BACKGROUND
the ancestors of modern land plants  colonized the terrestrial habitat about  <dig> to  <dig> million years ago .
this event was undoubtedly one of the most important steps in the evolution of life on earth  <cit> , thereby establishing the path to our current terrestrial ecosystems  <cit>  and significantly changing the atmospheric oxygen concentration  <cit> .
since this time three major groups of land plants evolved: bryophytes , pteridophytes  and spermatophytes with the latter dominating most habitats today.
it is widely accepted that embryophytes evolved from green algae, or more specifically, from a small but diverse group of green algae known as the streptophyte algae .
streptophyte algae and embryophytes together constitute the division streptophyta, which likely split from the chlorophyta  about 725- <dig> my ago  <cit> .
streptophyta and chlorophyta comprise the viridiplantae, one of the three evolutionary lineages derived from the single primary endosymbiosis of a cyanobacterium and a eukaryotic host cell  <cit> .
the streptophyta are characterized by several morphological , and physiological characters .
furthermore, several typical embryophyte traits have evolved within the streptophyte algae .
however, the streptophyte algae differ greatly in cellular organization and reproduction.
molecular phylogenies indicate that the mesostigmatales and chlorokybales form a clade that is a sister-group to all other streptophytes, currently containing only two genera: the biflagellate mesostigma and the sarcinoid  chlorokybus  <cit> .
the klebsormidiales, which is comprised of filamentous algae  <cit> , is the sister group to the remaining streptophyte algae and the embryophytes.
the phylogenetic position of the other three groups of streptophyte algae is currently controversial.
the conjugating green algae  today represent the most species-rich group of streptophyte algae and are characterized by their unique mode of sexual reproduction.
they have completely lost flagellate cells, using instead conjugation for sexual reproduction  <cit> .
the conjugating green algae include both filamentous and unicellular forms.
the last two groups of streptophyte algae, the coleochaetales and charales, are filamentous with apical growth and an oogamous mode of sexual reproduction.
based on morphological complexity, either of the latter two groups have been suggested to be the sister group of the embryophytes  <cit> .
in many illustrations referring to the evolution of streptophyte algae and embryophytes in textbooks  or review articles  <cit> , the charales  are depicted as the sister group of the embryophytes.
the strongest support for a sister group relationship between charales and embryophytes was obtained in a phylogenetic analysis using four genes .
in contrast, analyses using plastid lsu and ssu ribosomal rnas or whole chloroplast genomes support the zygnematales or a clade consisting of zygnematales and coleochaetales as sister group of the embryophytes  <cit> .
here we use ests from six different streptophyte algae for a phylogenomic analysis including  <dig> embryophytes.
we show that the charales are most likely not the sister group of the embryophytes, instead our analyses indicate that either the conjugating green algae or less likely a sister group formed by coleochaete and the zygnematales might be the closest living relatives of embryophytes, in agreement with previous phylogenetic analyses based on chloroplast genomes.
RESULTS
zygnematales alone or together with coleochaetales as the sister group of embryophytes
new ests were sequenced from the streptophyte algae klebsormidium subtile, coleochaete scutata, and chara vulgaris and the chlorophyte alga pyramimonas parkeae .
we assembled a data set of  <dig> expressed genes  for  <dig> viridiplant taxa including six streptophyte algae  using the chlorophytes as outgroup to root the trees.
the data set was analyzed by maximum likelihood  and bayesian inference  methods using several evolutionary models.
we first evaluated the fits of the models to our data set using cross validation .
the site-heterogeneous catgtr model is the best of the four models under study.
the site-heterogeneous cat model, which assumes uniform exchangeability rates among amino acids, has a much better fit than the site-homogeneous lg+f and gtr models, and is just slightly worse than the catgtr model.
interestingly, the data set is sufficiently large to accurately estimate the amino acid exchangeability rates, since the gtr model has a better fit to the data than the lg+f model, where these parameters were learned from numerous alignments  <cit> .
the simplifying assumption of equal rates of the cat model, albeit biologically unsound and rejected by cross validation , has the advantage of allowing a significant increase in computational speed  <cit> , and was therefore used for bootstrap analysis.
despite very different model fits, the same tree topology  was obtained in all analyses.
bootstrap support values were computed for both methods, using the site homogeneous gtr+Γ <dig> model  and the site heterogeneous cat+Γ <dig> model .
