BACKGROUND
tens of thousands of sequences of the small subunit ribosomal rna  gene of eukaryotes have accumulated in public databases such as ncbi genbank  <cit> , making this gene one of the first and most frequently used markers for molecular phylogenetics.
its popularity is due to a high degree of conservation in some regions of the molecule, in combination with a considerable amount of variability in others.
these features enable phylogenetic questions to be addressed between relatively closely related taxa, as well as between different domains of life  <cit> .
therefore, analyses of ssu rrna sequences have a long history, and new sequences are still being continuously generated.
ssu rrna molecules fold into a specific secondary structure, which is essential for maintenance of their three dimensional structure and their function within the ribosome  <cit> , but which also has consequences for the use of rrna molecules in phylogenetics.
the secondary structure of rrnas is maintained by hydrogen bonds between rna nucleotides, which form helices .
these helices are interleaved by regions consisting of unpaired nucleotides, forming loops at the end of a helix and bulges within different helices.
secondary structure of rnas is generally much more conserved than their primary sequence  <cit> .
therefore, considering this structure during multiple sequence alignment can greatly improve the assignment of homologous positions, consequently resulting in more probable phylogeny estimations  <cit> .
furthermore, paired nucleotides  frequently co-evolve in order to maintain rrna structure and function.
the co-evolution of doublets violates the assumption of independent evolution of sites made by most phylogenetic methods  <cit> .
consequently, specific evolutionary models have been proposed for paired sites and have been shown to outperform standard  nucleotide models  <cit> .
however, secondary structure models are still rarely used in phylogenetic analyses, presumably because establishing a secondary structure for a new sequence is still a time-consuming exercise even for the conserved core structure of ssu rrna, and very few software packages allow the simultaneous analysis of paired and unpaired rrna regions.
some rrna databases  <cit>  provide secondary structure information for a number of organisms, but their records are far from complete and structures of hypervariable insertions are usually not presented, or are only presented to a certain extent.
in particular, the lower metazoa, which are pivotal for the understanding of animal evolution, are still under-represented in databases.
one key taxon for early metazoan evolution is phylum porifera , in which the relationships are unresolved at all taxonomical levels, even between the three extant sponge classes demospongiae, calcarea  and hexactinellida .
within sponge classes, the results of molecular phylogenies are often incongruent with morphological expectations  <cit> .
in this study, we performed the first comprehensive survey of the complete ssu rrna secondary structures of representatives of the main lineages of phylum porifera, and evaluated how secondary structure information and features other than the primary sequence can contribute to improve phylogenetic reconstructions.
for these purposes, we considered all available ssu rrna sequences of porifera, inferred their secondary structures , and analyzed base compositions and sequence lengths.
we reconstructed a phylogeny with partitioned phylogenetic analyses using specific rrna models of nucleotide evolution for paired sites.
using this backbone, we assessed the phylogenetic value of secondary structures of unique insertions found in a specific demosponge clade , which would usually be disregarded as 'unalignable sites' and thus excluded from standard phylogenetic analyses.
methods
sequence acquisition, analyses and inference of secondary structures
we analyzed all  <dig> published full or nearly full-length ssu rrna sequences of porifera .
for taxonomy of the taxa included in our study we followed systema porifera  <cit>  and the world porifera database  <cit> , where also the species authorities are available.
the ssu rrna sequence of amphimedon queenslandica was reconstructed by performing a local blast search  <cit>  against data from genbank's trace archive.
traces from significant hits  were downloaded and assembled in codoncode aligner  <dig> .
<dig>  <cit> .
this resultant sequence can be downloaded from our database of ssu rrna secondary structures of porifera  <cit> .
for class hexactinellida, only limited data was available in genbank: all three full-length ssu rrna sequences belong to subclass hexasterophora.
two additional hexactinellid sequences were provided by martin dohrmann ahead of their publication in a comprehensive phylogenetic study of hexactinellida  <cit> : semperella schulzei  and aphrocallistes vastus .
all sequences were initially aligned with clustal w  <dig>   <cit>  and the preliminary alignments were manually improved in seaview  <cit> .
gblocks  <dig> b  <cit>  was used to identify and isolate the conserved sites of the alignment before clustering similar sequences using the neighbor joining  algorithm in paup*  <dig> b <dig>  <cit> .
secondary structures for resulting clades were established for certain representatives of the clade by aligning to known structures from the european rna database  <cit>  in separate alignments for each clade and considering compensatory base changes.
ssu rrna clade-alignments were then further refined according to secondary structure information.
the unusual structures of marine haplosclerida  and hexactinellida  were initially examined under minimum free energy predictions from the mfold-server  <cit> .
in most cases, only one structure was predicted by the algorithm.
if multiple structures were predicted, we chose the structure with either the minimal free energy or with the best compatibility to similar sequences.
a comparative approach  <cit>  was chosen if permitted by an appropriate level of sequence divergence.
for this approach, we used the alifold server  <cit>  to infer secondary structures of the insertions.
alifold infers secondary structures by considering both minimum free folding algorithms and compensatory base changes, and therefore includes additional information that provides hints for secondary structural motifs.
since this method requires a correct alignment, it could only be used if sequences were not too divergent from each other, such as with a subset of marine haplosclerida  and the insertions of hexasterophora  .
however, secondary structures inferred with both methods were identical, or only differed in a few positions .
therefore, while the comparative method is preferred, we still found that minimum free energy based predictions performed adequately to be used in cases where unambiguous alignments or missing comparative data does not allow inference of secondary structures based on compensatory base exchanges.
for taxa that were suitable for a comparative approach, compensatory base exchanges are presented together with the corresponding alignments in additional file  <dig>
we visualized selected structures by converting the sequence and structure information to a ct-format with a perl-script.
this format can be displayed in rnaviz  <dig>  <cit> .
helix names correspond to wuyts et al.
<cit> , with the exception of helices e23_ <dig> and e23_ <dig>  which together are referred to as e23_ <dig>  insertions are designated by the name of the conserved helix in which they occur, and a period plus the number of the additional helix is added: parts of conserved helices separated by insertions are named after the original helix followed by a letter .
base compositions and the lengths of the secondary structure features were calculated with a custom-made perl script.
to avoid biases introduced by missing data from the published sequences, we used a fragment  spanning from helix  <dig> until  <dig> bp before helix  <dig> , and only considered the  <dig> sequences without data missing within this region .
representative poriferan secondary structures are available as *.fasta-format  and in *.ct-format from our database for ssu rrna secondary structures of porifera  <cit> .
furthermore, several perl scripts  for format conversion are provided .
phylogenetic analyses
the secondary structure information from the previous step was used to generate a new alignment in seaview.
we generated a taxon-set comprising of  <dig> taxa  and focused on relationships of haplosclerid demosponges, in a similar way to redmond et al.
<cit> .
the ssu rrnas from this diverse taxon have been found to possess numerous insertions and extensions and our aim was to unravel their evolution.
sites with uncertain homology even after considering secondary structure were excluded from the phylogenetic analyses.
this was achieved by assigning sites to two groups and discarding those sites that were regarded as ambiguously aligned by the following criteria:
<dig>  unpaired sites: with length polymorphism and sequence divergence too high to identify homologous positions for all sequences.
