JSE. Evolution of the Chlorophyta: Insights from chloroplast phylogenomic analyses. Review. 1 Introduction

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1 JSE Review Journal of Systematics and Evolution doi: /jse Evolution of the Chlorophyta: Insights from chloroplast phylogenomic analyses Ling Fang 1, Frederik Leliaert 2,3, Zhen-Hua Zhang 1, David Penny 4, and Bo-Jian Zhong 1 * 1 College of Life Sciences, Nanjing Normal University, Nanjing , China 2 Botanic Garden Meise, 1860 Meise, Belgium 3 Phycology Research Group, Biology Department, Ghent University, 9000 Ghent, Belgium 4 Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand *Author for correspondence. bjzhong@gmail.com. Tel.: Received 6 January 2017; Accepted 4 March 2017; Article first published online 5 May 2017 Abstract Green plants comprise two main clades: the Streptophyta, which include charophyte green algae and the embryophytic land plants, and the Chlorophyta including a wide diversity of marine, freshwater, and terrestrial green algae. Establishing a robust phylogeny is important to provide an evolutionary framework for comparative and functional studies. During the last two decades our understanding of the evolution of green algae has profoundly changed, first by phylogenetic analyses of nuclear ribosomal sequence data (mainly 18S), and more recently by analyses of multi-gene and chloroplast genomic data. The phylogenetic relationships among the main streptophytan lineages have been extensively studied and are now relatively well resolved. Although a lot of progress has been made in the last few years, the phylogenetic relationships in the Chlorophyta are still less well established. Here we review how chloroplast genomic data have contributed to address relationships among the main chlorophytan lineages. We highlight recent progress and conflicts among different studies, and discuss future directions in chloroplast phylogenomics of green algae. Key words: Chlorophyta, chloroplast genome, green algae, model evaluation, phylogenomics. 1 Introduction Green algae are morphologically and ecologically diverse and ubiquitous in marine, freshwater, and terrestrial habitats (Graham et al., 2009). They are not a monophyletic group but belong to the green plants, an ancient lineage of eukaryotes comprising two main clades. One clade, the Streptophyta, include mostly freshwater green algae (known as charophytes) and the land plants. The other clade, the Chlorophyta, include marine, freshwater, and terrestrial green algae with a wide morphological diversity, ranging from planktonic unicellular organisms, to colonial, multicellular, and siphonous algae. The divergence between the Streptophyta and Chlorophyta is estimated to have taken place more than 1 billion years ago (Leliaert et al., 2011; Moczydłowska et al., 2011). The phylogenetic relationships among the main charophyte clades, and especially the relationship with the land plants, have been extensively investigated and reviewed (e.g., Cooper, 2014; Delwiche & Cooper, 2015; Zhong et al., 2015). It is now well established that the Zygnematophyceae are the sister clade to the land plants (Timme & Delwiche, 2010; Wodniok et al., 2011; Timme et al., 2012; Zhong et al., 2013; Wickett et al., 2014). The Chlorophyta include ecologically, morphologically, and cytologically diverse green algae. The early evolutionary history of the clade likely took place in the Meso- and 2017 Institute of Botany, Chinese Academy of Sciences Neoproterozoic era, where unicellular planktonic algae (known as prasinophytes) diversified in the oceans, and later gave rise to the core chlorophytes that radiated in coastal, freshwater, and terrestrial environments, and today contain most of the species (Leliaert et al., 2011, 2012). Extant prasinophytes form a paraphyletic assemblage of planktonic unicellular green algae with a wide variety of cell shapes, flagellar numbers and behavior, body scale morphologies, mitotic processes, and photosynthetic pigments, and are living both in oceanic and freshwater environments. This wide diversity in conserved phenotypic features supports the notion that prasinophytes are ancient lineages. The core Chlorophyta include three major classes, Chlorophyceae, Ulvophyceae, and Trebouxiophyceae, plus the two smaller lineages, the Chlorodendrophyceae, comprising the scaly quadriflagellates Tetraselmis and Scherffelia from freshwater, brackish water, marine, and hypersaline habitats (Norris et al., 1980; Arora et al., 2013) and Pedinophyceae, which include asymmetric, uniflagellate, mostly naked green algae from marine, freshwater, or soil habitats (Marin, 2012) (Fig. 1). The classes Chlorophyceae, Ulvophyceae, and Trebouxiophyceae are species-rich and morphologically and ecologically diverse. The Chlorophyceae include freshwater and terrestrial green algae displaying diverse cell organizations and variable ultrastructure of the flagellar apparatus (Lewis & McCourt, 2004). The Ulvophyceae July 2017 Volume 55 Issue

2 Chloroplast phylogenomics of the Chlorophyta 323 Chlorophyceae Ulvophyceae Core Trebouxiophyceae Chlorellales Chlorodendrophyceae Trebouxiophyceae Core Chloropyhta Chloropyhta Pedinophyceae Prasinophytes Charophyte Green Algae and Land Plants Fig. 1. Phylogeny of Chlorophyta inferred from chloroplast genomic data. Dashed and solid lines indicate uncertain and established relationships, respectively. include mostly macroscopic, multicellular, or siphonous species from coastal habitats, but also many microscopic unicellular or multicellular species from marine, freshwater, and terrestrial environments (Cocquyt et al., 2010). The Trebouxiophyceae, a class that was circumscribed mainly based on ultrastructural features and nuclear ribosomal DNA sequence data (Mattox & Stewart, 1984; Kantz et al., 1990), encompass motile and non-motile unicellular, colonial, and multicellular green algae from freshwater, terrestrial, and sometimes marine habitats, with several species engaging in symbiosis with a diversity of eukaryotes (Lewis & McCourt, 2004; Leliaert et al., 2012). A reliable phylogenetic tree of Chlorophyta is important to understand the early evolution of green algae. However, resolving the phylogenetic relationships among the major clades of the Chlorophyta has been shown to be a difficult task, because these