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Euglena: 2013

A Phylogenetic Analysis Using Heat Shock 90 () and Concatenated Small Subunit and Large Subunit Ribosomal RNA (18S and 28S) to Determine the Placement of the Hacrobiae within the Eukarya . Austin Iovoli, Steven Cole, Erica Meader, Danielle Reber.

Department of , Susquehanna University, Selinsgrove, PA 17870.

Abstract We investigated taxa of the Eukarya domain in order to determine the phylogentic placement of the Hacrobiae among the , , Unikonta, Alveolatae, and . In addition, we examined the different relationships between the phyla within the Hacrobiae. Using 90 (HSP90) and concatenated small and large subunit ribosomal RNA (SSU and LSU rRNA), a maximum likelihood tree for each was constructed using the Jones-Taylor-Thorton (JTT) model and the Jukes-Cantor model, respectively. These molecular trees were used to generate a consensus tree that defined the relationships between the Hacrobiae and other eukaryotic kingdoms. Our consensus tree indicated that the Hacrobiae taxa emerge in an association with the Archaeplastida. This suggests that the phyla of the Hacrobiae should be removed from the Supergroup and that their relationship with the archaeplastids should be redefined.

Please cite this article as: Iovoli, A., S. Cole, E. Meader, and D. Rever. 2013. A phylogenetic analysis using heat shock protein 90 (HSP90) and concatenated small subunit and large subunit ribosomal RNA (18S and 28S) to determine the placement of the Hacrobiae within the Eukarya domain. . doi:/euglena. 1(2): 66-73.

Introduction Cavalier-Smith (2003) united members by the The organization of the phylogenetic presence of that were obtained from red relationships of is a very controversial in a secondary endosymbiotic relationship topic (Burki et al. 2007). The taxa are (Burki et al. 2007; Hackett et al. 2007). More recent difficult to place within supergroups. The evidence also suggests that most chromalveolates supergroups are a predicted classification in which (with the exception of cryptophytes) also contain multiple related kingdoms fall under. Burki et al. mitochondria with tubular cristae (Hackett et al. (2007) and Hackett et al. (2007) suggest six 2007). The organization of Chromalveolata has been supergroups: Opistakonta, , under much scrutiny for the past few with the Archaeplastida, Chromalveolata, , and biggest concern being which kingdoms fall into this Excavata. The suggestion of Bauldauf (2008) uses category (Hackett et al. 2007; Harper et al. 2005). only four supergroups. This hypothesis places the Chromalveolata is now typically associated Rhizaria in the supergroup Chromalveolata and with four member kingdoms: Stramenopile (also suggests that Amoebozoa is related to Animalia and known as Herokontae), Alveolatae, Rhizaria, and Fungi; thus, belonging in the supergroup Unikonta Hacrobiae (Holt and Iudica 2013). The major (Holt and Iudica 2013). There are many different problem with this supergroup is the lack of molecular theories about the of eukaryotes with most support for the of its member kingdoms only having moderate support (Burki et al. 2007). (Baurain 2010). Nuclear-based analyses have The number and membership of kingdoms in the suggested that the and are Chromalveolate supergroup has caused one of the sister taxa, and that they are closely related to the most disputed of these supergroup classifications Rhizaria (Burki et al. 2012). Burki et al. (2007) (Hackett et al. 2007). dubbed this relationship the SAR group. Burki et al. The supergroup Chromalveolata is a (2012) have also shown a close relationship between grouping of eukaryotes that arise from both members of the phyla Haptophyta and Cryptophyta, photosynthetic and heterotrophic lineages (Baurain which have been proposed as members of the 2010). The Chromalveolate hypothesis developed by Hacrobiae by Cavalier-Smith (2010).

