Search

Robust resolution of controversial higher-level groupings within Arthropoda requires additional sources of characters. Toward this end, elongation factor-2 sequences (1899 nucleotides) were generated from 17 arthropod taxa (5 chelicerates, 6 crustaceans, 3 hexapods, 3 myriapods) plus an onychophoran and a tardigrade as outgroups. Likelihood and parsimony analyses of nucleotide and amino acid data sets consistently recovered Myriapoda and major chelicerate groups with high bootstrap support. Crustacea + Hexapoda (= Pancrustacea) was recovered with moderate support, whereas the conflicting group Myriapoda + Hexapoda (= Atelocerata) was never recovered and bootstrap values were always <5%. With additional nonarthropod sequences included, one indel supports monophyly of Tardigrada, Onychophora, and Arthropoda relative to molluscan, annelidan, and mammalian outgroups. New and previously published sequences from RNA polymerase II (1038 nucleotides) and elongation factor-1alpha (1092 nucleotides) were analyzed for the same taxa. A comparison of bootstrap values from the three genes analyzed separately revealed widely varying values for some clades, although there was never strong support for conflicting groups. In combined analyses, there was strong bootstrap support for the generally accepted clades Arachnida, Arthropoda, Euchelicerata, Hexapoda, and Pycnogonida, and for Chelicerata, Myriapoda, and Pancrustacea, whose monophyly is more controversial. Recovery of some additional groups was fairly robust to method of analysis but bootstrap values were not high; these included Pancrustacea + Chelicerata, Hexapoda + Cephalocarida + Remipedia, Cephalocarida + Remipedia, and Malaocostraca + Cirripedia. Atelocerata (= Myriapoda + Hexapoda) was never recovered. Elongation factor-2 is now the second protein-encoding, nuclear gene (in addition to RNA polymerase II) to support Pancrustacea over Atelocerata. Atelocerata is widely cited in morphology-based analyses, and the discrepancy between results derived from molecular and morphological data deserves greater attention., (Copyright 2001 Academic Press.)

Phylogenetic utility for the nuclear gene encoding dopa decarboxylase (DDC), little used in systematics, was recently demonstrated within the noctuid moth subfamily Heliothinae. Here we extend the test of the utility of a 709-bp DDC fragment to deeper levels, analyzing 49 species representing major groups across the superfamily Noctuoidea. Parsimony, distance, and maximum-likelihood analyses recover all or nearly all of a set of "test clades" supported by clear morphological synapomorphies, spanning a wide range of taxonomic levels. DDC also upholds a recent proposal that the Noctuidae are paraphyletic. Nt3 contributes a majority of the signal and recovers the basal split between Notodontidae and all other noctuoids, despite a plateau of nt3 divergence at this level. However, nonsynonymous changes also support groups at all levels, and in contrast to nt3, amino acid divergence shows no plateau. The utility of DDC promises to extend back to the early Tertiary and Cretaceous, a time span for which few suitable genes have been identified., (Copyright 2000 Academic Press.)

A central question concerning data collection strategy for molecular phylogenies has been, is it better to increase the number of characters or the number of taxa sampled to improve the robustness of a phylogeny estimate? A recent simulation study concluded that increasing the number of taxa sampled is preferable to increasing the number of nucleotide characters, if taxa are chosen specifically to break up long branches. We explore this hypothesis by using empirical data from noctuoid moths, one of the largest superfamilies of insects. Separate studies of two nuclear genes, elongation factor-1 alpha (EF-1 alpha) and dopa decarboxylase (DDC), have yielded similar gene trees and high concordance with morphological groupings for 49 exemplar species. However, support levels were quite low for nodes deeper than the subfamily level. We tested the effects on phylogenetic signal of (1) increasing the taxon sampling by nearly 60%, to 77 species, and (2) combining data from the two genes in a single analysis. Surprisingly, the increased taxon sampling, although designed to break up long branches, generated greater disagreement between the two gene data sets and decreased support levels for deeper nodes. We appear to have inadvertently introduced new long branches, and breaking these up may require a yet larger taxon sample. Sampling additional characters (combining data) greatly increased the phylogenetic signal. To contrast the potential effect of combining data from independent genes with collection of the same total number of characters from a single gene, we simulated the latter by bootstrap augmentation of the single-gene data sets. Support levels for combined data were at least as high as those for the bootstrap-augmented data set for DDC and were much higher than those for the augmented EF-1 alpha data set. This supports the view that in obtaining additional sequence data to solve a refractory systematic problem, it is prudent to take them from an independent gene.

