BACKGROUND
the green algae represent an ancient lineage of photosynthetic eukaryotes; molecular clock analyses estimate their origin between  <dig> and  <dig>  millions years ago  <cit> .
this lineage  split very early into two major divisions: the chlorophyta, containing the majority of the described green algae, and the streptophyta, containing the charophyte green algae and their land plant descendants.
in the last decade, substantial advances have been made in our understanding of the broad-scale relationships among the streptophytes, in particular the land plants  <cit> , and references therein; however, progress has lagged behind concerning the chlorophytes.
early hypotheses on green algal phylogeny were based on morphology and ultrastructural data derived from the flagellar apparatus and processes of mitosis and cell division  <cit> .
these ultrastructural features, which apply to most green algae, supported the existence of the streptophyta and chlorophyta and revealed four distinct groups within the chlorophyta that were recognized as classes: the predominantly marine, unicellular, prasinophyceae; the predominantly marine and morphologically diverse ulvophyceae; and the freshwater or terrestrial, morphologically diverse trebouxiophyceae  and chlorophyceae  <cit> .
it was hypothesized that the prasinophyceae gave rise to the ulvophyceae, trebouxiophyceae and chlorophyceae .
later, phylogenetic analyses based on the nuclear-encoded small subunit rrna gene  largely corroborated these hypotheses  <cit> .
it was found, however, that the prasinophyceae are paraphyletic, with the nine main lineages of prasinophytes identified so far representing the earliest branches of the chlorophyta  <cit> .
for the ulvophyceae and trebouxiophyceae, the limited resolution of 18s rdna trees made it impossible to assess the monophyly of these classes  <cit> .
analyses of 18s rdna data uncovered a myriad of lineages within each of the three utc classes, but could not resolve their precise branching order.
despite these uncertainties, many taxonomic revisions have been implemented: new species not distinguished by light microscopy were described, new genera were erected, the circumscription of several main lineages was modified, and existing orders were elevated to the class level .
a recurrent theme that emerged from such studies is the finding that multiple genera containing taxa with reduced morphologies  are polyphyletic, with members often encompassing more than one class e.g.
for chlorella,  <cit> .
for ancient groups of eukaryotes such as the green algae, a large number of genes from many species need to be analyzed using reliable models of sequence evolution to resolve relationships at higher taxonomic levels  <cit> .
multi-gene data sets can be assembled by concatenating the sequences of protein-coding genes that are shared by the chloroplast or nuclear genomes.
the chloroplast phylogenomic studies reported so far for green algae have provided valuable insights into the phylogeny of prasinophytes  <cit> , streptophytes  <cit>  and the chlorophyceae  <cit> , but only limited information is currently available regarding the relationships within the trebouxiophyceae.
for the ulvophyceae, an analysis of ten concatenated gene sequences from both the nuclear and chloroplast genomes enabled cocquyt et al.
<cit>  to resolve the branching pattern of the main lineages of this class.
in this context, it is worth mentioning that datasets of concatenated nuclear and chloroplast genes have also proved very useful to reconstruct phylogenetic relationships within specific green algal orders  <cit> .
the present investigation is centered on the trebouxiophyceae as delineated by frield  <cit> .
this species-rich class displays remarkable variation in both morphology  and ecology   <cit> .
no flagellate vegetative form has been identified in this class.
several species  participate in symbioses with fungi to form lichens  <cit>  and others  occur as photosynthetic symbionts in ciliates, metazoa and plants  <cit> .
the trebouxiophyceae also comprises species that have lost photosynthetic capacity and have evolved free-living or parasitic heterotrophic lifestyles   <cit> .
aside from their intrinsic biological interest, trebouxiophycean algae have drawn the attention of the scientific community because of their potential utility in a variety of biotechnological applications such as the production of biofuels or other molecules of high economic value  <cit> .
phylogenies based on 18s rdna data have identified multiple lineages within the trebouxiophyceae, and these include the chlorellales, trebouxiales, microthamniales, and the prasiola, choricystis/botryococcus, watanabea, oocystis and geminella clades  <cit> .
while the majority of the observed monophyletic groups are composed of several genera, a number of lineages consist of a single species or genus .
the interrelationships between most of the trebouxiophycean lineages are still unresolved.
interestingly, taxa with highly different morphologies  have been recovered in the same clade, demonstrating that vegetative morphology can evolve relatively rapidly.
polyphyly has been reported not only in morphologically simple genera  <cit> , but also in those with colonial forms  <cit> .
in this study, we have sought to decipher the relationships among the main trebouxiophycean lineages and to evaluate the monophyly of the trebouxiophyceae.
toward these goals, we have analyzed data sets of  <dig> chloroplast dna -encoded proteins and genes spanning the broad range diversity of the trebouxiophyceae.
twenty-nine chlorophyte chloroplast genomes were newly sequenced to generate these data sets.
the trees we inferred using the maximum likelihood  and bayesian inference methods enabled us not only to clarify the internal structure of the trebouxiophyceae but also to gain insights into their ancestral status with regards to the type of environment they first colonized and their subsequent adaptations to different ecosystems.
RESULTS
in the course of this study, we generated the chloroplast genome sequences of  <dig> trebouxiophycean taxa, thus bringing to  <dig> the total number of trebouxiophyceans sampled in our phylogenetic analyses .
these taxa represent the variety of trebouxiophycean lineages that had been recognized prior to january 2013; at least two representatives were examined for each of the lineages ncluding multiple genera.
the chloroplast genome sequences of two flagellates belonging to the pedinophyceae  were also determined because pedinomonas minor, the previously sampled taxon from this group had been found to be related to the chlorellales and a member of the oocystis lineage in an earlier phylogenomic study  <cit> .
only the results of our phylogenetic analyses are presented here; in a separate article, we will report the salient features of the newly sequenced chloroplast genomes and discuss how these structural data advance understanding of chloroplast genome evolution in the chlorophyta.table  <dig>
pedinophycean and trebouxiophycean taxa used in the chloroplast phylogenomic analyses
taxa
source
a
accession no.
b
sequencing method
c
pedinophyceae
pedinomonas minor
pedinomonas tuberculata
trebouxiophyceae
chlorellales
pseudochloris wilhelmii
chlorella variabilis
chlorella vulgaris
dicloster acuatus
marvania geminata
parachlorella kessleri
choricystis/botryococcus
clade
botryococcus braunii
choricystis minor
coccomyxa subellipsoidea
elliptochloris bilobata
paradoxia multiseta
trebouxiophyceae sp.
