BACKGROUND
taxonomists face inconsistent or even contradictory clues when they examine the affiliation of organisms to higher taxonomic groupings.
several characters may yield alternative hypotheses explaining their evolutionary background.
this also applies to the taxonomic position of the sphaeropleaceae  <cit> .
different authors affiliate the green algal family by morphological characters to either ulvophytes or chlorophytes, until amendatory deason et al.
<cit>  suggested that the neochloridaceae, the hydrodictyaceae and the sphaeropleaceae should be grouped as sphaeropleales within the chlorophytes, since all of them have motile biflagellate zoospores with a direct-opposite  confirmation of basal bodies.
subsequently, other taxonomic lineages  were added to this biflagellate do group, because they show molecular affiliation to either neochloridaceae or hydrodictyaceae  <cit> .
although nowadays most authors agree that the do group is monophyletic, until now no study pinpointed the taxonomic linkage of the name-giving 'sphaeroplea' clade to the remaining 'core sphaeropleales' persuasively with genetic evidence  <cit> , i.e.
the sister clade remains unclear  <cit> .
likewise, with respect to morphology, studies of 18s and 26s rrna gene sequences neither resolve the basal branching patterns within the chlorophyceae with high statistical power nor corroborate a monophyletic biflagellate do group without ambiguity  <cit> .
müller et al.
<cit>  obtained moderate statistical support for the close relationship of the 'sphaeroplea' clade and the 'core sphaeropleales' with profile distances of 18s and 26s rdna.
in this study we followed and expanded their methodology with a very different phylogenetic marker.
the internal transcribed spacer  <dig> , the region of ribosomal rna between the  <dig> s rrna gene and the large subunit  has proven to be an appropriate marker for the study of small scale phylogenies of close relatives  <cit> .
the sequence is in contrast to the bordering regions of ribosomal subunits evolutionary not conserved, thus genetic differentiation is detectable even in closely related groups of organisms.
by contrast, the secondary structure seems to be well conserved and thus provides clues for higher taxonomic studies  <cit> .
secondary structure information is furthermore especially interesting within the chlorophyceae, because van hannen et al.
<cit>  described an uncommon branching of its <dig> helix  <dig> within the genera desmodesmus, hydrodictyon  <cit>  and scenedesmus.
it is not known when this feature evolved and whether it is, as we expect, an apomorphic feature for the do-group.
it is obvious that phylogenetic statements should be improvable by inclusion of structural information in common sequence analysis.
for example, grajales et al.
<cit>  calculated morphometric matrices from its <dig> secondary structures for phylogenetic analyses, but treated information of sequence and structure as different markers.
here we combine sequence with structural information in just one analysis.
aside from the biological problem, we address the pivotal question of a methodological pipeline for sequence-structure phylogenetics using rdna data.
methods
dna extraction, amplification and sequencing
extraction of genomic dna from cultured cells of ankyra judayi, atractomorpha porcata and sphaeroplea annulina was done using dynabeads®  according to the manufacturer's protocol.
pcr reactions were performed in a  <dig> μl reaction volume containing  <dig> μl faststart pcr master ,  <dig> μl gdna and  <dig> nm of the primers its <dig>  and its <dig>  designed by white et al.
<cit> .
cycling conditions for amplification consisted of 94°c for  <dig> min,  <dig> cycles of 94°c for  <dig> s, 50°c for  <dig> s and 72°c for  <dig> s, followed by a final extension step of  <dig> min at 72°c.
pcr products were analysed by 3% agarose gel electrophoresis and ethidium bromide staining.
pcr probes where purified with the pcr purificaton kit  and where quantified by spectrometry.
each sequencing probe was prepared in an  <dig> μl volume containing  <dig> ng dna and  <dig>  μm primer.
sequencing was carried out using an annealing temperature of 50°c with the sequencer applied biosystems qst  <dig> genetic analyzer by the institute of hygiene and microbiology .
its <dig> secondary structure prediction
its <dig> secondary structures of the three newly obtained sequences were folded with the help of rnastructure  <cit>  and afterwards manually corrected.
all available  <dig> chlorophycean its <dig> sequences were obtained from the ncbi nucleotide database.
the its <dig> secondary structure of atractomorpha porcata was used as template for homology modelling.
homology modelling was performed by using the custom modelling option as provided with the its2-database  <cit>  .
forty-nine species representing the chlorophycean diversity were retained and used as comparative taxa in inferring phylogenies .
for this taxon sampling, accurate secondary structures of sequences were now folded by rnastructure and additionally corrected using pseudoviewer  <dig>  <cit> .
we standardized start and end of all helices according to the optimal folding of the newly obtained sequences.