the posterior probabilities of all nodes for both the cat+Γ <dig> and catgtr+Γ <dig> models were  <dig> except for three nodes .
the molecular phylogeny of embryophytes and chlorophyte algae  is in agreement with other recently published phylogenies  <cit>  and supports the monophyly of liverworts and mosses which is however still a matter of debate  <cit> .
the phylogeny of the streptophyte algae is asymmetrical.
mesostigma is sister to all the remaining streptophytes, as in other studies without chlorokybus  <cit> .
there is a long, highly supported, branch at the base of the clade uniting the other streptophyte algae and embryophytes, likely indicating the elapse of a substantial amount of time.
in contrast, the phylogeny of the remaining streptophyte algae resembles an adaptive radiation, with the five major lineages, including the embryophytes, appearing serial, but with relatively short internal branches.
within this clade, klebsormidium is sister to all the other species.
the charales are sister to a clade comprising coleochaetales, zygnematales and embryophytes and are therefore an unlikely candidate as the sister-group of the embryophytes .
the latter clade is moderately well supported  and its support is lower than for the clade klebsormidium+chara+coleochaetales+zygnematales+embryophytes .
however, the sister-group relationship of embryophytes and zygnematales  is less well supported, especially in the analysis under the site-homogeneous gtr+Γ <dig> model.
a fraction of the bootstrap replicates supports the alternative topology that unites coleochaete with the zygnematales .
the data indicate that mesostigma, chara, klebsormidium and coleochaete are evolving at a comparable and moderate rate, with embryophytes and zygnematales evolving faster.
for instance, the zygnematales appear to have evolved twice as fast as coleochaete.
this difference in evolutionary rate suggests that the grouping of embryophytes and zygnematales could be due to a long-branch attraction  artifact  <cit> .
to explore this possibility, we analyzed two reduced taxon samples, where the  <dig> fastest-evolving land plants have been discarded  and the  <dig> long-branched chlorophytes and mesostigma were not used as an outgroup .
the impact of lba should be reduced in both cases.
again, the four models and the two methods  lead to identical topologies for both data sets.
interestingly, in both cases, the topology differs from the one of the complete data set  by the appearance of a sister-group relationship between coleochaete and zygnematales.
however, this grouping receives non-significant support .
in both cases, the zygnematales plus coleochaete clade is the closest relative to embryophytes .
it is noteworthy, that in none of the analyses was chara recovered as a sister clade to the embryophytes .
another cause of systematic errors in phylogenetic inference is the compositional heterogeneity across taxa  <cit> .
a principal component analysis of the amino acid composition  demonstrates that coleochaete, chara and spirogyra and most embryophytes have a similar composition.
other organisms  show much larger compositional differences, but are correctly placed in a phylogenetic tree due to the presence of a strong phylogenetic signal, as the strongly supported monophyly of closterium+spirogyra and selaginella+huperzia illustrate.
we explored the potential impact of compositional heterogeneity on our inference by using the dayhoff recoding, an approach known to be efficient  <cit> .
interestingly, the three models  and the three taxon samples in figures  <dig> and  <dig> a, b all lead to the same topology as in figure  <dig>  since the dayhoff recoding reduces not only compositional heterogeneity but also saturation, the sister-group relationship between the zygnematales and embryophytes as observed in figure  <dig> is less likely to result from systematic error.
discussion
previous studies of the phylogeny of streptophyta were restricted mainly to ribosomal rna or sequences of organellar origin  <cit> .
we now, used for the first time, large data sets of nuclear-encoded proteins for phylogenetic studies in this important evolutionary lineage.
our phylogenetic analyses are in agreement with both phylogenies obtained using a data set of concatenated plastid proteins or ribosomal rnas  <cit> , but are in conflict with the  <dig> gene tree mentioned above  <cit> .
in contrast, coleochaete was found to be sister to embryophytes  <cit>  in a recent analysis based on  <dig> nuclear encoded ribosomal proteins .
however, as this study failed to recover the monophyly of the coleochaetales  the conclusions from this study should be treated with caution.
in the  <dig> gene analysis the topology )) was observed.
this analysis suggested that the streptophyte algae regularly  evolved towards increasing morphological complexity .
in contrast, our results and the results obtained using chloroplast data  <cit>  suggest that most likely the morphologically simpler zygnematales  is the sister group of embryophytes, rather than the charales.
