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© 2004 Society of Systematic Biologists
Accepting Partnership by Submission? Morphological Phylogenetics in a Molecular Millennium
Edited by Mike Steel: Associate Editor
University Museum of Zoology, University of Cambridge Downing Street, Cambridge CB2 3EJ, United Kingdom; E-mail: raj35{at}cam.ac.uk
Received July 31, 2003; Revised October 5, 2003; Accepted November 26, 2003 Organismic and morphological approaches to biology have lately come under attack from a variety of corners in both teaching and research. However, the main motivations for these attacks are not scientific but are instead reflective of economic pressures on teaching and the current fashionability of molecular approaches in research. For example, over the last few decades the number of students taking courses in invertebrate zoology in North American universities has declined significantly. This decline has largely been abetted by the abandonment of classes with small enrolments as increasingly more students are encouraged to take classes in the more fashionable disciplines of molecular and cell biology (Fautin and Watling, 1999). More recently, the fact that systematics is a time-consuming science dependent upon the mastering of specialist knowledge has been cited as a motivation for redirecting research toward DNA taxonomy (Pennisi, 2003; Tautz et al., 2003), and the utility of morphology for phylogeny reconstruction has recently been questioned on several grounds (see Hillis and Wiens, 2000; Baker and Gatesy, 2002).
The most recent challenge to the value of morphology for phylogenetics was published in a recent issue of this journal (Scotland et al., 2003). After a critical look at morphological phylogenetics, Scotland et al. (2003:543) reached a strong conclusion: "We disagree that morphology offers any hope for the future to resolve phylogeny at lower or higher taxonomic levels." Instead, they advocated a "more limited role for morphological data in phylogeny reconstruction" (2003:545), which consists chiefly of studying fewer morphological characters in the context of molecular phylogeny. Because the validity of these arguments would impinge upon morphological phylogenetic research ranging from algae to elephants, a detailed response to Scotland et al. (hereinafter SEA) is necessary.
SEA adduced several arguments in support of their contention that morphology has little if anything to offer to the future of phylogeny reconstruction and contended that we should be investing our time and effort into obtaining more molecular phylogenetic data. Here, I evaluate the main arguments of SEA and expose several weaknesses in their reasoning, the correction of which leaves the importance and future promise of morphology for phylogenetics fully intact.
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Numbers of Characters, Phylogenetic Accuracy, and Clade Support |
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The coordinating themes of the critique of SEA are their concerns for phylogenetic accuracy and quantitative clade support. SEA merged these two aspects of phylogenetic analysis into a universal yardstick that they used to assess the relative value of molecules and morphology. SEA (2003:539–540) claimed that in general both accuracy and support are positively correlated with the number of phylogenetic characters. First, SEA (2003:539) concluded that "the number of characters needed in simulation studies to recover accurate trees is an order of magnitude greater than that available from morphology." Second, SEA (2003:540) claimed that "the low character/taxon ratio in many morphological studies itself precludes high support values." Do these findings provide an irrefutable basis for discarding morphological evidence from phylogeny reconstruction?
Phylogenetic Accuracy
SEA judged the relative worth of molecular and morphological data with respect to the accuracy of phylogeny reconstruction. They asserted that increasing phylogenetic accuracy generally depends on increasing the number of characters, as is summarized in their figure 1a. As SEA made clear throughout their article, their main contention is that morphological data are inferior to molecular data in contributing accurate phylogenetic signal.
Despite comparing the performance of morphology versus that of molecules with respect to phylogenetic accuracy, SEA failed to discuss how phylogenetic accuracy can be estimated. Phylogenetic accuracy can only be assessed when the true phylogeny is known prior to the analysis. Phylogenetic accuracy is therefore assessed either by simulation studies or by study of known phylogenies (laboratory strains) (Hillis et al., 1994a, 1994b; Hillis, 1995; Givnish and Sytsma, 1997). Consequently, given the typical limitations of available molecular or morphological evidence for a particular set of taxa for which we do not know the phylogeny, we simply cannot know whether our phylogenetic estimate is accurate, i.e., represents the true phylogeny. This realization seriously compromises the value of phylogenetic accuracy as an empirical guide in most phylogenetic studies (Siddall and Kluge, 1997; Siddall, 1998; Farris, 1999).
