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Phylogenetics of Eggshell Morphogenesis in Antheraea (Lepidoptera: Saturniidae): Unique Origin and Repeated Reduction of the Aeropyle Crown

Posted on: Friday, 20 May 2005, 03:00 CDT

Abstract.-

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. [Aeropyle crown; Antheraea; development; Dollo's Law; eggshell; Lepidoptera; morphogenesis.]

Despite spectacular advances in our understanding of basic mechanisms of development (Gilbert, 2003), ignorance about the developmental-genetic basis of the interspecific variation routinely used by systematists still limits our ability to extract phylogenetic information from morphology. This problem is now being addressed through coordinated phylogenetic and developmental analyses of morphological characters (e.g., Rogers and Kaufman, 1997; Stark et al., 1999; Geeta, 2003; Sucena et al., 2003), but case studies are few.

The present study examines character phylogeny/development in a system that has proven unusually experimentally tractable, namely, formation of the lepidopteran eggshell (Regier and Hatzopoulos, 1988). Protein and ultrastructural studies have shown that the predominant eggshell layer, called the chorion, is assembled extracellularly from proteins synthesized by overlying follicular epithelial cells. The rich internal and surface structure of the chorion is assembled through a series of distinct phases of morphogenesis (Fig. 1; Mazur et al., 1989). The organization, expression, and evolution of approximately 100 structural genes involved in choriogenesis, belonging mostly to a single, highly structured gene superf amily, have been described (Goldsmith and Kafatos, 1984; Leclerc and Regier, 1993; Xiong et al, 1988).

Chorion morphology shows broad apparent phylogenetic trends (Fehrenbach, 1995), although definitive resolution of these will require much better understanding of deep-level lepidopteran phylogeny (Kristensen and Skalski, 1999). A complex structure known as lamellar chorion, completely absent in primitive lepidopterans, is characteristic of the advanced clade Ditrysia (Regier et al., 1995). Lamellar chorion is especially prominent, comprising most of the eggshell, in the silkmoths and allies (superfamily Bombycoidea), which exhibit much variation in the modes of lamellogenesis (Regier and Hatzopoulos, 1988; Regier et al., 1995).

The present study focuses on a much finer evolutionary scale, examining a morphological novelty, arising through a terminal phase of choriogenesis, that appears restricted to a subset of the genus Antheraea in the bombycoid family Saturniidae. The structures in question, called aeropyle crowns, are circular vertical projections of the lamellar chorion that surround the surface openings of aeropyles (Figs. 2 to 4; Regier et al., 1980). Aeropyles are tiny breathing tubes through the chorion interior, lying under three- cell junctions of follicle cells, that are passively sculpted during choriogenesis by elongated microvilli and the "filler" proteins they secrete. Previous work (Mazur et al., 1980) has shown that the vertical orientation of aeropyle crowns results from lamellar protein deposition by follicle cells that have been locally uplifted by underlying extracellular accumulation of filler protein. Developing aeropyle crowns have prominent filler as well as lamellar components, but after ovulation the filler collapses onto the chorion surface, leaving only the lamellar component visible. Species ofAntheraea vary both in the presence or absence of aeropyle crowns and in their spatial distribution: aeropyle crowns, when present, may be distributed over the entire egg surface (except for the small micropyle region) or localized to a circumferential band(s) (Figs. 3,4).

FIGURE 1. Schematic view of choriogenic stages in Antheraea polyphemus. left to right (early to very late periods): trabecular layer formation, lamellar framework formation, lamellar expansion, lamellar densification, aeropyle crown formation. Relative heights of the trabecular layer, lamellar zone, and aeropyle crowns are not to scale. Similarly, lamellar number is underrepresented. (See Regier et al, 1980; Mazur et al, 1980, 1989; and Regier et al., 1982.)

FIGURE 2. Transmission electron micrographs of a very latestage choriogenic follicle from Antheraea polyphemus. (A) Oblique cross- sectional view of a developing aeropyle crown at the folliclecell/ chorion interface, showing the secretory microvillar surface (S), lamellar chorion (L), and filler (F) localized to the aeropyle crown interior and underlying channel. Arrows identify the highest projection of lamellar chorion within the developing crown. Scale bar = 1 m (B) Oblique cross-sectional view of simple aeropyle opening in the flat region (lacking aeropyle crowns). Scale bar = 0.5 m. (C) Cross sectional view of an aeropyle channel traversing the interior of lamellar chorion. Special, elongated microvilli (V) (two small sections also visible in A extend from follicle cells through opening at base of aeropyle crown into chorion interior, where they secrete filler (F)). Scale bar = 0.5 m.

FIGURE 3. Schematic view of three chorion surface patterns. AC, aeropyle crowns ubiquitous except in the small micropylar zone (near top); AC reg, aeropyle crowns localized to circumferential band (sometimes split), absent from micropylar zone; A, aeropyle crowns absent.

The aeropyle crown exhibits variation typical of that fashioned into discrete characters by morphological systematists, and yet its evolutionary trajectory, like those of many other complex structures, is unclear. Is the origin of such a structure, as is often suggested (e.g., Simpson, 1953), a much rarer event than changes leading to its reduction or loss? If there are intermediate conditions between present and absent, do these reflect a stepwise sequence of gain or loss, or can any condition arise abruptly from any other?

Comparative developmental studies are potentially invaluable for resolving these issues by clarifying the underlying genetic basis of morphological change. To serve this purpose, however, they must be placed into a phylogenetic perspective, so that comparisons can be directed at the specific evolutionary steps for which there is evidence. Prior studies of aeropyle crown formation have focused on just a few species and lack the requisite phylogenetic context.