listed is the current clade classification of the species  <cit>  and the genbank accession numbers of the analyzed sequences.
the four newly obtained sequences are of the 'sphaeroplea' clade.
alignment and phylogenetic analyses
using 4sale  <cit>  with its its <dig> specific scoring matrix, we automatically aligned sequences and structures simultaneously.
sequence-structure alignment is available at the its <dig> database supplements page.
for the complete alignment we tested for appropriate models of nucleotide substitution using the akaike information criterion  as implemented in modeltest  <cit> .
the following paupblock was used for all maximum likelihood based phylogenetic analyses with paup*  <cit> : lset base =  nst =  <dig> rmat =  rates = gamma shape =  <dig>  pinvar =  <dig> ;.
a maximum likelihood  analysis was performed with a heuristic search  and nearest neighbour interchange   <cit> .
maximum parsimony   <cit>  was accomplished with gaps treated as missing data and all characters coded as "unordered" and equally weighted.
additionally, we clustered taxonomic units with neighbour-joining   <cit>  using maximum likelihood distances.
furthermore, with mrbayes  <cit>  a bayesian analysis  was carried out for tree reconstruction using a general time reversible substitution model   <cit>  with substitution rates estimated by mrbayes .
moreover, using profdist, a profile neighbour-joining  tree  <cit>  was calculated using the its <dig> specific substitution model available from the its <dig> database.
pnj was also performed with predefined profiles  of all the clades given in table  <dig>
for clade 'scenedesmus' two profiles were used for groups 'true scenedesmus'  and 'desmodesmus' .
we performed a sequence-structure profile neighbour-joining  analysis with a developmental beta version of profdist .
the tree reconstructing algorithm works on a  <dig> letter alphabet comprised of the  <dig> nucleotides in three structural states .
based on a suitable substitution model  <cit> , evolutionary distances between sequence structure pairs have been estimated by maximum likelihood.
all other applied analyses were computed only on the sequence part of the sequence-structure alignment.
for mp, nj, pnj, prepnj and strpnj analyses  <dig>  bootstrap pseudoreplicates  <cit>  were generated.
one hundred bootstrap replicates were generated for the ml analysis.
additionally we used raxml at the cipres portal to achieve  <dig>  bootstraps with a substitution model estimated by raxml  <cit> .
all methods were additionally applied to a 50% structural consensus alignment cropped with 4sale .
the individual steps of the analysis are displayed in a flow chart .
RESULTS
new its <dig> sequences
genbank accession numbers for newly obtained nucleotide sequences are given in table  <dig> .
the two its <dig> sequences of sphaeroplea annulina  strain sag 377-1a and strain sag 377-1e were identical and thus only the first one was used for further analysis.
according to folding with rnastructure, its <dig> secondary structures of the three newly obtained sequences did not exhibit any branching in their helix i  as it is described for the 'core sphaeropleales', i.e.
helix i was more similar to those of the cw-group and the 'oedogonium' clade.
helix i of sphaeroplea annulina was explicitly longer  than those of the other newly obtained algae.
due to this insertion, for sphaeroplea, a branching pattern was enforceable, but would have lower energy efficiency.
however, the additional nucleotides are not homologous to the insertion capable of making an additional stem  found in the 'scenedesmus' and the 'hydrodictyon' clade .
its <dig> sequence and secondary structure information
its <dig> sequence lengths of all studied species ran from  <dig> to  <dig> nucleotides ,  <dig> nt on average.
the gc contents of its <dig> sequences ranged from  <dig> % to  <dig> %, with a mean value of  <dig> %.
the number of base pairs  varied between  <dig> and  <dig> bp and averaged  <dig> bp.
the cropped alignment  showed that 23% of the nucleotides had at least a 50% consistency in their pairings.
compensatory base changes  as well as hemi-cbcs  range from  <dig> to  <dig> with a mean of  <dig>  cbcs .
sequence pairs lacking cbcs were exclusively found within the same major clade.
characteristics in a conserved part of alignment
in agreement with coleman  <cit> , the 5' side part near the tip of helix iii was highly conserved including the uggu motif  <cit> , likewise the ugggu motif in case of chlorophyceae.
we selected a part of the alignment at this position with adjacent columns  to verify the suggested conservation.
having a closer look at this part of helix iii, in our case, it showed typical sequence and structural characteristics for distinct groups.
studied species of the 'oedogonium' clade possess at position  <dig> in the selected part of the alignment an adenine and in addition at positions 3– <dig> paired bases.
in contrast, the cw-group solely possessed three consecutively paired bases in this block, but not the adenine.
