The Role of Functional Analysis in Phylogenetic Inference: Examples from the History of the Xiphosura 1

Transcription

1 AMER. ZOOL., 21:47-62 (1981) The Role of Functional Analysis in Phylogenetic Inference: Examples from the History of the Xiphosura 1 fc DANIEL C. FISHER Museum of Paleontology, University of Michigan, Ann Arbor, Michigan SYNOPSIS. Conventional cladistic analyses of phylogeny can be interpreted as operating at the level of phylogenetic trees. They assume that all "evolutionary steps" (transitions from one character state to the next, along a morphocline) are independent and equal, and, on that basis, select the cladogram which is consistent with the most parsimonious trees. Evaluation of the assumptions of independence and equality requires consideration of hypotheses at the level of scenarios. In some cases, arguments based on functional analysis can suggest revised interpretations of either homology or polarity. If properly formulated, these arguments can alter the evaluation of parsimony for trees to the extent that even the choice of cladogram is affected. The structure of scenario level arguments is identical to that of arguments operating at tree level. Examples of phylogenetic inference in the context of xiphosurans (horseshoe crabs), using both comparative morphological and functional analysis, illustrate this approach. In different cases, orthodox interpretations of relationship are either challenged or corroborated. Although the introduction of functional analysis into the process of phylogenetic inference may appear to compromise the usefulness of the reconstructed phylogeny for testing hypotheses concerning the role of natural selection in evolution, it actually increases the strength of such tests. INTRODUCTION Of the many difficulties which we might conceivably face in the context of the controversy alluded to in the title of this symposium, it seems clear that a lack of distinct alternative interpretations is not one of them. It has been argued both that explicit functional analysis must be, and also that it cannot be, applied to problems of phylogenetic reconstruction. Although these positions would seem to admit no middle ground, other interpretations are not in fact prohibited unless we actually accept one of these extremes. In the following discussion, I will argue that: (1) all attempts to reconstruct phylogeny involve at least minimal assumptions that can be construed as pertaining to issues subsumed under the broad heading of "adaptation"; (2) we may, however, choose to reconstruct phylogeny without specific evaluation of arguments concerning adaptation, by adopting a methodological convention equivalent to a ceteris paribus clause; and (3) if testable hypotheses concerning ad- 1 From the Symposium on Functional-Adaptive Analysis in Systematics presented at the Annual Meeting of the American Society of Zoologists, December, at Tampa, Florida. 47 aptation can be made, they can in some cases assist substantially in choosing among alternative reconstructions of phylogeny. These arguments will take a form that, on one level, has been suggested many times previously (e.g., Bock, 1977): namely, that functional morphology can offer important arguments in the context of character analysis, by suggesting initial interpretations of both homology and polarity, and by helping to resolve cases of incongruent apparent synapomorphies. However, my approach will be unusual in that I will argue that this use of functional morphology is a natural extension of the best of cladistic methodology. Examples of this approach will be drawn from work on the phylogeny of horseshoe crabs. I have chosen to circumvent the issue of constructing classifications. This decision is based primarily on constraints of space and the opinion that classification is less directly related to the fundamental concerns of evolutionary biology than is phylogenetic inference. In addition, the controversy over methods of classification seems less susceptible to objective resolution. This is not to say that the properties of different methods cannot be investigated empirically (Farris, 1977, 1979, has

2 48 DANIEL C. FISHER CLADOGRAM, CLAOOGRAM, TREE, SCENARIO, FIG. 1. Correspondence relationships between cladograms, trees, and scenarios. done so, for instance, and found phenetic methods wanting by some of their own performance criteria), but rather, that any resolution will require some concensus on the relative importance of a variety of potential evaluative criteria, all of which will depend on how we choose to use classifications. It would be rather futile to attempt to evaluate the relative usefulness of a hammer and a saw without first having agreed upon what job was to be performed with the chosen tool. PHYLOGENETIC INFERENCE Since the ideas and procedures basic to a cladistic approach to phylogenetic inference have been discussed in this symposium and elsewhere (e.g., Hecht et ai, 1977; Cracraft and Eldredge, 1979; Nelson, 1979), I need not belabor them here. It is important, however, to emphasize the very useful distinction of three aspects of the reconstruction of phylogeny (Eldredge, 1979): (1) cladograms (hypotheses concerning the distribution of synapomorphies among the included taxa, with implications for relative recency of common ancestry); (2) phylogenetic trees (hypotheses concerning the genealogical history of the included taxa); and (3) evolutionary scenarios (hypotheses concerning the historical-ecological context of the evolutionary history of the included taxa). These three classes of hypotheses involve increasingly specific statements on the phylogenetic relationships of a particular group of organisms. Furthermore, if we assume that evolution is a branching (not anastomosing) process, if we deal only with well defined phenons or OTUs, and if we do not demand that cladograms be exclusively dichotomous, then the correspondence relationships between cladograms, trees, and scenarios are as mapped in Figure 1. Any hypothesis at the level of scenarios entail^ a unique hypothesis at the level of tree&r and any tree entails a unique cladogram. Working in the other direction, for a finite number of taxa under consideration, there is certainly a finite number of possible cladograms, but whether or not there is only a finite number of trees derivable from any one cladogram depends on exactly what information is included in the tree (Platnick, 1977; Felsenstein, 1978; see below). In any case, there is an effectively infinite number of scenarios associated with any one tree. Although phylogenetic trees may be defined minimally as statements on the nature of evolutionary relationships between taxa (ancestor-descendent relationships, or descendants of a common ancestor, known or hypothetical), additional information may be included. For the purposes of this discussion, 1 will exclude from detailed consideration trees which specify stratigraphic or temporal data, since this type of data is not relevant to considering the role of functional morphology in phylogenetic inference. I will, however, want to consider trees which specify the character states associated with their terminal and interior nodes. Hereafter, I will use the term "phylogenetic tree" to refer to these somewhat more complex hypotheses, which are compounded from trees representing only the topology of relationships (to be called genealogical trees) and from trees representing character transformations (character state trees; Farris et al, 1970). Many phylogenetic systematists have claimed to be dealing exclusively with hypotheses at the level of cladograms and have eschewed explicit treatment of trees on the basis of arguments that trees do not (always/ever) represent falsifiable hypotheses. Even when trees or scenarios have been considered justifiable (Eldredge, 1979), it has been argued that the construction (i.e., testing) of cladograms occurs prior to, and is logically independent