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The Hacrobiae , established by Analysis (Tamura et al. 2011) to generate molecular Cavalier-Smith (2010), includes the , phylogenetic trees. The sequences for SSU and LSU Haptomonads and Centrohelomonads although other were then aligned separately by ClustalW and phyla (Telonemids) have also been suggested for the trimmed repeatedly to 1709 and 560 base pairs, kingdom (Okamoto et al. 2009). Nikolaev et al. respectively. This ensured the sequences were well (2004), Okamoto et al. (2009) and Burki et al. (2009) aligned and trimmed them all to the same length for also formally established Hacrobiae as a diverse each . The sequenced data was then concatenated monophyletic clade in their molecular analyses. The to create a larger sequence for every by inclusion of Hacrobiae in the Chromalveolates combining the SSU and LSU trimmed and aligned expands the supergroup to encompass several sequences. Following this, the newly concatenated eukaryotic lineages that were originally not expected SSU and LSU sequences were aligned again by to be related to the Chromalveolates (Burki et al. ClustalW to ensure they were appropriately paired. 2012) allowing this supergroup to now account for Once the sequences were aligned, the species about 50% of the known and algae species sampled were ready to be analyzed by statistical (Harper et al. 2005). The Hacrobiae represents the estimation methods. For the HSP90 presence of a second of Chromalveolates sequences, the same process was repeated to ready separate from the SAR group. The plastids in the sequences for analysis except without the members of this group (mainly and concatenation step. The trimmed and aligned HSP90 Cryptophytes) have a unique lateral gene transfer sequence included 570 amino acids. which is not present in Stramenopiles, Alveolates, or With the aligned concatenated SSU and LSU nucleic Rhizarians (Burki et al. 2012). The Cryptophytes also acid sequences for every sampled species, a have the derived feature of flattened cristae, which Maximum Likelihood (Figure 1) phylogentic tree separates these species (Hackett et al. 2007). was generated using MEGA5. We constructed Figure Because many of the species belonging to the phyla 1 using the Jukes-Cantor model. With the aligned for this kingdom are seemingly unrelated to the other HSP90 sequences, another ML tree (Figure 2) was Chromalveolates, the placement of the Hacrobiae generated using the Jones-Taylor-Thorton model. In kingdom in this supergroup has received some debate each phylogenetic analysis, a bootstrap method of (Burki et al. 2012; Harper et al. 2005; Hackett et al. 1000 bootstrap replications was used. The bootstrap 2007). The purpose of this paper is to determine the values generated through this aid in supporting the phylogenetic placement of the Hacrobian taxa among accuracy of the results. Using MEGA5, a consensus the Eukaryotes and to examine the relationships of tree based on Figure 1 and 2 was constructed that phyla within the Hacrobiae. defines the phylogenetic relationships between the Hacrobiae and the rest of the Eukarya Domain. Materials and Methods The sample size consisted of 29 species Results (Table 1) from within the Eukarya Domain for the Figure 1 is split into eight clearly defined concatenated small subunit and large subunit clades. In the rRNA tree there are 29 species listed. ribosomal RNA analysis and 39 species (Table 2) for Figure 1 places the phyla of the Hacrobiae in a the heat shock protein 90 analysis. Within the polytomy with the Rhizaria, Stramenopiles, and Eukaryotes, the study included eight species from the Archaeplastida. This is suggested by the high support Archaeplastida, eight species from the for this polytomy emerging as a clade. Within the Cryptomonada, two species from the Telonemids, Hacrobiae phyla, the divisions of the Cryptomonada, seven species from the Haptomonada, four species Haptomonada, and Centrohelomonada clades are from the Centrohelomonada, eight species from the shown to be equally related to the Rhizaria, Stramenopiles, seven species from the Alveolatae, six Archaeplastida, and Stramenopiles. The Alveolatae species from the Unikonta, six species from the appear as a sister clade to this polytomy, suggesting a Excavata, and two species from the Rhizaria (Tables more separate relationship to the Hacrobiae than the 1 and 2). groups from within the polytomy. The Unikonta and Using the National Center for Excavata emerge separate to the Alveolatae and the Information (NCBI) online database, polytomy consisting of the Hacrobiae, Rhizaria, sequences for concatenated small subunit and large Stramenopiles, and Archaeplastida. subunit ribosomal RNA (SSU and LSU) and heat Similar to Figure 1, HSP90 tree suggests shock protein 90 (HSP90) were found for each of the that the Hacrobiae appear in a polytomy alongside species analyzed. For the nucleotide analysis, the the Archaeplastida. Figure 2 is separated into six sequences for SSU and LSU were then individually major clades and displays several relationships uploaded to Molecular Evolutionary Genetic between them. The Cryptomonada, Haptomonada,

