Content uploaded by Juan M. Lopez-Bautista
Author content
All content in this area was uploaded by Juan M. Lopez-Bautista on May 10, 2016
Content may be subject to copyright.
1
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
www.nature.com/scientificreports
Chloroplast phylogenomic analyses
reveal the deepest-branching
lineage of the Chlorophyta,
Palmophyllophyceae class. nov.
Frederik Leliaert1,2,*, Ana Tronholm1,3,*, Claude Lemieux4, Monique Turmel4,
Michael S. DePriest1, Debashish Bhattacharya5, Kenneth G. Karol6, Suzanne Fredericq7,
Frederick W. Zechman8 & Juan M. Lopez-Bautista1
The green plants (Viridiplantae) are an ancient group of eukaryotes comprising two main clades: the
Chlorophyta, which includes a wide diversity of green algae, and the Streptophyta, which consists of
freshwater green algae and the land plants. The early-diverging lineages of the Viridiplantae comprise
unicellular algae, and multicellularity has evolved independently in the two clades. Recent molecular
data have revealed an unrecognized early-diverging lineage of green plants, the Palmophyllales, with
a unique form of multicellularity, and typically found in deep water. The phylogenetic position of this
enigmatic group, however, remained uncertain. Here we elucidate the evolutionary anity of the
Palmophyllales using chloroplast genomic, and nuclear rDNA data. Phylogenetic analyses rmly place
the palmophyllalean Verdigellas peltata along with species of Prasinococcales (prasinophyte clade VI)
in the deepest-branching clade of the Chlorophyta. The small, compact and intronless chloroplast
genome (cpDNA) of V. peltata shows striking similarities in gene content and organization with the
cpDNAs of Prasinococcales and the streptophyte Mesostigma viride, indicating that cpDNA architecture
has been extremely well conserved in these deep-branching lineages of green plants. The phylogenetic
distinctness of the Palmophyllales-Prasinococcales clade, characterized by unique ultrastructural
features, warrants recognition of a new class of green plants, Palmophyllophyceae class. nov.
e green plants or Viridiplantae are an ancient and diverse group of photosynthetic eukaryotes. Molecular phy-
logenetic and ultrastructural data indicate that the green plants split early in their evolution (estimated between
800 and 1200 Mya) into two main clades: the Chlorophyta and Streptophyta1.
e Streptophyta include a diverse array of unicellular and multicellular green algae from freshwater environ-
ments (collectively termed the charophytes), and the land plants2,3. Phylogenomic analyses indicate that morpho-
logically simple species of the Mesostigmatophyceae (Mesostigma viride, biagellate unicells), Chlorokybophyceae
(Chlorokybus atmophyticus, packets of non-motile cells), and Klebsormidiophyceae (packets of cells or simple
laments), characterized by cell division by furrowing, form the earliest-diverging clades of the Streptophyta4,5.
e later-diverging clades of the Streptophyta evolved a new mechanism of cell division that involved the produc-
tion of a phragmoplast, and cell-walls with plasmodesmata, which facilitated communication between cells, and
ultimately development of complex tissues and plant bodies6.
1Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35484-0345, USA. 2Department of
Biology, Ghent University, 9000 Ghent, Belgium. 3Southeast Environmental Research Center, Florida International
University, Miami, FL 33199, USA. 4Institut de biologie intégrative et des systèmes, Département de biochimie, de
microbiologie et de bio-informatique, Université Laval, Québec (QC) Canada. 5Department of Ecology, Evolution and
Natural Resources and Department of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ 08901,
USA. 6Lewis B. and Dorothy Cullman Program for Molecular Systematic Studies, The New York Botanical Garden,
Bronx, New York 10458, USA. 7Department of Biology, University of Louisiana at Lafayette, LA 70504-3602, USA.
8College of Natural Resources and Sciences, Humboldt State University, Arcata, CA 95521, USA. *These authors
contributed equally to this work. Correspondence and requests for materials should be addressed to F.L. (email:
frederik.leliaert@gmail.com) or A.T. (email: ana@tronholm.com)
Received: 15 February 2016
Accepted: 15 April 2016
Published: 09 May 2016
OPEN
www.nature.com/scientificreports/
2
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
e Chlorophyta include a large diversity of marine, freshwater and terrestrial green algae with a wide vari-
ety of morphologies, ranging from unicellular to complex multicellular morphologies. A paraphyletic assem-
blage of planktonic unicellular, mainly marine green algae forms the early-diverging clades of Chlorophyta,
collectively called the prasinophytes. e early-diverging nature of these clades is reected in a wide diver-
sity of cellular architectures (including agellate and coccoid cells that are naked, or covered by cell walls or
organic body scales), agellar behaviour, mitotic and cytokinetic processes, biochemical features, and photo-
synthetic pigments7–13. About ten prasinophyte clades have been identied based on nuclear-encoded small
subunit (18S) rDNA sequences9–11,13–15. e anities among these clades, however, were poorly resolved in 18S
rDNA phylogenies, and only recently have phylogenetic relationships been elucidated with more condence
using multi-gene datasets16. e prasinophytes gave rise to the core Chlorophyta, which include unicellular and
multicellular species that abound in marine, freshwater and terrestrial habitats. is clade includes the three
species-rich classes Ulvophyceae, Trebouxiophyceae and Chlorophyceae, and two smaller classes Pedinophyceae
and Chlorodendrophyceae1,17,18.