3 HORSESHOE CRAB PHYLOGENY 49 of, any consideration of hypotheses on the level of trees. Trees are only to be reconstructed by selecting from the alternatives that remain, subsequent to, and consequent on, the selection of the single most Satisfactory (or least unsatisfactory) cladogram, and scenarios similarly follow only upon the choice of a tree. Given this protocol, it is easy to understand why cladists have been hesitant to use stratigraphic information (which would consist of statements at tree level) or the results of functional analysis (statements at no less than scenario level) in the process of testing competing cladograms. I will suggest, however, that phylogenetic analysis, even when performed explicitly at the level of cladograms, is not in fact organized as in the above description. Character analysis, leading to the recognition of particular synapomorphies, is based on hypotheses of homology (tested through morphologic analysis) and hypotheses of polarity (tested primarily by outgroup comparisons). It is generally portrayed as preceding the testing of cladograms, but I believe few workers would insist that this is the whole story. In practice, whenever a character incongruity (the conflict in cladistic implications of different apparent synapomorphies) arises, we accept the implication that at least one decision on homology or polarity has been mistaken, and, using parsimony criteria, we modify our hypotheses of synapomorphy. A hypothesized synapomorphy is taken as falsifying all cladograms (for the taxa under consideration) which do not include the "component" which that synapomorphy defines, or one that is "combinable" with it (Nelson, 1979). After performing this operation for all hypothesized synapomorphies, our parsimony criterion allows us to accept that cladogram which has been least frequently rejected. It could be argued that explicit character analysis is unnecessary that simply hypothesizing similarities to be synapomorphous and following the cladogram testing procedure above would result in the same cladogram selection. While this is entirely possible, it is also possible, as Eldredge (1979) has discussed, that such a cladogram would cluster taxa by symplesiomorphies. In order to reject this possibility, character analysis is required. Although this explication makes no mention of trees, the arguments which underlie both character analysis and cladogram selection can be shown to involve evaluation of the relative parsimony of all phylogenetic trees that are consistent with the observed morphologic data. This can perhaps be most readily appreciated through consideration of a three-taxon problem involving cladogram selection. Associated with the four possible cladograms for three taxa (A, B, and C) are 22 possible genealogical trees (Platnick, 1977). If we consider only one character with two states (x and x'), there will be 41 possible phylogenetic trees (the higher number arising from the possibility of assigning either character state to a hypothetical common ancestor). We will also assume that A-B-C is a monophyletic group, that x is the only character state found outside of A-B-C, and that states are assigned to the three OTUs as follows: A, x; B, x'; C, x'. If we consider the hypothesis that x' is synapomorphous in B and C relative to x in A, it may seem unnecessary to think of the consequences of the hypothesis as being felt at the level of trees, rather than cladograms. After all, not one of the possible phylogenetic trees associated with cladograms other than the one showing B and C as sisters is consistent with such a hypothesis. Since cladograms are (in a sense) classes of trees, can we not simply say that these cladograms have been rejected? The first hint of a problem in such an approach comes through noting that even some of the phylogenetic trees associated with the "successful" cladogram are not consistent with the above hypothesis of synapomorphy (though this results from the characters assigned to hypothetical common ancestors; it is not a necessary consequence of their topology). More directly to the point is a reminder that a hypothesis of synapomorphy is actually a compound hypothesis one of homology and one of polarity. If these are taken separately, it is clear that their effects cut across boundaries between the groups of

4 50 DANIEL C. FISHER phylogenetic trees associated with particular cladograms. Some, but not all, phylogenetic trees associated with each cladogram are consistent with a given hypothesis of homology (or polarity). Hypotheses of homology and polarity thus operate, not with respect to single cladograms or single phylogenetic trees, but rather with respect to classes of phylogenetic trees. These classes are not the same as the classes of trees consistent with individual cladograms. The connection with parsimony is clear if we consider what underlies a given hypothesis of homology, for example. What we actually observe, in the example above, is a similarity between x' in B and x' in C, and between x in A and x in the taxa not included in A-B-C. We adopt a hypothesis of homology because we recognize it as more parsimonious, in terms of the number of evolutionary "steps" required (Camin and Sokal, 1965), to conceive of a given character state as having arisen once, prior to the most recent common ancestor of the taxa showing the similarity, than it would be to have it arise independently, in the lineages subsequent to that most recent common ancestor. This judgement is explicitly a comparison of alternative phylogenetic trees. In general, our evaluation of similarities and differences consists of asking which phylogenetic trees (necessarily plural, since we are dealing, at present, with only one character) account most parsimoniously for the distribution of character states among known taxa. We say that a hypothesis of synapomorphy falsifies cladograms because all of the phylogenetic trees associated with those cladograms are less parsimonious than some of the phylogenetic trees associated with the cladogram that is said to be corroborated. This general approach to cladistic analysis, through evaluation of the relative parsimony of trees, is, of course, explicitly developed in numerical cladistic techniques (though their methods may vary in detail; e.g., Farris, 1978; Felsenstein, 1979) and has even been related to the basic principles of phylogenetic systematics by Farris et al. (1970). Much of it is also implied in discussions of post-cladogram tree selection (e.g., Platnick, 1977). Yet its implications for extending methods of phylogenetic analysis appear not to have been appreciated. As shown in Figure 1, the correspondence between scenarios and trees is essentially like that between trees and clado-# grams. If we reject cladograms by rejecting classes of trees (as less parsimonious than competitors), might we not reject trees (and thence cladograms) by rejecting classes of scenarios (also as less parsimonious than competitors)? In cladistic analysis using only comparative morphological data this is effectively prevented by considering each evolutionary step as independent and of equal weight in the computation of parsimony. This convention amounts to a ceteris paribus clause which conditions the evaluation of parsimony on the absence of significant additional unbalanced constraints on the plausibility of various trees. Within this approach to cladistic analysis, the convention has axiomatic status; it is unfalsifiable by the morphologic data it is designed to handle. It need not imply that all other factors are in fact assumed to be equal only that we do not understand enough about their importance and effects to take them into account. As such, it is a necessary methodological tool. However, using it conscientiously demands that we review those factors under its purview to determine whether or not their effects remain unresolvable. Along these lines, Kluge and Farris (1969) have used data on character variability (because of its hypothesized effects on the rate of character transformation) to weight the contribution of different characters to the total evaluation of parsimony. Hecht (1976) has suggested another approach to character weighting, involving some consideration of functional morphology, but it operates in a different fashion from what I will suggest. If functional morphology is to be used in analysis at the level of cladograms and trees, I would argue that it must contribute testable hypotheses applicable to all taxa under consideration, and all characters involved in incompatible hypotheses of synapomorphy. Hypotheses concerning adaptation will be used to evaluate the relative parsimony (plausibility) of competing

