Phylogenomic Analysis of Transcriptomic Sequences Of

Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 86–93 DOI: 10.1007/s13131-014-0444-3 http://www.hyxb.org.cn E-mail: [email protected]

Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts for marine red algae (Rhodophyta) in China JIA Shangang1,3†, WANG Xumin1,3†, QIAN Hao2, LI Tianyong2, SUN Jing1,3,4, WANG Liang1,3,4, YU Jun1,3, LI Xingang1,3, 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 2013

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

Abstract The chloroplast and mitochondrion of red algae (Phylum Rhodophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using red algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Rhodophyta. We have found that red algae were divided into three clades of orders, Florideophyceae, Bangiophyceae and Cyanidiophyceae. Taxonomy resolution for Class Florideophyceae showed that Order Gigartinales was close to Order Halymeniales, while Order Graci- lariales was in a clade of Order Ceramials. We confirmed Prionitis divaricata (Family Halymeniaceae) was closely related to the clade of Order Gracilariales, rather than to genus Grateloupia of Order Halymeniales as reported before. Furthermore, we found both mitochondrial and chloroplastic genes in Rhodophyta under negative selection (Ka/Ks < 1), suggesting that red algae, as one primitive group of eukaryotic algae, might share joint evolutionary history with these two organelles for a long time, although we identified some differences in their phylogenetic trees. Our analysis provided the basic phylogenetic relationships of red algae, and demonstrated their potential ability to study endosymbiotic events. Key words: red algae, Rhodophyta, phylogenetic trees, mitochondrion, chloroplast Citation: Jia Shangang, Wang Xumin, Qian Hao, Li Tianyong, Sun Jing, Wang Liang, Yu Jun, Li Xingang, Yin Jinlong, Liu Tao, Wu Shuangxiu. 2014. Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts for marine red algae (Rho- dophyta) in China. Acta Oceanologica Sinica, 33(2): 86–93, doi: 10.1007/s13131-014-0444-3

1 Introduction Some missing genes had relocated to the nucleus, and many Red algae (Rhodophyta) are a distinct eukaryotic lineage genes were lost eventually (Martin et al., 1998). In subsequent which lacks chlorophyll b/c, centrioles and flagella in all life endosymbiosis, also called second or third endosymbiosis, eu- stages, but contains allophycocyanin, phycocyanin and phyco- karyotic hosts engulfed red algae or green algae, so that their erythrin in the form of phycobilisomes on unstacked thylakoids plastids finally contained three or four bounding membranes (Kawai et al., 2007). They are a morphologically diverse group (Green, 2011). In contrast, eukaryotes acquired algal mitochon- that consists of 6 000 unicellular and multicellular species in at dria long before the establishment of plastids. It was suggested least 12 orders (Burger and Nedelcu, 2012). For a long time, it is that mitochondria originated from an α-proteobacterial ances- believed that red algae diverged early from the primary endo- tor (Gray et al., 2001), and co-evolved together with their host, symbiosis. The ancient red algae share a common plastid with which means mitochondrial DNA probably could predict the the ancient green algae (Cavalier-Smith, 1998). evolutionary history of nuclear genome as a whole (Burger and Based on the primary endosymbiosis theory, the origin of Nedelcu, 2012). Although similar evolutionary events may be plastids would be derived from cyanobacterium-like prokary- shared in one single algal group, algal phylogenies based on otes (Rodríguez-Ezpeleta et al., 2005), leading to the origins of genes of plastid and mitochondrion may not be identical due three major host lineages: green algae and land plants, red algae to their potential differences in evolutionary history. Such dif- and glaucophytes (Moreira et al., 2000; McFadden, 2001). The ferences reflect the distinct endosymbiotic events of these two primary endosymbiosis resulted into the substantial reduc- organelles. For example, we had suggested that in the phyloge- tion of plastid genomes which have two bounding membranes. netic trees built with algal ESTs and genomes of mitochondria

