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Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101 DOI: 10.1007/s13131-014-0445-2 http://www.hyxb.org.cn E-mail: [email protected]

Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown (Phaeophyceae) in

JIA Shangang1,3†, WANG Xumin1,3†, LI Tianyong2, QIAN Hao2, SUN Jing1,3,4, WANG Liang1,3,4, YU Jun1,3, REN Lufeng1,3, YIN Jinlong1, LIU Tao2*, WU Shuangxiu1,3* 1 CAS Key Laboratory of Genome Sciences and Information, Beijing Key Laboratory of Genome and Precision Medicine Technologies, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China 2 College of Marine Life Science, Ocean University of China, Qingdao 266003, China 3 Beijing Key Laboratory of Functional Genomics for Dao-di Herbs, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China 4 University of Chinese Academy of Sciences, Beijing 100049, China

Received 22 March 2013; accepted 13 August 2013

©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2014

Abstract The chloroplast and mitochondrion of (Class Phaeophyceae of Phylum ) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others (Phylum Cryptophyta and Haptophyta) in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. resolution for Class Phaeophyceae showed that it was divided into Laminariales- clade and clade, and phylogenetic positions of Kjellmaniella crassi- folia, Hizikia fusifrome and okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events. Key words: Phaeophyceae, brown algae, Chromista, phylogenetic trees, mitochondrion, chloroplast Citation: Jia Shangang, Wang Xumin, Li Tianyong, Qian Hao, Sun Jing, Wang Liang, Yu Jun, Ren Lufeng, Yin Jinlong, Liu Tao, Wu Shuangxiu. 2014. Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown algae (Phaeophyceae) in China. Acta Oceanologica Sinica, 33(2): 94–101, doi: 10.1007/s13131-014-0445-2

1 Introduction genera, exhibit many unusual and interesting metabolic, devel- Algae comprise a large number of most diverse unicellular opmental and cell-biological features (Cho et al., 2004; Wynne and multi-cellular taxa that virtually populate all the ecosys- and Loiseaux, 1976). tems on Earth. It covers a large variety of about 20 taxonom- The origins of mitochondria and plastids, the most impor- ic groups. The best-known ones are red, green, brown, and tant organelles in algae, fundamentally explain the evolution- , , glaucophytes, , crypto- ary nature of , which draw much attention from the phytes, , chlorarachniophytes, dinoflagellates and researchers all over the world. It was suggested that plastid/ euglenids (Katz, 2012). Kingdom Chromista, nominated by chloroplast, the algal photosynthetic organelle, may originate Cavalier-Smith, comprises of Cryptophyta, Heterokonta and directly from a cyanobacterium-like prokaryote via primary en- Haptophyta, which contain chlorophyll a/c and four mem- dosymbiosis about 1 to 1.5 billion years ago (Rodríguez-Ezpele- branes surrounding the plastids and indicate the presence of a ta et al., 2005). Based on the recent hypothesis, there are three secondary endosymbiotic event (Medlin et al., 1995). Pigments major host lineages, red algae, green plants, and glaucophytes, and chlorophyll make them appear in brown or golden color. whose plastids have two bounding membranes (McFadden, Many Chromista algae are of great significance. For example, 2001). In addition, the second endosymbiosis refers to the en- brown algae (Class Phaeophyceae of Order Ochrophyta of Het- gulfment of red algae (Green, 2011) or green algae by eukary- erokonta) are a large group of mostly marine multicellular algae otic hosts, and their plastids finally have three or four bound- and are one of the most productive ecosystems in the world (Sil- ing membranes. In contrast, algal mitochondria experienced a berfeld et al., 2010), from microscopic filaments (for example, different evolutionary history. Gray et al. (2001) concluded that Ectocarpus) to the giant structurally-complex thalli of . All mitochondria originated from α-proteobacterial ancestor, en- of the estimated brown algal 1 811 species, belonging to ca. 285 tered eukaryotes long before plastids and co-evolved together