SEA's arguments about phylogenetic accuracy are based on simulation studies, summarized in their figure 1a. SEA (2003:542) stated that "DNA sequence data at least offer the unique potential of scoring large numbers of unambiguous characters and character states." SEA stated (2003:541) that in contrast to this bounty of sequence data, simulations show that "the number of unambiguously coded morphological characters for any study is finite ... and less than the number typically required to accurately reconstruct phylogenies in simulation studies." However, simulation studies can reliably assess phylogenetic accuracy only when the data evolve precisely according to the assumed model of evolution (Hillis et al., 1994a; Hillis, 1995). If the data do not evolve according to the specified model, then the phylogeny may not be accurately reconstructed, and the number of characters necessary to accurately reconstruct the phylogeny cannot be correctly estimated. The number of characters needed to accurately reconstruct a phylogeny varies according to the nature of the data at hand, the number of taxa, and the chosen model of evolution (Hillis et al., 1994a, 1994b, 2003; Hillis, 1996, 1998; Rosenberg and Kumar, 2003). Therefore, it is very difficult to generalize the number of characters needed to accurately reconstruct phylogeny in given instances. Models for sequence evolution have been selected as much (or more) for their computational convenience as for their approximation to reality (Farris, 1999:201, 202; but see Sullivan and Swofford, 2001). SEA wrote (2003:541) that "the exact processes underlying nucleotide substitution are more complex than the simple models used in phylogenetic reconstruction." This admission makes SEA's appeal to accuracy largely a moot point because widely employed methods cannot guarantee phylogenetic accuracy. With respect to morphology, SEA (2003:542) emphasized "our current inability to incorporate models of morphological evolution into phylogeny reconstruction methods," which makes it extremely difficult to determine the number of morphological characters necessary to accurately reconstruct phylogeny in specific cases. However, the development of new models for dealing with morphological data in a likelihood context (Lewis, 2001) may improve our ability to combine morphological and molecular data in single phylogenetic analyses.
In repeatedly contrasting molecules and morphology, SEA assumed that these are two distinct and clearly alternative categories of data for phylogeny reconstruction. The greater potential of molecules to contribute to increased phylogenetic accuracy is then chiefly a result of the genome representing a much bigger reservoir of potentially useful unambiguous phylogenetic data. However, is this anything more than a truism?
Millions or billions of genomic nucleotides offer a vastly bigger pool of potentially useful phylogenetic variation than the phenotype of even the morphologically most complex eukaryotes. By the same argument, one might as well conclude that because even entire mitochondrial genomes are typically dwarfed by the size of the nuclear genome, they are therefore inferior phylogenetic indicators. However, the quality of a data set and its size are two distinct attributes, and even limited data sets may contribute phylogenetic accuracy. In practice, molecular phylogenetic studies are typically limited to one or a few genes, the analysis of which is far more problematic than hinted at by SEA. Such limited data frequently fail to provide enough information for a robust and accurate phylogenetic estimate. For example, despite intense taxon sampling, the 18S ribosomal RNA locus has so far yielded no stable and maximally accurate hypothesis of metazoan relationships (Peterson and Eernisse, 2001; Giribet, 2002). This result is not surprising because different genes may evolve at different rates and may consequently be suited best for resolving different levels of a phylogeny. In a recent study, Rokas et al. (2003) made this potential particularly clear. They performed a phylogenetic analysis of 106 orthologous genes from seven species of yeast from the genus Saccharomyces. Their results (Rokas et al., 2003:798) suggested that "data sets consisting of single or a small number of concatenated genes have a significant probability of supporting conflicting topologies." Thus, single molecular data sets are potentially equally fallible with respect to accurate phylogeny reconstruction as are morphological data sets. Moreover, "the support for a given branch was strongly dependent on the gene analysed" (Rokas et al., 2003:799), which indicates that different genes are complementary in providing support for different parts of the phylogeny. In line with these findings, workers increasingly have adopted multigene approaches to phylogeny reconstruction to benefit from complementing data partitions that provide resolution at different levels in the phylogeny (Colgan et al., 1998; Edgecombe et al., 2000, 2002; Baker et al., 2001; Giribet et al., 2001; Giribet, 2002; Collin, 2003; Rokas et al., 2003), whereas each data set alone, be it molecular or morphological, may have only limited resolving power. So far, having a large proportion of the genome sequenced is an ideal that has only been realized for a few species.
