Our case study stems from an ongoing effort to resolve poorly understood deeper relationships in the large clade Ditrysia ( > 150,000 species) of the insect order Lepidoptera (butterflies and moths). Seeking to remedy the overall weak support for deeper divergences in an initial study based on five nuclear genes (6.6 kb) in 123 exemplars, we nearly tripled the total gene sample (to 26 genes, 18.4 kb) but only in a third (41) of the taxa. The resulting partially augmented data matrix (45% intentionally missing data) consistently increased bootstrap support for groupings previously identified in the five-gene (nearly) complete matrix, while introducing no contradictory groupings of the kind that missing data have been predicted to produce. Our results add to growing evidence that data sets differing substantially in gene and taxon sampling can often be safely and profitably combined. The strongest overall support for nodes above the family level came from including all nucleotide changes, while partitioning sites into sets undergoing mostly nonsynonymous versus mostly synonymous change. In contrast, support for the deepest node for which any persuasive molecular evidence has yet emerged (78-85% bootstrap) was weak or nonexistent unless synonymous change was entirely excluded, a result plausibly attributed to compositional heterogeneity. This node (Gelechioidea + Apoditrysia), tentatively proposed by previous authors on the basis of four morphological synapomorphies, is the first major subset of ditrysian superfamilies to receive strong statistical support in any phylogenetic study. A "more-genes-only" data set (41 taxa×26 genes) also gave strong signal for a second deep grouping (Macrolepidoptera) that was obscured, but not strongly contradicted, in more taxon-rich analyses.

Sources of signal in 62 protein-coding nuclear genes for higher-level phylogenetics of arthropods.

BACKGROUND: This study aims to investigate the strength of various sources of phylogenetic information that led to recent seemingly robust conclusions about higher-level arthropod phylogeny and to assess the role of excluding or downweighting synonymous change for arriving at those conclusions. METHODOLOGY/PRINCIPAL FINDINGS: The current study analyzes DNA sequences from 68 gene segments of 62 distinct protein-coding nuclear genes for 80 species. Gene segments analyzed individually support numerous nodes recovered in combined-gene analyses, but few of the higher-level nodes of greatest current interest. However, neither is there support for conflicting alternatives to these higher-level nodes. Gene segments with higher rates of nonsynonymous change tend to be more informative overall, but those with lower rates tend to provide stronger support for deeper nodes. Higher-level nodes with bootstrap values in the 80% - 99% range for the complete data matrix are markedly more sensitive to substantial drops in their bootstrap percentages after character subsampling than those with 100% bootstrap, suggesting that these nodes are likely not to have been strongly supported with many fewer data than in the full matrix. Data set partitioning of total data by (mostly) synonymous and (mostly) nonsynonymous change improves overall node support, but the result remains much inferior to analysis of (unpartitioned) nonsynonymous change alone. Clusters of genes with similar nonsynonymous rate properties (e.g., faster vs. slower) show some distinct patterns of node support but few conflicts. Synonymous change is shown to contribute little, if any, phylogenetic signal to the support of higher-level nodes, but it does contribute nonphylogenetic signal, probably through its underlying heterogeneous nucleotide composition. Analysis of seemingly conservative indels does not prove useful. CONCLUSIONS: Generating a robust molecular higher-level phylogeny of Arthropoda is currently possible with large amounts of data and an exclusive reliance on nonsynonymous change.

Increased gene sampling strengthens support for higher-level groups within leaf-mining moths and relatives (Lepidoptera: Gracillariidae).