a typical pattern for clades of the do-group was a twofold motif of  <dig> bases: uracile, adenine and guanine at positions 7– <dig>  which is repeated at positions 11– <dig>  this could be a duplication, which results in a modified secondary structure.
in addition, the 'core sphaeropleales'  showed an adenine base change at position  <dig>  compared to all other clades.
phylogenetic tree information
the paup* calculation applying maximum parsimony included a total of  <dig> characters, whereas  <dig> characters were constant,  <dig> variable characters were parsimony-informative compared to  <dig> parsimony-uninformative ones.
the resulting trees  of all performed analyses  yielded six major clades: the 'dunaliella', the 'hydrodictyon', the 'oedogonium', the 'reinhardtii', the 'scenedesmus', and the 'sphaeroplea' clade.
all of them were separated and – except for the 'scenedesmus' clade – highly supported by bootstrap values of 83–100%, respectively by bayesian posterior probabilities of  <dig> – <dig> .
the table supplements fig.
<dig> and fig.
<dig>  node "g" supports a monophyletic do group and is printed in bold letters.
software used: profdist and paup*.
models of substitution: its <dig> = gtr with its <dig> substitution matrix, modeltest: tvm+i+g with estimated parameters.
phylogenetic analysis: nj = neighbour-joining, pnj = profile neighbour-joining, prepnj = profile neighbour-joining with predefined profiles, strpnj = sequence-structure profile neighbour-joining, ml = maximum likelihood, b = bayesian analysis , mp = maximum parsimony.
1predefined profiles for profile neighbour-joining.
the 'hydrodictyon' clade, the 'scenedesmus' clade and the 'sphaeroplea' clade form one cluster that was strongly supported by high bootstrap values of 67–96% .
the three clades composed the do-group.
the opposite cluster included the 'dunaliella' and the 'reinhardtii' clade, forming the cw-group.
the 'oedogonium' clade was chosen as the outgroup  <cit> .
both clusters  were strongly supported by bootstrap values of 84–100% .
except for the bayesian analysis , all applied methods yielded node "e" as the weakest point within the basal  branches , which presents the relationship between the 'hydrodictyon' and the 'scenedesmus' clade on the one hand and the 'dunaliella', the 'oedogonium', the 'reinhardtii' and the 'sphaeroplea' clade on the other hand.
the phylogenetic tree resulting from neighbour-joining analysis by paup*  did not support node "e" at all, but strongly supported the remaining labelled branches.
the maximum likelihood analysis by paup*  did not encourage node "e" either.
both maximum likelihood methods did not even support nodes "a"  and "c" .
all other basal branches were supported by this method.
varying neighbour-joining analyses by profdist  supported all basal branches – except for the weakest node "e"  – with very high bootstrap support values of 84–100%.
the maximum parsimony method gave average support  for node "c" and "e" and high bootstrap values  for the remaining basal clades.
the bayesian analysis offered posterior probabilities of  <dig>  for node "c" and  <dig> – <dig>  for the remaining basal nodes.
for further sister group relations see fig.
<dig> and  <dig>
in comparison, the topology of the phylogenetic tree based on the 50% cropped alignment did not change, but the bootstrap support values were lower in all cases .
discussion
the internal transcribed spacer  <dig>  is required in ribosome biogenesis  <cit>  and its gradual removal from mature rrna is driven by its specific secondary structure  <cit> .
using three newly obtained its <dig> sequences from ankyra judayi, atractomorpha porcata and sphaeroplea annulina  in this study we aimed to pursue two consecutive questions concerning the phylogenetic relationships within chlorophyceae.
what is the phylogenetic position of the newly sequenced algae relative to the 'core sphaeropleales' and could the biflagellate do-group be regarded as monophyletic?
how does the secondary structure of the new its <dig> sequences look like and is an autapomorphic feature of the secondary structure associated with the monophyletic do-group?
considering the question  buchheim et al.
<cit>  and wolf et al.
<cit>  approached the problem with 18s + 26s rdna and 18s rdna data, but the relationship between the 'core sphaeropleales' and the sphaeropleaceae remained unclear.
however, in their studies, ankyra, atractomorpha and sphaeroplea clustered in a monophyletic clade named sphaeropleaceae.
we confirm this 'sphaeroplea' clade with all three genera being strongly separated from other clades.
as a result of a bayesian analysis on a combined 18s and 26s rdna dataset shoup and lewis  <cit>  also found the sphaeropleaceae as the most basal clade within the sphaeropleales, but again the analysis lacked a strong backing.
beside these difficulties the 'core sphaeropleales' were already shown to be monophyletic with high certainty  <cit> .
the do-group  as emended by deason et al.