It is generally accepted that that the ancestral green plants were unicellular green algae with characters typical
of most extant prasinophytes, such as the presence of agella and organic body scales1,19. e nature of this hypo-
thetical ancestral green agellate, however, has been a matter of debate9,19. A better understanding of the diversity
and phylogenetic relationships among early-diverging clades of Chlorophyta and Streptophyta is thus central to
understanding the evolution of green plants.
e viewpoint that the earliest-diverging green plant lineages comprise green algae with simple morphologies
was recently challenged by a molecular phylogenetic study by Zechman et al.20, which identied a deep-branching
clade of macroscopic algae, the Palmophyllales. e clade includes the genera Palmophyllum, Verdigellas and
Palmoclathrus, which occur in marine deep water and other dimly lit environments (Verdigellas has been recorded
from depths down to 200 m)20. Species of Palmophyllales exhibit a unique type of multicellularity, forming mac-
roscopic plants that are composed of isolated and undierentiated spherical cells embedded in an apparently
amorphous gelatinous matrix21–24. Although the Palmophyllales were identied as a distinct clade of green algae,
the exact phylogenetic placement could not be determined with certainty. Analysis of the plastid genes atpB
and rbcL placed the Palmophyllales sister to the Chlorophyta. On the other hand, analysis of nuclear 18S rDNA
sequences allied the Palmophyllales with the Prasinococcales (a group of coccoid prasinophytes) in a clade of
uncertain position.
e multiple genes encoded in the chloroplast genome (cpDNA) represent an invaluable source of data for
resolving dicult phylogenetic questions, including deep relationships in green plants16,17,25–27. In addition, com-
parative analysis of chloroplast genomes from early-diverging green plants (prasinophytes and early-diverging
streptophytes) provides important insights into the ancestral architecture and evolution of plastid genomes in the
green plants15,16,28.
e aim of this study was to resolve the evolutionary anities of the enigmatic Palmophyllales using a phylog-
enomic approach. We obtained the complete nucleotide sequence of the chloroplast genome of Verdigellas peltata,
and performed multi-gene phylogenetic and comparative genomic analyses. In addition, we inferred phylogenies
based on nuclear-encoded small and large subunit rDNA sequences, providing independent phylogenetic evi-
dence. Our phylogenomic analyses rmly placed the Palmophyllales together with the Prasinococcales in the
earliest-diverging lineage of the Chlorophyta. Comparative chloroplast genomic analyses provided new insights
into the ancestral plastid genome of the green plants.
Results and Discussion
The cpDNA of Verdigellas peltata is small and highly compacted. e circular chloroplast genome
of Verdigellas peltata (Fig.1) is 79,444 bp long, which is smaller than most chloroplast DNAs (cpDNAs) of free-liv-
ing 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. e 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.
e 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. e pres-
ence 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 picoplank-
tonic species.
We identied 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 identied
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). e presence of bacterial genes in plastid
genomes, possibly acquired through horizontal gene transfer, has only been observed in a few algal species, includ-
ing 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.
is 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
www.nature.com/scientificreports/
3
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
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 strepto-
phytes Chlorokybus atmophyticus (111 shared genes) and Mesostigma viride (110 shared genes) and the prasino-
phyte Prasinococcus sp. CCMP 1194 (110 shared genes). Verdigellas and several species of Prasinococcales share
a unique set of ve genes that is not found in other Chlorophyta cpDNAs: ndhJ, rpl21, rps15, rps16 and ycf66.
is 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.
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
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. ick 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.
www.nature.com/scientificreports/
4
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
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.
e black circles denote the genes shared exclusively between the Streptophyta and at least one species of
Palmophyllophyceae (Palmophyllales-Prasinococcales). e grey square indicates a pseudogene. e 68 genes
present in all compared cpDNAs are not shown in the gure: 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.
Figure 3. Shared gene pairs in the chloroplast genomes of early-diverging green algae. e gene pairs shared
by at least three taxa were identied among all possible signed gene pairs in the compared genomes. Note that
the Verdigellas gene pairs shared with only one taxon were not excluded. e 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.
www.nature.com/scientificreports/
5
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
dierent from those observed in the comparisons with Prasinophyceae sp. MBIC10622 (21 blocks, 68 genes) and
Prasinoderma coloniale (19 blocks, 62 genes).
e Verdigellas/Mesostigma gene clusters clearly encompass a larger portion of the Verdigellas genome than
the Verdigellas/Prasinococcus clusters (Fig.1). e 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.
ese 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)
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. e Bayesian majority-rule consensus tree resulting from the analysis of the AA
alignment (13,730 amino acid positions) under the cpREV + Γ 4 + F model is represented. Bayesian pp and ML
bs values are shown above the branches for the analyses of the AA alignment; from le to right are indicated
the pp and bs values for the analyses under the cpREV + Γ 4 + F model, and the pp values for the PhyloBayes
analyses under the CAT + Γ 4 and CATGTR + Γ 4 models, and the analysis of the Dayho6 recoded AA dataset
using a homogeneous GTR + Γ 4 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 + Γ 4 + 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.
www.nature.com/scientificreports/
6
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
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 prasino-
phyte 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. e Bayesian
phylogeny inferred from amino acid (AA) sequences under the cpREV + Γ 4 + F model is shown in Fig.4 with
indication of Bayesian posterior probability (pp) and maximum likelihood (ML) bootstrap support (bs) val-
ues, branch support from the analysis using the site-heterogeneous CAT + Γ 4 and CATGTR + Γ 4 models of
evolution, and the analysis of the Dayho6 recoded AA dataset using a homogeneous GTR + Γ 4 model, and
branch support from the analyses of the nucleotide sequences (rst 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.
e 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. ese results are similar to chloroplast and 18S rDNA phylog-
enies11,16,17, 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 sup-
port 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). is dif-
ference 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.
e 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 congru-
ent 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). us, the phylogenetic posi-
tion 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.
