BACKGROUND
the small subunit  18s rrna gene is one of the most frequently used genes in phylogenetic studies  and an important marker for random target pcr in environmental biodiversity screening  <cit> .
in general, rrna gene sequences are easy to access due to highly conserved flanking regions allowing for the use of universal primers  <cit> .
their repetitive arrangement within the genome provides excessive amounts of template dna for pcr, even in smallest organisms.
the 18s gene is part of the ribosomal functional core and is exposed to similar selective forces in all living beings  <cit> .
thus, when the first large-scale phylogenetic studies based on 18s sequences were published - first and foremost field et al.
's  <cit>  phylogeny of the animal kingdom - the gene was celebrated as the prime candidate for reconstructing the metazoan tree of life.
and in fact, 18s sequences later provided evidence for the splitting of ecdysozoa and lophotrochozoa , thus contributing to the most recent revolutionary change in our understanding of metazoan relationships.
methodological innovation within the last years came from the incorporation of secondary structure into phylogenetic analyses.
in particular rna specific substitution models considering paired sites in rrna genes have been shown to outperform standard dna models  <cit> .
during recent years and with increased numbers of taxa included into molecular phylogenies, however, two problems became apparent.
first, there are prevailing sequencing impediments in representatives of certain taxa, such as the mollusk classes solenogastres and tryblidia  <cit> , selected bivalve taxa , and the enigmatic crustacean class remipedia .
failure to obtain 18s sequences of single taxa is considered a common phenomenon but is rarely ever reported.
secondly, in contrast to initially high hopes, 18s cannot resolve nodes at all taxonomic levels and its efficacy varies considerably among clades.
this has been discussed as an effect of rapid ancient radiation within short periods  <cit> .
multigene analyses are currently thought to give more reliable results for tracing deep branching events in metazoa but 18s still is extensively used in phylogenetic analyses.
considering the wide range of studies based on the 18s gene as a molecular marker, both sequencing problems and the applicability of 18s for phylogenetic inferences need to be scrutinized.
to address these questions, we focus on the mollusca, the second largest animal phylum.
there are eight higher taxa  defined within mollusca: the aplacophoran solenogastres  and caudofoveata , two small clades with about  <dig> and  <dig> currently described species; polyplacophora ; and the conchiferan clades tryblidia , scaphopoda , cephalopoda , bivalvia , and gastropoda .
the monophyly of the phylum is well established based on morphological characters, but 18s phylogenies often show the mollusca as polyphyletic or paraphyletic with low resolution of the deeper nodes .
one of the main deficiencies of all published studies on mollusk phylogeny is the underrepresentation of the minor taxa solenogastres, caudofoveata, and monoplacophora.
the aplacophoran solenogastres and caudofoveata together with polyplacophora  are traditionally thought to represent early branching taxa within mollusca, but their relative position is still controversially discussed .
despite of their key position in mollusk phylogeny and evolution, the number of molecular phylogenetic studies including any aplacophoran mollusk species is low: winnipeninckx et al.
<cit> ; okusu et al.
<cit> ; lindgren et al.
<cit> ; passamaneck et al  <cit> ; giribet et al.
<cit> ; dunn et al.
<cit> ; wägele et al.
<cit> ; wilson et al.
<cit> .
to date there are no more than four caudofoveate and five solenogaster  18s sequences published in genbank, the taxon sampling spanning no more than two genera of caudofoveata and solenogastres, respectively.
in solenogasters, okusu and giribet  <cit>  described severe contamination issues caused by cnidarian prey.
here, we specifically address contamination issues and point out technical problems hampering amplification during pcr.
this is important not only for prompting representative taxonomic sampling for phylogenetic analyses, but also for avoiding under- or overestimation of biodiversity in environmental screening programs.
moreover, we evaluate the usefulness of the 18s gene for phylogenetic inferences by combining our new authentic solenogaster 18s data with published molluscan sequences and - based on an extended taxon sampling - analyze sequence divergence and compositional heterogeneity within the phylum.