However, even a multigene approach may not necessarily solve all problems when the number of genes is limited. Different genes may yield conflicting phylogenetic signals on all taxonomic levels across the tree of life for a variety of biological and analytical reasons, including choice of optimality criterion, comparison of paralogous genes, taxon sampling, mutational saturation and long branch attraction, changing substitution rates of a site across a gene and/or across a phylogeny, compositional biases, lateral gene transfer, similar selective regimes leading to molecular convergence, and lineage sorting, and the same gene may yield conflicting phylogenies when analyzed on the nucleotide or amino acid levels (Miyamoto and Fitch, 1995; Maddison, 1997; Naylor and Brown, 1998; Hillis and Wiens, 2000; Lockhart et al., 2000; Sota and Vogler, 2001; Sullivan and Swofford, 2001; Taggart et al., 2001; Gribaldo and Philippe, 2002; Lopez et al., 2002; Rydin et al., 2002; Shaw, 2002; Simmons et al., 2002a, 2002b; Machado and Hey, 2003; Rokas et al., 2003).
Consequently, not all parts of the genome necessarily uniformly and positively contribute to phylogenetic accuracy, and their combined use in a single phylogenetic analysis may therefore not lead to increased accuracy. This makes SEA's dichotomy between all molecular sequence data on the one hand and morphology on the other not particularly meaningful. SEA's conclusion (2003:539) "that a main constraint of morphology-based phylogenetic inference concerns the limited number of unambiguous characters available for analysis" is equally applicable to many available molecular data sets. Clearly, the well-known limitations of individual molecular data sets have not led to the general dismissal of molecular evidence. The acknowledged limitations of morphological data sets should therefore no more be used as an argument to dismiss the value of morphology for phylogeny reconstruction. Even considering the potential of large amounts of genomic information, we do not know whether we can reasonably expect genomes to furnish the required amount of data to resolve all phylogenetic problems on all phylogenetic levels. SEA (2003:543) acknowledged as much: "an honest observer would have to agree that even whole genomes for all species will probably not yield a fully resolved, highly confident tree."
Quantitative Clade Support
SEA (2003:540) argued that morphological phylogenetic studies generally have "too few morphological characters to provide confidence in any given estimate of phylogeny." SEA generalized the findings of Bremer et al. (1999) and a few other studies in their figure 1b and claimed (2003:540) that these results "demonstrated explicitly that the character/taxon ratio for morphological studies is such that bootstrap percentages are likely to be low." This statement holds true for the morphological data sets of two plant families analyzed by Bremer et al. (1999). These data sets had low numbers of characters: 3.2 and 2.6 characters/taxon for Rubiaceae and Apocynaceae, respectively. Bremer et al. (1999) cited the low number of available characters as the cause of the low support measures for the phylogenetic analyses of these data sets. The bigger molecular data sets had better support values. I appreciate the potential value of statistical support measures, leaving aside difficulties of interpreting commonly used support values such as bootstrap percentages (reviewed by Sanderson, 1995; Siddall, 2002) and difficulties of justifying the use of probabilistic arguments in historical inference in the first place (Siddall and Kluge, 1997; Swofford et al., 2001; Kluge, 2002). Morphology may often provide more coarse grained phylogenetic resolution than molecular data because of lower character/taxon ratios, but this does not necessarily lead to lower quantitative support measures. It is not only the number of characters but the distribution of homoplasy that will determine how well a given data set supports a phylogeny. Therefore, one cannot uncritically generalize the findings of Bremer et al. (1999) that small morphological data sets are poorer performers than larger molecular data sets.
Kress et al. (2001) analyzed a morphological data set for the Zingiberales (a group of monocotyledons including bananas and gingers) with 2.8 characters/taxon and found that 57% of the ingroup nodes were supported with bootstrap values >75%. In contrast, two of three analyzed molecular data sets for this group generated lower percentages of ingroup nodes with bootstrap values >75%, despite a much larger number of characters. It is unwarranted to generalize from a few morphological data sets discussed by SEA to the conclusion (2003:540) that high support values are precluded "in many morphological studies." Whether high support values are generated depends entirely on the morphological data set under consideration. In a recent comprehensive morphological analysis of snake phylogeny, Lee and Scanlon (2002) found that >70% of the ingroup nodes had bootstrap values of >75%. Similarly, in a morphological phylogenetic analysis of arthropod relationships Edgecombe et al. (2000) also found that >70% of the ingroup nodes had bootstrap support values of >75%, and high quantitative support values for morphological analyses are certainly not rare (Wiens and Hollingsworth, 2000; Damgaard and Sperling, 2001; Kress et al., 2001; Gatesy et al., 2003). SEA presented no convincing evidence that morphological phylogenetic evidence is generally undesirable because "the low character/taxon ratio in many morphological studies itself precludes high support values" (2003:540). Data sets of single molecules may provide contributions to clade support as limited at that provided by morphological data sets. The important point is that the available character space should be sampled as comprehensively as possible, and morphology may contribute valuable evidence in combination with molecular data.
