Syst. Biol. 46(1):145-179, 1997

EVOLUTION OF MALE TAIL DEVELOPMENT IN RHABDITID RELATED TO

DAVID H. A. FITCH Department of Biology, New York University, Room 1009 Main Building, 100 Washington Square East, New York, New York 10003, USA; E-mail: [email protected] Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 Abstract.—The evolutionary pathway that has led to male tails of diverse morphology among species of the family was reconstructed. This family includes the well- studied model species Caenorhabditis elegans. By relating the steps of male tail morphological evo- lution to the phenotypic changes brought about by developmental mutations induced experimen- tally in C. elegans, the goal is to identify genes responsible for morphological evolution. The varying morphological characters of the male tails of several rhabditid species have been described previously (Fitch and Emmons, 1995, Dev. Biol. 170:564-582). The developmental events preceding differentiation of the adult structures have also been analyzed; in many cases the origins of vary- ing adult morphological characters were traced to differences during ontogeny. In the present work, the evolutionary changes producing these differences were reconstructed in the context of the four possible phylogenies supported independently by sequences of 18S ribosomal RNA genes (rDNA). Two or more alternative states were defined for 36 developmental and adult morpholog- ical characters. These characters alone do not provide sufficient data to resolve most species re- lationships; however, when combined with the rDNA characters, they provide stronger support for one of the four rDNA phylogenies. Assuming a model of ordered transformations for multistate developmental characters generally results in greater resolution. Transformations between char- acter states can be assigned unequivocally by parsimony to unambiguous branches for most of the characters. Correlations are thereby revealed for some of the developmental characters, indi- cating a probability of a shared developmental or genetic regulatory pathway. Four of the un- equivocal character state changes on unambiguously supported branches closely resemble the phenotypic changes brought about by known mutations in C. elegans. These mutations define genes that are known to act in genetic regulatory hierarchies controlling pattern formation, differentia- tion, and morphogenesis. Although these studies are still at an early stage, these results strongly suggest that parallel studies of developmental mutants in C. elegans and of morphological and developmental evolution among related nematodes will help define genetic changes underlying the evolution of form. [Caenorhabditis elegans; character analysis; evolution of development; ontog- eny; phylogeny.]

Several long-standing controversies Achaete-Scute and scabrous) that have been among evolutionary theorists can poten- defined by developmental genetic analysis tially be addressed by including well-stud- to be important in the expression of such ied genetic model systems in phylogeneti- traits (Mackay and Langley, 1990; Long et cally representative empirical studies alv 1995). Macroevolutionary variation is (Kellogg and Shaffer, 1993). One such con- likely to arise from changes affecting such troversy concerns the question of whether developmentally important genes as well, macroevolutionary change is essentially but there is remarkably little empirical ev- saltational (i.e., fueled by mutations with a idence (Barton and Turelli, 1989; Orr and large phenotypic effect) or proceeds by the Coyne, 1992). accumulation of many changes that indi- Empirical data on the genetic regulatory vidually have a small effect on the organ- hierarchies underlying morphological (and ism (Gould and Eldredge, 1977; Charles- therefore developmental) evolution should worth et al., 1982; Wallace, 1985; Barton similarly improve our understanding of and Turelli, 1989; Orr and Coyne, 1992; how significantly developmental or other Weber, 1992). In microevolutionary studies constraints bias or limit phenotypic vari- including melanogaster, variation ability (Maynard Smith et al., 1985; Hall, in quantitative traits such as bristle num- 1992:75). Such constraints may augment ber has been directly traced to loci (e.g., homoplastic morphological changes. An 145 146 SYSTEMATIC BIOLOGY VOL. 46

understanding of how morphological char- ent alleles in interspecific transgenic ani- acters may be transformed by changes at mals for their effects on morphogenesis. genetic and developmental levels should The similarity between the effects of cer- eventually help us in building models for tain developmental mutants in C. elegans morphological character transformations and the evolved differences among nema- that would empower phylogenetic recon- tode species has been pointed out by sev- structions using such characters. eral authors (Steinberg and Horvitz, 1981, The definition of and criteria for recog- 1982; Ambros and Fixsen, 1987; Sternberg, nizing homology continue to be highly 1991; Sommer et al, 1994; reviewed by Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 controversial subjects (Hall, 1994). In mod- Fitch and Thomas, 1997). Several mutant el systems such as the nematode Caeno- screens have focused on isolating muta- elegans, the connections among ge- tions with an effect on the morphology of netic, developmental, and organogenetic the male tail (reviewed by Emmons and levels can be empirically defined. There- Sternberg, 1997). Interpretation of the phe- fore, parallel studies in genetics, develop- notypes of these mutations is facilitated ment, and evolution should help define (1) because the adult and developing stages of how morphological homology is manifest the male tail have been analyzed by elec- at the level of shared genetic and devel- tron microscopical reconstruction (Sulston opmental mechanisms (e.g., explore the bi- et al., 1980), and the origin of the constit- ological foundation for the hypothetical uent cells from largely invariant cell lin- morphostatic mechanisms of Wagner, eages has been described (Sulston and 1994), (2) how homologous morphological Horvitz, 1977; Sulston et al., 1980). Because characters are modified by evolutionary of these invariant cell lineages and small changes in (homologous or analogous) de- cell numbers, rhabditid nematodes are velopmental or genetic regulatory mecha- uniquely suited for identifying individual nisms (e.g., the morphogenetic mecha- genetic loci involved in the evolution and nisms of Wagner, 1994), and (3) how development of forms that would be diffi- homologous mechanisms may be deployed cult (or impossible) to map in organisms to form analogous or entirely different with larger tissues and variable cell lin- characters (e.g., Hall, 1995). eages. In response to this need to establish a Another advantage of the male tail for fundamental, mechanistic understanding evolutionary studies is that it is one of the of character evolution, I have initiated a most variable morphological features of long-term study of the developmental and nematodes (Chitwood and Chitwood, genetic basis of morphological evolution 1950). The systematics of many nematode designed to take advantage of the well- groups classically and currently depends studied developmental genetic model Cae- heavily on male tail characters (Chitwood norhabditis elegans. The focus of this study and Chitwood, 1950; Andrassy, 1976,1983, is the external genital specialization of the 1984; Sudhaus, 1974, 1976, 1980), many of male tail. My approach is to compare the which are analyzed in this work. Fitch and steps of the historical pathway of male tail Emmons (1995) previously compared the evolution in a group of species related to development of the male tail in C. elegans C. elegans to the functions and mutant phe- with that in several other species in the notypes of developmental genes in C. ele- family Rhabditidae (order , gans. The aim is to identify genes in which phylum Nematoda) and were able to trace mutational changes may have contributed differences in certain morphological fea- to morphological evolution. Eventually, I tures between species to differences in the will explore this possibility for the partic- rearrangement of individual cells in the ular genes identified by this "candidate developing epidermis of preceding larval gene" approach by reconstructing their stages. Such differences in cell rearrange- molecular evolution and by testing differ- ment were postulated to arise from differ- 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 147 Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021

"Eurhabditis"

*//// ////////

cr o* ^

FIGURE 1. The set of statistically supported cladograms (I-FV) inferred from 18S rDNA sequences of rhab- ditid nematodes (Fitch et al., 1995) and the strict consensus of these trees (P). Numbers designate branches that are referenced in the text. Branch 4 in trees III and IV corresponds to species group "Eurhabditis" (Sudhaus, 1976). Branch 2 in trees II and IV corresponds to Rhabditis monophyly. In tree P, O = the two nodes that have been collapsed because of uncertainty in the branching order. However, this consensus tree suggests more uncertainty than actually exists: Rhabditis blumi and Rhabditella axei are not each other's closest relatives in any of trees I-IV and Pellioditis typica is not more closely related to the Caenorhabditis group than to the Rhabditis- Rhabditella group. TABL E1. Parsimony analysis of nematode 18S rDNA sequenc e data alone or in combination with male tail character data using PAUP (Swofford, 1993) for different character sets, types, and weighting schemes.

Alternative trees1 Branches in MP tree % bootstrap support)d I II III IV Anal- MP ( ysis Character setd s.b treec 1 2 3 4 5 Rank Length CI Rank Length CI Rank Length CI Rank Length CI 1 A (1.0 un) -1.258 I 100 74 98 87 100 1 1,644 0.726 2 1,650 0.721 4 1,656 0.717 3 1,652 0.720 2 A (ng 1.0 un) -1.258 I 100 63 93 70 100 1 1,508 0.710 3 1,514 0.705 4 1,517 0.703 2 1,513 0.706 3 A (1.0 gp) -0.996 II 99 74 92 94 75 4 141 0.730 1 139 0.771 40 145 0.659 10 143 0.692 4 A (0.8 un) -1.260 I 100 66 96 84 100 1 7,048 0.725 2 7,074 0.721 4 7,101 0.717 3 7,083 0.720 5 A (ng 0.8 un) -1.261 I 100 65 92 72 100 1 6,449 0.709 3 6,476 0.704 4 6,489 0.702 2 6,472 0.705 6 A (0.5 un) -1.261 I 100 73 93 84 100 1 2,116 0.725 2 2,124 0.720 4 2,133 0.715 3 2,127 0.719 7 C (1.0 un) -1.242 HI 100 94 67 50 100 2 771 0.721 19 780 0.705 1 769 0.724 5 774 0.715 8 C (ng 1.0 un) -1.237 III 100 94 67 50 100 2 735 0.711 19 744 0.696 1 733 0.715 5 738 0.706 9 C (0.8 un) -1.255 III 100 92 65 41 100 2 3,313 0.722 17 3,352 0.706 1 3,308 0.724 4 3,329 0.715 10 M (un) -1.463 II, 100 100 39s 38 65 259 65 0.768 4 56 0.915 221 64 0.782 11 57 0.912 U-W 11 M (or) -1.301 Yh 100 42 51 100 48 573 89 0.544 49 77 0.642 461 88 0.551 35 76 0.652 12 M (un) A (1.0 un) -1.298 II 100 64 92 75 100 3 1,709 0.728 1 1,706 0.730 5 1,720 0.720 3 1,709 0.728 13 M (un) A (ng 1.0 un) -1.300 IV, II 100 70 92 40j 100 3 1,573 0.713 2 1,570 0.715 5 1,581 0.707 2 1,570 0.715 14 M (or) A (1.0 un) -1.263 II 100 87 86 66 100 3 1,733 0.712 1 1,727 0.716 8 1,744 0.705 2 1,728 0.716 15 M (or) A (ng 1.0 un) -1.263 IV 100 88 85 50 100 3 1,597 0.696 2 1,591 0.701 7 1,605 0.691 1 1,589 0.702 16 M (un) C (1.0 un) -1.291 IV 100 62 90 74 100 4 836 0.725 4 836 0.726 2 833 0.731 1 831 0.734 17 M (un) C (ng 1.0 un) -1.288 IV 100 62 90 74 100 4 800 0.718 4 800 0.718 2 797 0.723 1 795 0.726 18 M (or) C (1.0 un) -1.241 IV 100 82 85 86 99 6 860 0.692 5 857 0.696 5 857 0.696 1 850 0.706 19 M (or) C (ng 1.0 un) -1.237 IV 100 82 85 86 99 6 824 0.684 5 821 0.688 5 821 0.688 1 814 0.698 20 M (un) C (0.8 un) -1.306 IV 100 69 93 77 100 4 3,638 0.728 3 3,632 0.730 2 3,628 0.732 1 3,614 0.737 21 M (un) C (ng 0.8 un) -1.304 IV 100 69 93 77 100 4 3,482 0.720 3 3,476 0.722 2 3,472 0.724 1 3,458 0.729 22 M (or) C (0.8 un) -1.250 IV 100 89 77 85 100 6 3,758 0.689 3 3,737 0.696 5 3,748 0.692 1 3,709 0.705 23 M (or) C (ng 0.8 un) -1.245 IV 100 89 77 85 100 6 3,602 0.681 3 3,581 0.688 5 3,592 0.684 1 3,553 0.697 24 M (un) -1.625 II, 100 99 46 44 70 187 53 0.795 4 47 0.921 125 52 0.814 11 48 0.897 u-w 25 M (or) -1.302 100 100 48 40 74 144 67 0.614 12 60 0.700 115 66 0.625 9 59 0.714 k u 26 M (un) -1.757 II, 100 99 39' 38 70 217 49 0.775 4 43 0.912 151 48 0.795 11 44 0.886

u-w Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 September 30 on guest by https://academic.oup.com/sysbio/article/46/1/145/1685502 from Downloaded TABLE 1. Continued.