RESULTS
solenogastres sequences
initial experiments using standard pcr protocols and genomic dna from starved specimens resulted in sequences from prey organisms and epibionts, or in chimerical pcr products.
at best, cdna templates led to shortened 18s sequences.
finally, authentic solenogaster 18s sequences for three species were obtained via isolation of total rna from starved specimens, followed by reverse transcription and utilizing additives for gc-rich templates.
10% dmso was applied in sequencing reactions.
strong secondary structures and gc clamps were observed analyzing the wirenia argentea , simrothiella margaritacea , and micromenia fodiens  18s sequences.
stitch profiles  <cit>  computes the temperature-dependent melting probability and location of dsdna  and ssdna regions and thus can be used to locate the formation of dna bubbles at a predefined temperature.
we determined three to four helical sections in solenogaster dsdna .
these regions additionally bear prominent stems in ssdna, as inferred with the secondary structure probability plot in mfold at 72°c .
elevated gc contents above 60% were observed in the helical regions, with a maximum of 82% in the second helix of s. margaritacea.
a previously published complete caudofoveate  18s sequence showed only one short helical region , which could be aligned to the second helix of our solenogaster sequences .
all previously published solenogaster 18s sequences lack exceptional secondary structures and the gc contents are below 60%.
the epimenia sp.
sequence includes a  <dig> bp fragment with 100% cnidarian sequence identity .
the two nearly complete helicoradomenia sp.
sequences show  <dig> % identity to each other.
analyses of selected sections from these sequences using blastn searches showed significant identities of >95% with the polychaete amphisamytha galapagensis and other polychaetes .
sections with missing data or exogenous dna in previously published sequences match the regions of strong secondary structures determined within the sequences of w. argentea, s. margaritacea and m. fodiens .
sequence composition and alignment
among the  <dig> mollusk taxa sampled for 18s, the cephalopoda, solenogastres and patellogastropoda  show a combination of high gc content  and elongated gene length .
the variability within the selected taxa is measured with the disparity index , which has been shown to be more powerful than the commonly used χ <dig> test to assess substitution patterns  <cit> .
the compositional heterogeneity varies within the selected mollusk taxa and is not necessarily correlated with the sample size.
for example, the disparity index within the  <dig> caenogastropoda  is remarkably low whereas heterobranchia is highly variable .
the estimation of average maximum likelihood  branch lengths required the recoding of  <dig>  ambiguously aligned and highly variable positions as binary presence/absence data from  <dig>  overall positions.
the highest-ranking ml branch length values are found for cephalopoda, solenogastres and patellogastropoda .
* brls and σ values were multiplied with  <dig> for better readability.
we used all sequences available for the mollusca, sipuncula and kamptozoa with a length over  <dig>  bp, supplemented by some other lophotrochozoa and priapulus caudatus.
sequence composition of mollusca within selected taxa measured using the disparity index , compositional distance , gc content , gene length in base pairs  and average branch lengths  and their standard deviation .
brls and σ values were multiplied with  <dig> for better readability.
we used all sequences available for the mollusca, sipuncula and kamptozoa above  <dig>  bp supplemented by several other lophotrochozoa and priapulus caudatus.
this initially analyzed large dataset also guided the taxon selection for the phylogenetic analyses.
the resulting  <dig> taxa prank alignment comprises  <dig>  aligned positions and  <dig>  positions past trimming.
the proportion of gaps or completely undetermined characters  in the final alignment is  <dig> %.
the average disparity indices between the groups of sequences from this alignment show solenogastres distinct from all other mollusks except cephalopods  and are depicted in the lower triangle of table  <dig>  additionally, we tested the homogeneity of substitution patterns calculating all pair-wise sequence comparisons in mega  <dig>   <cit> .
significance with α≤ <dig>  was tested with  <dig>  monte carlo replicates using the pair-wise deletion option.
the substitution pattern of the three solenogastres sequences from this study are significantly different from all taxa except for cephalopoda, most patellogastropoda and some heterobranchs .
