The cpDNA of Verdigellas peltata is small and highly compacted

The circular chloroplast genome of Verdigellas peltata (Fig. 1) is 79,444 bp long, which is smaller than most chloroplast DNAs (cpDNAs) of free-living green algae1,29, but in the range of published prasinophyte cpDNAs15,16. GC content is 27.7%, which is the lowest value observed among the early-diverging chlorophytes examined so far. The cpDNA of the clade VI prasinophyte Prasinococcus sp. CCMP 1194 displays the second lowest value (32.1%)16. Similar to the situation in most prasinophytes and several other green algae, the V. peltata cpDNA lacks a large inverted repeat encoding the rRNA operon.

Figure 1 Gene map of the chloroplast genome of Verdigellas peltata. Genes shown on the outside of the circle are transcribed counterclockwise. Genes are coloured according to the functional categories shown in the legend inside the gene map. Thick lines in the inner rings represent conserved gene clusters between the cpDNAs of V. peltata and Mesostigma viride28 and between V. peltata and Prasinococcus sp. CCMP 119416. Full size image

The genes of the V. peltata cpDNA are densely packed, with intergenic spacers accounting for only 13% of the total genome. Introns are absent, similar to the situation in the cpDNAs of the clade VI prasinophytes, Prasinophyceae sp. CCMP 120516, Nephroselmis olivacea30 and Micromonas sp. RCC 29931. Chloroplast genomes of similar compactness have been found in small-celled prasinophytes and this has been attributed to a strong selection pressure to maintain a small and compact chloroplast genome in picoplanktonic species15,16,32. The presence of a small and gene-dense cpDNA in Verdigellas and the observation of compact cpDNAs in marine green macro-algae of the class Ulvophyceae33,34 indicate that highly compacted cpDNAs are not restricted to picoplanktonic species.

We identified 113 unique genes, including 85 protein-coding genes, 25 tRNA genes (trnG(ucc) is duplicated) and three rRNA genes. In addition, one freestanding open reading frame (ORF) of 1032 bp (orf1) was identified that did not show any relationship with known plastid genes. A blastp search indicated that this ORF contains a DNA polymerase III-like domain of bacterial origin (E-value 4e-25). The presence of bacterial genes in plastid genomes, possibly acquired through horizontal gene transfer, has only been observed in a few algal species, including the prasinophyte Nephroselmis olivacea15,30,33,35. It is relevant to note that Verdigellas harbours endophytic cyanobacteria (and probably a diverse community of other bacteria) in the gelatinous matrix of the thallus24. This close association may facilitate gene transfer from the endophytic bacteria to the host genome.

The V. peltata cpDNA shows high similarities in genome organization and gene content with the cpDNAs of Prasinococcales and early-diverging Streptophyta

A comparison of gene repertoires between V. peltata and a representative selection of published cpDNAs from prasinophytes, core Chlorophyta and early-diverging Streptophyta is shown in Fig. 2. A total of 68 genes are shared among these 18 cpDNAs (see legend Fig. 2). Verdigellas shares the largest number of genes with the early-diverging streptophytes Chlorokybus atmophyticus (111 shared genes) and Mesostigma viride (110 shared genes) and the prasinophyte Prasinococcus sp. CCMP 1194 (110 shared genes). Verdigellas and several species of Prasinococcales share a unique set of five genes that is not found in other Chlorophyta cpDNAs: ndhJ, rpl21, rps15, rps16 and ycf66. This set of chloroplast genes that was previously only known from the Prasinococcales and some Streptophyta was seen as support that these lineages maintain some ancestral genomic features of green algae16. Besides ndhJ, the Verdigellas cpDNA contains genes coding for 10 other subunits homologous to the mitochondrial NADH:ubiquinone oxidoreductase. In the Chlorophyta, the latter set of ndh genes has until now only been found in Prasinococcus sp. CCMP 1194, Pyramimonas parkeae, two Nephroselmis species and Picocystis salinarum16. Common green algal chloroplast genes that are apparently absent from the V. peltata and prasinophyte clade VI cpDNAs include psbM, infA and petL.