<dig>  paired sites: with length polymorphisms in helices and/or structural homologies that could not be unambiguously assigned .
furthermore, taxon-specific insertions within helices , as well as nucleotide insertions found only in single sequences were excluded.
doublet positions were only regarded as pairings in the consensus secondary structure if the two involved nucleotides formed a watson-crick  or g-u wobble pairing in at least five sequences within the alignment.
corresponding sites falling below this five-sequence threshold were treated as unpaired.
for phylogenetic reconstructions, sites were allocated to one of the following two partitions: partition 'stem'  or partition 'loop' .
we used mrbayes  <dig> .
<dig>  <cit>  and phase  <dig>   <cit>  for the phylogenetic analyses, as both programs allow the simultaneous analysis of a partitioned dataset with both rrna models for paired sites and standard models for unpaired sites.
mrbayes only allows the usage of a doublet model corresponding to the sh model  <cit> .
this is a  <dig> state-rna model, which considers all possible doublets as characters and assumes that compensatory base exchanges result from at least two substitution events.
a gtr + g + i model  <cit>  was assigned for the loop partition.
the markov chain monte carlo  analysis comprised two runs  for  <dig>  million generations, with the sample frequency set to  <dig> and the temperature for the heated chains set to  <dig> .
sampled trees were summarized using the sumt command in mrbayes with a burn-in set to the first  <dig> million generations.
sufficient convergence of chains for the mr bayes runs was monitored by observing log-likelihood values, the standard derivation of split frequencies , and diagnostics provided by awty  <cit> .
in phase, we applied the rna7a model  <cit>  and rna7d model  <cit>  for stem regions in independent runs.
rna7a is the most general 7-state rna model.
rna7d  is a simplification of rna7a .
the 7-state rna models treat all mismatches as one single state.
this simplification increases the risk of loss of phylogenetic information, but the occurrence of mismatch-pairs in rrna data is small, therefore, an estimation of mismatch substitution parameters from the data is probably not accurate  <cit> .
furthermore, by pooling mismatches into a single character, the number of parameters to be estimated in a phylogenetic analysis, and consequently the computational demands are significantly decreased.
for loop regions, the rev model  <cit>  was chosen.
in addition, a gamma distribution accounting for rate heterogeneity among sites and a proportion of invariant sites were assigned to each model for both partitions.
independent runs were performed in phase 2: two runs with the rna7a model  and one run  with the rna7d model for stem positions.
every 100th generation a sample was taken from the mcmc chains .
tracer v <dig>   <cit>  was used to monitor sufficient parameter stabilization.
to create readable input files for tracer from the phase runs, we used a slightly modified version of the perl script 'phase2tracer.pl' , which is available upon request.
the presented tree topology is based upon one of the  <dig> million phase runs with the rna7a model for stem partition .
to obtain branch-lengths for the tree, we conducted an additional analysis  under the same models, and tree topology was fixed to the consensus tree from the original  <dig> million generation analysis as suggested in the phase manual .
RESULTS
ssu rrna length differences and base composition
to avoid biases due to missing data, we analyzed base composition and sequence length for a fragment of ssu rrna that covers about  <dig> % of the gene .
base composition and fragment length differed considerably among the  <dig> poriferan sequences .
the gc content varied between  <dig>  and  <dig>  %.
calcarea posses the lowest gc contents with a modest variation from  <dig>  to  <dig>  %.
in this aspect they are clearly separated from demosponges, which display significantly higher gc contents, since the lowest demosponge value  still exceeds the highest gc content  of calcarea .
most demosponge ssu rrnas show modest length variations in a range comparable to those of calcarea.
notable exceptions are the extraordinary large rrna molecules found in several marine haplosclerids.
the highest gc contents of porifera are also found within this group .
the high gc pattern is independent of the presence of insertions in these large molecules, since members of marine haplosclerida with smaller rrna molecules also possess similar gc contents .
ssu rrnas of the few available hexactinellid sponges are approximately equal in length to large molecules of several haplosclerids .
in contrast to haplosclerids, hexactinellid sponges have lower gc contents, with base compositions in the range of those of demospongiae and calcarea.
secondary structure
porifera have the typical eukaryotic core ssu rrna structure .
the moderate length variation between calcarea and most demosponges is primarily caused by insertions in unpaired regions or by elongation of helices  <dig>  e10_ <dig> and  <dig> .
in hexactinellida, on average, these three helices are largely elongated compared to calcarea and demospongiae , but the lengths of the e10_ <dig> helices of some demosponge sequences fall into the same range.
*marine haplosclerids not included in helix 43; **farrea occa not included for helix  <dig>
in addition, we observed extra-helical insertions in hexactinellida and in several marine haplosclerid demosponges that are not part of the eukaryote core structure.
in marine haplosclerids, these extra sequences were inserted within helices e23_ <dig>  e23_ <dig> and  <dig>  and in hexactinellida, the insertions only occurred within helix e23_ <dig> at a different position than in marine haplosclerids .
all of the helices where sequence elongations and/or insertions occur belong to regions that are known to be highly variable within eukaryotes  <cit> .
calcarea
the ssu rrna of this sponge class comprises all of the typical eukaryote helices and lacks unusual structural features.
a calcarean ssu rrna consensus sequence and structure is shown in fig.
<dig>  several synapomorphies for the two calcinea and calcaronea subclasses were detected in the secondary structure.
in calcinea, helices  <dig> and e23_ <dig> are shorter by at least one base pair when compared to calcaronea .
in helix e10_ <dig>  calcaronea typically have three pairs at the helix end, whereas calcaronea dominantly possess four pairs .
however, independent elongations of this helix can be found in both subclasses .
these elongations are homoplasies as is evident when considering the subclass-specific compensatory base change  at the beginning of inset ii : the a-u pair in the corresponding structure of the calcaronean leucascandra caveolata  supports a secondary loss of a pair compared to other calcaronea.
differences in helix nucleotides between both subclasses occur in helices  <dig>  e23_ <dig>  e23_ <dig> and  <dig> .
most of these changes maintain the helix-relevant pairings , but a few cause mismatches in at least some sequences .
base changes and insertions in unpaired regions are also specific for the calcinea-calcaronea split.
this is indicated in fig.
<dig> for three bases in the bulge between helices  <dig> and  <dig>  one base within the loop of e23_ <dig>  and a calcaronean-specific insertion of one adenosine between helix  <dig> and  <dig>
hexactinellida
the ssu secondary structure of acanthascus dawsoni is presented in fig.
<dig> representatively for hexactinellida.
in all hexactinellid sequences, specific insertions were observed .
as mentioned previously, hexactinellid ssu rrna sequences are considerably longer than in other poriferans, except in some marine haplosclerid demosponges .
the additional nucleotides occur in extensive elongations of common helices , and a helical insertion in helix e23_ <dig>  the insertions in helix e23_ <dig> occur at a unique position among sponges and may form two helices  in all the studied ssu rrna molecules of subclass hexasterophora.
helix e23_1b.
<dig> contains  <dig> doublets and is much more conserved within hexasterophora than helix e23_1b.
<dig>  which varies in length from  <dig> to  <dig> bp.
in contrast to hexasterophora, semperella schulzei  has a helical insertion of  <dig> bp within e23_ <dig>  which is predicted to form a single helix e23_1b.
