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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–1α (EF–1α) 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–1α and 372 nucleotides (124 amino acids) of Pol II from 30 arthropods and three lobopods. The other data set contained the same EF––lα 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.

We described the organization of two silkmoth chorion genes, called E1 and E2, whose expression is largely restricted in time to the very late period of choriogenesis and in space to one of two major subpopulations of follicle cells. Using E1 and E2 clone cDNAs as probes, we showed that gene copy numbers per haploid genome remain constant throughout silkmoth development despite major changes in total DNA content per nucleus. Furthermore, gene copy numbers are the same in both cellular regions of the choriogenic follicle despite differences in nuclear size and levels of E gene expression. Southern analysis indicated between two and four copies each for E1 and E2 genes. Analysis of chromosomal clones showed that single copies of E1 and E2 are separated by about 7.5 kilobases and are transcribed from the same DNA strand. Two distinct pairs of cloned E1 and E2 genes were characterized. No other chorion genes were in their immediate vicinity.

We have sequenced DNA clones corresponding to the entire coding and 5' untranslated regions and almost all of the 3' untranslated region of a silkmoth chorion RNA which is expressed largely in a subpopulation of follicular epithelial cells (aeropyle crown region). This RNA encodes the E1 protein, one of two components of the prominent "filler" that helps mold the shape of aeropyle crowns. The conceptually translated E1 sequence reveals an alternation in hydrophobic and hydrophilic stretches of amino acids that correlates with certain predictions about its secondary structure. E1 is unusual in revealing no sequence homology with other known chorion sequences and in having an unusually long 3' untranslated region. Sequence analysis of the 5' end of the E1 gene has identified an intron near the end of the signal peptide-encoding region, a feature shared with other chorion genes.

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.

The silkmoth chorion has provided a stimulating model for the study of evolution and developmental regulation of gene families. Previous attempts at inferring relationships among chorion sequences have been based on pairwise comparisons of overall similarity, a potentially problematic approach. To remedy this, we identified the alignable regions of low sequence variability and then analyzed this restricted database by parsimony and neighbor-joining methods. At the deepest level, the chorion sequence tree is split into two branches, called "alpha" and "beta." Within each branch, early- and late-expressing genes each constitute monophyletic groups, while the situation with middle-expressing genes remains uncertain. The HcB gene family appears to be the most basal beta-branch group, but this conclusion is qualified because the effect of gene conversion on branching order is unknown. Previous studies by Eickbush and colleagues have strongly suggested that ErA, HcA, and HcB families undergo gene conversion within a gene family, whereas the ErB family does not. The occurrence of conversion correlates with a particular tree structure; namely, branch lengths are much greater at the base of the family than at higher internodes and terminal branches. These observations raise the possibility that chorion gene families are defined by gene conversion events (reticulate evolution) rather than by descent with modification (synapomorphy).

We have constructed a cDNA clone library from poly(A)+ RNA of very-late-period choriogenic silkmoth follicles. Clone DNAs that hybridize preferentially to RNA from the aeropyle crown region of the follicle (versus the flat region) were selected, and all could be placed in one of two homology groups. The two groups represent sequences encoding the very-late-period chorion proteins E1 and E2; this was established by hybrid-selected translation coupled with specific antibody precipitation. Regionalized synthesis of chorion proteins is restricted to the very late period, and its control can now be studied at the nucleic acid level.

We have characterized a new family of silkmoth chorion genes, called C, which is distinct from previously characterized A and B families. The amino acid compositions of 18 purified C proteins have been determined. Three subgroups are recognized on the basis of compositional similarities and may correspond to distinct gene families or subfamilies. The sequences of two overlapping cDNA clones have been determined in their entirety and shown to correspond to a C-specific sequence. Obvious homology is observed between the middle portions of the C sequence and previously characterized B sequences. By contrast, the arms of the C sequence share no significant similarities either with each other or with the corresponding arms of B sequences. Thus, the same tripartite structure originally observed in A and B family sequences is also present in the C family and may have functional significance. Secondary structure prediction of the C sequence is presented and supports this conclusion. The observed homology between C and B family sequences clearly establishes that silkmoth chorion multigene families constitute a superfamily.

Five polypeptide components have been isolated from the eggshell (chorions) of a silkmoth. Two are homogeneous on sodium dodecyl sulfate and isoelectric focusing gels, and three contain predominantly two proteins each. Amino acid analyses show that all five components are similar to each other. These proteins have been sequenced from the amino terminus. Homogeneous components yielded single sequences; heterogeneous components yielded two residues at some positions, consistent with their containing two major electrophoretic components. Striking similarities are apparent among all these sequences. These similarities can be increased dramatically by separating each of the three protein mixtures into two sequences and introducing a small number of gaps or insertions. This is due in part to bringing into register a portion that contains short repeating subunits found in all sequences. All proteins are also characterized by a region of high cysteine content near the amino terminus followed by a longer low-cysteine region. The data suggest that these proteins share a common evolutionary origin and are encoded by a multigene family.

Discrete changes in silkmoth choriogenesis have occurred during evolution, as exemplified in the present report in Antheraea polyphemus and Hyalophora cecropia. At the level of morphology, the chorion of A. polyphemus has surface structures, called aeropyle crowns, that are absent from H. cecropia. Aeropyle crowns form during the very late period of choriogenesis and consist of two substructures--lamellae and filler. Filler is present in H. cecropia in greatly reduced amounts. At the level of protein synthesis, overall similarities in the two species are maintained until the very late period of choriogenesis, when synthesis of aeropyle crown components is maximal. In H. cecropia, very late period-specific proteins are reduced in number and abundance. Several of these minor proteins are candidates for E1 and E2, the components of filler. E1 and E2 RNAs are about 35 times more abundant in A. polyphemus, despite very similar gene copy numbers and times of expression in the two species. These results support the hypothesis that evolutionary changes in chorion morphology have resulted from regulatory changes in the expression of chorion genes, either at the level of transcription or mRNA decay. The hypothesis that evolutionary changes in chorion morphology are based on terminal addition onto a preexisting developmental program is discussed.