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. 86–93 87

and chloroplasts, Ulva prolifera was placed in a sister position major marine red algae, especially at the molecular genetic to Ulva linza and shared a similar chloroplast origin with Pseu- level. dendoclonium akinetum (Jia et al., 2011). Furthermore, chloroplastic and mitochondrial origins are 2 Materials and methods mostly determined based on the shared conserved regions or genes in the algal genomes, DNA or protein sequences. Protein 2.1 Sequence datasets from 1kp Project sequences are more preferred because they are more conserved The assembled transcriptomic sequencing datasets were than DNA. Many genes can be used to have a close look at al- downloaded from the website of 1KP Project, including 22 algal gal phylogenies, such as small subunit (SSU) (Tan and Druehl, species of Phylum Rhodophyta (Table 1). In total, 476 583 455 1994) and large subunit (LSU) of rDNA (Phillips et al., 2008) in base pairs as a candidate database were used to search for or- nucleus, rbcL, psaA and psbA (Cho et al., 2004) in plastids, and thologs in phylogenetic analysis. Cytochrome c (Danne et al., 2012) in mitochondria. Meanwhile Up to date, ten red algal species' complete mitochondrial the available whole-genome data have been valuable resources genomes have been sequenced, belonging to five distinct in algal research, especially phylogenomic analysis which is al- Rhodophyte orders, which are Cyanidiales, Gigartinales, Plo- ways performed for phylogenetic relationships. Phylogenomics camiales, Gracilariales and Bangiales. In contrast, only Order was first introduced in 1998 (Eisen, 1998), and widely accepted Gracilariales, Cyanidiales, and Bangiales have complete chloro- for phylogenetic inference. It is noted that the concatenated plastic genomes sequenced in five species. The gene numbers genes provide more comprehensive information than a single range 22–33 for mitochondria and 196–208 for plastids (Table one, and expressed-sequence-tags (ESTs) and genomes from 2). These complete genome sequences are available in NCBI. mitochondria and plastids can be used to construct phylogenetic trees (Hallstrom and Janke, 2009). 2.2 Construction of phylogenetic trees In this study, as part of the 1 000 Plants (1KP) Project (http:// We collected all protein sequences of ten mitochondrial www.onekp.com/) which covers more than 1 000 different spe- genomes and five chloroplastic ones (including the out- cies of plants by generating large scale gene sequence informa- group species, Cyanophora paradoxa of Phylum Glauophyta, tion, we focused on 22 Chinese marine red alga species, includ- NC_001675.1) from NCBI website as references. Considering ing 21 species of class Florideophyceae and one species of class possible horizontal gene transfer or gene exchanges between Bangiophyceae. Based on the expressed gene orthologs from nucleus and organelles, we identified 18 typical genes shared the 1KP Project database and the publicly available complete by ten complete mitochondrial genomes and 139 genes shared red algal genomes of mitochondria and chloroplasts in NCBI, by five chloroplastic genomes in order to exclude potential we conducted a phylogenomic analysis, and our results showed nucleusoriginated genes. Using these typical and shared refer- that they are useful for development and utilization of Chinese ence genes, we performed local TBLASTN search against the as-