it seems plausible that the simpler morphology of extant zygnematales represents a secondary simplification, similar to the loss of flagellate cells in this group, which may actually represent an adaptation to ensure sexual reproduction in the absence of free water  <cit> .
alternatively, the morphological complexity of the charales and coleochaetales might have evolved independently after the three evolutionary lines  diverged.
this kind of scenario was already proposed by stebbins and hill  <cit> : they suggested that the early evolution of streptophyte algae took place in a moist terrestrial habitat and involved rather simple unicellular types.
they considered the extant coleochaetales, charales, klebsormidiales and zygnematales to be derived forms with a secondary aquatic life style.
a fast initial radiation at the time of colonization of the terrestrial habitat by the ancestors of modern streptophyte algae as proposed by stebbins and hill  <cit>  may also explain the relative difficulty to infer the phylogenetic relationships among the four groups of streptophyte algae, enhanced by the accelerated evolutionary rate of the zygnematales.
however, in contrast to stebbins and hill  <cit> , we argue that at least some of the morphological complexity had evolved prior to the early radiation of the streptophyte algae for the following reasons:  based on the available fossil record, the charales already had a morphology similar to that of extant forms in the silurian period  <cit> ,  the available est data indicate that zygnematales, coleochaetales and charales possess homologues of a number of proteins that are involved in the development of morphologically complex structures in embryophytes, such as gnom, wuschel, meristemlayer  <dig>  mikc-type mads-box protein .
taken together, these data lend support to the idea that the extant morphological complexity of the charales and coleochaetales is an ancient trait that may have been secondarily lost in the zygnematales.
alternatively, as was found by the comparison of volvox, a multicellular system, with its close relative chlamydomonas reinhardtii, the evolution of multi-cellular structures seems to rely mainly on the reorganization and differential regulation of already existing genes  <cit> .
from this point of view, the complex morphologies of the charales and coleochaetales could have evolved completely independently by using the toolbox already present in the common ancestor of these streptophyte algae.
although our results, based on the complete data set, favor the zygnematales as the sister group of the embryophytes, the results from the two alternative taxon sampling tests, in which we tried to reduce as much as possible potential disturbing influences of the lba artifacts, seem to point rather to a sister group relationship between coleochaete and the zygnematales.
in contrast, dayhoff coding favors the grouping of the zygnematales with the embryophytes, whatever the taxon sampling.
since this recoding is expected to reduce several sources of systematic errors, this topology is more likely.
however, the support in this part of the tree remains limited, and large-scale genomic data from more streptophyte algae  are needed to resolve this question.
whatever the relative position of coleochaete and the zygnematales, our analysis supports the scenario of a secondary loss of morphological complexity in the zygnematales.
the first land plants encountered a more extreme environment compared to a freshwater habitat, with large fluctuations in water content , radiation intensity  and nutrient supply.
potentially, the last common ancestor of the zygnematales and embryophytes was better adapted to these types of environmental stressors than other streptophyte algae.
the more variable environmental conditions might have also favored the evolution of more complex signaling pathways  <cit> .
rensing et al.
<cit>  discussed several proteins likely to be important for the adaptation of embryophytes to their terrestrial habitat.
preliminary analysis of the available ests from streptophyte algae indicate that expressed genes similar to most of the proteins listed by rensing et al.
<cit>  can be found in various streptophyte algae .
for example, proteins similar to major light harvesting complex ii proteins , which were considered to be missing from green algae  <cit> , are clearly found in ests from streptophyte algae except mesostigma .
expressed genes similar to late embryo abundant  proteins known to protect spermatophyte seeds from desiccation  <cit>  are found in several streptophyte algae .
blast analyses were performed to identify putative homologues in the indicated streptophyte algae.
the number of contigs and the best e-value obtained are given.
n.d. not detected, ?
no clear result.
1)for most species ests showing similarity to several different lea proteins were found
the probable fast radiation of the derived lineages of streptophyte algae  in conjunction with the secondary morphological simplification of the zygnematales makes it difficult to find any synapomorphies for the possible sister group relationship of the zygnematales and embryophytes.
we note two complex traits that might potentially support this relationship.
firstly, components of the "auxin signaling machinery" are highly conserved in embryophytes  <cit> , but appear to be absent in streptophyte algae, except for the auxin binding protein , which can be found in various green algae including chlorophytes  <cit> .
however, as also noted by de smet et al.