ancient lineages radiated rapidly, and possible multiple extinction events occurred from ancient lineages (Cocquyt et al., 2010). Ultrastructural data, and nuclear ribosomal, chloroplast, and mitochondrial sequence data have yielded ambivalent results (reviewed in Leliaert et al., 2012). Adding to the complexity, the monophyly of the Ulvophyceae and Trebouxiophyceae has been questioned. Initial molecular phylogenetic studies investigated the relationships among green algal lineages using a single gene, mostly the nuclear small subunit ribosomal RNA gene (18S) (Fawley et al., 2000; Krienitz et al., 2001; M uller et al., 2004; Bock et al., 2013). It is now apparent that multiple genes from many species are the premise to fully resolve ancient phylogenetic relationships (Philippe & Telford, 2006; Philippe et al., 2011). Chloroplast genomes are particularly useful for phylogenetic reconstruction because of their highly conserved and condensed gene content and their typical mode of uniparental inheritance. Moreover, chloroplast genes are typically single-copy, in contrast to nuclear genes that are multi-copy in nature, which can mislead phylogenetic inference. Another advantage is that chloroplast genes lack the multiple and often long introns typically found in nuclear genes. Chloroplast genomic data is dramatically expanding due to the availability of high-throughput sequencing technology, and have been extensively used in phylogenomic studies aiming to resolve ancient relationships in land plants (Wu et al., 2013; Ruhfel et al., 2014; Lu et al., 2015). In addition, more realistic substitution models of sequence evolution are applied for chloroplast phylogenomic analyses. For example, in the streptophytan lineage, Cox et al. (2014) reported the monophyletic phylogeny of the bryophytes (an early diverging land plant lineage) using the chloroplast genomes by correcting the phylogenetic artifacts, which is caused by mutation-driven compositional biases and synonymous substitutions. Zhong et al. (2014) analyzed the chloroplast genomes of land plants and charophytes, and phylogenomic analyses supported that Zygnematophyceae are the closest relatives to the land plants, which is congruent with recent large-scale phylotranscriptomic analyses (Wodniok et al., 2011; Timme et al., 2012; Wickett et al., 2014). In this review, we summarize recent progress in chloroplast phylogenomic analyses of Chlorophyta, discuss potential errors/biases limiting the resolution of phylogenomic inference, and provide further directions to improve the phylogenetic accuracy of Chlorophyta both by adding critical taxa and using more appropriate evolutionary models. 2 Chloroplast Phylogenomics of Chlorophyta 2.1 Phylogenetic relationship of the prasinophytes Approximately 10 clades of the prasinophytes have been identified based on 18S rdna sequences (Leliaert et al., 2011). Although 18S data clearly showed that prasinophytes comprise the earliest diverging lineages of the Chlorophyta, the affinities among prasinophyte clades are difficult to resolve (Fawley et al., 2000; Guillou et al., 2004; Marin & Melkonian, 2010). Chloroplast genomic data and increased taxon sampling of prasinophytes have greatly improved our understanding of early chlorophytan relationships (Turmel et al., 2009a; Lemieux et al., 2014a; Leliaert et al., 2016). Current understanding of phylogenetic relationships and uncertainties is summarized in Fig. 2. Although these recent chloroplast phylogenomic analyses provided high statistical support for J. Syst. Evol. 55 (4): , 2017

3 324 Fang et al. Core Chlorophyta Prasinophyceae sp. CCMP 1205 Picocystis salinarum Pycnococcus provasolii Nephroselmis astigmatica Nephroselmis olivacea Micromonas sp. RCC 299 Ostreococcus tauri Monomastix sp. OKE-1 Pyramimonas parkeae Prasinophyte clade VIIA Prasinophyte clade VIIC Pycnococcaceae (clade V) Nephroselmidophyceae (clade III) Mamiellophyceae (clade II) Pyramiminadales (clade I) Verdigellas peltata Prasinococcus capsulatus Prasinococcus sp. CCMP 1194 Prasinoderma coloniale Prasinophyceae sp. MBIC Palmophyllales Prasinococcales (clade VI) Palmophyllophyceae Fig. 2. Phylogeny of prasinophytes inferred from chloroplast genomic data. Dashed and solid lines indicate uncertain and established relationships, respectively. many branches, the phylogenetic positions of a number of lineages remain uncertain because of low support and/or contradicting relationships in different analyses. In general, the Prasinococcales (clade VI) together with the Palmophyllales (Palmophyllophyceae) occupied the deepest branch of the prasinophytes, and the Pyramimonadales (clade I) þ Mamiellophyceae (clade II) formed the second-deepest clade. The more recently diverging groups included the Nephroselmidophyceae (clade III), Pycnococcaceae (clade V) and a number of other lineages (clades VIIA and VIIC) (Fig. 2). The Pycnococcaceae (clade V) diverged before Prasinophyceae sp. CCMP 1205 (clade VIIA) and Picocystis (clade VIIC) (Lemieux et al., 2014a, 2014b; Turmel et al., 2016). The position of the Nephroselmidophyceae (clade III) is unstable Several chloroplast genomic datasets supported that the Nephroselmidophyceae (clade III) emerged after the Pycnococcaceae (clade V) (Lemieux et al., 2014a, 2014b; Leliaert et al., 2016). However, Turmel et al. (2016), using the chloroplast nucleotide datasets, supported a sister relationship between the Nephroselmidophyceae (clade III) and the Pycnococcaceae (clade V). It is likely that the diversity of prasinophytes is still underestimated, and that deep branching lineages remain to be discovered. For example, the Palmophyllales, a group of macroscopic algae living in dimly lit habitats, have only recently been recognized as an early diverging lineage of prasinophytes, affiliated to the Prasinococcales (Leliaert et al., 2016). In addition, 18S environmental sequencing of photosynthetic picoeukaryotic communities has identified prasinophyte lineages with uncertain affinities (Viprey et al., 2008; Lepere et al., 2009; Shi et al., 2009). Elucidating the phylogenetic affinities of these (and yet to be discovered) lineages may provide new insights into the early evolution of the Chlorophyta. 