Euglena: 2013
Evolutionary relationships within Ambulacraria (Peterson and Eernisse 2001) and Chordata (Bateson 1885): Examining Tunicata (Lamarck 1816), and Xenoturbellida (Bourlat et al. 2003) using the 18S rDNA gene Hannah Airgood, Jason Long, Kody Hummel, Alyssa Cantalini, and DeLeila Schriner
Susquehanna University, Selinsgrove, PA 17870.
Abstract Ambulacraria and Chordata are both part of Deuterostomata. The focus of this paper is the analysis of the evolutionary relationships found from our data within and between Ambulacraria and Chordata by examining the phyla within them, and more specifically the relationship of Tunicata to Vertebrata and Xenoturbellida to all deuterostome phyla. The gene 18S rDNA was used for molecular analysis. The characters notochord, hermaphroditic mode of reproduction, notochord extending into head, filter feeding, symmetry, separate anus, and stomochord were used for morphological analysis. Our study concluded that Vertebrata are a sister group to Cephalochordata, and Tunicata are basal to that clade. All three share a common ancestor with Ambulacraria. Also, according to the gene that we examined, Echinodermata are the sister group to Hemichordata. Xenoturbellida were shown to be basal to the Ambulacraria and Deuterostomata.
Please cite this article as: Airgood, H., J. Long, K. Hummel, A. Cantalini, and D. Schriner. 2013. Evolutionary relationships within Ambulacraria (Peterson and Eernisse 2001) and Chordata (Bateson 1885): Examining Tunicata (Lamark 1816), and Xenoturbellida (Bourlat et al. 2003) using the 18S rDNA gene. Euglena. doi:/euglena. 1(1): 10-16.
Introduction (Holt and Iudica 2012, Bourlat et al. 2006). Deuterostomes include Hemichordata, Xenoturbellids were thought to be members of Echinodermata, Xenoturbellida, and Chordata. Mollusca due to contamination in their DNA from Chordates are further divided into Cephalochordata, the consumption of mollusks, but Telford (2008) Tunicata, and Vertebrata (Holt and Iudica 2012). Five concluded that xenoturbellids are deuterostomes, not characters were investigated within these phyla. They mollusks. Cephalochordata was found to be a group include the presence of a support structure, contained within Chordata. reproductive organs, the way in which their food is Using different genes than the 18S rDNA obtained, the organisms’ symmetry, and the gene has yielded conflicting results concerning the development of a separate anus from the mouth. Each relationships within Deuterostomata. Zhong et al. character was chosen to be analyzed because they (2009) used mitochondrial DNA, and concluded that represent the synapomorphies of each clade. tunicates are more closely related to vertebrates than Cameron et al. (2000) studied the 18S rDNA cephalochordates. Since the entire mtDNA genome gene and indicated the relationships within could be used, the bias of using just 18S rDNA in the deuterostomes. Their analysis implies a close phylogenetic tree was removed (Zhong et al. 2009). relationship between cephalochordates, vertebrates Zhong et al. (2009) also confirmed that each extant and tunicates. deuterostome phylum and subphylum within The position of Tunicata within Chordata is Chordata is monophyletic. somewhat questionable as the larval forms have a Another study with a similar purpose, using notochord that is lost later in development. However, 59 proteins, also concluded that tunicates are more when the organism reaches adulthood, the notochord closely related to vertebrates (Blair and Hedges is reabsorbed into the body (Cole 2011). The 2005). Like the study by Zhong et al. (2009), Blair topology of tunicates within Vertebrata has been and Hedges (2005) used the evolutionary time scale dramatically changed and questioned in the last two when considering possible reorganization of decades, and the answers to their origins may reveal relationships among the subphyla of Deuterostomata. answers to the origins of all vertebrates. Bourlat et al. (2006) also support the idea that Ambulacraria is further divided into tunicates are most closely related to vertebrates Xenoturbellida, Hemichordata and Echinodermata instead of cephalochordates. There are two striking
10 Euglena: 2013 results from this study. One was the conclusion that xenoturbellids is not separate from the mouth as in all cephalochordates are more closely related to of the other deuterostome organisms. Although some echinoderms, and tunicates are more closely related studies support each other, there is still much to vertebrates (Bourlat et al. 2006). The other is that ambiguity on the topology within deuterostomes, based on mitochondrial DNA, Xenoturbellida is not a especially the position of Xenoturbellida and subphylum within Ambulacraria, but a basal group to Tunicata. all deuterostomes (Bourlat et al. 2006). There are two The purpose of this paper is to provide more major characters that separate Xenoturbellida from support for the topology of Deuterostomata, the other phyla. Xenoturbellids do not have a separate especially the dispute of Tunicata and anus nor do they have a support structure. Israelsson Cephalochordata in relation to Vertebrata, and (2008) makes it clear that the anus of the Xenoturbellida in relation to all deuterostomes.