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. e 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 capsulatus37–39. 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 spe-
cies 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 dierent morphospecies and a wide geographical sam-
pling 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).
Evolution and systematics of the new class Palmophyllophyceae. Our phylogenetic and compar-
ative 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.
e phylogenetic position of the Palmophyllales among the unicellular prasinophytes indicates an independ-
ent origin of macroscopic growth and multicellularity outside of the core Chlorophyta. Species of Palmophyllales
form well-dened, attached macroscopic plants (thalli) composed of small, isolated, undierentiated coccoid
cells (3.2–10 μ m) in a gelatinous matrix (palmelloid organisation)22–24. is type of macroscopic growth is rather
atypical, as multicellularity in green algae usually involves cell–cell contact and cellular dierentiation40. 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) pra-
sinophytes, 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 agellates (“ancestral green agellate”) with
characters typical of extant prasinophytes such as the presence of organic body scales7,8. Although it is indeed
www.nature.com/scientificreports/
7
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
probable that agella 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 agellar stages9. Alternatively, coccoid forms may have evolved
multiple times independently.
e 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 agellar stages, presence of a mucus-secreting sys-
tem, and similarities in cell division21,37,39,41,42. e morphological heterogeneity is not surprising given the large
sequence distances within the clade, which likely reects a great age of the divergences. Although dating the
phylogeny of green plants is a dicult 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 diversied somewhere in the late Proterozoic and Paleozoic.
e Prasinococcales include only a few described species from marine environments, characterized by small
(2.2–5.5 μ m) coccoid, scale-less cells (Supplementary Table S1)37–39,42. Sexual reproduction has not been observed.
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 + Γ 4 + I model with a partitioning
strategy in which the 18S and 28S rDNA were treated separately. e 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. e red arrow indicates the position of the
Palmophyllophyceae clade in the ML phylogeny (Supplementary Fig. S10).
www.nature.com/scientificreports/
8
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
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. e
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.
e relationship of the Palmophyllales with the Prasinococcales is supported by a number of shared cytologi-
cal features (Supplementary Table S1). Species of Palmophyllales and Prasinococcus both have a mucus-secreting
system originating from a large Golgi body21,42. 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 ssion. In Prasinococcus and Prasinoderma, one of the daughter
cells retains the parent wall, while the other is released with a newly produced cell wall37–39. 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 agella
or ultrastructural traces from agella (basal bodies, centrioles)21,23,41. is feature, however, is not unique to the
clade.
Taken together, the phylogenetic distinctness of the Palmophyllales-Prasinococcales clade, and the pres-
ence of some unique phenotypic features warrant recognition of a new class of Chlorophyta. In the currently
accepted classication of the Viridiplantae, the major clades of the Streptophyta and core Chlorophyta are clas-
sied 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 ts 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 agella and organic body
scales, with a single cup-shaped chloroplast enclosing a mitochondrion, nucleus, and large Golgi body. Cell sur-
rounded by a cell wall, with or without pores. Chloroplast surrounded by two membranes, with chlorophylls
Figure 6. Phylogenetic tree illustrating the diversity within the Palmophyllophyceae based on nuclear 18S
rDNA sequences. e best ML tree recovered under the GTR + Γ 4 + 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.
www.nature.com/scientificreports/
9
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
a and b, with or without pyrenoid. Cell division by unequal binary ssion. 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 sub-
spherical, lacking agella and organic body scales, with a thin cell wall surrounded by a thick ellipsoidal gelati-
nous 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 sur-
rounded by a starch sheath; pyrenoid matrix penetrated by a bifurcate extension of the cytoplasm and the mito-
chondrion. Cell division by unequal binary ssion 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, prasinoxan-
thin, 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 sin-
gle 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 dierent from Chadefaud,
Prasinococcales is an automatically typied 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 agged 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.
Conclusion
We provide solid phylogenetic evidence that the enigmatic Palmophyllales together with the Prasinococcales
form the deepest-branching clade of the Chlorophyta, which we describe as a new class, the Palmophyllophyceae.
Our phylogenetic results improve our understanding of morphological evolution in the green plants. Until pres-
ent, the early-diverging lineages of the Chlorophyta (the prasinophytes) were only known to comprise unicellular
planktonic algae. Our results point to an independent origin of macroscopic growth and multicellularity out-
side of the core Chlorophyta. Our study also contributes to a better understanding of plastid genome evolution
in green plants. e small, compact and intronless cpDNA of Verdigellas peltata shows remarkable similarities
in gene content and organization with the cpDNAs of Prasinococcales and the streptophyte Mesostigma viride,
indicating that cpDNA architecture has been extremely well conserved in the early-branching lineages of green
plants.