FIGURE 4. SEMs of mature and nearly mature chorions from three Antheraea species. (A) Chorion surface, A. pernyi. Note central micropylar region, lacking aeropyle crowns. Scale bar = 200 m. (B) Aeropyle crowns of A. pernyi. Scale bar = 20 m. (C) Cross section of ripped, nearly mature A. pernyi chorion. Follicle cells removed but filler still ful\ly hydrated inside aeropyle crowns. Aeropyle channels, generally obliquely oriented, are not in plane of ripping. Scale bar = 10 m. (D) A. polyphetmts chorion showing flat (F), stripe (S), and micropyle (M) regions. Flat region surface reticulations are imprints from previously overlying follicle cells. Scale bar = 200 m. (E) Aeropyle crowns, A. polyphemus. Scale bar = 10 m. (F) Chorion surface, A. paukstadtorum. Scale bars = 100 m and 30 m (insert).

The present study takes three steps toward an integrated developmental-phylogenetic understanding of aeropyle crowns. First, we present a broad survey of chorion surface structure in Antheraea and related genera to rigorously document the distribution of aeropyle crown occurrence and spatial patterning and to test whether the putatively binary nature of that variation is an artifact of previously sparse taxon sampling. second, we use cladogram mapping on a robust molecular phylogeny for these taxa, based on three nuclear genes previously shown to be informative in this moth family (Regier et al., 1998, 2002), to estimate the rate and direction of transitions among the states of aeropyle crown characters. Third, we draw on previous studies of choriogenesis in wild silkmoth species (Hatzopoulos and Regier, 1987; Regier et al., 1993), supplemented by new comparative data on gene expression patterns, to hypothesize developmental-genetic bases for the inferred evolutionary changes in aeropyle crown characters.

MATERIALS AND METHODS

Abbreviations

BP, bootstrap percentage; EF-1α, elongation factorla; DDC, dopa decarboxylase; MP, maximum parsimony; ML, maximum likelihood; NNI, nearest neighbor interchange; PCR, polymerase chain reaction; SEM, scanning electron microscopy; TBR, tree bisection and reconnection.

Gene, Taxon, and Eggshell Sampling

EF-1α (1227 nucleotides in length each), DDC (1302 nucleotides each), and period (948 nucleotides each) sequences were obtained from 16 species of Antheraea, representing all three subgenera and 8 of the 10 species groups/subgroups recognized by Paukstadt et al. (2000) (Table 1). Exemplars of two contribal genera (Saturniinae: Saturniini), Antherina and Saturnia, were chosen as outgroups, based on previous studies (Regier et al., 2002); substitution of six other genera of Saturniini as outgroups gave similar results (unpublished results). EF-1α was also sequenced from a specimen identified as Antheraea hartii, but its sequence was identical to that from A. pernyi, corroborating their conspecific status (Paukstadt et al., 2000). Antheraea jana (Bali), A. jana (Lombok), and A. pasteuri are treated here as separate species, although their status is not certain.

Mature ovulated eggshells have been viewed by SEM for a broad range of moths within Bombycoidea, and we have used this information to delimit the taxonomic distribution of aeropyle crowns (summarized in Table 2 for species of Antheraea and at http://www. umbi.umd.edu/ users/jcrlab/eggshells.pdf for nonAntheraea species as well). The non-Antheraea species examined, all of which were found to lack aeropyle crowns, are as follows (asterisks denote our own observations): Bombycidae: Bombyx mori; Lasiocampidae: Lasiocampa quercus; Saturniidae, Arsenurinae: Arsenura armida*, Dysdaemonia boreas*; Saturniidae, Ceratocampinae: Eacles imperalis*, Citheronia sepulcralis*; Saturniidae, Hemileucinae: Automeris ??*, Automeris zephyria*, Colomdia pandora*, Lonomia achdous*, PoIythysana apollina*; Saturniidae, Saturniinae, Micragonini: Holocerina smilax*; Saturniidae, Saturniinae, Bunaeini: Imbrasia cytherea*; Saturniidae, Saturniinae, Attacini: Archaeoattacus edwardsii*, Archaeoattacus staudingeri*, Attacus caesar*, Attacus lorquinii*, Callosamia securifera*, Coscinocera hercules*, Eupackardia colleta*, Hyalophora cecropia, Rothschildia forbesi*, Rothschildia lebeau*, Rothschildia orizaba*, Samia cynthia*, Samia abrerai, Samia ricini; Saturniidae, Saturniinae, Saturniini (not including species of Antheraea): Actias selene, Antherina suraka*, Argema mittrei*, Copaxa cydippe*, Copaxa multifenestrata*, Cricula andrei*, Graellsia isabellae*, Loepa katinka*, Rhodinia fugax*, Rhodinia jankowskii*, Saturnia (Caligula) japonica*, Saturnia (Eudia) pavonia, Saturnia (Saturnia) pyri*, Syntherata leonae*; Saturniidae, Saturniinae, Urotini: Pseudaphelia apollinaris; Sphingidae: Manduca sexta.

TABLE 1. Species sampled for EF-1α, DDC, and period sequences.

A previous report of aeropyle crowns in Archaeoattacus edwardsii, in the same subfamily as Antheraea but different tribe (Peigler and Stephens, 1986), was based on misidentification of an eggshell now thought to represent Antheraea polyphemus (Peigler, 2003). We confirmed this conclusion with SEMs from Archaeoattacus edwardsii and Archaeoattacus staudingeri.

