Records of the Western Australian Museum Supplement No. 69: 23-32 (2006).

Comparison and cost benefit analysis of different data sets used for determining mollusc evolution: a case study of the cryptobranch Halgerda (: )

Shireen J. Fahey Department of Invertebrate Zoology and Geology, California Academy of Sciences, 875 Howard Street, San Francisco, California, 94103 USA. email: [email protected]

Abstract - Several approaches to examining the evolution of organisms have been tested over the past several decades. Specifically, morphological and molecular (DNA sequences) data sets are the most widely used methods to test a phylogenetic hypothesis. A cost benefit analysis of previously published studies of nudibranch mollusc phylogeny is presented. The previous studies utilized different types of data sets to derive hypotheses of relationships. The present cost benefit analysis examines the usefulness of the different data sets to verify an existing level phylogeny. The existing hypothesis had been derived from morphological data only. Results of the present analysis indicate that two of the four data sets used, DNA sequence data and morphological characters, are the most effective when measured in terms of robustness of the resultant phylogeny and considering cost and time spent on the analysis. The other two data sets: sperm morphology and natural product chemistry were found to be valuable at higher taxonomic levels. Conclusions from the cost benefit analysis indicate that combinations of data can provide valuable insight into evolutionary relationships, allowing the researcher to further examine a hypothesis from various perspectives. Those perspectives include the molecular evolution of the organism, morphological character evolution, chemical evolution and reproductive evolution at a cellular level. Using a multidisciplinary approach to systematics reveals the complexities of evolution.

INTRODUCTION tree can be identified and examined so that future Evolutionary biologists have proposed that research can be designed to assist in resolving those phylogenetic hypotheses based on as many nodes (Hillis 1987; Ponder and Lindberg 1995). If independent lines of evidence as possible provide phylogeny is intended as the basis of classification the strongest support for the hypothesis (Farris and hypothesis testing (de Queiroz and Gauthier 1983; Avise 1994; Ponder and Lindberg 1995; Moritz 1990), the highest level of resolution is a desirable and Hillis 1996). Lines of evidence that have been and necessary goal. However, there can be a trade­ considered in previous phylogenetic studies include off between pursuing an ever more robust morphological (using gross anatomy as well as hypothesis and efficiency of time as well as finite cellular level data, such as sperm ultrastructure and research dollars, as found during the present histological examination of organs), molecular analysis. (using a variety of mitochondrial and nuclear Robustness, as defined for the present analysis, genes) and ecological. Since evolution occurs in a refers to strength of support for a phylogenetic tree variety of ways, as evidenced from the many as measured by Bremer support (Bremer 1994) for previous studies (See for example the studies by the example. Penny et al. (1991) noted that tree-building above referenced authors and Bremer and Struwe methods must not only be robust and efficient, but 1992; Ballard and Whitlock 2004), it would seem fast and falsifiable. So, for the present analysis, that a multi-dimensional approach would yield efficiency means the ability to accomplish one's verified phylogenies. Certainly, more refined tools goal (of having a reliable test or measure in this are now available to resolve evolutionary case) in a reasonable amount of time, with hypotheses, such as natural product chemistry, minimum expense or waste. The definition of cost combinations of gene sequences and phyloecology for the present analysis is in terms of actual dollars (Cimino and Ghiselin 2001; Wagele et al. 2003; required to complete the analyses. Valdes 2004). The present cost benefit analysis examines One particularly beneficial use of multiple data phylogenetic robustness achieved by each of the sets is that poorly resolved nodes in a phylogenetic data sets (both individually and combined) 24 S.]. Fahey discussed herein, the efficiency of using each of the The literature contains considerable disagreement different data sets individually and of combining over combined versus separate analysis of these data sets to examine a phylogenetic molecular and morphological data (see Hillis 1987; hypothesis of an organism. Swofford 1991; de Queiroz et al. 1995; Nixon and Although comparative morphology has Carpenter 1996). However, Ponder and Lindberg historically formed the basis of , it is now (1995) state that whether or not the data are considered by some investigators to be a rather analyzed together or separately to test congruence, crude means of discriminating organisms. See for is a matter of personal choice of the researcher. example the discussion in Hillis (1987); Cracraft and Ponder and Lindberg believe that examining the Helm-Bychowski (1991) and de Queiroz et al. (1995). degree of