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Euglena: 2013

Defining Phylogenetic Relationships of 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 , 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. . 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 (Medlin is a 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 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 and (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 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 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 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 . Ochrophyta when using SSU rRNA and 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 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 2-parameter and Tamura 3-parameter models, these

53 Euglena: 2013 two taxa are displayed in a sister relationship. In to one another. The corresponding bootstrap values Figure 3, the tree produced using the Tamura-Nei of this relationship are fairly weak in all three model, T. eccentrica and L. borealis are paraphyletic Figures.

Figure 1: Maximum Likelihood cladogram displaying the phylogeny of the Ochrophyta, generated with the best fit model Kimura 2-parameter in MEGA 5.1, using the 18s rRNA gene for all thirty-seven taxa. Using MEGA 5.1, the Kimura 2-parameter model was determined to be statistically the best choice for the gene in question. This cladogram illustrates three major clades; A, B, and C. The branching within Clade A is the least supported due to the low BP value of 38. Pinfuiophyceae, clade B, is strongly separated from the other clades with a BP value of 89. Clade C is the most basal group within the Ochrophyta. The BP values within clade C are not all within the accepted range.

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Discussion photosynthetic heterokonts. This placement agrees Figures 1-3 display the same phylogenetic with the work of Adl et al. (2005), Brown and relationships, summarized in Figure 4. This is Sorhannus (2010), and Yang et al. (2012). Anderson significant because three different evolutionary (2004) does not support this placement of the models produced essentially the same result. diatoms. Although supported with acceptable Figure 4 suggests that the diatoms, Clade bootstrap values, the organization of taxa within the C, are paraphyletic, yet basal within Ochrophyta, the diatoms , suggested by Figures 1-3, disagrees with

Figure 2: Maximum Likelihood cladogram displaying the phylogeny of the Ochrophyta, generated with the best fit model Tamura 3- parameter in MEGA 5.1, using the 18s rRNA gene for all thirty-seven taxa. Using MEGA 5.1, the Tamura 3-parameter model was determined to be statistically the second best choice for the gene in question. This cladogram illustrates three major clades; A, B, and C. The branching within clade A is the least supported due to the low BP value of 32. Pinfuiophyceae, clade B, is strongly separated from the other clades with a BP value of 88. Clade C is the most basal group within the Ochrophyta. The BP values within clade C are not all within the accepted range.

55 Euglena: 2013 previous work (Medlin and Kaczmarska, 2004). (2004), although it is still applicable for phylogenetic However, Medlin and Kaczmarska (2004) used a studies at the phylum level. The relationships of taxa multi gene analysis, unlike our single gene analysis. within the diatoms shown in Figures 1-3 do not affect It is probable that 18s rRNA does not resolve the the placement of Bacillariophyta within diatoms with the same resolution achieved by the Heterokontae, suggested by Figure 4. multi-gene analysis of Medlin and Kaczmarska

Figure 3: Maximum Likelihood cladogram displaying the phylogeny of the Ochrophyta, generated with the best fit model Tamura-Nei in MEGA 5.1, using the 18s rRNA gene for all thirty-seven taxa. Using MEGA 5.1, the Tamura- Nei model was determined to be statistically the third best choice for the gene in question. This cladogram illustrates three major clades; A, B, and C. The branching within clade A is the least supported due to the low BP value of 31. Pinfuiophyceae, clade B, is strongly separated from the other clades with a BP value of 84. Clade C is the most basal group within the Ochrophyta. The BP values within clade C are not all within the accepted range.