geminella
clade
geminella minor
geminella terricola
gloeotilopsis sterilis
microthamniales
fusochloris perforata
microthamnion kuetzingianum
oocystis
clade
oocystis solitaria
planctonema lauterbornii
prasiola
clade
“chlorella” mirabilis
koliella longiseta
pabia signiensis
stichococcus bacillaris
trebouxiales
myrmecia israelensis
trebouxia aggregata
watanabea
clade
dictyochloropsis reticulata
watanabea reniformis
other lineages
pleurastrosarcina brevispinosa
d
“koliella” corcontica
leptosira terrestris
lobosphaera incisa
neocystis brevis
parietochloris pseudoalveolaris
xylochloris irregularis
athe taxa originate from the culture collections of algae at the university of goettingen , the university of texas at austin , the provasoli-guillard national center for marine algae and microbiota , the national institute of environmental studies in tsukuba , and charles university in prague .
bthe genbank accession number of the chloroplast genome is given for each taxon.
the asterisks denote the genomes that were sequenced during the course of this study.
csequencing methods are given only for the chloroplast genomes sequenced in this study.
dthis taxon, originally classified in the genus chlorosarcina, was assigned to the new genus pleurastrosarcina by sluiman and blommers  <cit> .
all data sets analyzed in our study were assembled from  <dig> cpdna-encoded proteins and taxon sampling included up to  <dig> green algal taxa, i.e.
the  <dig> trebouxiophyceans and pedinophyceans listed in table  <dig>   <dig> additional chlorophytes  and two streptophyte algae .
we favored the use of amino acid rather than nucleotide sequences in our phylogenomic study because, in analyses of ancient divergences, amino acid data sets are less prone than nucleotide data sets to saturation problems, convergent compositional biases and convergent codon-usage biases  <cit> .
we initiated our phylogenomic study by analyzing the amino acid data set comprising all  <dig> taxa .
note that some of the genes coding for the proteins analyzed are missing from a number of taxa, in particular from prasinophytes and chlorophyceans ; however, the proportion of missing data in the analyzed data sets does not exceed 6%.figure  <dig>
phylogeny of  <dig> chlorophytes inferred using a data set of  <dig>  positions assembled from  <dig> cpdna-encoded proteins.
the tree presented here is the best-scoring ml tree inferred under the gtr + Γ <dig> model.
support values are reported on the nodes: from top to bottom, or from left to right, are shown the posterior probability  values for the phylobayes catgtr + Γ <dig> analyses and the bootstrap support  values for the raxml gtr + Γ <dig>  lg4x and gcprev + Γ <dig> analyses.
black dots indicate that the corresponding branches received bs and pp values of 100% in all four analyses.
shaded areas identify the clades that are well supported in 18s rdna phylogenies.
the histograms on the left indicate the proportion of missing data for each taxon.
the scale bar denotes the estimated number of amino acid substitutions per site.
even though amino acid phylogenies are more robust to compositional effects than nucleotide phylogenies, they may still suffer from a general mutational pressure acting at the nucleotide level  <cit> .
for this reason, we also inferred trees from nucleotide data sets corresponding to the 63-taxon amino acid data set and examined whether they are congruent with those derived from amino acid data sets.
analysis of the amino acid data sets
the amino acid data set comprising all  <dig> taxa was analyzed with phylobayes using the site-heterogeneous catgtr + Γ <dig> model and also with raxml using the site-homogeneous gtr + Γ <dig> and gcprev + Γ <dig> models as well as the lg4x model .
gcprev is an empirical amino acid substitution model that has been recently developed for use with green plant chloroplast protein data  <cit> ; it proved to be the best-scoring empirical model among those we tested using raxml .
lg4x is a mixture model based on four substitution matrices  <cit> .
the fits of the gcprev + Γ <dig>  gtr + Γ <dig> and catgtr + Γ <dig> models to the 63-taxon data set were assessed using cross-validation .
catgtr + Γ <dig> was found to be the best-fitting model; this finding was expected considering that site-heterogeneous models are known to provide a better fit than site-homogeneous models and minimize the impact of systematic errors arising from the difficulties to detect and interpret multiple substitutions  <cit> .
because it was also found that the gtr + Γ <dig> model has a better fit than the gcprev + Γ <dig> model , it appears that the size of the 63-taxon data set is sufficiently large to estimate a gtr amino acid substitution matrix that models more accurately our data than the empirical gcprev matrix.table  <dig>
comparison of evolutionary models using cross validation and the chloroplast data set of  <dig>  positions
models compared
likelihood difference
the majority-rule consensus trees inferred from the 63-taxon amino acid data set using ml and bayesian inference methods displayed essentially the same topology .
as expected, the prasinophyte lineages represent the first branches and their divergence order is identical to that reported for a recent phylogenomic tree with the same sampling of prasinophyte taxa  <cit> .
the trebouxiophyceans are recovered as a non-monophyletic assemblage.
the monophyletic group formed by the six members of the chlorellales is sister to the pedinophyceae and the chlorellales + pedinophyceae clade is sister to all other utc algae.
the rest of the trebouxiophyceans, designated hereafter as core trebouxiophyceans, form a strongly supported clade that shares a sister relationship with the ulvophyceae + chlorophyceae clade.
the deep node of the trees coinciding with the common ancestor of the utc and pedinophycean algae received maximal support in all analyses, but the following node corresponding to the divergence of the core trebouxiophyceans from the chlorellales + pedinophyceae received lower support, especially in the ml analyses as indicated by the bs values of  <dig>   <dig> and 45%.
the  <dig> taxa within the core trebouxiophyceans are resolved as a grade of several strongly supported lineages.
three monophyletic groups containing multiple genera can be distinguished .
clade a, which consists of koliella corcontica and members of the previously recognized geminella and oocystis clades, represents the earliest-diverging lineage of the core trebouxiophyceans.
clade b includes neocystis brevis and representatives of the highly diversified prasiola clade.
clade c, the largest of the three identified monophyletic groups, consists of  <dig> taxa belonging to the xylochloris, microthamniales, trebouxiales, lobosphaera, watanabea, choricystis and elliptochloris clades.
clades a and b as well as clades b and c are separated from one another by a lineage consisting of a single taxon, i.e.
the pleurastrosarcina brevispinosa and the parietochloris pseudoalveolaris lineage, respectively.
considering that heterogeneity in amino acid composition may violate the stationarity assumption made by the evolutionary models in the analyses presented above, we explored whether the inferred relationships were affected by compositional-related artifacts.
as a first approach, we examined the amino acid composition of the data set by plotting the first two components of a correspondence analysis of the  <dig> amino acid frequencies  but identified no large deviation in composition of the chloroplast proteins among the taxa examined.
we also used the dayhoff recoding strategy, which recodes the  <dig> amino acids into six groups on the basis of their physical and chemical properties.
we found that the tree inferred from the dayhoff-recoded data set under the catgtr + Γ <dig> model exhibits the same topology as that obtained using standard  <dig> state models, except that the chlorellales are not affiliated with the pedinophyceae .
in this bayesian analysis, which showed convergence problems , the position of the chlorellales relative to the core trebouxiophyceans is unresolved, whereas the pedinophyceae is sister to the utc clade .
these observations together with the finding that the chlorellales and pedinophyceae are grouped in the correspondence analysis  suggest a possible compositional attraction between these two groups.figure  <dig>
correspondence analysis of amino acid usage in the data set of  <dig>  positions.