congruence in two independently However the question is, will more refined acquired data sets is the way to test the robustness methods, such as DNA sequencing render of a hypothesis; whether the data sets are morphological studies as obsolete? Although morphological and molecular, or by comparing molecular analysis has given us a new approach to different sequence data, or by conducting a total study evolutionary relationships, even the cleverest evidence analysis (e.g. Swofford 1991; Ponder and observer cannot determine a DNA sequence Lindberg 1995; Moritz and Hillis 1996; Wagele et al. without sophisticated tools and techniques. Thus 2003). there are limitations to some researchers due to cost Morphological characters have traditionally been and skill level. In the present analysis, four data used to develop phylogenies for opisthobranchs sets (morphology, sperm ultrastructure, natural (including at the species level). Indeed, several product chemistry and one mitochondrial gene phylogenies of dorid nudibranchs have been sequence) are examined as used in taxonomy and proposed that utilize morphological characters systematics. Each data set is rated for robustness, exclusively (e.g. Gosliner and Johnson 1994; cost and efficiency as defined above. The Gosliner 1996; Gosliner and Johnson 1999; Valdes nudibranch mollusc Halgerda Bergh, 1880 and Gosliner 1999; Fahey and Gosliner 2001; (Discodorididae Bergh, 1891) serves as the model Garovoy et al. 2001; Valdes and Gosliner 2001; organism in the cost benefit analysis. Halgerda was Dorgan et al. 2002; Valdes 2002; Gosliner 2004). The chosen because a reasonably well-resolved phylogenetic trees derived from these analyses phylogenetic hypothesis, based on morphological were not compared to trees developed from other data is available for this group (Fahey and Gosliner data sets such as natural product chemistry or 2001). The existing morphological hypothesis serves sperm ultrastructure. as the starting point for the present analysis, and the three additional data sets (sperm ultrastructure, DNA sequence data natural product chemistry and one mitochondrial More recent publications have hypothesized gene) are applied for the cost benefit analysis opisthobranch phylogenies based exclusively on discussed above. For further details on the various molecular (DNA sequence) characters (e.g. components used for the analysis (Halgerda Thollesson 1998, 1999a and b; Medina and Walsh phylogenetics using morphology, natural product 2000; Wollscheid-Lengeling et al. 2001; Grande et al. chemistry, sperm morphology and/or 2004). These publications did not include mitochondrial DNA sequences) see Fahey and comparisons of the molecular-derived trees to Garson, 2002; Fahey 2003a and b; Fahey and Healy, phylogenies developed from morphological data. 2003. A review of the published literature However, Wollscheid-Lengeling et al. (2001) and describing applications of the data sets for the Grande et al. (2004) did discuss their results in view present cost benefit analysis is presented. of previous cladistic analyses based on morphology.

Gastropoda, Opisthobranchia systematics Sperm ultrastructure data combining traditional morphological and Sperm ultrastructure has proven very instructive molecular data sets for resolving gastropod and other molluscan Previous authors have recommended that a systematics and phylogeny below species level combination of molecular and morphological (Giusti 1971; Giusti and Selmi 1982; Healy 1984; methods be used to provide robust phylogenetic Healy and Willan 1984; Kohnert and Storch 1984; hypotheses (e.g. Hillis 1987; Ponder and Lindberg Koike 1985; Healy 1988a and b; Hodgson and 1995; Wagele et al 2003). More specifically, Ponder Bemard 1988). However, previous studies have not and Lindberg encouraged a collaborative effort to demonstrated that sperm ultrastructure has a test the current phylogenies of the Gastropoda, one comparable phylogenetic signal at the species level that uses an integrated approach. Particularly, they of nudibranchs (Fahey and Healy 2003; Fahey suggested that this effort would include new and 2003b). existing cytological, morphological and molecular There have been a number of transmission data in analyses of phylogenetic relationships. electron microscopical (TEM) studies dealing with Cost benefit analysis, mollusc evolution 25 sperm or spermiogenesis of nudibranchs classification. Past taxonomic classifications were (Thompson 1966; Schmekel 1971; Holman 1972; often based only on external appearance or Thompson 1973; Eckelbarger and Eyster 1981; inadequate descriptions of internal features (see for Eckelbarger 1982; Medina et al. 1985 and 1986; example Bergh 1891). This lack of taxonomic Healy and Willan 1991; Wilson and Healy 2002). information leads to confusion and disagreement on The majority of these authors either limited their the identity of particular groups (e.g. Fahey and study to a single group of Nudibranch (Thompson Gosliner 2003). Karuso (1987) stated that because 1966 and 1973; Holman 1972; Eckelbarger and the taxonomy of opisthobranchs is still fluid, it is Eyster 1981; Medina et al. 1985 and 1986) and/ or currently difficult to make any definitive statements confined their study to a single structure within the about the use of chemicals as taxonomic markers. sperm (Schmekel1971). No previous phylogeny has However, Cimino and Ghiselin (2001) comment on examined sperm ultrastructure across a genus of recent studies using marine natural products to nudibranch. However, both Healy and Willan reveal evolutionary patterns in opisthobranchs (1991) and Wagele and Willan (2000) acknowledged (Cimino and Ghiselin 1998; Cimino and Ghiselin sperm morphology as a very valuable character. 