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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 affinity of the Palmophyllales using chloroplast genomic, and nuclear rDNA data. Phylogenetic analyses firmly 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.
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Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
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 anity 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, biagellate 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: or A.T. (email:
Received: 15 February 2016
Accepted: 15 April 2016
Published: 09 May 2016
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 reected 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 identied based on nuclear-encoded small
subunit (18S) rDNA sequences9–11,13–15. e anities among these clades, however, were poorly resolved in 18S
rDNA phylogenies, and only recently have phylogenetic relationships been elucidated with more condence
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 identied 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 undierentiated spherical cells embedded in an apparently
amorphous gelatinous matrix21–24. Although the Palmophyllales were identied 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 dicult 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 anities 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 identied 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 identied
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
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.
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
Figure 3. Shared gene pairs in the chloroplast genomes of early-diverging green algae. e gene pairs shared
by at least three taxa were identied 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.
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
dierent 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 Dayho6 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.
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 Dayho6 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 dierent 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-dened, attached macroscopic plants (thalli) composed of small, isolated, undierentiated 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 dierentiation40. 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
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 reects a great age of the divergences. Although dating the
phylogeny of green plants is a dicult 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 diversied 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).
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
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 classication of the Viridiplantae, the major clades of the Streptophyta and core Chlorophyta are clas-
sied 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.
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 dierent from Chadefaud,
Prasinococcales is an automatically typied 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: and in AlgaeBase (, the name has never been described nor validly published, hence
the formal description in this paper.
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
Sampling. Material of Verdigellas peltata was obtained from a dredged sample oshore 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 identied as Verdigellas peltata, and
DNA-conrmed 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! (
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 identied by blastn
similarity searches (E-value < 106) 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, e resulting extended contigs were visually examined,
Scientific RepoRts | 6:25367 | DOI: 10.1038/srep25367
and consensus sequences re-assembled to obtain a large scaold 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 identied by mapping the abovementioned 1305 green algal chloroplast gene sequences
against the cpDNA contigs using the read mapper in Geneious. e annotations were veried by identifying
ORFs in Geneious, followed by blastp searches against the NCBI nonredundant database (http://blast.ncbi.nlm., last accessed December 7, 2015). e boundaries of the rRNA genes were identied based on
a dataset of aligned complete rRNA genes from published green algal cpDNAs. tRNA genes were detected and
identied using tRNAscan-SE 1.2149. e circular genome map was drawn with OGDRAW50.
e contig containing the nuclear-encoded rDNA cistron was identied 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 identied 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:// 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 aer 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 Dayho6 recoding scheme:
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 aer 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 ( bioinformatique_
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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.
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
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
... The phylogenetic relationships within Prasinodermophytes and prasinophytes are difficult to resolve, as has been exemplified by the fact that different data types and analytical approaches have produced considerable nuclear-nuclear and plastid-nuclear discordance 29 . Plastid phylogenies consistently recovered the Prasinodermophyta as sister to Chlorophyta 11,29,30 , while nuclear analyses yielded inconsistent positions concerning the Prasinodermophyta 14,29 . Nuclear rDNA analyses and multigene concatenated analyses generally supported the Prasinodermophyta as sister to all other green plants 11,14,29 . ...
... Plastid phylogenies consistently recovered the Prasinodermophyta as sister to Chlorophyta 11,29,30 , while nuclear analyses yielded inconsistent positions concerning the Prasinodermophyta 14,29 . Nuclear rDNA analyses and multigene concatenated analyses generally supported the Prasinodermophyta as sister to all other green plants 11,14,29 . Conversely, in the coalescent analyses from 1KP initiative 29 , Prasinodermophyta was allied with Streptophyta, whereas Li et al. 14 recovered Prasinodermophyta as sister to all other green plants. ...
... The determination of whether to recognize one or two classes (Palmophyllophyceae and Prasinodermophyceae) is primarily a matter of taxonomic preference. However, considering the unresolved position of Prasinococcus within the clade (Supplementary Figs. 3 and 4) and the comparable genetic divergence observed within the Prasinodermophyta and the Mamiellophyceae, it may be preferable to adopt a single class, Palmophyllophyceae (including Prasinoderma, Prasinococcus, Palmophyllum, and Verdigellas), as initially defined by Leliaert et al. 11 . ...