Table 1. The 22 red algal species information with assembled contigs based on RNA-seq datasets Order Family Species Contig# Tot/Mb Avg/bp Lon/bp Ceramiales Dasyaceae Heterosiphonia pulchra 39 345 23.4 595 18 563 Ceramiaceae Ceramium kondoi 22 997 18.2 790 15 965 Rhodomelaceae Polysiphonia japonica 24 377 18.0 739 11 665 Symphyocladia latiuscul 37 839 18.5 490 15 887 Gigartinales Dumontiaceae Dumontia simplex 18 665 18.4 987 14 732 Endocladiaceae Gloiopeltis furcata 20 184 19.5 965 18 192 Gigartinaceae Chondrus crispus 25 526 20.3 797 16 329 Mazzaella japonica 26 703 21.2 795 18 268 Phyllophoraceae Gymnogongrus ftabelliformis 21 986 25.5 1 161 19 290 Solieriaceae Kappaphycus alvarezii 54 852 26.3 480 8 409 Eucheuma denticulatum 25 025 22.1 885 16 577 Betaphycus gelatinae 24 161 23.1 955 16 487 Gracilariales Gracilariaceae Gracilaria asiatica 13 339 19.5 1 462 21 985 Gracilaria blodgettii 20 988 18.3 873 14 612 Gracilaria chouae 12 072 19.7 1 630 20 513 Gracilaria lemaneiformis 25 880 24.6 951 20 939 Halymeniales Halymeniaceae Grateloupia filicina 56 234 30.3 538 15 467 Grateloupia livida 14 254 17.9 1 253 14 948 Grateloupia turuturu 15 113 20.1 1 333 18 557 Prionitis divaricata1) 17 289 19.0 1 097 15 261 Sinotubimorpha guangdongensis1) 28 269 22.8 808 13 780 Bangiales Bangiaceae Porphyra yezoensis 72 974 29.7 406 11 005 Notes: Tot, Avg and Lon indicate total length/Mb, average length/bp and longest length/bp of contigs, respectively. # indicates the numbers of contigs. 1) Prionitis divaricata and Sinotubimorpha guangdongensis are also named as Grateloupia chiangii and Grateloupia catenata, respectively. 88 JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 86–93 NA NA 25 836 25 161 25 883 25 894 32 211 29 123 37 023 27 036 41 688 36 753 Size/bp 28 22 25 23 33 25 27 26 31 30 NA NA Gene# Mitochondrion NA NA Accession JQ071938.1 NC_001677.2 NC_014771.1 NC_014773.1 NC_000887.3 NC_018544.1 NC_017751.1 NC_014772.1 NC_017837.1 NC_002007.1 NA NA NA NA NA NA NA 183 883 164 921 149 987 191 952 191 028 Size/bp NA NA NA NA NA NA NA 202 196 206 208 208 Gene# Chloroplast NA NA NA NA NA NA NA Accession NC_006137.1 NC_001840.1 NC_004799.1 NC_007932.1 NC_000925.1 Species Gracilariopsis Gracilariopsis lemaneiformis Chondrus crispus Pyropia yezoensis Porphyra purpurea Porphyra Pyropia haitanensis Porphyra umbilicalis Porphyra Cyanidium caldarium Cyanidium Gracilaria tenuistipitata Gracilaria Plocamiocolax pulvinata Plocamiocolax Gracilariopsis andersonii Gracilariopsis Gracilariophila oryzoides Gracilariophila Cyanidioschyzon merolae Cyanidioschyzon Family Bangiaceae Cyanidiaceae Plocamiaceae Gigartinaceae Gracilariaceae Order Bangiales Cyanidiales Plocamiales Gigartinales Gracilariales The complete genome information of mitochondria and chloroplasts of reference red algae in NCBI red The complete genome information of mitochondria of reference and chloroplasts Class 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: Bangiophyceae Florideophyceae Cyanidiophyceae Table 2. Table JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 86–93 89