Table 1. The characters that were examined among deuterostome phyla. The states of those characters and the phyla from Appendix A are also listed. The character evolution is shown in Figure 3. Character Trait Examined Character States Phylum Containing Character Support Structure Stomochord, Notochord, No Support Echinodermata, Hemichordata, Vertebrata, Cephalochordata, Structure Tunicata, Xenoturbellida Hermaphroditic Mode of Contain both sex organs or Contain Tunicata Reproduction one sex organs Filter Feeders Filter Feeder or Non Filter Feeder Tunicata, Echinodermata, Cephalochordata, Hemichordata Symmetry Radial or Bilateral Symmetry Radial: Echinodermata Bilateral: Vertebrata, Cephalochordata, Tunicata, Hemichordata, Xenoturbellida Separate Anus Separate Anus or No Separate Anus Vertebrata, Cephalochordata, Tunicata, Echinodermata, and Hemichordata
Materials and Methods was generated using this model, and is based on the A selection of 26 in-group species (seen in program choosing the best tree according to the gene. Appendix A) was used to analyze the evolutionary Figure 2, in contrast, is the Maximum Parsimony relationship between Vertebrata, Cephalochordata tree, where the program chooses the shortest tree. and Echinodermata. The taxa that were used in this Figures 1 and 2 were combined to form a study were 4 species of hemichordates, 7 species of consensus tree, Figure 3. Both trees showed the same tunicates, 1 species of cephalochordates, 7 species of topology and relationships between the major groups echinoderms, 7 species of vertebrates, and 1 species in question. of xenoturbellids (Appendix A). Acharax sp. was The characters that were examined (seen in used as an out-group. The cladistic analysis began by Table 1) were the presence of filter-feeding, the using an accession number (AF236798) for the 18S presence of a separate anus, the presence of a support rDNA gene, based on the results by Cameron et al. structure, symmetry, and reproductive organs. All of (2000), to obtain gene sequences of the species listed the selected organisms have some characters from a in Appendix A. These sequences were then aligned common ancestor and evolve with new character using the ClustalW option and trimmed using MEGA states forming over time. 5.1 (Tumura et al. 2011) to obtain gene sequences of 1873 bases. The aligned sequences were then made Results into a maximum likelihood tree and a maximum In Figure 1, the maximum likelihood (ML) parsimony tree using the ML and MP methods both tree displays the relationships of the chosen species with 1000 bootstrap replications, Figures 1 and 2 based on the maximum probability of observing the respectively. data provided, in this case, genetic sequence 18S A model analysis was first performed for the rDNA. Figure 1 displays vertebrates and maximum likelihood that yielded the Tamura 3- cephalochordates stemming from the same common parameter model and Gamma distributed with ancestor, with moderate confidence due to a Invariant sites (G+I) model which was used for the bootstrap value of 62. These vertebrates and tree shown in Figure 1. The bootstrap values seen in cephalochordates share a common ancestor with Figures 1, 2, and 3 were obtained by creating 1000 tunicates, at the moderate bootstrap value of 63. iterations of a tree and analyzing the clades in each Tunicata was also found to be a monophyletic group. tree . The values given are the frequency that the Tunicata, Vertebrata, and Cephalochordata share a particular clade is seen in those 1000 iterations. common ancestry with Echinodermata and Figure 1 shows the Maximum Likelihood tree that Hemichordata, with high confidence. The