5 HORSESHOE CRAB PHYLOGENY 51 classes of scenarios in such a way as to have an effect at tree level. Cladistic relationships among xiphosurans In the following examples, I will consider only glimpses of a more comprehensive treatment of relationships among xiphosurans, or horseshoe crabs (Fisher, 1975a, in preparation), dealing with as few taxa as possible and considering only those characters which have the clearest implications for relationships. It will be necessary to present the results of functional analysis as only sparsely supported assertions. They have been discussed in greater detail elsewhere (Fisher, 1975a, b, 19776, 1979), but in the present context, their general plausibility is all that I will try to defend. Morphological terms and diagrammatic summaries of the morphology of the species being used are presented in Figures 2 and 3. Belinurus koenigianus Woodward-Ewproops danae Meek and Worthen-Pafeo/imulus avitus Dunbar. These three species are representatives of what are generally treated as three superfamilies of horsehoe crabs, the Belinuracea, Euproopacea, and Limulacea, respectively. These superfamilies are included within the infraorder Limulicina by Eldredge (1974). The conventional interpretation of their relationships at the level of a genealogical tree is that the Limulacea were derived from the Euproopacea, which in turn were derived from the Belinuracea. The only more cautious statement on their relationships was made by Eldredge (1974), who argued for a cladogram which included additional taxa, but was consistent with this conventional tree. Using species-level taxa, this interpretation of relationships is shown as the dashed cladogram of Figure 4A. I will eventually argue that the solid cladogram of Figure 4A is a better representation of their cladistic relationships, but I will do this in two stages. I will begin by considering only comparative morphological evidence, showing how the conventional use of this evidence can be understood as operating at the level of trees. I will then consider the applications of functional morphological evidence. In this particular FIG. 2. Morphology of representative xiphosurans; dorsal aspect on left; partial ventral aspect on right. A. Limulus polyphemus; Recent. B. Euproops danae; Carboniferous. Abbreviations: ar, axial ridge; ga, genal angle; ios, interophthalmic spine; mas, marginal spine of opisthosoma; mvs, moveable spine of opisthosoma; C>7_9, tagma formed of fused opisthosomal segments 7-9; oa, region occupied by opisthosomal appendages; ob, occipital band; od, opisthosomal doublure; Op, opisthosoma; op, opercular pleurite; or, ophthalmic ridge; pa, region occupied by prosomal appendages; Pr, prosoma; tas, terminal axial spine; Te, telson. three-taxon problem, comparative morphological arguments are entirely adequate for distinguishing the best of the competing cladograms, but functional arguments provide effective corroboration of certain points. The most conspicuous character in support of the dashed cladogram in Figure 4A is the fusion of opisthosomal segments into one solid tagma, shown in E. danae and P. avitus. The lines suggestive of opisthosomal segmentation in Figures 2B and 3B are only topographic indications of the position of segmental boundaries; they are not functional articulations. B. koenigianus, in contrast, has opisthosomal segments 7-9 (O 7 _ 9 ) fused, but Oi_ 6 are freely articulated along both their anterior and posterior boundaries. A similar condition is seen in other members of the Limulicina, but even more important is that it is almost ubiquitous among outgroups, such as the Pseudoniscina, Synziphosurina, or Aglaspida, and is common among even other merostomes (for the purposes of this analysis, I accept as unproblematic the higherlevel relationships suggested by Eldredge, 1974, and illustrated in Fig. 4D). Ordinarily, it would simply be said that this pattern leaves little doubt that, in general, fused B

6 52 DANIEL C. FISHER FIG. 3. Diagrammatic sketches of additional taxa used in examples; dorsal aspect, left; partial ventral aspect, right; approximately natural size. A. Belinurus koenigianus; Carboniferous. B. Paleolimulus avitus; Permian. C. Anacontium brevis; Permian. D. Limulitella bronni; Triassic. segments are derived with respect to freely mobile ones. While I agree with this proposition, I understand it as representing a more complex argument. With respect to the mobile-fused morphocline, we have two choices as to polarity. If mobile segments are derived relative to fused ones, then any two occurrences of these derived, mobile segments (let us say, once within synziphosurans and once within aglaspids) may be either homologous or not homologous. If they are not homologous, then we are, by definition of homology, dealing with a phylogenetic tree in which the character in question was not present in the most recent common ancestor of the synziphosuran and the aglaspid, and was derived independently somewhere along the lineages leading from that ancestor to each of them. If, on the other hand, this presumably derived, mobile segmented condition is homologous, then we are dealing with another class of phylogenetic trees, namely ones which specify a common FIG. 4. Cladograms representing relationships among selected xiphosuran taxa; solid lines: cladogram corroborated by this analysis; dashed lines: one of the cladograms rejected by this analysis. A-C. Cladograms evaluated in text. D. Relationships among xiphosuran merostomes; after Eldredge (1974). ancestor for the synziphosuran and the aglaspid which did have the derived, mobile segmented condition. But if this is true (i.e., if we were to accept any of this class of phylogenetic trees), then, since that ancestor is also an ancestor of the limulicines showing the fused condition (within the constraints of the cladogram accepted as the higher-level analysis of relationships), there must have been a transformation from the unfused to the fused condition in other words, a reversal of the assumed polarity. The only possibilities left are that the higher-level cladogram is wrong, or that fusion is a derived condition. We accept this latter hypothesis of polarity because the phylogenetic trees with which it is associated account for character evolution without requiring the reversals or convergences required by all trees associated with other hypotheses of polarity. This procedure involves the comparison, explicitly or implicitly, of hypotheses at the level of phylogenetic trees. In order to use the fused condition of opisthosomal segments in E. danae and P. avitus as a synapomorphy for resolving the relationships of these three taxa, two additional points must be covered. First, we

