GUO Li, and YANG Guanpin

?

Predicting the Reproduction Strategies of Several Microalgae Through Their Genome Sequences

GUO Li, and YANG Guanpin*

,,266003,

Documenting the sex and sexual reproduction of the microalgae is very difficult, as most of the results are based on the microscopic observation that can be heavily influenced by genetic, physiological and environmental conditions. Understanding the reproduction strategy of some microalgae is required to breed them in large scale culture industry. Instead of direct observation of sex and sexual reproduction under microscope, the whole set or the majority of core meiosis genes may evidence the sex and sexual reproduction in the unicellular algae,as the meiosis is necessary for maintaining the genomic stability and the advantages of genetic recombination. So far, the available genome sequences and bioinformatic tools (in this study, homolog searching and phylogenetic analysis) allow us to propose that at least 20 core meiosis genes (among them ≥6 must be meiosis specific) are enough for an alga to maintain its sexual reproduction. According to this assumption and the genome sequences, it is possible that sexual reproduction was carried out byand, while asexual reproduction was adopted by,,,,,and. This understanding will facilitate the breeding trials of some economic microalgae (,,,and). However, the reproduction strategies of these microalgae need to be proved by further biological experiments.

microalga; sexual reproduction; meiosis; core meiosis gene; meiosis specific gene; homolog searching; phylogenetic analysis

1 Introduction

Microalgae consist of a wide range of autotrophic unicellular organisms which grow through photosynthesis and convert solar energy into chemical energy. They have emerged into the lime light because they can be used for different purposes (Harun., 2010). Microalgae contain diverse pigments that can be used for food and cosmetic purposes. They can also be used in pharmaceutical industries to synthesize bioactive compounds such as antioxidants, antibiotics and toxins. Besides, microalgae are used as nutrient supplements for human consumption, as they produce proteins, vitamins and polysaccharides. Some microalgae contain very rich lipids which can be extracted and further converted into biofuels (Brown., 1997; de Jesus Raposo., 2013; Gouveia., 2008; Priyadarshani and Rath, 2012). Microalgae have also displayed the potential of remediating emerging environmental problems, such as the increase of carbon dioxide and industrial water pollution, as they can fix carbon dioxide released from power plants and produce nutrients efficiently (Chisti, 2007). In addition, some species of microalgae are able to fix nitrogen and absorb heavy metals and phosphorus (Romera., 2008; Shi., 2007). All these applications have excited our interest in culturing microalgae on large scales to refine these material treasures.

Presently, the microalgal culture industry is just in its infancy, as we have not witnessed any microalgal breeding trial yet. Of a wide range of microalgal species, only a few have been cultured on relatively large scales, while no domestication and genetic improvement have been conducted. Such a scenario has further been worsened by the poor germplasm collection of microalgal species and the unclearness of their reproductive strategy and nuclear ploidy. The isolation and characterization of new microalgal species being potentialycultivable on large scales and their culturing trials may continue to dominate scientific efforts on microalgae; however, it is difficult to pursue the microalgal genetic improvement without a full understanding of their reproductive strategy.

The mutable genes of, mainly cytoplasmic, and those ofsp., both nuclear and cytoplasmic, have been identified in early mutation trial (Galloway, 1990). The mutated gene in monoploidsp. determines the phenotype directly, while any of two alleles in diploidwill never become homologous during asexual propagation. Thus, understanding the ploidy and reproductive strategy is important for the genetic improvement of a selected microalga through mutation. Expressing foreign genes in both monoploid and diploid genomes will cause an obvious phenotype in(Siaut., 2007; Zaslavskaia., 2000) andsp. (Cha., 2011; Chen., 2008; Kilian., 2011); however, such a phenotype will disappear in some daughter cells during sexual reproduction. Additionally, genes that were knocked out rather than knocked down (de Riso., 2009) will not cause phenotypic change in an asexually reproducing diploid organism. Therefore, genetic modification of a microalga needs the knowledge of its reproductive strategy. To our knowledge, sexual reproduction has never been recorded insp. andsp., the industrially most important species. Such a scenario makes it impossible to initiate genetic improvement of microalgae as was widely and intensively carried out in crops.