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The existence of Clade A is supported by designated Clade A. On the other hand Anderson large bootsrap values. However, some of the (2004) shows Clade B as the most derived phylum. relationships within this clade are weakly supported. However, Figure 4 shows that Xanthophyta and Still, our study, Adl et al. (2005), Brown and Phaeophyta as the most derived phyla. It is clear that Sorhannus (2010), Yang et al. (2012), and Cavalier- more studies need to be conducted to investigate the Smith (1989), all support the general relationship that phylogenetic position of Pinguiophyta. Despite that, is depicted in Figure 4 using a wide variety of it is very likely that this phylum is closely related to molecular sequences. However, the relationships in Clade A since all of the taxa within both Clade A and Figure 4 do not agree with recent studies regarding B share similar morphological characters, including the placement of the silicoflagellates. Recent the pigments chlorophyll a and c1,2 (Adl et al. molecular investigations (Brown and Sorhannus 2005). 2010; Yang et. al. 2012; Andersen 2004) suggest that Diatoms have consistently been difficult to Silicoflagellata are in a clade with Actinophrydia and place phylogenetically within Heterokontae. Our Bacillariophyta. Still, the location of Silicoflagellata study, summarized in Figure 4, agrees with the recent presented in our study, sister to Eustigmatophyta and molecular work that suggests diatoms are basal, but Chrysophyta, is not unfounded. Silicoflagellates are not monophyletic within Ochrophyta. Figures 1-3 very similar to Chrysophytes, and were even are not conclusive in regards to the relationships of traditionally thought to be within Chrystophyta (Bold taxa within Bacillariophyta because Figures 1-3 show and Wynne 1985; Kristiansen 1990). Clearly, the this phylum to be paraphyletic. Other phyla that have phylogenetic placement of Silicoflagellata remains proven difficult to classify are Silicoflagellata and uncertain and further study is needed to remedy this Pinguiophyta. While Figure 4 displays a well- disagreement. Likely, characters of Silicoflagellata supported placement of Silicoflagellata that agrees are similar to both clades, so the determined with Bold and Wynne (1985) and Kristiansen (1990), relationship depends on the character examined. this phylum remains inconclusive and in need of Clade B, the Pinguiophyta (shown in further study. Additionally, Figure 4 suggests that Figure 4), is strongly supported by high bootstrap Pinguiophyta is a distinct monophyletic clade, but values on Figures 1-3. There is no accepted there is no consensus regarding the monophyly of consensus on the phylogenetic placement of this this phylum (Brown and Sorhannus 2010; Yang et al. phylum. The studies by Brown and Sorhannus (2010) 2012; Anderson 2004). and Yang et al. (2012) show Clade B within our

Figure 4: The summary tree that displays the phylum relationships suggested in Figures 1-3. The phyla have been separated into three major clades: A, B and C. Although Bacillariophyta has been labeled Clade C for simplification, Figures 1-3 suggest this phylum is paraphyletic.

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Submitted 22 March 2013 Accepted 7 April 2013

Appendix A: The Latin binomial, accession number for 18S and authority with corresponding year including all 37 heterokont species and 1 outgroup species. Latin Binomial Accession Number (18S) Authority Thermalis AY485458 Kutzing, 1862 Vacuolaria virescens U41651 Cienk, 1870 marina AY788928 Hada, 1980 Botrydium granulatum HQ710587 Greville, 1874 Botrydium stoloniferum U41648 Mitra, 1950 Bumilleriopsis pyrenoidosa AJ579332 Deason & Bold, 1978 AB512123 Ehrenberg, 1866 Chattonella subsalsa U41649 Biecheler, 1936 granulata KC128500 Karlson & Potter, 1996 Nannochloropsis oceanica AB183587 Suda & Miyashita, 2002 Nannochloropsis limnetica DQ977726 Krienitz et al., 2000 Thalassiosira eccentrica X85396 Ehrenberg, 1904 Lauderia borealis X85399 Gran,1900 Aulacoseira ambigua X85404 Simonsen, 1979 Coscinodiscus radiatus X77705 Ehrenberg, 1840 Cymatosira belgica X85387 Grunow, 1881 Papiliocellulus elegans X85388 Hasle, 1973 Grammonema striatula X77704 Lyngbye, 1819 Asterionellopsis glacialis AY216904 Round, 1990 Asteroplanus karianus Y10568 (Grunow) Gardner & Crawford, 1997 paxillifer M87325 Marsson, 1901 Cylindrotheca closteriva M87326 (Ehrenberg) Reimann & Lewin, 1964 Polypodochrysis teissieri HQ710581 Magne, 1975) Pinguiochrysis pyriformis HQ710580 Kawachi et al., 2002 Phaeomonas parva AB042204 Honda & Inouye, 2002 Bolidomonas pacifica HQ912557 Guillou & Chrétiennot-Dinet, 1999 Dictyocha fibula AB096710 Ehrenberg, 1839 Oochromonas moestrupii U42382 Andersen, 2011 Bolidomonas mediterranea AF123596 Guillou & Chrétiennot-Dinet,1999 Dictyocha speculum U14385 Ehrenberg, 1839 Dictyocha globosa HQ646558 Hara & Chihara, 1994 faculiferum JN934680 Willén, 1992 Synura curtispina EF165128 (Petersen & Hansen) Asmund 1968 Pirsonia diadema AJ561114 Kuhn, 1996 siliculosus L43062 (Dillwyn) Lyngbye, 1819 Phaeurus antarcticus HE866932 Skottsberg, 1907 aculeata HE866893 (Linnaeus) Lamouroux, 1813

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