Alternative tree; Branches in MP tree (% bootstrap support)d I II III IV Anal- MP ysis Character set" treec 1 2 3 4 5 Rank Length CI Rank Length CI Rank Length CI Rank Length CI 27 M (Olf -1.303 U 100 99 38 49 81 109 59 0.633 10 53 0.721 80 58 0.646 6 52 0.738 28 M (un)i C (1.0 un) -1.292 III 100 65 89 71 100 3 824 0.728 4 827 0.723 1 821 0.733 2 822 0.731 29 M (or? C (1.0 un) -1.278 IV 100 60 90 79 100 3 838 0.708 6 840 0.704 2 835 0.712 1 833 0.715 30 M (un)) C (0.8 un) -1.309 III 100 56 89 64 100 3 3,578 0.730 4 3,587 0.727 1 3,568 0.734 2 3,569 0.733 31 M (or) C (0.8 un) -1.293 IV 100 64 90 78 100 3 3,648 0.707 4 3,652 0.705 2 3,638 0.710 1 3,624 0.715 32 M (un>m C (1.0 un) -1.358 IV 100 66 91 73 100 5 869 0.730 3 866 0.734 2 865 0.736 1 862 0.740 " A = all positions included in the 18S rDNA alignment (alignment 3 of Fitch et al., 1995); C = only highly conserved, unambiguously aligned positions of the 18S rDNA alignment (containing very few gaps [indels]); M = male tail characters, excluding the nonindependent adult characters 2-5, 7, and 8 (characters 31-34 were also excluded); ng = indels ignored; un = character transformations assumed to be unordered; or = multistate character transformations assumed to be linearly ordered; gp = indels only were counted. Positions involved in stem secondary structures (Caenorhabditis elegans model; see Fitch et al., 1995) in the 18S rRNA were weighted 1.0, 0.8, or 0.5 times as much as loop positions. Because integer weights are required by PAUP, relative weights were increased to achieve the appropriate ratio (e.g., a ratio of 0.8 was achieved by weighting stems by 4 and loops by 5). This integer weighting therefore results in greater total tree lengths. When male tail characters were combined with 18S rDNA characters (analyses 12-23), male tail characters were weighted as much as the 18S rDNA loop characters. b Skewness of the distribution of tree lengths for the set of all possible unrooted trees. All values are significant at a level greater than 99% confidence limit (Hillis and Huelsenbeck, 1992). However, the phylogenetic signal may be limited to only a few clades (e.g., in analyses 10 and 11, only two branches show high bootstrap support). c Topology shown in Figure 1 (except as noted) of the most-parsimonious (MP) tree(s) found in an exhaustive search. d Percentage of 2,000 bootstrap replications supporting a particular branch in the MP tree (or the first MP tree listed). e Listed for each of the four alternative trees shown in Figure 1 are the rank (i.e., number of trees with a length less than or equal to that of the tree evaluated), the length (i.e., total number of substitutions and indels inferred by parsimony; if included, indels were counted as one substitution, regardless of lejigth), and the consistency index (CI, excluding "uninformative" characters, such as autapomorphies). 'Four MP trees were produced by the unordered male tail characters, one of which was tree II in Figure 1. The other three trees may be designated in parenthetical notation, where clades are designated as nested groups in parentheses with numbers designating corresponding branches, as follows: tree U, (1:(C. briggsae, C. elegans)5:(4:(3:(2:(R. myriophila, R. blumi)P. typica)Rh. axei))(T. palmamm, Pe. strongyloides)Y, tree V, ((((C. briggsae, C. elegans)({R. myriophila, R. blumi)P. typica))Rh. axei)(T. palmarum, Pe. strongyloides)Y, tree W, ((((C. briggsae, C. elegans)Rh. axei)((R. myriophila, R. blumi)P. typica))(T. palmarum, Pe. strongyloides)). Tree U is congruent with the phylogeny proposed by Sudhaus (1976). * Although the bootstrap value for this branch was 39%, a bootstrap value of 41% was obtained for branch 3 of tree U. h Tree Y (not shown in Fig. 1) was only produced by ordered male characters and does not correspond to any classification system or phylogeny proposed on the basis of either morphological or molecular evidence. In parenthetical notation, where clades are designated as nested groups in parentheses, this tree may be described as follows: (5:(4:(R. myriophila, R. blumi)(l:(C. briggsae, C. elegans)3:(2:(P. typica, Pe. strongyloides)Rh. axei)))T. palmarum). ' Although the bootstrap value for this branch was 40%, a bootstrap value of 60% was obtained for an alternative branch supporting a clade containing the two Rhabditis species, Rhabditella axei, and the Caenorhabditis species (i.e., as in tree II). i Because changes in characters 31-33 are each strongly correlated with changes in character 30 (see text and Appendix 5), parsimony analyses were repeated with characters 31- 33 excluded. k Because changes in character 34 may also be correlated with changes in character 30 (see text and Appendix 5), parsimony analyses were repeated with characters 31-34 excluded. ' Although the bootstrap value for this branch was 39%, a bootstrap value of 46% was obtained for branch 3 of tree U.

m Each male tail character that was included had twice the weight of a nucleotide character. Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 September 30 on guest by https://academic.oup.com/sysbio/article/46/1/145/1685502 from Downloaded 150 SYSTEMATIC BIOLOGY VOL. 46 ences in the expression or specificity of cel- (Sudhaus and Schulte, 1988) Fitch, Bugaj- lular recognition and adhesion proteins. Gaweda, and Emmons, 1995. All of the In the character analysis presented here, strains are wild type except C. elegans I have described these differences in mor- strain CB4088, which carries the him- phology and development as discrete char- 5(el490) mutation, and C. briggsae strain acter states and reconstructed their evolu- PB102, which carries the mih-l(bdlO2) mu- tionary changes in the context of the tation (S. Baird, pers. comm.); both of these phylogenetic relationships of the species. mutations result in a high incidence of The species relationships were previously males of wild type morphology in selfing Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 inferred from nucleotide sequences of 18S populations of these hermaphroditic spe- ribosomal RNA (rRNA) genes (rDNA; Fig. cies. All of the strains used in this work 1; Fitch et alv 1995). I further examined have been made available from the Cae- how sensitive the multistate character re- norhabditis Genetics Center (University of constructions are to possible ontogenetic Minnesota). Cultures were maintained at constraints on the ordering of changes. 20°C on NGM plates preseeded with Esch- These well-defined molecular and mor- erichia coli OP50 (Brenner, 1974). Except for phological character sets also allow an op- Teratorhabditis palmarum (maintained by portunity to determine whether or not the monthly transfers), worms were cryogeni- morphological and molecular data are con- cally preserved as described for C. elegans gruent and if so whether or not data com- (Sulston and Hodgkin, 1988). bination can in this case help resolve phy- logenetic ambiguities. Phylogenetic Analyses These reconstructions provide informa- PAUP 3.1.1 (Swofford, 1993) was used to tion not only about the polarity of char- estimate phytogenies from the Fitch et al. acter state changes but also about the min- (1995) 18S rDNA sequence data (EBI align- imum number of evolutionary steps ment accession number DS19607) alone or underlying particular character state dif- in combination with the male tail character ferences. By combining this information data presented in this work (see Appen- with existing knowledge about the roles of dices 1, 7). Details of the various character particular genes or genetic pathways in sets, character weights, and character pattern formation or morphogenesis, spe- types used are presented in Table 1. When cific hypotheses can be formulated for the male tail character data were used in these role of particular genes and genetic parsimony analyses, the nonindependent changes in male tail evolution. characters were excluded (see Table 1). Multistate male tail characters were as- MATERIALS AND METHODS sumed to be either unordered or linearly ordered (Tables 1-3; see Appendix 2). Nematodes and Culture Conditions Nematode strains used in this work have Reconstruction of Character State been described previously (Fitch and Em- Transformations mons, 1995; Fitch et al., 1995): PB102 = Morphological and developmental data (Dougherty and Ni- used in this study were taken from previ- gon, 1949) Dougherty, 1955; CB4088 = Cae- ous work (Fitch and Emmons, 1995) and norhabditis elegans (Maupas, 1899) Dough- used to define characters and character erty, 1953; EM435 = Rhabditis myriophila states as discussed in Appendix 1. All pos- Poinar, 1986; DF5010 = Rhabditis blumi sible evolutionary character state transfor- Sudhaus, 1974; DF5006 = Rhabditella axei mations were reconstructed on each of the (Cobbold, 1884) Chitwood, 1933; DF5026 four alternative trees by parsimony using = Pellioditis typica (Stefanski, 1922) An- MacClade 3.04 (Maddison and Maddison, drassy, 1983; DF5019 = Teratorhabditis pal- 1992) with the character states defined in marum Gerber and Giblin-Davis, 1990; Appendix 6 and the data matrix shown in DF5022 = Pelodera strongyloides dermatitica Appendix 7. Many changes could be re- 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 151 constructed unequivocally with respect to an ancestral condition (see Panchen, 1994: both state change and branch (i.e., such a 28). In contrast to Owen's application of the change remained on the same single term, however, the rhabditid archetype has branch for all possible most-parsimonious an explicit developmental basis (Fig. 2). reconstructions and all four alternative An understanding of the development of trees); only these unequivocal changes are male tail characters is particularly relevant listed in Table 2. Details concerning the to understanding their homologies and treatment of particular characters are pre- evolution. For example, assigning ray ho- sented in Appendices 1, 5, 7, and 8. To de- mologies, as reflected in this case through Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 termine the relative costs (i.e., effects on common developmental origins, is impor- the parsimony score) of assuming partic- tant for reconstructing evolutionary ular states at ancestral nodes (see Table 3), changes to individual rays and thus to the alternative states were fixed at certain pattern of rays as a whole. Previous at- nodes using MacClade. tempts to identify ray homologies (e.g., Sudhaus, 1974, 1976) were insufficient RESULTS AND DISCUSSION (e.g., they misidentified particular rays or mistook phasmids for rays) because they Development of the Rhabditid Male Tail: described patterns only in adults (appro- Characters and Ray Homologies priate tools for developmental analysis The eight nematode species studied here were unavailable). Appendix 1 therefore represent six genera of one of the largest presents a brief description of the devel- families of free-living nematodes, Rhabdi- opment of the male tail as requisite back- tidae, and were chosen in part because ground for understanding the evolution of they present a range of male tail morphol- the male tail characters studied in this ogies. Most of the morphological and de- work. For example, my assignments of ray velopmental features that are considered homologies are based on the specific pat- here have been described in detail else- tern of relative positions in which the ray where (Fitch and Emmons, 1995). These cells originate in the lateral hypodermis features are associated with the external (Fig. 2a). This specific pattern is symple- genitalia present in the posterior region of siomorphic for the species studied and the male. The most prominent of these fea- suggests homologous relationships be- tures are an acellular fan extending bilat- tween rays whose cells originate at the erally outwards from the body and a set same relative position (Fitch and Emmons, of nine bilateral pairs of sensilla, called 1995; Appendix 1). The assignment of ray rays, lying within the fan with their distal homologs with respect to the archetype tips opening to the exterior at various (Fig. 2) would probably have been regard- points. An archetypal arrangement of ed by Owen as a case of general homology these features is depicted in Figure 2. (see Panchen, 1994:36). However, my defi- In this paper, I reserve the term archetype nition of ray homologs in different taxa as for the hypothetical, least-differentiated having the same relative topological ori- state (i.e., the default state that would re- gins during development is perhaps more sult from the absence of ray cell migrations similar to von Baer's notion of homology after the cells originated in the lateral hy- being rooted "in the potential expressed podermis) and the term ground state for the by shared ontogenetic primordia" (Riep- hypothetical ancestral state. My use of the pel, 1994:94). These shared primordia are term archetype in the sense of a Platonic, determined according to their relative po- idealized generalization that is not actual- sitions as formulated by Remane (1952; ized in any taxon is somewhat similar to Sudhaus and Rehfeld, 1992:70-76) as a cri- Richard Owen's (1848) use of the term in terion of homology (adumbrated by Geof- his depiction of an idealized vertebrate ar- frey's [1818] "principle of connexions"). chetype and contrasts with Darwin's (1859: Regardless of how homology itself is de- 435) subsequent application of the term to fined, the symplesiomorphic pattern of ray 152 SYSTEMATIC BIOLOGY VOL. 46 Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021

3 4

stage hours Rn (d) " 30 I

L3 R/7.a

Rn.p (hyp)