<cit> , for which the directly opposed basal body orientation and basal body connection features are verified  <cit> , is now strongly supported by molecular phylogenetic analyses.
there was already evidence of an extended do-group  <cit> , however, for some groups ultrastructural results are still lacking, and even though the collective basal body orientation and connection imply a monophyletic do-group, until now no molecular phylogenetic analysis could show this with solid support  <cit> .
we demonstrate for the first time with robust support values for the equivocal nodes that the 'core sphaeropleales', the 'sphaeroplea' clade, and the sphaeropleales are monophyletic.
regarding question , for all structures of the 'hydrodictyon' and the 'scenedesmus' clade, helix i shows the typical branching .
initially, an et al.
<cit>  proposed a secondary structure model with an unbranched helix i for its <dig> sequences of 'scenedesmus' clade members.
thereafter, van hannen et al.
<cit>  updated the model by folding the nucleotide sequences based upon minimum free energy and found a branched helix i as the most energetically stable option.
the branching is result of an insertion of approximately  <dig> nucleotides capable of folding as an individual stem within the 5' end of the first helix.
however, its <dig> sequence and secondary structure information of further 'core sphaeropleales' members, e.g.
the 'ankistrodesmus' clade and the 'bracteacoccus' clade, lacks hitherto.
in contrast, the y-structure is absent within the 'sphaeroplea' clade and any other investigated group so far.
thus this feature is – contrary to our expectation – not an autapomorphic character for the biflagellate do-group as a whole but for the 'core sphaeropleales'.
regarding future work, the resolution among the main clades of chlorophyceae was statistically poorly supported in previous studies  <cit> .
pröschold and leliaert  <cit>  reviewed the systematics of green algae by applying a polyphasic approach, but did not yield a clear resolution regarding a sister taxon to the sphaeropleales.
since they are not yet available, its <dig> sequences of chaetopeltidalean and chaetophoralean taxa could not be included in the present study and therefore the phylogenetic relationships between the main chlorophyceae clades remain open.
we recommend involving sequence and secondary structure information of chaetopeltidalean and chaetophoralean its <dig> sequences in future studies to find out if the monophyletic biflagellate do-group could be further extended to a general monophyletic do-group containing quadri- and biflagellate taxa.
a genome-wide approach indicates that sphaeropleales and chlamydomonadales are sister taxa, however only a few organisms are included in this study  <cit> .
an additional uprising question is when the y has evolved within the 'core sphaeropleales'.
this could be resolved by inclusion of other members  in further studies.
the two major reasons contributing to the robust results presented here are the change of the phylogenetic marker and the inclusion of secondary structure information.
in contrast to previous phylogenetic work concerning chlorophyceae, this study is based on the its <dig>  which offers a resolution power for relationships from the level of subspecies up to the order level, because of their variable sequence but conserved secondary structure  <cit> .
hitherto commonly used markers in contrast are a lot more restricted.
using 4sale  <cit>  with implemented structure consideration, we could achieve for the first time a global simultaneously generated sequence-structure alignment  yielding specific sequence and structural features distinguishing different algae lineages .
CONCLUSIONS
in summary, the powerful combination of the its <dig> rrna gene marker plus a multiple global alignment based synchronously on sequence and secondary structure yielded high bootstrap support values for almost all nodes of the computed phylogenetic trees.
thus, the relationship of sphaeropleaceae is here resolved, being a part of the sphaeropleales representing the monophyletic biflagellate do-group.
furthermore, we could elucidate a branched helix i of its <dig> as an autapomorphic feature within the do-group.
this feature could be found only in the 'hydrodictyon' and the 'scenedesmus' clade.
our results corroborate the presented methodological pipeline, the fundamental relevance of secondary structure consideration, as well as the elevated power and suitability of its <dig> in phylogenetics.
for a methodological improvement it is suitable to ameliorate the alignment algorithm in further considering horizontal dependencies of paired nucleotides, and moreover in future its <dig> studies it is suggested to include sequence and secondary structure information of hitherto not regarded taxa to resolve the chlorophycean phylogeny.
authors' contributions
mw designed the study.
ff determined the new sequences in the laboratory.
br implemented the strpnj within profdist.
ts and ak performed sequence analyses, structure prediction and phylogenetic analyses.
tm developed the its <dig> sequence-structure substitution model and the its <dig> sequence-structure scoring matrix.
ts, ak and mw drafted the manuscript.
all authors contributed to writing the paper, read the final manuscript and approved it.

