Euglena: 2013 Defining Phylogenetic Relationships of Ochrophyta Using 18S rRNA: Existence of Three Major Clades in Which Bacillariophyta is Basal Kaitryn Ronning, Emily Beliveau, Emily McCaffery, Cierra Omlor, and Ellie Rosenblum Department of Biology, Susquehanna University, Selinsgrove, PA 17870. Abstract This paper presents an analysis of the phylogenetic relationships amongst the Ochrophyta, the photosynthetic heterokonts, using 18S rRNA gene sequences and maximum likelihood analysis. Three maximum likelihood (ML) cladograms were produced using Kimura 2- parameter, Tamura-Nei and Tamura 3-parameter best fit models. These cladograms were nearly identical, and strongly suggest that Bacillariophyta is a basal group within the Ochrophyta. Additionally, we raise questions regarding the phylogenetic placement of Silicoflagellata and Pinguiophyta. Please cite this article as: Ronning, K., E. Beliveau, E. McCaffery, C. Omlor, and E. Rosenblum. 2013. Defining phylogenetic relationships of Ochrophyta using 18S rRNA: existence of three major clades in which Bacillariophyta is basal. Euglena. doi:/euglena. 1(2): 52-59. Introduction 1980’s, when Medlin began to use 18s rRNA gene Heterokontae, also called the straminopiles, sequences to resolve the grouping of diatoms (Medlin is a Kingdom containing many diverse taxa, ranging et al.1988 and Beszteri et al. 2001). Using from large multicellular species to small unicellular morphological characters alone has been misleading species. They can be found in freshwater, marine, because convergent evolution may have played a role and terrestrial habitats. Cavalier-Smith (1986) in the similarities of certain taxa, which could lead to established Heterokontae as a phylum in 1986. Then, misidentification (Medlin et al. 2000). For example, Cavalier-Smith (1986) raised Heterokontae to an silica frustules of diatoms appear to be very similar infrakingdom split into two main groups: the among species, which would suggest that the species Ochrophyta (a photosynthetic group consisting of are closely related. However, molecular data have not mainly autotrophic heterokonts) and a purely supported the same relationships (Yang et al. 2012). heterotrophic group that was subdivided into the Similar morphological characters, such as silica Bigyra and Pseudofungi (Riisberg et al. 2009). This frustules have made it very challenging to determine infrakingdom included all eukaryotic biflagellate the position of diatoms within the heterokonts . In cells that have a forward directed flagellum with addition to challenges with morphological characters, tripartite tubular hairs and also a smooth, trailing there have also been difficulties with molecular flagellum, and this taxonomy is still accepted today research as a result of a limited number of gene (Riisberg et al. 2009; Anderson 2004; Cavalier-Smith sequences available. This may restrict the possibility and J. M. Scoble 2012). In some taxa one or both to accurately determine the phylogeny of the diatoms flagella have been secondarily lost. (Kooistra 2007). Bacillariophyta, also known as the diatoms, Phylogenetic branching within is a phylum within the kingdom Heterokontae. This is Heterokontae has remained controversial, which has a very successful phylum of micro alga that thrive in made it difficult to properly understand their both aquatic and terrestrial habitats. Diatoms are evolution (Cavalier-Smith and Chao 2006; Riisberg usually recognized by the siliceous cell walls they et al. 2009; Yang et al. 2012). Cavalier-Smith and contain, which are made up of two valves. The Chao (2006), through a combined analysis of genes structure and processes of the valves have been including 18S rRNA, determined that the important morphological characters used to classify Bacillariophyceae with the Bolidophyceae formed a diatoms (Round et al. 1990;;Medlin and Kaczmarska basal group within Ochrophyta. Riisberg et al. 2004). The diatoms also have an unusual process of (2009) combined nucleotide SSU and ISU rDNA cell division that involves a reduction in one of the sequences with amino acid sequences from the daughter cells after mitosis (Mann and Marchant Ochrophyta, Bigyra, and Pseudofungi. Their study 1989). Morphological characters have been used to used 35 taxa, representing ten different heterokont determine the phylogeny of diatoms up until the classes. The cladogram that was constructed from the 52 Euglena: 2013 rDNA gene alone supported Cavalier-Smith and comparison to the rest of the Ochrophyta. One Chao (2006) illustrating the Bacillariophyta to be a outgroup taxon, Pirsonia diadema (from the phylum basal group within the Ochrophyta. Yang et al. Oomycota), was also included in the analysis so that (2012) also found similar results illustrating the