11 Euglena: 2013 echinoderms are closely related to hemichordates seen to be linked by a common ancestor with and, from what was found, Echinodermata arose as a Vertebrata, Tunicata, and Cephalochordata with high sister group. However it is important to note that confidence. Hemichordata were again seen as a sister some of the bootstrap values were fairly low group to Echinodermata, similar to Figure 1. Also, designating low support. Xenoturbellids were also Xenoturbellida was shown as being a basal group to shown as having shared an ancestor with the rest of the other deuterostomes. It is important to note that in Ambulacraria and Chordata. both Figures 1 and 2, Hemichordata appear with a In Figure 2, the maximum parsimony (MP) problematic outlier, the Ptychodera bahamenisis. tree displays the relationships of the chosen species Figure 3 represent our best estimate, using based on the shortest possible tree, or the least 18S rDNA gene, of the evolution of the taxa in number of changes. Similar to Figure 1, Vertebrata question. Figures 1 and 2 were similar in how and Cephalochordata arose from a common ancestor Vertebrata related to Cephalochordata and how these with a moderate confidence value of 57. The related to Tunicata. Both figures also revealed monophyletic Tunicata was also seen as a basal Hemichordata as a sister group to Echinodermata. group to Vertebrata and Cephalochordata, but with Xenoturbellida were seen as being a basal group to low confidence, similarly seen in Figure 1. Chordata and Ambulacraria. Furthermore, Hemichordata and Echinodermata were
Figure 1. The Maximum Likelihood (ML) tree was assembled using MEGA 5.1 and a Tamura 3-parameter model and Gamma distributed with Invariant sites (G+I) (Tamura et al. 2011). Maximum Likelihood involves the program choosing the best tree that provides the maximum chance of producing the matrix created using gene sequences from NCBI (see Appendix A). Phylum names are listed at the right and common names are given, if available. This figure shows that Vertebrata and Cephalochordata as sister taxa. Tunicata appear as the basal group in the clade. This clade shares a common ancestor with Hemichordata (note the problematic outlier: Ptychodera bahamenisis (Spengel 1893) ) and Echinodermata. Xenoturbellida is basal to the entire clade made of Hemichordata, Echinodermata, and Chordata. It is important to note that there were 3 major clades in the tree, chordates, ambulacrarians and xenoturbellids.
12 Euglena: 2013
Figure 2. The Maximum Parsimony (MP) tree was assembled using MEGA 5.1 (Tamura et al. 2011). A few important things to note are the Cephalochordata position as a sister group to Vertebrata and that Tunicata are basal to these two taxa. Phyla are listed at the right and available common names are also listed. Hemichordata were shown as a sister group to the Echinodermata. Hemichordata also showed to have a problematic outlier. It is important to note that there were 3 major clades in the tree, chordates, ambulacrarians and xenoturbellids.
Discussion represents high certainty of the evolution of the taxa Vertebrata, Tunicata, Cephalochordata, and in question and also the characters that were Echinodermata were shown to agree with Cameron et examined in Table 1. By placing the characters on the al. (2000) as monophyletic groups. tree, we were able to see the evolution that most Although the species within Figure 2 have a likely matches current ideas about the taxa (Cameron different topology than in Figure 1, the overall et al. 2000). topology of the clades in each are the same. This The ancestor of deuterostomes shared many similarity provides high confidence in the overall of the same characters as the organisms, but some organization provided in Figure 3. The topology of have developed different characters than the others. our clades is supportive of Bourlat et al. (2006) in The common ancestor had bilateral symmetry, as do that xenoturbellids are basal to Ambulacraria and all evolved taxa except echinoderms (Pechenik Chordata and Cameron et al. (2000) in that 2005). The common ancestor was a filter feeder, Cephalochordata are more closely related to shown in Figure 3. This character can be seen in all Vertebrata. Cameron et al. (2000) did not discuss the of the organisms on the tree with the exception of relationship of xenoturbellids as a sister group to xenoturbellids and vertebrates. These two phyla have chordates and ambulacrarians, due to the lack of lost this ability. Although Israelsson (2008) indicates information available about the xenoturbellids at the that the mechanism xenoturbellida obtain their food time of publication. is unknown, Bourlat et al. (2006) suggests that they Figure 3 was obtained due to the extreme get their food from feeding on bivalve mollusks and similarity between Figures 1 and 2. This similarity not through filter feeding.