2.2 Phylogenetic relationships of core Chlorophyta The core Chlorophyta comprises the species-rich classes, Chlorophyceae, Ulvophyceae, and Trebouxiophyceae, and two smaller classes, Pedinophyceae and Chlorodendrophyceae (Fig. 1). Chloroplast phylogenomic analyses are concordant with nuclear ribosomal DNA data in recovering the class Chlorophyceae as a monophyletic group with strong support. Nonmonophyly of the Trebouxiophyceae was already suggested based on 18S data (Krienitz et al., 2010; Luo et al., 2010) and this is now confirmed by chloroplast genomic data (Lemieux et al., 2014b). Although 18S data was unable to resolve relationships among the main trebouxiophycean lineages, chloroplast genomic data recovered two well-supported clades: the Chlorellales, and the remainder of the trebouxiophycean lineages (termed core Trebouxiophyceae). The Chlorellales were resolved near the Pedinophyceae, whereas the core Trebouxiophyceae were related to the Chlorophyceae and Ulvophyceae (Lemieux et al., 2014a, 2014b, 2015; Melton et al., 2015) (Fig. 1). Monophyly of the Ulvophyceae has long been questionable because of the lack of synapomorphic characters. Although most 18S studies were unable to provide strong support for an Ulvophyceae clade, a phylogeny inferred from eight nuclear and two plastid genes recovered the class as monophyletic with high support and resolved many of the early divergences within the clade (Cocquyt et al., 2010). As will be discussed below, chloroplast phylogenomic data are contesting the J. Syst. Evol. 55 (4): ,

4 Chloroplast phylogenomics of the Chlorophyta 325 monophyly of the Ulvophyceae class (Fucıkova et al., 2014; Leliaert & Lopez-Bautista, 2015; Melton et al., 2015). The phylogenetic position of the classes Pedinophyceae and Chlorodendrophyceae has been unstable in previous phylogenetic analyses. Initial chloroplast phylogenomic studies allied the Pedinophyceae with the Chlorellales (Turmel et al., 2009b). In subsequent studies with richer taxon sampling, the Pedinophyceae was inferred as the earliest diverging lineage of the core Chlorophyta (Fucıkova et al., 2014; Melton et al., 2015), or as a sister group with the order Chlorellales (Trebouxiophyceae) (Lemieux et al., 2014a, 2014b, 2015; Leliaert & Lopez-Bautista, 2015; Leliaert et al., 2016). The relationship supporting Pedinophyceae close to Chlorellales, however, may be an artificial result because of the similar compositional bias and low taxon sampling of the two groups (Turmel et al., 2016). The Chlorodendrophyceae have been inferred as an early diverging clade of the core Chlorophyta, but the precise phylogenetic position remains ambiguous (Marin, 2012; Leliaert & Lopez-Bautista, 2015; Melton et al., 2015). Even in the most recent chloroplast phylogenomic analysis with rich taxon sampling, the phylogenetic position of the Chlorodendrophyceae was unstable and received low support in analyses using different datasets and methods (Turmel et al., 2016). 2.3 Phylogenetic relationships within the Chlorophyceae Molecular and ultrastructural (mainly orientation of the flagellar root system) data have identified five major orders of Chlorophyceae: Chlamydomonadales, Sphaeropleales, Chaetophorales, Chaetopeltidales, and Oedogoniales (Buchheim et al., 2002; Wolf et al., 2002; Turmel et al., 2008; Brouard et al., 2010) (Fig. 3). The phylogenetic relationship among the five main orders has proven difficult to resolve based on single-gene analyses. Phylogenetic analyses of 18S rdna data have suggested a sister relationship between the Chlamydomonadales and Sphaeropleales, with the Oedogoniales, Chaetophorales, Chaetopeltidales (OCC clade) forming early diverging clades of Chlorophyceae but with uncertain interrelationships (Booton et al., 1998; Shoup & Lewis, 2003; M uller et al., 2004; Brouard et al., 2010). These interrelationships were also difficult to resolve using chloroplast genomic data with limited taxon sampling (Turmel et al., 2008). Recent chloroplast phylogenomic analyses with richer taxon sampling recovered that the Chlamydomonadales were sister to the Sphaeropleales with strong support and the Chlamydomonadales Sphaeropleales clade was allied with the OCC clade (Lemieux et al., 2015; Fucıkova et al., 2016; Turmel et al., 2016). An intriguing phylogenetic result was that a new lineage was recently recognized consisting of the genera Jenufa, Golenkinia, and Treubarinia. This group was inferred as sister to the Sphaeropleales, but the affinity of Treubarinia within this lineage was not fully supported (Lemieux et al., 2015) (Fig. 3). The Chlamydomonadales are the most species-rich order of the Chlorophyceae and include a wide diversity of flagellate and non-flagellate unicellular algae, colonies, and filaments. Phylogenetic diversity and relationships within the order have been mainly investigated based on 18S data (Nakada et al., 2008). Several members of the order are important model systems for understanding the evolution of multicellularity and cellular differentiation, and the complete genomes of two species of the Chlamydomonadales: the unicellular Chlamydomonas reinhardtii P. A. Dangeard and colonial Volvox carteri F. Stein have been sequenced (Merchant et al., 2007; Prochnik et al., 2010). A solid phylogenetic framework is thus important for interpreting genomic characters in the light of evolution of multicellularity. Taxon sampling in chloroplast phylogenomic studies is still relatively low compared with the vast diversity of the group, but these analyses show great promise in resolving the relationships within the Chlamydomonadales. Chlamydomonas is a species-rich and non-monophyletic genus (at least 450 species) with various cell and chloroplast shapes, and pyrenoid positions and numbers (Buchheim et al., 1996; Nakada et al., 2008; Nakada & Tomita, 2011). In the analyses of 18S and 26S rdna with dense taxon sampling (more than 40 Chlamydomonas species), 21 strongly supported main clades of Chlamydomonadales were recovered, with Chlamydomonas appearing in nine of these clades (Nakada et al., 2008). Recent chloroplast phylogenomic analyses (including four Chlamydomonas species) showed that Chlamydomonas species were distributed in the Caudivolvoxa, Xenovolvoxa, and Reinhardtinia clades (Lemieux et al., 2015). The relationships of the early diverging chlamydomonadalean lineages (Crucicarteria, Hafniomonas, Radicarteria, and Chloromonadinia) inferred from chloroplast genomic data were not well supported, but largely congruent with earlier three-gene (18S rdna, atpb, and psab) phylogenies (Matsuzaki et al., 2010; Nozaki et al., 2010). 