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Centrohelomonada, and Telonemid groups within the appear in a large polytomy consisting of the proposed Hacrobiae emerge as closely related and are Excavata, Unikonta, Stramenopiles, Alveolatae, and represented in a polytomy with the Archaeplastida. Hacrobiae phyla + Archaeplastida clade. Each of This relationship is indicated with moderate support these monophyletic groups is supported with high and the individual clades are highly supported. bootstrap values. No further relationships were Following this, the rest of the Eukaryotic groups indicated with significant support.

Table 1: This displays the species, group, and accession number to the NCBI database for heat shock protein 90 (HSP90). The NCBI accession numbers were used to find an amino acid sequence for each species. The groups show where each species belongs within the Eukarya domain. Species Group HSP90 Archaeplastida BAF75926 lucimarinus Archaeplastida ABO98231 Pteroderma cristatum Archaeplastida ABJ80954 Archaeplastida AAL49788 Capsicum chinense Archaeplastida BAG16518 viride Archaeplastida ABJ80948 Glaucocystis nostochinearum Archaeplastida BAF75927 theta Cryptomonada AAX10949 sp. ATCC 50108 Cryptomonada AAP72158 Goniomonas truncata Cryptomonada ABJ80946 subtile Telonemids CAJ87182 Telonema antarcticum Telonemids CAJ87184 sp. NIES-1333 Haptomonada ABJ80934 galbana Haptomonada AAX10942 Pavlova lutheri Haptomonada AAX10944 contractilis Centrohelomonada BAF92012 rasilis Stramenopiles AAX10943 Plectospira myriandra Stramenopiles AAX10947 Achlya ambisexualis Stramenopiles AAM90674 Thraustotheca clavata Stramenopiles AAX10950 palmivora Stramenopiles AAX10946 Phaeodactylum tricornutum Stramenopiles AAX10945 Synura sphagnicola Stramenopiles ABJ80957 bovis Alveolatae AAF61428 intermedium Alveolatae AAR26656 Halteria grandinella Alveolatae AAR27539 Pseudourostyla cristata Alveolatae AAY67878 Alveolatae AAP44977 micrum Alveolatae ABI14419 Crypthecodinium cohnii Alveolatae AAM02974 Opistophthalmus carinatus Unikonta AAQ94359 Spodoptera frugiperda Unikonta AAG44630 Tetraodon nigroviridis Unikonta CAG03540 Monosiga brevicolis Unikonta AAP51213 Excavata AAQ24862 americana Excavata ABC54646 sp. ATCC50230 Excavata AAM93744 godoyi Excavata ACA34533 Rhynchobodo sp. ATCC50359 Excavata AAV66336

Discussion Chromalveolata supergroup. We interpret the Inferred Phylogeny relationships indicated by Figures 1 (LSU + SSU Figures 1 and 2 suggest that there was no ribosomal RNA) and Figure 2 (HSP90) to generate a monophyletic clade that constitutes the Hacrobiae. consensus tree (Figure 3) that does not conform to the The individual taxa of the Hacrobiae emerge supergroup analyses of Baldauf et al. (2003), Keeling alongside the Archaeplastids as a polytomy. (2004) and Cavalier-Smith (2010). Furthermore, they do not appear to be within the