13 Euglena: 2013
Figure 3. The consensus tree made from manually combining Figures 1 and 2 using the highest confidence bootstrap values. Character traits and states are listed in Table 1, marked in the tree with a slash for a change in state with the state listed. All of the taxa within this tree have filter feeding as their way to obtain food except for Vertebrata and Xenoturbellida. This character may have evolved after Xenoturbellida and then was lost by in vertebrates. A separate anus and support structure differentiate Ambulacraria and Chordata from Xenoturbellida. Ambulacraria are split from the Chordata by a stomochord. Echinodermata are listed as a sister group to Hemichordata. Due to these groups being sisters and the character that appears to differentiate these taxa from Chordata, Echinodermata may have lost the stomochord from a common ancestor. Chordata all possess a notochord however it is extended in the Cephalochordata. It is important to note that Ptychodera bahamensis seems to be a problem for Hemichordata and further research would need to incorporate more taxa within Enteropneusta (Gegenbaur 1870) that contain Ptychodera bahamensis. A problem could have occurred in the gene sequence that could have caused this.
Xenoturbellids also lack a support structure. the results by Cameron et al. (2000) that The organisms that have a support structure make up Cephalochordata is more closely related to Vertebrata two different clades, Chordata and Ambulacraria, than Tunicata. Also Tunicata was shown as further supporting our conclusion that xenoturbellids monophyletic, confirming results by Swalla et al. are basal and not part of Ambulacraria. The character (2000). state that separates the two clades is the presence of a The support structure in Ambulacraria, notochord or the presence of a stomochord. The similar to the notochord in chordates, is the notochord is a plesiomorphic character for Chordata. stomochord. This character, Pechenik (2005) This clade shows the presence of the notochord, indicates, is only found in Hemichordata and whereas Bateson (1885) describes that Ambulacraria, separates Hemichordata from Echinodermata. It is except hemichordates, do not. important to note that Ptychodera bahamensis seems The common ancestor of all of the to be a problem for Hemichordata and further organisms evaluated reproduced sexually. Each research would need to incorporate more taxa within gender of each organism only has one sex organ, with Enteropneusta (Gegenbaur 1870). This could be due only one exception. Swalla et al. (2000) explain that contamination of the DNA. tunicates are hermaphrodites. This character confirms
14 Euglena: 2013
From the analysis of the character evolution Cole, L. L. 2011. Diversity and Distribution of and cladistic analysis it is possible to conclude that Tunicata (Urochordata) in Tobago. Journal Cameron et al. (2000) have correct reasoning that of Life Sciences. 6: 221-232. Cephalochordata is a sister group to Vertebrata, and Dunn, C.W., A. Hejnol, D.Q. Matus, K. Pang, W.E. Tunicata are a basal group to these two taxa. Browne, S.A. Smith, E. Seaver, G.W. Furthermore Hemichordata and Echinodermata Rouse, M. Obst, G.D. Edgecombe, M.V. evolve as sister groups. However, similarly to Bourlat Sørensen, S.H.D. Haddock, A. Schmidt- et al. (2006), Xenoturbellida appear to be a sister Rhaesa, A. Okusu, R.M. Kristensen, W.C. group to Chordata and Ambulacraria. Future research Wheeler, M.Q. Martindale, and G. Giribet. inclusive of other genes, such as the work by Zhong 2008. Broad phylogenomic sampling et al. (2009), may help to remove the bias of using improves resolution of the animal tree of the singular 18S rDNA gene. However, the life. Nature. 