Sequence Data Collection and Assembly

Specimens were alive until frozen at -850C in 100% ethanol. Vouchers are stored in freezers at the Department of Entomology, University of Maryland. Total nucleic acids were isolated and specific sequences amplified by reverse transcription followed by the PCR (Regier et al., 1998). Specific bands were gel isolated and then, reamplified by PCR from one or two new internal primer sites. The desired band was gel isolated and sequenced. Visible bands that were too faint to sequence were reamplified using the M13 sites at the 5' ends of all primers (M13REV for forward or "F" primers, M13(- 21) for reverse or "R" primers; see Regier and Shi, 2005). For ease of locating primer sites on each of the three genes, we have placed in parentheses immediately after each primer name (see below), the position of the first encoded amino acid located immediately 3' to that particular primer site relative to that for the 5'-most primer listed, using Antheraea pernyi as the reference in the case of period, which has short indels in the unedited data matrix. Sequences of previously described primers for EF-Ia are 40.6F (33), 40.71F (46), 45.71R (135), 45.71F (143), 52R (236), 52F (244), 52.4F (284), 53.5R (347), 41.2R (409) (see Regier and Shultz, 1997); for DDC are 1.7sF (87), 1.9sF (106), 3.2sF (256), 3.3sR (276), 4sR (322) (see Mitchell et al., 2000); and for period are 177F (1), 197F (21), 341F (165), 397R (219), 402F (226), 514R (336), 532R (354) (see Regier et al., 1998).

The new primers used here, with M13 sequences omitted, are EF- 1α, 30F (1): CAY ATY AAY ATH GTS GTI ATH GG (I = deoxyinosine); EF-1α, 42.8R (82): ATC ATR TTY TTD ATR AAR TC; EF-1α, 42.8F (90): GAY TTY ATH AAR AAY ATG AT; EF-1α, 52.4R (277): TCR TGR TGC ATY TCN AC; DDC, 1.IvF (1): GAY TAY ATY RCR GAR TA; DDC, 1.1F (3): GGA CTA YAT CGC GGA ATA TTT GG; DDC, 1.2F (10): GAR AAY ATY AGA GAY AGR CAR GT; DDC, 1.9sR (97): CAT YTG RCC BAR CCA RTC NAD CAT; and DDC, 7.5sR (436): TCC CAN GAN ACR TGV ATR TC.

Data sets encompass sequences between the following terminal primer regions: 30F to 41.2R for EF-1α, 1.1F to 7.5sR for DDC, and 197F to 514R for period. Conditions for reverse transcription, PCR, and touchdown PCR have been described in detail for EF-1α (Regier and Shultz, 1997) and were very similar for DDC and period (see also Regier et al., 1998; Regier and Shi, 2005). At times, we found it useful to increase the magnesium and/or primer concentrations up to twofold.

TABLE 2. Compilation of eggshell surface structure patterns for the genus Antheraea.

Automated DNA sequencer chromatograms were edited and contiguous fragments were assembled using the GAP4 program within the Staden software package (Staden et al., 1999). Sequences were aligned using the Genetic Data Environment package (version 2.2, Smith et al., 1994). Across the ingroup, there was one informative indel (i.e., a six-nucleotide deletion) in period and none in EF-1α and DDC. This indel was shared between Antheraeci polyphemus and Antheraea godmani, which are strongly grouped by substitution data alone. The aligned data set (minus indels) in NEXUS format can be downloaded at http://www.umbi.umd.edu/users/jcrlab/Antheraea3gn2004.doc.

Electron Microscopic Analysis of Eggshells

For SEM analysis, mature ovulated eggshells (when available) were dissected from live adults, briefly rinsed in water to remove debris, and stored at room temperature in 100% ethanol. Otherwise, hatched eggshells, which had been stored dry at room temperature (in most cases, for years), were vigorously but briefly (i.e., up to 1 hour) shaken in 4 M guanidinium thiocyanate + 0.1 M Tris-HCl (pH 7.5) + 5 mM EDTA, rinsed in water, and stored at room temperature in 100% ethanol. Eggshells were air dried and mounted directly onto SEM stubs. The mounted eggshells were vacuum coated with platinum:palladium and viewed by SEM (Amray 1820D). For transmission electron microscopic analysis, sample preparation followed Regier et al. (1980) and samples were embedded in Epon.

Molecular Phylogenetic Analysis

Unweighted MP analyses of amino acid and total nucleotide data sets were conducted with PAUP*4.0 (Swofford, 1998). Analysis consisted of heuristic searches with TBR branch swapping and 100 random sequenceaddition replicates. Nonparametric bootstrap analyses (1000 bootstrap replications) differed only in having fewer sequence- addition replicates (10) per bootstrap replicate (Felsenstein, 1985). Partitioned Bremer support values (Baker and DeSaIIe, 1997) were calculated using TreeRot software (version 2c; Sorenson, 1999). Chisquare tests of base frequencies across taxa were conducted with PAUP*4.0 (Swofford, 1998).

Following optimal model selection using Modeltest 3.06 (Posada and Crandall, 1998), ML analysis and nonparametric bootstrap analysis (687 to 1000 replications depending on data set) of nucleotide data sets were performed with PAUP*4.0 using a general timereversible model of sequence evolution with amongsite rate heterogeneity modeled by a gamma function approximated by four discrete rate catego\ries. The ML tree search strategy started by optimizing characters on the MP all-nucleotide tree, followed by sequential branch swappings (NNI, TBR) and branch-length reoptimizations. Using the final ML-estimated parameters, a new ML search was then performed with random addition of taxa, NNI branch swapping, and 100 replications. Bootstrap analysis used fixed parameters based on the ML topology, random addition of taxa, NNI branch swapping, and 10 random-addition replicates per bootstrap data set (see also Regier and Shultz, 2001).