* we considered homogeneity as significantly rejected with p- values ≤  <dig> .
the pairwise deletion option was used to compare all possible species pairs .
anomalodesmata were treated separately and thus are excluded when showing results for the remaining heterodonta.
phylogenetic analyses
only the analysis using phase  <dig>   <cit>  with the time heterogeneous model found the mollusca monophyletic, albeit with low support .
we found full support for all class level taxa except bivalvia and confirmed all the well-established subgroups within the gastropoda, cephalopoda, and scaphopoda.
polyplacophora and the single monoplacophoran representative together form the first branch of the molluskan tree.
within bivalves, palaeoheterodonta and heterodonta form a robustly supported clade.
pteriomorpha and protobranchia are outside this clade.
protobranchia appears paraphyletic, with nuculoidea representing the sister group to pteriomorpha and with solemyoida included into a clade comprising scaphopoda, the aplacophoran clades, and cephalopoda.
both bayesian and ml analyses revealed an identical internal branching pattern of +caudofoveata)+scaphopoda).
the calculated ml bootstrap support values for this topology using different models and coding schemes are tabulated in figure  <dig>  all conducted analyses agree with large parts of the tree.
the ml topologies corresponding to the three best fitting rna doublet models offer only minor topological differences as judged by eye and using the weighted robinson foulds distance   <cit>  accounting for node support values .
rna doublet models of different complexity are not nested, thus we only determined the best fitting model within each class of substitution models using the akaike information criterion   <cit> .
the number of states describes the treatment of the possible ten non-watson-crick pairs, which account all together for 16% miss-matches in the  <dig> taxa alignment.
each model differs in the number of free parameters .
the favored six state model is s6a .
within the seven state models s7b  and within the  <dig> state models s <dig>  were selected.
we choose the s7b substitution model as a trade off between computational demand and complexity in the bayesian analyses.
the phase software is designed to infer phylogenies from rrna genes with paired sites and is able to model compositional heterogeneity  <cit> .
the likelihood traces and most of the model parameters entered the stationary phase between  <dig>  and  <dig> , <dig> generations .
all five chains using the time heterogeneous model resulted in the identical branching pattern of the respective consensus tree and only minor differences in branch lengths.
the consensus tree was calculated from a chain sampling  <dig> mio generations to maximize the effective sample size  for all estimated parameters.
from a total of  <dig> model parameters, six failed to extravagate the ess of  <dig>  and four parameters were only sparsely sampled .
to further investigate the possible influence of exceptional sequence composition of solenogastres  we modified the taxon sampling of gastropods in two analyses.
all patellogastropoda and heterobranchia in the alignment were excluded and replaced by three fast evolving aeolidina .
all gastropods were excluded but patellogastropoda.
both experiments resulted in a regrouping of the above mentioned +caudofoveata)+scaphopoda) clade placing patellogastropoda between scaphopoda and caudofoveata or proposing aeolidina as sistergroup to solenogastres and cephalopoda .
all trees and alignments are available from the authors.
discussion
gc rich 18s and the chimera problem
amplification problems are a frequent phenomenon, even if using standard markers such as the 18s rrna gene.
secondary structures and gc-rich sections as pronounced as for the solenogaster 18s rnas shown here, have not been reported earlier.
may gc-rich sequences cause hampered pcr reactions in other taxa, too?
based on our results, we assume this to be likely, but equally startling as the failure to obtain gene sequences is the danger to produce chimerical products.
gc-rich sequences demand higher melting temperatures to be converted into and kept as single stranded dna molecules.
under standard amplification conditions, sequence sections exclusively composed of gc residues will terminate elongation steps in pcr by forming highly stable stems or gc clamps causing amplification to be refractory.
incompletely extended primers can anneal to heterologous 18s sequences and, despite of some degree of nucleotide mismatching, they will often be completed in the subsequent polymerization step resulting in chimeras  <cit> .
alternatively, compatible priming sequences from genes with lower gc contents are favored and successfully amplified  <cit> .
single stranded sequences with pronounced gc-stem regions also may lead the taq polymerase to skip the 'locked' sections, which leads to shortened pcr products lacking the stem-loop regions .