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The Viridiplantae comprise two main clades, the Chlorophyta (including a diverse array of marine and freshwater green algae) and the Streptophyta (consisting of the freshwater charophytes and the land plants). Lineages sister to core Chlorophyta, informally refer to as prasinophytes, form a grade of mainly planktonic green algae. Recently, one of these lineages, Prasinodermophyta, which is previously grouped with prasinophytes, has been identified as the sister lineage to both Chlorophyta and Streptophyta. Resolving the deep relationships among green plants is crucial for understanding the historical impact of green algal diversity on marine ecology and geochemistry, but has been proven difficult given the ancient timing of the diversification events. Through extensive taxon and gene sampling, we conduct large-scale phylogenomic analyses to resolve deep relationships and reveal the Prasinodermophyta as the lineage sister to Chlorophyta, raising questions about the necessity of classifying the Prasinodermophyta as a distinct phylum. We unveil that incomplete lineage sorting is the main cause of discordance regarding the placement of Prasinodermophyta. Molecular dating analyses suggest that crown-group green plants and crown-group Prasinodermophyta date back to the Paleoproterozoic-Mesoproterozoic. Our study establishes a plausible link between oxygen levels in the Paleoproterozoic-Mesoproterozoic and the origin of Viridiplantae.
... RNA-seq has been used to investigate the evolutionary mechanism of Tigriopus californicus [88] and to reveal the population history dynamics of Nematostella vectensis [89]. Plastid genome studies have provided new insights into the early origins of green algae [90]. ...
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Seagrasses are higher flowering plants that live entirely in marine environments, with the greatest habitat variation occurring from land to sea. Genetic structure or population differentiation history is a hot topic in evolutionary biology, which is of great significance for understanding speciation. Genetic information is obtained from geographically distributed subpopulations, different subspecies, or strains of the same species using next-generation sequencing techniques. Genetic variation is identified by comparison with reference genomes. Genetic diversity is explored using population structure, principal component analysis (PCA), and phylogenetic relationships. Patterns of population genetic differentiation are elucidated by combining the isolation by distance (IBD) model, linkage disequilibrium levels, and genetic statistical analysis. Demographic history is simulated using effective population size, divergence time, and site frequency spectrum (SFS). Through various population genetic analyses, the genetic structure and historical population dynamics of seagrass can be clarified, and their evolutionary processes can be further explored at the molecular level to understand how evolutionary processes contributed to the formation of early ecological species and provide data support for seagrass conservation.
... Two of the four recognized Verdigellas species are known from Puerto Rico. Verdigellas represents a clade of ancient lineage within the Chlorophyta (Zechman et al. 2010;Leliaert et al. 2016). Verdigellas peltata D. L. Ballant. ...
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p>This treatment is a taxonomic study of the benthic species of Chlorophyta known from Puerto Rico, Caribbean Sea. In all, 2 classes, 8 orders, 25 families, 50 genera, and 150 species occur in the benthic marine communities in Puerto Rico. Along with date, place, and author(s) of valid publication for all genera, species, and infraspecific taxa, type locality information and descriptive accounts of vegetative morphological and reproductive anatomy are provided. Distribution of each species is given, and where relevant, comments on their habitat and taxonomic and nomenclature status are discussed. A key to the genera and keys to species within genera are included. Either an in situ image or an illustration accompanies most species. Two varieties each of Bryopsis pennata and Caulerpa cupressoides ; one variety each of Caulerpa chemnitzia, C. racemosa, and Udotea occidentalis ; and a single forma each of Halimeda opuntia and Udotea cyathiformis are reported for Puerto Rico for the first time. Avrainvillea mazei, Anadyomene howei, A. rhizoidifera, and Caulerpa parvifolia are also reported as new geographic records for Puerto Rico. A new section of Ulva , sect. Chaetomorphoides J. N. Norris et D. L. Ballant., is established. Descriptions of one new combination, Halimeda acerifolia (D. L. Ballant.) D. L. Ballant. et J. N. Norris, and two new forma, Udotea cyathiformis f. mesophotica D. L. Ballant, H. Ruiz, et J. N. Norris and U. occidentalis f. radiata D. L. Ballant. et J. N. Norris, are included. Smithsonian Contributions to Botany, number 117, x + 164 pages, 116 figures, 2023.</p
... Among green algae, Chlorophyta is one of the largest phyla with many species and a wide geographical distribution. Some can cause blooming in lentic systems during the summer season under favorable conditions [23][24][25]. Additionally, several microalgal strains of Chlorophyta have been used as biotechnological resources [26][27][28][29]. However, Chlorella-like species have at times been misidentified because of their morphological simplicity [30]. ...
