Evaluating Multiple Alternative Hypotheses for the Origin of Bilateria: an Analysis of 18S Rrna Molecular Evidence
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 15458–15463, December 1998 Evolution Evaluating multiple alternative hypotheses for the origin of Bilateria: An analysis of 18S rRNA molecular evidence ALLEN G. COLLINS† Department of Integrative Biology, Museum of Paleontology, University of California, Berkeley, CA 94720 Communicated by James W. Valentine, University of California, Berkeley, CA, October 28, 1998 (received for review October 1, 1998) ABSTRACT Six alternative hypotheses for the phylogenetic origin of Bilateria are evaluated by using complete 18S rRNA gene sequences for 52 taxa. These data suggest that there is little support for three of these hypotheses. Bilateria is not likely to be the sister group of Radiata or Ctenophora, nor is it likely that Bilateria gave rise to Cnidaria or Ctenophora. Instead, these data reveal a close relationship between bilaterians, placozoans, and cnidarians. From this, several inferences can be drawn. Morphological features that previously have been identified as synapomorphies of Bilateria and Ctenophora, e.g., mesoderm, more likely evolved independently in each clade. The endomeso- dermal muscles of bilaterians may be homologous to the endodermal muscles of cnidarians, implying that the original bilaterian mesodermal muscles were myoepithelial. Placozoans should have a gastrulation stage during development. Of the three hypotheses that cannot be falsified with the 18S rRNA data, one is most strongly supported. This hypothesis states that Bilateria and Placozoa share a more recent common ancestor than either does to Cnidaria. If true, the simplicity of placozoan body architecture is secondarily derived from a more complex ancestor. This simplification may have occurred in association with a planula-type larva becoming reproductive before meta- morphosis. If this simplification took place during the common history that placozoans share with bilaterians, then placozoan genes that contain a homeobox, such as Trox2, should be ex- plored, for they may include the gene or genes most closely related to Hox genes of bilaterians. FIG. 1. Six alternative hypotheses for the origin of the Bilateria. Despite numerous speculations and analyses concerning the forces us to look at old data in new ways. In the case of bilaterian evolutionary relationships of the major animal clades, until origins, molecular sequence data for the 18S rRNA gene show a recently only three distinct hypotheses have been offered possible relationship of Bilateria to Placozoa and Cnidaria, to the concerning the phylogenetic position of Bilateria within the exclusion of Ctenophora and Porifera (9–12). To this point, no other basal metazoan groups (Fig. 1 A–C). Two of these assessment of the likelihood of this possibility has been made. hypotheses are commonly found in zoology textbooks. One More importantly, almost nothing has been written about what view, an idea that goes back to Haeckel (1) and was champi- this phylogenetic arrangement implies if true. These 18S gene oned by Hyman (2, 3), is that Bilateria is the sister group to data prompt us to consider three additional hypotheses for the Radiata (Cnidaria and Ctenophora). A second hypothesis, origin of Bilateria (Fig. 1 D–F). Each of the six alternative preferred by Harbison (4) and Wilmer (5), holds that Bilateria hypotheses shown in Fig. 1 has different implications for char- and Ctenophora share a more recent common ancestor than acterizing the origin of Bilateria. Here I evaluate the strengths of either does to Cnidaria. Recent cladistic studies based on the six hypotheses with a data set that includes 10 additional morphological characters (6, 7) have supported this second complete 18S gene sequences and clarify inferences based on view. A third alternative, which appears to have fallen out of those hypotheses that appear to be the more robust. favor, has Bilateria as a nonmonophyletic group, with both cnidarians and ctenophorans derived from flatworm ancestors MATERIALS AND METHODS (8). The strength of these hypotheses is difficult to weigh on morphological evidence alone. The diploblastic groups have All primer sequences, 18S gene sequences, aligned data sets, disparate body plans with many derived features, only a few of and PAUP* data sets are publicly available at the archived data which are potentially informative as to their relative phyloge- web pages of the University of California Museum of Paleon- netic positions. Furthermore, homoplasies are difficult to infer tology (www.ucmp.berkeley.eduyarchdatayCollins98ybilateria. when dealing with such general features as mouths, mesoderm, html) as well as on request. and symmetry. Molecular sequence studies