35 L4 RAJA RrtB R/TSt

ray cells FIGURE 2. Archetype of rhabditid male tail development; view of the left lateral surface. Anterior is to the left, posterior is to the right. Thick lines represent the body outline. Ph = phasmid; se = body seam; hyp7 = main body hypodermal syncytium. (a) The boundaries of ray and hypodermal cells in the left lateral tail hypodermis are depicted for an archetypal early L4 male rhabditid after the ray cell lineages are completed. Thin lines represent cell boundaries. The groups of ray cells that arise as clusters of four cells each are numbered; the Rn.p hypodermal cells are designated individually. The relative positions in which ray cells and their associated Rn.p hypodermal cells originate in the lateral hypodermis are highly conserved (symplesiomorphic) in the rhabditids studied (Fitch and Emmons, 1995). (b) The boundaries of ray and hypodermal cells in the left lateral tail hypodermis are depicted for an archetypal late L4 male rhabditid. Thin lines represent cell bound- aries. For each cluster of four ray cells shown in a, one cell undergoes programmed cell death and the two neuronal cells sink below the hypodermal surface, leaving only the structural cell at the hypodermal surface (the single numbered cells), (c) Thin lines represent external features of the archetypal adult male tail. The rays are numbered. In the archetype, rays 5 and 7 open on the dorsal surface and the others open at the edge of the fan. If the ray structural cells remained at the same positions in which the ray cells originated, this hypo- thetical "default" pattern of rays in the archetypal adult would result from male tail morphogenesis. This archetype thus provides criteria for assigning hypothetical ray homologies, as denoted by the numerals near clusters of ray cells at early L4 (a), ray structural (Rnst) cells at late L4 (b), and adult rays (c). Ray patterns in the species represented in Figure 3 therefore result from shifts in the positions of the ray cells after they are born in the lateral hypodermis (Fitch and Emmons, 1995). (d) A typical ray cell lineage in Caenorhabditis elegans. At least in C. elegans, the four cells of each ray cluster are lineally related: one cell undergoes programmed cell death (X), and the two neuronal cells (RnA and RnB) sink below the hypodermal surface during L4 develop- ment, leaving only the structural cell (Rnst) at the surface. 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 153 cell origins provides a basis for comparing separating Teratorhabditis palmarum and ray development in the different species Pelodera strongyloides from the other species and for defining characters and character by parsimony using partial 18S rDNA se- states. quences from several ascaridoid species as I have divided the varying male tail fea- an outgroup (Fitch et al., 1995) and more tures of the eight species in the study recently using 18S rDNA sequences from group into a series of 36 characters, each representatives of several other groups of which assumes two or more character within the order Rhabditida as well as an states (Appendices 1 and 6). The distribu- anciently diverged adenophorean nema- Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 tion of character states among the species tode (Fitch and Thomas, 1997). The phy- is given in Appendices 1 and 7. Characters logenetic uncertainties represented by the 1-10 concern adult morphology, and char- set of alternative trees depicted in Figure acters 11-36 are developmental. A more 1 involve only two points (designated by detailed description of the characters and the open circles on tree P, the strict con- character states is provided in Appendices sensus): (1) either Pellioditis typica diverged 1 and 2. slightly before Caenorhabditis diverged Adult characters 2, 3, 5, 7, and 8 are de- from the Rhabditis species (R. blumi and R. pendent upon developmental characters myriophila, the latter referred to previously 25, 11, 12, 21, and 22-24, respectively. For as Rhabditis sp. br) and Rhabditella axei (i.e., example, the degree of adult fan extension as in trees I and II) or P. typica forms a (character 3) is ontogenetically related to monophyletic "Eurhabditis" species group the amount of overall retraction during L4 with Rhabditella axei and the Rhabditis spe- (the last larval stage) morphogenesis (char- cies (as in trees III and IV); (2) Rhabditis acter 11), and the displacement of the myriophila could be more closely related to openings of rays 1, 2, and 4 to the dorsal Rhabditella axei than to Rhabditis blumi or ventral surfaces of the adult fan (char- (trees I and III) or may be more closely acter 8) is correlated with the position of related to R. blumi (trees II and IV). the ray structural cell in the lateral hypo- To provide a baseline for comparing re- dermis of the L4 larva (characters 22-24). sults from analyses including the male tail Where I have used male tail characters in characters, I reanalyzed the 18S rDNA data phylogenetic analyses, the dependent set alone. Parsimony analysis of the entire adult characters have been excluded. 18S rDNA data set results in one most-par- simonious (MP) tree, tree I (Fig. 1); the oth- Phylogenetic Analysis: A Case for er three trees are next in rank (Table 1, Combining Data analysis 1). I also examined the effects on Previously (Fitch et al., 1995), 18S rDNA the four trees of (1) indels (gaps intro- sequences were used to infer a set of four duced into the sequences to allow align- alternative species phylogenies (Fig. 1) that ment), (2) nonindependence at stem posi- could not be statistically rejected in pair- tions (potentially paired bases that may wise parsimony tests (Templeton, 1983; undergo compensatory changes to main- Felsenstein, 1993), log-likelihood ratio tests tain secondary structures in rRNA), and (Kishino and Hasegawa, 1989; Felsenstein, (3) variable regions, which include posi- 1993), or ordinary least-squares tests tions that change more rapidly, have more (Rzhetsky and Nei, 1992, 1993). Relative to indels, and bear a higher probability of the Fitch et al. (1995) analysis, "C. vulgar- nonhomologous nucleotide comparisons is" and "C. remanei" (now C. remanei vul- than conserved regions. garis and Caenorhabditis sp. n., respectively; When indels are removed, the same MP W. Sudhaus, pers. comm.) were excluded tree is obtained as when they were includ- from the present character analysis be- ed, but bootstrap support drops for the cause they are essentially identical devel- clades defined by branches 2-4 (Fig. 1, tree opmentally and morphologically to C. ele- I; Table 1, analysis 2 compared with anal- gans. The root was placed on the branch ysis 1); the rank of tree IV is also in- 154 SYSTEMATIC BIOLOGY VOL. 46 creased. When indels are considered alone tree IV could be the "true" tree, such that (Table 1, analysis 3) it becomes clear that both "Eurhabditis" and Rhabditis represent indels are major contributors (94% of boot- monophyletic groups. Statistically, howev- strap replicates) to support for the branch er, the molecular data alone cannot distin- (branch 4, tree II, Fig. 1) that separates Pel- guish among the four alternative trees in lioditis from the other "Eurhabditis" taxa Figure 1 (Fitch et al., 1995). {Rhabditis and Rhabditella) and Caenorhab- Although assumptions of ordered ver- ditis. These effects are due to indels in vari- sus unordered models for male tail char- able regions, because removing the few in- acter state transformations yield different Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 dels in the conserved regions does not MP phytogenies, the bootstrap support is affect the topological results (even boot- very low for all but two clades, Caenorhab- strap percentages are unaffected; cf. anal- ditis and Rhabditis (Table 1, analyses 10 yses 8 and 7, Table 1). [100% support for branches 1 and 2, re- Possible nonindependence at stem posi- spectively] and 11 [100% support for tions does not have a strong effect on the branches 1 and 4, respectively]). A high analysis. When nucleotide characters likely level of bootstrap support (99-100%) is to be involved in stem structures (C. ele- maintained for branches 1 and 2 even gans model; Ellis et al., 1986) are down- when characters 31-34 are excluded (Table weighted by either 20% (as suggested by 1, analyses 24-27; characters 31-34 are Dixon and Hillis, 1993; Table 1, analyses 4 probably correlated). Because of the strong and 5) or 50% (as suggested by Wheeler support for the Rhabditis clade, the consis- and Honeycutt, 1988; Table 1, analysis 6), tency indices (CIs) for trees II and TV are the MP tree is unchanged, as are the rela- substantially higher than those for trees I tive ranks of the four trees. Also, the boot- and IQ. Thus, if Rhabditis myriophila is strap values for different branches fluctu- more closely related to Rhabditella axei than ate only slightly (Table 1, cf. analyses 4 and to Rhabditis blumi, then there has been sub- 6 with analysis 1, and analysis 5 with anal- stantial homoplasy for these morphologi- ysis 2). cal characters (even more than for the mo- When the more variable regions are ex- lecular characters, if ordered states are cluded (Table 1, analyses 7-9), there is assumed). Furthermore, character recon- greater relative support for the inclusion of structions show that there are no potential Pellioditis with Rhabditis and Rhabditella synapomorphies (from the male tail data into a monophyletic "Eurhabditis" clade. set) that could support the monophyly of That is, tree HI becomes the MP tree. This Rhabditis myriophila and Rhabditella axei but topology is also supported when stems are that several synapomorphies unequivocal- down-weighted (Table 1, analysis 9), al- ly support Rhabditis monophyly (i.e., the though bootstrap support for the "Eurhab- open bars in Fig. 3). I therefore favor the ditis" clade (branch 4 of tree HI, Fig. 1) is view that Rhabditis is not paraphyletic with not strong. respect to Rhabditella. These analyses suggest that when ac- The insufficient bootstrap support for count is taken of characters that are likely clades other than Caenorhabditis and Rhab- to contribute the majority of homoplastic ditis when male tail characters are used changes (and thus to the greater portion of alone and the inability of the 18S rDNA the error in the phylogenetic inference), the data to significantly resolve the two points "Eurhabditis" clade is given greater sup- shown on tree P (Fig. 1) suggest that the port. Based on morphological data, Sud- two data sets are complementary. The haus (1976) suggested that Rhabditis could male tail data do not provide significant be paraphyletic; however, Rhabditis has bootstrap support for any clade not dis- never been considered to be paraphyletic played in the set of alternative rDNA trees. with respect to Rhabditella. On the basis of That is, the conflicts in the results from the similar parsimony results (and additional different data sets (i.e., as manifest in the analysis), Fitch et al. (1995) postulated that different MP trees) appear only in portions 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 155

of the trees where character support is male tail characters is to slightly decrease weak. This is one of the most appropriate the ranks of trees I, II, and HI relative to instances in which data sets should be tree IV and to increase bootstrap support combined (de Queiroz, 1993). I have there- for the Rhabditis and "Eurhabditis" clades fore excluded the nonindependent male in tree IV (Table 1, cf. analyses 14, 15, 18, tail characters and combined the data sets 19, 22, 23, 29, and 31 with analyses 12, 13, (Table 1, analyses 12-23, 28-32). 16, 17, 20, 21, 28, and 30, respectively). Combining the data augments support Bootstrap support decreases only for the for tree IV. Whereas tree IV was never the Rhabditis-Rhabditella branch (branch 3 of Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 MP tree when either data set was consid- tree IV). This decrease is due at least in ered alone, it became the MP tree for the part to the extreme posterior positioning combined data set (Table 1, analyses 12- of the ray 4 cell cluster during morphogen- 23), except when rDNA variable regions esis in Khabditella axei, a reversal in tree IV and indels were included (Table 1, analy- that has a high parsimony cost under the ses 12 and 14) or when an unordered mod- assumption of ordered transformations. el was used with characters 31-33 exclud- These results sustain the prediction of ed (Table 1, analyses 28 and 30). Slowinski (1993) that minimally connected Changing assumptions about the rDNA (linearly ordered) characters increase phy- characters in the combined set yields sim- logenetic resolution relative to maximally ilar results as when the rDNA characters connected (unordered) characters. were considered alone. First, excluding in- Taken together, the phylogenetic analy- dels in the variable regions resulted in in- ses summarized in Table 1 strongly sug- creased support for inclusion of Pellioditis gest that tree IV is likely to be the best rep- in a "Eurhabditis" clade, as the parsimony resentation of the phylogenetic history of rank of tree IV increased (Table 1, cf. anal- these species. yses 13 and 15 with analyses 12 and 14, respectively). (The few indels in the con- Reconstruction of Unambiguous, Unequivocal served regions have no topological effect; Transformations tree ranks and bootstrap values are iden- Because the main goal of this work was tical when analyses 17, 19, 21, and 23 are to reconstruct the evolutionary changes to compared with analyses 16,18, 20, and 22, see if underlying genetic changes could be respectively.) Second, excluding the vari- suggested and targeted for later molecular able regions (analyses 16-23) resulted in study, I was interested in the least ambig- stronger support for a "Eurhabditis" clade uous, least equivocal set of reconstructions (e.g., comparing analyses 16 and 18 with possible. (I have reserved the terms ambig- analyses 12 and 14 shows that tree IV be- uous and equivocal to refer respectively to comes the MP tree instead of tree II); boot- the uncertainty in the branching order in a strap support for "Eurhabditis" (branch 4 cladogram and the uncertainty about the of tree IV) also increases (cf. analyses 17 branch on which a transformation occurs; and 19 with analyses 13 and 15, respec- uncertainty about the states in a transfor- tively). Third, down-weighting nucleotide mation will be specifically described as characters likely to be involved in forming such.) Using MacClade (Maddison and stem secondary structures (and thus could Maddison, 1992) with the male tail data undergo interdependent compensatory matrix (Appendix 7), character state trans- changes) results in slightly greater boot- formations were reconstructed for the in- strap support for the monophyly of both ternodes of all four possible cladograms Khabditis (branch 2 of tree IV) and "Eurhab- under the assumption that multistate char- ditis" (branch 4 of tree TV; cf. analyses 20- acters were unordered. This assumption is 23 with analyses 16-19). predicted to lead to a conservative esti- Finally, the most interesting effect of im- mate of the number of unequivocal trans- posing the constraint of linearly ordered formations because resolution is generally transformations on appropriate multistate lower than under the assumption of a lin- 156 SYSTEMATIC BIOLOGY VOL. 46 early ordered (minimally connected) TABLE 2. Unequivocal transformations recon- transformation model (Slowinski, 1993). Of structed for all four possible nematode cladograms in Figure 1. Transformations are listed for branches the 62-71 total transformations (the total numbered on the consensus tree (tree P, Fig. 1). Char- number depending on the tree topology), acters and character states are designated as in Ap- 26 could be reconstructed (for 25 of the 36 pendix 6. The reconstruction was made under the characters) that were unequivocal and oc- model of unordered transformations for multistate characters. Transformations that become equivocal for curred on the same unambiguous branch at least one tree under the ordered model are noted. in all four possible trees (Table 2). These The ordered model results in two additional unequiv- transformations were therefore deemed the ocal reconstructions over all four topologies: 14: c —» Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 most reliable representations of historical d and 15: c —» d on branch 5. changes; I selected from this pool the evo- lutionary changes that I have targeted for Branch no. Character no. State change further study. 1 la a -» ba a Although an ordered model does seem 2 7a a -> b to provide a higher phylogenetic resolution 3 3 a —> b 4 a —> b for the male tail characters (only one MP 11 a —> b tree results; Table 1, analysis 11), evolu- 4 20 a ->bb tionary reconstructions for some characters 5 20 a —> cb actually become less resolved, depending 26 a -> b on the tree. For example, the primitive state 27 a -> b for several of the transformations listed in 30 d -^bc Table 2 becomes uncertain under an or- 31 d ->bc 32 d ->bd dered model, resulting in an equivocal as- 34 b -» ce signment for some of these transforma- 35 d->b tions in particular trees. Only two 7 1 a -> b transformations that were equivocal under 2 a —> b 8 a —> b an unordered model became unambigu- a ously and unequivocally resolved under 9a a —> b 13 a —> b an ordered model. Only the transforma- 22 a -» b tions that did not change under different 23 a -» b models were targeted for further study. 24 a —» b A large portion of the unambiguous, un- 25 a -> b equivocal transformations listed in Table 2 29 a -> b 8 5a a -» ba occur in the Caenorhabditis lineage, sug- 12* a -> ba gesting that C. elegans may be a particular- a Chosen for further genetic study. ly suitable model for understanding the b If ordered, the primitive state could be either a or b for genetic basis of evolutionary change, es- trees I and II. pecially the aquisition of novel features. c If ordered, the primitive state could be either c or d for trees I, II, and IV. According to Table 2, an ancestor of this d If ordered, the primitive state could be either c or d for acquired a number of distinctive trees I, II, and IV but becomes unequivocally c for tree III (thereby imposing an unequivocal c —» d change on branch 6 characteristics, including a closed fan po- for tree III). sitioned more posteriorly (characters 1, 2, e If ordered, the primitive state could be either b or c for 25: a —» b), fusion of the terminal tail tip trees II, III, and IV. cells (13: a —> b), dorsoventral displace- ment of the openings of rays 1, 2, and 4 (8, 22-24: a —> b), and extension of the R9.p a novel tapered morphology for ray 6 (9: a cells dorsally to meet at the dorsal midline —> b); atavistic mutations result in a prim- (29: a —> b). Of particular significance to itive cylindrical morphology, pointing to understanding the genetic basis of some of genes required for the tapered morpholo- these evolutionary transformations are at- gy that therefore may have been involved avistic mutations in C. elegans. For example, in the evolution of this morphological nov- an ancestor of Caenorhabditis also acquired elty. 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 157