7 HORSESHOE CRAB PHYLOGENY 53 must argue that the opisthosomai segments of B. koenigianus are not secondarily free. Since they are, in all apparent respects, similar to the free segments of other xiphosuran groups, this hypothesis *vould require an "extra" reversal for which there is no independent evidence. It therefore seems unlikely. Secondly, we must argue that the fused condition is homologous in E. danae and P. avitus. There are certainly conspicuous differences between their opisthosomata, but without particular evidence to the contrary (in the form of incongruent synapomorphies), it is certainly most parsimonious to consider the character homologous. This has been the choice of all previous workers (Raymond, 1944; StOrmer, 1952, 1955; Eldredge, 1974), and on this basis, the dashed cladogram would be accepted. I am aware of no good arguments for synapomorphies shared by B. koenigianus and P. avitus that are not also shared by E. danae. The only other competitor among dichotomous cladograms for these three taxa is thus the solid one in Figure 4A, which, although it has never even been discussed by other workers, has a number of points to recommend it. Comparative morphologic analysis (which I now need not spell out in detail) strongly suggests that the rounded profile of the opisthosoma of B. koenigianus and E. danae is synapomorphic relative to the more triangular profile shown by P. avitus. In addition, the fused tagma formed in B. koenigianus by O 7 _ 9, complete with its single, large, rounded, axial swelling and dorsally directed spine, is present in E. danae in almost identical form (except that in E. danae the most posterior marginal opisthosomal spine is considerably smaller). This condition appears to be synapomorphic relative to that of P. avitus, where O 7 _ 9 have boundaries that are clearly demarcated topographically, with no axial swelling or single, long spine. We now are left with the problem of choosing between incongruent apparent synapomorphies. If the totally fused opisthosoma is accepted as a synapomorphy, then either P. avitus has secondarily lost all indication of a fused O 7 _ 9 tagma (and all its associated structures) that was present in one of its ancestors (at least as recently as its common ancestor with E. danae), or else the O 7 _ 9 complex in B. koenigianus and E. danae is convergent. An exactly analogous set of choices is presented with respect to the rounded opisthosomal profile. All of the trees represented by these options involve multiple reversals and/or convergences. In contrast, if we accept the synapomorphies declaring B. koenigianus and E. danae to be sisters, our choice is: either B. koenigianus has secondarily reverted to a mobile segmented condition, or P. avitus has independently fused up its entire opisthosoma. The trees consistent with either of these alternatives would be more parsimonious than any of the ones considered previously. Therefore, I am led to prefer the only cladogram with which they are consistent, the solid one in Figure 4A. While comparative morphological evidence has proven adequate for cladogram construction (or testing) in this case, it is important to consider the contribution that could have been made by functional morphology. In xiphosurans, opisthosomal fusion has the consequence of increasing the proportion of body volume devoted to extrinsic appendage musculature, by reducing the space occupied by longitudinal musculature controlling intersegmental posture. Through this effect, opisthosomal fusion is associated with more diverse behavioral repertoires and the capacity for higher levels of activity. In all known cases of opisthosomal fusion, the loss of the ability to adjust intersegmental posture (potentially important during locomotor and righting activities) is counterbalanced by the greater effectiveness of opisthosomal appendages and is allowed by the development of increased excursion at the prosoma-opisthosoma and opisthosoma-telson articulations (in fact, one or both of these must precede complete fusion). It therefore seems that the fused condition would always be at a selective advantage relative to mobile opisthosomal segments. Accepting these statements concerning the functional significance of opisthosomal fusion is tantamount to rejecting,

8 54 DANIEL C. FISHER as functionally implausible, those scenarios which would involve contradictory transformations. Since the ontogenetic development of the fused opisthosoma precedes any sclerotization (i.e., an individual does not develop mobile segments which lose their mobility later in ontogeny), I would not even expect reversal of this morphocline as an indirect effect of selection for a paedomorphic condition important in some other context. Taken together, these arguments corroborate the hypothesis of polarity. In this case, however, the parsimony criterion is more broadly based than the simple counting of evolutionary steps. Functional analysis also helps to corroborate previous decisions on homology. While having a strong explanation of the adaptive significance of fusion would certainly not, by itself, suggest that fusion had occurred on multiple occasions, it renders that interpretation more plausible (or less implausible) when it is suggested on other grounds. Since the benefits of fusion would accrue regardless of the particular tagmatic structure preceding full fusion, and regardless of the particular means by which the mobility of segments is restricted, it is particularly easy to explain the differences between the fused opisthosomata of P. avitus and E. danae as the result of fusion occuring within the context of differing tagmatic structures. As will be seen below, no comparable argument is available to suggest that the similarities between B. koenigianus and E. danae might only be homeomorphic. The rounded profile of the opisthosoma in B. koenigianus and E. danae is an adaptation for precise occlusion with the prosoma during enrollment (Fisher, 1977a). While there are tentative arguments confirming the polarity suggested above, they are not strong enough for me to consider them significant at present. In other words, the classes of scenarios involving alternative hypotheses of polarity have members which appear to be about equally plausible. However, functional arguments do have implications for homology here. What is most important about opisthosomal profile with respect to occlusion with the prosoma is the precise topography of the opisthosomal doublure; other aspects of the morphology of this region are not constrained by this adaptation. That the details of the opisthosomal margin should still be so similar in these two constitutes an argument that its modification is mologous. Exactly the same situation obtains with respect to functional analysis of the O 7 _ 9 tagma. It is related to the righting mechanism of these animals (as an area of origin for muscles operating the telson, and as a topographic prominence which controls the orientation of an individual when overturned), and although it offers only tentative arguments for polarity, it is strongly indicative of homology (again, since there is a very strong similarity even between aspects of morphology that are under relatively weak biomechanical constraint). In this particular three-taxon problem, we thus have a case where conventional cladistic methodology is adequate to resolve character conflict. At certain points in this reconstruction of relationships, functional arguments can stand on their own in support of certain interpretations of homology or polarity, and they can indicate which of two sets of apparent synapomorphies is more subject to convergence. An even stronger example of this use of functional analysis could be offered here, but will instead be dealt with below, where its application is more critical. B. koenigianus E. danae-anacontium brevis Raymond-P. avitus. This second example concerns the relationships of Anacontium brevis (known only from its prosoma), relative to the group of three species discussed above. The only possibility that has been considered previously is that A. brevis was "closely related" to E. danae, and the two have always been placed in the same family (Raymond, 1944; Stdrmer, 1955). This decision has been based on the striking similarity of their ophthalmic ridges, and although it leaves unspecified their exact relationships at the level of a tree or cladogram, a cladogram can be easily derived from it (dashed; Fig. 4B), assuming the validity of the solid cladogram of Figure 4A. In this case however, comparative morphologic analysis of