Although SEA acknowledged (2003:545) the potential value of combined analyses of both molecular and morphological data, they failed to mention that in the study of Bremer et al. (1999), from which they adapted their Figure 1a, the support measures of the combined analyses of molecules and morphology were significantly better than those of either the morphological or molecular data sets alone. Similar positive contributions of morphology to quantitative clade support measures in combined analyses have been observed for a host of other taxa across the tree of life (Flores-Villela et al., 2000; Kress et al., 2001; Winterton et al., 2001; Edgecombe and Giribet, 2002; Giribet et al., 2002; Stach and Turbeville, 2002; Jeffery et al., 2003; Schulmeister, 2003; Wahlberg and Nylin, 2003). The positive contribution of morphology to combined analyses may also become apparent from other measures, such as partitioned branch support values (Baker et al., 1998; Gatesy and Arctander, 2000; Damgaard and Sperling, 2001; Murrell et al., 2001; Gatesy, 2002; Meier and Baker, 2002; Remsen and O'Grady, 2002; Gatesy et al., 2003; Wahlberg and Nylin, 2003).
Even though morphological data sets may contain relatively small numbers of characters in comparison to the total amount of included molecular data, morphology may contribute more support and stability to the combined analysis when considered per character (Baker et al., 1998; Gatesy, 2002; Meier and Baker, 2002). For example, Baker et al. (1998) and Meier and Baker (2002) found that morphology may be up to twice as informative as molecules per included character in a combined analysis across a wide range of different taxa.
Morphology also may contribute to combined data analyses in the form of hidden clade support that becomes apparent only when data sets are combined (Gatesy and Arctander, 2000; Damgaard and Sperling, 2001; Murrell et al., 2001; Gatesy, 2002; Gatesy et al., 2003). Thus, morphology may be partially incongruent with molecular data when analyzed separately, but when combined the total evidence may generate a well-supported phylogeny. For the same reason combination of apparently conflicting molecular data sets may lead to a single well-supported phylogeny (Rokas et al., 2003).
In some cases, morphology and molecules supply complementary evidence, where each type of data may contribute to different parts of the phylogeny (Giribet and Wheeler, 2002). A strength of morphology is that characters informative at many different levels in the phylogeny are habitually included in a single data set, whereas the average substitution rate of a given gene may limit its utility to particular levels in a phylogeny. One or a few genes may not provide sufficient phylogenetic signal across all levels in a phylogeny, especially when the included taxa span a broad range of divergence dates. In other cases, morphology may contribute unique phylogenetic signal where molecules are completely mute (Mattern and McLennan, 2000). In some combined analyses, morphology may increase support of certain nodes while lowering that of others (Klompen et al., 2000; Stach and Turbeville, 2002), and in other cases morphology may not add any significant support to combined data analyses (Sorhannus, 2001). Although conflict between morphological and molecular evidence may occur and some workers remain skeptical about combining morphological and molecular data into a single phylogenetic analysis (SEA did not object to combined analysis of morphological and molecular data; 2003:545), these examples clearly show that morphological data may improve clade support values.
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Homology Assessment and Character Coding |
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SEA nominated the limited number of unambiguous characters as the main shortcoming of morphological phylogenetic data. To make matters even worse, SEA indicated that any attempt to solve this problem by increasing the number of morphological characters will be doomed to failure. First, SEA (2003:545) concluded that "much of the useful morphological diversity has already been scrutinized." Although this statement may be true for well-studied taxa such as the seed plants discussed by SEA, there is no indication that it holds true for other poorly studied taxa. The widespread use of new morphological data in phylogenetic analyses of many different taxa leaves no doubt that much phenotypic variation remains to be explored (Lee, 1995; Klass, 2001; Hooge et al., 2002; Giribet et al., 2002; Sørensen, 2002; Strong, 2003).
Second, SEA (2003:541) argued that the need to properly code and define morphological characters will only add "a level of subjectivity and interpretation" to the analysis, which merely "increases the level of ambiguous or problematic characters." SEA (2003:542) concluded: "For morphological studies comprising a relatively high number of characters, both character coding and character conceptualization become increasingly important variables that may have a negative impact on a study as more characters are added (Fig. 1)." SEA buttressed their conclusions with figures 1c and 1d, which show that ambiguity of both homology assessment and character coding will quickly increase with the addition of more characters.