Foundation item: The National Natural Science Foundation of China under contract Nos 31140070, 31271397 and 41206116; the algal transcrip- tome sequencing was supported by 1KP Project (www.onekp.com). *Corresponding author, E-mail: [email protected], [email protected] †Contributed equally. JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101 95

with their host. So mitochondrial DNA can show evolutionary ista algae, especially for brown algae. history of the nuclear genome (Burger and Nedelcu, 2012). The current knowledge on the origins of chloroplast and 2 Materials and methods mitochondrion is mostly determined based on the shared conserved regions or genes in the algal chloroplastic and mi- 2.1 Sequence datasets from 1KP project tochondrial genomes, respectively. DNA sequences are widely The assembled transcriptomic sequencing datasets were used to study on the algal phylogenetic relationships, such as, downloaded from the website of 1KP Project, including 19 brown small subunit (SSU) (Tan and Druehl, 1994) and large subunit algae species (Phaeophyceae) (Table 1). Totally, 786 783 495 (LSU) of rDNA (Phillips et al., 2008) in nucleus, rbcL, psaA and base pairs are used to search for orthologs. The 18 complete psbA (Cho et al., 2004) in plastids, and Cytochrome c (Danne mitochondrial and 13 chloroplastic genomes of Chromista et al., 2012) in mitochondria. However, protein sequences are algae, belonging to Ochrophyta, Cryptophyta and Haptophyta, preferred because they are more conserved than DNA ones, respectively, were downloaded from NCBI (Table 2). Their gene and the concatenated genes provide more comprehensive in- number ranges from 19 to 51 for mitochondria and 81 to 147 formation than a single one. Phylogenetic trees are construct- for plastids. ed in several software programs (for example, MEGA, PAML, Mrbayes, Tree-Puzzle, etc.) based on different models of evolu- 2.2 Constructing phylogenetic trees tion (Green, 2011). We performed several procedures to construct maximum The available whole-genome data has been a spotlight in al- likelihood trees which are described as follow. First, all gene gal research, which enables us to perform phylogenomic analy- DNA/protein sequences of 18 mitochondrial and 13 chloro- sis. Phylogenomics refer to large-scale data mining and analy- plastic genomes collected from NCBI website were used as ses of massive amounts of genome sequences. It is noted that references (including the outgroup Cyanophora paradoxa, expressed-sequence-tags (ESTs) and genomes from mitochon- NC_001675.1, Phylum Glaucophyta). Second, considering pos- dria and plastids can be used to construct phylogenetic trees sible horizontal gene transfer or gene exchanges between nu- (Hallstrom and Janke, 2009). The same set of genomic sequence cleus and organelles, we identified 17/41 typical genes shared data can potentially result into different taxonomic relation- by the complete mitochondrion/chloroplast genomes, aiming ships based on the unique orthologous genes of mitochondria at excluding potential nucleus-originated genes. Third, the pro- and chloroplasts. The differences reflect the distinct endosym- tein sequences of typical reference genes were used to perform biotic events of these two organelles. We had suggested that in local TBLASTN searches against the assembled transcript data- phylogenomic trees built with algal ESTs and genomes of mito- sets of our 19 brown algae from 1KP Project to obtain putative chondria and chloroplasts, Ulva prolifera was placed in a sister algal orthologs. The best hits were selected and their alignment position to Ulva linza but shared a similar chloroplast origin information was also stored to do pairing of references and with Pseudendoclonium akinetum (Jia et al., 2011). samples. We compared the filtering power of different cutoff In this study, as a part of the 1 000 Plants (1KP) Project E-values at 10−5, 10−10, 10−20, 10−30, 10−40 and 10−50, to achieve (http://www.onekp.com/) which covers more than 1 000 differ- reliable orthologs. Fourth, phylogenetic trees were constructed ent species of plants by generating large scale gene sequence in- by MEGA 5.1 based on Jones-Taylor-Thornton (JTT) model with formation, we focused on the phylogenomic analysis of Chrom- bootstrap method 1 000.

