Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19 DOI: 10.1007/s13131-014-0436-3 http://www.hyxb.org.cn E-mail: [email protected]

Transcriptome-wide evolutionary analysis on essential brown (Phaeophyceae) in China SUN Jing1,3,4†, WANG Liang1,3,4†, WU Shuangxiu1,3†, WANG Xumin1,3, XIAO Jingfa1,3, CHI Shan2, LIU Cui2, REN Lufeng1,3, ZHAO Yuhui1,4, LIU Tao2*, YU Jun1,3* 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 1 April 2013; accepted 18 July 2013

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

Abstract (Chromista, , Phaeophyceae) are a large group of multicellular algae that play im- portant roles in the ocean's ecosystem and biodiversity. However, poor molecular bases for studying their phylogenetic evolutions and novel metabolic characteristics have hampered progress in the field. In this study, we sequenced the de novo transcriptome of 18 major species of brown algae in China, covering six orders and seven families, using the high-throughput sequencing platform Illumina HiSeq 2000. From the transcriptome data of these 18 species and publicly available genome data of siliculosus and Phaeodactylum tricornutum, we identified 108 nuclear-generated orthologous genes and clarified the phy- logenetic relationships among these brown algae based on a multigene method. These brown algae could be separated into two clades: Clade - and Clade -Laminariales-Desmares- tiale-. The former was at the base of the phylogenetic tree, indicating its early divergence, while the latter was divided into two branches, with Order Fucales diverging from Orders Ectocarpales, Laminariales, and Desmarestiale. In our analysis of -contentious species, Sargassum fusiforme and Saccharina sculpera were found to be closely related to genera Sargassum and Saccharina, respectively, while Petalonia fascia showed possible relation to genus Scytosiphon. The study provided molecular evidence for the phylo- genetic taxonomy of brown algae. Key words: Phaeophyceae, transcriptome sequencing, multigene, phylogeny Citation: Sun Jing, Wang Liang, Wu Shuangxiu, Wang Xumin, Xiao Jingfa, Chi Shan, Liu Cui, Ren Lufeng, Zhao Yuhui, Liu Tao, Yu Jun. 2014. Transcriptome-wide evolutionary analysis on essential brown algae (Phaeophyceae) in China. Acta Oceanologica Sinica, 33(2): 13–19, doi: 10.1007/s13131-014-0436-3

1 Introduction these questions are issues of taxonomy and phylogeny in Pha- Brown algae are a large group of multicellular algae of Class eophyceae. For example, Sargassum fisiforme has a debated Phaeophyceae, Phylum Ochrophyta, Chromista. membership in genus Sargassum or Hizikia; Orders Ishigeales They usually live in cold and temperate oceans (Charrier et al., and Dictyotales were considered to have diverged early in Pha- 2012; Zambounis et al., 2012) and distribute very broadly in the eophyceae, despite a lack of evolutionary evidence. Moreover, Northern Hemisphere, ranging from the North Pacific to North among Phaeophyceae, only one species, Ectocarpus siliculo- Atlantic regions (Coyer et al., 2011). , Dictyotales, sus, has a completely sequenced genome (Cock et al., 2010), Ishigeales, Laminariales, Ectocarpales and Fucales are major or- and only three species, E. siliculosus, Saccharina japonica and ders of Phaeophyceae found in China and are dominant sea- S. latissima, have transcriptome data, showing poor molecular weeds along the western Pacific shore. They play important bases for the study of phylogenetic relationship within Phaeo- roles in marine ecosystems both as the predominant primary phyceae (Deng et al., 2012; Dittami et al., 2009; Heinrich et al., producers in the food chain and as underwater canopies for 2012). Fortunately, with the development of the next generation marine organisms (Eom et al., 2012). sequencing technology with the improvement of high through- From Linnaeus' first descriptions in his 1 753 book Species put and large decreasing of cost, such as 454 GS Junior (Roche) Plantarum to the present day, brown algae have continued to be and Hiseq 2000 (Illumina), de novo transcriptome sequencing a source for research. But despite the wealth of existing brown on a broad range of non-model species could realize to provide algae research, a number of unanswered evolutionary ques- more gene information to meet the demands for phylogenetic tions still persist (Dorrell and Smith, 2011; Green, 2011). Among analysis.