the members of the chlorellales and pedinophyceae are found within the circled area.
given the possibility that the affiliation between the chlorellales and pedinophyceae is caused by systematic errors of tree reconstruction, we tested whether removal of the three members of the pedinophyceae affects the position of the chlorellales.
as shown in figure 3a, the raxml tree inferred under the gtr + Γ <dig> model still identifies the chlorellales as sister to the chlorophyceae + ulvophyceae + core trebouxiophyceans .
to determine whether the two other possible positions occupied by the chlorellales  can be dismissed with statistical confidence, we carried out the approximately unbiased  test of phylogenetic tree selection  <cit> .
both topologies were found to be significantly different  from the best tree  and were thus rejected by the au test .figure  <dig>
influence of the pedinophyceae on the placement of the chlorellales.
phylogeny of chlorophytes inferred under the gtr + Γ <dig> model using the amino acid data set of  <dig>  positions after exclusion of the pedinophyceae.
the best-scoring raxml tree is presented and support values are reported on the nodes, with black dots indicating 100% bs values.
the scale bar denotes the estimated number of amino acid substitutions per site.
confidence assessment of the three possible topologies  for the placement of the chlorellales under the gtr + Γ <dig> model.
the Δlnl value indicates the difference in log likelihood relative to the best tree .
local bootstrap probabilities were estimated by resampling of the estimated log-likelihood .
pau, p value for the approximately unbiased  test  <cit>  as implemented in consel  <dig>   <cit> .
analysis of the nucleotide data sets
we analyzed two nucleotide data sets corresponding to the 63-taxon amino acid data set, both of which were designed to minimize deleterious effects of rapid sequence evolution and/or heterogeneous composition.
the degen <dig> data set comprises all three codon positions  that were degenerated using the degen <dig> pl script  <cit> , whereas the nt1 +  <dig> data set contains only the first and second codon positions .
the raxml trees inferred from these data sets under the gtr + Γ <dig> model display essentially the same trebouxiophycean relationships as in the 63-taxon amino acid tree , except that the marvania clade is sister to the chlorella + parachlorella clade  and that parietochloris pseudoalveolaris is recovered as sister to the prasiola clade .
as observed for the amino acid phylogenies, the chlorellales remained sister to the chlorophyceae + ulvophyceae + core trebouxiophyceans when the three algae belonging to the pedinophyceae were excluded from the sampled taxa .figure  <dig>
phylogeny of  <dig> chlorophytes inferred using nucleotide data sets assembled from  <dig> cpdna-encoded genes.
the tree presented here is the best-scoring ml tree inferred using the degen <dig> data set under the gtr + Γ <dig> model.
support values are reported on the nodes: from top to bottom, or from left to right, are shown the bs values for the analyses of the degen <dig> and nt1 +  <dig> data sets.
black dots indicate that the corresponding branches received bs values of 100% in the two analyses.
shaded areas identify the trebouxiophycean lineages uncovered in this study.
open and filled squares denote aquatic and terrestrial/aeroterrestrial habitats, respectively; an open square containing a star indicates that the taxon is a symbiont.
the scale bar denotes the estimated number of nucleotide substitutions per site.
discussion
identifying the relationships among the main lineages of the trebouxiophyceae is crucial for understanding the evolutionary history of this morphologically and ecologically diversified class of chlorophytes.
for the first time, a robust phylogeny of trebouxiophyceans with sampling of most of the lineages recognized on the basis of 18s rdna data is inferred using a phylogenomic approach.
our study reveals that the class trebouxiophyceae sensu stricto  <cit>  is not a monophyletic group.
in the chloroplast phylogenies we inferred from both amino acid and nucleotide data sets, the chlorellales and a core group containing all other  <dig> trebouxiophyceans constitute two distinct, strongly supported monophyletic groups that emerge before the chlorophyceae and ulvophyceae .
prior to our investigation, a number of multi-gene trees with sparse sampling of trebouxiophyceans had recovered with little support the trebouxiophyceae as nonmonophyletic  <cit> , thus casting doubt on the monophyletic status of this class.
to our knowledge, no morphological features can be invoked to support or refute the phylogenetic relationship we observed between the chlorellales and the core trebouxiophyceans.
mattox and stewart  <cit>  defined the class pleurastrophyceae  based on the ultrastructure of the flagellar apparatus  and features related to cytokinesis and mitosis .
because all members of the chlorellales lack motile stages and divide by autosporulation, the ultrastructural characters used by mattox and stewart are not available for this algal group, thus precluding an evaluation of the monophyletic status of the trebouxiophyceae sensu stricto  <cit> .
the phylogenetic relationships inferred in this study provide insights into the type of ecosystems colonized by the core trebouxiophyceans in their early evolutionary history .
considering that, like most of the chlorellaleans, the earliest-diverging core trebouxiophyceans  are predominantly planktonic species and that the core trebouxiophyceans occupying more derived lineages are mostly terrestrial algae, it appears that the first core trebouxiophyceans lived in aquatic ecosystems and that very early during evolution they evolved strategies to avoid desiccation  <cit>  and conquered the land.
this early transition from aquatic to terrestrial environments likely occurred just after the emergence of the oocystis/geminella clade.
in this context, it is worth mentioning that a subaerial lifestyle has been inferred for the last common ancestor of the early-diverging clade prasiola, which comprises terrestrial as well as aquatic species  <cit> .
therefore, the early evolution of desiccation tolerance undoubtedly accounts for the success of the core trebouxiophyceans in terrestrial/aeroterrestrial environments, and once this trait was acquired, reversals to aquatic habitats probably involved only minor molecular changes, explaining why transitions from terrestrial to aquatic habitats were frequent during the evolution of core trebouxiophyceans.
the main lineages of the core trebouxiophyceans
the core trebouxiophyceans form a grade of lineages, with several containing two or more genera and some containing a single known genus or taxon.
although the short internal branches separating the major clades of core trebouxiophyceans suggest that lineage diversification occurred rapidly, it is remarkable that only the placement of the single-taxon lineage occupied by the terrestrial alga parietochloris pseudoalveolaris is supported by modest bs values in both the amino acid and nucleotide analyses .
we highlight below the main evolutionary relationships uncovered for the core trebouxiophyceans in our chloroplast phylogenomic study.
the strongly supported assemblage formed by the oocystis and geminella clades represents the deepest branching trebouxiophycean lineage in both the protein- and dna-based phylogenies .
the placement of the oocystis clade within the core trebouxiophyceans contrasts sharply with the sister relationship of the oocystaceae and chlorellales observed in a number of 18s rdna studies  <cit> .
with regards to the geminella clade, we found that the “koliella” corcontica taxon is robustly allied with this clade and thus should be considered to be a bona fide member; this association was previously observed in a phylogeny inferred from 18s rdna, albeit with no support  <cit> .
the sarcinoid green alga pleurastrosarcina brevispinosa, for which no 18s rdna sequence is currently available in public databases, occupies the next branch after the oocystis/geminella lineages.
this desert crust alga, originally designated as chlorosarcina brevispinosa, was assigned to the genus pleurastrosarcina by sluiman and blommers  <cit> .
the phylogenies reported here confirm that this taxon belongs to the trebouxiophyceae and indicate that it represents a novel lineage of this class.
in a very recent study, fučíková et al.