 taxonomists face inconsistent or even contradictory clues when they examine the affiliation of organisms to higher taxonomic groupings. several characters may yield alternative hypotheses explaining their evolutionary background. this also applies to the taxonomic position of the sphaeropleaceae. different authors affiliate the green algal family by morphological characters to either ulvophytes or chlorophytes, until amendatory deason et al. suggested that the neochloridaceae, the hydrodictyaceae and the sphaeropleaceae should be grouped as sphaeropleales within the chlorophytes, since all of them have motile biflagellate zoospores with a direct-opposite confirmation of basal bodies. subsequently, other taxonomic lineages were added to this biflagellate do group, because they show molecular affiliation to either neochloridaceae or hydrodictyaceae. although nowadays most authors agree that the do group is monophyletic, until now no study pinpointed the taxonomic linkage of the name-giving'sphaeroplea' clade to the remaining'core sphaeropleales' persuasively with genetic evidence, i.e. the sister clade remains unclear. likewise, with respect to morphology, studies of 18s and 26s rrna gene sequences neither resolve the basal branching patterns within the chlorophyceae with high statistical power nor corroborate a monophyletic biflagellate do group without ambiguity. müller et al. obtained moderate statistical support for the close relationship of the'sphaeroplea' clade and the'core sphaeropleales' with profile distances of 18s and 26s rdna. in this study we followed and expanded their methodology with a very different phylogenetic marker. the internal transcribed spacer, the region of ribosomal rna between the s rrna gene and the large subunit has proven to be an appropriate marker for the study of small scale phylogenies of close relatives. the sequence is in contrast to the bordering regions of ribosomal subunits evolutionary not conserved, thus genetic differentiation is detectable even in closely related groups of organisms. by contrast, the secondary structure seems to be well conserved and thus provides clues for higher taxonomic studies. secondary structure information is furthermore especially interesting within the chlorophyceae, because van hannen et al. described an uncommon branching of its helix within the genera desmodesmus, hydrodictyon and scenedesmus. it is not known when this feature evolved and whether it is, as we expect, an apomorphic feature for the do-group. it is obvious that phylogenetic statements should be improvable by inclusion of structural information in common sequence analysis. for example, grajales et al. calculated morphometric matrices from its secondary structures for phylogenetic analyses, but treated information of sequence and structure as different markers. here we combine sequence with structural information in just one analysis. aside from the biological problem, we address the pivotal question of a methodological pipeline for sequence-structure phylogenetics using rdna data. methods dna extraction, amplification and sequencing extraction of genomic dna from cultured cells of ankyra judayi, atractomorpha porcata and sphaeroplea annulina was done using dynabeads® according to the manufacturer's protocol. pcr reactions were performed in a μl reaction volume containing μl faststart pcr master, μl gdna and nm of the primers its and its designed by white et al.. cycling conditions for amplification consisted of 94°c for min, cycles of 94°c for s, 50°c for s and 72°c for s, followed by a final extension step of min at 72°c. pcr products were analysed by 3% agarose gel electrophoresis and ethidium bromide staining. pcr probes where purified with the pcr purificaton kit and where quantified by spectrometry. each sequencing probe was prepared in an μl volume containing ng dna and μm primer. sequencing was carried out using an annealing temperature of 50°c with the sequencer applied biosystems qst genetic analyzer by the institute of hygiene and microbiology. its secondary structure prediction its secondary structures of the three newly obtained sequences were folded with the help of rnastructure and afterwards manually corrected. all available chlorophycean its sequences were obtained from the ncbi nucleotide database. the its secondary structure of atractomorpha porcata was used as template for homology modelling. homology modelling was performed by using the custom modelling option as provided with the its2-database. forty-nine species representing the chlorophycean diversity were retained and used as comparative taxa in inferring phylogenies. for this taxon sampling, accurate secondary structures of sequences were now folded by rnastructure and additionally corrected using pseudoviewer. we standardized start and end of all helices according to the optimal folding of the newly obtained sequences. listed is the current clade classification of the species and the genbank accession numbers of the analyzed sequences. the four newly obtained sequences are of the'sphaeroplea' clade. alignment and phylogenetic analyses using 4sale with its its specific scoring matrix, we automatically aligned sequences and structures simultaneously. sequence-structure alignment is available at the its database supplements page. for the complete alignment we tested for appropriate models of nucleotide substitution using the akaike information criterion as implemented in modeltest. the following paupblock was used for all maximum likelihood based phylogenetic analyses with paup*: lset base = nst = rmat = rates = gamma shape = pinvar =;. a maximum likelihood analysis was performed with a