1999). Healy and Willan (1991) examined sperm Not only have natural products been used ultrastructure of 27 nudibranchs representing the infrequently as taxonomic tools in opisthobranchs two suborders (Anthobranchia and Cladobranchia) in general, even fewer recent systematic studies and four superfamilies (Doridoidea, have included chemical characters in modern Dendronotoidea, Arminoidea, Aeolidoidea) of the phylogenetic analyses of dorid nudibranchs Nudibranchia. Although Healy and Willan's specifically. The exceptions to this are the studies examination found no related sperm by Brunckhorst (1993) and Valdes and Gosliner synapomorphies uniting the Nudibranchia, their (1999). In these two publications, the absence or data did shed new light on possible relationships presence of chemicals, used presumably for within the Doridoidea. Wilson and Healy (2002) defense, is used as a taxonomic character or examined a family of Nudibranchia characters. Valdes and Gosliner (1999) also (Chromodorididae) and found a sperm characterize the chemicals derived from a synapomorphy uniting the group. These authors nudibranch as being either diet derived or from de suggested that acrosomal morphology might be novo synthesis. taxonomically informative among nudibranch Cimino and Ghiselin (1999) conclude that there is genera. No phylogenetic analyses have been a weak correlation between the lineage of sponge­ published at the nudibranch species level using eating dorid nudibranchs and the defensive sperm morphology. chemicals isolated from them. However, these authors present evidence that combining chemical Natural product chemistry as a taxonomic data with morphological data can offer insight into indicator the evolution of the Nudibranchia and their closest Natural products have infrequently been used as relatives (Cimino and Ghiselin 1998 and 1999). But, taxonomic tools to characterize marine mollusc no phylogenetic analysis was provided in these groups (Brunckhorst 1993; Valdes; Gosliner 1999). publications. In summary, it is not clear at the Indeed, most researchers to date, with some present time that natural product chemistry will be significant exceptions (Cimino et al. 1999; Cimino able to elucidate evolutionary relationships at the and Ghiselin 1999; Cimino and Ghiselin 2001), species level (e.g. Fahey, 2003b). utilize the biochemistry of opisthobranchs not as an evolutionary tool, but as a method by which to determine the origin of various compounds within METHODS the mollusc, e.g. dietary derived vs. de novo The model taxon used for this study is the synthesis. Since the 1970's, the literature describing tropical, cryptobranch nudibranch genus Halgerda natural product chemistry of marine molluscs has Bergh, 1880. The systematics and phylogeny of this expanded considerably (for thorough reviews, see discodoridid have previously been described using Faulkner 1996, 1997, 1998, 1999, 2000a and b, 2001). traditional morphology-based data (Fahey and Since Idler et al. (1978) discussed the possibilities of Gosliner 2001). No systematic studies of the using sterols from marine molluscs for taxonomy, nudibranchs published to date have completed a only a few authors have followed with chemical species-level comparison between morphology­ studies of opisthobranchs that included taxonomic derived and molecular-derived (mtDNA sequences) considerations (Faulkner and Ghiselin 1983; Karuso phylogenies to resolve taxonomic or phylogenetic 1987; Avila 1992). A major reason for this lack of questions. Further, no taxonomic or systematic taxonomically related biochemical studies of study of the Nudibranchia has combined three opisthobranchs according to Cimino and Sodano independently acquired data sets to test the (1994) is the lack of a definitive or error free robustness of a phylogenetic hypothesis. 26 S.J. Fahey

The present cost benefit analysis first reviews and/or exporting organisms. Thus there is a recent literature for examples of each of the four possible cost associated with the application and/or data sets considered for the present analysis permit fees. (morphology, sperm ultrastructure, DNA sequences Proper preservation and storage of organisms for and natural product chemistry). data acquisition are other considerations for cost. Secondly, four recently published nudibranch­ For example, when preserving specimens for related data sets, DNA sequences (cytochrome ultrastructural (TEM) examination, glutaraldehyde oxidase I (COl)) (Fahey 2003b), histological data is required. However, only the organism's gonad (sperm ultrastructure) (Fahey and Healy 2003) and needs to be collected and stored in this way, so biochemical data (natural product chemistry) small amounts of the chemical