Methods
Sampling. Material of Verdigellas peltata was obtained from a dredged sample oshore Louisiana, Ewing
Bank, Gulf of Mexico, at ca. 70 m depth (Supplementary Fig. S1). A voucher specimen was deposited in the
Herbarium of the University of Louisiana at Lafayette (LAF-8-26-12-6-1), and a portion of the specimen was
dried in silica gel for molecular analysis. e specimen was morphologically identied as Verdigellas peltata, and
DNA-conrmed based on the chloroplast rbcL (NCBI-megablast: 98.8% identity with V. peltata, GenBank acces-
sion: EU586183) and nuclear 18S rRNA genes (99.7% identity with V. peltata, GenBank accession: FJ619277),
which were the only sequences publicly available for V. peltata previous to this study.
Sequencing, assembly and annotation of the chloroplast genome and nuclear rDNA cistron.
Total genomic DNA was extracted using the E.Z.N.A. Plant DNA Kit (OMEGA Bio-tek, Norcross, GA, USA).
e sequencing library was prepared using the Nextera DNA Sample Prep Kit (Illumina, San Diego, CA,
USA). Sequencing was performed using Illumina MiSeq technology, generating 5.9 million paired-end reads
of 2 × 250 bp and 6.6 million paired-end reads of 2 × 150 bp. Low-quality ends of the reads (Phred score <30)
and adapters were trimmed using Trim Galore! (www.bioinformatics.babraham.ac.uk/projects/trim_galore).
De novo assembly of paired-end reads was performed using Velvet v. 1.2.1048 and a k-mer length of 91, as well
as the CLC Genomics Workbench (CLC Bio, Aarhus, Denmark) de novo assembler with default parameters
(word size = 23). Contiguous DNA sequences (contigs) of the chloroplast genome were identied by blastn
similarity searches (E-value < 10−6) against a custom-built database of 1305 gene sequences from published
cpDNAs of green algae. Raw reads were iteratively mapped (10×) to these putative cpDNA contigs under
stringent conditions (no gaps allowed, minimum overlap of 25 nucleotides, and minimum overlap identity of
98%) in Geneious (Biomatters, www.geneious.com). e resulting extended contigs were visually examined,
www.nature.com/scientificreports/
10
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
and consensus sequences re-assembled to obtain a large scaold that could be closed into a circle by an overlap
of 456 bp. e assembly had an average coverage of 28× (min. 4× , max. 67×). e sequence with 4× coverage
was 9 bp long and situated between the trnR(ucu) and chlI genes.
Genes were initially identied by mapping the abovementioned 1305 green algal chloroplast gene sequences
against the cpDNA contigs using the read mapper in Geneious. e annotations were veried by identifying
ORFs in Geneious, followed by blastp searches against the NCBI nonredundant database (http://blast.ncbi.nlm.
nih.gov/Blast.cgi, last accessed December 7, 2015). e boundaries of the rRNA genes were identied based on
a dataset of aligned complete rRNA genes from published green algal cpDNAs. tRNA genes were detected and
identied using tRNAscan-SE 1.2149. e circular genome map was drawn with OGDRAW50.
e contig containing the nuclear-encoded rDNA cistron was identied by blastn similarity search of a
custom-built blast database of complete small and large subunit rRNA genes from green algae. e boundaries of
the rRNA genes were identied based on an alignment of published complete rRNA genes.
Comparative analyses of chloroplast gene order. A custom-built program was used to identify syn-
tenic regions between the chloroplast genomes of Verdigellas and early-diverging green algae. is program was
also employed to convert gene order in each of 15 selected green algal cpDNAs to all possible pairs of signed genes
(i.e. taking into account gene polarity). e presence/absence of the signed gene pairs in three or more genomes
were coded as binary characters using Mesquite 3.0451.
Phylogenetic analyses. Phylogenetic analyses were based on four datasets. Gene and protein align-
ments were produced from 71 chloroplast genes and their predicted protein products; these sequences were
obtained from complete (or in few cases partial) chloroplast genomes of 59 taxa of Archaeplastida, including 51
Viridiplantae and eight outgroup taxa (seven Rhodophyta and one Glaucophyta). A third alignment consisted
of (near) complete sequences for the nuclear small (18S) and large (28S) subunit rRNA genes of 63 Viridiplantae
and three outgroup taxa. A fourth alignment consisted of 18S rDNA sequences from 27 taxa of Prasinococcales
and Palmophyllales, and four prasinophyte outgroup taxa. Taxon lists are provided in Supplementary Tables S2
and S3.
Taxon and gene sampling for the 71-chloroplast gene and protein alignments was largely based on Lemieux
et al.16, with selected genes: accD, atpA, B, E, F, H, I, ccsA, cemA, chlB, I, L, N, P, sH, infA, petA, B, D, G, L,
psaA, B, C, I, J, M, psbA, B, C, D, E, F, H, I, J, K, L, M, N, T, Z, rbcL, rpl2, 5, 14, 16, 20, 23, 32, 36, rpoA, B, C1,
C2, rps2, 3, 4, 7, 8, 9, 11, 12, 14, 18, 19, tufA, ycf1,3,4,12. e dataset was 71% lled at the taxon × gene level.