heuristic search and nearest neighbour interchange. maximum parsimony was accomplished with gaps treated as missing data and all characters coded as "unordered" and equally weighted. additionally, we clustered taxonomic units with neighbour-joining using maximum likelihood distances. furthermore, with mrbayes a bayesian analysis was carried out for tree reconstruction using a general time reversible substitution model with substitution rates estimated by mrbayes. moreover, using profdist, a profile neighbour-joining tree was calculated using the its specific substitution model available from the its database. pnj was also performed with predefined profiles of all the clades given in table for clade'scenedesmus' two profiles were used for groups'true scenedesmus' and'desmodesmus'. we performed a sequence-structure profile neighbour-joining analysis with a developmental beta version of profdist. the tree reconstructing algorithm works on a letter alphabet comprised of the nucleotides in three structural states. based on a suitable substitution model, evolutionary distances between sequence structure pairs have been estimated by maximum likelihood. all other applied analyses were computed only on the sequence part of the sequence-structure alignment. for mp, nj, pnj, prepnj and strpnj analyses bootstrap pseudoreplicates were generated. one hundred bootstrap replicates were generated for the ml analysis. additionally we used raxml at the cipres portal to achieve bootstraps with a substitution model estimated by raxml. all methods were additionally applied to a 50% structural consensus alignment cropped with 4sale. the individual steps of the analysis are displayed in a flow chart.  new its sequences genbank accession numbers for newly obtained nucleotide sequences are given in table. the two its sequences of sphaeroplea annulina strain sag 377-1a and strain sag 377-1e were identical and thus only the first one was used for further analysis. according to folding with rnastructure, its secondary structures of the three newly obtained sequences did not exhibit any branching in their helix i as it is described for the'core sphaeropleales', i.e. helix i was more similar to those of the cw-group and the'oedogonium' clade. helix i of sphaeroplea annulina was explicitly longer than those of the other newly obtained algae. due to this insertion, for sphaeroplea, a branching pattern was enforceable, but would have lower energy efficiency. however, the additional nucleotides are not homologous to the insertion capable of making an additional stem found in the'scenedesmus' and the'hydrodictyon' clade. its sequence and secondary structure information its sequence lengths of all studied species ran from to nucleotides, nt on average. the gc contents of its sequences ranged from % to %, with a mean value of %. the number of base pairs varied between and bp and averaged bp. the cropped alignment showed that 23% of the nucleotides had at least a 50% consistency in their pairings. compensatory base changes as well as hemi-cbcs range from to with a mean of cbcs. sequence pairs lacking cbcs were exclusively found within the same major clade. characteristics in a conserved part of alignment in agreement with coleman, the 5' side part near the tip of helix iii was highly conserved including the uggu motif, likewise the ugggu motif in case of chlorophyceae. we selected a part of the alignment at this position with adjacent columns to verify the suggested conservation. having a closer look at this part of helix iii, in our case, it showed typical sequence and structural characteristics for distinct groups. studied species of the'oedogonium' clade possess at position in the selected part of the alignment an adenine and in addition at positions 3– paired bases. in contrast, the cw-group solely possessed three consecutively paired bases in this block, but not the adenine. a typical pattern for clades of the do-group was a twofold motif of bases: uracile, adenine and guanine at positions 7– which is repeated at positions 11– this could be a duplication, which results in a modified secondary structure. in addition, the'core sphaeropleales' showed an adenine base change at position compared to all other clades. phylogenetic tree information the paup* calculation applying maximum parsimony included a total of characters, whereas characters were constant, variable characters were parsimony-informative compared to parsimony-uninformative ones. the resulting trees of all performed analyses yielded six major clades: the'dunaliella', the'hydrodictyon', the'oedogonium', the'reinhardtii', the'scenedesmus', and the'sphaeroplea' clade. all of them were separated and – except for the'scenedesmus' clade – highly supported by bootstrap values of 83–100%, respectively by bayesian posterior probabilities of –. the table supplements fig. and fig. node "g" supports a monophyletic do group and is printed in bold letters. software used: profdist and paup*. models of substitution: its = gtr with its substitution matrix, modeltest: tvm+i+g with estimated parameters. phylogenetic analysis: nj = neighbour-joining, pnj = profile neighbour-joining, prepnj = profile neighbour-joining with predefined profiles, strpnj = sequence-structure profile neighbour-joining, ml = maximum likelihood, b = bayesian analysis, mp = maximum parsimony. 