Recent phylogenetic analyses using molecular data suggest that hexapods are more closely related to crustaceans than to myriapods, a result that conflicts with long-held morphology-based hypotheses. Here we contribute additional information to this debate by conducting phylogenetic analyses on two nuclear protein-encoding genes, elongation factor-1 alpha (EF-1 alpha) and the largest subunit of RNA polymerase II (Pol II), from an extensive sample of arthropod taxa. Results were obtained from two data sets. One data set comprised 1092 nucleotides (364 amino acids) of EF-1 alpha and 372 nucleotides (124 amino acids) of Pol II from 30 arthropods and three lobopods. The other data set contained the same EF-1 alpha fragment and an expanded 1038-nucleotide (346-amino-acid) sample of Pol II from 17 arthropod taxa. Results from maximum-parsimony and maximum-likelihood analyses strongly supported the existence of a Crustacea + Hexapoda clade (Pancrustacea) over a Myriapoda + Hexapoda clade (Atelocerata). The apparent incompatibility between the molecule-based Pancrustacea hypothesis and morphology-based Atelocerata hypothesis is discussed.

Compared to the number of genes available for study of both younger and older divergences, few genes have yet been identified that can strongly resolve phylogenetic splits of Mesozoic age ( approximately 65-250 mya). Thus, reconstruction of Mesozoic-age phylogenies, exemplified by basal divergences within the major orders of holometabolous insects, is likely to be especially dependent on combining multiple lines of evidence. This study tests the potential of the 18S ribosomal RNA gene for reconstructing Mesozoic-aged divergences within the insect order Lepidoptera and its ability when combined with a second, previously analyzed nuclear gene (phosphoenolpyruvate carboxykinase, PEPCK) to strongly resolve these relationships. 18S sequences were obtained for 21 taxa, representing major clades of Lepidoptera plus outgroups from the other "panorpoid orders. A well-corroborated morphology-based "test phylogeny was used to evaluate the effects of partitioning the 18S gene according to variable versus conserved domains, paired versus unpaired sites in the secondary structure, and transition versus transversion substitutions. Likelihood and unweighted parsimony analyses of the 18S data recover the "test phylogeny" almost completely, with no improvement of agreement or support provided by any form of weighting or partitioning. No conflict in signal between 18S and PEPCK was detected by the partition homogeneity test. Combined parsimony analysis yielded strong bootstrap support for nearly all relationships, much higher than for either gene alone, thereby also providing strong evidence on several hypotheses about the early evolution of lepidopteran-plant interactions. These genes in combination may be widely useful for resolving insect divergences of comparable age., (Copyright 2000 Academic Press.)

To extend initial characterizations of their phylogenetic utility, sequences from the nuclear genes for elongation factor-1 alpha (EF-1 alpha) and dopa decarboxylase (DDC) are tested for phylogenetic concordance with each other and with previous morphological evidence within the giant silk moth tribe Attacini (Lepidoptera: Saturniidae). The sampling of DDC is expanded from the 690 basepairs of previous studies to 1051 basepairs in the current study. All nine attacine genera are sampled. EF-1 alpha and DDC agree in the placement of seven of nine genera, with placement of the other two not in strong conflict. Combination of the gene sequences results in a nearly fully resolved tree that is consistent with EF-1 alpha alone and agrees with morphology in five of eight groups. Conflict between molecules and morphology is confined to deeper-level relationships within Attacini, where node support for the molecular hypotheses, but not the morphological hypotheses, is generally very strong. A strong signal is contributed by synonymous substitutions in both genes, and by nonsynonymous change particularly in DDC. The molecular phylogeny supports a revision of attacine biogeography in that neither East Asian nor New World genera form monophyletic groups.

The lack of a readily accessible roster of nuclear genes informative at various taxonomic levels is a bottleneck for molecular systematics. In this report, we describe the first phylogenetic application of the sequence that encodes the enzyme dopa decarboxylase (DDC). For 14 test species within the noctuid moth subfamily Heliothinae that represent the previously best-supported groupings, a 690-bp fragment of DDC resolved relationships that are largely concordant with prior evidence from elongation factor-1 alpha (EF-1 alpha), morphology, and allozymes. Although both synonymous and nonsynonymous changes occur in DDC substantially more rapidly than they do in EF-1 alpha, DDC divergences within Heliothinae are below saturation at all codon positions. Analysis of DDC and EF-1 alpha in combination resulted in increased bootstrap support for several groupings. As a first estimate of previously unresolved relationships, DDC sequences were analyzed from 16 additional heliothines, for a total of 30 heliothine species plus outgroups. Previous relationships based on DDC were generally stable with increased taxon sampling, although a two- to eightfold downweighting of codon position 3 was required for complete concordance with the 14-species result. The weighted strict consensus trees were largely resolved and were congruent with most although not all previous hypotheses based on either morphology or EF-1 alpha. The proposed phylogeny suggests that the major agricultural pest heliothines belong to a single clade, characterized by polyphagy and associated life history traits, within this largely host-specific moth subfamily. DDC holds much promise for phylogenetic analysis of Tertiary-age animal groups.