sembled transcript datasets of 22 red algae from 1KP Project to respectively. After aligning orthologs, we got concatenated se- obtain putative algal orthologs. We tested best hits with cutoff quences of 22 (totally 8 366 aa) and 4 (totally 1 295 aa) genes for E-values of 10−5, 10−10, 10−20, 10−30, 10−40 and 10−50 to achieve re- chloroplast and mitochondrion, respectively, which were used liable orthologs, and stored their alignment information to do to build phylogenetic trees. pairing of references and samples. Phylogenetic trees were con- structed in MEGA 5.1 based on Jones-Taylor-Thornton (JTT) 3.1 Chloroplastic tree model with bootstrap method 1 000 (Tamura et al., 2011), and In the maximum likelihood tree of 22 chloroplastic genes MrBayes 3.1.2 software with two independent runs, each with (rpoB, rps11, cpcB, rpl14, psbB, rpl3, rps7, tufA, rps12, atpB, four incrementally heated Metropolis-coupled Monte-Carlo psaA, psbA, apcA, rpl5, rpl16, psbC, psbD, atpA, rpl2, rps3, rps19 Markov Chains running for 5 000 000 generations (Ronquist and and psaB) with the total length of 8 366 aa and 2 443 variation Huelsenbeck, 2003). Bayesian trees were sampled every 100 sites, red algae were clustered into three clades, a large single generations. clade of Class Florideophyceae and two other clades of Class Bangiophyceae and Class Cyanidiophyceae, with an outgroup 2.3 Ka/Ks calculation of C. paradoxa (Fig. 1). Furthermore, within Class Florideophy- Ka/Ks calculation was performed by using MA method ceae, algae were split into orders of Ceramials, Gracilariales, and standard code of KaKs_Calculator 1.2 (Zhang et al., 2006) Gigartinales and Halymeniales. Order Gigartinales was close against the reference Cyanidioschyzon merolae. We used simi- to Order Halymeniales, while Order Ceramials was placed in lar methods to search for DNA orthologs with TBLASTX, rather the base position of Class Florideophyceae as a sister group to than TBLASTN. Order Gracilariales. We also confirmed that Class Cyanidiophy- ceae, in the base position of the tree, was comprised of some 3 Results and discussion unicellular algae, such as C. merolae and Cyanidium caldarium. Restricting E-value resulted in a reduction of shared gene C. merolae is thought to be one of the most primitive red algal numbers, and in order to get reliable results, we chose E-value species according to morphological proofs since it is unicellular of 10−5 and 10−30 for mitochondrial and chloroplastic analysis, and lacks a vacuole and a cell wall (Kuroiwa, 1998).C. caldari-

100 Eucheuma denticulatum 100 Betaphycus gelatinus Gigartinales Kappaphycus alvarezii Sinotubimorpha guangdongensis Halymeniales 72 Grateloupia turuturu Dumontia simplex Gloiopeltis furcata Chondrus crispus Gigartinales Gymnogongrus ftabelliformis Mazzaella japonica

80 Grateloupia livida Halymeniales 94 Grateloupia filicina Florideophyceae 58 Ceramium kondoi Ceramiales 0.05 Gracilaria lemaneiformi 100 Gracilaria blodgettii 100 Gracilaria asiatica 100 Gracilariales 99 Gracilaria tenuistipitata_r 83 Gracilaria chouae 100 Prionitis divaricata 100 Heterosiphonia pulchra 94 Symphyocladia latiuscul Ceramiales 93 Polysiphonia japonica Porphyra purpurea_r 100 Porphyra yezoensis_r Bangiophyceae 100 Porphyra yezoensis Cyanidioschyzon merolae_r Cyanidiophyceae 100 Cyanidium caldarium_r Cyanophora paradoxa_r

Fig.1. Maximum likelihood tree based on 22 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 method 1000. Bootstrap values lower than 50% are not shown. 90 JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 86–93