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. 14 SUN Jing et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19

In this study, a total of 18 major species of brown algae in ligase. Fragment size selection was performed using agarose China, covering six orders and seven families, were collected in gel, and fragments of 200–250 bp were extracted from agarose the Bohai Bay. Except S. japonica, most brown algal transcrip- gel. The selected cDNA fragments were amplified by PCR. The tomes were for the first time sequenced (RNA-seq) using high- constructed cDNA library was sequenced by Illumina HiSeq throughput sequencing technology on an Illumina HiSeq 2000 2000. platform. A multi-gene evolutionary analysis was applied based on these transcriptome sequencing data to clarify the phylo- 2.3 De novo assembly genetic relationship among these brown algae. The research Strict reads filtering was performed before the assembly. not only provided molecular evidence for species taxonomy in Pair-end reads with primer or adaptor sequences were removed. brown algae but also provided valuable gene and methodology Reads with more than 10% of bases below Q20 quality or more information for further molecular research of brown algae. than 5% unknown nucleotides (Ns) were filtered from the total reads. De novo assembly was carried out using SOAPdenovo- Trans with the following parameters: -p 1, -K 25, -e 2, -r, -F, -L 2 Material and methods 100, -t 5 (http://soap.genomics.org.cn/SOAPdenovo-Trans. 2.1 Algal samples and total RNA extraction html) (Li et al., 2008). Gapcloser was then used for gap filling of All the brown algal samples (Table 1) were provided by the the scaffolds (Luo et al., 2012). laboratory of the Culture Collection of Seaweed in the Ocean 2.4 Predicting protein sequence and coding sequence (cds) of University of China. Total RNA was extracted using an improved CTAB method (Ghangal et al., 2009; Li and Ren, 2012; Xu et al., transciptome 2010; Yao et al., 2009). The quality and quantity of extracted RNA A protein sequence for each transcript was predicted by were assessed using Nanodrop ND 1000 spectrophotometer FASTX search (Pearson, 1996; Pearson and Lipman, 1988; Pear- (Labtech International Ltd, Lewes, UK) and Agilent 2100 bio- son et al., 1997) against the proteome of E. siliculosus from Eu- analyzer (Agilent Biotechnologies, Santa Clara, USA). ropean Nucleotide Archive (ENA) (http://www.ebi.ac.uk/ena/) with E-value less than 10−5 (Deng et al., 2012; Lu et al., 2012). 2.2 Transcriptome sequencing Then, coding sequences (cds) were obtained by getting the DNA cDNA library construction and sequencing were performed sequence between the start and end position of the alignment by the BGI (Shenzhen, China) on Illumina (San Diego, USA) and replacing insertions and deletions in the alignment; the in- HiSeq instruments according to the manufacturer's instruc- sertion of cds was signified by “N” while the insertion of protein tions. Briefly, mRNA was isolated from total RNA by Sera-mag sequence was signified by “X”. The predicted protein sequences Magnetic Oligo (dT) Beads. The mRNA with fragment buffer were filtered by length and identity to improve protein identi- was sheared into short fragments of about 200 bp. Using these fication accuracy and maintain consistent sequencing quality mRNA fragments as templates, the first-strand cDNA was syn- between each species. Protein sequences with length over 90% thesized by random hexamers-primers and reverse transcrip- of the aligned protein and identity over 30% were retained. tase. The second-strand cDNA was synthesized using DNA polymerase I, together with RNase H and dNTPs, and was pu- 2.5 Getting orthologs between 20 species rified by QiaQuick PCR purification kit (Qiagen). The double- We searched for gene orthologs from the 18 brown algal stranded cDNA was end-repaired and phosphorylated using transcriptome sequencing data of this study and two genome T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK. PE data of E. siliculosus and Phaeodactylum tricornutum CCMP adapter was added to the repaired cDNA fragments by T4 DNA 1055 (http://www.jgi.doe.gov/) according to the following pro-