Fifteen unique chorion protein-encoding cDNAs from gypsy moth have been completely sequenced. These sequences are encoded by a family of genes, based on pairwise similarity values of 78-100% within a 225-nt region. Pairwise comparisons and maximum parsimony analysis strongly support the existence of two clusters of 11 and four sequences each, called noc1 and noc2. While noc2 consists of two subclusters, there is little character support for subclusters within noc1. The highly localized character-state distribution on the parsimony tree in gypsy moth is reminiscent of that in Bombyx mori, specifically for those chorion families that have been shown to undergo gene conversion. Gene conversion thus becomes a reasonable explanation for the homogeneity of noc1 sequences and for their distinctness from noc2. The relationship between the two major clusters of chorion sequences in gypsy moth (noc1, noc2) and Bombyx mori (Bm alpha, Bm beta) has been addressed through mixed-species tree construction. All four groups cluster separately, thus providing no direct evidence of orthologous sequences. However, the occurrence of gene conversion could have eliminated such evidence. The relationship between the chorion gene tree and the species cladogenic event is discussed, as are biases in codon usage, base composition, and nucleotide transformations.

Choriogenesis in silkmoths has been used as a model for relating developmental processes and evolutionary change. We now describe parallel studies in a nonsilkmoth lepidopteran species, the gypsy moth. Choriogenesis is described at the levels of ultrastructure, protein composition and synthesis, and specific mRNA accumulation. One complete chorion cDNA sequence is presented and features of its primary structure are discussed. This sequence is shown to be homologous to those of silkmoths.

Choriogenesis (eggshell formation) within the silkmoth Antheraea polyphemus proceeds in parallel for the two major subpopulations of follicle cells, diverging only during the very late period when aeropyle crown surface structures form in one region but not in the other. Correlated with their appearance is the synthesis of a set of region-specific proteins. In this report, aeropyle crowns are physically isolated and their protein composition is shown to consist of those same region-specific proteins. A cDNA clone, called pcvl 16, has been selected and shown to encode a lamellar-forming, aeropyle crown-specific protein, probably of the previously described C3,4 group. These conclusions are based on hybrid-selected translation, Northern analysis, and sequence analysis. pcvl 16 was used to isolate two distinct cloned copies of the 16 gene. Both 16 genes are closely paired with another region-specific gene but the proximity of the two gene pairs to each other is uncertain. Non-region-specific chorion genes expressed at earlier times in choriogenesis surround the 16 gene pairs, suggesting that cis sequences necessary for regionalized expression may be closely linked to coding sequences. To test this hypothesis, 5'-flanking sequences from eight region-specific genes are compared and shown to share two oligonucleotide sequences. One is a known regulatory element found in virtually all moth and fly chorion genes examined. The other, located just upstream from the TATA box, is not found in non-regionally expressed chorion genes and, thus, is a candidate for specifying regional expression.

Genomic and cDNA clones have been sequenced that encode the E2 silkmoth chorion protein. E2 assembles with E1 [Regier, J.C. and Pacholski, P. (1985) Proc. Natl. Acad. Sci. USA, 82, 6035-6039] to form the 'filler' that helps mold prominent chorion surface structures called aeropyle crowns. E2 has two distinct domains. The amino terminal domain consists of four alternating stretches of hydrophobic and hydrophilic residues, the first three of which are homologous in sequence to about half of the E1 protein. Comparison of predicted secondary structures provides further support for the localized homology of E2 and E1. The carboxy terminal domain of E2 is much longer, is hydrophilic and consists entirely of multiple tandem copies of a single, variant hexapeptide repeat sequence that is absent from E1. Numbers of hexapeptide repeat sequences differed dramatically in two animals. The types of events required for such variation are discussed. Finally, we have elaborated our earlier model for how E proteins may assemble in vivo to form filler.

The protein products from one of the two major silkmoth chorion multigene families contain blocked NH2 termini. The blocked residue has been identified as pyrrolidonecarboxylic acid. Its formation occurs post-translationally, by modification of NH2-terminal glutamine, after removal of a 20-residue-long "signal peptide." Pyrrolidonecarboxylic acid formation begins within minutes after polypeptide chain termination and is largely, although not entirely, complete by 60 min. Pyrrolidonecarboxylic acid thus appears to be formed both within the cell and in the extracellular chorion. Another class of minor chorion proteins also appears to contain pyrrolidonecarboxylic acid.