are all that is (Fahey and Garson 2002; Fahey 2003a) to determine required. But refrigeration (~ 4 0c) is recommended their particular usefulness (individually and in for at least the first 24 hours after immersion in the combinations) to provide a more resolved or robust gluteraldehyde. phylogenetic hypothesis at the species level. Each Collection is further encumbered by having to data set was examined separately and then deal with either toxic chemicals out of a laboratory compared to the existing morphologically derived environment, or the unavailability of cold-storage phylogenetic hypothesis of the model organism equipment. Specimens preserved for chemical taken from Fahey and Gosliner 200l. analysis require either freezing (-20-70°C) or The present cost benefit analysis answers the preservation in acetone or 100% methanol. following questions. Does combining these different Refrigerators with a capacity to go below 4°C or data sets provide further insight into the freezers below -20°C are not always available in the evolutionary relationships among the species of a field. model organism? Can a researcher achieve a more It is assumed that the researcher would have robust hypothesis by combining data sets? What access to a modern lab with the necessary can be said about the efficiency and cost of deriving equipment to perform the analyses for each of the these particular data sets within the time constraints three data sets, whether it is a molecular laboratory, of a typical research project, such as specified by microscopy facility, or organic chemical laboratory. most grants or postgraduate programs? The cost The laboratory should be evaluated in terms of benefit analysis can be applied to other nudibranch availability of high-speed computers, with adequate mollusk research projects to assist future storage to run phylogenetic analysis programs. researchers in determining the usefulness of each of Some of the analysis software such as MacClade© the data sets. and Sequencher© can also be quite expensive. There Each of the four criteria of the cost benefit analysis is however, some freeware available, for example (cost, time investment, efficiency and robustness of Clustal (Thompson et al. 1994). the phylogeny) is first defined and examples For the present cost benefit analysis, laboratory provided. The method for determining the assigned equipment, including computers and software has ratings shown in Table 1 is outlined. not been factored in, as it was available. Cost Ratings (in ADD per year) Definition of terms, considerations, examples and 1= > $10,000. 2 >$3000 and <$10,000. 3 under rating scales = = $3000. 1. Cost = Expense in terms of dollars for resources such as consumables, required fieldwork, 2. Investment in Time = Time is defined in terms transportation of collected specimens and some of days, weeks, months to gather the data, to equipment and software. train the researcher to use the methodology and The cost rating in Table 1 assumes that some to perform the analysis. specimens are available from institute collections. Time for collection of specimens for any study Having some or all material available will increase will depend largely on the project scope as (i.e. improve) the number rating due to the cost of addressed above. If a project is limited to a single the fieldwork component. If the organism has not geographic locality, and the specimens are known been previously studied and/or is rare, then the to be available at a certain time, then the time fieldwork component will be a major expense. investment can be minimal. However, if a project Therefore, unless a researcher has access to material includes a wide geographic range, or a large that has been stored in EtOH without formalin, (for sampling of populations, with scuba diving sequence data) or either frozen, stored in acetone or required for example, then the time investment will methanol (for chemical extraction) or stored in be far greater than with a more narrowly glutaraldehyde (for ultrastructural examination), constrained project. then fieldwork will be a major cost consideration. Another investment in time for acquiring each of Collection permit requirements can increase costs. these data sets is for training in each of the areas of Permits are required in most localities for collection research. In order to utilize any of the data sets, it is Cost benefit analysis, mollusc evolution 27 necessary for the researcher to have some basic and time for acquisition of each data set [(a) some advanced skills. Specific examples follow. morphology, (b) sperm ultrastructure (c) DNA (a) Gross Morphology: Morphology is perhaps one sequence data and (d) biochemistry (natural of the most time-consuming methods to learn product chemistry)]. and perform well enough for taxonomic studies. Dissection techniques, basic anatomy Time Ratings and biological illustration are tools acquired 1= Most time consuming, probably not possible during most biology course work. Perfecting (unless using a small sample size) if combined these skills and learning the techniques with any other newly developed data set, such required for specific organisms could be quite as a natural products or morphological data set. time consuming especially for small organisms. 