Table 1. Brown algal (Class Phaeophyceae, Order Ochrophyta) species information with assembled contigs from 1KP Project Order Family Species Contig# Tot/Mb Avg/bp Lon/bp viridis 53 140 24.8 467 11 018 Dictyotaceae Dictyopteris undulata 100 199 49.6 495 14 604 Ishigeaceae Ishige okamurai 78 583 48.2 614 14 784 Ectocarpales Scytosiphonaceae Colpomenia sinuosa 80 884 43.5 538 1 509 Petalonia fascia 89 229 52.6 590 12 350 Scytosiphon dotyo 69 680 40.9 587 10 507 Scytosiphon lomentaria 75 666 47.0 621 13 047 Laminariales Laminariaceae Kjellmaniella crassifolia 88 084 41.9 476 8 676 Laminaria japonica 76 104 31.7 417 10 968 Laminaria japonica-2 75 714 33.4 441 12 165 Alariaceae Undaria pinnatifida 72 256 34.9 483 8 628 Fucales Hizikia fusifrome 116 790 48.0 411 8 731 hemiphyllum 87 021 44.9 516 12 861 Sargassum henslowianum 77 474 38.0 491 10 826 75 127 39.0 519 11 559 Sargassum integerrimum 82 503 37.5 455 8 371 81 102 43.3 534 9 887 Sargassum thunbergii 106 596 49.0 459 10 151 Sargassum vachellianum 69 871 38.5 550 12 856 Notes: Tot, Avg and Lon indicate total length/Mb, average length/bp and longest length/bp of contigs, respectively. # indicates the numbers of contigs. 96 JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101 NA NA NA NA NA NA NA NA Size 28 660 60 553 29 013 60 553 48 063 37 657 37 657 37 656 37 638 37 657 37 605 37 500 37 657 36 392 58 507 38 007 31 617 39 049 19 43 20 43 43 37 37 37 37 37 37 37 37 37 51 38 37 38 NA NA NA NA NA NA NA NA Gene# Mitochondrion NA NA NA NA NA NA NA NA JN022704.1 EU651892.1 NC_005332.1 NC_010637.1 NC_002572.1 NC_013477.1 NC_013484.1 NC_013478.1 NC_015669.1 NC_013476.1 NC_013473.1 NC_013475.1 NC_013482.1 NC_007683.1 NC_003055.1 NC_004024.1 NC_007685.1 NC_007684.1 NCBI Accession NA NA NA NA NA NA NA NA NA NA NA NA NA Size 95 281 77 717 77 717 105 651 105 651 105 297 105 309 135 854 135 854 121 524 130 584 124 986 139 954 99 99 81 81 NA NA NA NA NA NA NA NA NA NA NA NA NA 110 109 118 145 145 146 138 138 147 Gene# Chloroplast NA NA NA NA NA NA NA NA NA NA NA NA NA JN117275.1 JN022705.1 EF508371.1 GQ358203.1 NC_016703.1 NC_020371.1 NC_007288.1 NC_009573.1 NC_000926.1 NC_013703.1 NC_018523.1 NC_016735.1 NC_013498.1 NCBI Accession Species Pavlova lutheri lutheri Pavlova theta Guillardia Fucus vesiculosus Fucus Emiliania littoralis Laminaria digitata Desmarestia Dictyota dichotoma salina Emiliania huxleyi- 2 Emiliania Saccharina coriacea Saccharina religiosa Saccharina japonica Saccharina diabolica Saccharina angustata Rhodomonas salina- 2 Ectocarpus siliculosus Saccharina ochotensis Phaeocystis antarctica Phaeocystis Saccharina japonica- 2 andersenii Hemiselmis Saccharina longipedalis 2 antarctica- Phaeocystis Hemiselmis andersenii- 2 Hemiselmis paramecium 2 Cryptomonas paramecium- Family Fucaceae Pavlovaceae Pavlovaceae Dictyotaceae Ectocarpaceae Laminariaceae Phaeocystaceae Desmarestiaceae Chroomonadaceae Chroomonadaceae Order Fucales Pavlovales Dictyotales Ectocarpales Laminariales Phaeocystales Desmarestiales Pyrennomonadales Class Phaeophyceae Pavlovophyceae Coccolithophyceae The complete genome information of mitochondria and chloroplasts of reference algae in NCBI The complete genome information of mitochondria of reference and chloroplasts Notes: # and size indicate the gene numbers of the mitochondrial and chloroplastic genomes, respectively. indicate the gene numbers of mitochondrial genomes, # and size and chloroplastic Notes: Phylum Haptophyta Ochrophyta Cryptophyta Table 2. Table JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101 97