The distribution of the other unambig- relative cost of assuming that the archetyp- uous, unequivocal transformations listed al form (state a for all of these characters) in Table 2 further suggests why the male is ancestral to the rhabditid species stud- tail characters support few relationships: ied here is generally higher than for any these changes are autapomorphic, except alternative hypothesis (Table 3). If trans- for the 10 changes supporting the Caeno- formations are assumed to be unordered, rhabditis clade (branch 7 of tree P, Fig. 1) the parsimony cost of assuming that the and the 2 changes supporting the Rhabdi- archetype is ancestral for most trees is tis-Rhabditella clade (branch 8; these 2 small (5-11 additional changes over 10 Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 changes are actually developmentally cor- characters need to be postulated, depend- related and were counted as a single ing on the tree; Table 3). In most cases, change in the phylogenetic analyses). Al- however, the cost of assuming the arche- though not shown in Table 2 (which only typal state at the root node is higher than shows changes on branches shared among assuming some other state (the archetypal all four cladograms), there are no potential state is not even represented for characters synapomorphies (from pools of either 35 and 36). If transformations are assumed equivocal or unequivocal changes) sup- to be ordered, the archetype is the single porting a Rhabditis myriophila-Rhabditellaworst hypothesis at most characters and axei relationship (trees I and III). However, requires 18-21 additional changes relative there are seven potential synapomorphies to the MP reconstructions (Table 3). The that are unequivocal on trees II and IV archetype is therefore unlikely to be rep- (nine under the ordered model) support- resentative of the most recent rhabditid an- ing Rhabditis monophyly (e.g., see Fig. 3). cestor. That is, the least-differentiated state Reconstruction of the Male Tail in the Most (i.e., the archetype, Fig. 2) is unlikely to Recent Common Ancestor represent the evolutionary ground state (ancestral state) for the Rhabditidae. This The archetype (Fig. 2), in which ray cells conclusion may apply more generally and do not shift position from where they orig- may be underappreciated in many com- inate and Rw.p cells do not fuse with their parative developmental studies that fail to neighbors, provides one hypothesis for the incorporate an explicit phylogenetic frame- ground state of Rhabditidae that can be work. evaluated phylogenetically. Accordingly, the ray positions in this archetypal ances- Table 3 also shows that the effect of as- tor would appear relatively evenly spaced, suming an ordered model of transforma- as in Figure 2c. Evolution from this arche- tions generally increases resolution (i.e., re- typal ancestor would have proceeded by duces the number of MP reconstructions of addition of steps during the ontogeny of ancestral states), but not for all characters. the rays between the time the ray cells For example, assuming an ordered model originate and ray morphogenesis. In these increases resolution over all trees for char- steps, the ray cells would move to new (de- acter 15 and over some trees for characters velopmentally derived) epidermal posi- 14, 16, 33, and 36. Whereas a single MP tions from where they originated; the re- reconstruction for characters 30-32 results sulting ray pattern would have displaced under an unordered model, these charac- and unevenly arranged rays. Sudhaus ters become less resolved in all the trees (1976:74-76) suggested a similar primitive under an ordered model. In cases where Urform with evenly spaced rays, although resolution is increased by assuming an or- he did not regard this form as represen- dered model, there are no states in the MP tative of the most recent ancestor of the reconstruction that were not in the MP re- Rhabditidae. construction under the unordered model. By fixing the states of the multistate de- The single exception is character 33, where velopmental characters corresponding to state b becomes the single MP ancestral ray positioning and Rn.p cell fusions, the state under an ordered model but was not 158 SYSTEMATIC BIOLOGY VOL. 46

TABLE 3. Consequences of assuming particular ancestral states and a linear model of ordered transforma- tions for appropriate nematode multistate morphological characters. I-IV and P refer to dadograms in Figure 1.

Relative parsimony costf" Ances- Unordered Ordered tral Character no. state" I II in IV P I II m IV P Ray cell associations 14. ray 1 a 0 0 0 0 0 1 1 1 1 1 b 1 1 0 0 0 1 1 0 0 0 c 0 0 0 0 0 1 1 0 0 0 Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 d 0 0 0 0 0 2 2 1 1 1 15. ray 2 a 1 1 1 1 1 2 2 2 2 2 b 0 0 0 0 0 0 0 0 0 0 c 0 0 0 0 0 0 0 0 0 0 d 0 0 0 0 0 1 1 1 1 1 16. ray 3 a 0 1 0 1 1 3 2 3 3 3 b 0 0 0 0 0 1 1 1 1 1 c 0 1 0 0 0 1 1 0 0 0 d 0 0 0 0 0 1 1 0 0 0 17. ray 4 a 0 1 0 1 1 5 5 5 5 5 b 0 1 0 1 1 3 3 3 3 3 c 0 1 0 1 1 2 2 3 2 2 d 0 0 0 0 0 2 2 3 2 2 18. ray 5 a 0 0 0 0 0 1 1 1 1 1 b 0 1 1 1 1 0 1 1 1 1 c 0 1 1 1 1 1 2 2 2 2 Rn.p cell fusions 30. Rl.p a 1 2 1 2 2 7 6 7 6 6 b 1 1 1 1 1 5 4 5 4 4 c 1 2 1 1 1 4 3 4 3 3 d 0 0 0 0 0 4 3 5 3 3 31. R2.p a 1 2 1 2 2 7 6 7 6 6 b 1 1 1 1 1 5 4 5 4 4 c 1 2 1 1 1 4 3 4 3 3 d 0 0 0 0 0 4 3 5 3 3 32. R3.p a 1 2 1 2 2 7 6 7 6 6 b 1 1 1 1 1 5 4 5 4 4 c 1 2 1 1 1 4 3 4 3 3 d 0 0 0 0 0 4 3 5 3 3 33. R4.p a 0 1 0 1 1 3 3 3 3 3 b 1 2 1 1 1 2 2 2 2 2 c 0 0 0 0 0 2 2 3 2 2 34. R5.p a 1 1 1 1 1 6 5 6 5 5 b 0 0 0 0 0 4 3 4 3 3 c 1 1 1 1 1 5 3 4 3 3 d 1 1 1 1 1 7 4 5 4 4 e 2 1 2 1 1 9 6 7 6 6 35. R6.p b 1 1 1 1 1 3 3 3 3 3 c 2 2 2 2 2 2 2 2 2 2 d 0 0 0 0 0 1 1 1 1 1 36. R7.p b 0 0 0 0 0 1 1 2 2 1 c 1 1 0 0 1 1 1 1 1 1 d 1 1 0 0 1 2 2 1 1 2 Total cosr= 5 11 5 11 11 20 18 20 21 21 a Different states were fixed for the root node using MacClade (Maddison and Maddison, 1992). b For each tree (P is a "soft" polytomy consistent with all four alternative trees), the number of additional changes that must be postulated if the root node is assumed to be a particular state, and transformations are either unordered (i.e., can occur directly from any state to any other state) or ordered (i.e., can only occur in the linear order where a is the most developmentally primitive state according to the archetype depicted in Fig. 2). The results are shown for all multistate characters except characters 20 and 21, for which a natural order for the alternative states was not apparent. c Total increase in parsimony score if state a is fixed at the root node for all characters listed except 35 and 36. Under the ordered model, the cost shown is the number of additional steps required over the lowest parsimony score assuming ordered characters, not over the lowest parsimony score assuming unordered characters. 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 159 an MP reconstruction under the unordered 14-16 and 36, respectively) in the common model (Table 3). ancestor. This analysis at the root node exempli- These five equivocally reconstructed fies features of the character reconstruc- characters define a significant morpholog- tions at other nodes. Specifically, the major ical-developmental division of the species qualitative differences in the character re- in the study group that corresponds to the constructions made for a particular tree earliest phylogenetic divergence (Fig. 3). under ordered as opposed to unordered For all the species on the left branch lead- models are in the numbers of step changes ing from the root (i.e., the Caenorhabditis Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 assigned to particular branches and in the and "Eurhabditis" clades, Fig. 3), the phas- resolution of nodal states or branch assign- mid is located posteriorly, ray 1 contacts ments. Rarely are there conflicting sets of Rl.p and lies near the anterior boundary MP reconstructions of nodal character of the fan, ray 2 contacts R2.p, ray 3 con- states between the different transformation tacts R3.p and is anterior to ray 5, and R5.p models. and R7.p fuse independently with other cells or remain unfused. For both of the The Best Reconstruction: Ancestral Features species on the right branch leading from and Developmental Constraints the root (i.e., T. palmarum and Pelodera strongyloides), the phasmid is located lat- Tree IV is currently my best hypothesis erally, ray 1 contacts R3.p and lies near ray for the relationships of the taxa studied. 2, ray 2 does not contact R2.p, ray 3 con- Specifically, although the phylogenetic tacts R6.p and is posterior to ray 5, and analysis of the 18S rDNA data alone could R5.p and R7.p fuse to each other. This dis- not significantly resolve the branching or- tribution of character states is consistent der at the two nodes marked in tree P (Fig. with the phylogeny and suggests that the 1), (1) the trends in the 18S rDNA data corresponding apomorphic changes oc- suggest that "Eurhabditis" is likely to be curred on one of the two branches leading monophyletic, and (2) the male tail char- from the root node. Because these charac- acters augment support for Rhabditis ters are equivocal at the root node, I cannot monophyly (as well as "Eurhabditis" confirm the prediction of Sudhaus (1976: monophyly to some extent). 73-75) that the rhabditid ancestor had (1) Therefore, to obtain a more focused pic- 2 rays lying precloacally (the positions of ture of male tail evolution, I reconstructed the anterior 3 rays [characters 14-16] could the evolutionary transformations specifi- not be unequivocally reconstructed) and cally for tree IV (only unequivocal changes (2) had 10 "rays" on each side (if the phas- are shown in Fig. 3). For this tree, 44 of the mid position [character 6] were anterior, total 63 transformations could be unequiv- between rays 6 and 7, then the ancestor ocally reconstructed, assuming unordered would appear to have 10 rays) (for a fur- multistate characters. Using these unequiv- ther comparison of these results with those ocal transformations, a reasonably well-re- of Sudhaus, see Appendix 4). solved picture of the ancestor at the root An ordered transformation model helps node can be reconstructed (Fig. 3). The to resolve many changes but also results only characters that have equivocal states in lower resolution for other changes and at the root node are 6,14-16, and 36. Thus, some nodal states. Specifically, assuming parsimony could not unequivocally pre- an ordered model results in the resolution dict whether the most recent common an- of 11 transformations that were equivocal cestor of the study group had a phasmid under the unordered model (Fig. 3). How- placed posteriorly or laterally (character 6). ever, five unequivocal transformations un- Likewise, parsimony could not unequivo- der the unordered model become equivo- cally predict the ray cell associations (and cal under the ordered model (Fig. 3). hence the positions of the rays) for rays 1- Moreover, there is no longer a single MP 3 or the fusion behavior of R7.p (characters state at the root node for ray cell position 534 12 5346 Ph T. palmarum Pe. strongyloides (14: (15:, Rhabditis §77:d->c 118: a-»c Ancestor: 20: a->b (36: c->b) *28: a->b (36: c/d-»b) 130: d-»b 13/: d->b

t *33: c-»a b/c-»e) b/c^a) 34: Rhabditis / Rhabditella Ancestor 35: d-»b

Transformation on an unambiguous branch is unequivocal for all trees in Fig. 1 Transformation on an unambiguous branch is unequivocal for this tree but equivocal on at least one other tree in Fig. 1 Transformation on an ambiguously supported branch is unequivocal on this tree

Unequivocal change on this tree only if an ordered model of transformation is assumed Homoplasy Ancestor at the Root Node: Ph?> Primitive state may differ for this unequivocal change if ordered transformations assumed Transformation becomes equivocal if an ordered model of transformation is assumed Transformation is absent if an ordered

46 model of transformation is assumed Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 September 30 on guest by https://academic.oup.com/sysbio/article/46/1/145/1685502 from Downloaded 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 161 characters 17 and 18 and Rn.p cell fusion 1990) is also inappropriate in this case be- characters 30-34, although the alternative cause the kinds of changes appear to be states of these developmental characters do correlated. My test of dependence is, how- not affect the inferred adult male tail mor- ever, based on a method similar to that of phology of the ancestor at the root node. Maddison (1990) for estimating the prob- There is very little homoplasy for the abilities of changes occurring in particular male tail morphological characters in this branches. I arbitrarily picked one of the tree. There are only two pairs of parallel characters as the "independent" character changes among the unequivocal transfor- (i.e., character 30) and asked what the Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 mations (Fig. 3) and three cases of parallel probability is for a change in a "depen- or reversal changes among the equivocal dent" character (e.g., 31) occurring in the characters (not shown). The major differ- same branch as that marked by the inde- ence in the parsimony scores between trees pendent character transformation (further I or III and II or IV (Table 1, analyses 10 details of this method are presented in Ap- and 11) results from the large number of pendix 5). homoplastic changes (parallels or rever- The results of these tests demonstrate sals) that must be postulated to maintain that characters 30-32 change coordinately Rhabditis paraphyly (trees I and III, Fig. 1); in three different branches of tree IV (i.e., these changes are apomorphic for a Rhab- those defining the taxa Caenorhabditis, ditis clade in trees II and IV (Fig. 3). Rhabditis, and Teratorhabditis palmarum). For Although there is little homoplasy in each of characters 31 and 32, the probabil- tree IV, there is an interesting case of char- ity of observing, by chance alone, three dif- acter dependence. This dependence in- ferent changes in the three branches distin- volves the characters (30-33) for the fu- guished by changes in character 30 is sions of the anterior four Rn.p cells and 6/837 (i.e., of 837 different ways to distrib- possibly the character (34) for R5.p fusions. ute three different changes in the clado- Because these are multistate characters, the gram, in only 6 do these changes fall on concentrated-changes test of Maddison the branches distinguished by changes in (1990) could not be used to demonstrate character 30) = 0.0072, a significantly this dependence. A binary recoding of small probability. (The assumptions in this multiple states (as suggested by Maddison, case are actually quite conservative, re-