um, because it is also a very small unicellular and primitive red teins, they were shown to be closer to the unicellular species C. alga with a single, simple-shaped plastid, is closely related to C. merolae and C. caldarium when compared to the clade of Class merolae (Takahara et al., 2000). In addition, we used publicly- Florideophyceae. available algal rbcL sequences to build up the phylogenetic tree It was reported that morphologically all Chinese species of for red algae and got similar results (Fig. 2), suggesting the reli- genus Sinotubimorpha should be included in synonymy un- ability of the results in Fig. 1. der genus Grateloupia (Sheng et al., 2011). In Fig. 1, we found Red algae share some common characteristics, such as a S. guangdongensis was placed in a single monophyletic sub- two-membrane “simple” plastid without chlorophyll b/c, un- clade of G. turuturu. In contrast, other Grateloupia species were stacked thylakoids with phycobilisomes, and photosynthetic separated, away from this phylogenetically unclear algal genus. reserves as floridean starch (Müller et al., 2001). In the tradi- More interestingly, in our study based on chloroplastic genes, tional view, red algae are classified into two classes: Bangiophy- S. guangdongensis and G. turuturu were close to Order Gigar- ceae and Florideophyceae (Cavalier-Smith and Chao, 2006). Ac- tinales rather than to Order Ceramials. These phenomena are cording to the AlgaeBase (http://www.algaebase.org/), Phylum confirmed by the results based on mitochondrial genes (Fig. 3). Rhodophyta belongs to Kingdom Plantae (Eukaryota), and is classified into eight classes. However, the relationships within 3.2 Mitochondrial tree red algae remain unclear. Class Florideophyceae and Bangio- In the maximum likelihood tree of 4 mitochondrial genes phyceae were thought to be monophyletic according to the (rpl16, rps12, cob and rps11) with the total length of 1 295 aa morphological and molecular data, and Class Florideophyceae and 18 variation sites, we also identified three main lineages, is shown to have diverged from an ancestor of bangiophycean Classes Florideophyceae, Bangiophyceae and Cyanidiophyceae and florideophycean alga (Yoon et al., 2006). In this study, we (Fig. 3). However, it should be noted that there is one split for showed that some algae of Class Florideophyceae were placed Class Florideophyceae with diverse distribution, which is a little in the clade of Class Bangiophyceae (Fig. 2). Class Bangiophyce- bit different from the results based on chloroplastic proteins ae is considered being ancient, with some morphological char- (Fig. 1). It is probably due to limited sequence variations, which acters that is present in the ancestral pool of red algae (from may be caused by slower evolutionary rates of mitochondrial unicells to multicells). In our results based on chloroplastic pro- genes. More discussions can be found in the section of Ka/Ks

Cyanophora paradoxa NC_001675.1 100 Ceramium codicola FJ795540.1 100 Ceramium sinicola GQ179818.1 89 Ceramium pacificum FJ795539.1 GQ252482.1 99 Microcladia coulteri Ceramium californicum GQ179819.1 69 61 Campylaephora californica GQ252449.1 Polysiphonia fucoides JX828163.1 Ceramiales 65 Corallophila eatoniana GQ252472.1 60 Centroceras clavulatum GQ252451.1 57 Rhodoptilum plumosum GQ252577.1 Caloglossa rotundata JN845523.1 95 Heterosiphonia pulchra GQ252549.1 100 Gracilaria vermiculophylla JQ768774.1 94 NC_006137.1 100 Gracilaria tenuistipitata Gracilariales Gracilaria cuneata EU380717.1 55 Gelidium serrulatum U01042.1 Gelidiales Ptilothamnionopsis lejolisea GQ252576.1 Ceramiales Florideophyceae 66 Schizymenia dubyi AB564324.1 Nemastomatales 59 Mazzaella splendens U03385.2 51 Sarcothalia stiriata U03089.2 U03378.2 100 Mazzaella flaccida Mazzaella laminarioides U03380.2 99 Mazzaella convoluta U03084.2 U02985.2 56 Chondrus elatus 81 Chondrus ocellatus U02987.2 100 Chondrus giganteus U02986.2 Chondrus verrucosus U02988.2 Gigartinales 100 Gigartina muelleriana U03427.2 Chondracanthus corymbiferus U02941.2 100 55 Mastocarpus papillatus AY135154.1 0.05 92 Stenogramme interrupta U22509.1 Ahnfeltiopsis glomerata AF388552.1 84 Phyllophora crispa U02990.2 Cryptosiphonia woodii GQ252545.1 100 Porphyra yamadae AB671540.1 Pyropia haitanensis AB118585.1 100 Wildemania sp HQ605700.1 100 65 Bangiales 76 Pyropia yezoensis AGH27551.1 Bangiophyceae Porphyra umbilicalis AB118584.1 98 52 Porphyra purpurea NC_000925.1 Palmaria palmata U28421.1 Palmariales Thorea bachmannii GU953247.1 Thoreales Florideophyceae 100 Nemalionopsis tortuosa AB159659.1 Galdieria partita AB018008.1 NC_004799.1 71 Cyanidioschyzon merolae Cyanidiales Cyanidiophyceae 100 Cyanidium caldarium NC_001840.1