Bayesian phylogenetic analysis was performed on amino acids using MrBayes 2.01 (Huelsenbeck and Ronquist, 2001) with the following parameters: aamodel = jones, rates = gamma, neat = 4, ngen = 106, samplefreq = 100, burn-in = 10^sup 5^. Posterior probabilities reached stationarity (estimated by averaging blocks of values from the .p file in Excel) at approximately 4000 generations, but to be safe the burn-in was extended to 105 generations. Starting trees were randomly selected and identical results were obtained in a near- duplicate run (see also Regier and Shultz, 2001).

Phylogenetic Mapping of Aeropyle Crown Variation

To reconstruct the evolutionary history of aeropyle crowns, we first coded the observed variation into discrete characters. We recognized two characters, one expressing variation in the maximal degree of aeropyle crown elaboration (presence/absence and possible modifications or antecedents) and one expressing variation in the spatial distribution of aeropyle crowns or mounds. (Described in the first section of Results.) We treat the second character as unscorable when crowns or mounds are lacking. We then mapped these characters onto the molecular phylogeny by hand using the unordered parsimony criterion. Because the molecular phylogeny was almost entirely in agreement with, although more resolved than, the morphology-based classification of Paukstadt et al. (2000; see Results), we included nine additional taxa for which chorion observations, but not sequence data, were available. For the sequenced taxa only, we also mapped the first character by ML using the computer program Multistate (Pagel, 1999). Forward and reverse transition rates were set equal, leading to six rate parameters (listed in the legend to Fig. 9). ML branch lengths were separately estimated from the nucleotide data of the period gene. Character 2 was not mapped under ML because of missing data.

Northern Analysis of Developmental RNA from Antheraea polyphemus and Hyalophora cecropia

Poly A+ RNA isolated from developmentally staged choriogenic follicles of A. polyphemus has previously been hybridized with conspecific probes for the lamellar and filler components of aeropyle crowns and, as a control, for a lamellar component that is not expressed preferentially in the aeropyle crown region (16c, 11, and 401, respectively; see fig. 5 in Regier et al., 1993). Total developmental RNA from H. cecropia has previously been hybridized with a conspecific probe for the filler component found in aeropyles (2; see figs. 3,4 in Hatzopoulos and Regier, 1987). These previous hybridizations are reproduced here in modified form. New to this report is a Northern analysis of poly A+ developmental RNA isolated from H. cecropia and hybridized with A. polyphemusspecific probes for lamellar components, both aeropyle crown-region-specific and non- specific (the complete insert from pcvl 16 and the pc401 plasmid, respectively; see Regier et al., 1993, and references therein). Hybridization conditions were similar to those for the E2 Northern analysis of RNA from H. cecropia (Hatzopoulos and Regier, 1987) except that the temperature was 60C to 61C. Higher temperatures and more stringent washes gave similar, although fainter, patterns of hybridization.

RESULTS

Taxonomic Survey of Eggshell Surface Structure

Eggshell surface patterns were obtained for 24 of the 72 species of Antheraea (Table 2), representing all three subgenera and all but one of the species groups and subgroups recognized (Paukstadt et al., 2000; Paukstadt and Paukstadt, 2001b). Aeropyle crowns were seen in 17 species, representing all subgenera and all but three species groups or subgroups. In eight of these, the aeropyle crowns were uniformly distributed across the eggshell except immediately surrounding the region of the micropyle at the anterior pole (Figs. 4A-C, 5A-F; character codings: AC, -reg; see Materials and Methods for a description of the two eggshell characters and below for a description of their character states). In the other nine, they were restricted to a banded zone that circumscribes the anterior- posterior axis (Figs. 4D, E; 5G-I; character codings: AC, regA). The aeropyle crown region varies in size among species, being, for example, much larger in A. polyphemus than in A. mylitta (Figs. 4D, 5G), and it is frequently bisected by another region (called the stripe region in A, polyphemus, Fig. 4D) that lacks aeropyle crowns. The extent of the stripe region varies dramatically within A. polyphemus-from completely circumscribing the eggshell to nearly absent (Regier et al, 1980). This variation, however, does not obscure the sharp distinction between species with uniform versus regionalized distributions of aeropyle crowns.

Eight species of Antheraea lacked aeropyle crowns of the typical form. Of these, A. paukstadtorum, A. exspectata, and A. rosieri have aeropyle openings with only the slightest surrounding elevation where aeropyle crowns are normally located (Figs. 4F; 6A, B; character codings: A', ?). However, aeropyle openings in A. lampei, A. cordifolia, and A. diehli are surrounded by protruding mounds that, although much smaller than aeropyle crowns and without prominent prongs, appear to represent an intermediate condition, which we term "aeropyle mounds" (Fig. 6C, F; character codings: AM, - reg). A. yamamai and A. roylii also have aeropyle mounds, and a previously published view suggests that A. yamamai may even have a very few, small aeropyle crowns (Table 2). In these two species, however, the aeropyle mounds are restricted to a narrow band surrounding the micropyle region (Fig. 6D, E; character codings: AM, regB). This observation of different patterns of regionalization reinforced our decision to treat spatial restriction of the expression of aeropyle crowns and mounds as an independent character (= character 2; character states = regA, regB, -reg), distinct from the maximal degree of aeropyle crown elaboration (= character 1; character states = AC, AM, A', A).