2.3 Ka/Ks calculation rpl2 and rps3) with the total length of 4 292 aa and 1 210 varia- Ka/Ks calculation was performed by using MA method tion sites, it revealed two lineages: Phylum Ochrophyta group, and standard code of KaKs_Calculator 1.2 (Zhang et al., 2006) and the others including Order Cryptomonadales, Pyrenno- against the reference Fucus vesiculosus. We used similar meth- monadales, Isochrysidales, Phaeocystales and Pavlovales in ods to search for DNA orthologs with TBLASTX, rather than Phylum Cryptophyta, and Haptophyta. All species in Phylum TBLASTN. Ochrophyta (brown algae) were clustered in a single clade, and were further divided into two sub-clades. One encom- 3 Results and discussion passes Order Laminariales and Ectocarpales, and the other in- To achieve the most reliable results and keep all sample cludes Order Fucales (Family Sargassaceae) (Fig. 1). The result species, we have evaluated the powers of different filtering E- is consistent with previous results from Silberfeld et al. (2010). values of 10−5, 10−10, 10−20, 10−30 and 10−50, and achieved fewer In addition, Order Desmarestiales and Asterocladales are also genes with higher E-values. Finally, we chose E-values of 10−5 included in Laminariales–Ectocarpales clade, along with Order and 10−30 for mitochondrial and chloroplastic analysis, respec- Ascoseiriales, and Sporochnales (Charrier et tively. After alignment, we got the concatenated gene orthologs al., 2012), while some brown algae which are not covered in this of 4 292 aa and 4 053 aa for chloroplast and mitochondrion, re- study were found in Fucales clade, for example, Order Nemo- spectively, which were used to build phylogenetic trees. dermatales, and (Silberfeld et al., 2010). Brown algal ancestor was estimated to date back to more 3.1 Brown algal taxonomy based on the chloroplastic tree than 200 million years ago, with a high diversity (Silberfeld et al., In the maximum likelihood tree of 13 chloroplast genes 2010). There are many differences in the morphology of brown (rpoB, rpl14, rpl3, rpl6, tufA, rps12, atpB, rps5, rpl5, rpl16, atpA, algae, such as pneumatocysts, receptacles and conceptacles in

Sargassum integerrimum 98 Sargassum vachellianum 78 Sargassum henslowianum 97 Sargassum hemiphyllum Hizikia fusiformis Fucales Sargassum horneri 100 Sargassum muticum 99 Sargassum thunbergii Ishige okamurai Ishigeales 100 100 Laminaria japonica Laminariales Laminaria japonica2 Petalonia fascia Ochrophyta Scytosiphon lomentaria 100 Ectocarpales (Phaeophyceae) 78 Colpomenia sinuosa 100 Scytosiphon dotyo 0.05 92 Desmarestia viridis Desmarestiales 77 Undaria pinnatifida Saccharina japonica_r 100 99 Laminariales 96 Kjellmaniella crassifolia 100 Ectocarpus siliculosus_r Ectocarpales Fucus vesiculosus_r Fucales Dictyopteris undulata Dictyotales 100 Rhodomonas salina2_r 100 Rhodomonas salina_r Pyrennomonadales 90 Guillardia theta_r Cryptophyta Cryptomonas paramecium2_r Cryptomonadales 100 Cryptomonas paramecium_r Pavlova lutheri_r Pavlovophyceae 100 Emiliania huxleyi2_r 64 Emiliania huxleyi_r Coccolithophyceae Haptophyta 100 Phaeocystis antarctica2_r 100 Phaeocystis antarctica_r Cyanophora paradoxa_r