Fig.2. MrBayes tree based on the rbcL protein sequences from NCBI. Species names are followed by their NCBI accession number. Bayesian analysis was performed in MrBayes 3.1.2 software, with two independent runs, each with four incrementally heated Metropolis-coupled Monte-Carlo Markov Chains running for 5 000 000 generations. Trees were sampled every 100 generations. JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 86–93 91

Heterosiphonia pulchra Ceramium kondoi Ceramiales Mazzaella japonica Gigartinales Grateloupia turuturu Halymeniales Chondrus crispus Gigartinales Sinotubimorpha guangdongensis Halymeniales Dumontia simplex Gigartinales Gracilaria lemaneiformi Gracilariales Gymnogongrus ftabelliformis Gigartinales 0.1 Symphyocladia latiuscul Florideophyceae Polysiphonia japonica Ceramiales Kappaphycus alvarezii Gigartinales 81 Betaphycus gelatinus Gracilaria chouae Prionitis divaricata Gracilariales Gracilaria blodgettii Gloiopeltis furcata Gigartinales 87 Porphyra purpurea_r Porphyra umbilicalis_r 87 Porphyra haitanensis_r Bangiophyceae 48 Pyropia yezoensis_r Porphyra yezoensis Gracilariophila oryzoides_r 50 52 Gracilaria asiatica Gracilariales Gracilariopsis andersonii_r Gracilariopsis lemaneiformis_r 52 Chondrus crispus_r Florideophyceae Eucheuma denticulatum Gigartinales 61 Grateloupia filicina Halymeniales Plocamiocolax pulvinata_r Grateloupia livida Halymeniales Cyanidioschyzon merolae_r Cyanidiophyceae

Fig.3. Maximum likelihood tree based on 4 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 method 1 000. Bootstrap values lower than 45% are not shown. calculation. from RNA-seq datasets. Though it was difficult to tell whether It was reported that Prionitis divaricata, now nominated as these transcripts were from chloroplast/mitochondrion or Grateloupia chiangii (Family Halymeniaceae) was closely relat- pseudogenes because of “endosymbiotic gene transfer” (EGT), ed to genus Grateloupia, both morphologically and phylogenet- we could still harvest the relevant genes/pseudogenes' diversity ically (Wang et al., 2001). However, in our results, P. divaricata information because EGT has been shown to leave a footprint was always in the clade of Order Gracilariales, as a neighbor of ancient photosynthetic activity in the nuclear genome (Burki of genus Grateloupia, based on both chloroplastic (Fig. 1) and et al., 2012). In our studies, algal species number is reversely as- mitochondrial genes (Fig. 3). The reason might be that in Wang sociated with the length or diversities of consensus sequences. and his group's study (Wang et al., 2001), they did not use the To assess evolutionary rates for individual genes, we cal- data of genus Gracilariales and just showed the relationships culated nonsynonymous (Ka) substitution rate, synonymous with limited samples. (Ks) substitution rate, and Ka/Ks values by using MA method and standard code of KaKs_Calculator 1.2 (Zhang et al., 2006) 3.3 Ka/Ks for chloroplastic and mitochondrial genes against the reference C. merolae. The ratio (Ka/Ks) indicates In this study, we first made good use of RNA-seq datas- neutral mutation (Ka/Ks = 1), negative (purifying) selection ets to have a look at phylogenies of these red algae, under the (Ka/Ks < 1) and positive (diversifying) selection (Ka/Ks > 1). Our condition that they were without complete gene and genome results showed that Ks values are much more than Ka ones (av- information. As we all know, molecular taxonomic results in erage Ks vs Ka: 4.19 vs 0.28 for chloroplast, and 4.15 vs 0.28 for phylogenetic trees depend on genes' number and their vari- mitochondrion, respectively), suggesting the conservation of ous evolution rates. In fact, we identified complicated phylo- their genes (Tables 3 and 4). On the other hand, these Ka and Ks genetic structures in the trees based on individual genes (data values showed that there were no genes which might be under not shown). There are several facts which must be considered positive selection, suggesting their long evolutionary history when we try to understand this issue. A single gene may not be and adaptation. powerful enough to distinguish one algal species from another, Most interestingly, the average Ka/Ks values, 0.077 for chlo- because of its length and limited diversity information. Our roplastic genes and 0.076 for mitochondrial ones, are almost phylogenomic analysis was conducted with assembled contigs the same for red algae, indicating that their evolution rates in 92 JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 86–93