FIGURE 3. Reconstruction of only the unequivocal character state transformations for the rhabditid nematode cladogram IV of Figure 1 (branch lengths are arbitrary) under the assumption of unordered transformations; no optimizations for equivocal changes are shown. For each species and for several hypothetical ancestors at internal nodes are shown schematics of late L4 and adult male tails (corresponding to the stages shown in Figs. 2b, 2c) The early L4 stage shown in Figure 2a is conserved in all the extant taxa and is thus inferred at all nodes. The upper drawing for each taxon depicts the positions of the ray structural cells (small numbered circles), the phasmid (Ph), and the Rn.p and other hypodermal cells in the left lateral hypodermis. Dashed lines at cell boundaries represent cell fusions. The lower drawing for each taxon shows a left lateral view of the adult male tail with the distinctive adult patterns of the rays (homologs numbered according to the arche- type in Fig. 2). For the hypothetical ancestor at the root node, dashed lines represent alternative positions for the phasmids. The positions of the phasmids and rays 1-3 in this ancestor could not be unequivocally recon- structed (see Appendix 8). See Appendix 6 for definitions of characters (italicized numbers) and associated states (a-e). Only the unequivocal transformations for these characters are shown. Branch assignments could not be resolved for any state changes for characters 6, 19, or 36; these characters are not represented on the tree. Although most of the transformations reconstructed are unequivocal on corresponding branches of the other possible cladograms shown in Figure 1 (heavy bars), some are not (hatched bars); other transformations are unequivocal on this cladogram but occur on a branch that is not supported in all four trees in Figure 1 (open bars). The effects of assuming an ordered model of transformations are also indicated: only 5 transfor- mations become equivocal (J), 1 becomes unsupported altogether (§), the primitive state could or does become different for 12 (t), and 11 additional transformations become resolved (unequivocal, thin bars with transfor- mation listed in parentheses). Only four transformations are homoplastic (*). C. = Caenorhabditis; R. = Rhabditis; Rh. = Rhabditella; P. = Pellioditis; T. = Teratorhabditis; Pe. = Pelodera. 162 SYSTEMATIC BIOLOGY VOL. 46 quiring that the same state, d, is primitive weakly) than a Rhabditis myriophila-Rhab- for all three changes.) Moreover, of the six ditella axei clade (branch 2 of tree III, anal- alternative ways that the three different yses 28 and 30) under two conditions: (1) changes can be distributed in the three transformations are assumed to be ordered branches, only one is identical to the par- or (2) rDNA stem positions are weighted ticular pattern of changes in character 30. less than loop positions (Table 1, cf. branch The probability of the observed identity 2 bootstrap values and tree III and IV between the distribution patterns of lengths in analyses 29 and 31 with those changes in character 30 and those in char- in analyses 28 and 30). In these analyses, I Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 acters 31 or 32 (see Fig. 3) is thus 1/837 = have conservatively weighted each male 0.0012, an even smaller (more significant) tail character equal to a single nucleotide probability. If I remove the constraint that character (instead of equally weighting the the primitive state must be the same for two sets of data, for example); doubling each change (but keep the total number of the weight of the male tail characters re- changes in the cladogram the same and as- sults in markedly increased bootstrap sup- sume unordered transformations), then the port for the Rhabditis clade and for tree IV three changes in characters 31 or 32 would as the MP tree (Table 1, cf. analyses 32 and occur in the three branches at a probability 28). Thus, even when male tail characters of 6/2184 = 0.0027. Similarly, a distribu- that are probably interdependent are ex- tion of changes (as observed in Fig. 3 for cluded, support is still augmented for tree characters 31 and 32) with the identical IV more than for the alternative trees. pattern as character 30 would be predicted at a probability of only 1/2184 = 0.0005. CONCLUSIONS AND SPECULATIONS As detailed in Appendix 5, a different The main objective of this work was to situation exists for characters 33 and 34, seek correlations between the elementary which cannot share an identical pattern of (i.e., minimally reconstructed) steps of changes with character 30. Although morphological evolution and the changes changes in character 33 are also strongly in morphology brought about by known correlated with changes in character 30, mutations. By this means, I hope to gen- changes in character 34 may or may not be, erate hypotheses regarding genetic loci depending on the assumptions used (see that could have been involved in morpho- Appendix 5). I did not attempt to deter- logical evolution. In this paper, I have mine probabilities under the complicating taken the first step toward this goal by re- assumptions of ordered transformations constructing the evolutionary pathway and unequal branch lengths. leading to diverse male tails of eight spe- Because changes in characters 31-33 cies in the nematode family Rhabditidae. (and possibly 34) are correlated with This reconstruction must be considered changes in character 30 (and with each preliminary for at least two reasons. First, other), they were excluded from the data the number of species in the study group set in a parsimony reanalysis (Table 1, is small. The number of described species analyses 24-31). When the male tail char- in the three main clades studied (i.e., Cae- acters are considered alone under unor- norhabditis, "Eurhabditis," and Pelodera-Ter- dered or ordered models, bootstrap sup- atorhabditis, not yet representative of the port remained high (99-100%) for a entire family Rhabditidae) is around 150 Rhabditis clade (analyses 24-27). However, (Sudhaus, 1976, 1991), and the number of with the combined data set I was unable existing species in these groups is un- to distinguish between trees III and IV: un- doubtedly far higher (Sudhaus, 1991). der the unordered model, the parsimony Therefore, I have examined but a fraction scores of these trees differ by a single of the whole, and the study group could change (analyses 28 and 30). However, the be unrepresentative. Second, the branches Rhabditis clade (branch 2 of tree IV, analy- of the tree are long; the 18S rRNA genes ses 29 and 31) is better supported (albeit of the different genera studied here are 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 163 about eight times more divergent than the nutiae that attain significance for classifi- 18S rRNA genes of tetrapod classes (Fitch cation, in this case for correlation between et alv 1995). It is not known whether these a mutation and an evolutionary change). long branches are due to the great age of Mutations in C. elegans define genes whose these groups and/or to rapid evolution. molecular products are known or may be Small sample size and long branch determined and where further information lengths affect both the molecular phylog- regarding interacting genes, molecular eny and the character reconstruction upon functions, and a possible evolutionary role which my conclusions regarding the evo- may be sought. Although only a small Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 lutionary pathway are based. The molec- number of male tail genes have as yet been ular phylogeny for these species is unre- studied in C elegans, and in spite of the solved at two nodes (Fig. 1) that are fairly fact that this reconstruction involves few deep (Fitch et alv 1995), although including species, the parallel analysis of mutations the morphological data does aid resolution and evolution has already resulted in the (Table 1). Also, the actual pathway of mor- identification of candidate genes for fur- phological evolution that gave rise to the ther study. Furthermore, I have been able species considered here need not have to infer the polarity of the evolutionary been the same as the hypothetical MP changes (Table 2), providing further pre- pathway(s) I have deduced. Because the dictions about the kinds of genetic changes branches are long, there is no reason to be- that may have occurred in these genes. lieve that multiple transformations be- Two of the four evolutionary changes tween character states have not occurred that are similar to those in mutants oc- along each branch. It is, however, also curred on terminal branches (Table 2, probable that evolutionary changes in transformations in characters 10 and 7). rhabditid morphology are slow in compar- The tendency in C. briggsae of ray 3 to ison with rates of genetic divergence; the move posteriorly towards ray 4 and to fuse Caenorhabditis species studied are geneti- with ray 4 is an autapomorphy of this spe- cally as divergent as different mammalian cies (character 10: a -» b). This change orders (Fitch et al, 1995; Fitch and Tho- from the ancestral condition of the Elegans mas, 1997), yet they are morphologically species group resembles the phenotype of nearly indistinguishable. This slower rate mab-5 gain-of-function {gf) mutations in C. of morphological than of genetic evolution elegans (Chow and Emmons, 1994). The is supported by the fact that there are few mab-5 gene encodes a homeodomain-con- morphological homoplasies (Fig. 3). The taining putative transcription factor simi- reconstruction presented here is therefore lar to Drosophila Antennapedia (Costa et al., useful as a phylogenetic hypothesis to be 1988); these genes are probably ortho- tested by analysis of additional species. logues (Schubert et al., 1993). In G elegans, mab-5 helps to determine the separate Candidate Genetic Changes Underlying identities of the rays required for their Evolutionary Changes in Male Tail proper assembly into a species-specific ar- Morphology rangement in the fan (Chow and Emmons, Although this analysis is at an early 1994). In a single known mab-5 gf mutant stage, it already suggests genes and genet- (Hedgecock et al., 1987), the identity of ray ic changes that are promising candidates 3 appears to be reassigned to that of ray 4, for further study in an evolutionary con- giving a ray 3-4 fusion phenotype identi- text. Of the unambiguous, unequivocal cal to that seen in C. briggsae (Chow and transformations that I have reconstructed Emmons, 1994). A mutation with a similar (Table 2), four are similar to the pheno- effect, at a mab-5 homolog or at another lo- types of mutants presently known in C. ele- cus involved in specifying the identity of gans; a couple of these are similar to the ray 3, may have occurred along the lineage mutants in intriguingly trifling ways (tri- leading to C. briggsae. fling sensu Darwin [1859:417]: shared mi- A second autapomorphy that is similar 164 SYSTEMATIC BIOLOGY VOL. 46

in trifling ways to a C. elegans mutant phe- mary function are not known, although notype is the loss of ray 8 in Rhabditis blumi cloning of this gene is in progress (Ying (Table 2; Fig. 3, character 7: a -> b). In C. Yang and Fitch, unpubl.). A similar low- elegans, loss of the ability to express the ge- ered-function change could thus have pro- neric ray sublineage is associated with mu- duced the leptoderan tail tips in the Rhab- tations at the lin-32 locus (Zhao and Em- ditis-Rhabditella clade. Such a change could mons, 1995). This gene encodes a putative have become fixed in an ancestral popu- transcription factor of the basic helix-loop- lation, as indicated by the fact that neither helix (bHLH) family of genes and has both mutant allele of lep-1 demonstrates signif- Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 molecular and genetic properties similar to icant loss of siring capacity or mating ef- the Achaete-Scute genes of Drosophila. Ex- ficiency with regard to the wild type in pression of lin-32 is both necessary and preliminary mating tests (Fitch, unpubl.). sufficient to allow expression of neuroblast The appearance of a morphologically cell fate for all the rays; hence, regulation specialized ray 6 along the lineage leading of its action is central to the development to Caenorhabditis (Table 2; Fig. 3, character of rays in the posterior region of the male. 9: a -» b) may have involved changes in a Known mutations in C. elegans affecting genetic pathway presently under study in the level of wild type lin-32 gene product C. elegans. Perhaps more than the previous or the amino acid sequence of the lin-32 examples, this change appears to represent gene product can affect the probability of the evolutionary appearance of a novel fea- generating some rays more than they can ture. The C. elegans genes known to act in affect the probability of generating other a pathway leading to the unique morphol- rays (Zhao and Emmons, 1995). In fact, ray ogy of ray 6 include (1) daf-4 (S. Baird, 8 is always the most susceptible to these pers. comm.), which encodes a TGF-p re- mutations. That is, although loss of the ceptor homolog (Estevez et al., 1993), (2) other rays is variable in all three of the egl-5 (Chow and Emmons, 1994), a hom- known alleles of lin-32, ray 8 is lost 100% eobox-containing transcription factor of of the time, even in the weakest allele, the HOM-C/Hox gene cluster that is a ho- el926 (C. Zhao, pers. comm.). Thus, a mu- molog of Drosophila AbdominalB (Wang et tation in a lin-32 homolog, or one that af- al., 1993), (3) mab-18 (Baird et al., 1991), a fects regulation of a lin-32 homolog, may homolog of mammalian Pax-6 (Zhang and have occurred in the lineage leading to Emmons, 1995), which is a homeobox-con- Rhabditis blumi, resulting in the specific taining transcription factor of a different loss of ray 8. family (Chalepakis et al., 1993), and (4) The appearance of the leptoderan tail tip mab-21 (Baird et al., 1991), which encodes along the lineage leading to Rhabditis and a protein with a novel sequence (Chow et Rhabditella could have resulted from a loss- al., 1995). Mutations in these genes cause of-function (//) mutation or a change in loss or gain of the unique morphology nor- regulation affecting a homolog of the lep-1 mally associated with ray 6 in Caenorhab- gene of C. elegans (Table 2; Fig. 3, character ditis, and they cause adjacent subsets of 5: a -» b, character 12: a —> b). Postulated rays to fuse; thus, they control not only the // mutations at the lep-1 locus result in a morphology but also the identity of ray 6 leptoderan tail tip in C. elegans that closely and possibly of other rays (Chow and Em- resembles (in trifling ways at the cellular mons, 1994). Because mutations in these level) the small tail tip in Rhabditis (Fitch, genes cause ray fusions, they do not pre- unpubl). Specifically, these alleles prevent cisely mimic the ray 6 phenotype of non- the retraction and fusion of the tail tip hy- Caenorhabditis species. Nevertheless, evo- podermal cells in a manner identical to the lutionary changes in any of these genes, as failure of retraction and fusion of tail tip well as in others in the pathway known hypodermal cells in at least one Rhabditis (Baird et al., 1991) and others yet to be de- species (Fitch, unpubl.). The molecular na- scribed, might have contributed to the ap- ture of the lep-1 gene product and its pri- pearance of novel ray 6 morphology in 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 165