These arguments erect nothing but a straw man. SEA fabricated these graphs without marshalling any evidence to support them. There is nothing to support SEA's contention that increasing the number of characters in a morphological data set will necessarily lead to increasingly ambiguous homology determination and character coding. The only example SEA (2003:542) presented where an increase in the number of morphological characters "made no significant difference to the results" scarcely provides the basis for these generalizations applicable across the tree of life. In contrast, I expect an increase in the quality of morphological phylogenetic data with time as the development of new analytical techniques opens up unexplored sources of data and facilitates the validation of older information. For example, in metazoan phylogenetics powerful techniques such as transmission electron microscopy, molecular developmental biology, and cell lineage tracing are contributing to an improvement of the morphological data sets as new data are added and old mistakes are corrected (e.g., Giribet, 2002; Jenner, 2004).
The main problem of SEA's interpretation here is that they assumed that ease of homology assessment and character coding can be regarded as proxies of a single, general phylogenetic signal in a data set and are as such related as single values to the number of characters in an analysis. However, data sets do not contain a single phylogenetic signal. The phylogenetic signal in a data set is hierarchically structured, and different characters are significant on different phylogenetic levels. Certain characters are therefore relevant for placing certain taxa but are irrelevant for placing others. Consequently, there is no simple relationship between an increase in the number of morphological characters and the ambiguity of character coding and homology assessment that can be captured in a graph, such as figures 1c and 1d, and that is valid throughout the whole phylogeny.
SEA's arguments about defining and coding characters are reflective of a deplorable appreciation of the morphological scholarship that is at the heart of phylogenetics. This attitude mirrors a worrying statement in the chapter on animal phylogeny in the recently published new edition of the widely used textbook on invertebrate zoology by Brusca and Brusca (2003:874): "The process of a priori character assessment is perhaps the weakest link in morphological phylogenetic biology." However, character assessment is the only empirical anchor of phylogenetics. If properly conducted, comparative morphological study will be the greatest strength of a phylogenetic analysis, and when performed less than critically it may be the greatest weakness. The objectivity of morphological phylogenetic analyses lies in intersubjective testing, in which previously compiled data are reanalyzed and reevaluated for their congruence with other characters (Kluge, 1997, 1998; Ax, 1999). SEA's recommendations of greater explicitness in character selection and care in morphological studies will naturally contribute to the quality of morphological phylogenetic analyses.
With respect to character coding and character conceptualization in molecular phylogenetics, SEA (2003:541) concluded that for "aligned sequence data, there is no ambiguity in assessing character states," and "there is no ambiguity that the unit of comparison is the nucleotide." In contrast, the definition of homologous characters and the coding of character states in morphological phylogenetics is much more problematic. However, the important issue is not only whether two nucleotides at the same position are identical but also whether they are historically identical, i.e. homologous. Nucleotides are characters of relatively low complexity, and the character state space for nucleotides is much more restricted than that for morphology. In certain circumstances, this restriction creates a considerable danger that the same nucleotide has evolved independently in the same position. This realization has been an incentive to develop models of evolution that estimate the probability that the same nucleotides at a site are historically identical, to explore the value of more complex molecular characters (Swofford et al., 1996; Rokas and Holland, 2000), and to develop alignment methods that use comparative molecular anatomical information (secondary and tertiary structure) to improve the probability of recovering homology (Kjer, 1995; Hickson et al., 2000). In contrast, morphology generally presents a richer space of more complex characters that allows a more fine-grained comparison of potential homology, which may help explain why in certain cases morphology may be qualitatively superior to molecules when considered per character (Baker et al., 1998; Gatesy, 2002; Meier and Baker, 2002).
The definition and coding of morphological characters is certainly a difficult subject, and for a given set of taxa alternative approaches to homology assessment and character coding may indeed lead to different phylogenies. However, as the articles cited by SEA indicate, there is no easy solution to this problem, and subjective choices are unavoidable. One way to deal with this issue is to perform a sensitivity analysis with different character codings to see how the phylogeny changes with changing character codings (Jenner, 2002; Rieppel and Kearney, 2002; Simmons and Geisler, 2002). This approach at least allows a better understanding of the relationship between assumptions and hypothesis.