<dig> .
within the sequence of farrea occa , we found deletions in conserved regions.
helices  <dig> and  <dig> are missing completely, as are the 3' strand of helix  <dig>  parts of helix  <dig> and the 5' strand of helix  <dig> .
such complete or partial deletion of conserved helices has been shown to be typical for non-functional rrna pseudogenes  <cit> .
potential paralogs like this one are not necessarily subject to concerted evolution, and are therefore not suitable for phylogenetic inference.
in this context, the consideration of secondary structure is crucial for identification of such non-functional sequences, and prevents biases in phylogenetic reconstruction due to potentially misleading data.
nonetheless, predictions of insertions for this sequence are displayed in fig.
<dig> , since no suspicious modifications were found within this part of the molecule and no other sequence of farreidae was available.
however, the results for this species should be treated with caution.
demospongiae
most demosponges possess a ssu rrna molecule with the common metazoan secondary structure.
remarkable exceptions are only found within the marine haplosclerida , which possess insertions that are long enough to be predicted to form additional helices.
those helices are found within known variable regions for eukaryotes and appear in the 5' strand of helix e23_1/ <dig>  the 5'strand within helix e23_ <dig> and the 3' strand of helix  <dig>
phylogenetic analyses
we inferred the phylogeny of marine haplosclerids to compare the evolutionary history of helical insertions found in this group of demospongiae .
results from the phase- and mrbayes analyses of  <dig> taxa are shown in fig.
<dig>  although more general 7-state models have been shown to result in higher likelihood values for phylogenies than less parameter-rich models for real rrna data  <cit> , our analyses with phase with the rna7a model and the less complex rna7d model yielded identical tree topologies .
independent runs in phase and mrbayes resulted in similar, almost identical topologies, and differences in demosponge relationships were only observed in the positions of clades with weak support values.
namely these are the relationship of dictyoceratida to the myxospongiae , the position of scopalina ruetzleri and relationships within freshwater sponges .
additionally, differences were observed in calcarea and cnidaria.
the order haplosclerida was not resolved as monophyletic, since suborder spongillina  fell into other distantly related demosponge clades, rather than into marine haplosclerida.
the two suborders, haplosclerina  and petrosina  were not supported as monophyla .
these results are congruent with results from former analyses of ssu rrna, 28s rrna and cytochrome oxidase subunit i  <cit> .
according to our analysis, amphimedon queenslandica  is most closely related to oceanapia sp.
, with xestospongia muta  as sister taxon, and both nodes in the tree are very highly supported by posterior probability  values in both bayesian analyses.
other species of the family niphatidae  are not closely related to each other or to amphimedon queenslandica .
in addition, other members of families petrosiidae and phloeodictyidae are not found in a closer relationship to the three species clade.
our results were mostly concordant with redmond et al.
<cit> , but with higher support values in several clades.
we did not find any monophyletic haplosclerid families or genera in our taxon set.
differences between our results and the previous study are highlighted at the nodes in fig.
<dig>  we could not recover monophyletic petrosiidae in clade i, and relationships of several clade iii taxa differed.
furthermore, xestospongia muta and oceanapia sp.
cluster in one clade  .
successive evolution of additional helices in marine haplosclerids
within haplosclerids, the evolution of additional helices can be reconstructed by plotting structures to the well-supported phylogenetic backbone .
primary sequences of these motifs were not included in the tree construction  due to ambiguous alignment, but can be regarded as additional phylogenetic characters.
the helical insertions apparently evolved in at least two steps, which fits the findings of the ssu rrna gene tree strikingly well.
the relationships within marine haplosclerids can be described as four well-supported  nested clades i-iv that display different stages of secondary structure evolution .
clade i contains all marine haplosclerida.
the basal diverging taxa lack any large insertions that are typical for other marine haplosclerida.
however, the predicted structure within helix  <dig> differs from the standard structure in this region found in other porifera  and displays a larger bulge of unpaired bases at the insertion point of the larger helical structures found within all taxa in clade ii.
this bulge may be the precursor for the extensions at this position observed in clade ii.
within basal diverging taxa of clade ii , a similar bulge is found for xestospongia muta in helix e23_ <dig> at the insertion-site of subsequent extensions in clade iii, but not the other sequences lacking e23_ <dig> .
larger insertions appeared in helices e23_ <dig> and  <dig> 'simultaneously'  in the common ancestor of clade ii taxa.
the three taxa of clade iv according to our minimum free energy calculations share an additional helix 43b.
<dig> as synapomorphy .
after the introduction of helical insertions in helices e23_ <dig> and  <dig>  a long extension evolved within helix e23_ <dig> as a synapomorphy in clade iii.
an autapomorphy for dasychalina fragilis is an additional helix formed b insertions within e23_ <dig>  within the complete taxon of marine haplosclerids, no loss of formerly gained additional helical insertions has been documented, therefore, no ssu rrna molecule from a descendent of a taxon with extraordinary features has returned to the ancestral basic metazoan core structure.
discussion
unusual patterns within poriferan ssu rrna secondary structure
we reported the secondary structures of a variety of poriferan ssu rrna sequences, and suggest structure predictions for secondary structure motifs that are specific for some lineages, i.e.
marine haplosclerida  and hexactinellida.
such additional helical insertions occur in a variety of eukaryotes and are known to be homoplasies, because they occur in several, not closely related taxa  <cit> .
our data shows that such structures are also present in early diverging metazoa .
insertions in helix e23_ <dig> evolved independently in hexactinellida and the marine haplosclerida , which is evident  from our phylogenetic analyses that captured 'snapshots' of the evolution of helices within the marine haplosclerida  and  from the observations that insertions appear at different positions within helix e23_ <dig> .
although additional helical insertions are present within the e23-extension fragment in various eukaryotic taxa, to our knowledge, none have been reported within helix e23_ <dig>  which is therefore synapomorphic for clade iii haplosclerids.
interestingly, helical insertions within haplosclerids first appeared in the typical regions for such insertions, namely within helices e23_ <dig> and  <dig>  before they evolved within e23_ <dig> .
therefore, the evolution of extensions at more common insertion sites might be a prerequisite for the evolution of additional helical structures within e23_ <dig>
higher metazoa with unusual ssu rrna structures also contain unusual motifs in their large ribosomal subunit , e.g., in branchiopod crustacea  <cit> .
in sponges, additional motifs in a fragment of the lsu have previously been reported for hexactinellida and marine haplosclerid demosponges  <cit> , but not for non-haplosclerid demosponges or calcarea.
this is in striking accordance with our ssu rrna findings, and encourages further studies of the complete lsu secondary structure of these taxa.
since both rrna units are encoded in one translational unit, the same mechanisms may be responsible for the formation of extra helical features in both rrna molecules.
remarkably, not only the nuclear rrnas display unusual secondary structure motifs in marine haplosclerida.
in the recently published mitochondrial genome of the haplosclerid amphimedon queenslandica, both of the mitochondrial  rrna genes  also contain additional helices that are not found in other demosponges  <cit> .
although this may be a coincidental observation and needs to be verified by data from additional haplosclerid mt rrna sequences, it is possible that the same selection mechanisms act on the nuclear and mt rrna in this taxon.
however, such correlations do not exist in all taxa, since the recently sequenced mitochondrial genomes of hexactinellida  <cit>  contain extremely short rrnas , in contrast to the large insertions in the hexactinellid nuclear rrnas .
for the nuclear ssu rrna, the fact that extra-helical structures are found in the e23-extension region and helix  <dig> in various taxa indicates that these regions are under less functional constraints than are the core regions of ssu rrna; wuyts et al.