Table 1. Brown algal species selected for the transcriptome sequencing analysis Class Order Family Species Phaeophyceae Dictyotales Dictyotaceae Dictyopteris undulata Ishigeales Ishigeaceae okamurai Desmarestiales viridis Laminariales Laminariaceae Saccharina japonica S. sculpera Alariaceae Undaria pinnatifida Ectocarpales sinuosa Petalonia fascia S. dotyi Fucales Sargassaceae Sargassum fusiforme S. hemiphyllum var.chinense S. henslowianum S. horneri S. integerrimum S. muticum S. thunbergii S. vachellianum SUN Jing et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19 15

cedure. First, gene orthologs between any two species among ML-tree was calculated by RAXML with 1 000 runs. Finally, the the 20 species were searched for with the Inparanoid program consensus tree was built by RAXML based on the bootstrap tree (O'Brien et al., 2005). Next, gene orthologs among the 20 species and best ML-tree. were searched for using the perl program Multiparanoid (Alex- eyenko et al., 2006). Genes with paralogs were then filtered. Fur- 3 Results and discussion thermore, mitochondiral and chloroplastic genes were filtered from obtained orthologs. Last, KEGG (Kyoto Encyclopedia of 3.1 Eighteen species were representatives for different groups Genes and Genomes) analysis of the 108 orthologous genes was In order to clarify evolutionary relationships in brown algae, performed using KAAS (KEGG Automatic Annotation Server) we collected 18 species covering six orders and seven families (http://www.genome.jp/tools/kaas/) (Moriya et al., 2007). P. tri- in Class Phaeophyceae. , Dictyopteris undu- cornutum CCMP 1055 were used as an outgroup of brown algae. late, and Ishige okamurai were of Orders Desmarestiales, Dic- tyotales, and Ishigeales, respectively, while Orders Laminari- 2.6 Sequence alignment among 20 species ales, Ectocarpales, and Fucales contained three, four, and eight Protein sequences among 20 species for each gene were aligned with Probcons 1.12 (Do et al., 2005); Cds alignment was representative species, respectively (Table 1). With its genome obtained from the result of protein sequence alignment. For published in 2010, E. siliculosus has served as a representative each gap in the protein sequence alignment, three nucleotide species in Order Ectocarpales (Cock et al., 2010). As such, the E. gaps were inserted at the corresponding sites in the cds. After siliculosus genome was used as the reference for annotation of alignment, all positions with gaps were removed before multi- the algal transcriptomes sequenced in this study. Additionally, gene phylogenetic analysis. P. tricornutum, a species in Class Bacillariophyceae with com- plete genome sequencing data, was chosen as an outgroup of 2.7 Construction of phylogeny tree the phylogenetic tree. ProtTest 3.0 was used to search for the best AA substitu- tion model (Darriba et al., 2011; Guindon and Gascuel, 2003). 3.2 Transcriptome sequencing and assembly RAXML was used to reconstruct phylogenetic trees of cds and A total 45G sequencing data of approximately 250 million proteins by using the maximum likelihood algorithm (ML-tree) paired-end reads were generated. These reads were 90 bp in (Stamatakis et al., 2005). First, a bootstrap phylogenetic tree length with high quality after strict reads filtering (more than was calculated by RAXML with 1 000 bootstraps. Second, best 90% bases with Q20 quality). On average 10–15 million reads

120 000

100 000

80 000 r

60 000 fold numbe Scaf

40 000

20 000

0 a a ii stia viridis gassum horneri Ishige okamurai Petalonia fascia gassum muticu m gassum fusiforme Scytosiphon dotyi gassum thunbe rg Sar Desma re Undaria pinnatifid Sar Colpomenia sinuos a Saccharina sculpera Saccharina japonica Sar gassum hemiphyllu m gassum vachellianum gassum integerrimu m Sar Dictyopteris undulat gassum henslowianum Scytosiphon lomentaria Sar Sar Sar Sar

Fig.1. Scaffold numbers of each algal library after assembly. 16 SUN Jing et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19