2= Can be completed within a typical research Developing well thought out characters and period, but the researcher will need extensive then coding them for a phylogenetic analysis is mentoring if combining another data set. 3= Can also extremely time consuming. A mentor is be completed even in conjunction with another important for this component. data set, i.e. morphological or histological Cb) Ultrastructural investigations: With regard to (ultrastructure investigations). training for sperm morphological studies, it is 3. Efficiency = Gaining a reliable test or measure assumed that the researcher has a basic of evolutionary relationships. knowledge of microscopy skills. Learning the For the purposes of the present cost benefit more advanced methodology required for analysis, efficiency (gaining a reliable measure of transmission electron microscopy, such as evolutionary relationships) is measured by the processing, sectioning, staining and use of TEM microscopes is required. Accumulating enough ability to illustrate evolutionary relationships by data for a study on the cellular level, such as means of a phylogenetic tree. Published studies for comparative sperm morphology, can have used each of the four components to examine require a large investment of time. Detailed evolutionary relationships with mixed results. (See guidance from a microscopist is also invaluable the examples in the Introduction section). for TEM work. It should be noted that molecular and natural (c) DNA sequences (COl): A researcher trained in product chemistry are both relatively new laboratory competence can acquire molecular applications for evolutionary biology as compared laboratory skills in a reasonable amount of to morphological studies dating back to Linnaeus time. That is, a researcher is able to extract, (1767). Thus both disciplines are in the testing phase amplify, sequence and align at least a 20-40 for elucidating evolution (Cimino and Ghiselin 2001). specimen sample over a few months. The analysis and interpretation of the data takes Efficiency ratings in Table 1 are based on additional time, as it requires a solid examination of the results of each data set used to background in genetics, molecular systematics infer a species-level phylogeny. and advanced phylogenetic methods. Keeping Efficiency Ratings up with and understanding the molecular 1= Not apparent at present that this data will systematics literature is also very time­ provide an efficient means of elucidating consuming because molecular analysis evolutionary relationships at species level. 2= literature is expanding considerably. Efficiencies demonstrated at higher levels, though (d) Natural Product Chemistry: With regard to more studies are needed. 3= A reliable means of chemistry a biology researcher with a limited testing evolutionary relationships even at species biochemistry background would be hard­ level of organism evolution as determined by a pressed to interpret the sophisticated robust phylogeny (see 4. below). chemical analyses required for complex chemical compounds. Reliance on a 4. Robustness = Strength of support for a chemistry mentor for the interpretive aspects hypothesis. of the chemical data set is necessary. The present cost benefit analysis defines robustness However, learning the equipment and as few, if any branch trichotomies and including high technical skills for the chemical extractions retention and consistency indices for a phylogenetic and isolations and scanning (flash columns, tree. Thus, in the present analysis, robustness ratings TLC, GC, HPLC and NMR) can be closely mirror the efficiency ratings. accomplished within a year or two, even Support, for the purposes of this analysis, is while acquiring skills in other areas of a evaluated by consistency index (Cl), retention index multi-discipline research project. (RI), Bremer support for morphology-derived trees In the present paper, published results are (Bremer 1994) and bootstrap values for DNA analyzed to determine the required investment in sequence-derived trees (Felsenstein 1985) and 28 S.]. Fahey resolution of trichotomies. Computer analysis data. Cost and time issues are greatly reduced by programs such as PAUP* provide these scores. having adequate material available for examination The data sets and published results are reviewed at the onset of a project. for strength of support for the hypotheses offered. The scores assigned to the morphology data set are: Cost=2, Time investment=3, Efficiency=3, Robustness Ratings Robustness=3. Total score=l1. 1= Has not yet been shown to resolve (b) Ultrastructural data: No sperm characters that ambiguous branching patterns, or to increase could be used to unite the clade Halgerda were support, especially at higher taxonomic levels. found (See Fahey and Healy 2003). Considerable 2= Some additional resolution has been diversity was observed in sperm morphology demonstrated, but more work is required. 