Table 3. Average Ka and Ks values for chloroplastic genes of red algae Gene # Ka Ks Ka/Ks Gene # Ka Ks Ka/Ks Gene # Ka Ks Ka/Ks secA 28 0.379 3.139 0.126 menB 26 0.342 3.259 0.106 rbcR 19 0.136 4.738 0.029 groEL 28 0.369 3.378 0.123 chlI 26 0.267 3.839 0.084 ccsA 18 0.253 3.667 0.069 rpoB 28 0.291 4.148 0.091 rps4 26 0.332 4.405 0.076 apcE 18 0.266 5.079 0.054 rps9 28 0.354 4.254 0.084 rpl3 26 0.307 4.123 0.076 ycf3 18 0.215 5.190 0.042 rps7 28 0.317 4.116 0.077 rps8 26 0.255 3.477 0.074 chlN 17 0.467 2.948 0.176 rpoA 28 0.304 3.988 0.077 petA 26 0.285 4.959 0.058 rpl12 17 0.323 4.946 0.068 dnaK 28 0.264 4.447 0.068 ycf24 26 0.200 5.051 0.040 rpl11 17 0.255 4.141 0.062 atpA 28 0.219 4.236 0.062 psaD 26 0.193 5.010 0.039 rps2 16 0.349 3.986 0.089 cbbX 28 0.221 4.130 0.060 rbcL 26 0.099 4.548 0.024 tsf 15 0.489 3.693 0.134 ftsH 28 0.158 4.047 0.042 preA 25 0.389 3.347 0.134 ycf45 15 0.315 4.173 0.083 clpC 28 0.154 4.479 0.038 rpl1 25 0.404 3.494 0.132 accD 15 0.275 4.079 0.068 atpB 28 0.116 4.487 0.029 rpl4 25 0.425 3.754 0.120 thiG 15 0.240 4.114 0.059 rps12 28 0.128 4.585 0.028 petB 25 0.127 3.975 0.032 accA 15 0.244 4.308 0.059 tufA 28 0.121 4.620 0.026 cpcA 25 0.121 4.387 0.028 orf27 15 0.151 4.332 0.036 psaB 28 0.125 5.128 0.024 apcB 25 0.064 6.614 0.010 psbW 14 0.329 4.190 0.081 psbA 28 0.080 4.434 0.018 secY 24 0.489 3.060 0.161 rpoC2 14 0.236 4.485 0.053 psbD 28 0.035 4.003 0.009 carA 24 0.334 3.864 0.095 argB 13 0.396 3.303 0.126 glmS 27 0.639 2.225 0.291 atpE 24 0.336 3.931 0.086 rps19 13 0.213 4.737 0.045 odpA 27 0.426 3.536 0.173 infC 24 0.351 4.280 0.083 ilvH 13 0.133 4.756 0.028 rpoC1 27 0.300 3.686 0.112 cobA 23 0.471 2.555 0.187 trpG 12 0.471 4.156 0.116 rpl13 27 0.375 3.950 0.096 menD 23 0.460 2.562 0.182 rps14 12 0.354 4.027 0.089 apcD 27 0.33 4.046 0.087 psbV 23 0.219 4.878 0.045 thdF 10 0.555 2.287 0.247 odpB 27 0.261 4.036 0.082 ycf59 23 0.159 5.061 0.032 ycf53 10 0.531 3.142 0.171 rpl2 27 0.321 4.144 0.079 psaF 22 0.335 4.329 0.078 ycf39 9 0.368 3.434 0.109 rps11 27 0.289 3.823 0.076 ilvB 22 0.244 4.491 0.063 psaL 9 0.176 4.719 0.038 rpl6 27 0.322 4.444 0.073 rbcS 22 0.254 4.596 0.056 cemA 8 0.153 5.100 0.032 rpl5 27 0.290 4.014 0.073 petF 22 0.215 4.879 0.046 tatC 6 0.390 3.230 0.122 rps13 27 0.307 4.360 0.072 psbE 22 0.071 5.725 0.013 rpl31 6 0.243 4.379 0.055 rps3 27 0.297 4.461 0.067 psaC 22 0.050 5.760 0.009 ycf23 5 0.531 3.224 0.177 rps5 27 0.253 4.168 0.061 trxA 21 0.231 4.111 0.058 atpG 3 0.524 3.338 0.158 rpl16 27 0.232 4.076 0.058 ycf16 20 0.366 3.955 0.093 trpA 3 0.446 3.468 0.129 rpl14 27 0.243 4.759 0.052 apcF 20 0.293 5.141 0.057 ompR 2 0.478 3.676 0.130 cpcB 27 0.118 4.653 0.026 cpcG 20 0.239 4.855 0.051 ccs1 1 0.563 3.399 0.166 apcA 27 0.103 4.999 0.025 gltB 20 0.183 4.487 0.044 ycf85 1 0.512 3.245 0.158 psaA 27 0.104 4.648 0.022 petD 20 0.144 4.214 0.034 rpl27 1 0.234 4.998 0.047 psbC 27 0.068 4.392 0.015 atpI 19 0.224 4.303 0.053 rpl20 1 0.131 5.169 0.025 psbB 27 0.063 4.549 0.014 ftrB 19 0.218 5.033 0.044 Average 0.278 4.189 0.077 Notes: # indicates the algal species numbers used for calculating Ka and Ks.