were obtained in each algal library, indicating the consistency distribution of these genes in the KEGG database (Moriya et al., in all algal sample libraries. About 50 000–100 000 scaffolds were 2007). Of these 108 genes, 82 could be mapped to the KEGG da- generated after short reads assembly using SOAPdenovo-trans tabase and distributed among 22 pathway categories with dif- (Fig. 1). The N50 of each library ranged from 756 bp to 1 709 bp. ferent functions. In the first-level categories of the KEGG path- way, Category “B: genetic information processing” was mapped 3.3 Gene annotation and prediction of protein sequence and by 37 genes, the highest gene number of our dataset. Within cds this category, most mapped to the subcategory “folding, sorting We annotated the sequenced transcripts and obtained their and degradation” (22 genes); others mapped to “translation” (12 protein sequences using the global alignment program FASTA. genes), “transcription” (5 genes) (Fig. 2). The second was Cat- Based on the results of global alignment, over 50% of scaffolds egory “A: metabolism” and most of genes in it mapped to the were annotated to E. siliculosus genes. In order to avoid mis- subcategory “energy metabolism” (9 genes) and “carbohydrate takes caused by partial sequences during phylogenetic analy- metabolism” (7 genes). Category “C: environmental informa- sis, we filtered the protein sequences by length (at least 90% tion processing” was mapped by only 9 genes, the least gene the length of reference proteins) and identity (higher than 30% numbers of our dataset; furthermore all of its genes mapped to of reference proteins) to obtain the high-quality sequences for the subcategory “signal transduction”. This analysis showed the phylogenetic study. Table 2 shows the percentage of high-qual- shared orthologous genes were not limited to a single pathway; ity protein sequences that were retained. We concluded that on the contrary, they covered 22 categories of variant pathways. Library Scytosiphon dotyi had the highest proportion (7.39%) Therefore these orthologous genes could reduce the bias of of high-quality protein sequences; the assembled sequences in genes' different evolutionary speed in certain pathways and this library thus were of higher integrity and quality than others. produce objective phylogenetic results.

3.4 Orthologs of 20 species in Ochrophyta 3.5 Phylogeny tree Of the same phylum (Ochrophyta) as brown algae but of a A phylogenetic tree based on 108 orthologs of these 20 algal different class (Bacillariophyceae), P. tricornutum was species was constructed based on their cds using the maximum chosen as the outgroup for phylogenetic analysis. Through likelihood method (Fig. 3). As shown in Fig. 3, bootstrap values the perl program Multiparanoid, we were able to search for or- were mostly greater than 90%. Verifying our outgroup choice, thologous genes in the 18 species sequenced in this study, as P. tricornutum's Class Bacillariophyceae was shown to have well as published genomes for E. siliculosus and P. tricornutum; an early separation from Class Phaeophyceae. The base clade we found 325 orthologous genes. Thereafter, we filtered genes of Phaeophyceae contained two orders, Ishigeales and Dictyo- with paralogs, leaving 114 genes. Among these genes, six mito- tales, showing early divergence from other brown algae. The chondiral and chloroplastic genes were identified and removed other clade contained four orders and they grouped into two due to a possibility of differing evolutionary speed from nuclear branches, Branch Ectocarpales-Laminariales-Desmarestiales genes. As a result, 108 nuclear-generated orthologous genes, and Branch Fucales, showing Fucales' separation from other shared among these 20 species (Phylum Ochrophyta), were left brown algae into a different direction of evolution. for the phylogenetic study. The study of Kawai's group based on the plastid gene rbcL We further analyzed the function of these 108 orthologous sequence showed the close relationship of Orders Ishigeales genes using KAAS of KEGG to identify the function and pathway and Dictyotales and their early divergence from the phaeophy-

Table 2. Predicted protein sequence numbers of 18 brown algal species Protein sequence Library Total scaffold Percentage/% of high quality Desmarestia viridis 3 176 53 140 5.98 Dictyopteris undulata 3 924 100 199 3.92 Ishige okamurai 4 625 78 583 5.89 Saccharina japonica 3 180 98 627 3.22 Saccharina sculpera 4 197 88 084 4.76 Undaria pinnatifida 3 143 72 256 4.35 4 940 80 884 6.11 Petalonia fascia 5 923 89 229 6.64 Scytosiphon dotyi 5 149 69 680 7.39 Scytosiphon lomentaria 5 264 75 666 6.96 Sargassum fusiforme 3 243 116 790 2.78 Sargassum hemiphyllum 4 668 87 021 5.36 Sargassum henslowianum 4 534 77 474 5.85 Sargassum horneri 4 385 75 127 5.84 Sargassum integerrimum 4 213 82 503 5.11 Sargassum muticum 4 656 81 102 5.74 Sargassum thunbergii 4 962 106 596 4.65 Sargassum vachellianum 5 009 69 871 7.17 SUN Jing et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19 17

KEGG pathway analysis

25 A: metabolism B: genetic information processing C: environmental information processing 20 D: cellular processes E: organismal systems r 15

