Three-dimensional reconstruction of the stomatostylet and anterior epidermis in the nematode Aphelenchus avenae (Nematoda: Aphelenchidae) with implications for the evolution of plant parasitism

Molecular phylogenies often raise questions of morphological evolution where they impose a new paradigm on classical scenarios reliant on opposing ideas of phylogeny. One pressing case of incongruence has been shown in nematodes regarding the evolution of the Tylenchomorpha, which include most species of plant parasitic nematodes, as well as many insect parasites and fungivores. With finer phylogenetic resolution based on molecular analyses, it is apparent that evolution of feeding structures for a parasitic lifestyle can no longer be explained by traditional scenarios, with which new hypotheses of relationships are incongruent. Accumulating evidence suggests that Tylenchomorpha, with a specialized stomatostylet, are sister taxon to or nested within the Cephalobomorpha (Aleshin et al., 1998; Blaxter et al., 1998; Holterman et al., 2006; Meldal et al., 2007; Nadler et al., 2006; Smythe and Nadler, 2006), which lack a stylet and instead have an open stoma characteristic of microbivores. Our understanding of morphological evolution necessarily begins with addressing homology questions. Statements of homology are confounded in these nematodes by the seemingly huge morphological leap from the relatively simple open stoma of microbivores to the complex hypodermic needle-like feeding apparatus of plant parasites. Classical hypotheses of the evolution of the stomatostylet in Tylenchomorpha considered the entire stoma of free-living nematodes to be homologous with the entire stylet in various formulations, usually following a transformation series including several other taxa as intermediates between cephalobids and tylenchids (Thorne, 1961; Andrassy, 1962, 1976; Goodey, 1963; De Grisse, 1972). These character transformations have now been challenged by revised hypotheses in light of new phylogenies, based on limited available fine structural data of the stomatostylet (Baldwin et al., 2004a). New hypotheses of relationships preclude an obvious morphological intermediate between stylet bearing tylenchids and most closely related open stoma bearing nematodes. The potential for difficulties in interpreting feeding structure homologies in these groups demands the choice of an appropriate model taxon for stylet reconstruction. Many Tylenchomorpha are assumed to be poor representatives for understanding stylet origins, because their highly specialized feeding adaptations further confound the morphological disparity between them and outgroups. Recent molecular evidence suggests that the family Aphelenchidae1, including Aphelenchus avenae, assumes a sister taxon relationship to all other non-aphelenchid Tylenchomorpha (Blaxter et al., 1998; Holterman et al., 2006; Meldal et al., 2007). Aphelenchus avenae thus occupies a phylogenetic position well suited to its use as a model system for comparison to free-living outgroups and it has been selected as a model herein. Homologies are supported by their adherence to three criteria: congruence with phylogeny based on independent characters; conjunction, or the non-existence of putative homologs together in the same taxon; and similarity (Patterson, 1982). Categories of similarity include topological, compositional, functional, developmental, and genetic (Panchen, 1994), which can serve individually as recognition criteria and tests. Hypotheses of homology for cuticular feeding structures, which in many respects are divergent between tylenchids and their closest free-living outgroups, are tested by their relationships to complex underlying tissues. These tissues are presumably the secretory source of the extracellular stylet and associated cuticle (Wright and Thomson, 1981; Endo, 1985). Identities of tissues, including syncytia and muscles, are confirmed by several lines of evidence. Their topological relationships to each other provide initial homology proposals, which can then be scrutinized based on other aspects of similarity and ultimately according to their congruence with independent phylogeny. The anterior epidermal syncytia comprise a stack of successive toroids (rings) that line the cuticle of the feeding apparatus, lips, and body wall. The identities and positions of at least some stoma elements, such as the arcade syncytia and stomatal muscles, are known to be highly conserved from reconstructions of representatives of rhabditids (Albertson and Thomson, 1975; Wright and Thomson, 1981), diplogastrids (Baldwin et al., 1997a), and cephalobids (Van de Velde et al., 1994; Baldwin and Eddleman, 1995; De Ley et al., 1995; Bumbarger et al., 2006). The arrangement of most epidermal syncytia is also conserved between representative cephalobids and rhabditids (Bumbarger et al., 2006). The numbers, positions, and interrelationships of syncytial somal extensions, which extend relatively far posteriad to include the nuclei of the syncytia, are highly conserved and confirm hypotheses of tissue identities. The relationships of epidermal syncytia to sensory structures, such as is inferred by relative locations of sensillum socket cells, are in many respects conserved and thus offer additional support to homology statements. Studies employing TEM have afforded a much clearer understanding of homologies at a more fundamental (i.e., cellular) level, as some structures are only barely or not at all visible by light microscopy. Yet even serial thin sectioning has been limited in its resolution of complex cells, with minute processes and contacts being difficult to recognize as associated with a particular cell body. Baldwin et al. (2004a) recognized a body of fine structural research of the tylenchid stylet that predates our understanding of stoma homologies in Chromadorea, but which has nevertheless stimulated questions for more sophisticated examination. In this work, morphology is analyzed using three-dimensional (3D) reconstruction of more complete serial thin sections, with the aid of digital imaging and recently developed computer software for alignment and model building and rendering. These tools have made visualization of elusive but significant features more tractable. By establishing homologies of tissues in the level of detail afforded by 3D reconstruction, this work builds upon comparably detailed reconstruction in a free-living out-group, Acrobeles complexus (Cephalobomorpha) (Bumbarger et al., 2006), as part of a larger scale laboratory effort. With respect to what is also known of corresponding tissues in the model nematode Caenorhabditis elegans (Rhabditomorpha), a more distant outgroup, we test homologies of the stylet and associated cuticular structures with specific components of the cephalobid stoma. The regions making up the stoma of cephalobids and rhabditids are the units of analysis for stomatostylet homologies herein. Stoma regions were originally defined by their apparent division, in light microscopy observations, into a series of distinct sets of cuticular thickenings or rhabdia (Steiner, 1933); however, the terminology was later expanded based on fine structure to reflect a clearer understanding of stoma components, including homologies across several microbivorous taxa (De Ley et al., 1995). Thus, three basic divisions are identified as the units of a comparative study between cephalobids and tylenchids (see Fig. 1): i) the cheilostom, which is the cuticle continuous with the body wall and secreted by underlying epidermis; ii) the gymnostom, which is an area of thickened cuticle surrounded by two syncytial arcade rings; iii) the stegostom, which is the part of the stoma surrounded by the anterior end of the pharynx, including muscle and epithelia, and characterized by a triradiate lumen, although homologies implied for stegostom components at a finer level (e.g., pro-, meso-, meta-, and telo-stegostom) between cephalobids and rhabditids have since been challenged (Dolinski et al., 1998). The goal of this study is to test whether the cheilostom, gymnostom, and stegostom are represented by readily identifiable homologs in the tylenchid stomatostylet. Fig. 1 Diagrammatic representation of stoma components of rhabditids and cephalobids, with hypotheses of homologies extended to tylenchids. Generalized rhabditid is redrawn from De Ley et al. (1995); cephalobid and tylenchid are redrawn from Baldwin et al. (2004a) ...