1predefined profiles for profile neighbour-joining. the'hydrodictyon' clade, the'scenedesmus' clade and the'sphaeroplea' clade form one cluster that was strongly supported by high bootstrap values of 67–96%. the three clades composed the do-group. the opposite cluster included the'dunaliella' and the'reinhardtii' clade, forming the cw-group. the'oedogonium' clade was chosen as the outgroup. both clusters were strongly supported by bootstrap values of 84–100%. except for the bayesian analysis, all applied methods yielded node "e" as the weakest point within the basal branches, which presents the relationship between the'hydrodictyon' and the'scenedesmus' clade on the one hand and the'dunaliella', the'oedogonium', the'reinhardtii' and the'sphaeroplea' clade on the other hand. the phylogenetic tree resulting from neighbour-joining analysis by paup* did not support node "e" at all, but strongly supported the remaining labelled branches. the maximum likelihood analysis by paup* did not encourage node "e" either. both maximum likelihood methods did not even support nodes "a" and "c". all other basal branches were supported by this method. varying neighbour-joining analyses by profdist supported all basal branches – except for the weakest node "e" – with very high bootstrap support values of 84–100%. the maximum parsimony method gave average support for node "c" and "e" and high bootstrap values for the remaining basal clades. the bayesian analysis offered posterior probabilities of for node "c" and – for the remaining basal nodes. for further sister group relations see fig. and in comparison, the topology of the phylogenetic tree based on the 50% cropped alignment did not change, but the bootstrap support values were lower in all cases. discussion the internal transcribed spacer is required in ribosome biogenesis and its gradual removal from mature rrna is driven by its specific secondary structure. using three newly obtained its sequences from ankyra judayi, atractomorpha porcata and sphaeroplea annulina in this study we aimed to pursue two consecutive questions concerning the phylogenetic relationships within chlorophyceae. what is the phylogenetic position of the newly sequenced algae relative to the'core sphaeropleales' and could the biflagellate do-group be regarded as monophyletic? how does the secondary structure of the new its sequences look like and is an autapomorphic feature of the secondary structure associated with the monophyletic do-group? considering the question buchheim et al. and wolf et al. approached the problem with 18s + 26s rdna and 18s rdna data, but the relationship between the'core sphaeropleales' and the sphaeropleaceae remained unclear. however, in their studies, ankyra, atractomorpha and sphaeroplea clustered in a monophyletic clade named sphaeropleaceae. we confirm this'sphaeroplea' clade with all three genera being strongly separated from other clades. as a result of a bayesian analysis on a combined 18s and 26s rdna dataset shoup and lewis also found the sphaeropleaceae as the most basal clade within the sphaeropleales, but again the analysis lacked a strong backing. beside these difficulties the'core sphaeropleales' were already shown to be monophyletic with high certainty. the do-group as emended by deason et al., for which the directly opposed basal body orientation and basal body connection features are verified, is now strongly supported by molecular phylogenetic analyses. there was already evidence of an extended do-group, however, for some groups ultrastructural results are still lacking, and even though the collective basal body orientation and connection imply a monophyletic do-group, until now no molecular phylogenetic analysis could show this with solid support. we demonstrate for the first time with robust support values for the equivocal nodes that the'core sphaeropleales', the'sphaeroplea' clade, and the sphaeropleales are monophyletic. regarding question, for all structures of the'hydrodictyon' and the'scenedesmus' clade, helix i shows the typical branching. initially, an et al. proposed a secondary structure model with an unbranched helix i for its sequences of'scenedesmus' clade members. thereafter, van hannen et al. updated the model by folding the nucleotide sequences based upon minimum free energy and found a branched helix i as the most energetically stable option. the branching is result of an insertion of approximately nucleotides capable of folding as an individual stem within the 5' end of the first helix. however, its sequence and secondary structure information of further'core sphaeropleales' members, e.g. the'ankistrodesmus' clade and the'bracteacoccus' clade, lacks hitherto. in contrast, the y-structure is absent within the'sphaeroplea' clade and any other investigated group so far. thus this feature is – contrary to our expectation – not an autapomorphic character for the biflagellate do-group as a whole but for the'core sphaeropleales'. regarding future work, the resolution among the main clades of chlorophyceae was statistically poorly supported in previous studies. pröschold and leliaert reviewed the systematics of green algae by applying a polyphasic approach, but did not yield a clear resolution regarding a sister taxon to the sphaeropleales. since they are not yet available, its sequences of chaetopeltidalean and chaetophoralean taxa could not be included in the present study and therefore the phylogenetic relationships between the main chlorophyceae clades remain open. we recommend involving sequence and secondary structure information of chaetopeltidalean and chaetophoralean its sequences in future studies to find out if the monophyletic biflagellate do-group could be further extended to a general monophyletic do-group containing quadri- and biflagellate taxa. a genome-wide approach indicates that sphaeropleales and chlamydomonadales are sister taxa, however only a few organisms are included in this study. an additional uprising question is when the y has evolved