2.4 Phylogenetic relationships among the main clades of Ulvophyceae The evolution of the Ulvophyceae is interesting from a morphological and ecological point of view. Morphologically, the class is highly diverse and comprises a wide range of thallus organizations and cytological types. Thallus architectures range from microscopic unicellular thalli to macroscopic multicellular or siphonous thalli (Leliaert et al., 2015). Cellular organization is equally diverse, ranging from cells with a single nucleus and chloroplast, to multinucleate cells with regularly spaced nuclei, and no cytoplasmic streaming (siphonocladous cell types) to giant tubular cells with thousands of nuclei and chloroplasts that are transported throughout the siphon by cytoplasmic streaming (siphonous cell type) (McNaughton & Goff, 1990; Nakayama et al., 1996; Vroom & Smith, 2001; Friedl & O Kelly, 2002; Watanabe & Nakayama, 2007). A 10-gene (eight nuclear and two plastid) phylogenetic analysis provided a relatively robust phylogenetic framework for interpreting morphological and cytological evolution in this group (Cocquyt et al., 2010). The ancestral ulvophyte was likely a unicellular uninucleate organism and descendant lineages may have evolved into multicellular, siphonocladous, and siphonous types independently. Importantly, Cocquyt et al. (2010) suggested a monophyletic Ulvophyceae. However, recent chloroplast phylogenomic analyses have shown significant conflict with the study of Cocquyt et al. (2010), indicating possible nonmonophyly of the Ulvophyceae, thus potentially requiring a reevaluation of the interpretation of cytomorphological evolution in this group. A second important evolutionary aspect in the Ulvophyceae is the apparent multiple transitions between marine, freshwater, and terrestrial habitats. An interesting clade in this context is the Trentepohliales, an ultrastructurally atypical clade that is entirely restricted to terrestrial habitats J. Syst. Evol. 55 (4): , 2017

5 326 Fang et al. Caudivolvoxa Xenovolvoxa Reinhardtinia Oogamochlamydinia Chloromonadinia Radicarteria Crucicarteria Chlamydomonadales Hafniomonas Sphaeropleales Chlorophyceae Jenufa Golenkinia Treubarinia Chaetophorales Chaetopeltidales Oedogoniales Fig. 3. Phylogeny of Chlorophyceae inferred from chloroplast genomic data. Dashed and solid lines indicate uncertain and established relationships, respectively. (Chapman et al., 2001). A phylogenetic position of the Trentepohliales embedded in marine clades would suggest an evolutionary sea-to-land transition (Cocquyt et al., 2010). However, the phylogenetic position of the Trentepohliales in chloroplast phylogenomic data is uncertain possibly due to long branch attraction artifact (Felsenstein, 1978; Fucıkova et al., 2014). Although the phylogenetic relationships among the main clades of Ulvophyceae were incongruent among different phylogenetic analyses, some common patterns have emerged. The two main clades of Ulvophyceae: the Oltmannsiellopsidales þ Ulvales Ulotrichales clade, and a clade consisting of the Trentepohliales, Cladophorales, Bryopsidales, and Dasycladales (TCBD clade) have been recovered in phylogenetic analyses inferred from nuclear and chloroplast genes (Cocquyt et al., 2010; Skaloud et al., 2013) (Fig. 4). In contrast to the study of Cocquyt et al. (2010), in which the relationships within the TCBD clade were resolved with moderate support, chloroplast phylogenomic analyses have, until now, failed to recover well-resolved relationships among these clades. For example, the sister relationship between Dasycladales and Trentepohliales was recovered in some chloroplast phylogenomic analyses (Fucıkova et al., 2014; Melton et al., 2015), whereas another analysis weakly supported the Dasycladales close to the Oltmannsiellopsidales þ Ulvales Ulotrichales clade (Leliaert & Lopez-Bautista, 2015). The phylogenetic position of Bryopsidales was also uncertain, either supported as the sister group to the Chlorophyceae (Smith et al., 2011; Leliaert & Lopez-Bautista, 2015; Leliaert et al., 2016), or the Bryopsidales þ Dasycladales þ Trentepohliales clade close to the core Trebouxiophyceae (Fucıkova et al., 2014). Interestingly, the Cladophorales, which include a wide diversity of species from marine and freshwater habitats, have until now not been included in chloroplast phylogenetic analyses (Fucıkova et al., 2014; Boedeker et al., 2016). In general, chloroplast gene data from this group is largely lacking from public DNA sequence databases. The only trustworthy rbcl sequence published thus far is highly divergent from those of other green algae, hampering accurate reconstruction of the phylogenetic position of the Cladophorales based on this gene (Deng et al., 2014). Although the chloroplast genome in Cladophorales remains elusive, La Claire et al. (1998) suggested that abundant plasmid-like DNA molecules, which have been found in several species of Cladophorales, might have originated from chloroplast DNA. Other smaller clades in the Ulvophyceae, such as the Scotinosphaerales, Ignatius, and Blastophysa clades (Cocquyt et al., 2010; Skaloud et al., 2013), and denser sampling in the main ulvophyte clades, may yield important insights into the phylogenetic relationships of the Ulvophyceae; however, their chloroplast genomes have not been sequenced. 2.5 Phylogenetic relationships among the main clades of Trebouxiophyceae The Trebouxiophyceae include flagellate and non-flagellate unicellular green algae, as well as some colonial and multicellular species, from freshwater to terrestrial habitats, with some members penetrating in brackish and even marine J. Syst. Evol. 55 (4): ,