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Graesiella emersonii is a commercially exploitable source of bioactive compounds and bio-fuels with potential applications in microalgae-based industries. Despite this, little taxonomical information is available. Therefore, proper identification and characterization are needed for the sustainable utilization of isolated microalgae. In this study, an axenically isolated unicellular green alga from the Geumgang Estuary, Korea was investigated for its morphological, molecular, and biochemical characteristics. The morphological characteristics were typical of G. emersonii. Molecular phylogenetic analysis of the 18S rDNA sequence verified that the isolate belonged to G. emersonii and was subsequently named G. emersonii GEGS21. It was isolated from brackish water, and its optimal growth temperature, salinity, and light intensity were at 28-32 °C, 0 M NaCl, and 130-160 µ mol m −2 s −1 , respectively. The strain thrived over a range of temperatures (5-40 °C) and withstood up to 0.5 M NaCl. The isolate was rich in omega-6 linoleic acid (C18:2 n-6, 26.3%) and palmitic acid (C16:0, 27.5%). The fuel quality properties were determined, and biodiesel from GEGS21 could be used as a biodiesel blend. Value-added carotenoids lutein (1.5 mg g −1 dry cell weight, DCW) and neoxanthin (1.2 mg g −1 DCW) were biosynthesized as accessory pigments by this microalga. The biomass of this microalga may serve as feedstock for biodiesel production as well as producing valuable ω-6 and carotenoids.
... Until 2021, members of a third Viridiplantae phylum, the Prasinodermophyta, were considered prasinophytes (i.e., the Prasinococcales), despite the fact that they often branched deep within the Chlorophyta based on 18S rRNA gene (35) and plastidial gene analyses (94). Prasinodermophytes are now divided into two orders, the Palmophyllales and Prasinococcales (represented herein by the genome of Prasinoderma coloniale) (100). ...
The colonization of land by plants generated opportunities for the rise of new heterotrophic life forms, including humankind. A unique event underpinned this massive change to earth ecosystems—the advent of eukaryotic green algae. Today, an abundant marine green algal group, the prasinophytes, alongside prasinodermophytes and nonmarine chlorophyte algae are facilitating insights into plant developments. Genome-level data allow identification of conserved proteins and protein families with extensive modifications, losses, or gains and expansion patterns that connect to niche specialization and diversification. Here, we contextualize attributes according to Viridiplantae evolutionary relationships, starting with orthologous protein families, and then focusing on key elements with marked differentiation, resulting in patchy distributions across green algae and plants. We place attention on peptidoglycan biosynthesis, important for plastid division and walls; phytochrome photosensors that are master regulators in plants; and carbohydrate-active enzymes, essential to all manner of carbohydrate biotransformations. Together with advances in algal model systems, these areas are ripe for discovering molecular roles and innovations within and across plant and algal lineages. Expected final online publication date for the Annual Review of Plant Biology, Volume 73 is May 2022. Please see for revised estimates.
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In the evolution of eukaryotes, the transition from unicellular to simple multicellular organisms has happened multiple times. For the development of complex multicellularity, characterized by sophisticated body plans and division of labor between specialized cells, symplasmic intercellular communication is supposed to be indispensable. We review the diversity of symplasmic connectivity among the eukaryotes and distinguish between distinct types of non-plasmodesmatal connections, plasmodesmata-like structures, and ‘canonical’ plasmodesmata on the basis of developmental, structural, and functional criteria. Focusing on the occurrence of plasmodesmata (-like) structures in extant taxa of fungi, brown algae (Phaeophyceae), green algae (Chlorophyta), and streptophyte algae, we present a detailed critical update on the available literature which is adapted to the present classification of these taxa and may serve as a tool for future work. From the data, we conclude that, actually, development of complex multicellularity correlates with symplasmic connectivity in many algal taxa, but there might be alternative routes. Furthermore, we deduce a four-step process towards the evolution of canonical plasmodesmata and demonstrate similarity of plasmodesmata in streptophyte algae and land plants with respect to the occurrence of an ER component. Finally, we discuss the urgent need for functional investigations and molecular work on cell connections in algal organisms.