<cit>  showed by considering the tertiary structure of rrna that nucleotide variability increases with distance from the ribosome centre.
eukaryotic insertion sites for additional helices are therefore located in the same, or similar regions at the  periphery of rrna molecules.
the authors concluded that these insertions do not interfere with the ribosomal function of the ribosome and can therefore arise independently within different lineages, similar to our observations in hexactinellida and marine haplosclerids.
phylogenetic value of rrna features
we demonstrated different applications of ssu rrna features for phylogenetic analyses:
base composition and synapomorphic base exchanges
base compositions of ssu rrna differ strikingly between demospongiae and calcarea.
the gc contents of the  demospongiae are always higher and show a wider range of variability than the ones of calcarea.
for hexactinellida, only five sequences were available , therefore general conclusions regarding their gc contents should be interpreted with care.
however, for the few sequences available the gc contents fell into the ranges observed in calcarea and demospongiae.
several apomorphic positions identified in calcarean ssu rrna allow to unambiguously distinguish between the two subclasses calcinea and calcaronea, thus supporting other morphological and molecular data  <cit> .
rna models for phylogeny estimation and evolution of additional helical structures as evolutionary markers
the bayesian phylogenetic reconstructions using structure-defined partitions with different rrna models for doublets in mrbayes  <dig> .
<dig> and phase  <dig> yielded very similar tree topologies with increased support for several nodes compared to the maximum parsimony  and maximum likelihood  analyses presented by redmond et al.
<cit>  .
partitioned analyses using rrna models as applied in our analysis, have been reported to result in better-supported topologies .
however, other factors may have contributed to our findings, e.g., it is known that bayesian posterior probability values are often higher than corresponding nonparametric bootstrap values and may even provide support for the 'wrong' clades in studies with simulated data  <cit> .
the relevant important haplosclerid clades were supported with very high pp values .
these high values should overcome eventual problems of support overestimation.
also, support for 'wrong' clades is unlikely to be a problem in our results, since the topologies from redmond et al.
<cit>  are mostly concordant with ours.
regarding the general differences in bootstrap and pp values, the different software packages used and the difference in the data set  may have contributed to the higher support found for several clades.
although standard models of nucleotide evolution violate the assumption of independent evolution of all sites when also applied to paired sites as done by redmond et al.
<cit> , this seems to have little impact on nodes with high support values in the case of the demosponge dataset studied .
this suggests that the biases introduced by the use of less well fitting 'standard' rrna models may have a higher impact on clades that are difficult to resolve , whereas a strong phylogenetic signal will be recovered even if a sub-optimal evolutionary model is used for analyses.
amphimedon queenslandica, the target of the sponge genome sequencing project, did not cluster with any other representative of its family niphatidae.
likewise, neither the other haplosclerid families , nor the genera  could be recovered as monophyletic,  in accordance with the ssu-rrna based findings of redmond et al.
<cit> .
strikingly, these inferred relationships are supported by the presence or absence of secondary structure motifs within haplosclerida: different members of the families niphatidae, petrosiidae and phloeodictyidae show a different number of specific insertions that are congruent with the phylogenetic relationships that we previously inferred without the inclusion of these extended regions .
the presence and absence of such helices are therefore good phylogenetic indicators for these relatively closely related taxa, even though alignment of the primary sequence of these helices  between all taxa is difficult due to high evolutionary rates.
homology inference of sites in hypervariable regions according to their secondary structure is known to be problematic.
functional constraints are probably more relaxed in these regions, and the observed evolution of insertions is driven by unknown mutational mechanisms, which might tend to produce similar motifs by homoplasy  <cit> .
in contrast, within marine haplosclerids, no loss of helical insertions that arose at some earlier point of their evolutionary history occurred .
furthermore, no independent homoplasic helical insertions ever appeared in the same positions within ssu rrna.
considering our findings, the presence and absence of large helical insertions appears to provide strong phylogenetic information at selected taxonomical levels.
a similar phylogenetic information value of additional helical structures may be present for insertions in helix e23_ <dig> of hexactinellida, although generalized conclusions are limited by the small sample size.
nonetheless, the insertions in hexasterophora  are predicted to possess two additional helices, while the insertion found in the only considered amphidiscophoran, semperella schulzei, forms only one helix .
it is evident that integration of secondary structure information in sequence alignment and analyses  will optimize rrna phylogenies considerably.
CONCLUSIONS
the ssu rrna provides far more valuable phylogenetic information than just its primary sequence.
even simple features like base composition already bear enough information to distinguish between the two higher sponge taxa calcarea and demospongiae .
unusual secondary structures can lend further support to results from independent phylogenetic inferences, as we showed for helical insertions in marine haplosclerid demosponges  and for a small number of hexactinellid sponges .
in this way, otherwise neglected hypervariable insertions can yield further support to a given topology.
although we only explored additional structures of ssu rrna in sponges, our results should encourage further studies.
especially the study of lsu rrna structure seems promising in this regard, since this gene is more variable than ssu rrna and contains large extension regions that strongly differ among higher taxa  <cit> .
on the intraspecific level, the even more variable internal transcribed spacer regions  can provide secondary structure features of phylogenetic value .
relating secondary structure to sequence information will allow the phylogenetic signal of the huge numbers of rrna sequences currently available in genbank to be considerably increased.
our newly generated database of porifera ssu rrna secondary structures will facilitate the inclusion of secondary structure information in phylogenetic analyses.
authors' contributions
ov contributed to the conception and design of the study, acquired, analyzed  and interpreted the data, and wrote the manuscript.
de and gw contributed to the conception and design of the study, and critical revision of the manuscript.
all authors read and approved the final version of the manuscript.
supplementary material
additional file 1
taxonomy and genbank accession numbers of downloaded poriferan ssu rrna sequences.
click here for file
additional file 2
trace ids from genbank trace archive used to assemble the amphimedon queenslandica ssu rdna sequence.
click here for file
additional file 3
compensatory base changes and alignments of predicted secondary structures of clade iii marine haplosclerida  and hexasterophora .
click here for file
additional file 4
base composition and fragment lengths of examined poriferan ssu rrna fragment .
click here for file
additional file 5
alignment with secondary structure information and character set.
secondary structure is provided for regions that are included in the phylogenetic analyses only.
additional secondary structure information is available from our poriferan ssu rrna database.
click here for file
acknowledgements
this work was financially supported by the german research foundation  and is also a contribution from the dfg priority program spp <dig> 'deep metazoan phylogeny' .
we thank martin dohrmann for providing hexactinellid sequences ahead of publication and for useful comments on the manuscript and catherine vogler for commenting on the manuscript and for very productive discussions.
ov would like to thank and the organizers and participants of the  <dig> workshop on molecular evolution in woods hole for inspiring discussions and the support of the boehringer ingelheim fonds to cover travel expenses and tuition fees.
d.e.