<cit>  reported that most major trebouxiophycean lineages contain desert-dwelling taxa and presented evidence for three new lineages of free-living trebouxiophyceans found in north american desert soil crusts.
while the desertella lineage is nested within the watanabea clade, the eremochloris and xerochlorella lineages represent independent clades of the trebouxiophyceae.
in future studies, it will be interesting to investigate whether the sarcinoid pleurastrosarcina brevispinosa belongs to one of the latter lineages.
another lineage that should examined for a possible affinity with pleurastrosarcina is the leptochlorella clade, which was recently discovered by neustupa et al.
<cit>  and further delineated by fučíková et al.
<cit> .
the branching order observed for the representatives of the prasiola clade is mostly congruent with 18s rdna phylogenies  <cit> , and in agreement with the studies of krienitz et al.
<cit>  and gaysina et al.
<cit> , the crescent-shaped green alga neocystis brevis is recovered as sister to this clade.
given that this affiliation is supported with maximal bs values in all analyses, the neocystis lineage clearly represents a basal branch of the prasiola clade.
chlorella mirabilis shares a sister relationship with the pabia + koliella clade in all our analyses ; in contrast, 18s rdna trees frequently identify c. mirabilis as sister to all other lineages of the prasiola clade  <cit> .
the coccoid soil alga parietochloris pseudoalveolaris forms an independent lineage between the prasiola clade and the monophyletic group uniting the microthamniales and the xylochloris clade in the amino acid-based phylogeny .
parietochloris is allied with the microthamniales in a number of published 18s rdna trees  <cit> , but this alliance is weakly supported.
the xylochloris clade is a newly identified assemblage of two lineages for which no sister groups were previously identified; it consists of the coccoid subaerial alga xylochloris irregularis and the filamentatous soil alga leptosira terrestris.
the recent discovery of a coccoid soil alga  belonging to a lineage related to the genus leptosira suggests that the xylochloris clade likely represents a diversified group of trebouxiophyceans  <cit> .
the five remaining clades of core trebouxiophyceans consist of the trebouxiales and the lobosphaera, watanabea, choricystis and elliptochloris clades.
members of all these clades, except the lobosphaera lineage, include algae that occur as symbionts; the trebouxiales, in particular, are the most common photobionts in lichens.
the branching order reported here for the five clades of core trebouxiophyceans was not observed in 18s rdna trees, even though these clades were often found as neighboring lineages.
only the most recent divergence of core trebouxiophycean lineages we identified  was also recivered in 18s rdna studies  <cit> , but with no support.
in contrast to 18s rdna trees where the trebouxiales and the lobosphaera clade display an unsupported sister relationship  <cit> , the lobosphaera clade consistently emerges with strong support as an independent lineage after the trebouxiales in all chloroplast trees.
the chlorellales and their relationship with other core chlorophytes
three distinct clades of chlorellales were recovered in this study: the parachlorella, chlorella and marvania clades .
as observed by somogyi et al.
<cit>  in 18s rdna trees , we found that the parachlorella clade is sister to the other two lineages in most amino acid-based trees; however, this position is occupied by the marvania clade in the phylogenies inferred from nucleotide data.
a recent 18s rdna study  <cit>  recovered pseudochloris wilhelmii and the parachlorella and chlorella clades as part of a large assemblage that is sister to marvania, a topology that contrasts with the finding that marvania and pseudochloris are sister taxa in all our analyses.
the results presented here reveal an affinity between the chlorellales and the pedinophyceae, although support is weak in the bayesian analysis under the catgtr + Γ <dig> model .
this finding is consistent with previous chloroplast phylogenomic studies with scarce sampling of trebouxiophyceans, wherein the freshwater flagellate pedinomonas minor was found to be sister to the clade formed by members of the chlorellales  <cit> .
but subsequently, marin  <cit>  identified no association between the pedinophyceae and the chlorellales using nuclear and chloroplast rrna operon data sets, the pedinophyceae being placed as an independent lineage that is sister to the chlorodendrophyceae + utc.
note that the clade formed by the chlorellales and other trebouxiophyceans was not supported with high confidence in these rrna operon trees and that the branching order of most trebouxiophycean lineages was unresolved.
given the conflicting positions of the chlorellales and pedinophyceae in the aforementioned analyses, the weak pp support that the chlorellales + pedinophyceae clade received in the phylobayes analyses of the amino acid data set and the basal position occupied by the pedinophyceae in trees inferred from the dayhoff-recoded data set, we conclude that the question as to whether the chlorellales and pedinophyceae form a monophyletic group remains unsettled.
it is possible that the chlorellales + pedinophyceae affiliation is the result of systematic errors of phylogenetic reconstructions.
solving this issue will require sampling of the chlorodendrophyceae and the inclusion of additional taxa from the ulvophyceae and the lineage represented by the prasinophyte ccmp  <dig>  the two ulvophycean taxa used in our study represent distinct basal lineages of the ulvophyceae ; taxa from the bcdt  and ignatius clades will need to be examined for a more representative sampling of ulvophycean diversity  <cit> .
we expect that resolving the ancient and rapid radiations of the core chlorophyte lineages  using a chloroplast phylogenomic approach will be challenging and will require optimized models of sequence evolution.
CONCLUSIONS
the phylogeny reported in this study forms a solid basis for future studies aimed at advancing knowledge about the nature of the morphological and ecological diversification of the trebouxiophyceae.
it provides important insights into the origins and adaptations of terrestrial and symbiotic lifestyles.
members of this group clearly occupy a pivotal position in the viridiplantae and display considerable genetic diversity.
a fundamental understanding of the molecular mechanisms underlying their adaptations to changing environments will require the analysis of genomes from key trebouxiophycean taxa.