this potentially explains the lack of data for the gc-rich regions in the previously published solenogaster sequences, in contrast to the wirenia argentea, simrothiella margaritacea, and micromenia fodiens sequences described herein.
this interpretation is corroborated by the severe amplification problems we experienced when using recombinant plasmids as templates under standard pcr conditions .
the cloned wirenia argentea 18s inserts could not be amplified using conventional pcr protocols but resulted in blank agarose gels , although the number of templates accessible from clone amplification usually exceeds the gene copy number of genomic dna extractions by far.
failure to amplify solenogaster 18s under such conditions demonstrates that clean starting material is just the first step.
in addition to ours, a number of protocols for difficult or gc rich templates are published  <cit> , supplemented by several commercial kits.
contamination
the solenogaster midgut is extremely voluminous and can hold undigested food that may provide considerable amounts of dna templates leading to non-target or chimerical amplification products .
to avoid contamination with prey organisms, okusu and giribet  <cit>  suggested the use of gonad tissue, larval material or of species that are not predators of metazoan organisms.
the first two options are feasible where material is available, but the third option can be misleading when the diet is unknown.
a prey-predator relationship between helicoradomenia and non-cnidarian metazoan organisms, probably polychaetes, has been proposed earlier based on transmission electron microscopy data  <cit> .
starving animals in the laboratory for several days can reduce the amount of contaminating prey tissue considerably  <cit> , but exogenous dna was amplified even after starvation times of up to three months when using standard pcr protocols.
this may be due to residues of prey cnidocysts held back in the midgut epithelial cells.
thus, we found isolation of total rna from starved specimens, followed by reverse transcription, to be most effective to avoid contamination.
isolation of total rna followed by dnase digestion makes up to 95% of rrna available for rt-pcr and destroys contaminating non-target dna templates.
due to the ubiquitous presence of rnases causing instability of rna outside of living cells, the presence of considerable amounts of exogenous rna within an isolate is highly unusual, but one always has to bear in mind the possibility of contamination with parasites, epibionts, and undigested prey tissue.
phylogenetic inferences
considering the pitfalls of phylogenetic analyses based on a single gene and taking into account well-established knowledge on mollusk relationships, a number of groupings in our tree are ambiguous.
this concerns for example the clade composed of cephalopoda and solenogastres, where molecular inferences may reflect a shared bias in base composition rather than a sistergroup relationship.
this assumption is corroborated by the two experiments testing the influence of taxon sampling by manipulating the gastropod dataset .
in patellogastropoda and heterobranchia there are species with substitution patterns similar to solenogastres .
for both taxa, the exclusion of species breaking up their long branches resulted in a placement next to species with a similar sequence composition.
the branching pattern including all major gastropod lineages is robust in all analyses both within the gastropoda and the clade +caudofoveata)+scaphopoda).
when we add the heterobranch taxa from the original analysis to the three extremely fast evolving aeolidina, the latter taxon is correctly placed inside the heterobranch radiation .
within solenogastres our species selection aimed to reflect the taxon diversity by selecting representatives from two different orders , but the trimming of the alignment adapted to the broad taxon sampling diminished the phylogenetic signal at the species level.
our new and more representative dataset of  <dig> taxa covered for the first time all mollusk classes, but our analyses across the mollusca resulted in short internal branches combined with single long branching clades.
this scenario is known to be critical in phylogenetic tree reconstructions  <cit> .
the structure aware and non-stationary models greatly improved the results by detecting "problematic" taxa and by resulting in increased node support for well established subgroups.
thus we were able to recover nearly all mollusk classes, even though problematic fast evolving taxa, such as patellogastropoda, were included.
in rejecting the major mollusk clade conchifera , however, our phylogenetic tree does not reflect traditional views.
we detected a sister group of polyplacophora and monoplacophora as proposed by giribet et al.
<cit>  and wilson et al.