6 Chloroplast phylogenomics of the Chlorophyta 327 Chlorophyceae Bryopsidales Cladophorales Dasycladales Trentepohliales TCBD clade Scotinosphaerales Ignatius clade Blastophysa clade Ulvophyceae Ulotrichales Ulvales Oltmannsiellopsidales Trebouxiophyceae Fig. 4. Phylogeny of Ulvophyceae inferred from chloroplast genomic data. Dashed and solid lines indicate uncertain and established relationships, respectively. waters. Several species are known as symbionts with a diverse array of eukaryotes, most notably with fungi to form lichens, and some species are heterotrophic free-living or parasitic (Leliaert et al., 2012). A reliable phylogenetic tree would enhance our understanding of morphological and ecological evolution in this group, and in particular, provide clues about the origins of symbiotic lifestyles. Previous studies based on 18S sequence data were not able to strongly support monophyly of the Trebouxiophyceae, and the relationships among the main clades have remained largely unresolved (Darienko et al., 2010; Krienitz et al., 2010; Neustupa et al., 2011, 2013; Bock et al., 2013). Chloroplast genomic data partially resolved the relationships among the main trebouxiophyte lineages, and additional taxon sampling will likely improve our understanding of phylogenetic diversity and relationships within the group. Phylogenies based on 18S have identified multiple clades within the Trebouxiophyceae, including the Chlorellales, Geminella, Oocystis, Prasiola, Microthamniales, Trebouxiales, Watanabea, Choricystis, and Ellipotochloris clades, as well as several other lineages consisting of a single species or genus (e.g., Pleurastrosarcina, Xylochloris, and Lobosphaera). The relationship among these clades, however, was largely unresolved based on 18S data (Karsten et al., 2005; Elias et al., 2008; Darienko et al., 2010; Neustupa et al., 2011, 2013; Bock et al., 2013). Chloroplast phylogenomic data and increased taxon sampling have significantly improved support for trebouxiophycean relationships (Lemieux et al., 2014b). The Trebouxiophyceae were recovered as a non-monophyletic assemblage including two unrelated clades, the Chlorellales and core Trebouxiophyceae (Fucıkova et al., 2014; Lemieux et al., 2014b) (Fig. 5). Three distinct clades of the Chlorellales were discovered using 79 chloroplast genes and 18S rdna: the Parachlorella, Chlorella, and Marvania clades (Somogyi et al., 2011; Lemieux et al., 2014b). However, the phylogenetic relationships among the three clades are not clear (Lemieux et al., 2014b, 2015; Turmel et al., 2016). As for the relationship within core Trebouxiophyceae, the Oocystis is close to the Geminella clade, and Neocystis was recovered as a sister relationship with the Prasiola clade (Lemieux et al., 2014b, 2015; Turmel et al., 2016). The phylogenetic relationships of other remaining clades (e.g., Xylochloris, Microthamniales, Trebouxiales, Lobosphaera, Watanabea, Choricystis, and Ellipotochloris) were basically established as shown in Fig. 5 (Lemieux et al., 2014b, 2015; Turmel et al., 2016). The phylogenetic position of the Parietochloris clade was ambiguous. It was either sister to the Prasiola Neocystis clade based on chloroplast nucleotide data or close to the monophyletic lineage including the Ellipotochloris þ Choricystis þ Watanabea þ Lobosphaera þ Trebouxiales þ Microthamniales þ Xylochloris clades using the same amino acid data (Lemieux et al., 2014b; Turmel et al., 2016). The possible factors causing conflicting phylogenetic signals between nucleotide and amino acid data may be substitutional saturation (Jeffroy et al., 2006; Philippe et al., 2011), among-lineage compositional heterogeneity (Foster, 2004; Inagaki & Roger, 2006; Rota- Stabelli et al., 2013), or codon-usage bias (Gouy & Gautier, 1982; Stenøien, 2005). Approaches to mitigate these problems will be discussed in the next section. 3 The Promise of Applying Realistic Evolutionary Models for Resolving Ancient Relationships The Chlorophyta likely originated in the Neoproterozoic ( Mya), and such ancient phylogenetic relationships are difficult to resolve. The Markov substitution models are J. Syst. Evol. 55 (4): , 2017

7 328 Fang et al. Chlorophyceae Ulvophyceae Elliptochloris clade Choricystis clade Watanabea clade Lobosphaera clade Trebouxiales Microthamniales clade Xylochloris clade Core Trebouxiophyceae Parietochloris clade Prasiola clade Neocystis Pleurastrosarcina clade Oocystis clade Geminella clade Chlorella clade Marvania clade Chlorellales Parachlorella clade Fig. 5. Phylogeny of Trebouxiophyceae inferred from chloroplast genomic data. Dashed and solid lines indicate uncertain and established relationships, respectively. commonly used to describe substitution processes that generate DNA and protein sequences in phylogenetic inference, but the Markov models are mathematically expected to lose information at the ancient divergences (Mossel & Steel, 2004). Ancient branches tend to be long because of multiple substitutions, which Markov substitution models have difficulty accurately describing. Inaccurate phylogenetic relationships are often inferred when the nucleotide or amino acid data poorly fit to the substitution model (Philippe et al., 2011; Whelan et al., 2015). There are several reasons causing poor fit to the model and a number of approaches can improve the goodness of fit between the model and data. 3.1 Fast substitution rate of sequence evolution Fast substitution rate could cause substitutional saturation. Saturation is a function of time and mutation rate that has been shown to affect reconstruction of ancient phylogenetic relationships of animals and land plants (e.g., Philippe et al., 2011; Liu et al., 2014). The rate of substitutional saturation at the third codon position is typically higher than at the first and/ or second codon positions (Cox et al., 2014; Liu et al., 2014). In order to reduce the influence of substitution saturation, a common approach is to remove the third codon positions from protein-coding genes. However, some third codon positions are conserved, while some first and second codon positions may be fast-evolving (Goremykin et al., 2009; Cocquyt et al., 2010). Several fast-site removal approaches have been suggested to reduce the impact of saturation in plant phylogenomic studies, such as resolving the root of flowering plants (Goremykin et al., 2013), the phylogenetic position of Gnetales (Zhong et al., 2011), and the Chlorophyta phylogeny (Fucıkova et al., 2014). These analyses showed that removing the fastest-evolving sites dramatically improves the accuracy of ancient phylogenetic relationships. 3.2 Codon-usage bias Lineage-specific codon-usage biases have been observed in many groups (Liu et al., 2004; Wang et al., 2010; Plotkin & Kudla, 2011), and have important effects on phylogenetic inferences (Foster, 2004; Inagaki & Roger, 2006; Regier et al., 2010). Nucleotide ambiguous synonymous codon families are often used to undertake codon-degenerated phylogenetic analyses. For example, the synonymous codons AAA and AAG encoding lysine were recoded as AAR; similarly, six synonymous codons for serine (i.e., TCT, TCC, TCA, TCG, AGT, and AGC) were all replaced with WSN (e.g., Cox et al., 2014; Liu et al., 2014; Fucıkova et al., 2016). Phylogenetic inference using J. Syst. Evol. 55 (4): ,