FIGURE 5. SEMs of mature Anthcraea chorions with aeropyle crowns. (A) A. rosemariac. (B) A. halconensis. (C) A. kelimutuensis. (D) A. youngi. (E) A. godmani. (F) A. helferi. (G) A. jana (Lombok). (H) A. platessa. (I) A. mylitta. Scale bars = 20 m.

Outside Antheraea, aeropyle crowns were found to be absent in our sampling of 11 of the 15 other genera of Saturniini (e.g., see Fig. 7A-G), the four other tribes of Saturniinae (e.g., see Fig. 7H, I), three of the other eight subfamilies of Saturniidae, and three of the other eight families of Bombycoidea (character codings: A, ?). From the SEM photos in Figure 7, it is apparent that the simple aeropyle openings in eggshells from non-Antheraea species are less prominent than even those species in Antheraea without aeropyle crowns or mounds (cf., Figs. 4F; 6A, B). We therefore scored the latter as having a "rudimentary" condition for the aeropyle crown character, as distinguished from the "absent" condition outside Antheraea.

Molecular Phytogeny of Antheraea

The combined analyses of EF-1α, DDC, and period for 16 species of Antheraea yielded well-resolved trees (Fig. 8A), with 12 of 15 nodes strongly supported under both MP and ML criteria (BP > 80%.) Overall, the molecular phylogeny is strongly congruent with the morphology-based classification of Paukstadt et al. (2000), which is based on an examination of additional species. The one clear departure is the strongly supported placement of A. larissa outside (and also not sister group to) thefrithi subgroup (see Table 2 for composition of frithi subgroup). The conflict may be apparent rather than real, however, as no explicit morphological apomorphies for thefrithi subgroup are known.

The most problematic aspect of the molecular phylogeny is the position of A. yamamai as sister to the pernyi group. Although this placement is strongly supported by nucleotide analyses (BP: 81- 89%), the partitioned Bremer support values indicate strong conflict between EF-1α + DDC and period. Separate analyses of these two partitions (Fig. 8B, C) differed strongly in the placement of A. yamamai (and no other species): EF-1α + DDC strongly supported the same resolution as all three genes combined (BP: 99%), whereas period strongly grouped A. yamamai with the helferi subgroup of the helferi group (BP: 95% to 96%), in agreement with the Paukstadt et al. (2000) morphological classification (Table 2). Period also yielded higher node support for grouping A. lampei + A. roylii to the exclusion of A. pernyi (cf. Fig. 8B, C).

FIGURE 6. SEMs of mature chorions from Antheraea species with aeropyle crowns missing or rudimentary. (A) A. exspectata. Aeropyle crowns missing (character coding: A', ?). (B) A. rosieri. Aeropyle crowns missing (character coding: A', ?). Surface contaminated with moth scales. (C) A. lampei. Small mound-like protuberances or very small crowns-"aeropyle mounds"-ubiquitous except in micropylar region (character coding: AM, -reg). (D) A. yamamai. "Aeropyle mounds" form narrow band around micropylar region (see insert), absent elsewhere (character coding: AM, regB). Insert specimen poorly preserved. See Kawakami et al. (1980) and Hinton (1981) for other views of A. yamamai. (E) A. roylii. "Aeropyle mounds" in narrow band around micropylar region (right panel), absent elsewhere (left panel) (character coding: AM, regB). (F) A. cordif\olia. "Aeropyle mounds" ubiquitous except in micropylar region. Surface contaminated with moth scales (character coding: AM, -reg). Scale bars = 50 m.

The anomalous placement of A. yamamai seems unlikely to result either from long-branch attraction (cf. Fig. 8B, C) or base compositional heterogeneity. Concerning the latter, chi-square tests of base frequencies (all codon positions included) across taxa could not reject homogeneity for the three genes, either combined or individually (all P values = 1.00).

Phylogeny Mapping ofAeropyle Crown Characters

Because the molecular phylogeny was almost entirely in agreement with, although more resolved than, the morphology-based classification of Paukstadt et al. (2000; cf. Fig. 8A and Table 2), we included in the cladogram mapping nine taxa for which chorion observations, but not sequence data, were available. Each of these nine was placed on the molecular tree at the base of the least- inclusive grouping to which it belongs in the morphological classification. In choosing a tree on which to depict aeropyle crown mapping (Fig. 9), we have somewhat arbitrarily favored the placement of A, yamamai favored by period (see above, Fig. 8C). This resolution agrees better with morphology and is among the MP trees for amino acids when all three genes are combined. The inferred history of chorion characters is only slightly different if the alternative placement of A. yamamai is adopted.

The cladogram mapping of the two aeropyle crown characters is summarized in Figure 9. Evolution of the character expressing maximal degree of elaboration of the aeropyle crown shows clear evidence of directionality. With or without the inclusion of nonsequenced species, a fully developed aeropyle crown is inferred (by parsimony and ML) to have arisen just once in Bombycoidea, in the ancestor of Antheraea. Subsequently, this structure is inferred to have been reduced to the "aeropyle mound" condition three times and to "rudimentary" twice. The data are equivocal on the possibility that aeropyle mounds are an evolutionary intermediate between fully developed and rudimentary aeropyle crowns. Stepwise reduction is one of several equally parsimonious histories within the cordifolia subgroup, but there is no indication of close relationship between A. rosieri, which has rudimentary crowns, and any species bearing aeropyle mounds.