BACKGROUND: Researchers conducting molecular phylogenetic studies are frequently faced with the decision of what to do when weak branch support is obtained for key nodes of importance. As one solution, the researcher may choose to sequence additional orthologous genes of appropriate evolutionary rate for the taxa in the study. However, generating large, complete data matrices can become increasingly difficult as the number of characters increases. A few empirical studies have shown that augmenting genes even for a subset of taxa can improve branch support. However, because each study differs in the number of characters and taxa, there is still a need for additional studies that examine whether incomplete sampling designs are likely to aid at increasing deep node resolution. We target Gracillariidae, a Cretaceous-age (~100 Ma) group of leaf-mining moths to test whether the strategy of adding genes for a subset of taxa can improve branch support for deep nodes. We initially sequenced ten genes (8,418 bp) for 57 taxa that represent the major lineages of Gracillariidae plus outgroups. After finding that many deep divergences remained weakly supported, we sequenced eleven additional genes (6,375 bp) for a 27-taxon subset. We then compared results from different data sets to assess whether one sampling design can be favored over another. The concatenated data set comprising all genes and all taxa and three other data sets of different taxon and gene sub-sampling design were analyzed with maximum likelihood. Each data set was subject to five different models and partitioning schemes of non-synonymous and synonymous changes. Statistical significance of non-monophyly was examined with the Approximately Unbiased (AU) test. RESULTS: Partial augmentation of genes led to high support for deep divergences, especially when non-synonymous changes were analyzed alone. Increasing the number of taxa without an increase in number of characters led to lower bootstrap support; increasing the number of characters without increasing the number of taxa generally increased bootstrap support. More than three-quarters of nodes were supported with bootstrap values greater than 80% when all taxa and genes were combined. Gracillariidae, Lithocolletinae + Leucanthiza, and Acrocercops and Parectopa groups were strongly supported in nearly every analysis. Gracillaria group was well supported in some analyses, but less so in others. We find strong evidence for the exclusion of Douglasiidae from Gracillarioidea sensu Davis and Robinson (1998). Our results strongly support the monophyly of a G.B.R.Y. clade, a group comprised of Gracillariidae + Bucculatricidae + Roeslerstammiidae + Yponomeutidae, when analyzed with non-synonymous changes only, but this group was frequently split when synonymous and non-synonymous substitutions were analyzed together. CONCLUSIONS: 1) Partially or fully augmenting a data set with more characters increased bootstrap support for particular deep nodes, and this increase was dramatic when non-synonymous changes were analyzed alone. Thus, the addition of sites that have low levels of saturation and compositional heterogeneity can greatly improve results. 2) Gracillarioidea, as defined by Davis and Robinson (1998), clearly do not include Douglasiidae, and changes to current classification will be required. 3) Gracillariidae were monophyletic in all analyses conducted, and nearly all species can be placed into one of six strongly supported clades though relationships among these remain unclear. 4) The difficulty in determining the phylogenetic placement of Bucculatricidae is probably attributable to compositional heterogeneity at the third codon position. From our tests for compositional heterogeneity and strong bootstrap values obtained when synonymous changes are excluded, we tentatively conclude that Bucculatricidae is closely related to Gracillariidae + Roeslerstammiidae + Yponomeutidae.

Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences.

The remarkable antiquity, diversity and ecological significance of arthropods have inspired numerous attempts to resolve their deep phylogenetic history, but the results of two decades of intensive molecular phylogenetics have been mixed. The discovery that terrestrial insects (Hexapoda) are more closely related to aquatic Crustacea than to the terrestrial centipedes and millipedes (Myriapoda) was an early, if exceptional, success. More typically, analyses based on limited samples of taxa and genes have generated results that are inconsistent, weakly supported and highly sensitive to analytical conditions. Here we present strongly supported results from likelihood, Bayesian and parsimony analyses of over 41 kilobases of aligned DNA sequence from 62 single-copy nuclear protein-coding genes from 75 arthropod species. These species represent every major arthropod lineage, plus five species of tardigrades and onychophorans as outgroups. Our results strongly support Pancrustacea (Hexapoda plus Crustacea) but also strongly favour the traditional morphology-based Mandibulata (Myriapoda plus Pancrustacea) over the molecule-based Paradoxopoda (Myriapoda plus Chelicerata). In addition to Hexapoda, Pancrustacea includes three major extant lineages of 'crustaceans', each spanning a significant range of morphological disparity. These are Oligostraca (ostracods, mystacocarids, branchiurans and pentastomids), Vericrustacea (malacostracans, thecostracans, copepods and branchiopods) and Xenocarida (cephalocarids and remipedes). Finally, within Pancrustacea we identify Xenocarida as the long-sought sister group to the Hexapoda, a result confirming that 'crustaceans' are not monophyletic. These results provide a statistically well-supported phylogenetic framework for the largest animal phylum and represent a step towards ending the often-heated, century-long debate on arthropod relationships.