9 HORSESHOE CRAB PHYLOGENY 55 this and another character proves inadequate for choosing between two competing cladograms. On the basis of morphologic analysis the course of the ophthalmic ridge system of 0l brevis and E. danae seems synapomorphous relative to the more narrow, parallel-to-anteriorly-convergent ophthalmic ridge system of P. avitus and B. koenigianus. However, a different light is thrown on this problem by consideration of the character alluded to at the end of the last example. All four of these horseshoe crabs (and all others as well) have an occipital band along the posterior margin of their prosoma. In all of these taxa, the occipital band is more or less vertically oriented between the ophthalmic ridges. However, lateral to the ophthalmic ridges, its orientation varies. In E. danae and B. koenigianns it turns to face ventrally along the posteromedial margin of the genal angle. In P. avitus and A. brevis it faces dorsally along the same stretch. In most other belinuraceans and in all other non-limulicine xiphosurans, it retains its vertical orientation until it thins out near the tip of the genal angle. Outgroup comparison suggests that the vertical occipital band is primitive, and that both the condition seen in B. koenigianus and E. danae, and that of P. avitus and A. brevis, are derived with respect to the most primitive state. However, what is the polarity of the two "derived" conditions relative to each other} This is what we need to know if we are to use either of them in the present argument. There are three possible answers to this question: 1) Both the dorsally oriented and the ventrally oriented condition of the occipital band could be independently derived from the primitive vertical condition. This would make each of them derived with respect to the other and allow each to be used as a synapomorphy in the present problem (as long as the homology of multiple occurrences of each condition is assumed). 2) The dorsally oriented condition could be derived from the ventrally oriented condition, via an intermediate, vertical stage, homeomorphic to the overall primitive condition. With this interpretation, only the dorsally oriented condition would be indicative of relationships among these taxa. 3) The ventrally oriented condition could be derived from the dorsally oriented condition, leading to the opposite of the second possibility with only the ventrally oriented condition being indicative of relationships here. If the approach to this problem is restricted to purely morphological analysis, there is little basis for choosing among these possibilities. We might give a slight preference to the first, since the total transformation it requires can be accomodated in only two steps, while both the second and third possibility involve one additional step an initial reversal to the superficially primitive condition. However, if this possibility and its implied synapomorphies are accepted, we must also accept either convergence in ophthalmic ridge design by E. danae and A. brevis, or an independent reversal to the primitive ophthalmic ridge condition by both B. koenigianus and P. avitus. Although this latter interpretation might be rejected on grounds of parsimony, the remaining classes of phylogenetic trees are equally complex, and thus do not allow us to choose between cladograms in Figure 4B. The intuition of most workers might well favor the synapomorphy of ophthalmic ridges, as a more "complex" character than occipital bands, and in doing so, concur with the orthodox interpretation of relationships. However, there would be little rigorous basis for such a decision. A very different result can be obtained if functional arguments are allowed. The ophthalmic ridge system is primarily significant as a corrugation of the dorsal prosomal exoskeleton, stiffening it against deformation (particularly during the interval after molting and prior to appreciable sclerotization) by muscles originating on it. Since many of these muscles are associated with the prosomal appendages, the course of the ophthalmic ridges bears a close relationship to the arrangement and relative size of these appendages. The type of ophthalmic ridge system seen in P. avitus

10 56 DANIEL C. FISHER and B. koenigianus is associated with the use of the prosomal appendages in: swimming; walking or scuttling along a relatively planar substrate; and burrowing. The ophthalmic ridge system seen in E. danae or A. brevis, while not eliminating these activities from the behavioral repertoire, represents a specialization for clinging to and walking along relatively narrow, cylindrical substrates, probably represented by plant axes (Fisher, 1979). This interpretation does not suggest any strong arguments for polarity; it is at least possible that a transition from one system to the other could have occurred in either direction. With regard to homology, there is also no decisive verdict. Since the ophthalmic ridge system is tightly constrained by this adaptation, convergence is certainly a possibility. Yet there is no conspicuous difference between the ophthalmic ridges of A. brevis and E. danae that would, by itself, suggest such an interpretation. Turning to the other character, the primitive vertical occipital band forms a relatively blunt trailing edge to the genal angle during swimming, resulting in significant drag production. Rotating this band either dorsally or ventrally produces a much more evenly tapered trailing edge and reduces drag production. I know of no counterbalancing advantage of a vertical occipital band. Therefore, scenarios and trees suggesting its derivation from either the dorsally oriented or the ventrally oriented condition seem unlikely. The dorsally and ventrally oriented bands are thus best interpreted as independently derived conditions representing alternative, functionally equivalent modifications of the primitive vertical condition. Their appearance as modifications of the primitive condition is not surprising, but I would not expect either one of them to be derived from the other (since this would require either an initial increase in drag production or a complete reduction and redevelopment of the occipital band). Functional analysis thus favors the first interpretation of polarity mentioned above, by falsifying, if you will, the second and third possibilities. Therefore, unless we want to interpret the occipital band of each of these species as independently derived from the primitive condition, A. brevis cannot be a sister to E. danae. The most parsimonious interpretation is the solid cladogram of Figure 4B. Limulitella bronni Schimper^Lmm/«j polifa phemus Linne-P. avitus. As a final example, consider these taxa. There are no characters that appear to be derived and shared by Limulitella bronni and P. avitus, but not by Limulus polyphemus. The solid cladogram of Figure 4C is consistent with the conventional genealogical tree, reconstructing P. avitus as ancestral to Limulitella bronni, and it in turn as ancestral to Limulus polyphemus (St0rmer, 1952, 1955), but this interpretation is based on little more than the occurence of P. avitus in the Permian, Limulitella bronni in the Triassic, and Limulus polyphemus in the Recent. More careful character analysis turns up apparent synapomorphies arguing for both cladograms in Figure 4C. P. avitus and Limulus polyphemus both exhibit the derived conditions of having an enlarged and elevated pleurite on the opercular (O 2 ) segment and of having lost the moveable marginal spine located just posterior to this. Though it has not been recognized previously, Limulitella bronni shows the complementary primitive conditions, with an opercular pleurite similar to more posteriorly located pleurites and with the associated spine present. Limulitella bronni and Limulus polyphemus, on the other hand, both exhibit the derived condition of having relatively widely spaced ophthalmic ridges and more complete effacement of topography on the opisthosomal axial and opisthophthalmic ridges. Morphologic analysis of this case offers no clear answer. A somewhat dubious argument can be made that the effacement of topography is the least dependable character. It has certainly proceeded to a different extent in Limulitella bronni and Limulus polyphemus, and if we were to bring in morphological evidence on other taxa, we would find clear examples of convergence in effacement. However, without additional synapomorphies, a choice between the cladograms of Figure 4C would be difficult.