It is essential for us to understand the frequency and nature of the sexual reproduction of microalgae. Unfortunately, it is hard to observe the life history of most species of microalgae in either field or cultures derived from a single cell. Alternatively, the identification of a whole set or a major portion of core meiosis genes that function during meiosis, some only during meiosis (meiosis-spe- cific), may imply the existence of sex and further sexual reproductive strategy in a microalga. In addition, the genetic recombinant consequence may also indicate the existence of sex in a species, as was in(Cooper., 2007). A core meiosis gene set (functioning during meiosis) with 17 genes has been distinguished from a massive collection of genes by Ramesh. (2005). The genes were expanded further to 29 by Malik. (2008), and 9 of them were meiosis specific (functioning only during meiosis). With the advancement of genome sequencing, more and more microalgal genomes have been or will be documented. The existence of either a core meiosis gene set or a specific meiosis gene set will prove the existence of sex and sexual reproduction in a microalga. The homologs of the core meiosis genes have been identified according to their similarity with a known core meiosis gene set that were well identified early in models (Donaldson and Saville, 2008; Schurko., 2009). They were assigned to phylogenetic families, groups, or clades if they showed similar meiosis or meiosis-related function. Sometimes they were sorted out as either pseudogenes or genes with unknown but novel functions, as was proposed by Schurko and Logsdon Jr. (2008). Although the whole set or the majority of core meiosis genes cannot prove definitely the existence of sex and sexual reproduction of an organism, they can be employed to predict the sex and sexual reproduction. In this study, we searched the genomes of a microalgae set for the homologs of core meiosis genes, and presented their potential reproductive strategies.

2 Materials and Methods

2.1 Genome Sequence Retrieval

The sequences of 29 core meiotic proteins which have been assigned to their phylogenetic trees by Malik. (2008) were retrieved from National Center for Biotechnology Information (NCBI) and used as the queries in researching for the members of core meiotic proteins newly identified after 2008 (e-value≥e?5). In order to update the core meiotic proteins, the members published after 2008 were integrated with those identified early by reconstructing the phylogenetic trees of these proteins. The sequences of proteins in the reconstructed phylogenetic trees were used as queries in searching the genomic sequences deposited from May 2006 to February 2013 with BLAST v 2.2.9 (Altschul., 1997). The genomes searched are listed in Table 1.(only nucleotide sequence is available) was searched with tBLASTn, and the others were searched with BLASTp. The results did not include the genome ofpublished very recently (Read., 2013).

Table 1 The source of algal genome sequences used in this study

Note: The data were downloaded from either Joint Genome Institute (JGI, http://genome.jgi-psf.org/) or Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/), or Online Resource for Community Annotation of Eukaryotes (ORCAE, http://bioinformatics. psb.ugent.be/webtools/bogas/), or Genome Project Solutions (GPS, http://nannochloropsis.genomeprojectsolutions-databases.com/) following the directions in references.

2.2 Phylogenetic Analysis

The evolutionary relationship among the putative core meiotic proteins found in each genome and those characterized early was phylogenetically influenced by the integration. To avoid such influences, the multiple alignment of each amino acid sequence set was created with ClustalX2.1 (Larkin., 2007) and Mesquite 2.75 (Maddison., 2011), which was then analyzed with MrBayes 3.2 (Ronquist, 2012). MrBayes 3.2 was run for 106generations by selecting 4 incremental Markov chains (3 heated and 1 cold), WAG model for amino acid substitutions and a sampling frequency of 500 generations, and setting the temperature at 0.2 and an invariable rate (Whelan., 2001). The mean and 95% credibility interval of model parameters were summarized, which included gamma distribution shape parameter () and proportion of invariable sites (i). The consensus tree topology with the posterior probabilities for each split and the arithmetic mean log-likelihood (ln) were estimated for each alignment. Phylogenetic trees were rooted into out- groups, for example, non-meiotic paralogs in a eukaryotic multigene family and prokaryotic orthologs. The sequences with a systematical problem were removed.

3 Results

The genomes subjected to the core meiosis genes searching can be divided into four groups, the multicellular and sexually reproductive (and); the unicellular and sexually reproductive (); the asexual cyanobacterium (sp.); and the sexually unknown (;;;;;;.; and) (Table 2). From asexually re- productive cyanobacterium to the sexually reproductive unicellular alga and then to the sexually reproductive multicellular alga, an evolving chain of reproductive strategy was represented. From these representatives, the minimum number of meiosis genes that support sexual reproduction may be determined and used to judge the reproductive strategy of the microalgae with their reproductive strategy unrecorded. Sexual reproduction evolved very early in eukaryotic evolution (Ramesh., 2005). Thus all asexual lineages have derived from sexual relatives, and obligate asexual lineages that appear to be ancient asexual should be scarce (Normark., 2003). However, as an asexually reproductive unicellular micro- alga with sequenced genome is unavailable currently, it cannot be decided the number of meiosis genes for maintaining a sexual reproduction.

As listed in Table 3, the core meiosis genes documented early may not be found in a genome in homologous searching (--), or may be found but not assigned to a known meiosis gene (-), or may be found and assigned to a known meiosis gene (+). Occasionally, a few copies of a meiosis gene were identified in a genome (.., 2 copies of). Of the 29 meiosis genes which were documented by Malik. (2008), ≥20 genes were found in,,,,andgenome. Among these species,andare sexually reproductive multicellular algae, whileis a sexually reproductive unicellular microalga. Accordingly, we assumed that ≥20 core meiosis genes support the existence of sex and sexual reproduction in microalgae. This number may vary with the sequencing data of microalgal genomes, especially those with known reproductive strategy. It is very difficult to set a threshold number for the core meiosis genes which support the sex of microalgae; however, ≥20 core meiosis genes may indicate an extremely high possibility of sexual reproduction.