Caenorhabditis. Transgenic experiments to kind of change this was require determi- examine the effect of expressing the wild nation of the branch assignment and thus type C. elegans mab-18 gene in species direction of this character transformation. without a specialized ray 6 are one ap- The addition of appropriate outgroup taxa proach to testing these hypotheses. should facilitate such a determination. As more species are added to the tree, the polarity of other character changes Developmental Constraints in the Evolution of may be determined, resulting in more and the Male Tail better hypotheses about the underlying ge- The correlations among characters 30, Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 netic changes. For example, the position of 32, and 32, which represent different cell the phasmid (character 6) relative to the fusion events involving the R(l-3).p cells, three posterior rays (rays 7-9) differs be- do not arise trivially from the requirement tween the two groups of species that are that cell fusions must involve at least two the first to diverge in the tree shown in cells (which thus share the same fate by Figure 3 (i.e., the Teratorhabditis-Pelodera definition). First, there is no a priori con- clade and the clade containing the other straint that a fusion must involve more species). In C. elegans, asymmetric division than two cells. Second, the cell fusions in- of the T blast cell produces two cell lin- volving R(l-3).p are likely to result from a eages, the anterior one giving rise to rays developmental pathway that is shared in 7-9 and the posterior one producing the the course of the aquisition of cellular phasmid (Sulston et al., 1980). The anterior identity by the three individual Rn.p cells position of the phasmid relative to rays 7- during their ontogeny. To a somewhat less- 9 in Teratorhabditis and Pelodera (and several er extent, R4.p and possibly R5.p (charac- other species groups) could result from re- ters 33 and 34) may share this pathway. versed polarity of the asymmetric T-cell One interpretation of the correlation division. Such polarity differences have among characters 30-32 is that it repre- been observed in other nematode species sents a developmental constraint in the comparisons (Steinberg and Horvitz, 1981, sense that the coordinated evolution of 1982). Lowered-function mutations in the these characters results from a shared de- gene lin-44 result in the failure to control velopmental or genetic regulatory hierar- the polarity of the T-cell division such that chy rather than from identical selective 68-79% of T-cell divisions are reversed rel- forces acting on independent characters. ative to wild type, generally resulting in Selection is an unlikely explanation for the displacement of phasmid cells anteri- these coordinated changes because there is orly (Herman and Horvitz, 1994). The no obvious morphological consequence of LIN-44 gene product is a cell-signaling the fusion or lack of fusion among these molecule homologous to vertebrate Wnt Rn.p cells (as demonstrated, for example, and Drosophila wingless proteins and is pro-by the variation in fusions without corre- duced by the tail tip cells (Herman et al., sponding variation in adult features such 1995). Downstream of lin-44 in the genetic as fan morphology; Fig. 3). However, a pathway controlling T-cell division asym- pleiotropic genetic regulatory hierarchy is metry is lin-17, required for establishing likely to influence the identity of several of 7the asymmetry itself (Sternberg and Hor- these developmentally similar Rn.p cells. vitz, 1988; Herman and Horvitz, 1994; Such a regulatory hierarchy involves Hox Chamberlin and Sternberg, 1995). It is genes in determining ray cell identities, for therefore possible that the difference in example (Chow and Emmons, 1994), al- phasmid position between Teratorhabditis- though no phylogenetically coordinated Pelodera and the other taxa resulted from a changes could be detected among the rays change in the regulation of the LIN-44 cell- of the species studied here. signaling pathway or a change in the com- Another type of developmental con- petence or response of the T cell to the straint may be predicted from the analysis LIN-44 signal. Hypotheses about what of ray development. In this case, the poten- 166 SYSTEMATIC BIOLOGY VOL. 46 tial constraint involves a strict limitation opmental evolution among related nema- imposed on the possible variation of ray todes provide a powerful model for defin- cell positions in the lateral hypodermis by ing developmental and genetic changes by the topology of the Rn.p cell boundaries. which diverse forms may arise through That is, the apical ends of the ray cells evolution. Specifically, the C. elegans model could well be restricted to the boundaries has provided candidate genes that can be of the Rn.p cells (the positions of which targeted for futher investigation into their appear to be determined by the cell divi- roles in the evolution of male tail morpho- sions producing the Rn.p cells). According- logical diversity. Although it is obviously Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 ly, evolutionary shifts in ray positions are too early to use this preliminary data to expected to be saltational, not gradualistic. settle the long-standing controversies, In Caenorhabditis species, the apparent ex- these preliminary results do provide a ba- ception, rays 1, 2, and 4 appear to move sis for constructive speculation and predic- slightly away from these boundaries and tion. are engulfed by specific Rn.p cells or the Is macroevolution gradualistic or salta- hyp7 syncytium (Fitch and Emmons, tional at the genetic level? At least in the 1995). The mechanism of this engulfment Rhabditidae, where differences can be de- is unknown but suggests a limited poten- scribed at the level of single cells, evolu- tial for ray cell patterns to escape from the tionary changes in form may be discrete topological constraints of the Rn.p bound- enough to suggest the underlying genetic aries. changes. This does not imply that the mi- Beyond this topological constraint, selec- croscopic level of analysis allowed by this tion could play a strong role in shaping ray system makes it a poor model for studying pattern if ray pattern influences copulatory the evolution of quantitative characters, a behavior and mate choice, for example. Al- criticism, for example, that recently has though most described species in the been leveled (I think mistakenly) against Rhabditidae are gonochoristic (with males the experimental evolution of cell size in E. and females), one model that derives an coli (see Mlot, 1996). Rather, the potential optimum /male sex ratio to elucidate the genetic regulatory net- predicts that the C. elegans mating system works that govern development provides will select for males with promiscuous an exceptional tool for identifying genetic copulatory behavior and largely "disinter- changes with large or subtle effects. Be- ested" (selective?) (Hed- cause single genetic changes can be pos- gecock, 1976). Although different copula- tulated for some of the evolutionary tory behaviors depend to a large extent on changes in the male tail, I predict that the presence of particular rays (reviewed many evolutionary changes in morphology by Emmons and Steinberg, 1997), it is not will have resulted mainly from changes at clear how specific patterns of rays influ- single loci, perhaps accompanied by ence copulatory behavior or what behav- changes at other loci with much lower iors might be selected by hermaphrodites. magnitude effects. This prediction agrees The ability to manipulate these patterns with population genetic analyses of several genetically in C. elegans should allow us to quantitative traits where the underlying address such questions and thus to empir- genetic changes involve few loci and differ ically discriminate between adaptive and markedly in both magnitude and quality nonadaptive explanations for ray patterns. of their effects (Mackay and Langley, 1990; Tanksley, 1993; Long et al, 1995). Because Toward a Developmental and Genetical of the hierarchical organization of the ge- Understanding of Macroewlutionary Change netic pathways and networks controlling Although these studies are at an early development, it can additionally be pre- stage, the results strongly suggest that par- dicted that many morphological changes allel studies of developmental mutants in result from changes at loci that occupy rel- C. elegans and of morphological and devel- atively high levels in this hierarchy, thus 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 167 preserving the developmental integration is the sharing of some pathways sufficient of complex structures while allowing co- for homology (e.g., because Pax-6 / eyeless ordinated morphological change (Futuy- gene patterning of eyes is shared between ma, 1986:439-440). vertebrates and flies, should these very dif- How significant are developmental or ferent eye organs be considered homolo- other constraints likely to be in biasing gous)? Could we then talk about percent variation? Although a clear picture is not homology as the number of morphostatic yet available of the entire developmental pathways shared out of the total pathways and genetic regulatory pathways leading forming the character, or are only mecha- Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 to ray patterning, two different kinds of nisms homologous (despite the fact that constraints are already suggested: (1) as in many mechanisms are themselves assem- the case of the R(l-3).p cells, cells may blies of parts that might be individually share a developmental or genetic regula- substituted)? Clearly, elucidating the infor- tory hierarchy and therefore evolve coor- mation pathways themselves is what is im- dinately (i.e., independent development is portant to understanding the elements of limited), or (2) as in the case of the cells macroevolutionary change, not converging that give rise to the rays, cell positions may to any particular conceptual definition of be constrained by the topology of neigh- homology. Developmental genetic models boring cells. In both cases, die develop- such as C. elegans allow information path- mental constraints do not result from in- ways to be defined; a phylogenetic frame- ternal selection against a potential variant work for C. elegans (to which the present but from the low probability of getting work is meant to contribute) will allow the variants that escape coordinate regulation evolution of these pathways to be studied. in the first case or topological constraint in Although a comprehensive definition of the second. Because the power of selection a homology concept is not required for is limited by variation, such developmental these studies, the character comparisons constraints could cause significant bias in necessary for reconstructing evolutionary the evolution of form. However, the rare changes require operational tools to define variant that allows escape from such a con- the same character in different species straint (as in the patterning of rays 1, 2, (e.g., the criteria for suggesting homology and 4 in Caenorhabditis) could allow signif- as opposed to homology itself). However, icant opportunity for selection to shape use of these operational tools (which can new forms. Thus, mapping out the devel- be refined as more phylogenetic and mech- opmental and genetic regulatory networks anistic information is accumulated) does in model systems such as C. elegans is like- not result in a foregone conclusion about ly to enable the identification of potential which features are actually homologous; constraints. phylogenetic analysis using character as- What is homology? The volumes that semblages can reveal convergences as well have been written on this subject alone at- as apomorphies. In the case of ray pattern test to the complexity of a problem that evolution, mechanistic information about cannot be solved here. But perhaps the ray development (formulated as a hypo- quest for a definition of homology that fits thetical archetype) allowed an operational all cases is somewhat misdirected. Mor- hypothesis for ray comparisons, thus facil- phological characters are assembled from itating evolutionary reconstructions. multiple information pathways (including Among the underlying genetic changes both genetic networks and epigenetic that I have hypothesized for the evolution mechanisms). Does homology require that of ray positioning are those that actually all information pathways underlying a modify ray identity, e.g., mab-5(gf). Thus, character be shared through descent, or even if rays can change their identities (ho- does it only require that some of those mologies?) into those of other rays within pathways (i.e., the morphostatic ones; Wag- the series, underlying genetic changes can ner, 1994) be shared through descent? If so, potentially be identified. 168 SYSTEMATIC BIOLOGY VOL. 46

SUMMARY build an ordered model of character state Combining independent morphological transformations, which does not necessar- and molecular characters increases phylo- ily depend on an archetypal state being an- genetic resolution in the case of the Rhab- cestral. This difference between archetype ditidae (see Table 1). Conflicts between the and ancestor emphasizes the importance MP trees resulting from separate analyses of including phylogenetic analysis in com- of the male tail and of rDNA data sets are parative studies (a typological view is not an evolutionary one). The present analysis