 the green algae represent an ancient lineage of photosynthetic eukaryotes; molecular clock analyses estimate their origin between and millions years ago. this lineage split very early into two major divisions: the chlorophyta, containing the majority of the described green algae, and the streptophyta, containing the charophyte green algae and their land plant descendants. in the last decade, substantial advances have been made in our understanding of the broad-scale relationships among the streptophytes, in particular the land plants, and references therein; however, progress has lagged behind concerning the chlorophytes. early hypotheses on green algal phylogeny were based on morphology and ultrastructural data derived from the flagellar apparatus and processes of mitosis and cell division. these ultrastructural features, which apply to most green algae, supported the existence of the streptophyta and chlorophyta and revealed four distinct groups within the chlorophyta that were recognized as classes: the predominantly marine, unicellular, prasinophyceae; the predominantly marine and morphologically diverse ulvophyceae; and the freshwater or terrestrial, morphologically diverse trebouxiophyceae and chlorophyceae. it was hypothesized that the prasinophyceae gave rise to the ulvophyceae, trebouxiophyceae and chlorophyceae. later, phylogenetic analyses based on the nuclear-encoded small subunit rrna gene largely corroborated these hypotheses. it was found, however, that the prasinophyceae are paraphyletic, with the nine main lineages of prasinophytes identified so far representing the earliest branches of the chlorophyta. for the ulvophyceae and trebouxiophyceae, the limited resolution of 18s rdna trees made it impossible to assess the monophyly of these classes. analyses of 18s rdna data uncovered a myriad of lineages within each of the three utc classes, but could not resolve their precise branching order. despite these uncertainties, many taxonomic revisions have been implemented: new species not distinguished by light microscopy were described, new genera were erected, the circumscription of several main lineages was modified, and existing orders were elevated to the class level. a recurrent theme that emerged from such studies is the finding that multiple genera containing taxa with reduced morphologies are polyphyletic, with members often encompassing more than one class e.g. for chlorella,. for ancient groups of eukaryotes such as the green algae, a large number of genes from many species need to be analyzed using reliable models of sequence evolution to resolve relationships at higher taxonomic levels. multi-gene data sets can be assembled by concatenating the sequences of protein-coding genes that are shared by the chloroplast or nuclear genomes. the chloroplast phylogenomic studies reported so far for green algae have provided valuable insights into the phylogeny of prasinophytes, streptophytes and the chlorophyceae, but only limited information is currently available regarding the relationships within the trebouxiophyceae. for the ulvophyceae, an analysis of ten concatenated gene sequences from both the nuclear and chloroplast genomes enabled cocquyt et al. to resolve the branching pattern of the main lineages of this class. in this context, it is worth mentioning that datasets of concatenated nuclear and chloroplast genes have also proved very useful to reconstruct phylogenetic relationships within specific green algal orders. the present investigation is centered on the trebouxiophyceae as delineated by frield. this species-rich class displays remarkable variation in both morphology and ecology. no flagellate vegetative form has been identified in this class. several species participate in symbioses with fungi to form lichens and others occur as photosynthetic symbionts in ciliates, metazoa and plants. the trebouxiophyceae also comprises species that have lost photosynthetic capacity and have evolved free-living or parasitic heterotrophic lifestyles. aside from their intrinsic biological interest, trebouxiophycean algae have drawn the attention of the scientific community because of their potential utility in a variety of biotechnological applications such as the production of biofuels or other molecules of high economic value. phylogenies based on 18s rdna data have identified multiple lineages within the trebouxiophyceae, and these include the chlorellales, trebouxiales, microthamniales, and the prasiola, choricystis/botryococcus, watanabea, oocystis and geminella clades. while the majority of the observed monophyletic groups are composed of several genera, a number of lineages consist of a single species or genus. the interrelationships between most of the trebouxiophycean lineages are still unresolved. interestingly, taxa with highly different morphologies have been recovered in the same clade, demonstrating that vegetative morphology can evolve relatively rapidly. polyphyly has been reported not only in morphologically simple genera, but also in those with colonial forms. in this study, we have sought to decipher the relationships among the main trebouxiophycean lineages and to evaluate the monophyly of the trebouxiophyceae. toward these goals, we have analyzed data sets of chloroplast dna -encoded proteins and genes spanning the broad range diversity of the trebouxiophyceae. twenty-nine chlorophyte chloroplast genomes were newly sequenced to generate these data sets. the trees we inferred using the maximum likelihood and bayesian inference methods enabled us not only to clarify the internal structure of the trebouxiophyceae but also to gain insights into their ancestral status with regards to the type of environment they first colonized and their subsequent adaptations to different ecosystems.  in the course of this study, we generated the chloroplast genome sequences of trebouxiophycean taxa, thus bringing to the total number of trebouxiophyceans sampled in our phylogenetic analyses. these taxa represent the variety of trebouxiophycean lineages that had been recognized prior to january 2013; at least two representatives were examined for each of the lineages ncluding multiple genera. the chloroplast genome sequences of two flagellates belonging to the pedinophyceae were also determined because pedinomonas minor, the previously sampled taxon from this group had been found to be related to the chlorellales and a member of the oocystis lineage in an earlier phylogenomic study. only the results of our phylogenetic analyses are presented here; in a separate article, we will report the salient features of the newly sequenced chloroplast genomes and discuss how these structural data advance understanding of chloroplast genome evolution in the chlorophyta.table pedinophycean and trebouxiophycean taxa used in the chloroplast phylogenomic analyses taxa source a accession no. b sequencing method c pedinophyceae     pedinomonas minor     pedinomonas tuberculata     trebouxiophyceae     chlorellales     pseudochloris wilhelmii     chlorella variabilis     chlorella vulgaris     dicloster acuatus     marvania geminata     parachlorella kessleri     choricystis/botryococcus clade     botryococcus braunii     choricystis minor     coccomyxa subellipsoidea     elliptochloris bilobata     paradoxia multiseta     trebouxiophyceae sp.     geminella clade     geminella minor     geminella terricola     gloeotilopsis sterilis     microthamniales     fusochloris perforata     microthamnion kuetzingianum     oocystis clade     oocystis solitaria     planctonema lauterbornii     prasiola clade     “chlorella” mirabilis     koliella longiseta     pabia signiensis     stichococcus bacillaris         trebouxiales     myrmecia israelensis     trebouxia aggregata     watanabea clade     dictyochloropsis reticulata     watanabea reniformis     other lineages     pleurastrosarcina brevispinosa d     “koliella” corcontica     leptosira terrestris     lobosphaera incisa     neocystis brevis     parietochloris pseudoalveolaris     xylochloris irregularis athe taxa originate from the culture collections of algae at the university of goettingen, the university of texas at austin, the provasoli-guillard national center for marine algae and microbiota, the national institute of environmental studies in tsukuba, and charles university in prague. bthe genbank accession number of the chloroplast genome is given for each taxon. the asterisks denote the genomes that were sequenced during the course of this study. csequencing methods are given only for the chloroplast genomes sequenced in this study. dthis taxon, originally classified in the genus chlorosarcina, was assigned to the new genus pleurastrosarcina