Ochrophyta taxa could be compared to other Bacillariophyta as a basal group among the species of algae. Ochrophyta when using SSU rRNA and chloroplast The phylogenetic analysis was completed by genes. However, other studies, based on morphology, obtaining the 18S rRNA sequenced data from an have suggested that the diatoms are not basal at all, NCBI BLAST search for each species, the accession but belong within or closely related to the numbers for which are listed in Appendix A. The Chrysophyta (Dodge 1973; Taylor 1976; Mann and sequences were aligned using CLUSTAL W in Marchant 1989). Clearly, the placement of the MEGA 5.1and then used to generate three ML diatoms within Heterokontae remains uncertain. phylogenetic trees using three best-fit models. Within Recent molecular investigations have MEGA 5.1, there are twenty-four Maximum also shown uncertainty in regards to other phyla Likelihood nucleotide substitution models available within the heterokonts. Brown and Sorhannus for phylogenetic analysis. The three models used in (2010), Yang et. al. (2012), and Andersen (2004) this study are the Kimura 2-parameter (K2P, Figure suggest that Silicoflagellata are in a clade with 1), Tamura 3-parameter (T92, Figure 2), and Tamura- Actinophrydia and Bacillariophyta. However, Bold Nei (TN93, Figure3). The K2P model considers that and Wynne (1985) and Kristiansen (1990) suggest rates of transitions and transversions along a gene that Silicoflagellates are taxa within Chrystophyta. may occur and may not be equal. The T92 model Another phylum that has remained uncertain is the accounts for bias that may have been created by Pinguiophyta. While Anderson (2004) suggests that mutations. The TN93 model accounts for differences Pinguiophyta are the most derived of the heterokonts, between the transitional substitution rates of purines Brown and Sorhannus (2010) and Yang et al. (2012) and the transversional substitution rates of place Pinguiophyta within other clades. There is not pyrimidines. These three models are statistically the yet a consensus concerning the placement of these best choices when using many genes, such as 18S phyla. rRNA, that often undergo transitions, transversions, The 18s rRNA gene is frequently used when and nucleotide changes (Hall 2011). The trees were determining the phylogeny of diatoms because the produced with bootstrapping set to 1000 replications. gene illustrates evolutionary relationships that are Finally, a summary tree (Figure 4) was constructed, independent of morphological characters (Woese showing the phylum relationships suggested by the 1987; Bhattacharya et al. 1992). Additionally, 18s three maximum likelihood cladograms. rRNA gene sequences are readily available for many diatom lineages as a result of early molecular work Results by Medlin (Medlin et al. 1988). Currently, the Figures 1-3, the three maximum likelihood determinations of phylogenetic relationships among cladograms generated, display identical organization most or all heterokont algal classes have been based except for the relationship between Thalassiosira on the 18S rRNA gene (Andersen 2004). eccentrica and Lauderia borealis. The relationships The purpose of this paper is to confirm the suggested by Figures 1-3 can be divided into three phylogenetic relationships amongst the Ochrophyta, main clades, shown in Figure 4. Clade A includes the photosynthetic Heterokonts. Specifically, we Xanthophya, Phaeophyta, Raphidiophyta, explore the basal location of the Bacillariophyta, the Silicoflagellata, Eustigmatophyta, and Chrysophya; uncertain position of the Silicoflagellata, and the Clade B only includes Pinguiophyta; and Clade C distinction of the Pinguiophyta from the rest of the includes Bacillariophyta, the diatoms. In all three Ochrophyta. Figures, the diatoms (clade C) are paraphyletic and forms the most basal group of the Ochrophyta, with Materials and Methods low bootstrap support. The three Figures also Thirty-six taxa of Ochrophyta were illustrate Pinguiophyta as a distinct monophyletic examined in this study, all of which are listed with clade (labeled Clade B), supported by high bootstrap authorities in Appendix A. Taxa were taken from values. The other Ochrophyta phyla are organized in phyla within the Ochrophyta including Xanthophyta, a more derived clade (labeled Clade A) in Figures 1- Phaeophyta, Raphidiophyta, Silicoflagellata, 3. Many of the nodes within this clade are Eustigmatophyta, Chrysophyta, Pinguiophyta and inadequately supported due to low bootstrap values. Bacillariophyta. A greater portion of the taxa were Figure 4 displays these phylum relationships. In taken from Bacillariophyta in order to more closely Figures 1 and 2, the trees produced using the Kimura analyze the systematics of these organisms in