Table 2 The reproductive strategy of the interogated algae as previously reported

Notes: –, a common understanding although no literature is available for verifying the reproductive strategy; Abbr., abbreviation; Euk., eukaryotic; Prok., prolaryotic.

It was interesting to note that ≥6 meiosis specific genes which function only in meiosis as was outlined by Malik. (2008) were also found in,,,,andgenme (Table 3; Fig.1). We assumed that ≥6 meiosis specific genes support the existence of sex and sexual reproduction. Similar to the whole core meiosis gene set, this number may vary according to the data from different microalgae, making it impossible to set a definite threshold number of meiosis specific genes which support the sex of microalgae. In addition, the number of the whole core meiosis gene set including those specific for meiosis was positively proportional to that of meiosis specific genes to some extent (=0.78), which implied that the meiosis specific genes evolve concertedly with other core meiosis genes (Fig.2). The above scenarios allowed us to propose that ≥20 core meiosis genes (≥6 must be meiosis specific) were enough for an alga to maintain its sexual reproduction. According to this assumption, either obligate or facultative sexual reproduction was carried out byand, while the asexual reproduction was adopted by.,.,.,,.,.andinvestigated in this study. The real reproductive strategies of these algae need to be finally proved by further reproduction experiments.

Table 3 The distribution of known core meiotic genes among interrogated algae

Notes: MSG, meiosis specific gene (in bold face) which functions only in meiosis; CMG, core meiosis gene including MSG, which associates with meiosis or functions during meiosis; +, gene which was found in BLASTp search against diverse databases and verified with phylogenetic analysis; +*, gene which was found in search, verified in phylogenetic analysis and previously annotated; -, either pseudogene or protein with non-meiotic function, which was found in search at e-value of ≥ e?5, but not assigned to the known protein group in phylogenetic analysis; --, gene which is absent in complete genome sequence, and not found in BLASTp search against diverse databases; The boldfaced are the gene encoding meiosis-specific proteins.

4 Discussions

Culturing microalgae may provide us diverse materials for a wide range of applications in comsumption and medicine. The genetic improvement of microalgae is in its infancy, and a majority of microalgae in culture have not experienced domestication and breeding. We may initiate microalgal breeding with gene modification and mutation when their reproductive strategies are understood. Although our endeavors have been made, the reproductive strategies of widely cultured microalgae, for example,,and, were still unclear. Core meiosis genes were identified from the ge- nome of microalgae with sexual reproduction, and more than 20 core meiosis genes were identified, including more than 6 meiosis-specific genes. Accordingly,,,,,andwere proposed to be asexual. Mutation should be an effective way of genetic improvement for these asexual microalgae as was practiced in(Cha., 2011; Chen., 2008; Galloway, 1990; Kilian., 2011; Qu., 2013) and(de Riso., 2009; Siaut., 2007; Zaslav- skaia., 2000).

Twenty nine core meiosis genes (9 were meiosis specific) have been selected as the tool of diagnosing the sexual reproduction of organisms with their reproductive strategy unknown due to different reasons (Malik., 2008; Ramesh., 2005). It was noticed that either the whole core genes set or the whole meiosis specific genes set do not always exist in a given genome, even in thegenome of sexually reproducing organisms like,and. Diverse possibilities may explain such a scenario, which include the incompleteness of genome sequence, the evolution of novel core meiosis gene (s) that has not been cognized in a species, the functional replacement of meiosis specific genes by other core meiosis genes, and so on. In fact the core meiosis genes including those meiosis-specific genes are identified in a wide range of organisms. The diversification of meiosis process among organisms may allow them share some but not all these genes. Therefore, it is very possible to miss a few but not a large portion of core meiosis genes. Based on the limited genome information, it is very hard to determine a common set of core meiosis genes. From the results of this study, ≥20 core meiosis genes (≥6 must be meiosis specific) may be enough for an alga to maintain the sexual reproduction. However, the genes may vary among the genomes in different microalgae.

Fig.1 Phylogenetic trees of the meiosis-specific proteins. The meiosis-specific proteins include Spo11, Hop1, Msh4, Msh5, Dmc1, Hop2, Mnd1, Mer3 and Rec8, 9 in total. All trees shown are the consensus tree topologies determined from ≥800 best trees inferred by Bayesian analysis using alignments of inferred proteins. Scale bars show the number of amino acid substitutions. Algae analyzed in this study are indicated in bold type. The full scientific name is shown if a genus contains >1 species. A, relationship among species used in this study. B, Rad51 and Dmc1 paralogs rooted into archaeal RadA. In total, 224 aligned amino acid sites are based in tree calculation. The consensus topology derives from 980 trees. α=1.03 (0.91<α<1.17), pi=0.0031 (0.0001