<cit>  the recently published ests from spirogyra  <cit>  include expressed genes similar to components  of the embryophyte-specific "auxin signaling machinery".
secondly, embryophytes generally show chloroplast movements in response to high  or low light , which has been shown to be of ecological importance  <cit> .
chloroplast movements in response to low or high light conditions have also been reported for several zygnematales  <cit>  as well as for some chlorophytes, diatoms and vaucheria  <cit> .
while the photoreceptor is not known for most algae, recent work has shown that, in mougoetia scalaris , phototropin and neochrome are used as photoreceptor similar to the situation in physcomitrella and adiantum  <cit> , suggestive of a common origin of this response.
CONCLUSIONS
knowledge of the phylogenetic relationships within streptophyte algae is of crucial importance for developing a realistic scenario for the colonization of the terrestrial habitat and the origin and early evolution of embryophytes.
phylogenomic analyses of nuclear and chloroplast data now indicate that the charales are most likely not the closest living extant relatives of the embryophytes despite their morphological complexity.
instead, the analyses favor either the zygnematales or, less likely, a clade consisting of the zygnematales and coleochaetales as the sister group of embryophytes.
an extended taxon sampling and/or analyses of larger data sets such as complete genomes/transcriptomes will likely be necessary to shed further light on the elusive sister group of the embryophyte plants

 the ancestors of modern land plants colonized the terrestrial habitat about to million years ago. this event was undoubtedly one of the most important steps in the evolution of life on earth, thereby establishing the path to our current terrestrial ecosystems and significantly changing the atmospheric oxygen concentration. since this time three major groups of land plants evolved: bryophytes, pteridophytes and spermatophytes with the latter dominating most habitats today. it is widely accepted that embryophytes evolved from green algae, or more specifically, from a small but diverse group of green algae known as the streptophyte algae. streptophyte algae and embryophytes together constitute the division streptophyta, which likely split from the chlorophyta about 725- my ago. streptophyta and chlorophyta comprise the viridiplantae, one of the three evolutionary lineages derived from the single primary endosymbiosis of a cyanobacterium and a eukaryotic host cell. the streptophyta are characterized by several morphological, and physiological characters. furthermore, several typical embryophyte traits have evolved within the streptophyte algae. however, the streptophyte algae differ greatly in cellular organization and reproduction. molecular phylogenies indicate that the mesostigmatales and chlorokybales form a clade that is a sister-group to all other streptophytes, currently containing only two genera: the biflagellate mesostigma and the sarcinoid chlorokybus. the klebsormidiales, which is comprised of filamentous algae, is the sister group to the remaining streptophyte algae and the embryophytes. the phylogenetic position of the other three groups of streptophyte algae is currently controversial. the conjugating green algae today represent the most species-rich group of streptophyte algae and are characterized by their unique mode of sexual reproduction. they have completely lost flagellate cells, using instead conjugation for sexual reproduction. the conjugating green algae include both filamentous and unicellular forms. the last two groups of streptophyte algae, the coleochaetales and charales, are filamentous with apical growth and an oogamous mode of sexual reproduction. based on morphological complexity, either of the latter two groups have been suggested to be the sister group of the embryophytes. in many illustrations referring to the evolution of streptophyte algae and embryophytes in textbooks or review articles, the charales are depicted as the sister group of the embryophytes. the strongest support for a sister group relationship between charales and embryophytes was obtained in a phylogenetic analysis using four genes. in contrast, analyses using plastid lsu and ssu ribosomal rnas or whole chloroplast genomes support the zygnematales or a clade consisting of zygnematales and coleochaetales as sister group of the embryophytes. here we use ests from six different streptophyte algae for a phylogenomic analysis including embryophytes. we show that the charales are most likely not the sister group of the embryophytes, instead our analyses indicate that either the conjugating green algae or less likely a sister group formed by coleochaete and the zygnematales might be the closest living relatives of embryophytes, in agreement with previous phylogenetic analyses based on chloroplast genomes.  