DNA sequences were aligned for each gene separately using the ClustalW translational alignment function52 in
Geneious with a BLOSUM cost matrix, a gap open penalty of 10, and a gap extension cost of 0.1. e separate
gene alignments were then concatenated, and poorly aligned codons removed using the Gblocks server53 (http://
molevol.cmima.csic.es/castresana/Gblocks_server.html) and the least stringent settings, allowing smaller nal
blocks, gap positions within the nal blocks, less strict anking positions and many contiguous non-conserved
positions. Gblocks reduced the alignment from 146,202 to 44,493 positions. Only the rst two codon positions
in the nucleotide alignment were included for phylogenetic analyses (29,662 positions). Gblocks with the least
stringent settings was also applied to the protein alignment, reducing the alignment from 48,759 to 13,730 amino
acid positions.
Taxon and gene sampling for the nuclear rDNA alignment was based on Marin18 and extended with species
of Streptophyta, Rhodophyta and Glaucophyta for which (near) complete small (18S) and large (28S) subunit
rRNA genes were available. 18S and 28S rDNA sequences were aligned separately using MUSCLE54. Both align-
ments (1876 and 3501 positions, respectively) were concatenated, and poorly aligned positions removed using the
Gblocks with the least stringent settings (see above). Gblocks reduced the alignment from 5377 to 4579 positions.
Plastid phylogenies were inferred from the amino acid and nucleotide datasets using Bayesian and ML anal-
yses. For the amino acid dataset, the cpREV + Γ 4 + F model of evolution was selected as the best-tting model
using ProtTest 3.255 based on the corrected Akaike Information Criterion. For the nucleotide dataset, we selected
the GTR + Γ 4 + I model and a partitioning strategy in which codon positions were treated separately (2 parti-
tions) based on the outcome of the BIC criterion in PartitionFinder56. Bayesian analyses of both the amino acid
and nucleotide datasets were conducted using MrBayes v.3.2.157 with a single partition for the amino acid analy-
sis, and two partitions with unlinked models for the nucleotide analysis. Two independent runs were performed
using 1 million (amino acid dataset) or 3 million (nucleotide dataset) generations, each with one cold and three
heated chains, and sampling every 1000 generations. e rst 20% (amino acid analysis) or 10% (nucleotide
analysis) of samples were discarded as burn-in based on assessment of convergence of the runs and stability of
parameters using Tracer v.1.558. ML trees were inferred using RAxML v. 8.2.459 using the same partitioning strat-
egies, and bootstrapping with 500 replicates to assess branch support.
In addition, the amino acid dataset was analysed using the site-heterogeneous CAT + Γ 4 and CATGTR + Γ 4
models of evolution with PhyloBayes v. 4.160,61. Five independent chains were run for 10,000 cycles for the
CAT + Γ 4 analysis and 2,000 cycles for the CATGTR + Γ 4 analysis and a consensus topology was calculated from
the saved trees using the BPCOMP program of PhyloBayes aer a burn-in of 2,000 and 500 cycles, respectively. A
maxdi of 1 was obtained in both analyses, indicating that at least one of the runs was stuck in a local maximum.
We also addressed possible phylogenetic artefacts due to potential biases in amino acid composition resulting
from the low GC content in the cpDNAs of Verdigellas and clade VI prasinophytes27,62. In order to overcome
non-phylogenetic signal due to compositional heterogeneity, we performed a phylogenetic analysis based on a
Dayho recoded dataset63,64 using PhyloBayes with a homogeneous GTR + Γ 4 model of evolution. e amino
acid data was recoded using the -recode option of PhyloBayes and the following Dayho6 recoding scheme:
www.nature.com/scientificreports/
11
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
(A,G,P,S,T) (D,E,N,Q) (H,K,R) (F,Y,W) (I,L,M,V) (C). Five independent chains were run for 3,000 cycles and a
consensus topology was calculated aer a burn-in of 600 cycles.
A provisional time-calibrated phylogeny was obtained with BEAST v. 1.8.265 with two nodes constrained in
time based on previous molecular clock analyses1: e root of the Viridiplantae was constrained using a normal
prior with mean 970 Mya, standard deviation 200, and a minimum and maximum age of 655 and 1280 Mya,
respectively; the root of the land plants was constrained using a normal prior with mean 475 Mya and standard
deviation 20. Details of the BEAST analysis are provided in Supplementary Fig. 11. Because the root age of the
Viridiplantae is highly uncertain, these results should be regarded as tentative.
Nuclear rDNA-based phylogenies were inferred using unlinked GTR + Γ 4 + I models for the 18S and 28S par-
titions selected based on the BIC criterion in PartitionFinder. MrBayes analyses included two independent runs
of 5 million generations (each with four chains). e rst 10% of samples were discarded as burn-in. ML trees
were inferred using RAxML v. 8.2.4 using the same partitioning strategy, and bootstrapping with 500 replicates
to assess branch support.
e nuclear 18S rDNA dataset of Prasinococcales and Palmophyllales was based on Viprey et al.66 and Jouenne
et al.39 and extended with sequences retrieved from blastn searches of Prasinococcus, Prasinoderma and Verdigellas
18S sequences. Phylogenetic analyses of this dataset (1778 positions) was performed under the GTR + Γ 4 + I
model with MrBayes (2 million generations, two runs of four chains each, and the rst 500 thousand samples
discarded as burnin) and RAxML with default settings.