Fig.1. Maximum likelihood tree based on 13 concatenated chloroplastic protein sequences. The reference species are marked with “_r” following the species name. The tree was built in MEGA 5.1 based on Jones-Taylor-Thornton (JTT) model with bootstrap meth- od 1000. Bootstrap values lower than 60% are not shown. 98 JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101

Order Fucales, and stipe or young holdfast in Order Laminaria- agreement with that proposed by Wynne and Loiseaux (1976) les. A two-step cell division process (polystichous construction) based on their studies on algal life cycles. Furthermore, it was with a terminal growth is synapomorphic for Order Syringoder- suggested that the earliest diverging phaeophycean orders matales, , Dictyotales and Onslowiales (SSDO) were , Ishigeales and Dictyotales (Kawai et (Charrier et al., 2012). al., 2007), and that Ishige okamurai and Dictyopteris undulata seemed to be apart from most of the brown algae in our results. 3.2 Brown algal taxonomy based on the mitochondrial tree The phylogenetic relationships were confirmed further by In the maximum likelihood tree of 5 mitochondrial genes the tree based on available complete cox1 sequences in NCBI (rpl16, cob, rps12, rps3, and nad1) with the total length 4 053 aa (Fig. 3). And we found that algae of Phylum Ochrophyta were and 200 variation sites, the algal relationships (Fig. 2) are similar divided into two subgroups, Class Phaeophyceae and the other to that in the tree built with nucleus genes (data not shown). 9 species belonging to Class Chrysophyceae, Synurophyceae, Phylum Ochrophyta is obviously separated with Phylum Cryp- Bacillariophyceae and Raphidophyceae. tophyta and Haptophyta. We identified a distinct division in Class Phaeophyceae: Laminariales-Ectocarpales clade vs Fu- 3.3 Other discoveries of brown algae cales clade, indicating that Fucales evolved independently from Kjellmaniella crassifolia was always described based on ap- Laminariales-Ectocarpales for a long period of time. This is in pearance (Patron et al., 2007). Here, we confirmed the previous

Saccharina ochotensis_r Saccharina religiosa_r Saccharina longipedalis_r 85 Saccharina japonica_r Saccharina japonica2_r Saccharina diabolica_r 82 Laminaria japonica Laminariales Laminaria japonica2 Saccharina angustata_r Saccharina coriacea_r

94 Undaria pinnatifida Kjellmaniella crassifolia 78 Laminaria digitata_r Pylaiella littoralis_r Petalonia fascia 61 82 Scytosiphon lomentaria 80 Ectocarpales Ochrophyta 78 Colpomenia sinuosa (Phaeophyceae) Scytosiphon dotyo 98 Desmarestia viridis_r Desmarestiales 100 Desmarestia viridis Fucus vesiculosus_r 0.1 Sargassum hemiphyllum 69 Sargassum thunbergii 99 Hizikia fusiformis 90 97 Sargassum henslowianum Fucales Sargassum vachellianum 83 Sargassum muticum

100 65 Sargassum integerrimum 64 Sargassum horneri Dictyota dichotoma_r Dictyotales 98 Dictyopteris undulata Ishige okamurai Ishigeales