Toward reconstructing the evolution of advanced moths and butterflies (Lepidoptera: Ditrysia): an initial molecular study.

BACKGROUND: In the mega-diverse insect order Lepidoptera (butterflies and moths; 165,000 described species), deeper relationships are little understood within the clade Ditrysia, to which 98% of the species belong. To begin addressing this problem, we tested the ability of five protein-coding nuclear genes (6.7 kb total), and character subsets therein, to resolve relationships among 123 species representing 27 (of 33) superfamilies and 55 (of 100) families of Ditrysia under maximum likelihood analysis. RESULTS: Our trees show broad concordance with previous morphological hypotheses of ditrysian phylogeny, although most relationships among superfamilies are weakly supported. There are also notable surprises, such as a consistently closer relationship of Pyraloidea than of butterflies to most Macrolepidoptera. Monophyly is significantly rejected by one or more character sets for the putative clades Macrolepidoptera as currently defined (P < 0.05) and Macrolepidoptera excluding Noctuoidea and Bombycoidea sensu lato (P < or = 0.005), and nearly so for the superfamily Drepanoidea as currently defined (P < 0.08). Superfamilies are typically recovered or nearly so, but usually without strong support. Relationships within superfamilies and families, however, are often robustly resolved. We provide some of the first strong molecular evidence on deeper splits within Pyraloidea, Tortricoidea, Geometroidea, Noctuoidea and others.Separate analyses of mostly synonymous versus non-synonymous character sets revealed notable differences (though not strong conflict), including a marked influence of compositional heterogeneity on apparent signal in the third codon position (nt3). As available model partitioning methods cannot correct for this variation, we assessed overall phylogeny resolution through separate examination of trees from each character set. Exploration of "tree space" with GARLI, using grid computing, showed that hundreds of searches are typically needed to find the best-feasible phylogeny estimate for these data. CONCLUSION: Our results (a) corroborate the broad outlines of the current working phylogenetic hypothesis for Ditrysia, (b) demonstrate that some prominent features of that hypothesis, including the position of the butterflies, need revision, and (c) resolve the majority of family and subfamily relationships within superfamilies as thus far sampled. Much further gene and taxon sampling will be needed, however, to strongly resolve individual deeper nodes.

Phylogeny and biogeography of hawkmoths (Lepidoptera: Sphingidae): evidence from five nuclear genes.

BACKGROUND: The 1400 species of hawkmoths (Lepidoptera: Sphingidae) comprise one of most conspicuous and well-studied groups of insects, and provide model systems for diverse biological disciplines. However, a robust phylogenetic framework for the family is currently lacking. Morphology is unable to confidently determine relationships among most groups. As a major step toward understanding relationships of this model group, we have undertaken the first large-scale molecular phylogenetic analysis of hawkmoths representing all subfamilies, tribes and subtribes. METHODOLOGY/PRINCIPAL FINDINGS: The data set consisted of 131 sphingid species and 6793 bp of sequence from five protein-coding nuclear genes. Maximum likelihood and parsimony analyses provided strong support for more than two-thirds of all nodes, including strong signal for or against nearly all of the fifteen current subfamily, tribal and sub-tribal groupings. Monophyly was strongly supported for some of these, including Macroglossinae, Sphinginae, Acherontiini, Ambulycini, Philampelini, Choerocampina, and Hemarina. Other groupings proved para- or polyphyletic, and will need significant redefinition; these include Smerinthinae, Smerinthini, Sphingini, Sphingulini, Dilophonotini, Dilophonotina, Macroglossini, and Macroglossina. The basal divergence, strongly supported, is between Macroglossinae and Smerinthinae+Sphinginae. All genes contribute significantly to the signal from the combined data set, and there is little conflict between genes. Ancestral state reconstruction reveals multiple separate origins of New World and Old World radiations. CONCLUSIONS/SIGNIFICANCE: Our study provides the first comprehensive phylogeny of one of the most conspicuous and well-studied insects. The molecular phylogeny challenges current concepts of Sphingidae based on morphology, and provides a foundation for a new classification. While there are multiple independent origins of New World and Old World radiations, we conclude that broad-scale geographic distribution in hawkmoths is more phylogenetically conserved than previously postulated.