Phylogenetic analyses were run on the CIPRES Science Gateway v3.367 and Katak server of the Institut de
Biologie Intégrative et des Systèmes of Université Laval (http://www.ibis.ulaval.ca/?pg= bioinformatique_
accesServeurs).
References
1. Leliaert, F. et al. Phylogeny and molecular evolution of the green algae. Crit. ev. Plant Sci. 31, 1–46 (2012).
2. McCourt, . M., Delwiche, C. F. & arol, . G. Charophyte algae and land plant origins. Trends Ecol. Evol. 19, 661–666 (2004).
3. Becer, B. & Marin, B. Streptophyte algae and the origin of embryophytes. Ann. Bot. 103, 999–1004 (2009).
4. Civáň, P., Foster, P. G., Embley, T. M., Séneca, A. & Cox, C. J. Analyses of charophyte chloroplast genomes help characterize the
ancestral chloroplast genome of land plants. Genome Biol. Evol. 6, 897–911 (2014).
5. Wicett, N. J. et al. Phylotranscriptomic analysis of the origin and early diversication of land plants. Proc. Natl Acad. Sci. USA 111,
E4859–E4868 (2014).
6. Graham, L. E., Coo, M. E. & Busse, J. S. e origin of plants: Body plan changes contributing to a major evolutionary radiation.
Proc. Natl Acad. Sci. USA 97, 4535–4540 (2000).
7. Melonian, M. Phylum Chlorophyta. Class Prasinophyceae in Handboo of Protoctista. e structure, cultivation, habitats and life
histories of the euaryotic microorganisms and their descendants exclusive of animals, plants and fungi (eds Margulis, L., Corliss, J. O.,
Melonian, M. & Chapman, D. J.) 600–607 (Jones and Bartlett Publishers, 1990).
8. Sym, S. D. & Pienaar, . N. e class Prasinophyceae in Prog. Phycol. es. (eds ound, F. E. & Chapman, D. J.) 281–376 (Biopress
Ltd., 1993).
9. Leliaert, F., Verbruggen, H. & Zechman, F. W. Into the deep: New discoveries at the base of the green plant phy logeny. BioEssays 33,
683–692 (2011).
10. Fawley, M. W., Yun, Y. & Qin, M. Phylogenetic analyses of 18S rDNA sequences reveal a new coccoid lineage of the Prasinophyceae
(Chlorophyta). J. Phycol. 36, 387–393 (2000).
11. Guillou, L. et al. Diversity of picoplantonic prasinophytes assessed by direct nuclear SSU rDNA sequencing of environmental
samples and novel isolates retrieved from oceanic and coastal marine ecosystems. Protist 155, 193–214 (2004).
12. Latasa, M., Schare, ., Le Gall, F. & Guillou, L. Pigment suites and taxonomic groups in Prasinophyceae. J. Phycol. 40, 1149–1155
(2004).
13. Naayama, T. et al. e basal position of scaly green agellates among the green algae (Chlorophyta) is revealed by analyses of
nuclear-encoded SSU rNA sequences. Protist 149, 367–380 (1998).
14. Marin, B. & Melonian, M. Molecular phylogeny and classication of the Mamiellophyceae class. nov. (Chlorophyta) based on
sequence comparisons of the nuclear- and plastid-encoded rNA operons. Protist 161, 304–336 (2010).
15. Turmel, M., Gagnon, M.-C., O’elly, C. J., Otis, C. & Lemieux, C. e chloroplast genomes of the green algae Pyramimonas,
Monomastix, and Pycnococcus shed new light on the evolutionary history of prasinophytes and the origin of the secondary
chloroplasts of euglenids. Mol. Biol. Evol. 26, 631–648 (2009).
16. Lemieux, C., Otis, C. & Turmel, M. 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 picoplantonic species. BMC
Genomics 15, 857 (2014).
17. Lemieux, C., Otis, C. & Turmel, M. Chloroplast phylogenomic analysis resolves deep-level relationships within the green algal class
Trebouxiophyceae. BMC Evol. Biol. 14, 211 (2014).
18. Marin, B. Nested in the Chlorellales or independent class? Phylogeny and classication of the Pedinophyceae (Viridiplantae)
revealed by molecular phylogenetic analyses of complete nuclear and plastid-encoded rNA operons. Protist 163, 778–805 (2012).
19. O’elly, C. J. e origin and early evolution of green plants in Evolution of primary producers in the sea (eds Falowsi, P. G. & noll,
A. H.) Ch. 13, 287–309 (Elsevier Academic Press, 2007).
20. Zechman, F. W. et al. An unrecognized ancient lineage of green plants persists in deep marine waters. J. Phycol. 46, 1288–1295
(2010).
21. Pueschel, C., Sullivan, . & Ballantine, D. Ultrastructure of Verdigellas peltata (Palmellaceae, Chlorophyta), a deep-water, palmelloid
alga with ferritin and trilaminar sheaths. Phycologia 36, 492–499 (1997).
22. Womersley, H. B. S. e Marine Benthic Flora of Southern Australia. Part I. (Government Printer, South Australia, 1984).
23. Nelson, W. A. & yan, . G. Palmophyllum umbracola sp. nov. (Chlorophyta) from oshore islands of northern New Zealand.
Phycologia 25, 168–177 (1986).