8 Chloroplast phylogenomics of the Chlorophyta 329 nucleotide sequences with reduced ambiguous codons has been found congruent with analysis of amino acid data in dinoflagellates (Inagaki & Roger, 2006), early diverging land plants (Liu et al., 2014), arthropods (Regier et al., 2010), and Pancrustacea (Rota-Stabelli et al., 2013). Nucleotide sequences evolve relatively faster than protein sequences in ancient lineages, so amino acid data can be considered as less affected by compositional biases driven from sequence convergence or similar mutation (Cox et al., 2014). 3.3 Compositional heterogeneity Among-site and among-branch compositional heterogeneity are important factors for causing poor fitness between the data and model, as it violates the assumption of stationarity over time for commonly used substitution models (Foster, 2004). Liu et al. (2014) reported that in the mitochondrial protein-coding genes, the GC content between vascular and non-vascular plants are significantly different, and the nonstationary composition model (e.g., NDCH model) can partially resolve the incongruence of inferences from stationary composition models between nucleotide and amino acid datasets. Sun et al. (2016) investigated chloroplast genomic data from 53 green algae showing strong among-branch and site compositional heterogeneity, and showed that applying a site-heterogeneous evolutionary model (CAT model) can reduce the effect of compositional heterogeneity. More importantly, they reported that fast evolutionary rates are positively correlated with strong among-site compositional heterogeneity, and the fastevolving sites also have poor fit to the evolutionary models, which leads towards the model misspecification problem. This correlation will help to evaluate and identify the potential errors for phylogenomic analyses of green algae relationships. 4 Perspectives Chloroplast genome data have greatly advanced our understanding of green algae evolutionary history. There are 20 complete chloroplast genomes of charophyte green algae published so far, but more than 100 complete chloroplast genomes of Chlorophyta have been sequenced (Table S1). These data have been shown to be valuable in resolving relationships among lower taxonomic ranks (families, genera, and species), but their use to resolve higher taxonomic levels (class and order) of Chlorophyta has been less explored. At lower taxonomic levels, the factors affecting accuracy of phylogenetic reconstruction would be substantially reduced (particularly substitutional saturation and compositional bias), but will be exacerbated for higher taxonomic levels. These problems are the barriers for resolving ancient phylogenetic relationships. The current low statistical support of some relationships and conflicting phylogenetic hypotheses from various studies impede our understanding of ancient green algae evolution. Applying better-fitting (more realistic) models that incorporate heterogeneity of the substitution process, and removing/recording data associated with fast evolutionary rate and compositional heterogeneity will have better fitness between the evolutionary model and data, and further improve the accuracy of phylogenetic inference. Increasing taxon sampling can also increase the accuracy of phylogenetic inference. Sampling more taxa can break up long branches to alleviate the long-branch attraction artifact (Hillis, 1998), and can improve the accuracy of parameter estimation for model-based phylogenetic approaches (Sullivan et al., 1999; Pollock & Bruno, 2000). New linages of green algae are still regularly being discovered (e.g., Caisova et al., 2015; Watanabe et al., 2016). These discoveries are often fortuitous, so it is difficult to foresee which critical taxa are still missing for the reconstruction of a comprehensive Chlorophyta phylogeny. With the increase of chloroplast genomic data, the better fitting site-heterogeneous model (CAT model of Phylobayes; Lartillot et al., 2009) and branch-heterogeneous models (NDCH model of p4 (Foster, 2004) and PhyloCTMC module of RevBayes (H ohna et al., 2014)) will dramatically improve the phylogenetic resolution and accuracy in deeplevel phylogenetic analyses. Using more genome-scale data and better-fitting heterogeneous evolutionary models, the phylogenetic errors can be comprehensively mitigated, and the phylogenetic relationships of Chlorophyta will be further updated and clarified. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant No ), the Natural Science Foundation of Jiangsu Province (BK ), the Jiangsu Province Key Project for Scientific Research (16KJA180002), the Six Talent Peaks Project of Jiangsu Province (2016-XNY-035), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Qing Lan Project. References Arora M, Anil AC, Leliaert F, Mesbahi JDE Tetraselmis indica (Chlorodendrophyceae, Chlorophyta), a new species isolated from salt pans in Goa, India. European Journal of Phycology 48: Bock C, Luo W, Kusber WH, Hegewald E, Pazoutova M, Krienitz L Classification of crucigenoid algae: Phylogenetic position of the reinstated genus Lemmermannia, Tetrastrum spp. Crucigenia tetrapedia, and C. lauterbornii (Trebouxiophyceae, Chlorophyta). Journal of Phycology 49: Boedeker C, Leliaert F, Zuccarello GC Molecular phylogeny of the Cladophoraceae (Cladophorales, Ulvophyceae), with the resurrection of Acrocladus N ageli and Willeella Børgesen, and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov. Journal of Phycology 52: Booton GC, Floyd GL, Fuerst PA Origins and affinities of the filamentous green algal orders Chaetophorales and Oedogoniales based on 18S rrna gene sequences. Journal of Phycology 34: Brouard JS, Otis C, Lemieux C, Turmel M The exceptionally large chloroplast genome of the green alga Floydiella terrestris illuminates the evolutionary history of the Chlorophyceae. Genome Biology and Evolution 2: Buchheim MA, Lemieux C, Otis C, Gutell RR, Chapman RL, Turmel M Phylogeny of the Chlamydomonadales (Chlorophyceae): A comparison of ribosomal RNA gene sequences from the nucleus J. Syst. Evol. 55 (4): , 2017