Figure 2 Comparison of gene contents between the cpDNA of Verdigellas peltata and a representative selection of published cpDNAs from prasinophytes, core Chlorophyta and early-diverging Streptophyta. The black circles denote the genes shared exclusively between the Streptophyta and at least one species of Palmophyllophyceae (Palmophyllales-Prasinococcales). The grey square indicates a pseudogene. The 68 genes present in all compared cpDNAs are not shown in the figure: atpA, B, E, F, H, I, clpP, petA, B, G, psaA, B, psbA, B, C, D, E, F, H, I, J, K, L, N, T, Z, rbcL, rpl2, 20, 36, rpoA, C1, C2, rps2, 4, 7, 8, 11, 12, 14, 18, 19, rrl, rrs, tufA, ycf1, 12 and 21 tRNA genes: trnA(ugc), C(gca), D(guc), E(uuc), F(gaa), H(gug), I(gau), K(uuu), L(uag), L(uaa), Me(cau), Mf(cau), N(guu), P(ugg), Q(uug), R(acg), R(ucu), S(gcu), S(uga), W(cca) and Y(gua). Data sources:4,15,16,25,28,30,32,33,36,68. Full size image

As highlighted by our analyses of chloroplast gene pairs shared between Verdigellas and early-diverging green plants, retention of ancestral gene order appears to be the most interesting feature of the Verdigellas genome (Fig. 3). Indeed, among the prasinophytes examined thus far, Verdigellas shares the most gene pairs with the streptophytes Mesostigma viride and Chlorokybus atmophyticus. It even exhibits a higher level of synteny with Mesostigma than with any other clade VI prasinophyte taxa. A total of 81 Verdigellas genes form 20 clusters with Mesostigma (Fig. 1), whereas only 59–62 genes present in 16 clusters are conserved in the three other clade VI taxa. Of the latter taxa, Prasinococcus sp. CCMP 1194 displays the most similar gene order to the Verdigellas genome, with 22 syntenic blocks involving 69 genes (Fig. 1); however, this conservation level is not much different from those observed in the comparisons with Prasinophyceae sp. MBIC10622 (21 blocks, 68 genes) and Prasinoderma coloniale (19 blocks, 62 genes).

Figure 3 Shared gene pairs in the chloroplast genomes of early-diverging green algae. The gene pairs shared by at least three taxa were identified among all possible signed gene pairs in the compared genomes. Note that the Verdigellas gene pairs shared with only one taxon were not excluded. The presence of a gene pair is denoted by a blue box; a grey box refers to a gene pair in which at least one gene is missing due to gene loss. Full size image

The Verdigellas/Mesostigma gene clusters clearly encompass a larger portion of the Verdigellas genome than the Verdigellas/Prasinococcus clusters (Fig. 1). The clusters in these two pairs of genomes have 23 endpoints in common; 15 of the 21 remaining Verdigellas/Prasinococcus endpoints interrupt Verdigellas/Mesostigma clusters, whereas only two of the 17 unique Verdigellas/Mesostigma endpoints interrupt Verdigellas/Prasinococcus clusters. These observations provide further evidence that ancestral gene order was disrupted more extensively in the Prasinococcales than in the Palmophyllales.

Like Prasinococcus, Verdigellas has not maintained an intact rDNA operon, but the two algal species do not share the same breakage site in this operon (Fig. 3, between rrl and rrf in Verdigellas and between rrs and trnI(gau) in Prasinococcus). While a number of IR-less green algal genomes have also been found to have a disrupted rDNA operon15,16,36, there are several cases of IR-less genomes that have preserved an intact operon (e.g. the prasinophyte Monomastix sp. OKE-1).

The Palmophyllales-Prasinococcales clade forms the deepest branch of the Chlorophyta

Phylogenies were inferred from 71 concatenated plastid genes and their translation products. The Bayesian phylogeny inferred from amino acid (AA) sequences under the cpREV + Γ4 + F model is shown in Fig. 4 with indication of Bayesian posterior probability (pp) and maximum likelihood (ML) bootstrap support (bs) values, branch support from the analysis using the site-heterogeneous CAT + Γ4 and CATGTR + Γ4 models of evolution and the analysis of the Dayhoff6 recoded AA dataset using a homogeneous GTR + Γ4 model and branch support from the analyses of the nucleotide sequences (first two codon-positions). All inferred trees are shown in the Supplementary Figs S2–8. Overall, the topologies of the AA and nucleotide trees were congruent. The topology of the tree shown in Fig. 4 and in particular the branching order of the prasinophyte clades is in general agreement with published plastid phylogenies of green algae16,17. In all plastid gene analyses, Verdigellas peltata (Palmophyllales) forms a fully supported clade with four species of Prasinococcales (prasinophyte clade VI): Prasinococcus capsulatus, Prasinococcus sp. CCMP 1194, Prasinoderma coloniale and Prasinophyceae sp. MBIC10622. The Palmophyllales-Prasinococcales clade was recovered as the sister group to all other Chlorophyta with high support in all analyses. These results are similar to chloroplast and 18S rDNA phylogenies11,16,17, which showed the early-diverging position of the Prasinococcales in the Chlorophyta. Within the Palmophyllales-Prasinococcales clade, the alliance of V. peltata with the Prasinococcales species received no support in the AA trees, while in the nucleotide trees V. peltata is sister to the four other species (pp = 0.95, bs = 94). Our phylogenomic results are thus in contrast with the plastid gene phylogeny of Zechman et al.20, who recovered the Palmophyllales as a sister clade to all other Chlorophyta with moderate support (pp = 0.97, bs = 77). This difference in topology is likely related to scarce phylogenetic information in two plastid genes (rbcL and atpB) and the missing atpB data for most prasinophytes in Zechman et al.20.