The "regionalization" character shows weaker evidence of evolutionary directionality than the aeropyle crown itself, but there does appear to be a trend toward reduced surface coverage. Uniform distribution of aeropyle crowns is inferred to be ancestral in Antheraea. Among species with fully developed aeropyle crowns, restriction of these to specific regions of the egg surface is inferred to have arisen twice, with subsequent reversal to a uniform distribution in A. kelimutuensis. If regionalization is considered a comparable condition when manifested by aeropyle mounds, two additional origins are inferred as well as one additional loss (in A. cordifolia), although interpretation of the former is clouded by the possibility of hybridization involving A. yamamai (see Discussion). Altogether, four of the inferred changes in distribution result in reduced coverage by aeropyle crown-like structures, while two result in increased coverage.

FIGURE 7. SEMs of mature chorions from moths in the same tribe (Saturniini) as or sister tribe (Attadni) to Antheraea. (A) Graellsia Isabellas (Saturniini). Numerous small aeropyle openings are visible. (B) Loepa katinka (Saturniini). (C) Saturnia pyri (Saturninii). (D) Rhodinia fugax (Saturniini). (E) Antherinn stirakn (Saturniini). (F) Syntherntn leonne (Saturniini). (G) Copaxa cydippe (Saturniini). (H) Archaeoattacus edwardsii (Attacini). (I) Archaeoattacus staudingeri (Attacini). Scale bars = 50 m.

Chorion mRNA Expression in Moths with and without Aeropyle Crowns

Previously, an aeropyle crown region- and very latepenod- specific, lamellar-chorion-encoding clone from Antheraea polyphemus was sequenced (Regier et al., 1993). We have now used the insert to this clone (called 16) to probe developmental RNAs from Hyalophora cecropia, which lacks aeropyle crowns, and A. polyphemus (Fig. 10). In A. polyphemus, the 16 probe hybridizes most strongly to RNA from choriogenesis stage 4, when aeropyle crowns are being constructed, while its hybridization is undetectable at stages 1 and 2. In contrast, in H. cecropia hybridization to the 16 probe is detectable throughout choriogenesis, weakly at stage 1 and approximately uniformly strongly from stages 2 to 4. The 16-probe pattern of hybridization in H. cecropia is similar to those of the 401 probe for both species. 402 encodes a non-region- and late-period- specific lamellar-chorion sequence (Regier et al., 1993). Parallel results using an E2-filler-specific probe (Hatzopoulos and Regier, 1987) are reproduced for comparison.

DISCUSSION

The Phylogenetic Context of Aeropyle Crown Evolution

The combination of these three genes provides a strongly resolved tree, in close accord with the morphology-based classification, that yields a firm basis for inferring the history of aeropyle crown variation. The main remaining phylogenetic puzzle is the conflict in placement of A. yamamai between the EF-1α + DDC and period partitions. We have never seen such striking discord in our previous studies of lepidopteran phylogeny (e.g., Regier et al., 2002). Although gene lineage sorting is a possible explanation, we cannot rule out the possibility of hybridization and subsequent introgression, which have been reported in other Saturniinae (Peigler, 1978). Particularly for this reason, we treat A. yamamai cautiously in our interpretation of aeropyle crown evolution.

Aeropyle Crown Evolution-Unique Innovation and Multiple Reductions

Our phylogenetic study indicates that the range of events to be explained in aeropyle crown evolution is as follows: Origin of fully developed, uniformly distributed acropylc crowns in an ancestor lacking any traces of these; reduction of the aeropyle crown, in multiple lineages, to the aeropyle mound and/or rudimentary condition; restriction of aeropyle crowns to only part of the chorion surface; somewhat different restriction, possibly nonhomologous, in the distribution of aeropyle mounds.

FIGURE 8. (A) ML topology for EF-1α + DDC + period nucleotides. In the cladogram format (left portion), support values (bootstrap percentages [BP] or posterior probabilities [PP]) above branches are from amino acid MP (MP aa) and amino acid Bayesian (MB aa) analyses (upper line) and from total nucleotide MP (MP nt) and total nucleotide ML (ML nt) analyses (lower line). Partitioned Bremer support values (PBS; EF-1α, DDC, period) below branches are from MP aa (upper line) and MP nt (lower line) analyses. Dashed lines denote weakly supported groupings. Bracketed values denote absence of the group from the best tree(s) under that criterion. Support values are also shown for selected alternative groupings (see arrows). In the phylogram format (right portion), branch lengths are estimated from ML analysis of total nucleotides (3477 nt). (B, C) Section of ML trees from analysis of EF-1α + DDC total nucleotides (2529 nt; B) versus period total nucleotides (948 nt; C) alone, showing strong conflict in placement of A. yamamai. Branch lengths are estimated from the ML analyses.

Aeropyle crowns seem to fit the description of complex structures following a version of Dollo's Law (see Simpson, 1953), originating just once, de novo, with repeated secondary reduction or loss (up to five times in our analyses) and no evidence for reappearance (Fig. 9). The causes for evolutionary trends of this type, often postulated but seemingly difficult to establish, merit further study (Cunningham, 1999 and references therein; Danforth et al, 2003).