3= among species of Halgerda. Thus the results could Resolves branching ambiguities and/or not be mapped onto a phylogeny to test robustness increases the Cl and RI. The higher the RI, the of the data. higher the amount of synapomorphy in the data It was necessary to recollect all specimens set. The Cl measures the amount of homoplasy examined in order to process them for histology in the data set. The higher the score, the lower (sperm ultrastructure investigations). Thus for this the homoplasy in the data set. component, fieldwork contributed to the overall cost as did the smaller costs associated with chemicals, processing and photography. RESULTS AND DISCUSSION The time to learn this aspect of the research was The results of the cost benefit analysis are achieved in two years. Skills were learned during summarized in Table 1. The higher the overall the first year with further material collected, rating (1 = low, 12 = high), the more likely the data processed and analyzed during the following year. set will accomplish the ultimate goal of illuminating In summary, by itself sperm ultrastructure on the a robust phylogenetic hypothesis for an acceptable species level within the Discodorididae does not cost, with a reasonable investment in time and with appear to provide enough discriminating the greatest efficiency. phylogenetic information. Thus, sperm The results of each data set are: ultrastructure was less useful as an analytical tool (a) Morphological data: Morphological data yielded for phylogenetic relationships for clades within the species level trees with Cl, RI and Bremer support study group. However, the results of this study values close, though not as high as those achieved provided other insights. For example, the study by DNA (COl) sequence data. supported the use of the outgroups, Asteronotus and Since most of the material was already available Discodoris for phylogenetic studies of Halgerda. The through museum collections and had been sperm morphology between the study group and examined for previous analyses (for example Fahey the outgroup taxa were more similar to each other and Gosliner 2001) no additional collecting costs than to other nudibranchs. Thus, succeeding were associated with gross morphology. phylogenetic analyses of the discodorids could Although morphology is one of the most time­ utilize these taxa as outgroup or ingroup species. consuming methods to learn and perform well The scores assigned to the ultrastructure data set enough for taxonomic studies, for the reviewed are: Cost=l, Time investment=3, Efficiency=2, project, morphological techniques had been Robustness=2. Total score=8. acquired in advance. (c) DNA sequence (COl) data: Phylogenetic trees In summary, the projects (Fahey 2003a and b) derived from molecular data resolved branch demonstrated that a well-constructed trichotomies in a morphological tree (from Fahey morphological analysis results in a very robust and Gosliner 2001). The molecular-based trees had phylogeny that is strongly supported by molecular higher Cl and RI scores than found in trees derived

Table 1 Results of cost benefit analysis. The higher the overall rating, the more likely the data set can be successfully applied to the reconstruction of phylogeny for an acceptable cost, investment in time and with the greatest efficiency. 1 = low score, 2 = mid-range, 3 = high score. *Assumes some amount of ethyl alcohol-preserved material is available.

Cost Time Investment Efficiency Robustness Overall rating (out of 12)

Gross Morphology 2* 3 3 3 11 Sperm Ultrastructure 1 3 2 2 8 DNA sequences (COl) 2* 3 3 3 11 Natural Product Chemistry 1 1 2 2 6 Cost benefit analysis, mollusc evolution 29 from a morphological data set (Fahey 2003a and b). Halgerda shares some common characteristics with Thus, an improved existing phylogenetic hypothesis other closely related cryptobranch dorids, specific of Halgerda was gained. When molecular data were chemical compounds could not be identified among combined with morphological data (Fahey 2003a) species of Halgerda and thus could not be mapped the resulting trees had lower Cl and RI scores than onto the phylogeny of the clade. Thus the for trees derived from either morphological or robustness of a phylogenetic tree (Fahey and molecular data alone. Gosliner 2001) could not be tested using natural For the cost considerations, specimens had to be products as characters. recollected to obtain fresh tissue. Small clippings of Specimen collection was also required for the mantle tissue were taken and voucher specimens chemistry component. Larger amounts of the were retained (See Fahey 2003a and b). organism were required for extractions than for the The time to learn the molecular laboratory other data sets. Thus the costs associated with protocols required the least amount of time (two sample collection contributed to the greatest extent months), with the data analysis portion taking for this aspect. (See Fahey and Garson 2002 and several additional months. Fahey 2003b). In summary, the results of the cost benefit With regard to time investment, the limited analysis show that COl is very useful for estimating conclusive chemical data obtained for Halgerda evolutionary relationships between species of a (Fahey 2003b) demonstrated that obtaining species­ clade, and indeed support the major clades of the specific phylogenetic information is not as morphology-based phylogenetic analyses (see achievable in the