acknowledges financial support from the european union under a marie-curie outgoing fellowship , gw acknowledges financial support from the marie-curie est hotspots.
the gesellschaft für wissenschaftliche datenverarbeitung göttingen  is acknowledged for access to their computer facilities.
we thank three anonymous reviewers for their constructive comments, especially reviewer  <dig>

 tens of thousands of sequences of the small subunit ribosomal rna gene of eukaryotes have accumulated in public databases such as ncbi genbank, making this gene one of the first and most frequently used markers for molecular phylogenetics. its popularity is due to a high degree of conservation in some regions of the molecule, in combination with a considerable amount of variability in others. these features enable phylogenetic questions to be addressed between relatively closely related taxa, as well as between different domains of life. therefore, analyses of ssu rrna sequences have a long history, and new sequences are still being continuously generated. ssu rrna molecules fold into a specific secondary structure, which is essential for maintenance of their three dimensional structure and their function within the ribosome, but which also has consequences for the use of rrna molecules in phylogenetics. the secondary structure of rrnas is maintained by hydrogen bonds between rna nucleotides, which form helices. these helices are interleaved by regions consisting of unpaired nucleotides, forming loops at the end of a helix and bulges within different helices. secondary structure of rnas is generally much more conserved than their primary sequence. therefore, considering this structure during multiple sequence alignment can greatly improve the assignment of homologous positions, consequently resulting in more probable phylogeny estimations. furthermore, paired nucleotides frequently co-evolve in order to maintain rrna structure and function. the co-evolution of doublets violates the assumption of independent evolution of sites made by most phylogenetic methods. consequently, specific evolutionary models have been proposed for paired sites and have been shown to outperform standard nucleotide models. however, secondary structure models are still rarely used in phylogenetic analyses, presumably because establishing a secondary structure for a new sequence is still a time-consuming exercise even for the conserved core structure of ssu rrna, and very few software packages allow the simultaneous analysis of paired and unpaired rrna regions. some rrna databases provide secondary structure information for a number of organisms, but their records are far from complete and structures of hypervariable insertions are usually not presented, or are only presented to a certain extent. in particular, the lower metazoa, which are pivotal for the understanding of animal evolution, are still under-represented in databases. one key taxon for early metazoan evolution is phylum porifera, in which the relationships are unresolved at all taxonomical levels, even between the three extant sponge classes demospongiae, calcarea and hexactinellida. within sponge classes, the results of molecular phylogenies are often incongruent with morphological expectations. in this study, we performed the first comprehensive survey of the complete ssu rrna secondary structures of representatives of the main lineages of phylum porifera, and evaluated how secondary structure information and features other than the primary sequence can contribute to improve phylogenetic reconstructions. for these purposes, we considered all available ssu rrna sequences of porifera, inferred their secondary structures, and analyzed base compositions and sequence lengths. we reconstructed a phylogeny with partitioned phylogenetic analyses using specific rrna models of nucleotide evolution for paired sites. using this backbone, we assessed the phylogenetic value of secondary structures of unique insertions found in a specific demosponge clade, which would usually be disregarded as'unalignable sites' and thus excluded from standard phylogenetic analyses. methods sequence acquisition, analyses and inference of secondary structures we analyzed all published full or nearly full-length ssu rrna sequences of porifera. for taxonomy of the taxa included in our study we followed systema porifera and the world porifera database, where also the species authorities are available. the ssu rrna sequence of amphimedon queenslandica was reconstructed by performing a local blast search against data from genbank's trace archive. traces from significant hits were downloaded and assembled in codoncode aligner.. this resultant sequence can be downloaded from our database of ssu rrna secondary structures of porifera. for class hexactinellida, only limited data was available in genbank: all three full-length ssu rrna sequences belong to subclass hexasterophora. two additional hexactinellid sequences were provided by martin dohrmann ahead of their publication in a comprehensive phylogenetic study of hexactinellida: semperella schulzei and aphrocallistes vastus. all sequences were initially aligned with clustal w and the preliminary alignments were manually improved in seaview. gblocks b was used to identify and isolate the conserved sites of the alignment before clustering similar sequences using the neighbor joining algorithm in paup* b. secondary structures for resulting clades were established for certain representatives of the clade by aligning to known structures from the european rna database in separate alignments for each clade and considering compensatory base changes. ssu rrna clade-alignments were then further refined according to secondary structure information. the unusual structures of marine haplosclerida and hexactinellida were initially examined under minimum free energy predictions from the mfold-server. in most cases, only one structure was predicted by the algorithm. if multiple structures were predicted, we chose the structure with either the minimal free energy or with the best compatibility to similar sequences. a comparative approach was chosen if permitted by an appropriate level of sequence divergence. for this approach, we used the alifold server to infer secondary structures of the insertions. alifold infers secondary structures by considering both minimum free folding algorithms and compensatory base changes, and therefore includes additional information that provides hints for secondary structural motifs. since this method requires a correct alignment, it could only be used if sequences were not too divergent from each other, such as with a subset of marine haplosclerida and the insertions of hexasterophora. however, secondary structures inferred with both methods were identical, or only differed in a few positions. therefore, while the comparative method is preferred, we still found that minimum free energy based predictions performed adequately to be used in cases where unambiguous alignments or missing comparative data does not allow inference of secondary structures based on compensatory base exchanges. for taxa that were suitable for a comparative approach, compensatory base exchanges are presented together with the corresponding alignments in additional file we visualized selected structures by converting the sequence and structure information to a ct-format with a perl-script. this format can be displayed in rnaviz. helix names correspond to wuyts et al., with the exception of helices e23_ and e23_ which together are referred to as e23_ insertions are designated by the name of the conserved helix in which they occur, and a period plus the number of the additional helix is added: parts of conserved helices separated by insertions are named after the original helix followed by a letter. base compositions and the lengths of the secondary structure features were calculated with a custom-made perl script. to avoid biases introduced by missing data from the published sequences, we used a fragment spanning from helix until bp before helix, and only considered the sequences without data missing within this region. representative poriferan secondary structures are available as *.fasta-format and in *.ct-format from our database for ssu rrna secondary structures of porifera. furthermore, several perl scripts for format conversion are provided. phylogenetic analyses the secondary structure information from the previous step was used to generate a new alignment in seaview. we generated a taxon-set comprising of taxa and focused on relationships of haplosclerid demosponges, in a similar way to redmond et al.. the ssu rrnas from this diverse taxon have been found to possess numerous insertions and extensions and our aim was to unravel their evolution. sites with uncertain homology even after considering secondary structure were excluded from the phylogenetic analyses. this was achieved by assigning sites to two groups and discarding those sites that were regarded as ambiguously aligned by the following criteria: unpaired sites: with length polymorphism and sequence divergence too high to identify homologous positions for all sequences. paired sites: with length polymorphisms in helices and/or structural homologies that could not be unambiguously assigned. furthermore, taxon-specific insertions within helices, as well as nucleotide insertions found only in single sequences were excluded. doublet positions were only regarded as pairings in the consensus secondary structure if the two involved nucleotides formed a watson-crick or g-u wobble pairing in at