100 Emiliania huxleyi2_r Emiliania huxleyi_r Haptophyta Rhodomonas salina_r 87 Hemiselmis andersenii2_r Cryptophyta 100 Hemiselmis andersenii

Fig.2. Maximum likelihood tree based on 5 concatenated mitochondrial protein sequences. The reference species are marked with “_r” following the species name. The tree was built in MEGA 5.1 based on Jones-Taylor-Thornton (JTT) model with bootstrap meth- od 1000. Bootstrap values lower than 60% are not shown. JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101 99 - ophyta ophyta Rhodophyta Glaucophyta Haptophyta Cryptophyta Ochr (Phaeophyceae) Ochr NC_016739. 1 estiales NC_013710. 1 Phaeodactylum tricornutum Laminariales Ectocarpales Dictyotales Desmar Fucales Synedra acus NC_007684.1 NC_007683.1 NC_003055.1 NC_010637. 1 NC_013484.1 NC_002174.1 NC_013478.1 NC_007405.1 NC_015669.1 NC_014773.1 NC_013473.1 NC_013482.1 NC_013477.1 NC_013476.1 2 a NC_004024.1 NC_013475.1 NC_001677.2 JN022704.1 JQ071938.1 NC_005332.1 NC_018544.1 NC_002571.1 NC_007685.1 2 eligiosa NC_002007.1 a NC_017837.1 NC_017751.1 NC_002572.1 Desmarestia viridis Fucus vesiculosu s NC_014771.1 NC_014772.1 Chattonellales i Hemiselmis anderseni i NC_000887.3 Saccharina r Saccharina ochotensi s Saccharina japonica Saccharina japonica Saccharina diabolica Saccharina longipedalis Saccharina angustata Laminaria digitata Chondrus crispus 1 Saccharina coriacea Plocamiocolax pulvinat Thalassiosira pseudonana Emiliania huxleyi Chrysodidymus synuroideus Emiliania huxleyi 1 1 Dictyota dichotom Ochromonas danica Pyropia yezoensis Porphyra purpurea Porphyra umbilicalis 17.1 Pyropia haitanensis 1 NC_013837.1 Rhodomonas salin a GQ222228.1 1 3 GQ222227.1 NC_016738.1 Gracilariopsis lemaneiformi s 2 NC_0151 Gracilariophila oryzoide s 1 Gracilariopsis andersoni 1 1 Cyanidioschyzon merola e 0.71 marina 1 1 1 Heterosigma akashiwo Heterosigma akashiwo NC_017836.1 1 1 1 ta is divided into the two subgroups, Phaeophyceae and the others. and the others. Phaeophyceae ta is divided into the two subgroups, Glaucocystis nostochinearum 1 1 1 Cyanophora paradox a 1 protein sequences exacted from publicly available mitochondrial genomes of Kingdom Chromista and Phylum Rhodophyta. Species names are names are mitochondrial Rhodophyta. Species publicly available genomes of Kingdomand Phylum sequences exacted from Chromista 1 protein 1 1 1 0.88 1 11 1 0.93 0.66 MrBayes tree based on the cox tree MrBayes

0.06 followed by their NCBI accession number. Topology shows that there are three groups (Ochrophyta & Haptophyta, CryptophytaOchrophy Phylum & Rhodophyta, and Glaucophyta). & Haptophyta, (Ochrophyta groups three are that there shows Topology their NCBI accession number. by followed Fig.3. 0.83 100 JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101