Fig.2Phylogenetic trees of other core meiosis proteins. A, Mlh1, Mlh3 and Pms1 paralogs rooted into prokaryotic MutL. In total, 308 aligned amino acid sites are based in analysis. The consensus topology derives from 560 trees.=1.13 (0.71 <<1.26),i=0.033 (0.021

The number of core meiosis genes searched out from a genome (e-value≥e?5) was larger than the number finally assigned to the known core genes. Some of the genes were regarded as pseudogenes or genes with unknown but novel functions. Homolog searching alone or in combina- tion with phylogenetic analysis is not enough for deter- mining the function of a gene. Homologous searching in combination with phylogenetic analysis may improve our understanding of a gene; however such trials cannot finally verify or decipher the function of a gene. Recognizing the presence of each meiotic gene is not necessary, considering some genes may be missed in a lineage due to our inability to detect them or simply due to evolutionary reality.

It is very interesting to note that 7 meiosis-specific genes were found in the genome of. Such an observation has also been described by Blanc. (2010) when they annotate the genome of. Regarding the number of the whole core meiosis genes set, we found that 19 (<20) core meiosis genes exist in its genome. According to our assumption,should be asexual, which was in accordance with the biological asexual process in this species. Additionally, loss of meiosis-related genes, even if they are not meiosis-spe- cific, may also cause asexual reproduction of.

The most complicated reproductive strategies are in diatoms which contain many species with large scale culturing potential (Holtermann., 2010). Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. However, the reproductive strategies of most diatoms remain unknown. Variation also exists in the vegetative portion of diatom life cycles. Several taxa have been shown to possess the ability of regenerating their maximum size through asexual vegetative enlargement or parthenogenesis. Other taxa, both centric and pennate, are able to prevent size diminution through modified girdle band arrangement. Morphologically distinct resting spores can also be present in the vegetative life cycle, but they do not perform size restoration (Chepurnov and Mann, 1997; Koester., 2007; Sabbe, 2004; von Dassow., 2006). Only fragmentary information is available because almost all studies are based on microscopic observation. Diatom sexuality is in fact limited to brief periods (minutes or hours) that may occur less than once a year in some species, which involves only a small number of vegetative cells within a population. Sometimes the sexual reproduction can be induced when vegetative cells are exposed to unfavorable growth conditions (Armbrust, 1999; Falciatore and Bowler, 2002). Sexual reproduction has never been recorded in. The genusis a major component of marine phytoplankton, which is known to form resting spores. According to the number of core meiosis genes, we believe thatandreproduce asexually, and gene modification should be the most appropriate breeding approach for them, as they are diploid at the same time.

is a hot spring red alga with the smallest genome (Nozaki., 2007). To date, the sexual reproduction has never been documented in it. It holds 24 core meiosis genes including 6 meiosis-specific genes. According to the standard established in this study, it should be able to reproduce sexually. This assumption may orient our further endeavor of unrevealing the reproductive secret of this alga, which is also expected for marine picoeukaryote(Worden., 2009).species have emerged as leading phototrophic microorganisms for the production of biofuels..has been recognized as oil producing model alga (Jinker- son., 2013). This scenario associates with a wide range of studies including its genetic modification, mutation and among others. We have demonstrated thatis monoploid and asexual (Pan., 2011). Asexual reproduction ofspecies in combination with their monoploidy and many other advantages (, high frequent homologous recombination and high lipid content) will make them more appropriate for being models.andhave preserved their nucleomorphs in evolution (Curtis., 2012). However, they may never evolve to sexual reproduction or have lost the ability of sexual reproduction. This is an interesting question which should be addressed in the future.

5 Conclusions

The whole set or the majority of core meiosis genes may decide the sex and sexual reproduction in an organism. The sequencing analysis results allow us to propose that ≥20 core meiosis genes (≥6 must be meiosis specific) are enough for an alga to maintain its sexual reproduction. According to this assumption, it can be predicted that ei- ther obligate or facultative sexual reproduction was carried out byand, while asexual reproduce- tion was adopted by,,,,,and.

Acknowledgements

We are very grateful to anonymous reviewers for their suggestions of data processing and organizing. We express our heartfelt thanks to English editors for their patience in polishing our writing, This work was finan- cially supported by National Science and Technology Supporting Program of China (2011BAD14B01), National Natural Science Foundation of China (31270408), and Key Laborotary of Marine Bioactive Substance of State Oceanic Administrationof China, The First Institute of Oceanography.

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J., 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs., 25: 3389-3402.

Armbrust, E. V., 1999. Identification of a new gene family expressed during the on-set of sexual reproduction in the centric diatom., 65: 3121-3128.

Blanc, G., Duncan, G., Agarkova, I., Borodovsky, M., Gurnon, J., Kuo, A., Lindquist, E., Lucas, S., Pangilinan, J., Polle, J., Salamov, A., Terry, A., Yamada, T., Dunigan, D. D., Grigoriev, I. V., Claverie, J. M., and van Etten, J. L., 2010. TheNC64A genome reveals adaptation to photosymbiosis, coevolution with viruses, and cryptic sex., 22 (9): 2943-2955.