Figure 4 Plastid tree of green plants showing the phylogenetic position of the new class Palmophyllophyceae. Bayesian and ML phylogenies were inferred from 71 concatenated plastid genes and their translation products. The Bayesian majority-rule consensus tree resulting from the analysis of the AA alignment (13,730 amino acid positions) under the cpREV + Γ4 + F model is represented. Bayesian pp and ML bs values are shown above the branches for the analyses of the AA alignment; from left to right are indicated the pp and bs values for the analyses under the cpREV + Γ4 + F model and the pp values for the PhyloBayes analyses under the CAT + Γ4 and CATGTR + Γ4 models and the analysis of the Dayhoff6 recoded AA dataset using a homogeneous GTR + Γ4 model. Bayesian pp and ML bs values are shown below the branches for the nucleotide analyses (1st and 2nd codon position: 29,662 positions) under the GTR + Γ4 + I model with a partitioning strategy in which codon positions were treated separately (2 partitions). Asterisks indicate full support in all analyses; dashes denote pp values <0.90 or bs values <50. All inferred plastid trees are shown in the Supplementary Figs S2–S8. Full size image

The phylogenetic trees resulting from the analyses of the nuclear rDNA data (concatenated small and large subunit rRNA gene sequences) are summarized in Fig. 5. In general, the phylogenetic relationships are congruent with the plastid trees, although relationships among several prasinophyte clades received less support. As observed in the plastid tree, the Palmophyllales (Verdigellas peltata and Palmophyllum umbracola) form a fully supported clade with species of Prasinococcales (Prasinococcus capsulatus and Prasinoderma coloniale). Within this clade, the Palmophyllales and Prasinococcales represent two distinct subclades. Unlike the plastid phylogeny, the position of the Palmophyllales-Prasinococcales clade could not be determined with certainty. In the Bayesian tree, this clade is sister to the Chlorophyta-Streptophyta, while in the ML tree the Palmophyllales-Prasinococcales clade forms the earliest-diverging clade of the Chlorophyta, as in the plastid trees (red arrow in Fig. 5); however, both relationships received no statistical support (Supplementary Figs S9 and S10). Thus, the phylogenetic position of the clade has to be interpreted as unresolved based on the nuclear rDNA data, similar to the 18S phylogeny of Zechman et al.20. It should be noted that in some published 18S-based phylogenies with larger taxon sampling (but without members of Palmophyllales), the Prasinococcales have been resolved as an early-diverging clade of the Chlorophyta with low to moderate support11,14,15.

Figure 5 Nuclear rDNA tree of green plants showing the phylogenetic position of the new class Palmophyllophyceae. Bayesian and ML phylogenies were inferred from concatenated small (18S) and large (28S) subunit rRNA genes (4,579 nucleotide positions) under the GTR + Γ4 + I model with a partitioning strategy in which the 18S and 28S rDNA were treated separately. The Bayesian majority-rule consensus tree is represented. Bayesian pp and ML bs values are shown at the nodes. Asterisks indicate full support in both analyses; dashes denote pp values <0.90 or bs values <50. The red arrow indicates the position of the Palmophyllophyceae clade in the ML phylogeny (Supplementary Fig. S10). Full size image