Most important in the context of this critique, however, is that despite the admitted difficulties of morphological character analysis, there is no evidence to suggest that morphology generally performs more poorly than molecules in estimating phylogeny (Hillis and Wiens, 2000; Baker and Gatesy, 2002). SEA (2003:543) cited an example where molecular evidence from three genes is taken to suggest that phylogenetic analyses of morphology and fossils have led to inaccurate phylogenetic hypotheses of the angiosperms. Although this statement may be true, we cannot automatically conclude that morphology always suggests the wrong answer whenever molecules and morphology conflict. In many cases different molecular analyses may conflict with each other (Sota and Vogler, 2001; Taggart et al., 2001; Rydin et al., 2002; Shaw, 2002; Machado and Hey, 2003; Rokas et al., 2003), and morphology may be more reliable than some molecular data partitions (Wiens and Hollingsworth, 2000; Damgaard and Sperling, 2001). The findings of Rokas et al. (2003) potently illustrate that data from one or a small number of different genes may lead to a robustly supported phylogeny that may be in direct conflict with an alternative phylogeny based on another set of genes, even for relatively closely related species. In the context of such limited amounts of data we simply cannot know whether we have accurately reconstructed the true phylogeny. We can only sample character space as comprehensively as possible and find the most well-corroborated hypothesis.
SEA (2003:541) claimed that these problems of "subjectivity and interpretation" are absent from molecular data, because "areas of ambiguity [in sequence alignment] can be excluded." As recent research has shown, to choose this way of least resistance may be thoroughly misleading, and this short statement seriously underplays the degree of subjectivity and interpretation associated with molecular phylogenetics. Apart from the choice of included taxa, "subjectivity and interpretation" in molecular phylogenetics may reside in the choice of the gene, the decisions of how to determine homology when insertion and deletion events or exon shuffling occur, the choice of phylogenetic analysis parameters, the choice of whether nucleotides or amino acids are analyzed for protein-coding genes, and the choice of the optimality criterion. Different decisions for these variables may result in different phylogenies (Giribet and Wheeler, 2002; Giribet et al., 2002; Simmons et al., 2002a, 2002b; Rokas et al., 2003). Horizontal gene transfer and gene duplication may add further difficulties to homology assessment in molecular phylogenetic analyses. Moreover, SEA's suggestion to simply exclude all information associated with ambiguously aligned sequences may also cause misleading results, as illustrated by the following example.
For more than a decade, 18S ribosomal DNA (rDNA) sequences suggested that birds were most closely related to mammals (hematotherm hypothesis). This hypothesis was in conflict with results derived from a large amount of traditional (morphological and paleontological) and other molecular data, which instead united birds with crocodilians. After different workers analyzed the 18S data in various different ways, they concluded that this was an example of different molecules giving significantly different estimates of phylogeny. However, in a recent study Xia et al. (2003) convincingly showed that the conflict between 18S data and the traditional and other molecular data was an artifact attributable to two main factors: misalignment of sequences and inappropriate estimation of base frequency parameters. Crucial to the resolution of this paradox was the incorporation of information from the most variable regions of the 18S molecule that were most difficult to align unambiguously. This study clearly showed that restricting the data set to only the least unambiguous sites might produce a thoroughly misleading phylogeny. The problem that "different workers will perceive and define characters in different ways" (2003:541) is therefore certainly not limited to morphological data. The recent development of methods to deal with ambiguously aligned regions or to circumvent multiple sequence alignment altogether (for review, see Lee, 2001) further undermines the fundamental dichotomy between the value and treatment of molecular and morphological data as portrayed by SEA.
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Value of Morphology in Phylogenetics |
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SEA (2003:544) stated that "a continued role for morphology in phylogeny reconstruction seems a reasonable expectation," and they envisioned the role of morphology in systematics in one of three ways. In the first form, which is the one strongly recommended by SEA, fewer morphological features are studied in depth and are then mapped onto a molecular phylogeny "on the basis that morphological characters can be diagnostic for nodes on molecular trees" (2003:545). Each morphological character is studied separately with respect to a node in the molecular phylogeny, and characters found to be incongruent "are not incorporated into the phylogenetic hypothesis" (2003:545). SEA (2003:545) claimed that this approach "is akin to Patterson's (1982) congruence test." If one adheres to cladistics as a falsificationist research program, the problem here is the claim of congruence. Patterson (1982) was clearly concerned about the congruence of characters against all others. Standard cladistic analysis operates on the basis of character congruence, in which the congruence of all characters is assessed simultaneously. Only this approach will guarantee finding the globally most-parsimonious cladogram that embodies maximal explanatory power. By comparing individual morphological characters against an already resolved molecular phylogeny, one is not assessing overall congruence of characters, despite SEA's confusing invocation of Patterson's congruence test. If 99 of 100 characters were incongruent with the molecular phylogeny but entirely congruent among themselves, SEA's approach would nevertheless disregard them as homoplasies. This approach is more akin to clique analysis, the logical basis of which for phylogeny reconstruction was long ago questioned (Farris, 1983). This approach purposely removes any testing power from the phylogenetic analysis, which is in direct conflict with the falsificationist premises of cladistics.