by sluiman and blommers. all data sets analyzed in our study were assembled from cpdna-encoded proteins and taxon sampling included up to green algal taxa, i.e. the trebouxiophyceans and pedinophyceans listed in table  additional chlorophytes and two streptophyte algae. we favored the use of amino acid rather than nucleotide sequences in our phylogenomic study because, in analyses of ancient divergences, amino acid data sets are less prone than nucleotide data sets to saturation problems, convergent compositional biases and convergent codon-usage biases. we initiated our phylogenomic study by analyzing the amino acid data set comprising all taxa. note that some of the genes coding for the proteins analyzed are missing from a number of taxa, in particular from prasinophytes and chlorophyceans; however, the proportion of missing data in the analyzed data sets does not exceed 6%.figure phylogeny of chlorophytes inferred using a data set of positions assembled from cpdna-encoded proteins. the tree presented here is the best-scoring ml tree inferred under the gtr + Γ model. support values are reported on the nodes: from top to bottom, or from left to right, are shown the posterior probability values for the phylobayes catgtr + Γ analyses and the bootstrap support values for the raxml gtr + Γ lg4x and gcprev + Γ analyses. black dots indicate that the corresponding branches received bs and pp values of 100% in all four analyses. shaded areas identify the clades that are well supported in 18s rdna phylogenies. the histograms on the left indicate the proportion of missing data for each taxon. the scale bar denotes the estimated number of amino acid substitutions per site. even though amino acid phylogenies are more robust to compositional effects than nucleotide phylogenies, they may still suffer from a general mutational pressure acting at the nucleotide level. for this reason, we also inferred trees from nucleotide data sets corresponding to the 63-taxon amino acid data set and examined whether they are congruent with those derived from amino acid data sets. analysis of the amino acid data sets the amino acid data set comprising all taxa was analyzed with phylobayes using the site-heterogeneous catgtr + Γ model and also with raxml using the site-homogeneous gtr + Γ and gcprev + Γ models as well as the lg4x model. gcprev is an empirical amino acid substitution model that has been recently developed for use with green plant chloroplast protein data; it proved to be the best-scoring empirical model among those we tested using raxml. lg4x is a mixture model based on four substitution matrices. the fits of the gcprev + Γ gtr + Γ and catgtr + Γ models to the 63-taxon data set were assessed using cross-validation. catgtr + Γ was found to be the best-fitting model; this finding was expected considering that site-heterogeneous models are known to provide a better fit than site-homogeneous models and minimize the impact of systematic errors arising from the difficulties to detect and interpret multiple substitutions. because it was also found that the gtr + Γ model has a better fit than the gcprev + Γ model, it appears that the size of the 63-taxon data set is sufficiently large to estimate a gtr amino acid substitution matrix that models more accurately our data than the empirical gcprev matrix.table comparison of evolutionary models using cross validation and the chloroplast data set of positions models compared likelihood difference the majority-rule consensus trees inferred from the 63-taxon amino acid data set using ml and bayesian inference methods displayed essentially the same topology. as expected, the prasinophyte lineages represent the first branches and their divergence order is identical to that reported for a recent phylogenomic tree with the same sampling of prasinophyte taxa. the trebouxiophyceans are recovered as a non-monophyletic assemblage. the monophyletic group formed by the six members of the chlorellales is sister to the pedinophyceae and the chlorellales + pedinophyceae clade is sister to all other utc algae. the rest of the trebouxiophyceans, designated hereafter as core trebouxiophyceans, form a strongly supported clade that shares a sister relationship with the ulvophyceae + chlorophyceae clade. the deep node of the trees coinciding with the common ancestor of the utc and pedinophycean algae received maximal support in all analyses, but the following node corresponding to the divergence of the core trebouxiophyceans from the chlorellales + pedinophyceae received lower support, especially in the ml analyses as indicated by the bs values of and 45%. the taxa within the core trebouxiophyceans are resolved as a grade of several strongly supported lineages. three monophyletic groups containing multiple genera can be distinguished. clade a, which consists of koliella corcontica and members of the previously recognized geminella and oocystis clades, represents the earliest-diverging lineage of the core trebouxiophyceans. clade b includes neocystis brevis and representatives of the highly diversified prasiola clade. clade c, the largest of the three identified monophyletic groups, consists of taxa belonging to the xylochloris, microthamniales, trebouxiales, lobosphaera, watanabea, choricystis and elliptochloris clades. clades a and b as well as clades b and c are separated from one another by a lineage consisting of a single taxon, i.e. the pleurastrosarcina brevispinosa and the parietochloris pseudoalveolaris lineage, respectively. considering that heterogeneity in amino acid composition may violate the stationarity assumption made by the evolutionary models in the analyses presented above, we explored whether the inferred relationships were affected by compositional-related artifacts. as a first approach, we examined the amino acid composition of the data set by plotting the first two components of a correspondence analysis of the amino acid frequencies but identified no large deviation in composition of the chloroplast proteins among the taxa examined. we also used the dayhoff recoding strategy, which recodes the amino acids into six groups on the basis of their physical and chemical properties. we found that the tree inferred from the dayhoff-recoded data set under the catgtr + Γ model exhibits the same topology as that obtained using standard state models, except that the chlorellales are not affiliated with the pedinophyceae. in this bayesian analysis, which showed convergence problems, the position of the chlorellales relative to the core trebouxiophyceans is unresolved, whereas the pedinophyceae is sister to the utc clade. these observations together with the finding that the chlorellales and pedinophyceae are grouped in the correspondence analysis suggest a possible compositional attraction between these two groups.figure correspondence analysis of amino acid usage in the data set of positions. the members of the chlorellales and pedinophyceae are found within the circled area. given the possibility that the affiliation between the chlorellales and pedinophyceae is caused by systematic errors of tree reconstruction, we tested whether removal of the three members of the pedinophyceae affects the position of the chlorellales. as shown in figure 3a, the raxml tree inferred under the gtr + Γ model still identifies the chlorellales as sister to the chlorophyceae + ulvophyceae + core trebouxiophyceans. to determine whether the two other possible positions occupied by the chlorellales can be dismissed with statistical confidence, we carried out the approximately unbiased test of phylogenetic tree selection. both topologies were found to be significantly different from the best tree and were thus rejected by the au test.figure influence of the pedinophyceae on the placement of the chlorellales. phylogeny of chlorophytes inferred under the gtr + Γ model using the amino acid data set of positions after exclusion of the pedinophyceae. the best-scoring raxml tree is presented and support values are reported on the nodes, with black dots indicating 100% bs values. the scale bar denotes the estimated number of amino acid substitutions per site. confidence assessment of the three possible topologies for the placement of the chlorellales under the gtr + Γ model. the Δlnl value indicates the difference in log likelihood relative to the best tree. local bootstrap probabilities were estimated by resampling of the estimated log-likelihood. pau, p value for the approximately unbiased test as implemented in consel. analysis of the nucleotide data sets we analyzed two nucleotide data sets corresponding to the 63-taxon amino acid data set, both of which were designed to minimize deleterious