due to low resolution of some clades in- Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 stead of nonindependence of characters cannot distinguish whether the archetype within one of the data sets. The data sets represents an ancestral state in a very early are complementary with regard to phylo- nematode (Urform), the reconstruction of genetic information (e.g., the male tail which will require the inclusion of much characters strongly support Rhabditis more divergent nematode taxa. monophyly whereas the rDNA data can- Reconstruction of the unequivocal trans- not distinguish between Rhabditis mono- formations on tree IV reveals little homo- phyly or paraphyly). Specifically, analysis plasy in the male tail characters but does of the combined male tail and rDNA char- uncover an interesting case of correlated acters results in augmented support for characters. The homoplasy in the morpho- tree IV (which was never the MP tree when logical-developmental characters is sub- the different data sets were analyzed sep- stantially less than in the rDNA characters arately). (see the CIs in Table 1), suggesting their potential usefulness in phylogenetic anal- Assuming a model of ordered as op- ysis (although the number of these char- posed to unordered transformations for acters will obviously be much smaller than the multistate male tail characters results the potential number of nucleotide char- (in combination with the rDNA data) in a acters). The developmental fates of some of higher resolution of the phylogeny (i.e., the Rn.p hypodermal cells are phylogenet- branching order) and in higher resolution ically correlated. Because selection on mor- for most (but not all) character transfor- phology is unlikely to have been a factor mations. Specifically, support for tree IV in this correlation, these cells probably always increases relative to that for the share a genetic or developmental regula- three alternative trees (I—III, Fig. 1) under tory hierarchy. an ordered as opposed to an unordered Most importantly, parallel consideration model (Table 1). Although most character of the evolutionary reconstruction and C. transformations become more highly re- elegans developmental genetics allows us to solved (with respect to branch and nodal target candidate genes possibly involved state assignments) under an ordered mod- in the evolution of form. Several unambig- el than under an unordered model, other uous, unequivocal evolutionary transfor- transformations may become less resolved mations are remarkably similar (even in (e.g., characters 30-34 at the root node; Ta- trifling aspects) to mutations in C. elegans. ble 3; Fig. 3). These mutations define genes that could The ancestral (ground) state for male tail have changed to produce the observed development and morphology (inferred evolutionary transformations. These can- from the phylogenetic reconstructions of didate genes have been targeted for further transformations that are both unambigu- analysis to understand their roles in de- ous and unequivocal) differs from the ar- velopment and evolution. chetype (Figs. 2b, 2c) based on a least-dif- ferentiated developmental model. The ACKNOWLEDGMENTS archetype is, in fact, the single worst hy- pothesis for a rhabditid ancestral form (see I thank S. Baird, D. Cannatella, S. Emmons, K. Kiontke, P. Mabee, W. Sudhaus, and two anonymous Table 3); however, this does not invalidate reviewers for strains, valuable discussions, and com- the archetype as a foundation on which to ments on the manuscript. I especially thank Scott Em- 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 169 mons at Albert Einstein College of Medicine, who sup- DARWIN, C. 1859. The origin of species by means of ported the initial part of this work both financially natural selection, or the preservation of favoured (through NSF grant IBN-9204895) and intellectually. races in the struggle for life. John Murray, London. This research was also supported by NSF grant IBN- [Facsimile edition, 1964 (E. Mayr, ed.). Belknap 9506844 and an NYU Research Challenge grant to Press, Cambridge, Massachusetts.] D.H.A.F. DE QUEIROZ, A. 1993. For consensus (sometimes). Syst. Biol. 42:368-372. REFERENCES DIXON, M. T, AND D. M. HILLIS. 1993. Ribosomal RNA secondary structure: Compensatory mutations AMBROS, V, AND W. FIXSEN. 1987. Cell lineage vari- and implications for phylogenetic analysis. Mol. ation among nematodes. Pages 39-159 in Develop- Biol. Evol. 10:256-267. 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MALE TAIL CHARACTERS AND CRITERIA SUDHAUS, W. 1991. Check list of species of Rhabditis FOR RAY HOMOLOGIES sensu lato (Nematoda: Rhabditidae) discovered be- tween 1976 and 1986. Nematologica 37:229-236. In all the rhabditid species studied, each ray is pro- duced from three cells of an initial four-cell cluster SUDHAUS, W., AND K. REHFELD. 1992. Einfuhrung in die Phylogenetik und Systematik. Gustav Fischer (Fig. 2a). In C. elegans (and probably in the other spe- Verlag, Stuttgart. cies, see Appendix 3), each cluster of four cells de- scends from a precursor cell called Rn.a (where n = SUDHAUS, W., AND F. SCHULTE. 1988. Rhabditis iPelo- {1, 2, ..., 9} for each side) in the lateral hypodermis dera) strongyloides (Nematoda) als Verursacher von (Fig. 2d). (Throughout this paper, cell nomenclature is Dermatitus, mit systematischen und biologischen that used for C. elegans; see Sulston et al., 1980; White, Bemerkungen iiber verwandte Arten. Zool. Jahrb. 1988:119.) One cell in each cluster undergoes a pro- Syst. 115:187-205. grammed cell death, and each ray is formed from the SULSTON, J. E., D. G. ALBERTSON, AND J. N. THOMSON. three remaining cells: a structural cell, which anchors 1980. The Caenorhabditis elegans male: Postembry-the distal tip of the ray to the fan cuticle, and the onic development of nongonadal structures. Dev. dendritic endings of two neurons (Fig. 2d). The sister Biol. 78:542-576. of Rn.a, called Rn.p, is a hypodermal cell. In C. elegans, SULSTON, J., AND J. HODGKIN. 1988. Methods. Pages Rl.p-R5.p fuse together and form part of the tail 587-606 in The nematode Caenorhabditis elegans (Wseam. , or set. Although the ray cell lineages of the oth- B. Wood, ed.). Cold Spring Harbor Laboratory er species are presently unknown, rays in these spe- Press, Cold Spring Harbor, New York. cies are derived from cells that originate during de- SULSTON, J. E., AND H. R. HORVITZ. 1977. Post-em- velopment of the last larval (L4) stage in the same bryonic cell lineages of the nematode Caenorhabditis relative positions as in C. elegans (Fig. 2a). elegans. Dev. Biol. 56:111-156. Because this pattern of relative positions and cellu- SWOFFORD, D. L. 1993. PAUP: Phylogenetic analysis lar connections is the same in all these species (and using parsimony, version 3.1.1. Illinois Natural His- thus is symplesiomorphic), homologous relationships tory Survey, Champaign. have been hypothesized between rays whose cells TANKSLEY, S. D. 1993. Mapping polygenes. Annu. originate at the same relative position (Fitch and Em- Rev. Genet. 27:205-233. mons, 1995). Accordingly, homologous rays bear the TEMPLETON, A. R. 1983. Phylogenetic inference from same identifying numerals in Figures 2 and 3. This restriction endonuclease cleavage site maps with hypothesis of ray homologies is therefore based on the particular reference to the evolution of humans and criteria whereby a special and complex pattern of con- the apes. Evolution 37:221-244. nections (in this case between cells in the lateral hy- WAGNER, G. P. 1994. Homology and the mechanisms podermis, Fig. 2a) suggests the homologies of the in- of development. Pages 273-299 in Homology: The dividual parts (Remane, 1952, in Sudhaus and hierarchical basis of comparative biology (Hall, B. Rehfeld, 1992:70-76). Specifically in this characteristic K., ed.). Academic Press, San Diego, California. arrangement (Fig. 2a), (1) nine Rn.p hypodermal cells WALLACE, B. 1985. Reflections on the still-"hopeful are arranged in a characteristic anteroposterior pat- monster." Q. Rev. Biol. 60:31-42. tern on each side, (2) each cluster of ray cells originates WANG, B. B., M. M. MULLER-IMMERGLUCK, J. AUSTIN, in a position adjacent to a particular Rn.p cell (which N. T. ROBINSON, A. CHISHOLM, AND C. KENYON. is the sister of the cell producing the ray cells in C. 1993. A homeotic gene cluster patterns the antero- elegans and probably in the other species, see Appen- posterior body axis of C. elegans. Cell 74:29-42. dix 3), (3) the cells for rays 5, 7, and 9 lie dorsal to WEBER, K. E. 1992. How small are the smallest se- the row of Rn.p cells, and (4) the cells for rays 1-6 lectable domains of form? Genetics 130:345-353. and 8 lie ventral to the row of Rn.p cells. Continuity WHEELER, W. C, AND R. L. HONEYCUTT. 1988. Paired of information, further supporting this hypothesis of sequence difference in ribosomal RNAs: Evolution homology, is implied by the inheritance of this specific and phylogenetic implications. Mol. Biol. Evol. 5:90- pattern by all the species in the study group (i.e., con- 96. vergence is highly unlikely). WHITE, J. 1988. The anatomy. Pages 81-122 in The It is not necessary to impose the restriction that all nematode Caenorhabditis elegans (W. B. Wood, ed.)o.f the underlying developmental mechanisms need be Cold Spring Harbor Laboratory Press, Cold Spring shared by homologous morphological characters (the Harbor, New York. aim is to understand the biological basis of changes 172 SYSTEMATIC BIOLOGY VOL. 46 to such characters). For example, ray precursor cell pendix 4). Analysis of the developmental basis of ray identities could be positionally (regulatively) or line- homologies (Fitch and Emmons, 1995) indicated that ally (mosaically) determined. Positional determination one pair of sensilla previously thought to be a pair of of early lineages leading to ray precursors is sup- rays was in fact a pair of phasmids lying laterally in- ported by laser ablation studies in C. elegans (Sulston stead of posteriorly as in the remaining species (la- et al., 1980). Therefore, observations of different cell beled "Ph" in Figs. 2, 3). Phasmids differ from rays lineages leading to specific ray precursor cells in dif- in developmental time of appearance, cellular struc- ferent species would not refute or weaken the hypoth- ture, and function (Sulston et al., 1980). Thus, all the esis of ray homology assignments on the basis of the species except one actually have 9 pairs of rays; no positions in which ray cells originate. species has 10. The exceptional species (Rhabditis blu- Whereas the patterns of ray cell originations are the mi) has eight pairs of rays because of a failure of the Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 same in different species, subsequent patterns of ray R8 cell homologue to produce the typical lineage lead- cell movement and clustering are different. After all ing to the formation of ray 8. of the ray cells have been generated during L4 larval I have broken down the variable characteristics of development, they begin to change shape and to mi- the 10 species of the study group into a series of 36 grate to and cluster at specific and reproducible junc- characters, each of which assumes two or more char- tions between particular Rn.p cells or near other ray acter states (Appendix 6). The distribution of character cell groups (Fitch and Emmons, 1995). This cell mi- states among the species is given in Appendix 7. gration (generally in a posterior direction) is the basis The adult characters mainly concern the fan and of my model for ordered transformations of multistate overall body shape. Four adult characters (7-10) con- ray cell position characters, such that more steps are cern the rays: presence of ray 8 (7), dorsoventral po- assumed to be required the further a cell migrates sitions of the tips of rays 1, 2, and 4 (8), conformation from its point of origin (see Appendix 2). The arche- of ray 6 (9), and position of ray 3 (10). I did not at- type represented in Figure 2 is a reconstruction of the tempt to define adult characters that described the hypothetical least-differentiated state, in which these most prominent variable property of the rays, i.e., cell migrations would not occur. During this morpho- their anteroposterior positions, because there was no genetic process, the dendritic endings of the two ray obvious axial coordinate system against which to neurons sink slightly into the hypodermis, leaving measure the positions of single rays in adults. The only the structural cells of each ray at the epidermal evolution of ray positions was instead only represent- surface (Fig. 2b); these cells become anchored in the ed by developmental characters. Adult ray positions cuticle in a species-specific pattern (this larval pattern are correlated with the final positions of the ray cell is represented for each species in Fig. 3). groups with respect to Rn.p cells during the late L4 This two-dimensional pattern of structural cells in larval stage (see Fitch and Emmons, 1995; Figs. 2b, 3). the larval surface prefigures the species-specific pat- The array of Rn.p cells provides an obvious point of tern of rays in the adult male tail (Fig. 2c). During the reference for rays 1-8, and the various character state next period of morphogenesis, all of the cells in the changes thus defined are represented by characters male tail change shape and retract inwardly and an- 14-21. For ray 9, it was not clear how to describe its teriorly, causing the lateral cuticle to collapse, fold, and position and whether or not this position was variable; flatten into the shape of the fan. Also at this time, it was therefore left out. particular Rn.p hypodermal cells may fuse together, A problem arises in describing the variable patterns fuse with the lateral body seam (se) hypodermis lying of the Rn.p cell fusions that occur during L4 morpho- anterior to the tail seam (set), or fuse with the main genesis. Specifically, the difference in the fusion be- body syncytium (hyp7) in which these seams are em- haviors of Rl.p-R5.p in Caenorhabditis (where they bedded (Baird et al., 1991; Fitch and Emmons, 1995). fuse together, forming the set syncytium) versus in These Rn.p cell fusions have no apparent morpholog- Rhabditis (where they remain unfused) could be de- ical consequence. Because the apical tips of the rays scribed as presence or absence of a set or could be are anchored in the cuticle, the rays are formed as described cell by cell as presence or absence of fusions tubes of cytoplasm in the wake of the retraction. Al- with particular neighbors. Because changes could con- though the process of retraction itself appears to be ceivably affect individual cells, I have chosen to de- very similar in all of the species, it progresses to var- scribe these fusions as if they were properties of the ious extents. The adult form thus results from the individual cells (characters 30-36), although pleiotro- morphogenetic retraction translating the two-dimen- pic changes could affect multiple cell groups. In this sional pattern of ray cells in the L4 lateral hypodermis way, the concentrated-changes test (see text and Ap- (Fig. 2b) into the three-dimensional pattern of rays in pendix 5) may be used to show whether pleiotropy is the adult fan (Fig. 2c). In the archetype (Fig. 2), which a likely explanation for the observed patterns of represents the hypothetical state in which ray cell mi- changes. The concentrated-changes test suggests that grations do not occur, the rays would arise at the same some of the Rn.p cells are coordinately regulated. positions at which the ray cells originate. APPENDIX 2 Prior to this work, the number of rays in different RATIONALE FOR THE ORDERED rhabditid species was thought to vary between 8 and 10; the evolution of differing arrangements was hy- TRANSFORMATION MODELS pothesized to occur by gain and loss of particular rays For several of the developmental characters, there as well as lateral movement (Sudhaus, 1976; see Ap- were more than two character states. In these in- 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 173 stances, it was possible to propose a sequence that eages). The lineages leading from the Rn precursor would represent an ordered evolutionary progression cells to generate the ray cells and the associated Rn.p from one state to the next. Character states for which cell are also identical to those in C. elegans. Further- ordered models could be proposed were (1) the cell more, the cell lineages leading to the Rn cells are very associations made by the ray cells, which govern ray similar to the corresponding lineages in C. elegans. cell position, and (2) the fusion partners of particular Specifically, there is one less asymmetric division in Rn.p cells. each of two lineages giving rise (from V6) to the P. Ray cell position (cell association) states may be or- redivivus homologues of rays 3-6; additionally, the ho- dered with respect to the anteroposterior axis. Specif- mologues to C. elegans rays 1 and 2 are absent in P. ically, when the Rn.p cell associations of a ray cell redivivus (this conclusion is suggested both by the group varied, it is possible that during evolution the similarity in cell lineages and by the similarity be- Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 position of the ray cell group progressed in a stepwise tween the spatial pattern of ray cell nuclei in the P. (linearly ordered) fashion to associate the ray cell redivivus L4 hypodermis and the spatial pattern of ray group first with one Rn.p cell and then with the next cells in the rhabditid comparisons). The lineages pro- in physical anteroposterior order. The states of such ducing rays 7-9 (from the T cell) are identical in the multistate characters were assigned the letters a, b, c, two species. The fact that these ray cell lineages are and d in the proposed order. State a is always asso- remarkably conserved between representatives of dif- ciated with the least-differentiated state, i.e., a ray cell ferent suborders predicts that the ray cell lineages group is associated with its respective Rn.p hypoder- within the family Rhabditidae will be highly con- mal cell, as in the archetype pictured in Figure 2b. served. Additional developmental steps appear to occur that Although phylogenetic analysis has not yet includ- produce in the displacement of ray cells to new po- ed Panagrellus redivivus, certain C. elegans mutants phe- sitions and associations with different Rn.p cells, re- nocopy this species' difference in ray number. The ray sulting in displaced rays and uneven ray spacing in lineages in this species are nearly identical to those in the different species. According to the ordered model, C. elegans, except that the anterior two rays are not the further the displacement away from the point of present (Sternberg and Horvitz, 1982). The same an- origin of the ray cells, the greater are the number of terior ray loss results in C. elegans from the hypo- steps away from the least-differentiated state (a). morphic bx54 mutation in the gene mab-5 (Chow and Similarly, the fate of a particular Rn.p cell may be Emmons, 1994) and from hypomorphic alleles of lin- ordered with respect to the kind of hypodermis with 32 (Zhao and Emmons, 1995). To test whether such which it fuses, which is somewhat related to the dis- hypomorphic changes were involved in the evolution tance of this hypodermis away from the Rn.p cell. For of the male tail of Panagrellus, transgenic C. elegans example, in order of increasing numbers of steps away wild type lin-32 might "rescue" the hypomorphic evo- from the unfused developmental ground state (state lutionary change and produce nine rays in Panagrellus. a), Rl.p may fuse with its adjacent R2.p neighbor (state b), may fuse with a cluster of Rn.p cells (the set; APPENDIX 4 state c), or may fuse with the body seam (se) or hyp7 (the main body syncytium) (state d). REINTERPRETATION OF RAY PATTERN EVOLUTION IN RHABDITIDAE APPENDIX 3 The conclusions presented here differ from those ON THE PROBABLE CONSERVATION OF previously published chiefly with regard to the num- RAY CELL LINEAGES ber of rays and the origin of differences in the posi- tions of the rays. Analysis of developmental events The ray cell sublineages (as well as the positions at leading to the rays allows an explicit definition of ho- which the ray cells originate) probably are conserved mologies among rays previously determined exclu- among the species studied. Specifically, because the sively from positions of rays in adult and numbers of cells forming each ray cell cluster are con- thus provides a reinterpretation of ray pattern evolu- served, the rays in all the species are probably made tion in Rhabditidae. This analysis has shown that one of three cells generated by a ray (Rn) sublineage sim- of the so-called rays in the Teratorhabditis-Pelodera ilar to that found in C. elegans (see Fig. 2d). Fitch and clade is in fact a phasmid (Fitch and Emmons, 1995). Emmons (1995) demonstrated that the putative pre- The phasmid is a characteristic posterior sensillum, cursors to the ray cells also originate in the identical apparently chemosensory, present from hatching in locations in which the ray cells originate. both males and females/hermaphrodites (Chitwood Also, the cell lineages producing the Rn cells prob- and Chitwood, 1950; Sulston and Horvitz, 1977). It is ably are conserved among the species studied here. positioned at the extreme posterior and does not ex- Sternberg and Horvitz (1982) determined the male tend significantly from the body surface in most of postembryonic cell lineages for Panagrellus redivivus (a the species of the study group. But in T. palmarum and species in a different suborder, Cephalobina, from the Pe. strongyloides, two species previously considered to Rhabditina species described here, therefore repre- have 10 rays, the phasmid is positioned laterally and senting an outgroup). In P. redivivus, which lacks a fan extends outwards in the fan in a manner identical to and rays, genital papillae are generated by a ray cell a ray. The phasmid is also the seventh papilla from sublineage identical to the corresponding lineages of the anterior in both species. In accord with this find- C. elegans (supporting the conservation of ray sublin- ing, Sudhaus (1976) suggested that either the sixth or 174 SYSTEMATIC BIOLOGY VOL. 46 the seventh papilla was the one that was "lost" (weg- APPENDIX 5 gefallen or reduziert) in the evolution of 9 rays in Me- PROBABILITY CALCULATIONS FOR sorhabditis, not represented in this study, and in a few Rhabditis species (Sudhaus postulated that the rhab- CORRELATED CHARACTERS ditid ancestor had 10 rays). I interpret this "loss" to The possible correlation of changes in characters 30- mean a relocation of the phasmid to a posterior loca- 33 (and possibly character 34) were tested by deter- tion, out of the domain of fan retraction, such that it mining the probability that the similarities in the dis- is not drawn into a raylike form during retraction. tribution of the changes in these characters could be However, in the evolution of nine rays in most of explained by chance alone. This probability calcula- the "Eurhabditis" species (Rhabditis, Rhabditella, Pellio-tion depends on being able to derive the number of ditis), Sudhaus (1976) suggested that the most poste- possible ways (W,) that these changes could be dis- Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 rior (10th) papilla was lost (although no clue was pro- tributed in a given cladogram. Maddison (1990) pro- vided as to which papilla was lost in the evolution of posed a method for deriving W, for binary state char- Caenorhabditis). This misinterpretation was probably acters (or a binary recoding of multistate characters). due to the fact that the phasmids in some "Eurhabdi- Although his concentrated-changes test is inappropri- tis" species are slightly papilliform or raylike (e.g., ate for testing the coordinated changes of the multi- Rhabditis blumi and Rhabditella axei, see Fig. 3) and arestate male tail characters, the same basic approach located posterior to the nine rays. Although Sudhaus may be used to count the number of possible ways (1980) correctly identified the phasmid for Rhabditella that multistate character changes can be distributed axei, he did not recognize the "reduced ninth ray" as under certain explicit assumptions and restrictions. the phasmid in Rhabditis blumi (Sudhaus, 1974) and Specifically, transformations were assumed to be thereby missed the fact that Rhabditis blumi generally unordered and the parsimony score was not allowed has only eight rays. The "10 rays" of other "Eurhab- to change (e.g., distributions of changes that required ditis" species may have been similarly posited on the extra reversals were excluded from consideration, as basis of a posterior, papilliform phasmid. The "atavis- in the concentrated-changes test). Additionally, the tic" aberrations called Schwanzknoten (tail-knots) in count was limited to combinations that showed the Rhabditis and Pellioditis alluded to by Sudhaus (1976: same kinds of changes as those in the observed pat- 73) cannot, therefore, be atavistic in the sense that they tern (as specified in each case, the primitive state for represent a reappearance of a 10-rayed ancestral form. each transformation was or was not kept the same as The natural variants of Rhabditis blumi observed by the corresponding transformation in the observed pat- Fitch and Emmons (1995) to have nine rays actually tern). Because of the complexities of accounting for originated by a novel duplication of a ray lineage an- different cases, a computer algorithm was not devel- terior of ray 1, and therefore these variants were not oped, and calculations were carried out manually. atavistic. Sudhaus (1974:453) may have also observed Once W, is calculated, the probability of the particular such a variant but considered it to be an atavistic reap- distribution of changes in a (dependent) character is pearance of the "seventh ray." 1 / W,. There may be w ways that these different kinds of The conclusion that the number of rays is remark- changes can be distributed in the same branches; the ably conserved (Sudhaus, 1976) is even stronger if we probability of these branches being occupied by changes consider that the "10-rayed" species (e.g., Teratorhab- is then w/Wt. For example, Figure Al shows the total ditis palmarum and Pelodera strongyloides) actually havnumbee r of ways (W, = 130) that two different kinds of 9 rays and one phasmid per side. Despite variation changes (b -» d and b -> c) in character 34 can be dis- within species, and with the exception of Rhabditis blu- tributed in the cladogram such that there are only two mi, this number seems to be highly conserved in changes in character 34 (e.g., no reversals) and that the Rhabditoidea (perhaps as conserved as digits in tet- primitive state of each change was only b. The latter re- rapods!). Perhaps future genetic and functional stud- striction yields a conservative estimate of the number of ies of the rays in C. elegans and the other species will possibilities and thus results in a conservative estimate provide insight into the possible adaptive, historical, of the significance of any correlation that may be in- or developmental constraints that may help to explain ferred. In six of these distributions of the two changes this conservation of ray number. (denoted by asterisks in Fig. Al), both changes fall on My explanation for the evolutionary basis for the dif- branches that are also occupied by changes in character ferences in ray pattern among species also differs from 30. The probability of this type of event is therefore those of previous authors. Previously, explanations for 6/130 = 0.046, just barely significant. In two of these six the presence of rays at different positions within the possibilities, the same kind of change in character 34 oc- fan were sought by postulating not only positional curs as occurs in character 30 in the Caenorhabditis lin- shifts but also evolutionary gain and loss of particular eage (i.e, fusion of an Rn.p cell with the set, or tail seam), rays (Osche, 1958; Sudhaus, 1976). The assignment of a significant probability, therefore, of 2/130 = 0.015. Un- homologies for the rays of different species based on der the alternative assumption that the primitive states the similarities in cellular arrangements during devel- are not necessarily the same as in the observed changes, opment has shown instead that all of the species have there are 182 different ways for these two different kinds the same set of rays (with the exception of Rhabditis of changes to occur on the tree without invoking extra blumi, which has lost ray 8). Differences in the ray ar- changes. In this case, the probability of observing the rangements have resulted from the shifting of these changes in branches also occupied by changes in char- rays to various positions in the fan during evolution. acter 30 by chance alone is 6/182 = 0.033, and the prob- 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 175