10 Unigene numbe

5

0 s s m m m m m n m n n

Translation Transcription Cell motility gy metabolis Development Immune system Nervous system Lipid metabolis Endocrine systemExcretory system Signal transduction Circulatory system Ener Cell communicatio Cell growth and death NucleotideAmino metabolis acid metabolis Carbohydrate metabolis Transport and catabolis Environmental adaptatio

Metabolism of other aminoFolding, acid sorting and degradatio

Metabolism of cofactors and vitamin

A B C D E

Fig.2. KEGG analysis of 108 orthologous genes of 20 species.

cean lineage, but did not apparently separated them from other 95 Scytosiphon dotyi 100 Petalonia fascia orders of the studies (Kawai et al., 2005). Another study based 100 Scytosiphon lomentaria Ectocarpales on ten mitochondrial and chloroplastic genes separated brown algae into two clades, and Dictyotales was in the clade differ- 100 Colpomenia sinuosa ently from that of Laminariales, Ectocarpales, Desmarestiales 100 and Fucales, but Ishigeales was not included (Silberfeld et al., Undaria pinnatifida 100 2010). Our results gave a clear and comprehensive phylogenetic 95 Saccharina sculpera Laminariales 100 taxonomic classification of Orders Ishigeales and Dictyotales, Saccharina japonica as well as other orders, by using more species and nuclear-ori- Desmarestia viridis Desmairestiales ented multigene analysis. 100 Sargassum muticum 90 In Fucales, eight Sargassums species were further divided 100 Sargassum hemiphyllum into two parts. One included five species, S. fusiforme, S. muti- 100 Sargassum thunbergii cum, S. hemiphyllum, S. thunbergii, and S. horneri, and the other 100 Sargassum fusiforme included three species, S. integerrimum, S. henslowianum, and Fucales 100 Sargassum horneri S. vachellianum. Previously, S. fusiforme was called Hizikia fusi- Sargassum integerrimum formis and there were arguments whether it belonged to genus 100 Sargassum or genus Hizikia (Cho et al., 2006). Setchell classified 100 Sargassum henslowianum Sargassum vachellianum it in Sargassum and named it as S. fusiforme (Harvey) Setchell (Setchell, 1931) but several Japanese algologists considered Dictyopteris undulata Dictyotales 55 S. fusiforme of genus Hizikia (Yoshida et al., 1990). In a phy- Ishige okamurai Ishigeales logenetic study based on ribosomal DNA internal transcribed P. tricornutum 0.5 spacer-2 (ITS-2), it was also classified into genus Sargassums in the phylogeny tree (Stiger et al., 2003). In our results, based on the whole transcriptome sequencing of eight Sargassum Fig.3. Phylogenetic analysis of 20 algal species using 108 orthologs with the maximum likelihood (ML) meth- species and multi-gene phylogenetic analysis, we found that od of RAXML program (Stamatakis et al., 2005). A diatom S. fusiforme was phylogeneticly close to S. thunbergii, S. muti- P. tricornutum belonging to Class Bacillariophyceae, was cum, and S. henslowianum from the point of nuclear-oriented selected as outgroup. Numbers indicated the bootstrap gene evolution. The bootstrap values of the branches in Fucales values in the ML analysis. were all 100%, supporting relationships among these species in 18 SUN Jing et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19

genus Sargassum. Therefore, our results support S. fusiforme's ics, 22(14): e9–e15 classification as genus Sargassum at the whole-genome-anal- Charrier B, Le Bail A, de Reviers B. 2012. Plant proteus: brown algal ysis level. morphological plasticity and underlying developmental mecha- nisms. Trends Plant Sci, 17(8): 468–477 In Ectocarpales, there were five species in four genera. In Cho G Y, Rousseau F, de Reviers B, et al. 2006. Phylogenetic relation- our study, Scytosiphon dotyi was closer to Petalonia fascia than ships within the Fucales (Phaeophyceae) assessed by the photo- to S. lomentaria (Fig. 3), with a bootstrap value of 95%. Previ- system I coding psaA sequences. Phycologia, 45(5): 512–519 ous studies found that P. fascia was a relative of S. lomentaria Cock J M, Sterck L, Rouze P, et al. 2010. The Ectocarpus genome and (Flores-Moya et al., 2002; Rhodes and Connell, 1973). 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