Resolving arthropod phylogeny: exploring phylogenetic signal within 41 kb of protein-coding nuclear gene sequence.

This study attempts to resolve relationships among and within the four basal arthropod lineages (Pancrustacea, Myriapoda, Euchelicerata, Pycnogonida) and to assess the widespread expectation that remaining phylogenetic problems will yield to increasing amounts of sequence data. Sixty-eight regions of 62 protein-coding nuclear genes (approximately 41 kilobases (kb)/taxon) were sequenced for 12 taxonomically diverse arthropod taxa and a tardigrade outgroup. Parsimony, likelihood, and Bayesian analyses of total nucleotide data generally strongly supported the monophyly of each of the basal lineages represented by more than one species. Other relationships within the Arthropoda were also supported, with support levels depending on method of analysis and inclusion/exclusion of synonymous changes. Removing third codon positions, where the assumption of base compositional homogeneity was rejected, altered the results. Removing the final class of synonymous mutations--first codon positions encoding leucine and arginine, which were also compositionally heterogeneous--yielded a data set that was consistent with a hypothesis of base compositional homogeneity. Furthermore, under such a data-exclusion regime, all 68 gene regions individually were consistent with base compositional homogeneity. Restricting likelihood analyses to nonsynonymous change recovered trees with strong support for the basal lineages but not for other groups that were variably supported with more inclusive data sets. In a further effort to increase phylogenetic signal, three types of data exploration were undertaken. (1) Individual genes were ranked by their average rate of nonsynonymous change, and three rate categories were assigned--fast, intermediate, and slow. Then, bootstrap analysis of each gene was performed separately to see which taxonomic groups received strong support. Five taxonomic groups were strongly supported independently by two or more genes, and these genes mostly belonged to the slow or intermediate categories, whereas groups supported only by a single gene region tended to be from genes of the fast category, arguing that fast genes provide a less consistent signal. (2) A sensitivity analysis was performed in which increasing numbers of genes were excluded, beginning with the fastest. The number of strongly supported nodes increased up to a point and then decreased slightly. Recovery of Hexapoda required removal of fast genes. Support for Mandibulata (Pancrustacea + Myriapoda) also increased, at times to "strong" levels, with removal of the fastest genes. (3) Concordance selection was evaluated by clustering genes according to their ability to recover Pancrustacea, Euchelicerata, or Myriapoda and analyzing the three clusters separately. All clusters of genes recovered the three concordance clades but were at times inconsistent in the relationships recovered among and within these clades, a result that indicates that the a priori concordance criteria may bias phylogenetic signal in unexpected ways. In a further attempt to increase support of taxonomic relationships, sequence data from 49 additional taxa for three slow genes (i.e., EF-1 alpha, EF-2, and Pol II) were combined with the various 13-taxon data sets. The 62-taxon analyses supported the results of the 13-taxon analyses and provided increased support for additional pancrustacean clades found in an earlier analysis including only EF-1 alpha, EF-2, and Pol II.

Phylogeny of pteromalid parasitic wasps (Hymenoptera: Pteromalidae): initial evidence from four protein-coding nuclear genes.