<cit> , but not with full support.
both taxa show only minor differences in sequence composition , which might mislead analyses of class level relationships.
the failure to detect monophyletic bivalvia probably best demonstrates the limits of 18s analyses in mollusca using contemporary methods .
bivalvia are known to show a number of exceptional morphological characters as well as several unusual structural features in the mitochondrial genome  <cit> .
thus, despite not obviously fast evolving, here the alignment methods may have failed to identify homologous positions correctly.
alternatively, the phylogenetic signal in bivalves may be largely eroded.
the close relationship between scaphopoda and protobranchia is worth mentioning because it  supports the so-called diasoma hypothesis, recently rejected by a number of molecular and morphological analyses, but here again proposing a sistergroup relationship between bivalvia and scaphopoda  <cit> ,
across all analyses we recovered a previously proposed clade composed of cycliophora and kamptozoa within the outgroup  <cit> .
sequence divergence in mollusca
elevated substitution rates are known to be gene specific but also characteristic for certain lineages across different genes  <cit> .
high substitution rates in the generally well-conserved 18s gene thus may point to fast evolving taxa.
to allow for general conclusions across the mollusca, we inferred substitution rates for all published molluscan 18s sequences longer than  <dig> bp.
the solenogaster 18s sequences obtained in this study are among the fastest evolving 18s sequences within the mollusca.
solenogastres bear a number of assumed ancestral mollusk features, but substitution rates in the 18s gene are nearly doubled in our selected species compared to other presumably "basal" mollusks, such as caudofoveata and polyplacophora.
if the assumed plesiomorphic morphological characters in solenogastres are in fact conserved ancestral features, then molecular and morphological rates of evolution are unlinked, at least for the 18s gene.
similar cases of possibly ancestral morphology combined with exceptionally high substitution rates as shown in table  <dig> are nautilus, the "living fossil" cephalopods, and patellogastropoda, the true limpets  <cit> .
a number of factors, for example functionality of proteins and rnas, generation time, metabolic rate, population size, and life histories, are thought to influence substitution rates  <cit> .
CONCLUSIONS
we show that the solenogaster 18s gene has an exceptional base composition, resulting in a number of technical difficulties.
amplification problems are a common phenomenon but can be overcome by combining known methods in a new framework and by employing alternative strategies.
we suggest to include assays with modified pcr methods and to creatively vary pcr conditions if amplification fails or if it leads to 'peculiar' results .
we show that the practical amplification issues of 18s are conquerable, whereas the future of mollusk class level phylogeny appears not to lie in this gene.
the incorporation of additional data, such as structural information to identify homologous positions between sequences, is a promising approach to improve alignments and phylogenetic analyses.
within the mollusca, where the sequence composition is highly variable between clades, both alignment methods and models of evolution used in phylogenetic analyses at date still remain the main bottlenecks in tracing deep phylogeny.
thus, multigene analyses are more effective to resolve such ancient splits, as recently demonstrated  <cit> .