9 330 Fang et al. and the chloroplast. Molecular Phylogenetics and Evolution 5: Buchheim MA, Michalopulos EA, Buchheim JA Phylogeny of the Chlorophyceae with special reference to the Sphaeropleales: A study of 18S and 26S rdna data. Journal of Phycology 37: Caisova L, Perez CP, Alamo VC, Quintana AM, Surek B, Melkonian M Barrancaceae: A new greeen algal lineage with structural and behavioral adaptations to a fluctuating environment. American Journal of Botany 102: Chapman RL, Borkhsenious O, Brown RC, Henk MC, Waters DA Phragmoplast-mediated cytokinesis in Trentepohlia: Results of TEM and immunofluorescence cytochemistry. International Journal of Systematic and Evolutionary Microbiology 51: Cocquyt E, Verbruggen H, Leliaert F, De Clerck O Evolution and cytological diversification of the green seaweeds (Ulvophyceae). Molecular Biology and Evolution 27: Cooper ED Overly simplistic substitution models obscure green plant phylogeny. Trends in Plant Science 19: Cox CJ, Li B, Foster PG, Embley TM, Civan P Conflicting phylogenies for early land plants are caused by composition biases among synonymous substitutions. Systematic Biology 63: Darienko T, Gustavs L, Mudimu O, Menendez CR, Schumann R, Karsten U, Friedl T, Pr oschold T Chloroidium, a common terrestrial coccoid green alga previously assigned to Chlorella (Trebouxiophyceae, Chlorophyta). European Journal of Phycology 45: Delwiche CF, Cooper ED The evolutionary origin of a terrestrial flora. Current Biology 25: Deng Y, Zhan Z, Tang X, Ding L, Duan D Molecular cloning and expression analysis of RbcL cdna from the bloom-forming green alga Chaetomorpha valida (Cladophorales, Chlorophyta). Journal of Applied Phycology 26: 1 9. Elias M, Neustupa J, Skalou P Elliptochloris bilobata var. corticola var. nov. (Trebouxiophyceae, Chlorophyta), a novel subaerial coccal green alga. Biologia 63: Fawley MW, Yun Y, Qin M Phylogenetic analyses of 18s rdna sequences reveal a new coccoid lineage of the prasinophyceae (Chlorophyta). Journal of Phycology 36: Felsenstein J Cases in which parsimony or compatibility methods will be positively misleading. Systematic Zoology 27: Foster PG Modeling compositional heterogeneity. Systematic Biology 53: Friedl T, O Kelly CJ Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): Evidence from 18S rdna sequence data and ultrastructure. European Journal of Phycology 37: Fucıkova K, Leliaert F, Cooper ED, Skaloud P, D hondt S, De Clerck O, Gurgel CFD, Lewis LA, Lewis PO, Lopez-Bautista JM New phylogenetic hypotheses for the core Chlorophyta based on chloroplast sequence data. Frontiers in Ecology and Evolution 2: 63. Fucıkova K, Lewis PO, Lewis LA Chloroplast phylogenomic data from the green algal order Sphaeropleales (Chlorophyceae, Chlorophyta) reveal complex patterns of sequence evolution. Molecular Phylogenetics and Evolution 98: Goremykin VV, Nikiforova SV, Biggs PJ, Zhong B, DeLange P, Martin W, Woetzel S, Atherton RA, McLenachan PA, Lockhart PJ The evolutionary root of flowering plants. Systematic Biology 62: Goremykin VV, Viola R, Hellwig FH Removal of noisy characters from chloroplast genome-scale data suggests revision of phylogenetic placements of Amborella and Ceratophyllum. Journal of Molecular Evolution 68: Gouy M, Gautier C Codon usage in bacteria: Correlation with gene expressivity. Nucleic Acids Research 10: Graham LE, Graham JM, Wilcox LW Algae. 2nd ed. San Francisco: Pearson Benjamin Cummings. Guillou L, Eikrem W, Chretiennot-Dinet MJ, Gall FL, Massana R, Romari K, Pedros-Alio C, Vaulot D Diversity of picoplanktonic Prasinophytes assessed by direct nuclear SSU rdna sequencing of environmental samples and novel isolates retrieved from oceanic and coastal marine ecosystems. Protist 155: Hillis DM Taxonomic sampling phylogenetic accuracy and investigator bias. Systematic Biology 47: 3 8. H ohna S, Heath TA, Boussau B, Landis MJ, Ronquist F, Huelsenbeck JP Probabilistic graphical model representation in phylogenetics. Systematic Biology 63: Inagaki Y, Roger AJ Phylogenetic estimation under codon models can be biased by codon usage heterogeneity. Molecular Phylogenetics and Evolution 40: Jeffroy O, Brinkmann H, Delsuc F, Philippe H Phylogenomics: The beginning of incongruence? Trends in Genetics 22: Kantz TS, Theriot EC, Zimmer EA, Chapman RL The Pleurastrophyceae and Micromonadophyceae: A cladistic analysis of nuclear rrna sequence data. Journal of Phycology 26: Karsten U, Friedl T, Schumann R, Hoyer K, Lembcke S Mycosporine-like amino acids and phylogenies in green algae: Prasiola and its relatives from the Trebouxiophyceae (Chlorophyta). Journal of Phycology 41: Krienitz L, Bock C, Luo W, Pr oschold T Polyphyletic origin of the Dictyosphaerium morphotype within Chlorellaceae (Trebouxiophyceae). Journal of Phycology 46: Krienitz L, Ustinova I, Friedl T, Huss VAR Traditional generic concepts versus 18S rrna gene phylogeny in the green algal family Selenastraceae (Chlorophyceae, Chlorophyta). Journal of Phycology 37: La Claire JW, Loudenslager CM, Zuccarello GC Characterization of novel extrachromosomal DNA from giant celled marine green algae. Current Genetics 34: Lartillot N, Lepage T, Blanquart S PhyloBayes 3: A Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics 25: Leliaert F, Lopez-Bautista JM The chloroplast genomes of Bryopsis plumosa and Tydemania expeditiones (Bryopsidales, Chlorophyta): Compact genomes and genes of bacterial origin. BMC Genomics 16: 204. Leliaert F, Lopez-Bautista JM, De Clerck O Class Ulvophyceae K.R. Mattox & K.D. Stewart. In: Frey W ed. Syllabus of plant families: A Engler s Syllabus der Pflanzenfamilien part 2/1: photoautotrophic eukaryotic algae. Stuttgart: Schweizerbart Leliaert F, Smith DR, Moreau H, Herron MD, Verbruggen H, Delwiche CF, De Clerck O Phylogeny and molecular evolution of the green algae. Critical Reviews in Plant Sciences 31: Leliaert F, Tronholm A, Lemieux C, Turmel M, De Priest MS, Bhattacharya D, Karol KG, Fredericq S, Zechman FW, Lopez- Bautista JM Chloroplast phylogenomic analyses reveal the deepest-branching lineage of the Chlorophyta, Palmophyllophyceae class. nov. Scientific Reports 6: Leliaert F, Verbruggen H, Zechman FW Into the deep: new discoveries at the base of the green plant phylogeny. BioEssays 33: J. Syst. Evol. 55 (4): ,