least five sequences within the alignment. corresponding sites falling below this five-sequence threshold were treated as unpaired. for phylogenetic reconstructions, sites were allocated to one of the following two partitions: partition'stem' or partition'loop'. we used mrbayes. and phase for the phylogenetic analyses, as both programs allow the simultaneous analysis of a partitioned dataset with both rrna models for paired sites and standard models for unpaired sites. mrbayes only allows the usage of a doublet model corresponding to the sh model. this is a state-rna model, which considers all possible doublets as characters and assumes that compensatory base exchanges result from at least two substitution events. a gtr + g + i model was assigned for the loop partition. the markov chain monte carlo analysis comprised two runs for million generations, with the sample frequency set to and the temperature for the heated chains set to. sampled trees were summarized using the sumt command in mrbayes with a burn-in set to the first million generations. sufficient convergence of chains for the mr bayes runs was monitored by observing log-likelihood values, the standard derivation of split frequencies, and diagnostics provided by awty. in phase, we applied the rna7a model and rna7d model for stem regions in independent runs. rna7a is the most general 7-state rna model. rna7d is a simplification of rna7a. the 7-state rna models treat all mismatches as one single state. this simplification increases the risk of loss of phylogenetic information, but the occurrence of mismatch-pairs in rrna data is small, therefore, an estimation of mismatch substitution parameters from the data is probably not accurate. furthermore, by pooling mismatches into a single character, the number of parameters to be estimated in a phylogenetic analysis, and consequently the computational demands are significantly decreased. for loop regions, the rev model was chosen. in addition, a gamma distribution accounting for rate heterogeneity among sites and a proportion of invariant sites were assigned to each model for both partitions. independent runs were performed in phase 2: two runs with the rna7a model and one run with the rna7d model for stem positions. every 100th generation a sample was taken from the mcmc chains. tracer v was used to monitor sufficient parameter stabilization. to create readable input files for tracer from the phase runs, we used a slightly modified version of the perl script'phase2tracer.pl', which is available upon request. the presented tree topology is based upon one of the million phase runs with the rna7a model for stem partition. to obtain branch-lengths for the tree, we conducted an additional analysis under the same models, and tree topology was fixed to the consensus tree from the original million generation analysis as suggested in the phase manual.  ssu rrna length differences and base composition to avoid biases due to missing data, we analyzed base composition and sequence length for a fragment of ssu rrna that covers about % of the gene. base composition and fragment length differed considerably among the poriferan sequences. the gc content varied between and %. calcarea posses the lowest gc contents with a modest variation from to %. in this aspect they are clearly separated from demosponges, which display significantly higher gc contents, since the lowest demosponge value still exceeds the highest gc content of calcarea. most demosponge ssu rrnas show modest length variations in a range comparable to those of calcarea. notable exceptions are the extraordinary large rrna molecules found in several marine haplosclerids. the highest gc contents of porifera are also found within this group. the high gc pattern is independent of the presence of insertions in these large molecules, since members of marine haplosclerida with smaller rrna molecules also possess similar gc contents. ssu rrnas of the few available hexactinellid sponges are approximately equal in length to large molecules of several haplosclerids. in contrast to haplosclerids, hexactinellid sponges have lower gc contents, with base compositions in the range of those of demospongiae and calcarea. secondary structure porifera have the typical eukaryotic core ssu rrna structure. the moderate length variation between calcarea and most demosponges is primarily caused by insertions in unpaired regions or by elongation of helices e10_ and. in hexactinellida, on average, these three helices are largely elongated compared to calcarea and demospongiae, but the lengths of the e10_ helices of some demosponge sequences fall into the same range. *marine haplosclerids not included in helix 43; **farrea occa not included for helix in addition, we observed extra-helical insertions in hexactinellida and in several marine haplosclerid demosponges that are not part of the eukaryote core structure. in marine haplosclerids, these extra sequences were inserted within helices e23_ e23_ and and in hexactinellida, the insertions only occurred within helix e23_ at a different position than in marine haplosclerids. all of the helices where sequence elongations and/or insertions occur belong to regions that are known to be highly variable within eukaryotes. calcarea the ssu rrna of this sponge class comprises all of the typical eukaryote helices and lacks unusual structural features. a calcarean ssu rrna consensus sequence and structure is shown in fig. several synapomorphies for the two calcinea and calcaronea subclasses were detected in the secondary structure. in calcinea, helices and e23_ are shorter by at least one base pair when compared to calcaronea. in helix e10_ calcaronea typically have three pairs at the helix end, whereas calcaronea dominantly possess four pairs. however, independent elongations of this helix can be found in both subclasses. these elongations are homoplasies as is evident when considering the subclass-specific compensatory base change at the beginning of inset ii: the a-u pair in the corresponding structure of the calcaronean leucascandra caveolata supports a secondary loss of a pair compared to other calcaronea. differences in helix nucleotides between both subclasses occur in helices e23_ e23_ and. most of these changes maintain the helix-relevant pairings, but a few cause mismatches in at least some sequences. base changes and insertions in unpaired regions are also specific for the calcinea-calcaronea split. this is indicated in fig. for three bases in the bulge between helices and one base within the loop of e23_ and a calcaronean-specific insertion of one adenosine between helix and hexactinellida the ssu secondary structure of acanthascus dawsoni is presented in fig. representatively for hexactinellida. in all hexactinellid sequences, specific insertions were observed. as mentioned previously, hexactinellid ssu rrna sequences are considerably longer than in other poriferans, except in some marine haplosclerid demosponges. the additional nucleotides occur in extensive elongations of common helices, and a helical insertion in helix e23_ the insertions in helix e23_ occur at a unique position among sponges and may form two helices in all the studied ssu rrna molecules of subclass hexasterophora. helix e23_1b. contains doublets and is much more conserved within hexasterophora than helix e23_1b. which varies in length from to bp. in contrast to hexasterophora, semperella schulzei has a helical insertion of bp within e23_ which is predicted to form a single helix e23_1b.. within the sequence of farrea occa, we found deletions in conserved regions. helices and are missing completely, as are the 3' strand of helix parts of helix and the 5' strand of helix. such complete or partial deletion of conserved helices has been shown to be typical for non-functional rrna pseudogenes. potential paralogs like this one are not necessarily subject to concerted evolution, and are therefore not suitable for phylogenetic inference. in this context, the consideration of secondary structure is crucial for identification of such non-functional sequences, and prevents biases in phylogenetic reconstruction due to potentially misleading data. nonetheless, predictions of insertions for this sequence are displayed in fig., since no suspicious modifications were found within this part of the molecule and no other sequence of farreidae was available. however, the results for this species should be treated with caution. demospongiae most demosponges possess a ssu rrna molecule with the common metazoan secondary structure. remarkable exceptions are only found within the marine haplosclerida, which possess insertions that are long enough to be predicted to form additional helices. those helices are found within known variable regions for eukaryotes and appear in the 5' strand of helix e23_1/ the 5'strand within helix e23_ and the 3' strand of helix phylogenetic analyses we inferred the phylogeny of marine haplosclerids to compare the evolutionary history of helical insertions found in this group of demospongiae. results from the phase- and mrbayes analyses of taxa are shown in fig. although more general 7-state models have been shown to result in higher likelihood values for phylogenies than less parameter-rich models for real rrna data, our analyses with phase with the rna7a model and the less complex rna7d model yielded identical tree topologies. independent runs in phase and mrbayes resulted in similar, almost identical topologies, and differences in demosponge relationships were only observed in the positions of clades with weak support