452: 745-749. relationships within Deuterostomata remain Geer LY, Marchler-Bauer A., Geer R. C., Han L, He complicated, and the exact topology debatable. J., He S., Liu C., Shi W., Bryant S. H. The NCBI BioSystems database. Nucleic Acids Conclusion Res. 2010 Jan; 38(Database issue):D492-6. From these results there are a few (Epub 2009 Oct 23) [PubMed PMID: conclusions that can be made from Figure 3. First, the 19854944] tree originally presented by Cameron et al. (2000) [http://www.ncbi.nlm.nih.gov/biosystems] was confirmed. Vertebrata were shown to be most Holt, J. R., C. A. Iudica. 2012. Diversity of Life. closely related to Cephalochordata with Tunicata
15 Euglena: 2013
Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Wang, W., H. Kim, Nam S., Rho B. J., Kang H. Nei, and S. Kumar. 2011. MEGA5: 2012. Antibacterial Butenolides from the Molecular Evolutionary Genetic Analysis Korean Pseudodistoma antinboja. Journal of using Maximum Likelihood, Evolutionary Natural Products. 75: 2049-2054. Distance, and Maximum Parsimony West-Eberhard, M. 2008. Toward a Modern Revival Methods. Molecular Biology and Evolution. of Darwin’s Theory of Evolutionary 28: 2731-2739. Novelty. Philosophy of Science. 31: 899- Tatian, M., C. Lagger, M. Demarchi, and C. Mattoni. 908. 2011. Molecular phylogeny endorses the Wray, G. A. 1999. Tree of Life. relationship between carnivorous and filter-
Submitted 8 February 2013 Accepted 27 February 2013
Appendix A. The species used with their authorities, phylum, and NCBI accession numbers. *Species authority unknown, genus authority is given. Taxon Authority Phylum NCBI Accession Number Acharax sp. A5-Aleuten Dall 1908 Mollusca AJ563760.1 Acontias percivali Loveridge 1935 Vertebrata AY217894.1 Branchiostoma lanceolatum Pallas 1774 Cephalochordata AY428817.1 Calocidaris micans Mortensen 1903 Echinodermata DQ073782.1 Cephalodiscus nigrescens Lankester 1905 Hemichordata EU728440.1 Channallabes sanghaensis Devaere, Adriaens, Verraes 2007 Vertebrata AJ876387.1 Chelyosoma siboja Oka 1906 Tunicata AB104872.1 Ciona intestinalis Linnaeus 1767 Tunicata AB013017.1 Clavelina meridonalis Herdman 1891 Tunicata FM244840.1 Eucidaris tribuloides Lamarck 1816 Echinodermata Z37127.1 Harrimania kupfferi von Willemoes-Suhm 1871 Hemichordata JF900487.1 Hemibagrus guttatus Lacepède 1803 Vertebrata GQ465845.1 Leiocassis longirostris Günther 1864 Vertebrata GQ465842.1 Luidia clathrata Say 1825 Echinodermata DQ060795.1 Megalodicopia hians Oka 1918 Tunicata AB075543.1 Melanoseps occidentalis Peters 1877 Vertebrata AY217921.1 Ophioderma brevispina Say 1825 Echinodermata DQ060803.1 Ophioplocus japonicas Clark 1911 Echinodermata D14361.1 Ophiopsammus maculate Verrill 1869 Echinodermata DQ060807.1 Perophora sagamiensis Tokioka 1953 Tunicata AB104873.1 Phallusia mammalliata Cuvier 1815 Tunicata AF236803.2 Potamotrygon hystrix Müller, Henle 1834 Vertebrata AY049846.1 Ptychodera bahamenisis Spengel 1893 Hemichordata JF900486.1 Pyrostremma spinosum Herdman 1888 Tunicata HQ015379.1 Rhinobatos productus Ayres 1854 Vertebrata AY049852.1 Saccoglossus bromophenolosus King 1994 Hemichordata AF236801.1 Stereocidaris excavates Pomel 1883* Echinodermata DQ073795.1 Xenoturbella westbladi Israelsson 1999 Xenoturbellida AF207993
16