A phylogeny based on currently available 18S rDNA sequences of Palmophyllales and Prasinococcales is shown in Fig. 6 and provides an indication of the known diversity within this group based on nuclear rDNA sequence data. The tree shows several well-supported clades that generally correspond to the two genera and three currently recognized species of Prasinococcales: Prasinoderma coloniale, Prasinoderma singularis and Prasinococcus capsulatus37,38,39. In addition, several clades represent undescribed diversity. DNA sequence data for the Palmophyllales are scarcer. Only three 18S rDNA sequences are currently available, representing the species Palmophyllum umbracola and Verdigellas peltata, which form a fully supported clade in the tree reported here. Only a few species have been described in the genera Palmophyllum, Verdigellas and Palmoclathrus (Supplementary Table S1), but sequence data from these different morphospecies and a wide geographical sampling will be needed to test generic boundaries and assess species diversity in the Palmophyllales. It is worth mentioning that genetic divergence between Palmophyllum and Verdigellas (max. p-distance 0.009) is much lower than between Prasinoderma and Prasinococcus (max. p-distance 0.108), or even between the two Prasinoderma species (max. p-distance 0.048).

Figure 6 Phylogenetic tree illustrating the diversity within the Palmophyllophyceae based on nuclear 18S rDNA sequences. The best ML tree recovered under the GTR + Γ4 + I model is shown with indication of ML bs and Bayesian pp values (pp values < 90 and bs values <50 are not shown); asterisks indicate full support in both the ML and Bayesian analyses. Full size image

Evolution and systematics of the new class Palmophyllophyceae

Our phylogenetic and comparative genomic analyses provide compelling evidence that the Palmophyllales and Prasinococcales group in a distinct and well-supported clade that forms the deepest branch of the Chlorophyta.

The phylogenetic position of the Palmophyllales among the unicellular prasinophytes indicates an independent origin of macroscopic growth and multicellularity outside of the core Chlorophyta. Species of Palmophyllales form well-defined, attached macroscopic plants (thalli) composed of small, isolated, undifferentiated coccoid cells (3.2–10 μm) in a gelatinous matrix (palmelloid organisation)22,23,24. This type of macroscopic growth is rather atypical, as multicellularity in green algae usually involves cell–cell contact and cellular differentiation40. However, palmelloid thalli are found in a number of core Chlorophyta, including the Tetrasporales (Chlorophyceae), although they never form the elaborate large thalli found in the Palmophyllales20,24,41.

As discussed by Leliaert et al.9, the broad phylogenetic distribution of non-motile (coccoid) prasinophytes, including Picocystis, Pycnococcus, some species of Mamiellales and the early-diverging Palmophyllales-Prasinococcales clade, may alter our understanding about the nature of the green plant ancestor. It is generally accepted that the ancestral green algae were unicellular flagellates (“ancestral green flagellate”) with characters typical of extant prasinophytes such as the presence of organic body scales7,8. Although it is indeed probable that flagella were present in a life cycle stage of the green plant ancestor, it is possible that this ancestor was a scale-less coccoid organism with transient flagellar stages9. Alternatively, coccoid forms may have evolved multiple times independently.

The sister relationship of the macroscopic Palmophyllales and the unicellular Prasinococcales is unusual from a morphological perspective, although, as will be discussed below, this relationship is supported by a number of shared cytological characteristics, such as cell size, lack of flagellar stages, presence of a mucus-secreting system and similarities in cell division21,37,39,41,42. The morphological heterogeneity is not surprising given the large sequence distances within the clade, which likely reflects a great age of the divergences. Although dating the phylogeny of green plants is a difficult task because of the sparse fossil record of the group, our tentative time calibrated phylogeny (Supplementary Fig. S11) indeed suggests that the Palmophyllophyceae are ancient, having originated and diversified somewhere in the late Proterozoic and Paleozoic.

The Prasinococcales include only a few described species from marine environments, characterized by small (2.2–5.5 μm) coccoid, scale-less cells (Supplementary Table S1)37,38,39,42. Sexual reproduction has not been observed. Cells of Prasinococcus are typically embedded in gelatinous capsules secreted by complex pores (“Golgi-decapore complex”)42. Prasinoderma has a thick multi-layered cell wall without pores and lacks a gelatinous envelope39. Traditionally, scale-less coccoid planktonic green algae were placed in the family Pycnococcaceae (Mamiellales), which initially included the genus Pycnococcus43 and subsequently Prasinococcus and Prasinoderma37,38. The grouping of Prasinococcus and Prasinoderma in a distinct clade (clade VI) separated from Pycnococcus (clade V) has been demonstrated by 18S rDNA phylogenetic data10,11.