values. namely these are the relationship of dictyoceratida to the myxospongiae, the position of scopalina ruetzleri and relationships within freshwater sponges. additionally, differences were observed in calcarea and cnidaria. the order haplosclerida was not resolved as monophyletic, since suborder spongillina fell into other distantly related demosponge clades, rather than into marine haplosclerida. the two suborders, haplosclerina and petrosina were not supported as monophyla. these results are congruent with results from former analyses of ssu rrna, 28s rrna and cytochrome oxidase subunit i. according to our analysis, amphimedon queenslandica is most closely related to oceanapia sp., with xestospongia muta as sister taxon, and both nodes in the tree are very highly supported by posterior probability values in both bayesian analyses. other species of the family niphatidae are not closely related to each other or to amphimedon queenslandica. in addition, other members of families petrosiidae and phloeodictyidae are not found in a closer relationship to the three species clade. our results were mostly concordant with redmond et al., but with higher support values in several clades. we did not find any monophyletic haplosclerid families or genera in our taxon set. differences between our results and the previous study are highlighted at the nodes in fig. we could not recover monophyletic petrosiidae in clade i, and relationships of several clade iii taxa differed. furthermore, xestospongia muta and oceanapia sp. cluster in one clade. successive evolution of additional helices in marine haplosclerids within haplosclerids, the evolution of additional helices can be reconstructed by plotting structures to the well-supported phylogenetic backbone. primary sequences of these motifs were not included in the tree construction due to ambiguous alignment, but can be regarded as additional phylogenetic characters. the helical insertions apparently evolved in at least two steps, which fits the findings of the ssu rrna gene tree strikingly well. the relationships within marine haplosclerids can be described as four well-supported nested clades i-iv that display different stages of secondary structure evolution. clade i contains all marine haplosclerida. the basal diverging taxa lack any large insertions that are typical for other marine haplosclerida. however, the predicted structure within helix differs from the standard structure in this region found in other porifera and displays a larger bulge of unpaired bases at the insertion point of the larger helical structures found within all taxa in clade ii. this bulge may be the precursor for the extensions at this position observed in clade ii. within basal diverging taxa of clade ii, a similar bulge is found for xestospongia muta in helix e23_ at the insertion-site of subsequent extensions in clade iii, but not the other sequences lacking e23_. larger insertions appeared in helices e23_ and'simultaneously' in the common ancestor of clade ii taxa. the three taxa of clade iv according to our minimum free energy calculations share an additional helix 43b. as synapomorphy. after the introduction of helical insertions in helices e23_ and a long extension evolved within helix e23_ as a synapomorphy in clade iii. an autapomorphy for dasychalina fragilis is an additional helix formed b insertions within e23_ within the complete taxon of marine haplosclerids, no loss of formerly gained additional helical insertions has been documented, therefore, no ssu rrna molecule from a descendent of a taxon with extraordinary features has returned to the ancestral basic metazoan core structure. discussion unusual patterns within poriferan ssu rrna secondary structure we reported the secondary structures of a variety of poriferan ssu rrna sequences, and suggest structure predictions for secondary structure motifs that are specific for some lineages, i.e. marine haplosclerida and hexactinellida. such additional helical insertions occur in a variety of eukaryotes and are known to be homoplasies, because they occur in several, not closely related taxa. our data shows that such structures are also present in early diverging metazoa. insertions in helix e23_ evolved independently in hexactinellida and the marine haplosclerida, which is evident from our phylogenetic analyses that captured'snapshots' of the evolution of helices within the marine haplosclerida and from the observations that insertions appear at different positions within helix e23_. although additional helical insertions are present within the e23-extension fragment in various eukaryotic taxa, to our knowledge, none have been reported within helix e23_ which is therefore synapomorphic for clade iii haplosclerids. interestingly, helical insertions within haplosclerids first appeared in the typical regions for such insertions, namely within helices e23_ and before they evolved within e23_. therefore, the evolution of extensions at more common insertion sites might be a prerequisite for the evolution of additional helical structures within e23_ higher metazoa with unusual ssu rrna structures also contain unusual motifs in their large ribosomal subunit, e.g., in branchiopod crustacea. in sponges, additional motifs in a fragment of the lsu have previously been reported for hexactinellida and marine haplosclerid demosponges, but not for non-haplosclerid demosponges or calcarea. this is in striking accordance with our ssu rrna findings, and encourages further studies of the complete lsu secondary structure of these taxa. since both rrna units are encoded in one translational unit, the same mechanisms may be responsible for the formation of extra helical features in both rrna molecules. remarkably, not only the nuclear rrnas display unusual secondary structure motifs in marine haplosclerida. in the recently published mitochondrial genome of the haplosclerid amphimedon queenslandica, both of the mitochondrial rrna genes also contain additional helices that are not found in other demosponges. although this may be a coincidental observation and needs to be verified by data from additional haplosclerid mt rrna sequences, it is possible that the same selection mechanisms act on the nuclear and mt rrna in this taxon. however, such correlations do not exist in all taxa, since the recently sequenced mitochondrial genomes of hexactinellida contain extremely short rrnas, in contrast to the large insertions in the hexactinellid nuclear rrnas. for the nuclear ssu rrna, the fact that extra-helical structures are found in the e23-extension region and helix in various taxa indicates that these regions are under less functional constraints than are the core regions of ssu rrna; wuyts et al. showed by considering the tertiary structure of rrna that nucleotide variability increases with distance from the ribosome centre. eukaryotic insertion sites for additional helices are therefore located in the same, or similar regions at the periphery of rrna molecules. the authors concluded that these insertions do not interfere with the ribosomal function of the ribosome and can therefore arise independently within different lineages, similar to our observations in hexactinellida and marine haplosclerids. phylogenetic value of rrna features we demonstrated different applications of ssu rrna features for phylogenetic analyses: base composition and synapomorphic base exchanges base compositions of ssu rrna differ strikingly between demospongiae and calcarea. the gc contents of the demospongiae are always higher and show a wider range of variability than the ones of calcarea. for hexactinellida, only five sequences were available, therefore general conclusions regarding their gc contents should be interpreted with care. however, for the few sequences available the gc contents fell into the ranges observed in calcarea and demospongiae. several apomorphic positions identified in calcarean ssu rrna allow to unambiguously distinguish between the two subclasses calcinea and calcaronea, thus supporting other morphological and molecular data. rna models for phylogeny estimation and evolution of additional helical structures as evolutionary markers the bayesian phylogenetic reconstructions using structure-defined partitions with different rrna models for doublets in mrbayes. and phase yielded very similar tree topologies with increased support for several nodes compared to the maximum parsimony and maximum likelihood analyses presented by redmond et al.. partitioned analyses using rrna models as applied in our analysis, have been reported to result in better-supported topologies. however, other factors may have contributed to our findings, e.g., it is known that bayesian posterior probability values are often higher than corresponding nonparametric bootstrap values and may even provide support for the'wrong' clades in studies with simulated data. the relevant important haplosclerid clades were supported with very high pp values. these high values should overcome eventual problems of support overestimation. also, support for'wrong' clades is unlikely to be a problem in our results, since the topologies from redmond et al. are mostly concordant with ours. regarding the general differences in bootstrap and pp values, the different software packages used and the difference in the data set may have contributed to the higher support found for several clades. although standard models of nucleotide evolution violate the assumption of independent evolution of all sites when also applied to paired sites as done by redmond et al., this seems to have