11 HORSESHOE CRAB PHYLOGENY 57 Functional analysis, on the other hand, opens another dimension for comparison. The widely spaced ophthalmic ridges of Limulitella bronni and Limulus polyphemus are related to an increase in the relative ize of the basal masticatory podomeres (gnathobases) of the prosomal appendages, and probably have to do with a trophic adaptation (coarser and/or larger food items). The loss of spines on the opisthosomal ridges has to do with drag reduction during swimming. The first of these explanations offers no strong independent evidence on either homology or polarity. The second strongly confirms the morphological analysis of polarity, but is indeterminate on homology. It is clear, however, that the two derived characters shared by Limulitella bronni and Limulus polyphemus represent functionally independent modifications. In contrast to this, the characters shared by P. avitus and Limulus polyphemus show a clear, though not mutual, dependence. The enlarged and elevated opercular pleurite is oriented in such a way that it roofs over a narrow channel, developed between the posteromedial margin of the genal angle and the anterolateral margin of the opisthosoma, which serves as the incurrent site for respiratory currents that are maintained during shallow burial (Eldredge, 1970). Some such "roof is required to keep this channel from being continually clogged by sediment (even with it, the channel must be occasionally cleared by protrusion of the distal end of the sixth prosomal appendage), and I would expect it to develop along with an increase in the importance or frequency of burial in a horseshoe crab behavioral repertoire. The moveable marginal spines are part of a very different system. They are mechanoreceptors which play a role in righting behavior, but whose distal tips also just touch the substrate during scuttling. Integration of information from them allows the opisthosoma to maintain very precisely a posture, relative to the substrate, which maximizes the thrust produced by the retraction of the flap-like opisthosomal appendages. The modification of the opercular pleurites associated with burrowing behavior displaces the site of the anteriormost moveable marginal spine far dorsally, making it difficult or impossible for it to maintain the same relationship to the substrate. In fact, its presence in any form would interfere with the flow of water through the incurrent aperture and with the channel maintenance movements of the sixth prosomal appendage. The reduction and loss of this spine is thus a predictable consequence of the differentiation of the opercular pleurite. Since these two characters are therefore not independent, the plausibility of them occurring together, through convergence, is greatly increased. Because of this functional dependence, they do not really represent two separate steps. Therefore, the characters shared by Limulitella bronni and Limulus polyphemus are more readily accepted as synapomorphous, and I am led to prefer the solid cladogram of Figure 4C. Phylogenetic trees involving xiphosurans It should be obvious by now that if cladograms are constructed by determining the set of most parsimonious trees, there is nothing left to do at tree level after the cladogram is finished unless, of course, we bring additional data to bear on the problem. If, and only if, each OTU has been attributed with one or more autapomorphies during the analysis, the parsimony criterion will have narrowed down the choice of phylogenetic trees to certain ones that are consistent with the one genealogical tree that is isomorphic to the chosen cladogram (otherwise, certain phylogenetic trees consistent with one or more other genealogical trees will be equally parsimonious). Even when autapomorphies are recognized in all OTUs and only one hypothesis of synapomorphy has been rejected by the parsimony criterion, two equally parsimonious trees will remain. In the case of the first horseshoe crab example considered above (where autapomorphies were not discussed), these two phylogenetic trees are representative respectively of two residual classes of equally parsimonious trees, which can be differentiated as follows: (1) ones in which opisthosomal fusion is considered convergent