Table 4. Average Ka and Ks values for mitochondrial genes of red algae might be similar. In contrast, in another analysis of red algae brown algae (Class Phaeophyceae of Phylum Ochrophyta) using 1KP Project data, mitochondrial genes have larger average Gene # Ka Ks Ka/Ks Ka/Ks values than chloroplastic ones. This finding seemed very rps12 28 0.211 4.931 0.044 important for red algae, and might be explained by the long cytb 26 0.353 3.755 0.112 shared evolutionary history of these two organelles, because cox1 26 0.175 4.204 0.045 red algal chloroplast, as a primitive endosymbiotic one, arose cox2 19 0.263 4.494 0.067 long before that of brown algae. It was predicted that the ancient primary symbiogenesis gave rise to photosynthetic plas- sdhB 17 0.268 4.438 0.066 tids, which are present in green plants, land plants, red algae cox3 16 0.353 3.467 0.112 and glaucophytes, even before 1 558 million years ago (MYA). nad1 14 0.258 3.849 0.077 Red algae split from green algae about 1 500 MYA, while the sec- nad5 13 0.357 3.469 0.117 ondary symbiogenesis of enslaving red algae occurred about 1 300 MYA which gave rise to plastids in Chromist algae. And nad4 8 0.298 4.225 0.075 then, there are stramenopiles (Ochrophyta) and haptophytes nad3 3 0.276 4.816 0.061 (Haptophyta) which split 1 047 MYA (Yoon et al., 2004). atp6 2 0.261 4.041 0.065 This phenomenon was consistent with the results of their Average 0.279 4.154 0.076 chloroplastic and mitochondrial genes which showed simi- Notes: # indicates the algal species numbers used for calculat- lar phylogenetic relationships at the order levels for red algae. ing Ka and Ks. However, there was still a division of clustered species in Class JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 86–93 93

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