Calculating amino-acid substitution models that are specific for individual protein data sets is often difficult due to the computational burden of estimating large numbers of rate parameters. In this study, we tested the computational efficiency and accuracy of five methods used to estimate substitution models, namely Codeml, FastMG, IQ-TREE, P4 (maximum likelihood), and P4 (Bayesian inference). Data-specific substitution models were estimated from simulated alignments (with different lengths) that were generated from a known simulation model and simulation tree. Each of the resulting data-specific substitution models was used to calculate the maximum likelihood score of the simulation tree and simulated data that was used to calculate the model, and compared with the maximum likelihood scores of the known simulation model and simulation tree on the same simulated data. Additionally, the commonly-used empirical models, cpREV and WAG, were assessed similarly. Data-specific models performed better than the empirical models, which under-fitted the simulated alignments, had the highest difference to the simulation model maximum-likelihood score, clustered further from the simulation model in principal component analysis ordination, and inferred less accurate trees. Data-specific models and the simulation model shared statistically indistinguishable maximum-likelihood scores, indicating that the five methods were reasonably accurate at estimating substitution models by this measure. Nevertheless, tree statistics showed differences between optimal maximum likelihood trees. Unlike other model estimating methods, trees inferred using data-specific models generated with IQ-TREE and P4 (maximum likelihood) were not significantly different from the trees derived from the simulation model in each analysis, indicating that these two methods alone were the most accurate at estimating data-specific models. To show the benefits of using data-specific protein models several published data sets were reanalysed using IQ-TREE-estimated models. These newly estimated models were a better fit to the data than the empirical models that were used by the original authors, often inferred longer trees, and resulted in different tree topologies in more than half of the re-analysed data sets. The results of this study show that software availability and high computation burden are not limitations to generating better-fitting data-specific amino-acid substitution models for phylogenetic analyses.
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The monograph is devoted to a review of the higher taxa of eu-karyotes and the approaches to the problem of their rank correlation. Significant milestones in the history of eukaryotic megasys-tematics are touched upon. The principles and approaches to the nomenclature of the higher taxa of eukaryotes are discussed. A revision of the system of eukaryotic organisms was carried out with re-ference to the original descriptions. The tendencies of general system transformation are considered and problems with some practical applications of eukaryotic megasystematics are discussed. The Obimoda subdomain, the Crumalia kingdom, the Mantamonadea, Rigifilidea, and Collodictyonidea phyla are described. A retrospective overview of basic systems of eukaryotes published in the period 1925—2022 and an extensive list of publications on megasystematics and microbiology of eukaryotes are presented. Tags: protozoans, algae, eukaryotes, megasystematics, taxon rank, eukaryotic supergroups, Cavalier-Smith, Ehrenberg, Opimoda, Loukozoa, Amoebozoa, Opisthokonta, Discoba, Euglenozoa, Archaeplastida, Cryptista, Haptista, Chromalveolata, Heterokonta, Provora, Telonemia, Oomycetes, Fungi, Florideae hypothesis
The diatoms evolved within the stramenopiles, an ecologically important and diverse assemblage of eukaryotes that includes both photosynthetic macrophytes and microalgae, as well as non-photosynthetic heterotrophs and parasites. The evolutionary history of the stramenopiles, which stretches back to the Palaeozoic, has been marked by the acquisition of chloroplasts in a recent common ancestor of their photosynthetic members, the ochrophytes; and progressive gains of genes in the nuclear genome by horizontal and endosymbiotic gene transfer. Here, we place diatoms in their actual evolutionary context within the stramenopiles; identify gene transfers that have shaped the coding content of the diatom nucleus; and profile sources of differences in chloroplast and mitochondrial genome content between different stramenopiles including diatoms. We underline the importance of considering diatoms as evolutionary mosaics, supported by genes of bacterial, red, green and other eukaryotic algal origins, as illustrated by multiple phylogenomic studies realised over the last two decades; and the relatively limited changes to organelle genome content in diatoms compared to other stramenopile lineages. We further identify a previously undocumented transfer of a novel open reading frame of the chloroplasts of green algae into the ochrophytes, underlining the importance of changes in organelle and nuclear gene content, in defining the current biology of diatoms.KeywordsHorizontal gene transferEndosymbiosisShopping bag modelCryptomonadsHaptophytesDinoflagellates
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Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but in- consistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.