effects of rapid sequence evolution and/or heterogeneous composition. the degen data set comprises all three codon positions that were degenerated using the degen pl script, whereas the nt1 +  data set contains only the first and second codon positions. the raxml trees inferred from these data sets under the gtr + Γ model display essentially the same trebouxiophycean relationships as in the 63-taxon amino acid tree, except that the marvania clade is sister to the chlorella + parachlorella clade and that parietochloris pseudoalveolaris is recovered as sister to the prasiola clade. as observed for the amino acid phylogenies, the chlorellales remained sister to the chlorophyceae + ulvophyceae + core trebouxiophyceans when the three algae belonging to the pedinophyceae were excluded from the sampled taxa.figure phylogeny of chlorophytes inferred using nucleotide data sets assembled from cpdna-encoded genes. the tree presented here is the best-scoring ml tree inferred using the degen data set under the gtr + Γ model. support values are reported on the nodes: from top to bottom, or from left to right, are shown the bs values for the analyses of the degen and nt1 +  data sets. black dots indicate that the corresponding branches received bs values of 100% in the two analyses. shaded areas identify the trebouxiophycean lineages uncovered in this study. open and filled squares denote aquatic and terrestrial/aeroterrestrial habitats, respectively; an open square containing a star indicates that the taxon is a symbiont. the scale bar denotes the estimated number of nucleotide substitutions per site. discussion identifying the relationships among the main lineages of the trebouxiophyceae is crucial for understanding the evolutionary history of this morphologically and ecologically diversified class of chlorophytes. for the first time, a robust phylogeny of trebouxiophyceans with sampling of most of the lineages recognized on the basis of 18s rdna data is inferred using a phylogenomic approach. our study reveals that the class trebouxiophyceae sensu stricto is not a monophyletic group. in the chloroplast phylogenies we inferred from both amino acid and nucleotide data sets, the chlorellales and a core group containing all other trebouxiophyceans constitute two distinct, strongly supported monophyletic groups that emerge before the chlorophyceae and ulvophyceae. prior to our investigation, a number of multi-gene trees with sparse sampling of trebouxiophyceans had recovered with little support the trebouxiophyceae as nonmonophyletic, thus casting doubt on the monophyletic status of this class. to our knowledge, no morphological features can be invoked to support or refute the phylogenetic relationship we observed between the chlorellales and the core trebouxiophyceans. mattox and stewart defined the class pleurastrophyceae based on the ultrastructure of the flagellar apparatus and features related to cytokinesis and mitosis. because all members of the chlorellales lack motile stages and divide by autosporulation, the ultrastructural characters used by mattox and stewart are not available for this algal group, thus precluding an evaluation of the monophyletic status of the trebouxiophyceae sensu stricto. the phylogenetic relationships inferred in this study provide insights into the type of ecosystems colonized by the core trebouxiophyceans in their early evolutionary history. considering that, like most of the chlorellaleans, the earliest-diverging core trebouxiophyceans are predominantly planktonic species and that the core trebouxiophyceans occupying more derived lineages are mostly terrestrial algae, it appears that the first core trebouxiophyceans lived in aquatic ecosystems and that very early during evolution they evolved strategies to avoid desiccation and conquered the land. this early transition from aquatic to terrestrial environments likely occurred just after the emergence of the oocystis/geminella clade. in this context, it is worth mentioning that a subaerial lifestyle has been inferred for the last common ancestor of the early-diverging clade prasiola, which comprises terrestrial as well as aquatic species. therefore, the early evolution of desiccation tolerance undoubtedly accounts for the success of the core trebouxiophyceans in terrestrial/aeroterrestrial environments, and once this trait was acquired, reversals to aquatic habitats probably involved only minor molecular changes, explaining why transitions from terrestrial to aquatic habitats were frequent during the evolution of core trebouxiophyceans. the main lineages of the core trebouxiophyceans the core trebouxiophyceans form a grade of lineages, with several containing two or more genera and some containing a single known genus or taxon. although the short internal branches separating the major clades of core trebouxiophyceans suggest that lineage diversification occurred rapidly, it is remarkable that only the placement of the single-taxon lineage occupied by the terrestrial alga parietochloris pseudoalveolaris is supported by modest bs values in both the amino acid and nucleotide analyses. we highlight below the main evolutionary relationships uncovered for the core trebouxiophyceans in our chloroplast phylogenomic study. the strongly supported assemblage formed by the oocystis and geminella clades represents the deepest branching trebouxiophycean lineage in both the protein- and dna-based phylogenies. the placement of the oocystis clade within the core trebouxiophyceans contrasts sharply with the sister relationship of the oocystaceae and chlorellales observed in a number of 18s rdna studies. with regards to the geminella clade, we found that the “koliella” corcontica taxon is robustly allied with this clade and thus should be considered to be a bona fide member; this association was previously observed in a phylogeny inferred from 18s rdna, albeit with no support. the sarcinoid green alga pleurastrosarcina brevispinosa, for which no 18s rdna sequence is currently available in public databases, occupies the next branch after the oocystis/geminella lineages. this desert crust alga, originally designated as chlorosarcina brevispinosa, was assigned to the genus pleurastrosarcina by sluiman and blommers. the phylogenies reported here confirm that this taxon belongs to the trebouxiophyceae and indicate that it represents a novel lineage of this class. in a very recent study, fučíková et al. reported that most major trebouxiophycean lineages contain desert-dwelling taxa and presented evidence for three new lineages of free-living trebouxiophyceans found in north american desert soil crusts. while the desertella lineage is nested within the watanabea clade, the eremochloris and xerochlorella lineages represent independent clades of the trebouxiophyceae. in future studies, it will be interesting to investigate whether the sarcinoid pleurastrosarcina brevispinosa belongs to one of the latter lineages. another lineage that should examined for a possible affinity with pleurastrosarcina is the leptochlorella clade, which was recently discovered by neustupa et al. and further delineated by fučíková et al.. the branching order observed for the representatives of the prasiola clade is mostly congruent with 18s rdna phylogenies, and in agreement with the studies of krienitz et al. and gaysina et al., the crescent-shaped green alga neocystis brevis is recovered as sister to this clade. given that this affiliation is supported with maximal bs values in all analyses, the neocystis lineage clearly represents a basal branch of the prasiola clade. chlorella mirabilis shares a sister relationship with the pabia + koliella clade in all our analyses; in contrast, 18s rdna trees frequently identify c. mirabilis as sister to all other lineages of the prasiola clade. the coccoid soil alga parietochloris pseudoalveolaris forms an independent lineage between the prasiola clade and the monophyletic group uniting the microthamniales and the xylochloris clade in the amino acid-based phylogeny. parietochloris is allied with the microthamniales in a number of published 18s rdna trees, but this alliance is weakly supported. the xylochloris clade is a newly identified assemblage of two lineages for which no sister groups were previously identified; it consists of the coccoid subaerial alga xylochloris irregularis and the filamentatous soil alga leptosira terrestris. the recent discovery of a coccoid soil alga belonging to a lineage related to the genus leptosira suggests that the xylochloris clade likely represents a diversified group of trebouxiophyceans. the five remaining clades of core trebouxiophyceans consist of the trebouxiales and the