little impact on nodes with high support values in the case of the demosponge dataset studied. this suggests that the biases introduced by the use of less well fitting'standard' rrna models may have a higher impact on clades that are difficult to resolve, whereas a strong phylogenetic signal will be recovered even if a sub-optimal evolutionary model is used for analyses. amphimedon queenslandica, the target of the sponge genome sequencing project, did not cluster with any other representative of its family niphatidae. likewise, neither the other haplosclerid families, nor the genera could be recovered as monophyletic, in accordance with the ssu-rrna based findings of redmond et al.. strikingly, these inferred relationships are supported by the presence or absence of secondary structure motifs within haplosclerida: different members of the families niphatidae, petrosiidae and phloeodictyidae show a different number of specific insertions that are congruent with the phylogenetic relationships that we previously inferred without the inclusion of these extended regions. the presence and absence of such helices are therefore good phylogenetic indicators for these relatively closely related taxa, even though alignment of the primary sequence of these helices between all taxa is difficult due to high evolutionary rates. homology inference of sites in hypervariable regions according to their secondary structure is known to be problematic. functional constraints are probably more relaxed in these regions, and the observed evolution of insertions is driven by unknown mutational mechanisms, which might tend to produce similar motifs by homoplasy. in contrast, within marine haplosclerids, no loss of helical insertions that arose at some earlier point of their evolutionary history occurred. furthermore, no independent homoplasic helical insertions ever appeared in the same positions within ssu rrna. considering our findings, the presence and absence of large helical insertions appears to provide strong phylogenetic information at selected taxonomical levels. a similar phylogenetic information value of additional helical structures may be present for insertions in helix e23_ of hexactinellida, although generalized conclusions are limited by the small sample size. nonetheless, the insertions in hexasterophora are predicted to possess two additional helices, while the insertion found in the only considered amphidiscophoran, semperella schulzei, forms only one helix. it is evident that integration of secondary structure information in sequence alignment and analyses will optimize rrna phylogenies considerably.  the ssu rrna provides far more valuable phylogenetic information than just its primary sequence. even simple features like base composition already bear enough information to distinguish between the two higher sponge taxa calcarea and demospongiae. unusual secondary structures can lend further support to results from independent phylogenetic inferences, as we showed for helical insertions in marine haplosclerid demosponges and for a small number of hexactinellid sponges. in this way, otherwise neglected hypervariable insertions can yield further support to a given topology. although we only explored additional structures of ssu rrna in sponges, our results should encourage further studies. especially the study of lsu rrna structure seems promising in this regard, since this gene is more variable than ssu rrna and contains large extension regions that strongly differ among higher taxa. on the intraspecific level, the even more variable internal transcribed spacer regions can provide secondary structure features of phylogenetic value. relating secondary structure to sequence information will allow the phylogenetic signal of the huge numbers of rrna sequences currently available in genbank to be considerably increased. our newly generated database of porifera ssu rrna secondary structures will facilitate the inclusion of secondary structure information in phylogenetic analyses. authors' contributions ov contributed to the conception and design of the study, acquired, analyzed and interpreted the data, and wrote the manuscript. de and gw contributed to the conception and design of the study, and critical revision of the manuscript. all authors read and approved the final version of the manuscript. supplementary material additional file 1 taxonomy and genbank accession numbers of downloaded poriferan ssu rrna sequences. click here for file additional file 2 trace ids from genbank trace archive used to assemble the amphimedon queenslandica ssu rdna sequence. click here for file additional file 3 compensatory base changes and alignments of predicted secondary structures of clade iii marine haplosclerida and hexasterophora. click here for file additional file 4 base composition and fragment lengths of examined poriferan ssu rrna fragment. click here for file additional file 5 alignment with secondary structure information and character set. secondary structure is provided for regions that are included in the phylogenetic analyses only. additional secondary structure information is available from our poriferan ssu rrna database. click here for file acknowledgements this work was financially supported by the german research foundation and is also a contribution from the dfg priority program spp'deep metazoan phylogeny'. we thank martin dohrmann for providing hexactinellid sequences ahead of publication and for useful comments on the manuscript and catherine vogler for commenting on the manuscript and for very productive discussions. ov would like to thank and the organizers and participants of the workshop on molecular evolution in woods hole for inspiring discussions and the support of the boehringer ingelheim fonds to cover travel expenses and tuition fees. d.e. acknowledges financial support from the european union under a marie-curie outgoing fellowship, gw acknowledges financial support from the marie-curie est hotspots. the gesellschaft für wissenschaftliche datenverarbeitung göttingen is acknowledged for access to their computer facilities. we thank three anonymous reviewers for their constructive comments, especially reviewer 
of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. we showed that analyses of primary rrna sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. however, information regarding its secondary structure is neglected in most phylogenetic analyses. information about secondary structure also supports the process of aligning rrna sequences across taxa. here, we explore ssu rrna secondary structures from the three extant classes of phylum porifera, a pivotal, but largely unresolved taxon of early branching metazoa. this is the first phylogenetic study of poriferan ssu rrna data to date that includes detailed comparative secondary structure information for all three sponge classes. to encourage and facilitate the application of rrna models in phylogenetics of early metazoans, we present ssu rrna secondary structures over the taxonomic range of porifera in a database, along with some basic tools for relevant format-conversion. we demonstrated that sophisticated secondary structure analyses can increase the potential phylogenetic information of already available rdna sequences currently accessible in databases and conclude that the importance of ssu rrna secondary structure information for phylogenetic reconstruction is still generally underestimated, at least among certain early branching metazoans. incorporation of this information is essential in order to apply specific rrna evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa. we found base compositional and structural differences in ssu rrna among demospongiae, hexactinellida and calcarea.
 the cytoplasmic ribosomal small subunit ribosomal rna is the most frequently-used gene for molecular phylogenetic studies. however, information regarding its secondary structure is neglected in most phylogenetic analyses. incorporation of this information is essential in order to apply specific rrna evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. information about secondary structure also supports the process of aligning rrna sequences across taxa. both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa. here, we explore ssu rrna secondary structures from the three extant classes of phylum porifera, a pivotal, but largely unresolved taxon of early branching metazoa. this is the first phylogenetic study of poriferan ssu rrna data to date that includes detailed comparative secondary structure information for all three sponge classes.  we found base compositional and structural differences in ssu rrna among demospongiae, hexactinellida and calcarea. we showed that analyses of primary rrna sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. therefore, these features can provide alternative support for sequence-based topologies and give insights into the evolution of the molecule itself. to encourage and facilitate the application of rrna models in phylogenetics of early metazoans, we present ssu rrna secondary structures over the taxonomic range of porifera in a database, along with some basic tools for relevant format-conversion.  we demonstrated that sophisticated secondary structure analyses can increase the potential phylogenetic information of already available rdna sequences currently accessible in databases and conclude that the importance of ssu rrna secondary structure information for phylogenetic reconstruction is still generally underestimated, at least among certain early branching metazoans. 