provide sets of characters that can Abbreviations: Bsi, Bremer support index; 18S, small subunit of be used to both evaluate previous phylogenetic hypotheses and rRNA; T-PTP, topology-dependent permutation tail probability. generate new ones. The challenge of testing new hypotheses Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF100940– AF100949). © 1998 by The National Academy of Sciences 0027-8424y98y9515458-6$2.00y0 †To whom reprint requests should be addressed. e-mail: allenc@ PNAS is available online at www.pnas.org. ucmp1.berkeley.edu. 15458 Downloaded by guest on September 25, 2021 Evolution: Collins Proc. Natl. Acad. Sci. USA 95 (1998) 15459 Table 1. List of species with Linnean classification and GenBank Table 1. (Continued) accession numbers GenBank GenBank Species and classification accession number Species and classification accession number Ctenophora, Lobata Sequences generated by the author Mnemiopsis leidyi L10826 Choanoflagellida Incertae setis Monosiga brevicolis AF100940 Dermocystidium salmonis U21337 Salpingoeca infusionum AF100941 Ichthyophonus hoferi U25637 Cnidaria, Scyphozoa Rosette agent of chinook Atolla vanhoeffeni AF100942 salmon L29455 Cnidaria, Anthozoa Placozoa Antipathes galapagensis AF100943 Trichoplax adhaerens L10828 Ctenophora, Pleurobrachidae Trichoplax sp. Z22783 Hormiphora sp. AF100944 Porifera, Calcarea Porifera, Calcarea Clathrina cerebrum U42452 Leucosolenia sp. AF100945 Scypha ciliata L10827 Porifera, Demospongiae Porifera, Demospongiae Mycale fibrexilis AF100946 Axinella polypoides U43190 Suberites ficus AF100947 Microciona prolifera L10825 Plakortis sp. AF100948 Tetilla japonica D15067 Porifera, Hexactinellida Rhabdocalyptus dawsoni AF100949 Genomic DNA was isolated from tissue samples of 10 Sequences culled from GenBank species (Table 1). The method that most consistently yielded Bilateria, Annelida high molecular weight genomic DNA consisted of pulveriza- Lanice conchilega X79873 2 Bilateria, Chordata tion of previously frozen ( 80°) tissue in the reagent DNAzol Herdmania momus X53538 (Molecular Research Center, Cincinnati), followed by centrif- Latimeria chalumnae L11288 ugation and ethanol precipitation. The complete sequence for Bilateria, Echinodermata the 18S coding region was amplified from genomic DNA Strongylocentrotus purpuratus L28055 preparations using eukaryotic-specific primers (13) via PCR Amphipholis squamata X97156 (30 cycles: 10 s at 94°, 60 s at 37°, and 180 s at 72°) after an initial Bilateria, Echiura 2-min 94° denaturation. The PCR products of three species Ochetostoma erythrogrammon X79875 (Atolla vanhoeffeni, Hormiphora sp., and Antipathes galapagen- Bilateria, Hemichordata sis) were directly sequenced with an Applied Biosystems Prism Balanoglossus carnosus D14359 377 DNA Sequencer, whereas the remaining PCR products Bilateria, Mollusca were cloned and pooled (minimum of eight clones) before Tresus nuttali L11269 sequencing with a Li-Cor (Lincoln, NE) model 4000L IR Limicolaria kambeul X66374 automated DNA sequencer. Bilateria, Nematomorpha Phylogenetic accuracy is increased by adding taxa that break Gordius aquaticus X87985 up branches (14–16). Thus, the maximum number of se- Bilateria, Nemertea quences was analyzed given computational limitations im- Lineus sp. X79878 posed by maximum likelihood searches, which were only Bilateria, Platyhelminthes feasible with data sets up to roughly 50 taxa. Forty-two Stenostomum sp. U95947 sequences not generated by me were culled from GenBank to Planocera multitentaculata D83383 create a 52-taxon data set with reasonably balanced sampling Schistosoma mansoni X53986 across the basal metazoan groups. Representatives from two Bilateria, Pogonophora nonmetazoan clades were included as outgroups: the single- Siboglinum fiordicum X79876 celled choanoflagellates, and an odd group of fish parasites Bilateria, Vestimentifera that include the common aquarium pest Ich. Previous work has Ridgeia piscesae X79877 established the phylogenetic proximity of these groups to the Choanoflagellida Metazoa (9, 17, 18). The 52 sequences (Table 1) used in this Acanthocoepsis unguiculata L10823 analysis were aligned by eye with well over 100 published and Diaphanoeca grandis L10824 unpublished metazoan 18S gene sequences by using the Ge- Cnidaria, Anthozoa netic Data Environment sequence editor (written by Steve Leioptilus fimbriatus Z92903 Smith, Millipore). Characters for which putative homology Anemonia sulcata X53498 could not be asserted were excluded to arrive at a final data set Haliplanella lucia Z86097 of characters for phylogenetic analysis. This data set consists Bellonella rigida Z49195 of 1,526 nucleotide characters for the 52 taxa. All subsequent Calicogorgia granulosa Z92900 analyses were carried out on this data set by using PAUP* 4.0 Virgularia