Bowler, C., Allen, A. E., Badger, J. H., Grimwood, J., Jabbari, K., Kuo, A., Maheswari, U., Martens, C., Maumus, F., Otillar, R. P., Rayko, E., Salamov, A., Vandepoele, K., Beszteri, B., Gruber, A., Heijde, M., Katinka, M., Mock, T., Valentin, K., Verret, F., Berges, J. A., Brownlee, C., Cadoret, J. P., Chiovitti, A., Choi, C. J., Coesel, S., de Martino, A., Detter, J. C., Durkin, C., Falciatore, A., Fournet, J., Haruta, M., Huysman, M. J., Jenkins, B. D., Jiroutova, K., Jorgensen, R. E., Joubert, Y., Kaplan, A., Kroger, N., Kroth, P. G., la Roche, J., Lindquist, E., Lommer, M., Martin-Jezequel, V., Lopez, P. J., Lucas, S., Mangogna, M., McGinnis, K., Medlin, L. K., Montsant, A., Oudot-Le Secq, M. P., Napoli, C., Obornik, M., Parker, M. S., Petit, J. L., Porcel, B. M., Poulsen, N., Robison, M., Rychlewski, L., Rynearson, T. A., Schmutz, J., Shapiro, H., Siaut, M., Stanley, M., Sussman, M. R., Taylor, A. R., Vardi, A., von Dassow, P., Vyverman, W., Willis, A., Wyrwicz, L. S., Rokhsar, D. S., Weissenbach, J., Armbrust, E. V., Green, B. R., van de Peer, Y., and Grigoriev, I. V., 2008. Thegenome reveals the evolutionary history of diatom genomes., 456: 239-244.

Brown, M. R., Jeffrey, S. W., Volkman, J. K., and Dunstan, G. A., 1997. Nutritional properties of microalgae for mariculture., 151: 315-331.

Cha, T. S., Chen, C. F., Yee, W., Aziz, A., and Loh, S. H., 2011. Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient-mediated transfor- mation of the unicellular green alga,sp., 84: 430-434.

Chen, H. L., Li, S. S., Huang, R., and Tsai, H. J., 2008. Conditional production of a functional fish growth hormone in the transgenic line of(Eustigmato- phyceae)., 44: 768-776.

Chepurnov, V. A., and Mann, D. G., 1997. Variation in the sexual behavior of natural clones of(Bacillariophyta)., 32: 2147- 2154.

Chepurnov, V. A., Mann, D. G., Sabbe, K., and Vyverman, W., 2004. Experimental studies on sexual reproduction in diatoms., 237: 91-154.

Chisti, Y., 2007. Biodiesel from microalgae., 25: 294-306.

Cock, J. M., Sterck, L., Rouzé, P., Scornet, D., Allen, A. E., Amoutzias, G., Anthouard, V., Artiguenave, F., Aury, J. M., Badger, J. H., Beszteri, B., Billiau, K., Bonnet, E., Bothwell, J. H., Bowler, C., Boyen, C., Brownlee, C., Carrano, C. J., Charrier, B., Cho, G. Y., Coelho, S. M., Collén, J., Corre, E., da, Silva, C., Delage, L., Delaroque, N., Dittami, S. M., Doulbeau, S., Elias, M., Farnham, G., Gachon, C. M., Gschloessl, B., Heesch, S., Jabbari, K., Jubin, C., Kawai, H., Kimura, K., Kloareg, B., Küpper, F. C., Lang, D., Le Bail, A., Leblanc, C., Lerouge, P., Lohr, M., Lopez, P. J., Martens, C., Maumus, F., Michel, G., Miranda-Saavedra, D., Morales, J., Moreau, H., Motomura, T., Nagasato, C., Napoli, C. A., Nelson, D. R., Nyvall-Collén, P., Peters, A. F., Pommier, C., Potin, P., Poulain, J., Quesneville, H., Read, B., Rensing, S. A., Ritter, A., Rousvoal, S., Samanta, M., Samson, G., Schroeder, D. C., Ségurens, B., Strittmatter, M., Tonon, T., Tregear, J. W., Valentin, K., von Dassow, P., Yamagishi, T., van de Peer, Y., and Wincker, P., 2010. Thegenome and the independent evolution of multicellularity in brown algae., 465: 617-621.

Coelho, S. M., Scornet, D., Rousvoal, S., Peters, N. T., Dartevelle, L., Peters, A. F., and Cock, J. M., 2012.: A model organism for the brown algae., 2012: 193-198.

Cooper, M. A., Adam, R. D., Worobey, M., and Sterling, C. R., 2007. Population genetics provides evidence for recombination in., 17 (22): 1984-1988.