24. Ballantine, D. L. & Norris, J. N. Verdigellas, a new deep-water genus (Tetrasporales, Chlorophyta) from the tropical western Atlantic.
Crypt. Bot. 4, 368–372 (1994).
25. Lemieux, C., Otis, C. & Turmel, M. A clade uniting the green algae Mesostigma viride and Chloroybus atmophyticu s represents the
deepest branch of the Streptophyta in chloroplast genome-based phylogenies. BMC Biol. 5, 2 (2007).
26. Fučíová, . et al. New phylogenetic hypotheses for the core Chlorophyta based on chloroplast sequence data. Front. Ecol. Evol. 2,
63 (2014).
27. Sun, L. et al. Chloroplast phylogenomic inference of green algae relationships. Sci. ep. 6, 20528 (2016).
28. Lemieux, C., Otis, C. & Turmel, M. Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant
evolution. Nature 403, 649–652 (2000).
www.nature.com/scientificreports/
12
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
29. Lang, B. F. & Nedelcu, A. M. Plastid genomes of algae in Genomics of chloroplasts and mitochondria Vol. 35 Advances in
Photosynthesis and espiration (eds Boc, . & noop, V.) Ch. 3, 59–87 (Springer Netherlands, 2012).
30. Turmel, M., Otis, C., Lemieux & C. e complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: Insights into
the architecture of ancestral chloroplast genomes. Proc. Natl Acad. Sci. USA 96, 10248–10253 (1999).
31. Worden, A. Z. et al. Green evolution and dynamic adaptations revealed by genomes of the marine picoeuaryotes Micromonas.
Science 324, 268–272 (2009).
32. obbens, S. et al. e complete chloroplast and mitochondrial DNA sequence of Ostreococcus tauri: organelle genomes of the
smallest euaryote are examples of compaction. Mol. Biol. Evol. 24, 956–968 (2007).
33. Leliaert, F. & Lopez-Bautista, J. M. The chloroplast genomes of Bryopsis plumosa and Tydemania expeditionis (Bryopsidales,
Chlorophyta): compact genomes and genes of bacterial origin. BMC Genomics 16, 204 (2015).
34. Melton, J. T. III, Leliaert, F., Tronholm, A. & Lopez-Bautista, J. M. e complete chloroplast and mitochondrial genomes of the green
macroalga Ulva sp. UNA00071828 (Ulvophyceae, Chlorophyta). PLos One 10, e0121020 (2015).
35. Huang, J. & Yue, J. Horizontal gene transfer in the evolution of photosynthetic euaryotes. J. Syst. Evol. 51, 13–29 (2013).
36. Turmel, M., Otis, C. & Lemieux, C. Dynamic evolution of the chloroplast genome in the green algal classes Pedinophyceae and
Trebouxiophyceae. Genome Biol. Evol. 7, 2062–2082 (2015).
37. Hasegawa, T. et al. Prasinoderma coloniale gen. et sp. nov., a new pelagic coccoid prasinophyte from the western Pacic ocean.
Phycologia 35, 170–176 (1996).
38. Miyashita, H., Iemoto, H., urano, N., Miyachi, S. & Chihara, M. Prasinococcus capsulatus gen. et sp. nov., a new marine coccoid
prasinophyte. J. Gen. Appl. Microbiol. 39, 571–582 (1993).
39. Jouenne, F. et al. Prasinoderma singularis sp. nov. (Prasinophyceae, Chlorophyta), a solitary coccoid prasinophyte from the South-
East Pacic Ocean. Protist 162, 70–84 (2011).
40. Umen, J. G. Green algae and the origins of multicellularity in the plant ingdom. Cold Spring Harb. Perspect. Biol. 6, a016170 (2014).
41. O’elly, C. J. Division of Palmoclathrus stipitatus (Chlorophyta) vegetative cells. Phycologia 27, 248–253 (1988).
42. Sieburth, J. M., eller, M. D., Johnson, P. W. & Mylestad, S. M. Widespread occurrence of the oceanic ultraplanter, Prasinococcus
capsulatus (Prasinophyceae), the diagnostic “Golgi-decapore complex” and the newly described polysaccharide “capsulan”. J. Phycol.
35, 1032–1043 (1999).
43. Guillard, . ., eller, M. D., O’elly, C. J. & Floyd, G. L. Pycnococcus provasolii gen. et sp. nov., a coccoid prasinoxanthincontaining
phytoplanter from the western north Atlantic and Gulf of Mexico. J. Phycol. 27, 39–47 (1991).
44. Lewis, L. A. & McCourt, . M. Green algae and the origin of land plants. Am. J. Bot. 91, 1535–1556 (2004).
45. Chadefaud, M. & Emberger, L. Traité de botanique systématique. Tome 1. Les végétaux non vasculaires (Cryptogamie). 1016 (Masson,
1960).
46. McNeill, J. et al. Internat ional Code of Nomenclature for algae, fungi, and plants (Melbourne Code) (2012).
47. Sym, S. D. Basal lineages of green algae: eir diversity and phylogeny in Marine Protists (eds Ohtsua, S. et al.) 89–105 (Springer, 2015).
48. Zerbino, D. . & Birney, E. Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome es. 18, 821–829
(2008).
49. Schattner, P., Broos, A. N. & Lowe, T. M. e tNAscan-SE, snoscan and snoGPS web servers for the detection of tNAs and
snoNAs. Nucleic Acids es. 33, W686–W689 (2005).