The main features of the B family of chorion proteins in saturniid moths were examined by partial sequencing of representative B proteins, seven from Antheraea polyphemus and two from A. pernyi. Comparisons were made to sequences derived from seven recombinant DNA clones representing three types of B family proteins of A. polyphemus. The central regions of the sequences are conservative, both within and between moth species, and differ largely by a few amino acid replacements, rather than deletions or insertions. By contrast, the amino-terminal third varies more substantially, in a manner which defines two protein subfamilies: within each subfamily sequences are similar, but the subfamilies differ by at least two multiresidue deletions as well as by amino acid replacements. These properties are analogous to features of the A family of chorion proteins.

Silkmoth eggshell (chorion) proteins have been characterized by electrophoresis on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels; up to 33 and 41 components, respectively, were detected from a single chorion. Some of these components are polymorphic, being absent from chorions of certain animals. A system of nomenclature for all chorion proteins is presented, based on their separation on sodium dodecyl sulfate and isoelectric focusing gels. The chorion is enriched in glycine, alanine, cysteine, and tyrosine and poor in methionine and histidine. The proteins were fractionated into four partially overlapping groups; all four are enriched in the above amino acids, although significant differences exist. Further fractionation by isoelectric focusing of one of the above groups, s/s, yielded seven components, two of which are homogeneous both on sodium dodecyl sulfate and isoelectric focusing gels. The amino acid compositions, molecular weights, and solubility properties of the components share certain features which distinguish s/s as a group from the other three groups. The sequence of the first 67 NH2-terminal residues of a homogeneous protein purified from s/s has been determined. The protein contains a cysteine-rich tail (3 cysteines in the first 18 residues) followed by a 49-residue segment which contains only a single cysteine residue. This latter segment also contains two different tetrapeptide sequences which are each repeated, one twice and the other four times.

The silkmoth chorion is a helicoidally layered, fibrous structure which is constructed in four sequential morphogenetic modes, beginning with the assembly of a thin, low density lamellar framework. Subsequently, the framework expands in height by the insertion of additional fiber sheets into the preexisting lamellae. This expansion mode begins farthest from the follicular secretory cells and progresses in reverse. Individual fibers then grow in thickness, presumably through accretion of newly synthesized proteins, and eventually fuse. This third mode, which also begins in the most distant lamellae and proceeds in reverse, is called densification, as it results in an approximately two fold increase in overall chorion density without further lamellar expansion. Finally, lamellogenesis is recapitulated in miniature in a region of the chorion's surface, where very-late-forming lamellae are molded into prominent surface structures, the aeropyle crowns. The densification and especially the expansion modes suggest considerable fluidity in the developing chorion, consistent with its proposed cholesteric liquid crystalline structure. Such a structure is also consistent with numerous deviations from the ideal helicoidal array. These distortions and defects are described and discussed in terms of their possible origin and function.

Using as criteria the size, abundance and developmental specificity of hybridizing mRNA sequences, we have selected from our chorion cDNA library a clone corresponding to a specific chorion protein, A4--cl. Comparison between the clone sequence and the largely known sequence of A4--cl validates the use of the cDNA library for sequence analysis of the chorion multigene families. The two major chorion protein families, A and B, share certain structural similarities.

Choriogenesis in silkmoths (superfamily Bombycoidea) and in a sphingid moth (superfamily Sphingoidea) differ in major, but discrete, ways. In silkmoths, the predominant lamellar component assembles early in choriogenesis to form a thin framework. Subsequently, the lamellar framework is modified, first by expansion, and then by densification. Finally, ornate surface structures called aeropyle crowns form in some silkmoths, but they are absent in the species described here. In the sphingid, lamellar framework formation occurs throughout choriogenesis rather than largely during the early stages as in silkmoths. Lamellar densification occurs, but lamellar expansion and aeropyle crown formation do not. An evolutionary model is presented that accounts for the observed morphogenetic differences. Patterns of chorion protein synthesis in the sphingid differ from those in silkmoths in ways that are interpretable in light of the observed morphogenetic differences and the previously postulated functions of the proteins in silkmoths.

Time-dependent and cell-specific changes in concentrations of total RNA, of poly A+ RNA, and of specific mRNAs have been measured throughout silkmoth choriogenesis. Levels of total RNA and of poly A+ RNA are maintained throughout much of choriogenesis, but decrease at least fourfold during the very late period, in parallel with a decrease in overall protein synthesis. Very late period changes in total RNA and in poly A+ RNA are less pronounced in the aeropyle crown region, where a subset of chorion proteins is preferentially synthesized, than in the flat region. Maximal accumulation of the E1 and E2 chorion mRNAs occurs preferentially in the aeropyle crown region during the very late period. Uridine pulse-labeling studies suggest that E1 and E2 transcription is similarly aeropyle crown region-specific and immediately precedes the time of maximal E1 and E2 RNA accumulation and protein synthesis. Sequences from the 5' flanking regions of E1 and E2 genes have been compared. Several oligonucleotide sequences are present in both genes, and some are duplicated. These are potential cis-acting, regulatory elements.