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Table 2: This displays the species, group, and accession number to the NCBI database for small subunit and large subunit ribosomal RNA (18S and 28S). The NCBI accession numbers were used to find a nucleotide sequence for each species. The groups show where each species belongs within the Eukarya domain. Species Group SSU 18S LSU 28S Karlodinium micrum Alveolatae JF791048 DQ898222 Crypthecodinium cohnii Alveolatae FJ821501 FJ939575 Halteria grandinella Alveolatae AY007443 M98371 Pseudourostyla cristata Alveolatae GU942569 HM122033 Toxoplasma gondii Alveolatae L37415 AF076865 Pterocystis tropica Centrohelomonada AY749603 AY752993 Sphaerastrum fockii Centrohelomonada AY749614 AY752991 Chlamydaster sterni Centrohelomonada AF534709 EF681909 andersenii Cryptomonada AM901350 AM901321 pochmannii Cryptomonada AM901369 AM901342 marina Cryptomonada AB193602 DQ980471 Cryptomonada AM901358 AM901329 japonica Cryptomonada AB231617 FJ973371 Haptomonada JF489946 EU729474 Haptomonada HQ877901 EU502880 parvum Haptomonada AJ246269 AJ876802 Pavlova viridis Haptomonada DQ075201 DQ202388 Exanthemachrysis gayraliae Haptomonada DQ531625 JF718764 Synura sphagnicola Stramenopiles U73221 DQ980475 Phytophthora palmivora Stramenopiles AY742745 HQ665195 pseudonana Stramenopiles HM991698 HM991683 Cryptococcus neoformans Unikonta AJ560332 AB744849 Coprinopsis cinerea Unikonta JN939895 JQ045869 Chlamydomonas reinhardtii Archaeplastida KC166137 JX839535 Mesostigma viride Archaeplastida AF408245 L49152 Pterosperma cristatum Archaeplastida AB017127 L43359 natans Rhizaria FJ973362 AB453004 Gymnophrys cometa Rhizaria AF411284 FJ973379 gruberi Excavata M18732 XM_002674199

In Figure 1, the Cryptomonada, of these taxa. Thus, our work confirms and Haptomonada and Centrohelomonada (taxa included is consistent with that of Zhao et al. (2012). in the Hacrobia of Cavalier-Smith 2010) do not Figure 3 indicates that the phyla of the emerge as a monophyletic clade. However, Figure 1 Hacrobia emerge alongside the Archaeplastids and does show that each within the hacrobian form a clade that shares a sister relationship to the clade forms a polytomy with the Rhizaria, Rhizarians and Stramenopiles. Each phylum included Stramenopiles and Archaeplastids, a group also in the Hacrobia individually emerge as distinct known as the (Nikolaev et al. 2004). This groups that share sister relationships with one suggests that the Hacrobiae may not be anything but another, as well as the archaeplastids. The analysis of a paraphyletic collection of taxa, associated Nikolaev et al. (2004) suggests that the cryptophytes with SAR and the Archaeplastids. Figure 1 also does and heliozoans emerge as sisters, and the not signify that the phyla of the Hacrobia belong in crptophytes are sisters to the archaeplastids; however either the Chromalveolata or Archaeplastida. there is no monophyletic hacrobian clade that Figure 2 unites the individual phyla of the emerges (Holt and Iudica 2013). Kim and Archibald Hacrobiae and the Archaeplastids together, forming a (2013) show the individual phyla of the Hacrobia polytomy, with rather low support. However, this forming a clade with Viridiplante and Glaucophyta phylogenetic arrangement could suggest that the within Archaeplastida, and sharing a sister phyla of the proposed Hacrobia clade are more relationship with the SAR group. There is no clear closely related to the Archaeplastids than the emergence of a monophyletic Hacrobia clade in this Chromalveolates. A molecular analysis by Zhao et al. analysis either. Another analysis by Burki et al. (2012) recognizes the Hacrobiae as individual phyla (2009) presented the Cryptomonads, , that share sister relationships with one another and Telonemids, and Haptophytes as a clade with also with Archaeplastids. Zhao et al. (2012) also unsupported branching patterns, as a sister to the suggest the monophyly of Hacrobia remains SAR group. Subsequently, Figure 3 implies that there controversial because it is dependent on the selection may not be a Hacrobiae clade, but rather the of taxa and gene data set, and evolutionary individual phyla belong in a new clade including the relationships are still uncertain due to the complex Archaeplastida. This also suggests that these phyla

69 Euglena: 2013 may be more closely related to Archaeplastids than classic placement of the Hacrobia phyla within the Chromalveolates, based on the Hacrobiae + Chromalveolata supergroup and the existence of a Archaeplastida group seen in Figure 3. The molecular Hacrobia as a clade all together. evidence summarized in Figure 3 challenges the