50. Lohse, M., Drechsel, O. & Boc, . OrganellarGenomeDAW (OGDAW): a tool for the easy generation of high-quality custom
graphical maps of plastid and mitochondrial genomes. Curr. Genet. 52, 267–274 (2007).
51. Mesquite: a modular system for evolutionary analysis Version 3.04, Available from http://mesquiteproject.org (2015).
52. Larin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).
53. Castresana, J. Selection of conserved blocs from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17,
540–552 (2000).
54. Edgar, . C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 1–19
(2004).
55. Darriba, D., Taboada, G. L., Doallo, . & Posada, D. ProtTest 3: fast selection of best-t models of protein evolution. Bioinformatics
27, 1164–1165 (2011).
56. Lanfear, ., Calcott, B., Ho, S. Y. W. & Guindon, S. PartitionFinder: Combined selection of partitioning schemes and substitution
models for phylogenetic analyses. Mol. Biol. Evol. 29, 1695–1701 (2012).
57. onquist, F. & Huelsenbec, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574
(2003).
58. ambaut, A., Suchard, M. A., Xie, D. & Drummond, A. J. Tracer v1.6, Available from http://beast.bio.ed.ac.u/Tracer (2014).
59. Stamatais, A. AxML Version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30,
1312–1313 (2014).
60. Lartillot, N., Lepage, T. & Blanquart, S. PhyloBayes 3: a Bayesian soware pacage for phylogenetic reconstruction and molecular
dating. Bioinformatics 25, 2286–2288 (2009).
61. Lartillot, N. & Philippe, H. A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process. Mol.
Biol. Evol. 21, 1095–1109 (2004).
62. Zhong, B. et al. Streptophyte algae and the origin of land plants revisited using heterogeneous models with three new algal
chloroplast genomes. Mol. Biol. Evol. 31, 177–183 (2014).
63. odríguez-Ezpeleta, N. et al. Detecting and overcoming systematic errors in genome-scale phylogenies. Syst. Biol. 56, 389–399
(2007).
64. Hrdy, I. et al. Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I. Nature 432,
618–622 (2004).
65. Drummond, A. J., Suchard, M. A., Xie, D. & ambaut, A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol.
29, 1969–1973 (2012).
66. Viprey, M., Guillou, L., Ferréol, M. & Vaulot, D. Wide genetic diversity of picoplantonic green algae (Chloroplastida) in the
Mediterranean Sea uncovered by a phylum-biased PC approach. Environ. Microbiol. 10, 1804–1822 (2008).
67. Miller, M. A., Pfeiffer, W. & Schwartz, T. Creating the CIPES Science Gateway for inference of large phylogenetic trees. In
Proceedings of the Gateway Computing Environments Worshop (GCE), New Orleans, 1–8 (2010).
68. Turmel, M., Otis, C. & Lemieux, C. e chloroplast genomes of the green algae Pedinomonas minor, Parachlorella essleri, and
Oocystis solitatia reveal a shared ancestry between the Pedinomonadales and Chlorellales. Mol. Biol. Evol. 26, 2317–2331 (2009).
Acknowledgements
is study was funded by e College of Arts & Sciences at e University of Alabama under the program
Research Stimulation Post-Doctoral Fellow, and the National Science Foundation through Assembling the
Tree of Life for Green Algae GrAToL (DEB 1036495), both to JLB, and NSF GrAToL (DEB 1036466) to KGK.
Additional funding was provided by the Department of Biological Sciences at UA to MSD. SF thanks the Coastal
Water Consortium of e Gulf of Mexico Research Initiative (GoMRI-I) for funding the collecting cruises.
www.nature.com/scientificreports/
13
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
is is contribution number 788 from the Southeast Environmental Research Center at Florida International
University. We thank Willem Prud’homme van Reine (Naturalis Biodiversity Center) for nomenclatural advice,
and Olivier De Clerck (Ghent University) and Heroen Verbruggen (Melbourne University) for useful discussion.
Author Contributions
F.L., A.T. and J.M.L.-B. conceived the study, S.F. and F.W.Z. provided samples. D.B. generated DNA sequence data
for Verdigellas. K.G.K. provided prasinophyte cpDNA data. F.L. and A.T. carried out the genome assemblies and
annotations, F.L., A.T., C.L., M.T. and M.S.D. performed the genomic and phylogenetic analyses, F.L., A.T., C.L.
and M.T. wrote the manuscript, and generated the gures, all authors contributed in discussing ideas, and data
interpretation, and read and approved the nal manuscript.
Additional Information
Accession numbers: e complete chloroplast genome and nuclear ribosomal DNA unit of Verdigellas peltata
have been deposited in the European Nucleotide Archive with INSDC (GenBank, EMBL-EBI/ENA, DDBJ)
accession numbers LT174527 and LT174528, respectively. Sequence alignments and trees are available from
TreeBase (study ID 19074).
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Leliaert, F. et al. Chloroplast phylogenomic analyses reveal the deepest-branching
lineage of the Chlorophyta, Palmophyllophyceae class. nov. Sci. Rep. 6, 25367; doi: 10.1038/srep25367 (2016).
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons
license, users will need to obtain permission from the license holder to reproduce the material. To view a copy
of this license, visit http://creativecommons.org/licenses/by/4.0/