Curtis, B. A., Tanifuji, G., Burki, F., Gruber, A., Irimia, M., Maruyama, S., Arias, M. C., Ball, S. G., Gile, G. H., Hirakawa, Y., Hopkins, J. F., Kuo, A., Rensing, S. A., Schmutz, J., Symeonidi, A., Elias, M., Eveleigh, R. J., Herman, E. K., Klute, M. J., Nakayama, T., Oborník, M., Reyes-Prieto, A., Armbrust, E. V., Aves, S. J., Beiko, R. G., Coutinho, P., Dacks, J. B., Durnford, D. G., Fast, N. M., Green, B. R., Grisdale, C. J., Hempel, F., Henrissat, B., H?ppner, M. P., Ishida, K. I., Kim, E., Ko?eny, L., Kroth, P. G., Liu, Y., Malik, S. B., Maier, U. G., McRose, D., Mock, T., Neilson, J. A., Onodera, N. T., Poole, A. M., Pritham, E. J., Richards, T. A., Rocap, G., Roy, S. W., Sarai, C., Schaack, S., Shirato, S., Slamovits, C. H., Spencer, D. F., Suzuki, S., Worden, A. Z., Zauner, S., Barry, K., Bell, C., Bharti, A. K., Crow, J. A., Grimwood, J., Kramer, R., Lindquist, E., Lucas, S., Salamov, A., McFadden, G. I., Lane, C. E., Keeling, P. J., Gray, M. W., Grigoriev, I. V., and Archibald, J. M., 2012. Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs., 492 (7427): 59-65.

de Jesus Raposo, M. F., de Morais, R. M. S. C., and de Morais, A. M. M. B., 2013. Bioactivity and applications of sulphated polysaccharides from marine microalgae., 11: 233-252.

de Riso, V., Raniello, R., Maumus, F., Rogato, A., Bowler, C., and Falciatore, A., 2009. Gene silencing in the marine diatom., 37: e96.

Donaldson, M. E., and Saville, B. J., 2008. Bioinformatic identification ofmeiosis genes., 45: S47-53.

Falciatore, A., and Bowler, C., 2002. Revealing the molecular secrets of marine diatoms., 53: 109-130.

Galloway, R. E., 1990. Selective condition and isolation of mutants in salt-tolerant, lipid-producing microalgae., 26: 752-760.

Gouveia, L., Batista, A. P., Sousa, I., Raymundo, A., and Bandarra, N. M., 2008. Microalgae in novel food products. In:. Papadopoulos, K. N. ed., Nova Science Publishers Inc., New York, 75-111.

Harun, R., Singh, M., Forde, G. M., and Danquah, M. K., 2010. Bioprocess engineering of microalgae to produce a variety of consumer products., 14: 1037-1047.

Hense, I., and Beckmann, A., 2006. Towards a model of cyanobacteria life cycle-effects of growing and resting stages on bloom formation of N2-fixing species., 195: 205-218.

Holtermann, K. E., Bates, S. S., Trainer, V. L., Odell, A., and Armbrust, E. V., 2010. Mass sexual reproduction in the toxigenic diatomsand(Bacillariophyceae) on the Washington coast, USA., 46: 41-52.

Huang, K., and Beck, C. F., 2003. Phototropin is the blue-light receptor that controls multiple steps in the sexual life cycle of the green alga., 100: 6269-6274.

Jinkerson, R. E., Radakovits, R., and Posewitz, M. C., 2013. Genomic insights from the oleaginous model alga., 4 (1): 37-43.

Kilian, O., Benemann, C. S. E., Niyogi, K. K., and Vick, B., 2011. High efficiency homologous recombination in the oil- producing algasp., 108: 21265-21269.

Koester, J. A., Brawley, S. H., Karp-Boss, L., and Mann, D. G., 2007. Sexual reproduction in the marine centric diatom(Bacillariophyta)., 42: 4351-4366.

Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., and Higgins, D. G., 2007. Clustal W and Clustal X version 2.0., 23: 2947-2948.

Maddison, W. P., and Maddison, D. R., 2011. Mesquite: A modular system for evolutionary analysis, version 2.75. http://mesquiteproject.org.

Malik, S. B., Pightling, A. W., Stefaniak, L. M., Schurko, A. M., and Logsdon, J. M., 2008. An expanded inventory of con- served meiotic genes provides evidence for sex in., 3 (8): e2879.

Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., Terry, A., Salamov, A., Fritz-Laylin, L. K., Maréchal-Drouard, L., Marshall, W. F., Qu, L. H., Nelson, D. R., Sanderfoot, A. A., Spalding, M. H., Kapitonov, V. V., Ren, Q., Ferris, P., Lindquist, E., Shapiro, H., Lucas, S. M., Grimwood, J., Schmutz, J., Cardol, P., Cerutti, H., Chanfreau, G., Chen, C. L., Cognat, V., Croft, M. T., Dent, R., Dutcher, S., Fernández, E., Fukuzawa, H., González-Ballester, D., González-Halphen, D., Hallmann, A., Hanikenne, M., Hippler, M., Inwood, W., Jabbari, K., Kalanon, M., Kuras, R., Lefebvre, P. A., Lemaire, S. D., Lobanov, A. V., Lohr, M., Manuell, A., Meier, I., Mets, L., Mittag, M., Mittelmeier, T., Moroney, J. V., Moseley, J., Napoli, C., Nedelcu, A. M., Niyogi, K., Novoselov, S. V., Paulsen, I. T., Pazour, G., Purtons, S., Ral, J. P., Ria?o-Pachón, D. M., Riekhof, W., Rymarquis, L., Schroda, M., Stern, D., Umen, J., Willows, R., Wilson, N., Zimmer, S. L., Allmer, J., Balk, J., Bisova, K., Chen, C. J., Elias, M., Gendler, K., Hauser, C., Lamb, M. R., Ledford, H., Long, J. C., Minagawa, J., Page, M. D., Pan, J., Pootakham, W., Roje, S., Rose, A., Stahlberg, E., Terauchi, AM., Yang, P., Ball, S., Bowler, C., Dieckmann, CL., Gladyshev V. N., Green, P., Jorgensen, R., Mayfield, S., Mueller-Roeber, B., Rajamani, S., Sayre, R. T., Brokstein, P., Dubchak, I., Goodstein, D., Hornick, L., Huang, Y. W., Jhaveri, J., Luo, Y., Martínez, D., Ngau, W. C., Otillar, B., Poliakov, A., Porter, A., Szajkowski, L., Werner, G., Zhou, K., Grigoriev, I. V., Rokhsar, D. S., and Grossman, A. R., 2007. Thegenome reveals the evolution of key animal and plant functions., 318 (5848): 245-250.

Moestrup, ?., and Sengco, M., 2001. Ultrastructural studies on, gen. et sp. nov., a chlorarachniophyte flagellate., 37: 624-646.

Normark, B. B., Judson, O. P., and Moran, N. A., 2003. Genomic signatures of ancient asexual lineages., 79: 69-84.

Nozaki, H., Takano, H., Misumi, O., Terasawa, K., Matsuzaki, M., Maruyama, S., Nishida, K., Yagisawa, F., Yoshida, Y., Fujiwara, T., Takio, S., Tamura, K., Chung, S. J., Nakamura, S., Kuroiwa, H., Tanaka, K., Sato, N., and Kuroiwa, T., 2007. A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga., 5: 28.

Qu, X. M., Mi, W. Y., Zhu, B. H., Yang, G. P., Li, S. D, and Pan, K. H., 2013. Applicability of berbicide quizalofop-p- ethyl to the screening of lipid rich., 43 (6): 25-28 (in Chinese with English abstract).

Pan, K., Qin, J., Li, S., Dai, W., Zhu, B., Jin, Y., Yu, W., Yang, G., and Li, D., 2011. Nuclear monoploidy and asexual propagation of(Eustigmatophycaea) as revealed by its genome sequence., 47: 1425-1432.

Priyadarshani, I., and Rath, B., 2012. Commercial and industrial applications of micro algae–A review., 3: 89-100.

Prochnik, S. E., Umen, J., Nedelcu, A. M., Hallmann, A., Miller, S. M., Nishi, I., Ferris, P., Kuo, A., Mitros, T., Fritz-Laylin, L. K., Hellsten, U., Chapman, J., Simakov, O., Rensing, S. A., Terry, A., Pangilinan, J., Kapitonov, V., Jurka, J., Salamov, A., Shapiro, H., Schmutz, J., Grimwood, J., Lindquist, E., Lucas, S., Grigoriev, I. V., Schmitt, R., Kirk, D., and Rokhsar, D. S., 2010. Genomic analysis of organismal complexity in the multicellular green alga., 329 (5988): 223- 226.

Radakovits, R., Jinkerson, R. E., Fuerstenberg, S. I., Tae, H., Settlage, R. E., Boore, J. L., and Posewitz, M. C., 2012. Draft genome sequence and genetic transformation of the oleaginous alga., 3: 686.

Ramesh, M. A., Malik, S. B., and Logsdon, J. M., 2005. A phylogenomic inventory of meiotic genes: Evidence for sex inand an early eukaryotic origin of meiosis., 15: 185-191.