Chalcidoidea (approximately 22,000 described species) is the most ecologically diverse superfamily of parasitic Hymenoptera and plays a major role in the biological control of insect pests. However, phylogenetic relationships both within and between chalcidoid families have been poorly understood, particularly for the large family Pteromalidae and relatives. Forty-two taxa, broadly representing Chalcidoidea but concentrated in the 'pteromalid lineage,' were sequenced for 4620 bp of protein-coding sequence from four nuclear genes for which we present new primers. These are: CAD (1719 bp) DDC (708 bp), enolase (1149 bp), and PEPCK (1044 bp). The combined data set was analyzed using parsimony, maximum likelihood, and Bayesian methods. Statistical significance of the apparent non-monophyly of some taxonomic groups on our trees was evaluated using the approximately unbiased test of Shimodaira [Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51(3), 492-508]. In accord with previous studies, we find moderate to strong support for monophyly of Chalcidoidea, a sister-group relationship of Mymaridae to the remainder of Chalcidoidea, and a relatively basal placement of Encarsia (Aphelinidae) within the latter. The 'pteromalid lineage' of families is generally recovered as monophyletic, but the hypothesis of monophyly for Pteromalidae, which appear paraphyletic with respect to all other families sampled in that lineage, is decisively rejected (P < 10(-14)). Within Pteromalidae, monophyly was strongly supported for nearly all tribes represented by multiple exemplars, and for two subfamilies. All other multiply-represented subfamilies appeared para- or polyphyletic in our trees, although monophyly was significantly rejected only for Miscogasterinae, Ormocerinae, and Colotrechninae. The limited resolution obtained in the analyses presented here reinforces the idea that reconstruction of pteromalid phylogeny is a difficult problem, possibly due to rapid radiation of many chalcidoid taxa. Initial phylogenetic comparisons of life history traits suggest that the ancestral chalcidoid was small-bodied and parasitized insect eggs.

Phylogenetics of eggshell morphogenesis in Antheraea (lepidoptera: saturniidae): unique origin and repeated reduction of the aeropyle crown.

Integrated phylogenetic and developmental analyses should enhance our understanding of morphological evolution and thereby improve systematists' ability to utilize morphological characters, but case studies are few. The eggshell (chorion) of Lepidoptera (Insecta) has proven especially tractable experimentally for such analyses because its morphogenesis proceeds by extracellular assembly of proteins. This study focuses on a morphological novelty, the aeropyle crown, that arises at the end of choriogenesis in the wild silkmoth genus Antheraea. Aeropyle crowns are cylindrical projections, ending in prominent prongs, that surround the openings of breathing tubes (aeropyle channels) traversing the chorion. They occur over the entire egg surface in some species, are localized to a circumferential band in many others, and in some are missing entirely, thus exhibiting variation typical of discrete characters analyzed in morphological phylogenetics. Seeking an integrated developmental-phylogenetic view, we first survey aeropyle crown variation broadly across Antheraea and related genera. We then map these observations onto a robust phylogeny, based on three nuclear genes, to test the adequacy of character codings for aeropyle crown variation and to estimate the frequency and direction of change in those characters. Thirdly, we draw on previous studies of choriogenesis, supplemented by new data on gene expression, to hypothesize developmental-genetic bases for the inferred chorion character transformations. Aeropyle crowns are inferred to arise just once, in the ancestor of Antheraea, but to undergo four or more subsequent reductions without regain, a pattern consistent with Dollo's Law. Spatial distribution shows an analogous trend, though less clear-cut, toward reduction of coverage by aeropyle crowns. These trends suggest either that there is little or no natural selection on the details of the aeropyle crown structure or that evolution toward functional optima is ongoing, although no direct evidence exists for either. Genetic, biochemical, and microscopy studies point to at least two developmental changes underlying the origin of the aeropyle crown, namely, reinitiation of deposition of chorionic lamellae after the end of normal choriogenesis (i.e., heterochrony), and sharply increased production of underlying "filler" proteins that push the nascent final lamellae upward to form the crown (i.e., heteroposy). Identification of a unique putative cis-regulatory element shared by unrelated genes involved in aeropyle crown formation suggests a possible simple mechanism for repeated evolutionary reduction and spatial restriction of aeropyle crowns.

Pancrustacean phylogeny: hexapods are terrestrial crustaceans and maxillopods are not monophyletic.