12 58 DANIEL C. FISHER in P. avitus and E. danae; and (2) ones in which the mobile opisthosomal segments of B. koenigianus are considered a reversal to the primitive condition. There are, of course, other phylogenetic trees that might be picked out by one of these criteria, but that are less parsimonious. In this case, although comparative morphological analysis cannot make any further headway in tree selection, the functional arguments available for homology and polarity clearly favor trees in the first group mentioned above. They allow this greater resolution by bringing in a different kind of evidence. Under the "best" circumstances, they would be able to select a single most parsimonious phylogenetic tree. On the vexed question of whether or not ancestors can be recognized, the present approach to phylogenetic trees does not provide the necessary resolution. That is, under no circumstances, with the parsimony criteria used thus far in this discussion, will a phylogenetic tree hypothesizing an OTU as an actual ancestor be more parsimonious than one (or more) of the phylogenetic trees isomorphic to the cladogram. However, the entry of other factors into the parsimony evaluation could significantly alter this (see below). Scenarios involving xiphosurans Since I have argued that scenarios are involved, implicitly if not explicitly, even in the construction of cladograms, it is not surprising to find that they, like trees, need not now be elaborated upon in any great detail. Functional analysis is, almost by definition, involved in the complex hypotheses which comprise scenarios, but the really important issue is whether it can be used rigorously to test and reject scenarios {i.e., render them less parsimonious). I have certainly argued as if it can be, but this is considered in greater detail below. DISCUSSION As noted by Cracraft (in this symposium) the distinction between adaptation as a result of evolution and adaptation as a process of evolution is both familiar and useful. It may also be an artifact of looking at systems in an insufficiently dynamic way, but it can still play a part in discussions such as this. Adaptation-as-result involves relationships between various features of an organism, and between the organism and the context in which it occurs (Bock and von Wahlert, 1965). TheS# relationships are just as much attributes of an organism as are spines and legs, though they do have a higher, or at least a different, dimensionality. For Recent organisms, they can be investigated by direct experimentation or observation. We can test our understanding of these relationships by using laws of geometry and physics to develop, from observations on one set of features (morphological, behavioral, etc.), predictions concerning the state of expression of other features. Although testing is usually more difficult in the context of fossil organisms (simply because fewer attributes can be observed directly); I have argued (Fisher, 19756, 19776, 1979) that the same type of hypothetico-deductive approach can be applied in studying their adaptations. All of this is quite independent of studying adaptation-as-process. It makes no assumption that adaptation-asprocess is the only important mechanism of evolution, or even that it is the dominant one. It is part of the nature, or structure, of relationships, including the relationships we call adaptation, to have consequences in a temporal as well as a spatial dimension. In any given case, often depending on its complexity, we may or may not be able to deduce what those consequences would be. However, through studies of both natural and artificial selection, using Recent organisms, any such predictions that can be made, can also be tested, at least "in the small." I do not at present see the necessity or the possibility of arguing that such observations give us direct insight into the longer term effects of selection, or into the importance (relative to other factors) of adaptation as a mechanism of evolution. Nevertheless, they do seem sufficient to test our understanding of the potential effects of adaptation-as-result, ceteris paribus. They give us a more or less informed basis on which to construct scenario level hypotheses concerning

13 HORSESHOE CRAB PHYLOGENY 59 the direction (not the magnitude) of the influence of adaptation-as-result on evolutionary history. In order to operate in the phylogenetic context I have proposed, these scenario level hypotheses take one w two forms. Hypotheses involving questions of polarity must be able to argue that relationships between features of an organism tend to constrain the direction of character transformation, allowing us to reject as less parsimonious those scenarios involving transformations inconsistent with those constraints. There are surely many cases in which this relatively stringent demand cannot be met and in which transformation in either direction thus appears equally plausible. Any hypothesis of polarity may be falsified at the scenario level (just as one hypothesis of synapomorphy may be said to falsify another), by an alternative hypothesis with contrary predictions (based, for instance, on physical principles not taken into account in the first). As discussed below, it may also be falsified at tree level, by a character distribution with conflicting implications. It may not, however, be falsified by finding that the condition hypothesized as primitive persists in some taxa. The polarity hypothesis is conditioned upon the occurrence of change. It suggests that the direction of change is constrained, without necessarily predicting that change will occur. Hypotheses involving questions of homology, on the other hand, must be able to argue that the interactions between particular features of an organism are such that the presence or absence of those features can or cannot be treated as independent events. Effective independence between two features can arise either through minimal interaction between them, or through a dependent relationship with the structure of a one-to-many mapping (i.e., the phenomenon of multiple solutions; the distinction between these two forms of "independence" disappears if several levels of organization are considered). Again, these hypotheses can be falsified at either scenario or tree level. Their interpretation, however, is somewhat more complex than for hypotheses of polarity. If it can be argued that features are independent, then this corroborates the parsimonious tree argument (which counts features as independent) by which their homology was hypothesized. If it is argued that features are not independent, this does not necessarily imply that their cooccurrence in two taxa is not homologous. It only means that trees which postulate an independent origin of the features do not thereby incur a "parsimony debt" as great as the number of features being considered. Hypotheses of homology and polarity based on functional analysis could be applied to the analysis of any character for which they have been developed. However, in the examples I have drawn upon here, I have allowed these hypotheses to be decisive only in situations where comparative morphological evidence was indeterminate. While this may raise the problem of phylogenetic reconstruction to a higher level of universality, it does not require an assumption that natural selection dominates as a mechanism of evolution. Even if the constraints imposed by adaptation on tree construction were treated as being much less important than those imposed by observed morphology itself, consideration of functional relationships could "tip the balance" if it were otherwise even. The ceteris paribus clause would still be necessary to cover yet other factors, and the strategy of inference would not have changed. Even if functional analysis can be used appropriately in phylogenetic reconstruction, it can also be misused. The most obvious place for it to go astray is at the beginning. The functional analysis itself may suffer in credibility as a result of: inadequate testing; failure to consider relevant geometrical, physical, or physiological principles; or failure to consider the whole animal and its whole behavioral repertoire. Even a credible functional analysis may be unsuccessfully applied, if it does not have the components or the structure necessary to exclude at least one group of competing scenarios. Manton (1977), for instance, has been outspoken in her claims for the relevance of functional analysis for phylogenetic reconstruction, but has produced arguments which have the form: feature x