finding that K. crassifolia (now as Saccharina crassifolia) should okamurai is very close to Fucales based on chloroplastic genes be placed in the clade of Laminariales (Charrier et al., 2012). It in Fig. 1, while in Fig. 2, we identified the mostly accepted an- should be noted that Saccharina and Laminaria are in a single cient phylogenetic position for I. okamurai and D. undulata. clade in the mitochondrial tree, but we identified Saccharina ja- Ectocarpales and Laminariales seem to be split. One of the rea- ponica in a distinct subgroup together with Ectocarpales in the sons might be that more genes were involved in building trees chloroplastic tree. It suggests that Saccharina and Laminaria (13 vs 5 genes used for chloroplast and mitochondrion, respec- are in the same Order Laminariales, but they still show some tively), and phylogenetic structures were complicated. Another differences in the evolution of mitochondrion and chloroplast. key reason might be that the taxonomies depend on extracted Hizikia fusifrome (Harvey) Okamura, which is shown as a origin-unknown genes, whose evolution rate can introduce taxon of Sargassaceae, Phaeophyta (Ochrophyta) in algaeBASE, modifications of phylogenetic tree. The following points should is a brown sea vegetable called . Even now, there is a dis- be addressed here. First, a single gene has relatively limited di- pute for its taxonomic position to be named as Sargassum fu- versity information, and phylogenetic trees must be construct- siforme (Burki et al., 2012; Sheng et al., 2011). Our results rec- ed on the consensus regions of concatenated gene sequences. ognize its close relationship to genus Sargassum (Figs 1 and 2). Second, more algae species result in short consensus sequences and less variations for building trees. Third, coding genes are 3.4 Non-brown Chromista algal taxonomy under various evolutionary pressures with diverse evolution- Notably, non-brown Chromista algae show the same sce- ary rates. Finally, “endosymbiotic gene transfer (EGT) translo- nario away from Phaeophyceae. However, it was interesting that cated chloroplast/mitochondrion-originated encoded protein the Haptophyta were within the Ochrophyta clade when ana- into the nucleomorph, and our extraction methods could not lyzed based on cox1 protein sequences (Fig. 3), while they were distinguish whether they were originally from chloroplast/mi- placed in the clade together with the Cryptophyta in Figs 1 and 2 tochondrion, or pseudogene transcripts in the nuclear genome. based on multiple genes of chloroplasts and mitochondria. The However, EGTs have been shown to leave a footprint of ancient Haptophyta is a phylum of chlorophyll a/c, which was ever con- photosynthetic activity in the nuclear genome (Burki et al., sidered as a single class ( Hibberd, or Hap- 2012). tophyceae Christensen ex Silva) and later was believed to be To assess evolutionary rates for individual genes, we cal- composed of two distinct classes (Prymnesiophyceae and Pav- culated nonsynonymous (Ka) substitution rate, synonymous lovophyceae) (Edvardsen et al., 2000). In algaeBASE, there are (Ks) substitution rate, and Ka/Ks values by using MA method three classes for Haptophyta: Prymnesiophyceae, Haptophyta and standard code of KaKs_Calculator 1.2 (Zhang et al., 2006) and Pavlovophyceae. The Prymnesiophyceae's against the reference F. vesiculosus. The ratio (Ka/Ks) indicates plastids are surrounded by four membrane layers indicating a neutral mutation (Ka/Ks = 1), negative (purifying) selection history of successive endocytosis. The Class Prymnesiophyceae (Ka/Ks < 1), and positive (diversifying) selection (Ka/Ks > 1). can be divided into four orders: Phaeocystales ord. nov., Prym- Our results showed that the average Ks is much more than the nesiales, Isochrysidales, and . average Ka (3.53 vs 0.13 for chloroplast, and 3.98 vs 0.20 for mi- Furthermore, we found that Rhodomonas salina and tochondrion), suggesting the genes' conservation (average Ka/ Hemiselmis andersenii (Cryptophyta) are sisters to the Rho- Ks: 0.043 and 0.057 for chloroplast and mitochondrion, respec- dophyta clade based on cox1 protein sequences (Fig. 3), sug- tively), and it seems the variances of Ka and Ks are associated gesting that their mitochondria have a close relationship to with their average values. red algae. It was shown that are sisters to the algae, which also have a red-algal secondary plas- References tid (Patron et al., 2007). 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