10 Chloroplast phylogenomics of the Chlorophyta 331 Lemieux C, Otis C, Turmel M. 2014a. Six newly sequenced chloroplast genomes from Prasinophyte green algae provide insights into the relationships among Prasinophyte lineages and the diversity of streamlined genome architecture in picoplanktonic species. BMC Genomics 15: 857. Lemieux C, Otis C, Turmel M. 2014b. Chloroplast phylogenomic analysis resolves deep-level relationships within the green algal class Trebouxiophyceae. BMC Evolutionary Biology 14: 211. Lemieux C, Vincent AT, Labarre A, Otis C, Turmel M Chloroplast phylogenomic analysis of Chlorophyte green algae identifies a novel lineage sister to the Sphaeropleales (Chlorophyceae). BMC Evolutionary Biology 15: 264. Lepere C, Vaulot D, Scanlan DJ Photosynthetic picoeukaryote community structure in the South East Pacific Ocean encompassing the most oligotrophic waters on Earth. Environmental Microbiology 11: Lewis LA, McCourt RM Green algae and the origin of land plants. American Journal of Botany 91: Liu Q, Feng Y, Xue Q Analysis of factors shaping codon usage in the mitochondrion genome of Oryza sativa. Mitochondrion 4: Liu Y, Cox CJ, Wang W, Goffinet B Mitochondrial phylogenomics of early land plants: Mitigating the effects of saturation compositional heterogeneity and codon-usage bias. Systematic Biology 63: Lu JM, Zhang N, Du, XY, Wen J, Li DZ Chloroplast phylogenomics resolves key relationships in ferns. Journal of Systematics and Evolution 53: Luo W, Pr oschold T, Bock C, Krienitz L Generic concept in Chlorella-related coccoid green algae (Chlorophyta, Trebouxiophyceae). Plant Biology 12: Marin B Nested in the Chlorellales or independent class? Phylogeny and classification of the Pedinophyceae (Viridiplantae) revealed by molecular phylogenetic analyses of complete nuclear and plastid-encoded rrna operons. Protist 163: Marin B, Melkonian M Molecular phylogeny and classification of the Mamiellophyceae class. nov. (Chlorophyta) based on sequence comparisons of the nuclear- and plastid-encoded rrna operons. Protist 161: Matsuzaki R, Nakada T, Hara Y, Nozaki H Light and electron microscopy and molecular phylogenetic analyses of Chloromonas pseudoplatyrhyncha (Volvocales, Chlorophyceae). Phycological Research 58: Mattox KR, Stewart KD Classification of the green algae: A concept based on comparative cytology. In: Irvine DEG, John DM eds. Systematics of the Green Algae. London: Academic Press McNaughton EE, Goff LJ The role of microtubules in establishing nuclear spatial patterns in multinucleate green algae. Protoplasma 157: Melton JT 3rd, Leliaert F, Tronholm A, Lopez-Bautista JM The complete chloroplast and mitochondrial genomes of the green macroalga Ulva sp. UNA (Ulvophyceae, Chlorophyta). PLoS ONE 10: e Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritzlaylin LK, Marechaldrouard L The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: Moczydłowska M, Landing E, Zang W, Palacios T Proterozoic phytoplankton and timing of Chlorophyte algae origins. Palaeontology 54: Mossel E, Steel M A phase transition for a random cluster model on phylogenetic trees. Mathematical Biosciences 187: M uller T, Rahmann S, Dandekar T, Wolf M Accurate and robust phylogeny estimation based on profile distances: A study of the Chlorophyceae (Chlorophyta). BMC Evolutionary Biology 4: 20. Nakada T, Misawa K, Nozaki H Molecular systematics of Volvocales (Chlorophyceae, Chlorophyta) based on exhaustive 18S rrna phylogenetic analyses. Molecular Phylogenetics and Evolution 48: Nakada T, Tomita M Chlamydomonas neoplanoconvexa nom. nov. and its unique phylogenetic position within Volvocales (Chlorophyceae). Phycological Research 59: Nakayama T, Watanabe S, Inouye I Phylogeny of wall-less green flagellates inferred from 18S rdna sequence data. Phycological Research 44: Neustupa J, Elias M, Skaloud P, Nemcova Y, Sejnohova L Xylochloris irregularis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel subaerial coccoid green alga. Phycologia 50: Neustupa J, Nemcova Y, Vesela J, Steinova JPS Leptochlorella corticola gen. et sp. nov. and Kalinella apyrenoidosa sp. nov.: Two novel Chlorella-like green microalgae (Trebouxiophyceae, Chlorophyta) from subaerial habitats. International Journal of Systematic and Evolutionary Microbiology 63: Norris RE, Hori T, Chihara M Revision of the genus Tetraselmis (Class Prasinophyceae). Journal of Plant Research 93: Nozaki H, Nakada T, Watanabe S Evolutionary origin of Gloeomonas (Volvocales, Chlorophyceae), based on ultrastructure of chloroplasts and molecular phylogeny. Journal of Phycology 46: Philippe H, Brinkmann H, Lavrov DV, Littlewood DT, Manuel M, W orheide G, Baurain D Resolving difficult phylogenetic questions: Why more sequences are not enough. PLoS Biology 9: e Philippe H, Telford MJ Large-scale sequencing and the new animal phylogeny. Trends in Ecology and Evolution 21: Plotkin JB, Kudla G Synonymous but not the same: The causes and consequences of codon bias. Nature Reviews Genetics 12: Pollock DD, Bruno WJ Assessing an unknown evolutionary process: Effect of increasing site-specific knowledge through taxon addition. Molecular Biology and Evolution 17: Prochnik SE, Umen J, Nedelcu AM, Hallmann A, Miller SM, Nishii I, Ferris P, Kuo A, Mitros T, Fritzlaylin LK Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science 329: Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R, Martin JW, Cunningham CW Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature 463: Rota-Stabelli O, Lartillot N, Philippe H, Pisani D Serine codonusage bias in deep phylogenomics: Pancrustacean relationships as a case study. Systematic Biology 62: Ruhfel BR, Gitzendanner MA, Soltis PS, Soltis DE, Burleigh JG From algae to angiosperms inferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes. BMC Evolutionary Biology 14: 23. Shi XL, Marie D, Jardillier L, Scanlan DJ, Vaulot D Groups without cultured representatives dominate eukaryotic picophytoplankton in the oligotrophic South East Pacific Ocean. PLoS ONE 4: Shoup S, Lewis LA Polyphyletic origin of parallel basal bodies in swimming cells of chlorophycean green algae (Chlorophyta). Journal of Phycology 39: J. Syst. Evol. 55 (4): , 2017