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The green algal phylum Chlorophyta has six diverse classes, but the phylogenetic relationship of the classes within Chlorophyta remains uncertain. In order to better understand the ancient Chlorophyta evolution, we have applied a site pattern sorting method to study compositional heterogeneity and the model fit in the green algal chloroplast genomic data. We show that the fastest-evolving sites are significantly correlated with among-site compositional heterogeneity, and these sites have a much poorer fit to the evolutionary model. Our phylogenomic analyses suggest that the class Chlorophyceae is a monophyletic group, and the classes Ulvophyceae, Trebouxiophyceae and Prasinophyceae are non-monophyletic groups. Our proposed phylogenetic tree of Chlorophyta will offer new insights to investigate ancient green algae evolution, and our analytical framework will provide a useful approach for evaluating and mitigating the potential errors of phylogenomic inferences.
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Previous studies of trebouxiophycean chloroplast genomes revealed little information regarding the evolutionary dynamics of this genome because taxon sampling was too sparse and the relationships between the sampled taxa were unknown. We recently sequenced the chloroplast genomes of 27 trebouxiophycean and two pedinophycean green algae to resolve the relationships among the main lineages recognized for the Trebouxiophyceae. These taxa and the previously sampled members of the Pedinophyceae and Trebouxiophyceae are included in the comparative chloroplast genome analysis we report here. The 38 genomes examined display considerable variability at all levels, except gene content. Our results highlight the high propensity of the rDNA-containing large inverted repeat (IR) to vary in size, gene content and gene order as well as the repeated losses it experienced during trebouxiophycean evolution. Of the seven predicted IR losses, one event demarcates a superclade of 11 taxa representing five late-diverging lineages. IR expansions/contractions account not only for changes in gene content in this region, but also for changes in gene order and gene duplications. Inversions also led to gene rearrangements within the IR, including the reversal or disruption of the rDNA operon in some lineages. Most of the 20 IR-less genomes are more rearranged compared to their IR-containing homologs and tend to show an accelerated rate of sequence evolution. In the IR-less superclade, several ancestral operons were disrupted, a few genes were fragmented, and a subgroup of taxa features a G+C-biased nucleotide composition. Our analyses also unveiled putative cases of gene acquisitions through horizontal transfer. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
The basal lineages of the green algae (Chlorophyta) were historically merged in a single class of usually scaly green algal members, the Prasinophyceae. While ultrastructural data were more or less supportive of this idea, modern molecular phylogenetics is showing that a single class is untenable and members are currently accommodated in up to five new classes, (the Chlorodendrophyceae, Mamiellophyceae, Mesostigmatophyceae, Nephroselmidophyceae and Pedinophyceae), with more still to come. This chapter considers the characteristics of, and phylogenetic relationships between, these new classes and other related groups, as well as their representatives and, briefly, their ecology.
This chapter discusses the origin and early evolution of green plants. The chapter defines green plants and discusses the green plant body plans, and the evolutionary history of some of the basic types. The unicellular flagellate is considered to be the most ancestral green plant body. There are three main sexual life history patterns expressed in green plants. Each type is determined both by the evolutionary history of the species and the environment in which it has evolved. The chapter discusses the core structure of the green plant phylogenetic tree; the archegoniate line, the chlorophyte line, and the prasinophytes. The unresolved nodes in the phylogenetic tree, with special reference to those areas that are most relevant to larger issues of phototroph evolution in aquatic environments are discussed. A brief note on green plants in today's marine environment is also presented. Certain branch points, especially at the base of the tree and at important nodes such as the ones associated with the evolution of the "seaweed" genera and with the early radiations of the archegoniate plants, remain to be resolved. As nuclear genome sequencing becomes more prevalent, comparative data from this source will need to be employed.