within the'core sphaeropleales'. this could be resolved by inclusion of other members in further studies. the two major reasons contributing to the robust results presented here are the change of the phylogenetic marker and the inclusion of secondary structure information. in contrast to previous phylogenetic work concerning chlorophyceae, this study is based on the its which offers a resolution power for relationships from the level of subspecies up to the order level, because of their variable sequence but conserved secondary structure. hitherto commonly used markers in contrast are a lot more restricted. using 4sale with implemented structure consideration, we could achieve for the first time a global simultaneously generated sequence-structure alignment yielding specific sequence and structural features distinguishing different algae lineages.  in summary, the powerful combination of the its rrna gene marker plus a multiple global alignment based synchronously on sequence and secondary structure yielded high bootstrap support values for almost all nodes of the computed phylogenetic trees. thus, the relationship of sphaeropleaceae is here resolved, being a part of the sphaeropleales representing the monophyletic biflagellate do-group. furthermore, we could elucidate a branched helix i of its as an autapomorphic feature within the do-group. this feature could be found only in the'hydrodictyon' and the'scenedesmus' clade. our results corroborate the presented methodological pipeline, the fundamental relevance of secondary structure consideration, as well as the elevated power and suitability of its in phylogenetics. for a methodological improvement it is suitable to ameliorate the alignment algorithm in further considering horizontal dependencies of paired nucleotides, and moreover in future its studies it is suggested to include sequence and secondary structure information of hitherto not regarded taxa to resolve the chlorophycean phylogeny. authors' contributions mw designed the study. ff determined the new sequences in the laboratory. br implemented the strpnj within profdist. ts and ak performed sequence analyses, structure prediction and phylogenetic analyses. tm developed the its sequence-structure substitution model and the its sequence-structure scoring matrix. ts, ak and mw drafted the manuscript. all authors contributed to writing the paper, read the final manuscript and approved it. 
a branched helix i is an apomorph characteristic within the monophyletic do-group. within chlorophyceae the its secondary structure shows an unbranched helix i, except for the'hydrodictyon' and the'scenedesmus' clade having a ramified first helix. in the present study we analysed the secondary structure of three newly obtained its sequences classified within the'sphaeroplea' clade and resolved sphaeroplealean relationships by applying different phylogenetic approaches based on a combined sequence-structure alignment. the newly obtained its sequences of ankyra judayi, atractomorpha porcata and sphaeroplea annulina of the'sphaeroplea' clade do not show any branching in the secondary structure of their helix i. all applied phylogenetic methods highly support the'sphaeroplea' clade as a sister group to the'core sphaeropleales'. in green algae, a branched helix i in the secondary structure of the its evolves past the'sphaeroplea' clade. our results corroborate the fundamental relevance of including the secondary structure in sequence analysis and phylogenetics. the latter two are classified within the sphaeropleales, characterised by directly opposed basal bodies in their flagellar apparatuses. furthermore, based on characteristics in the sequence-structure alignment one is able to distinguish distinct lineages within the green algae. previous studies could not resolve the taxonomic position of the'sphaeroplea' clade within the chlorophyceae without ambiguity and two pivotal questions remain open: is the do-group monophyletic and is a branched helix i an apomorphic feature of the do-group?
 within chlorophyceae the its secondary structure shows an unbranched helix i, except for the'hydrodictyon' and the'scenedesmus' clade having a ramified first helix. the latter two are classified within the sphaeropleales, characterised by directly opposed basal bodies in their flagellar apparatuses. previous studies could not resolve the taxonomic position of the'sphaeroplea' clade within the chlorophyceae without ambiguity and two pivotal questions remain open: is the do-group monophyletic and is a branched helix i an apomorphic feature of the do-group? in the present study we analysed the secondary structure of three newly obtained its sequences classified within the'sphaeroplea' clade and resolved sphaeroplealean relationships by applying different phylogenetic approaches based on a combined sequence-structure alignment.  the newly obtained its sequences of ankyra judayi, atractomorpha porcata and sphaeroplea annulina of the'sphaeroplea' clade do not show any branching in the secondary structure of their helix i. all applied phylogenetic methods highly support the'sphaeroplea' clade as a sister group to the'core sphaeropleales'. thus, the do-group is monophyletic. furthermore, based on characteristics in the sequence-structure alignment one is able to distinguish distinct lineages within the green algae.  in green algae, a branched helix i in the secondary structure of the its evolves past the'sphaeroplea' clade. a branched helix i is an apomorph characteristic within the monophyletic do-group. our results corroborate the fundamental relevance of including the secondary structure in sequence analysis and phylogenetics. 