Even if morphological characters were not directly used to reconstruct a phylogeny but, as suggested by SEA, were merely mapped onto a molecular phylogeny to "provide evidence for a more limited number of monophyletic taxa" (2003:545), we would still have to confront exactly the same problems of character definition and character coding that SEA nominated as the greatest weaknesses of morphological phylogenetic studies. Before characters can be mapped onto a molecular phylogeny as a limited number of additional synapomorphies, they must be defined. Just as decisions about character coding determine on what level in the phylogeny a character is informative, so too is the mapping of a character in a certain place in a phylogeny inextricably linked with particular assumptions about character coding. For example, SEA (2003:541) cited the famous "no tails, red tails, blue tails" example to illustrate that even for relatively simple characters it may be difficult to choose an appropriate coding method. For these data, multistate coding does not capture the variation of a tail being present versus absent (see discussion by Hawkins et al., 1997). Consequently, if we map the presence of tails as a synapomorphy on a particular level in a molecular phylogeny, we necessarily imply that the observed variation is not conceptualized as a multistate character. Thus, even though decisions about character coding are not explicitly made in a data matrix, implicit decisions cannot be avoided.
SEA recommended the study of fewer rather than more characters because rigorous character study is too time consuming. Today, the high pace of publication in the field of phylogenetics is principally set by molecular studies, where automated methods allow much faster data collection than can be achieved when studying comparative morphology. Unfortunately, this publication pressure may in some cases lead to the publication of phylogenetic studies based on hastily and uncritically compiled morphological data sets of low quality (Jenner, 2001, in press). However, we want to understand as much as we can about the evolution of morphology, and molecules, including features for which homology assessment is difficult. That difficult science can be time consuming can hardly be a scientific argument for studying fewer rather than more characters.
SEA's second and third proposed strategies for incorporating morphology into phylogenetics are actually the same. As a second approach, SEA advocated a continuation of current practice. That is, morphological and molecular data both have their value, and they are analyzed both separately and in combination to study evolution. The third strategy is similar except only "those characters that are unproblematic in terms of homology assessment and character coding are selected" (2003:545) and analyzed together with the molecular data. These two approaches actually provide the same possibilities as the first recommended approach, and as discussed with respect to combined analyses, more in addition. SEA realized that careful morphological study is valuable, and I could not agree more.
However, SEA's recommendation to select only unambiguously homologous morphological characters is problematic. All primary homology decisions and character state assignments are provisional and can in principle be corroborated and refuted. By concluding that the definition and coding of even simple characters can be problematic (the tails example), SEA themselves show that prior to a phylogenetic analysis there are no "unambiguously coded morphological characters" (2003:541). Workers who attempted to separate problematic from supposedly more reliable characters prior to a phylogenetic analysis confirm this. Their results show that the phylogenetic utility of characters is very difficult to predict a priori (Jenner and Schram, 1999: Table 3; Schander and Sundberg, 2001; Collin, 2003; Gatesy et al., 2003; Strong, 2003).
By concluding (2003:543) that morphology cannot offer "any hope for the future to resolve phylogeny at lower or higher taxonomic levels," SEA gave very little consideration to fossils. Although fossils were briefly discussed in connection with taxon sampling, SEA did not reach any firm conclusion about their value for phylogenetics other than that their contribution to phylogenetic accuracy is uncertain in view of the problems of homology assessment and character coding. However, the limited amount of molecular and morphological evidence typically available to us does not allow us to determine whether the true phylogeny has been accurately reconstructed in the first place. As an additional problem of including fossils in phylogenetic analyses, SEA (2003:543) cited the "large amounts of missing data." In contrast, Wiens (2003:536) concluded on the basis of simulation studies that "the proportion of missing data cells in the incomplete taxa is a poor indicator of their impact on phylogenetic accuracy." Accuracy is more related to the distribution of missing data in the matrix and the number of characters that can be scored for the incomplete taxa. As Baker and Gatesy (2002:171) concluded; "If morphological evidence is ignored, the phylogeny of over 99% of life is ignored." In certain groups, such as the certartiodactyls, >85% of the genera are known only from fossils (Gatesy, 2002). The importance of fossils for reconstructing phylogeny can perhaps best be illustrated by the importance of stem groups (Wills et al., 1998; Budd and Jensen, 2000; Budd, 2002; Holmer et al., 2002; Lee and Scanlon, 2002; Mallatt and Chen, 2003; Ruta et al., 2003). Stem groups exemplify the most basal divergences of extant taxa, and their value in bridging the often considerable morphological gaps between disparate body plans is of central importance to reconstructing the full diversity of the tree of life. With the exception of ancient DNA, rigorous morphological phylogenetic analysis is the only way in which fossils can be incorporated into the phylogeny of life. Failure to do so will leave phylogenies of extant organisms forever uprooted.