The relationship of the Palmophyllales with the Prasinococcales is supported by a number of shared cytological features (Supplementary Table S1). Species of Palmophyllales and Prasinococcus both have a mucus-secreting system originating from a large Golgi body21. In Prasinococcus, the polysaccharide (mucus) capsule is secreted through a complex structure perforating the cell wall, which is composed of a round collared lid with 8 to 14 pores (“Golgi-decapore complex”)42. Species of Palmophyllales lack the complex decapore structure and instead have simple pores in the cell wall21,23. Mode of cell division is also similar in species of Palmophyllales and Prasinococcales, characterized by unequal binary fission. In Prasinococcus and Prasinoderma, one of the daughter cells retains the parent wall, while the other is released with a newly produced cell wall37,38,39. In Palmoclathrus (the only species of Palmophyllales where cell division has been studied in detail), the parental cell wall is discarded and incorporated into the gelatinous matrix41. Finally, cells of Palmophyllales and Prasinococcales lack flagella or ultrastructural traces from flagella (basal bodies, centrioles)21,23,41. This feature, however, is not unique to the clade.

Taken together, the phylogenetic distinctness of the Palmophyllales-Prasinococcales clade and the presence of some unique phenotypic features warrant recognition of a new class of Chlorophyta. In the currently accepted classification of the Viridiplantae, the major clades of the Streptophyta and core Chlorophyta are classified at the class level, as are some of the major prasinophyte clades, including Nephroselmidophyceae and Mamiellophyceae1,14,44. Our proposal for a new class entirely fits this taxonomic scheme.

Class Palmophyllophyceae Leliaert et al. class. nov.

Description

Marine green algae. Cells planktonic, solitary or in loose colonies, or cells grouped in a gelatinous matrix forming benthic macroscopic thalli. Cells spherical or subspherical, lacking flagella and organic body scales, with a single cup-shaped chloroplast enclosing a mitochondrion, nucleus and large Golgi body. Cell surrounded by a cell wall, with or without pores. Chloroplast surrounded by two membranes, with chlorophylls a and b, with or without pyrenoid. Cell division by unequal binary fission. Strongly supported clade in plastid multi-gene and nuclear ribosomal DNA phylogenetic analyses.

Order Palmophyllales Zechman et al.20.

Family Palmophyllaceae Zechman et al.20.

Genera Palmophyllum Kützing (type genus), Verdigellas D.L. Ballantine & J.N. Norris, Palmoclathrus Womersley.

Order Prasinococcales Guillou et al.11.

Description

Marine planktonic green algae. Cells solitary or forming loose colonies. Cells spherical or subspherical, lacking flagella and organic body scales, with a thin cell wall surrounded by a thick ellipsoidal gelatinous capsule, or with a thick, multi-layered cell wall without gelatinous capsule. Cells with a single cup-shaped chloroplast enclosing a mitochondrion, nucleus and large Golgi body. Chloroplast with a large pyrenoid surrounded by a starch sheath; pyrenoid matrix penetrated by a bifurcate extension of the cytoplasm and the mitochondrion. Cell division by unequal binary fission in which one of the daughter cells retains the parent wall, while the other is released with a newly produced cell wall. Main pigments include chlorophylls a and b, prasinoxanthin, Mg-2,4-divinylphaeoporphyrin a5 monomethylester (MgDVP), uriolide and micromonol.

Family Prasinococcaceae Leliaert fam. nov.

Characters as for order.

Genera Prasinococcus H. Miyashita & M. Chihara (type genus) and Prasinoderma T. Hasegawa & M. Chihara.

Nomenclatural notes

The order Prasinococcales was originally described by Chadefaud45 for the single species Halosphaera viridis (descriptive order name according to article 16.1 of the International Code of Nomenclature (ICN)46). Since Halosphaera is now considered a member of the Pyramimonadales7,13,47, Prasinococcales Chadefaud is a synonym of Pyramimonadales. More recently, Guillou et al.11 used the name Prasinococcales to label “prasinophyte clade VI”10, which includes Prasinococcus (Miyashita et al. 1993)38 and Prasinoderma (Hasegawa et al. 1996)37. In the interpretation of Guillou et al.11, which is different from Chadefaud, Prasinococcales is an automatically typified name according to article 16.1 of the ICN, with type Prasinococcus. Because Guillou et al.11 did not provide a description for the order, we provide one here. Although the family Prasinococcaceae is flagged as an accepted family name in the Global Biodiversity Information Facility (GBIF: www.gbif.org) and in AlgaeBase (algaebase.org), the name has never been described nor validly published, hence the formal description in this paper.