Read, B. A., Kegel, J., Klute, M. J., Kuo, A., Lefebvre, S. C., Maumus, F., Mayer, C., Miller, J., Monier, A., Salamov, A., Young, J., Aguilar, M., Claverie, J. M., Frickenhaus, S., Gonzalez, K., Herman, E. K., Lin, Y. C., Napier, J., Ogata, H., Sarno, A. F., Shmutz, J., Schroeder, D., de Vargas, C., Verret, F., von Dassow, P., Valentin, K., de Peer, Y. V., Wheeler, G., Allen, A. E., Bidle, K., Borodovsky, M., Bowler, C., Brownlee, C., Cock, J. M., Elias, M., Gladyshev, V. N., Groth, M., Guda, C., Hadaegh, A., Iglesias-Rodriguez, D., Jenkins, J., Jones, B. M., Lawson, T., Leese, F., Lindquist, E., Lobanov, A., Lomsadze, A., Lucas, S., Malik, S. B., Marsh, M. E., Mock, T., Mueller-Roeber, B., Pagarete, A., Parker, M., Probert, I., Quesneville, H., Raines, C., Rensing, S. A., Riano-Pachon, D. M., Richier, S., Rokitta, S., Shiraiwa, Y., Soanes, D. M., van der Giezen, M., Wahlund, T. M., Williams, B., Wilson, W., Wolfe, G., Wurch, L. L., Dacks, J. B., Delwiche, C. F., Dyhrman, S. T., Gl?ckner, G., John, U., Richards, T., Worden, A. Z., Zhang, X., and Grigoriev, I. V., 2013. Pan genome of the phytoplanktonunderpins its global distribution., 499: 209-213.

Romera, E., Gonzalez, F., Balleste, A., Blazquez, M. L., and Munoz, J. A., 2008. Biosorption of Cd, Ni, and Zn with mix- tures of different types of algae., 25: 999-1008.

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., H?hna, S., Larget, B., Liu, L., Suchard, M. A., and Huelsenbeck, J. P., 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space., 61 (3): 539-542.

Sabbe, K., Chepurnov, V. A., Vyverman, W., and Mann, D. G., 2004. Apomixis in(Bacillariophyceae); develop- ment of a model system for diatom reproductive biology., 39: 3327-3341.

Schurko, A. M., and Logsdon, J. M., 2008. Using a meiosis detection toolkit to investigate ancient asexual ‘scandals’ and the evolution of sex., 30 (6): 579-589

Schurko, A. M., Logsdon, J. M., and Eads, B. D., 2009. Meiosis genes inand the role of parthenogenesis in genome evolution.,9: 78.

Shi, J., Podola, B., and Melkonian, M., 2007. Removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: An experimental study., 19: 417-423.

Siaut, M., Heijde, M., Mangogna, M., Montsant, A., Coesel, S., Allen, A., Manfredonia, A., Falciatore, A., and Bowler, C., 2007. Molecular toolbox for studying diatom biology in., 406: 23-35.

von Dassow, P., Chepurnov, V. A., and Armbrust, E. V., 2006. Relationship between growth rate, cell size, and induction of spermatogenesis in the centric diatom(Bacillariophyta)., 42: 887-899.

Welsh, E. A., Liberton, M., Stocke, L. J., Loh, T., Elvitigala, T., Wang, C., Wollam, A., Fulton, R. S., Clifton, S. W., Jacobs, J. M., Aurora, R., Ghosh, B. K., Sherman, L. A., Smith, R. D., Wilson, R. K., and Pakrasi, H. B., 2008. The genome of51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle., 105: 15094-15099.

Whelan, S., and Goldman, N., 2001. A general empirical model of protein172 BIBLIOGRAPHY evolution derived from multiple protein families using a maximum likelihood approach., 18: 691-699.

Worden, A. Z., Lee, J. H., Mock, T., Rouzé, P., Simmons, M. P., Aerts, A. L., Allen, A. E., Cuvelier, M. L., Derelle, E., Everett, M. V., Foulon, E., Grimwood, J., Gundlach, H., Henrissat, B., Napoli, C., McDonald, S. M., Parker, M. S., Rombauts, S., Salamov, A., Von, Dassow, P., Badger, J. H., Coutinho, P. M., Demir, E., Dubchak, I., Gentemann, C., Eikrem, W., Gready, J. E., John, U., Lanier, W., Lindquist, E. A., Lucas, S., Mayer, K. F., Moreau, H., Not, F., Otillar, R., Panaud, O., Pangilinan, J., Paulsen, I., Piegu, B., Poliakov, A., Robbens, S., Schmutz, J., Toulza, E., Wyss, T., Zelensky, A., Zhou, K., Armbrust, E. V., Bhattacharya, D., Goodenough, U. W., van de Peer, Y., and Grigoriev, I. V., 2009. Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes., 324 (5924): 268-272.

Zaslavskaia, L. A., Lippmeier, J. C., Kroth, P. G., Grossman, A. R., and Apt, K. E., 2000. Transformation of the diatom(Bacillariophyceae) with a variety of selectable marker and reporter genes., 36: 379-386.

(Edited by Qiu Yantao)

10.1007/s11802-015-2442-2

(July 29, 2013; revised December 25, 2013; accepted April 9, 2014)

. Tel: 0086-532-82031636 E-mail: yguanpin@ouc.edu.cn

ISSN 1672-5182, 2015 14 (3): 491-502

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015