Recent molecular analyses indicate that crustaceans and hexapods form a clade (Pancrustacea or Tetraconata), but relationships among its constituent lineages, including monophyly of crustaceans, are controversial. Our phylogenetic analysis of three protein-coding nuclear genes from 62 arthropods and lobopods (Onychophora and Tardigrada) demonstrates that Hexapoda is most closely related to the crustaceans Branchiopoda (fairy shrimp, water fleas, etc.) and Cephalocarida + Remipedia, thereby making hexapods terrestrial crustaceans and the traditionally defined Crustacea paraphyletic. Additional findings are that Malacostraca (crabs, isopods, etc.) unites with Cirripedia (barnacles, etc.) and they, in turn, with Copepoda, making the traditional crustacean class Maxillopoda paraphyletic. Ostracoda (seed shrimp)--either all or a subgroup--is associated with Branchiura (fish lice) and likely to be basal to all other pancrustaceans. A Bayesian statistical (non-clock) estimate of divergence times suggests a Precambrian origin for Pancrustacea (600 Myr ago or more), which precedes the first unambiguous arthropod fossils by over 60 Myr.

Phylogenetic analysis of Myriapoda using three nuclear protein-coding genes.

We assessed the ability of three nuclear protein-encoding genes-elongation factor-1alpha (EF-1alpha), RNA polymerase II (Pol II), and elongation factor-2 (EF-2)-from 59 myriapod and 12 non-myriapod species to resolve phylogenetic relationships among myriapod classes and orders. In a previous study using EF-1alpha and Pol II (2134 nt combined) from 34 myriapod taxa, Regier and Shultz recovered widely accepted classes, orders, and families but failed to resolve interclass and interordinal relationships. The result was attributed to heterogenous rates of cladogenesis (specifically, the inability of the slowly evolving sequences to capture phylogenetic signal during rapid phylogenetic diversification) but the possibility of inadequate taxon sampling or limited sequence information could not be excluded. In the present study, the myriapod taxon sample was increased by 25 taxa (73%) and sequence length per taxon was effectively doubled through addition of EF-2 (4318 nt combined). Parsimony and Bayesian analyses of the expanded data set recovered a monophyletic Myriapoda, all four myriapod classes and all multiply sampled orders, often with high node support. However, except for three diplopod clades (Colobognatha, Helminothomorpha, and a subgroup of Pentazonia), few interordinal relationships and no interclass relationships were well supported. These results are similar to those of the earlier study by Regier and Shultz, which indicates that taxon sample and sequence length alone do not readily explain the weakly supported resolution in the earlier study. We review recent paleontological evidence to further develop our proposal that heterogeneity in phylogenetic signal provided by our slowly evolving sequences is due to heterogeneity in the temporal structure of myriapod diversification.

Evidence for a role of the regulator of G-protein signaling protein CPRGS-1 in Galpha subunit CPG-1-mediated regulation of fungal virulence, conidiation, and hydrophobin synthesis in the chestnut blight fungus Cryphonectria parasitica.

We previously reported that the chestnut blight fungus Cryphonectria parasitica expresses at least three G-protein alpha subunits and that Galpha subunit CPG-1 is essential for regulated growth, pigmentation, sporulation, and virulence. We now report the cloning and characterization of a C. parasitica regulator of G-protein signaling (RGS) protein, CPRGS-1. The phylogenetic relationship of CPRGS-1 to orthologs from other fungi was inferred and found to be generally concordant with species relationships based on 18S ribosomal sequences and on morphology. However, Hemiascomycotine RGS branch lengths in particular were longer than for their 18S sequence counterparts, which correlates with functional diversification in the signaling pathway. Deletion of cprgs-1 resulted in reduced growth, sparse aerial mycelium, and loss of pigmentation, sporulation, and virulence. Disruption of cprgs-1 was also accompanied by a severe posttranscriptional reduction in accumulation of CPG-1 and Gbeta subunit CPGB-1 and severely reduced expression of the hydrophobin-encoding gene cryparin. The changes in phenotype, cryparin expression, and CPGB-1 accumulation resulting from cprgs-1 gene deletion were also observed in a strain containing a mutationally activated copy of CPG-1 but not in strains containing constitutively activated mutant alleles of the other two identified Galpha subunits, CPG-2 and CPG-3. Furthermore, cprgs-1 transcript levels were increased in the activated CPG-1 strain but were unaltered in activated CPG-2 and CPG-3 strains. The results strongly suggest that CPRGS-1 is involved in regulation of Galpha subunit CPG-1-mediated signaling and establish a role for a RGS protein in the modulation of virulence, conidiation, and hydrophobin synthesis in a plant pathogenic fungus.