Two levels of explanation for these events can be sought. First, we might ask how (or whether) natural selection acts on eggshell structure. There is no direct evidence on the function of aeropyle crowns, but variation in chorion structure ought to matter. The eggshell is the interface between the oocyte or embryo and the external environment. It serves as a barrier to stresses such as predators, desiccation, and flooding, while simultaneously permitting sperm penetration, gas exchange, and hatching (Hinton, 1981). Following Hinton's (1981) suggestion that "irregular" eggshell surfaces, including aeropyle crowns, might function as part of a "plastron," or physical gill, to trap air during brief periods of flooding, we attempted to find correlations between variation in aeropyle crown structure or surface distribution and geographical or altitudinal distributions of Antheraea species (see Paukstadt et al., 2003, for relevant data), but without success.

FIGURE 9. MP and ML mappings of aeropyle crown characters, based on phylogeny of Figure 8A, with conflict in placement of A. yamamai resolved in accord with morphological classification (Table 2) and period (Fig. 8C), and with pemyi group resolved in accord with period (see Results, Fig. 8C). Total number of species in each taxon is shown in parentheses beside the corresponding node. Dashed lines denote species with known chorion structures but not sequenced. These species have been positioned on the molecular tree at the base of the lowest level morphological group to which they belong (see Table 2). MP-based character state transformations of all taxa are shown on the tree and have been tabulated in the upper-left box; variable numbers reflect multiple alternative optimizations for the cordifolia subgroup. Four states for chorion character 1 (i.e., maximal degree of aeropyle crown elaboration): A, simple aeropyle openings only; A', rudimentary swellings around aeropyle openings; AM, aeropyle mounds; AC, full aeropyle crowns. Three states for character 2 (i.e., spatial distribution of aer\opyle crowns or mounds): -reg, everywhere except micropyle region; regA, restricted to circumferential (anterior [Lef-right arrow] posterior) band; regB, restricted to circum-micropylar band; ?, character unscorable. Inferred character state changes denoted by arrows. ML mapping using the Multistate program of chorion character 1 shows significant support at 11 of the 14 internal nodes (see nodes with filled circles, based on analysis of sequenced taxa only). State assignments for the other three nodes are partially ambiguous (two out of four character states each) but always include the MP- assigned state. The transition matrix estimated by Multistate is as follows: qA = AC = 0.038863; qA = AM = 0.000001; qA = A' = 0.000168; qAC = AM = 0.086036; qAC = A'=0.040009; qAM =A' = 0.000010.

FIGURE 10. Northern analysis of developmental RNA from Hyalophora cecropia and Antheraea polyphemus. RNA was isolated from follicles of progressively older choriogenic stages (1 to 4, both species), and in the aeropyle crown (AC) and flat (F) regions of very-late- period follicles (stage 4 or later; A. polyphemus only). Probes were: 16, an aeropyle-crown-region-specific lamellar sequence, and 402, a non-region-specific lamellar sequence, both from A. polyphemus; the filler component (E2) was conspecific in each case.

Explanations other than panglossian adaptation of aeropyle crown characters are also plausible, especially given the apparent evolutionary trend toward reduction in the size and spatial extent of aeropyle crowns. Of the 11 evolutionary changes inferred for these two traits together, 8 have reduced the size or spatial distribution of aeropyle crown-like structures, whereas just 3 have increased the overall degree of surface sculpturing of the egg. Our sampling, coupled with the relative species richness of groups in the morphological classification, suggests that only about a third of the extant species of Antheraea retain the ancestral condition of an egg covered with aeropyle crowns, whereas over half the species belong to a single subgenus typified by regionalization of the aeropyle crowns. Perhaps the aeropyle crown was a "failed evolutionary experiment," only briefly and ancestrally beneficial, and is now in various stages of being lost. Or, perhaps the intermediate degree of sculpturing seen in most extant species is the pervasive optimum but was not genetically achievable except through the ancestral condition as an intermediate. A third hypothesis is that natural selection might not (strongly) distinguish among the various types of partial or complete surface sculpturing we observed, allowing the "genetic lines of least resistance" (Schluter, 1996) to set the prevailing direction of evolution, as discussed in the next section.

Developmental-Genetic Basis of Aeropyle Crown Evolution

The second level of explanation for aeropyle crown variation lies in development and genetics. Our phylogenetic results immediately yield a new perspective on studies of aeropyle crown development because they specify which inter- and intraspecific comparisons correspond to which evolutionary events. For example, comparisons between Antheraea polyphemus and outgroups such as Hyalophora cecropia bear on the question of what genetic changes produced fully developed aeropyle crowns de novo in the ancestor of Antheraea. On the other hand, comparisons between the "flat" and aeropyle crown areas of the "regionalized" species A. polyphemus, or between this and species with uniformly distributed aeropyle crowns (e.g., A. pernyi), bear primarily on the changes that secondarily suppressed aeropyle crown formation on part of the egg surface in some Antheraea species. These two events did not necessarily involve the same genetic mechanisms. With this caution in mind, we sketch out what can thus far be inferred about the number and kind of genetic changes underlying each documented step in aeropyle crown evolution.

Aeropyle crown formation in A. polyphemus has been shown to involve very late-stage synthesis and deposition of large amounts of filler proteins, coupled with a final round of lamellogenesis (Regier et al., 1993). How, specifically, does this process differ from choriogenesis in the immediately pre-aeropyle crown ancestor? Comparison to H. cecropia, in which choriogenesis is otherwise similar, has been especially illuminating regarding evolution of the "filler" component. The copy number and timing of filler gene expression are very similar in these two species (see E2 lanes in Fig. 10), but the level of expression is about 35-fold greater in A. polyphemus (Hatzopoulos and Regier, 1987). These molecular findings coincide with the microscopy observation of small amounts of filler in aeropyle channels of H. cecropia, contrasting to the localization of abundant filler in A polyphemus mainly to the aeropyle crowns, although it is also found in the aeropyle channels. Thus, the contribution of filler to the origin of aeropyle crowns was in part an example of heteroposy, a change in the production level of some substance affecting development (Regier and Hatzopoulos, 1988).