Figure 1: A generated using concatenated small subunit and large subunit ribosomal RNA sequences from NCBI. This tree was generated through the Maximum Likelihood method under the Jukes-Cantor model using MEGA5. This method produces a tree based on the rate of nucleotide substitution being the same for all pairs of the four nucleotides A, T, C, and G. The phylogenetic relationship between the Hacrobiae, Stramenopile, Alveolatae, Unikonta, Rhizaria, and Excavata clades is shown. To support the accuracy of the data, 1000 bootstrap replications were conducted. The phyla of the Hacrobiae do not emerge as a monophyletic group, indicated by a question mark (?).

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Hampl et al. (2009) suggest that the construction of eukaryotes (Lane and Archibald uncertain phylogenetic position of haptomonads 2008). A possible explanation for the relationship (which are secondary algae) may be a result of the between the hacrobians and archaeplastids could be a acquired through secondary endosymbiosis. result of secondary endosymbiosis, which could Endosymbiotic gene transfer is one of the major cause part of the archaeplastid to exist within causes of lateral gene transfers in eukaryotic genomes several taxa of the hacrobians Even so, the complex (Ball et al. 2011). that acquired genomes of the Hacrobiae lead to unresolved via secondary endosymbiosis have a discrepancies related to the classification of nuclear genome made up of genes derived from two Hacrobiae. or more nuclei, complicating the phylogenic

Figure 2: A phylogenetic tree generated using heat shock protein 90 (HSP90) amino acid sequences from NCBI. This tree was generated through the Maximum Likelihood method under the Jones-Taylor-Thornton (JTT) model using MEGA5 (Tamura et al. 2011). This method produces a tree based on the divergence among sequences modeled using a . The phylogenetic relationship between the Hacrobiae, Stramenopile, Alveolatae, Unikonta, and Excavata clades is shown. To support the accuracy of the data, 1000 bootstrap replications were conducted. The phyla of the Hacrobiae do not emerge as a monophyletic group, indicated by a question mark (?).

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Figure 3: A consensus phylogenetic tree based on the data from the rRNA and HSP90 trees (Figure 1 and 2, respectively). This tree unites the phyla of the proposed Hacrobiae and the archaeplastids together within a polytomy. In addition, it indicates the Hacrobiae phyla and Archaeplastida share a close relationship with the Stramenopiles and Rhizaria. The Excavata, Alveolatae, and Unikonts emerge as separate sister groups. The phyla of the Hacrobiae do not emerge as a monophyletic group, indicated by a question mark (?).

Conclusion Burki et al. 2009). Our overall analysis suggests that The hacrobians are a difficult group of taxa the Hacrobia hypothesis (Cavalier-Smith 2010) is not to place among the Eukaryotes. They are made up of valid, that they be removed from the Chromalveolata several diverse groups and have been traced to supergroup, and that the individual hacrobian phyla belonging in multiple different relationships. When be associated with the archaeplastids. organizing them with molecular data, it is important to take into account lateral gene transfer and Literature Cited endosymbiotic events. The selection of genes and Baldauf, S. L. 2003a. The deep roots of eukaryotes. taxa for a molecular analysis can alter the results, due Science. 300 (5626): 1701-1703. to the intricate genomes they acquire by engulfing Baldauf, S. 2008. An overview of the phylogeny and other eukaryotes. Based on our results, the individual diversity of eukaryotes. Journal of hacrobian phyla emerge in a group alongside the and Evolution. 46(3): 263-273. Archaeplastida, and thus seem to not belong in the Ball, S., C. Colleoni, U. Cenci, J. N. Raj and C. Chromalveolata supergroup. This finding is Tirtiaux. 2011. The evolution of glucogen consistent with many recent published studies based and starch in eukaryotes gives on molecular data (Zhao et al. 2012, Kim and molecular clues to understand the Archibald 2013); however, it challenges several establishment of endosymbiosis. others (Cavalier-Smith 2010; Okamoto et al. 2009;

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Submitted 22 March 2013 Accepted 7 April 2013

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