same time frame as is possible Fahey, 2003a for specific details). A better­ using molecular-based information (See Fahey and supported hypothesis resulted, (more robust, higher Garson 2002; Fahey 2003a and b). Higher-level data bootstrap support), and in a very efficient manner however appear more within range of a shorter (as defined in the Methods section). It appears that project (See for example Cimino and Ghiselin 1998, with sound morphological characters, a well­ 1999,2001). If the nudibranch (or other organism of supported phylogeny can be verified and interest) and its prey are readily available and if the supplemented by the use of molecular data. chemical compounds are known compounds, then Furthermore, inter-specific analysis of the COl gene the task of doing a species-level phylogeny based can be easily accomplished within a relatively short on chemistry could be completed within a shorter time frame. Collection of fresh material for DNA timeframe. But, when the are uncommon extraction appears to be the major challenge in and the compounds are unknown in the literature, order to utilize molecular data across a lower-level or are difficult to isolate due to small sample size, clade, as well as taxon sampling for rare, but critical or complexity of the compound, as was the case for taxa in elucidating phylogeny (Fahey 2003a). this study, substantial difficulties arise in achieving Finally, analyses of molecular data allowed the the desired outcome within a reasonable period, say examination of morphological character evolution. two to three years. The time it takes to identify the The results of the COl analysis showed two myriad of signals in a small amount of material can examples of a change in the tree topology as exceed or exhaust a short timeframe. This is compared to analysis of morphological data (Fahey especially true if the researcher is developing 2003a). In these cases there were few morphological multiple data sets (such as those discussed herein) characters that supported the clade in question, (i.e. at the same time. Halgerda okinawa plus H. theobroma). A review of the In summary, even given the limitations and morphological characters supporting the clades constraints outlined above, some general insights indicated that color characters might not be were made possible from biochemical studies adequate to support the placement of these two (Fahey 2003b). For example, the cryptobranch species. The need to reassess characters or add nudibranch Halgerda was found to share feeding additional characters was illuminated. characteristics and compounds such as sterols, The results of the present study suggest that the carotinoids and fatty acids with other cryptobranch same combination of data (morphological and COl nudibranchs (See Fahey 2003b for specific details). sequence) could be used for other nudibranchs or These shared qualities can be used as characters in organisms to develop new or examine existing a phylogenetic analysis of the cryptobranch dorids. phylogenies. The scores assigned to the chemical data set are: The scores assigned to the DNA sequence data set Cost=l, Time investment=l, Efficiency=2, are: Cost=2, Time investment=3, Efficiency=3, Robustness=2. Total score=6. Robustness=3. Total score=l1. In conclusion, the results show that for the cost and (c) Natural product chemistry data: The results of time investment, morphological and molecular analyses of the natural products of the clade methods serve the nudibranch researcher well when Halgerdidae, (including Halgerda) (Fahey and undertaking a project that investigates phylogenetic Garson 2002; Fahey 2003b) showed that although relationships at the species level. Both methodologies 30 S.]. Fahey scored 11 out of a possible 12 points. Sperm Cimino, G. and Ghiselin, M. (2001). Marine Natural ultrastructure investigations (score 8 out of a possible Products Chemistry as an Evolutionary Narrative. In 12) and natural product chemistry (score 6 out of a J.A.B. McClintock (ed), Marine Chemical Ecology, 115­ possible 12) can be valuable at higher taxonomic 154. CRC Press, New York. levels and should be pursued, especially if time can Cimino, G. and Ghiselin, M. T. (1998). Chemical defense be devoted to developing those data sets exclusively. and evolution in the Sacoglossa (Mollusca: Gastropoda: Opisthobranchia). Chemoecology 8: 51-60. The present cost benefit analysis of the cited studies shows that examination of multiple data Cimino, G. and Ghiselin, M. T. (1999). Chemical defense sets can provide valuable insights into evolutionary and evolutionary trends in biosynthetic capacity among dorid nudibranchs (Mollusca: Gastropoda: relationships, allowing the researcher to further Opisthobranchia). Chemoecology 9: 187-207. examine the hypothesis from various perspectives. Cimino, G. and Sodano, G. (1994). Transfer of sponge In addition, a more robust phylogeny, as defined secondary metabolites to predators. In R. VanSoest, for the purposes of this study, can be achieved by T.M.G. VanKempen, J.c. Braekman. (eds), Sponges in combining data sets, particularly morphological Time and Space, 459-472. Balkema, Rotterdam. and DNA sequence data. 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