zygnematales alone or together with coleochaetales as the sister group of embryophytes new ests were sequenced from the streptophyte algae klebsormidium subtile, coleochaete scutata, and chara vulgaris and the chlorophyte alga pyramimonas parkeae. we assembled a data set of expressed genes for viridiplant taxa including six streptophyte algae using the chlorophytes as outgroup to root the trees. the data set was analyzed by maximum likelihood and bayesian inference methods using several evolutionary models. we first evaluated the fits of the models to our data set using cross validation. the site-heterogeneous catgtr model is the best of the four models under study. the site-heterogeneous cat model, which assumes uniform exchangeability rates among amino acids, has a much better fit than the site-homogeneous lg+f and gtr models, and is just slightly worse than the catgtr model. interestingly, the data set is sufficiently large to accurately estimate the amino acid exchangeability rates, since the gtr model has a better fit to the data than the lg+f model, where these parameters were learned from numerous alignments. the simplifying assumption of equal rates of the cat model, albeit biologically unsound and rejected by cross validation, has the advantage of allowing a significant increase in computational speed, and was therefore used for bootstrap analysis. despite very different model fits, the same tree topology was obtained in all analyses. bootstrap support values were computed for both methods, using the site homogeneous gtr+Γ model and the site heterogeneous cat+Γ model. the posterior probabilities of all nodes for both the cat+Γ and catgtr+Γ models were except for three nodes. the molecular phylogeny of embryophytes and chlorophyte algae is in agreement with other recently published phylogenies and supports the monophyly of liverworts and mosses which is however still a matter of debate. the phylogeny of the streptophyte algae is asymmetrical. mesostigma is sister to all the remaining streptophytes, as in other studies without chlorokybus. there is a long, highly supported, branch at the base of the clade uniting the other streptophyte algae and embryophytes, likely indicating the elapse of a substantial amount of time. in contrast, the phylogeny of the remaining streptophyte algae resembles an adaptive radiation, with the five major lineages, including the embryophytes, appearing serial, but with relatively short internal branches. within this clade, klebsormidium is sister to all the other species. the charales are sister to a clade comprising coleochaetales, zygnematales and embryophytes and are therefore an unlikely candidate as the sister-group of the embryophytes. the latter clade is moderately well supported and its support is lower than for the clade klebsormidium+chara+coleochaetales+zygnematales+embryophytes. however, the sister-group relationship of embryophytes and zygnematales is less well supported, especially in the analysis under the site-homogeneous gtr+Γ model. a fraction of the bootstrap replicates supports the alternative topology that unites coleochaete with the zygnematales. the data indicate that mesostigma, chara, klebsormidium and coleochaete are evolving at a comparable and moderate rate, with embryophytes and zygnematales evolving faster. for instance, the zygnematales appear to have evolved twice as fast as coleochaete. this difference in evolutionary rate suggests that the grouping of embryophytes and zygnematales could be due to a long-branch attraction artifact. to explore this possibility, we analyzed two reduced taxon samples, where the fastest-evolving land plants have been discarded and the long-branched chlorophytes and mesostigma were not used as an outgroup. the impact of lba should be reduced in both cases. again, the four models and the two methods lead to identical topologies for both data sets. interestingly, in both cases, the topology differs from the one of the complete data set by the appearance of a sister-group relationship between coleochaete and zygnematales. however, this grouping receives non-significant support. in both cases, the zygnematales plus coleochaete clade is the closest relative to embryophytes. it is noteworthy, that in none of the analyses was chara recovered as a sister clade to the embryophytes. another cause of systematic errors in phylogenetic inference is the compositional heterogeneity across taxa. a principal component analysis of the amino acid composition demonstrates that coleochaete, chara and spirogyra and most embryophytes have a similar composition. other organisms show much larger compositional differences, but are correctly placed in a phylogenetic tree due to the presence of a strong phylogenetic signal, as the strongly supported monophyly of closterium+spirogyra and selaginella+huperzia illustrate. we explored the potential impact of compositional heterogeneity on our inference by using the dayhoff recoding, an approach known to be efficient. interestingly, the three models and the three taxon samples in figures and a, b all lead to the same topology as in figure since the dayhoff recoding reduces not only compositional heterogeneity but also saturation, the sister-group relationship between the zygnematales and embryophytes as observed in figure is less likely to result from systematic error. discussion previous studies of the phylogeny of streptophyta were restricted mainly to ribosomal rna or sequences of organellar origin. we now, used for the first time, large data sets of nuclear-encoded proteins for phylogenetic studies in this important evolutionary lineage. our phylogenetic analyses are in agreement with both phylogenies obtained using a data set of concatenated plastid proteins or ribosomal rnas, but are in conflict with the gene tree mentioned above. in contrast, coleochaete was found to be sister to embryophytes in a recent analysis based on nuclear encoded ribosomal proteins. however, as this study failed to recover the monophyly of the coleochaetales