SEA (2003:545) admitted the value of reciprocal illumination (Hennig, 1966) in which independent data can shed unique light on a problem. However, they apparently fail to realize that by restricting morphological phylogenetic analyses, the power of reciprocal illumination is crippled. Phylogenetic analyses of different data sets provide valuable and unique independent perspectives on both phylogenetic questions and questions of character evolution. The existence of a dialogue between morphological and molecular hypotheses guarantees continued attention and excitement in phylogenetic research on many taxa on many different phylogenetic levels. For example, a recent phylogenetic analysis of 18S rDNA sequences in asellote isopods failed to yield results strongly expected on the basis of careful morphological study (Wägele et al., 2003). This discordance stimulated a more detailed look at the molecular data, yielding several interesting insights that may otherwise have remained invisible. Similarly, Vidal and Hedges (2002) recently analyzed snake phylogeny with nuclear and mitochondrial genes. They found that the morphologically strongly supported clade of macrostomatans (Lee and Scanlon, 2002) is contradicted by molecular evidence. Because some workers have claimed that most apparent conflict between molecular and morphological phylogenies is due to weak support for either or both of the estimates and that strongly supported and misleading morphological phylogenies are relatively rare (Hillis and Wiens, 2000; Wiens et al., 2003), such a potential case for strong conflict is worth exploring. It allows us to explore the possibility that in this case morphological phylogenetic analyses may suffer from the confounding effect of concerted homoplasy, possibly in the characters related to wide gape size. This reassessment may teach us something important about the extent of parallel evolution in a complex of adaptive features. These insights into evolution are dependent upon the compilation, analysis, and comparison of independently compiled molecular and morphological data sets.
Science derives its greatest strength from its multifaceted nature. Artificial restriction of approaches can lead only to impoverishment of science. Many researchers see the merits of analyzing molecular and morphological data sets separately and in combination to compare the results (Littlewood et al., 1999; Edgecombe et al., 2000; Flores-Villela et al., 2000; Janies, 2001; Kress et al., 2001; Wheeler et al., 2001; Winterton et al., 2001; Edgecombe and Giribet, 2002; Giribet and Wheeler, 2002; Giribet et al., 2002; Stach and Turbeville, 2002; Jeffery et al., 2003; Schulmeister, 2003; Wahlberg and Nylin, 2003). As SEA admitted, study of morphological characters is necessary anyway because one goal of phylogenetic analysis is to understand character evolution. We might as well submit the collected data to as many potentially fruitful uses as possible.
By discouraging morphological phylogenetics, we take away a main formal impetus for morphological comparative research. Combined with the continuing marginalization of organismic biology in academic curricula (Fautin and Watling, 1999), this attitude introduces the real danger that potentially interested students will not be attracted to systematic biology, and this dearth of students may seriously undermine the future and versatility of our science for largely nonscientific reasons.
SEA failed to present any convincing scientific arguments for narrowing the focus of phylogenetics to mainly molecular research. In writing that morphology "may not be able to resolve the full branch structure of the tree of life," SEA (2003:543) merely presented an empty statement, because no single data set, molecular or morphological, can reasonably be expected to be the Holy Grail of phylogenetics, as they themselves underscore by stating (2003:543) that even whole genomes "will probably not yield a fully resolved, highly confident tree." The only scientifically valid reason that I can think of for excluding or severely limiting the use of morphological evidence for phylogeny reconstruction as proposed by SEA is when we know a priori that these data will be positively misleading. SEA failed to show that we have this knowledge. Instead, they presented a few examples from botanical phylogenetics where the contribution of morphological data to resolving the phylogeny was limited. However, as shown here, these examples scarcely provide enough foundation to question the general value of morphology for other parts of the tree of life. In the absence of evidence to the contrary, I am convinced that an equal partnership between molecular and morphological phylogenetics is our best bet for future progress.
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I gratefully acknowledge helpful comments on the manuscript by Tim Collins, Chris Simon, Gavin Naylor, Richard Olmstead, Jonathan Bennett, and an anonymous reviewer. A Marie Curie Individual Fellowship of the European Community program Improving Human Potential under contract number HPMF-CT-2002-01712 supports my work.
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