14 60 DANIEL C. FISHER in A functions differently from the rather similar feature x' in B; x cannot be derived by a plausible modification of x', nor can x' be derived from x; therefore x and x' cannot be homologous. Even if we accept the two initial premises (which is not always possible), the conclusion does not follow, because the argument does not exclude the possibility that x and x' are homologous, but that their functional relationships (like details of their morphology) have been modified independently from the homologous condition x in their most recent common ancestor (see Platnick, 1978, for other criticisms). The case of the occipital band, discussed above, provides an example of such a transformation. Even when an argument of appropriate design is available, it may be misused. Eldredge has quite correctly pointed out that having a ready explanation of the functional significance of one character may result in our accepting a convergent or parallel origin for it more readily than for a character whose "functional significance remains obscure to us" (1979, p. 179). However, the functional arguments I have used are fundamentally comparative. They do not operate by virtue of one character "having function" and another incongruously distributed character "not having it." Rather, they rely on the perception of differences between the structure of functional relationships associated with each of the characters. Just as in comparative morphology, if one of the elements to be compared is "obscure," then no comparison can be made. I do not mean by this that perfect clarity is any more realizable a goal in functional analysis than in other pursuits only that testable hypotheses must be available on each side in order to make a meaningful comparison. Problems related to this appear to be associated with the a priori weighting scheme advocated by Hecht (1976) and Hecht and Edwards (1977). Their categories of characters seem to be far from mutually exclusive and to require comparison of very heterogeneous entities. For instance, I am not sure that 1 could argue that a given suite of characters was either developmentally integrated, or functionally integrated, or innovative and unique; and even if I could, I am not sure that their order of weighting would always be most appropriate. Finally, even well designed scenarios based on thorough comparative function^ analysis can be inappropriately construed if they are represented as privileged insight, unfalsifiable by comparative morphological data. Exactly when to consider them falsified is a more complex issue, but they must be potentially falsifiable on these grounds. The desirability of phylogenetic reconstructions that are minimally dependent on assumptions concerning particular mechanisms of evolution can hardly be doubted (Eldredge and Cracraft, 1979), since such reconstructions would provide some of the most powerful tests of hypotheses concerning those mechanisms of evolution. Although phylogenetic inference using only comparative morphology has frequently been represented as being independent of these "inappropriate" assumptions, I have argued that it actually proceeds by assuming that the effects of adaptation (or of any other constraints on the structure of trees) are negligible on the scale at which the reconstruction takes place. Given this, let us imagine the design of a test of natural selection. To begin with, let us assume that we reconstruct a reasonably well corroborated phylogeny, without recourse to data other than morphology (or analogous attributes). We then consider a character for which we are able to develop a strong hypothesis of polarity, based on functional analysis, and which also successfully passes tests for its homology in two or more OTUs. What happens if the distribution of this apparent synapomorphy turns out to be incongruent with the previously constructed hypothesis of relationships? If we consider ourselves limited to the cautious application of functional analysis exemplified above, there would be little choice but to consider the synapomorphy based on functional analysis to have been falsified. Ordinarily, we might consider only the specific hypothesis of polarity or homology to be mistaken, but if we are explicitly testing natural se-

15 HORSESHOE CRAB PHYLOGENY 61 lection, we will presumably have chosen the (compound) hypothesis of synapomorphy to be as strong as possible. Do we then accept natural selection as having been falsified? Should we scrap it and start again? ^r is it possible that the ceteris paribus were not paribus (if I may be excused a perversion of the ablative absolute)? This possibility obviously should not be invoked capriciously, but if we do not examine it rigorously to begin with, we will have to sooner or later. We could, of course, seek other ways of corroborating the original reconstruction of relationships. Stratigraphic and geographic data may also represent constraints on our reconstruction of trees and are certainly independent of adaptation. If they were incorporated into a model analogous to what I have discussed here, and if they were accompanied by an appropriately modified parsimony criterion (to be discussed elsewhere), these data could resume a role not totally unlike that which they have traditionally held in paleontological approaches to phylogenetic inference. Ultimately however, we must still come back to adaptation (along with any other potential influence on the pattern of evolution). If, in contrast to the strategy assumed above, our test of natural selection had involved corroboration of the original phylogenetic reconstruction, by functional analysis, we would now be in a position to speak much more definitively of falsification. In fact, we would have to choose between: rejecting the supposedly well tested homology of the various occurrences of the "test-synapomorphy"; rejecting the reconstructed phylogeny and its corroborating functional analysis; or rejecting either the test's analysis of polarity or the efficacy of natural selection itself. Common experiences of this sort could not speak well for the role of adaptation in influencing the larger scale course of evolution (or else for our perception of its influence). I therefore come to the conclusion, counter-intuitive though it may be, that we are in a better position to evaluate critically the role of adaptation when we do incorporate its analysis into our reconstruction of phylogeny than when we do not. This in turn would suggest an important role for functional analysis even when comparative morphological evidence is not indecisive: Through this strategy, comparative functional analysis can be seen to have a foundation analogous to that of comparative morphology and a degree of evolutionary significance that has often, of late, been thought beyond its reach. ACKNOWLEDGMENTS I have appreciated comments from a number of persons on the material presented here: J. Cracraft, N. Eldredge, J. S. Farris, S. J. Gould, G. Nelson, R. T. Schuh, and J. A. Slater. This work was completed during tenure of a grant (DEB ) from the National Science Foundation. I also thank C. S. Darling for technical assistance, D. Robins for typing the manuscript, and K. Steelquist for photographic assistance. REFERENCES Bock, W. J Foundations and methods of evolutionary classification. In M. K. Hecht, P. C. Goody, and B. M. Hecht (eds.), Major patterns in vertebrate evolution, pp Plenum Press, New York. Bock, W. J. and G. von Wahlert Adaptation and the form-function complex. Evolution 19: Camin, J. H. and R. R. Sokal A method for deducing branching sequences in phylogeny. Evolution 19: Cracraft, J. and N. Eldredge. (eds.) Phylogenetic analysis and paleontology. Columbia Univ. Press, New York. Eldredge, N Observations on burrowing behavior in Limulus polyphemus (Chelicerata, Merostomata), with implications on the functional anatomy of trilobites. Amer. Mus. Novit. No. 2436:1-17. Eldredge, N Revision of the suborder Synziphosurina (Chelicerata, Merostomata), with remarks on merostome phylogeny. Amer. Mus. Novit. No. 2543:1-41. Eldredge, N Cladism and common sense. In J. Cracraft and N. Eldredge (eds.), Phylogenetic analysis and paleontology, pp Columbia Univ. Press, New York. Eldredge, N. and J. Cracraft Introduction to the symposium. In J. Cracraft and N. Eldredge (eds.), Phylogenetic analysis and paleontology, pp Columbia Univ. Press, New York. Farris, J. S On the phenetic approach to vertebrate classification. In M. K. Hecht, P. C. Goody, and B. M. Hecht (eds.), Major patterns in vertebrate evolution, pp Plenum Press, New York.

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