the conclusions from this study should be treated with caution. in the gene analysis the topology )) was observed. this analysis suggested that the streptophyte algae regularly evolved towards increasing morphological complexity. in contrast, our results and the results obtained using chloroplast data suggest that most likely the morphologically simpler zygnematales is the sister group of embryophytes, rather than the charales. it seems plausible that the simpler morphology of extant zygnematales represents a secondary simplification, similar to the loss of flagellate cells in this group, which may actually represent an adaptation to ensure sexual reproduction in the absence of free water. alternatively, the morphological complexity of the charales and coleochaetales might have evolved independently after the three evolutionary lines diverged. this kind of scenario was already proposed by stebbins and hill: they suggested that the early evolution of streptophyte algae took place in a moist terrestrial habitat and involved rather simple unicellular types. they considered the extant coleochaetales, charales, klebsormidiales and zygnematales to be derived forms with a secondary aquatic life style. a fast initial radiation at the time of colonization of the terrestrial habitat by the ancestors of modern streptophyte algae as proposed by stebbins and hill may also explain the relative difficulty to infer the phylogenetic relationships among the four groups of streptophyte algae, enhanced by the accelerated evolutionary rate of the zygnematales. however, in contrast to stebbins and hill, we argue that at least some of the morphological complexity had evolved prior to the early radiation of the streptophyte algae for the following reasons: based on the available fossil record, the charales already had a morphology similar to that of extant forms in the silurian period, the available est data indicate that zygnematales, coleochaetales and charales possess homologues of a number of proteins that are involved in the development of morphologically complex structures in embryophytes, such as gnom, wuschel, meristemlayer mikc-type mads-box protein. taken together, these data lend support to the idea that the extant morphological complexity of the charales and coleochaetales is an ancient trait that may have been secondarily lost in the zygnematales. alternatively, as was found by the comparison of volvox, a multicellular system, with its close relative chlamydomonas reinhardtii, the evolution of multi-cellular structures seems to rely mainly on the reorganization and differential regulation of already existing genes. from this point of view, the complex morphologies of the charales and coleochaetales could have evolved completely independently by using the toolbox already present in the common ancestor of these streptophyte algae. although our results, based on the complete data set, favor the zygnematales as the sister group of the embryophytes, the results from the two alternative taxon sampling tests, in which we tried to reduce as much as possible potential disturbing influences of the lba artifacts, seem to point rather to a sister group relationship between coleochaete and the zygnematales. in contrast, dayhoff coding favors the grouping of the zygnematales with the embryophytes, whatever the taxon sampling. since this recoding is expected to reduce several sources of systematic errors, this topology is more likely. however, the support in this part of the tree remains limited, and large-scale genomic data from more streptophyte algae are needed to resolve this question. whatever the relative position of coleochaete and the zygnematales, our analysis supports the scenario of a secondary loss of morphological complexity in the zygnematales. the first land plants encountered a more extreme environment compared to a freshwater habitat, with large fluctuations in water content, radiation intensity and nutrient supply. potentially, the last common ancestor of the zygnematales and embryophytes was better adapted to these types of environmental stressors than other streptophyte algae. the more variable environmental conditions might have also favored the evolution of more complex signaling pathways. rensing et al. discussed several proteins likely to be important for the adaptation of embryophytes to their terrestrial habitat. preliminary analysis of the available ests from streptophyte algae indicate that expressed genes similar to most of the proteins listed by rensing et al. can be found in various streptophyte algae. for example, proteins similar to major light harvesting complex ii proteins, which were considered to be missing from green algae, are clearly found in ests from streptophyte algae except mesostigma. expressed genes similar to late embryo abundant proteins known to protect spermatophyte seeds from desiccation are found in several streptophyte algae. blast analyses were performed to identify putative homologues in the indicated streptophyte algae. the number of contigs and the best e-value obtained are given. n.d. not detected,? no clear result. 