ability of observing both Rl.p and R5.p fuse with the set by chance alone is 2/182 = 0.011. These calcula- tions do not take into account the fact that there is one observed lineage (Rhabditis) where R5.p does not change coordinately with Rl.p under the unordered model. Thus, it is possible that the fate of R5.p is cor- related to some extent with the fate of Rl.p. The correlation of change in character 33 with change in character 30 is slightly more complex than in the previous example. In this case, two of the three changes are the same (parallel, c -> a) and the other Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 change is different (c -» b). Of the 429 different ways that such changes could be arranged on the clado- gram, assuming that the primitive state is c for all the changes, 3 ways show changes in the three lineages distinguished by changes in character 30, a probabil- ity of 3/429 = 0.007. However, in one of these ar- rangements, the kind of change in character 33 (fusion with the set) in the Caenorhabditis lineage is identical to the kind of change in character 30 for this lineage. The probability of observing this similarity in pattern is thus 1/429 = 0.002. Allowing up to one reversal and removing the constraint that the primitive state must be the same for all changes results in probabil- ities of 3/936 = 0.003 and 1/936 = 0.001, respectively, 34: b->d for the same kinds of events described above. Again, this analysis ignores the fact that of the three changes • 34: b-»c in character 33 two are the same, but all three changes in character 30 are different. Nevertheless, it seems FIGURE Al. All possible ways that two different likely that the fate of R4.p (character 33) is correlated changes (b -> d and b —»c) could occur over the rhab- to some extent with the fate of Rl.p (character 30). ditid cladogram IV (Fig. 1). For each possible branch assignment for the b —> d change (•), the number of alternative ways (WH) that b -> c (•) could occur is shown, such that state b remains the primitive state for both changes and there are no additional changes (such as reversals) that would alter the parsimony score. Asterisks denote the six alternative ways that b -> c changes could occur, together with the b -> d change, in the branches where changes in character 30 also occur. The total number of ways that these two different changes could occur, given these assump- tions, is W, = XlU Wn = 130. 176 SYSTEMATIC BIOLOGY VOL. 46

APPENDIX 6. States of varying characters in male tails of rhabditid nematodes.

Character states Character Adult 1. Fan: anterior confor- open closed mation 2. Fan: position of an- near base of near cloaca terior edge spicules Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 3. Fan: extension broad narrow 4. Body shape: overall blunt elongate 5. Body shape: tail tip peloderan leptoderan 6. Phasmid: position posterior lateral 7. Rays: ray 8 present absent 8. Rays: tip position for at margin on dorsal or rays 1, 2, 4 ventral surface 9. Rays: morphology of narrow, uni- broad, ta- ray 6 form, pered, open closed 10. Rays: position of separate tendency to ray 3 fuse with ray 4 Developmental 11. Overall body retrac- strong weak tion 12. Terminal cells: re- present absent traction 13. Terminal cells: fu- do not fuse fuse sion 14. Ray cell associations: Rl.p Rl.p + R2.p R2.p + R3.p R3.p + R4.p ray 1 15. Ray cell associations: R2.p R2.p + R3.p R3.p R3.p + R4.p ray 2 16. Ray cell associations: R3.p R2.p + R3.p R3.p + R4.p R6.p ray 3 17. Ray cell associations: R4.p R4.p + R6.p R6.p R6.p + R7.p ray 4 18. Ray cell associations: R5.p R4.p + R6.p R5.p + R6.p ray 5 19. Ray cell associations: R6.p R6.p + R7.p ray 6 20. Ray cell associations: R7.p R7.p + R9.p migrates into ray 7 R7.p 21. Ray cell associations: R8.p R7.p (no ray 8) ray o 22. Dorsoventral posi- seam-hyp7 within set tion of Rnst: ray 1 border 23. Dorsoventral posi- seam-hyp7 within hyp7 tion of Rnst: ray 2 border 24. Dorsoventral posi- seam-hyp7 within hyp7 tion of Rnst: ray 4 border 25. se-set boundary near base of near cloaca position3 spicules 26. Rn.p position: Rl.p within seam ventral of seam 27. Rn.p position: R2.p within seam ventral of seam 28. Rn.p position: R6.p ventral of within seam seam 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 177

APPENDIX 6. Continued

Character states Character a b c d e 29. Rn.p position: R9.p strictly to dorsal lateral midline 30. Rn.p cell fusions: unfused fuses with fuses with set fuses with se Rl.p R2.p + R3.p 31. Rn.p cell fusions: unfused fuses with fuses with set fuses with se R2.p Rl.p + R3.p Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 32. Rn.p cell fusions: unfused fuses with fuses with set fuses with se R3.p Rl.p + R2.p 33. Rn.p cell fusions: unfused fuses with fuses with se R4.p set 34. Rn.p cell fusions: unfused fuses with fuses with R6.p fuses with set fuses with hyp7 R5.p R7.p + R7.p or se 35. Rn.p cell fusions: unfused fuses with fuses with se fuses with hyp7 JXO.p JXO.DC „p T_i_ JX/.p07 „ 36. Rn.p cell fusions: unfused fuses with fuses with R5.p fuses with hyp7 R7.p R5.p + R6.p 1 se = body seam; set = tail seam. APPENDI X7. Character states for each rhabditid nematode species.

Character no.a Species 12 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2 3 24 25 26 27 28 29 30 31 32 33 34 35 36 Caenorhabditis briggsae bbaaaaabbbaabbbcbabaabbbbaaabcccbdd d Caenorhabditis elegans bbaaaaabbaaabbbcbabaabbbbaaabcccbdd d Rhabditis myriophila aaaabaaaaaabaaaaabaabaaaaaaaaaaaaad d Rhabditis blumi aaaababaaaabaaaaabaacaaaaaaaaaaaaad d Rhabditella axei aabbbaaaaabbaabbdabaaaaaaaaaadddce c/d b d Pellioditis typica aaaaaaaaaaaaaabbcbabaaaaaaabadddcbd b Teratorhabditis palmarum aaaaabaaaaaaaddddabcaaaaabbbabbbacb c Pelodera strongyloides aaaaabaaaaaaaccddcbaaaaaaaaaadddcb d b

a See Appendix 6 for descriptions of character s (1-36) and states (a-e).

b Uncertainty in the character state assignment . Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 September 30 on guest by https://academic.oup.com/sysbio/article/46/1/145/1685502 from Downloaded 1997 FITCH—NEMATODE MALE TAIL EVOLUTION 179

APPENDIX 8. Possible features of the nematode at the root node of tree IV (Fig. 3), assuming an unordered model of character state transformation (the effects of assuming an ordered transformation model are noted).

Character State Description Adult 1. Fan: anterior conformation a open 2. Fan: position of anterior edge a near base of spicules 3. Fan: extension a broad 4. Body shape: overall a blunt 5. Body shape: tail tip a peloderan Downloaded from https://academic.oup.com/sysbio/article/46/1/145/1685502 by guest on 30 September 2021 6. Phasmid: position a or b posterior or lateral 7. Rays: ray 8 a present 8. Rays: tip position for rays 1, 2, 4 a at margin 9. Rays: morphology of ray 6 a narrow, uniform, open 10. Rays: position of ray 3 a separate Developmental characters 11. Overall body retraction a strong 12. Terminal cells: retraction a present 13. Terminal cells: fusion a do not fuse with Rl.p, Rl.p + R2.p, R2.p + R3.p, or 14. Ray cell associations: ray 1 a, b, c, or da R3.p + R4.p 15. Ray cell associations: ray 2 b, c, or da with R2.p + R3.p, R3.p, or R3.p + R4.p 16. Ray cell associations: ray 3 b, c, or db with R2.p + R3.p, R3.p + R4.p, or R6.p 17. Ray cell associations: ray 4 dc with R6.p + R7.p 18. Ray cell associations: ray 5 ad with R5.p 19. Ray cell associations: ray 6 b with R6.p + R7.p 20. Ray cell associations: ray 7 a with R7.p 21. Ray cell associations: ray 8 a with R8.p 22. Dorsoventral position of Rnst: ray 1 a seam-hyp7 border 23. Dorsoventral position of Rnst: ray 2 a seam-hyp7 border 24. Dorsoventral position of Rnst: ray 4 a seam-hyp7 border 25. se-set boundary position3 a near base of spicules 26. Rn.p position: Rl.p a within seam 27. Rn.p position: R2.p a within seam 28. Rn.p position: R6.p a ventral of seam 29. Rn.p position: R9.p a strictly lateral 30. Rn.p cell fusions: Rl.p df fuses with se 31. Rn.p cell fusions: R2.p df fuses with se 32. Rn.p cell fusions: R3.p df fuses with se 33. Rn.p cell fusions: R4.p c« fuses with se 34. Rn.p cell fusions: R5.p b« fuses with R7.p 35. Rn.p cell fusions: R6.p d fuses with hyp7 fuses with R5.p, fuses with R5.p + R6.p, or 36. Rn.p cell fusions: R7.p b, c, or db fuses with hyp7 " If a linearly ordered transformation model is assumed, the possible states are b or c, and the character reconstruction becomes more resolved at other nodes. b If transformations are ordered, the possible states are c or d, and the reconstruction becomes more resolved. c If transformations are ordered, the possible states are c or d, and the reconstruction becomes equivocal only at this node; plesiomorphic states at some later nodes become c. d If ordered, the assignment becomes equivocal (a or b), and the reconstruction becomes equivocal at two later nodes. e se = body seam; set = tail seam. ' If transformations are ordered, the assignment becomes equivocal (c or d), and the reconstruction becomes equivocal at several later nodes. * If ordered, the assignment becomes equivocal (b or c), and the reconstruction becomes equivocal at several later nodes.