The other major element of aeropyle crown formation is a final phase of lamellogenesis. In contrast to A. polyphemus, H. cecropia does not produce any additional lamellae in the final stages of choriogenesis. Protein and mRNA analyses in A. polyphemus have identified a number of presumed lamellar sequences whose expression is restricted to very late choriogenesis in the aeropyle crown region and have shown that the aeropyle crown itself is greatly enriched in these proteins (Regier et al., 1993). In this study we probed H. cecropia for expression of an abundant aeropyle crown- specific lamellar protein in A. polyphemus, labeled 16 in Figure 10. The 16-hybridizing sequence in H. cecropia is expressed throughout choriogenesis, and its expression level does not increase at the end, unlike the 16 gene in A. polyphemus but similar to the non- region-specific lamellar control sequence (labeled 401 in Fig. 10) in both species. A reasonable interpretation is that the timing of expression of a 26-like gene (and presumably other lamellar genes as well) changed in the ancestor of Antheraea to become specifically involved in aeropyle crown formation. Thus, the lamellar component of aeropyle crowns seems to have arisen by a second type of regulatory change, heterochrony (Gould, 1977).

Comparisons between the aeropyle and flat regions of the A. polyphemus chorion have yielded clues about the genetic basis of regionalization. For 23 of 178 known chorion proteins in this species, synthesis is largely restricted to the aeropyle crown region (Regier et al., 1980). Analysis of the 5' flanking region of 8 of these 23, including multiple copies of both the 16 gene and two filler genes, identified an element common to all 8 but absent from all non-region-specific genes (Regier et al., 1993). This element probably reflects a shared functional constraint rather than common ancestry, because the regionally expressed filler genes are not part of the large chorion gene superfamily to which all lamellar protein genes belong. Functional studies have yet to be done, but this element may well represent a means for simultaneous down-regulation of multiple genes specific to aeropyle crown formation. If so, discovery of this joint regulator, and determination of whether it also accounts for other independent origins of regionalization, including those associated with aeropyle mounds, become compelling issues.

No experimental comparisons are yet available that bear directly on the mechanism of aeropyle crown reduction to the aeropyle mound or rudimentary condition. However, the narrow boundary between the aeropyle crown and flat regions of several species showing regionalization displays a range of aeropyle morphologies comparable to the variation among species, with flat regions looking essentially identical to the "rudimentary" conditions (see fig. 2b, 8, in Mazur et al., 1980). This suggests overlap in mechanism with that underlying regionalization, which might help to explain the puzzling phylogenetic distribution of aeropyle mound regionalization.

A complete genetic model of aeropyle crown evolution may help to explain not only how structures arise or disappear, but also why some evolutionary changes take place so much more readily than others. Do developmental-genetic constraints help to explain why the aeropyle crown has originated just once but has been reduced and/or regionalized so many times, or why its origin apparently involved a large quantum step, whereas its reduction occurs in a series of stages? Given the limited evidence, we restrict ourselves to a single speculation. The conventional explanation for Dollo's Law is that creation de novo of a complex structure requires numerous simultaneous independent genetic changes, whereas loss of the structure might result from loss or suppression of any one of these genes. It appears that the origin of aeropyle crowns required at least two distinct kinds of regulatory change, acting on at least two independent types of genes. Loss of either regulatory innovation alone would presumably interfere with aeropyle crown formation. Alternatively, if the aeropyle crown-forming genes come eventually to share a common regulatory sequence, a single genetic change might inhibit them all. Either way, we might expect that evolutionary reduction of the aeropyle crown would be more probable than de novo origin, other things being equal. This and other possibilities await an expanded, phylogeny-guided set of developmental-genetic comparisons among Antheraea species and between these and closely related wild silkworm genera.

A more detailed protocol of the laboratory methods, and a complete listing of primer sequences, can be freely downloaded at http://www.umbi.umd.edu/users/jcrlab/PCR_primers.pdf.

ACKNOWLEDGMENTS

JCR dedicates \this report to F. C. Kafatos for his valued mentorship. We thank K. Budiamin (Indonesia), J. Fiedler (Germany), S. Naumann (Germany), D. Suparman (Indonesia), and K. L. Wolfe (USA) for providing specimens used in this study, and Diane Shi and Cynthia Cole for technical assistance. Financial support from the National Science Foundation (DEB-0212910) is gratefully acknowledged.

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First submitted 18 December 2003; reviews returned 29 August 2004; final acceptance 29 August 2004

Associate Editor: Ted Schultz

Editor: Chris Simon

JEROME C. REGIER,1 ULRICH PAUKSTADT,2 LAELA H. PAUKSTADT,2 CHARLES MITTER,3 AND RICHARD S. PEIGLER4

1 Center for Biosystems Research, University of Maryland Biotechnology Institute, 5140 Plant Sciences Building, College Park, Maryland 20742, USA; E-mail: regier@umd.edu

2 Knud-Rasmussen-Strasse 5, D-26389 Wilhelmshaven, Germany

3 Department of Entomology, University of Maryland, Plant Sciences Building, College Park, Maryland 20742, USA

4 Department of Biology, University of the Incarnate Word, 4301 Broadway, San Antonio, Texas 78209, USA

Copyright Society of Systematic Biologists Apr 2005

Source: Systematic Biology

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