lobosphaera, watanabea, choricystis and elliptochloris clades. members of all these clades, except the lobosphaera lineage, include algae that occur as symbionts; the trebouxiales, in particular, are the most common photobionts in lichens. the branching order reported here for the five clades of core trebouxiophyceans was not observed in 18s rdna trees, even though these clades were often found as neighboring lineages. only the most recent divergence of core trebouxiophycean lineages we identified was also recivered in 18s rdna studies, but with no support. in contrast to 18s rdna trees where the trebouxiales and the lobosphaera clade display an unsupported sister relationship, the lobosphaera clade consistently emerges with strong support as an independent lineage after the trebouxiales in all chloroplast trees. the chlorellales and their relationship with other core chlorophytes three distinct clades of chlorellales were recovered in this study: the parachlorella, chlorella and marvania clades. as observed by somogyi et al. in 18s rdna trees, we found that the parachlorella clade is sister to the other two lineages in most amino acid-based trees; however, this position is occupied by the marvania clade in the phylogenies inferred from nucleotide data. a recent 18s rdna study recovered pseudochloris wilhelmii and the parachlorella and chlorella clades as part of a large assemblage that is sister to marvania, a topology that contrasts with the finding that marvania and pseudochloris are sister taxa in all our analyses. the results presented here reveal an affinity between the chlorellales and the pedinophyceae, although support is weak in the bayesian analysis under the catgtr + Γ model. this finding is consistent with previous chloroplast phylogenomic studies with scarce sampling of trebouxiophyceans, wherein the freshwater flagellate pedinomonas minor was found to be sister to the clade formed by members of the chlorellales. but subsequently, marin identified no association between the pedinophyceae and the chlorellales using nuclear and chloroplast rrna operon data sets, the pedinophyceae being placed as an independent lineage that is sister to the chlorodendrophyceae + utc. note that the clade formed by the chlorellales and other trebouxiophyceans was not supported with high confidence in these rrna operon trees and that the branching order of most trebouxiophycean lineages was unresolved. given the conflicting positions of the chlorellales and pedinophyceae in the aforementioned analyses, the weak pp support that the chlorellales + pedinophyceae clade received in the phylobayes analyses of the amino acid data set and the basal position occupied by the pedinophyceae in trees inferred from the dayhoff-recoded data set, we conclude that the question as to whether the chlorellales and pedinophyceae form a monophyletic group remains unsettled. it is possible that the chlorellales + pedinophyceae affiliation is the result of systematic errors of phylogenetic reconstructions. solving this issue will require sampling of the chlorodendrophyceae and the inclusion of additional taxa from the ulvophyceae and the lineage represented by the prasinophyte ccmp the two ulvophycean taxa used in our study represent distinct basal lineages of the ulvophyceae; taxa from the bcdt and ignatius clades will need to be examined for a more representative sampling of ulvophycean diversity. we expect that resolving the ancient and rapid radiations of the core chlorophyte lineages using a chloroplast phylogenomic approach will be challenging and will require optimized models of sequence evolution.  the phylogeny reported in this study forms a solid basis for future studies aimed at advancing knowledge about the nature of the morphological and ecological diversification of the trebouxiophyceae. it provides important insights into the origins and adaptations of terrestrial and symbiotic lifestyles. members of this group clearly occupy a pivotal position in the viridiplantae and display considerable genetic diversity. a fundamental understanding of the molecular mechanisms underlying their adaptations to changing environments will require the analysis of genomes from key trebouxiophycean taxa. 
we determined the sequences of chlorophyte chloroplast genomes and assembled amino acid and nucleotide data sets derived from chloroplast genes of chlorophytes, including trebouxiophyceans. the amino acid- and nucleotide-based phylogenies inferred using maximum likelihood and bayesian methods and various models of sequence evolution revealed essentially the same relationships for the trebouxiophyceans. the assemblage composed of the oocystis and geminella clades is the deepest divergence of the core trebouxiophyceans. because a reliable backbone phylogeny is essential to understand the evolutionary history of the trebouxiophyceae, we sought to identify the relationships among the major trebouxiophycean lineages that have been previously recognized in nuclear-encoded 18s rrna phylogenies. the green algae represent one of the most successful groups of photosynthetic eukaryotes, but compared to their land plant relatives, surprisingly little is known about their evolutionary history. the trebouxiophyceae is a species-rich class of the chlorophyta that includes symbionts as well as free-living green algae. the core trebouxiophyceans form a grade of strongly supported clades that include a novel lineage represented by the desert crust alga pleurastrosarcina brevispinosa. the inferred trees reveal that this class is not monophyletic; they offer new insights not only into the internal structure of the class but also into the lifestyle of its founding members and subsequent adaptations to changing environments. like most of the chlorellaleans, early-diverging core trebouxiophyceans are predominantly planktonic species, whereas core trebouxiophyceans occupying more derived lineages are mostly terrestrial or aeroterrestrial algae. our phylogenomic study provides a solid foundation for addressing fundamental questions related to the biology and ecology of the trebouxiophyceae. to this end, we used a chloroplast phylogenomic approach.
 the green algae represent one of the most successful groups of photosynthetic eukaryotes, but compared to their land plant relatives, surprisingly little is known about their evolutionary history. this is in great part due to the difficulty of recognizing species diversity behind morphologically similar organisms. the trebouxiophyceae is a species-rich class of the chlorophyta that includes symbionts as well as free-living green algae. members of this group display remarkable ecological variation, occurring in aquatic, terrestrial and aeroterrestrial environments. because a reliable backbone phylogeny is essential to understand the evolutionary history of the trebouxiophyceae, we sought to identify the relationships among the major trebouxiophycean lineages that have been previously recognized in nuclear-encoded 18s rrna phylogenies. to this end, we used a chloroplast phylogenomic approach.  we determined the sequences of chlorophyte chloroplast genomes and assembled amino acid and nucleotide data sets derived from chloroplast genes of chlorophytes, including trebouxiophyceans. the amino acid- and nucleotide-based phylogenies inferred using maximum likelihood and bayesian methods and various models of sequence evolution revealed essentially the same relationships for the trebouxiophyceans. two major groups were identified: a strongly supported clade of taxa that is sister to the chlorophyceae + ulvophyceae and a clade comprising the chlorellales and pedinophyceae that represents a basal divergence relative to the former group. the core trebouxiophyceans form a grade of strongly supported clades that include a novel lineage represented by the desert crust alga pleurastrosarcina brevispinosa. the assemblage composed of the oocystis and geminella clades is the deepest divergence of the core trebouxiophyceans. like most of the chlorellaleans, early-diverging core trebouxiophyceans are predominantly planktonic species, whereas core trebouxiophyceans occupying more derived lineages are mostly terrestrial or aeroterrestrial algae.  our phylogenomic study provides a solid foundation for addressing fundamental questions related to the biology and ecology of the trebouxiophyceae. the inferred trees reveal that this class is not monophyletic; they offer new insights not only into the internal structure of the class but also into the lifestyle of its founding members and subsequent adaptations to changing environments. keywords chlorophytatrebouxiophyceaeplastid genomephylogenomicsissue-copyright-statement© the author 2014 