1)for most species ests showing similarity to several different lea proteins were found the probable fast radiation of the derived lineages of streptophyte algae in conjunction with the secondary morphological simplification of the zygnematales makes it difficult to find any synapomorphies for the possible sister group relationship of the zygnematales and embryophytes. we note two complex traits that might potentially support this relationship. firstly, components of the "auxin signaling machinery" are highly conserved in embryophytes, but appear to be absent in streptophyte algae, except for the auxin binding protein, which can be found in various green algae including chlorophytes. however, as also noted by de smet et al. the recently published ests from spirogyra include expressed genes similar to components of the embryophyte-specific "auxin signaling machinery". secondly, embryophytes generally show chloroplast movements in response to high or low light, which has been shown to be of ecological importance. chloroplast movements in response to low or high light conditions have also been reported for several zygnematales as well as for some chlorophytes, diatoms and vaucheria. while the photoreceptor is not known for most algae, recent work has shown that, in mougoetia scalaris, phototropin and neochrome are used as photoreceptor similar to the situation in physcomitrella and adiantum, suggestive of a common origin of this response.  knowledge of the phylogenetic relationships within streptophyte algae is of crucial importance for developing a realistic scenario for the colonization of the terrestrial habitat and the origin and early evolution of embryophytes. phylogenomic analyses of nuclear and chloroplast data now indicate that the charales are most likely not the closest living extant relatives of the embryophytes despite their morphological complexity. instead, the analyses favor either the zygnematales or, less likely, a clade consisting of the zygnematales and coleochaetales as the sister group of embryophytes. an extended taxon sampling and/or analyses of larger data sets such as complete genomes/transcriptomes will likely be necessary to shed further light on the elusive sister group of the embryophyte plants 
although this result is in agreement with a previously published phylogenetic study of chloroplast genomes, additional data are needed to confirm this conclusion. the terrestrial habitat was colonized by the ancestors of modern land plants about to million years ago. today it is widely accepted that land plants evolved from streptophyte algae, also referred to as charophycean algae. for a better understanding of the evolution of land plants, it is of prime importance to identify the streptophyte algae that are the sister-group to the embryophytes. our phylogenetic analyses indicate that either the zygnematales or a clade consisting of the zygnematales and the coleochaetales are the sister group to embryophytes. the streptophyte algae are a paraphyletic group of green algae, ranging from unicellular flagellates to morphologically complex forms such as the stoneworts. here, we use a large data set of nuclear-encoded genes from green plant taxa including embryophytes and six streptophyte algae, representing all major streptophyte algal lineages, to investigate the phylogenetic relationships of streptophyte algae and embryophytes. the charales, the coleochaetales or more recently the zygnematales have been considered to be the sister group of the embryophytes however, despite many years of phylogenetic studies, this question has not been resolved and remains controversial. instead, the zygnematales or a clade consisting of zygnematales and coleochaetales are most likely the sister group of embryophytes. a zygnematales/embryophyte sister group relationship has important implications for early land plant evolution.
 the terrestrial habitat was colonized by the ancestors of modern land plants about to million years ago. today it is widely accepted that land plants evolved from streptophyte algae, also referred to as charophycean algae. the streptophyte algae are a paraphyletic group of green algae, ranging from unicellular flagellates to morphologically complex forms such as the stoneworts. for a better understanding of the evolution of land plants, it is of prime importance to identify the streptophyte algae that are the sister-group to the embryophytes. the charales, the coleochaetales or more recently the zygnematales have been considered to be the sister group of the embryophytes however, despite many years of phylogenetic studies, this question has not been resolved and remains controversial.  here, we use a large data set of nuclear-encoded genes from green plant taxa including embryophytes and six streptophyte algae, representing all major streptophyte algal lineages, to investigate the phylogenetic relationships of streptophyte algae and embryophytes. our phylogenetic analyses indicate that either the zygnematales or a clade consisting of the zygnematales and the coleochaetales are the sister group to embryophytes.  our analyses support the notion that the charales are not the closest living relatives of embryophytes. instead, the zygnematales or a clade consisting of zygnematales and coleochaetales are most likely the sister group of embryophytes. although this result is in agreement with a previously published phylogenetic study of chloroplast genomes, additional data are needed to confirm this conclusion. a zygnematales/embryophyte sister group relationship has important implications for early land plant evolution. if substantiated, it should allow us to address important questions regarding the primary adaptations of viridiplants during the conquest of land. clearly, the biology of the zygnematales will receive renewed interest in the future. 