 the small subunit 18s rrna gene is one of the most frequently used genes in phylogenetic studies and an important marker for random target pcr in environmental biodiversity screening. in general, rrna gene sequences are easy to access due to highly conserved flanking regions allowing for the use of universal primers. their repetitive arrangement within the genome provides excessive amounts of template dna for pcr, even in smallest organisms. the 18s gene is part of the ribosomal functional core and is exposed to similar selective forces in all living beings. thus, when the first large-scale phylogenetic studies based on 18s sequences were published - first and foremost field et al.'s phylogeny of the animal kingdom - the gene was celebrated as the prime candidate for reconstructing the metazoan tree of life. and in fact, 18s sequences later provided evidence for the splitting of ecdysozoa and lophotrochozoa, thus contributing to the most recent revolutionary change in our understanding of metazoan relationships. methodological innovation within the last years came from the incorporation of secondary structure into phylogenetic analyses. in particular rna specific substitution models considering paired sites in rrna genes have been shown to outperform standard dna models. during recent years and with increased numbers of taxa included into molecular phylogenies, however, two problems became apparent. first, there are prevailing sequencing impediments in representatives of certain taxa, such as the mollusk classes solenogastres and tryblidia, selected bivalve taxa, and the enigmatic crustacean class remipedia. failure to obtain 18s sequences of single taxa is considered a common phenomenon but is rarely ever reported. secondly, in contrast to initially high hopes, 18s cannot resolve nodes at all taxonomic levels and its efficacy varies considerably among clades. this has been discussed as an effect of rapid ancient radiation within short periods. multigene analyses are currently thought to give more reliable results for tracing deep branching events in metazoa but 18s still is extensively used in phylogenetic analyses. considering the wide range of studies based on the 18s gene as a molecular marker, both sequencing problems and the applicability of 18s for phylogenetic inferences need to be scrutinized. to address these questions, we focus on the mollusca, the second largest animal phylum. there are eight higher taxa defined within mollusca: the aplacophoran solenogastres and caudofoveata, two small clades with about and currently described species; polyplacophora; and the conchiferan clades tryblidia, scaphopoda, cephalopoda, bivalvia, and gastropoda. the monophyly of the phylum is well established based on morphological characters, but 18s phylogenies often show the mollusca as polyphyletic or paraphyletic with low resolution of the deeper nodes. one of the main deficiencies of all published studies on mollusk phylogeny is the underrepresentation of the minor taxa solenogastres, caudofoveata, and monoplacophora. the aplacophoran solenogastres and caudofoveata together with polyplacophora are traditionally thought to represent early branching taxa within mollusca, but their relative position is still controversially discussed. despite of their key position in mollusk phylogeny and evolution, the number of molecular phylogenetic studies including any aplacophoran mollusk species is low: winnipeninckx et al.; okusu et al.; lindgren et al.; passamaneck et al; giribet et al.; dunn et al.; wägele et al.; wilson et al.. to date there are no more than four caudofoveate and five solenogaster 18s sequences published in genbank, the taxon sampling spanning no more than two genera of caudofoveata and solenogastres, respectively. in solenogasters, okusu and giribet described severe contamination issues caused by cnidarian prey. here, we specifically address contamination issues and point out technical problems hampering amplification during pcr. this is important not only for prompting representative taxonomic sampling for phylogenetic analyses, but also for avoiding under- or overestimation of biodiversity in environmental screening programs. moreover, we evaluate the usefulness of the 18s gene for phylogenetic inferences by combining our new authentic solenogaster 18s data with published molluscan sequences and - based on an extended taxon sampling - analyze sequence divergence and compositional heterogeneity within the phylum.  solenogastres sequences initial experiments using standard pcr protocols and genomic dna from starved specimens resulted in sequences from prey organisms and epibionts, or in chimerical pcr products. at best, cdna templates led to shortened 18s sequences. finally, authentic solenogaster 18s sequences for three species were obtained via isolation of total rna from starved specimens, followed by reverse transcription and utilizing additives for gc-rich templates. 10% dmso was applied in sequencing reactions. strong secondary structures and gc clamps were observed analyzing the wirenia argentea, simrothiella margaritacea, and micromenia fodiens 18s sequences. stitch profiles computes the temperature-dependent melting probability and location of dsdna and ssdna regions and thus can be used to locate the formation of dna bubbles at a predefined temperature. we determined three to four helical sections in solenogaster dsdna. these regions additionally bear prominent stems in ssdna, as inferred with the secondary structure probability plot in mfold at 72°c. elevated gc contents above 60% were observed in the helical regions, with a maximum of 82% in the second helix of s. margaritacea. a previously published complete caudofoveate 18s sequence showed only one short helical region, which could be aligned to the second helix of our solenogaster sequences. all previously published solenogaster 18s sequences lack exceptional secondary structures and the gc contents are below 60%. the epimenia sp. sequence includes a bp fragment with 100% cnidarian sequence identity. the two nearly complete helicoradomenia sp. sequences show % identity to each other. analyses of selected sections from these sequences using blastn searches showed significant identities of >95% with the polychaete amphisamytha galapagensis and other polychaetes. sections with missing data or exogenous dna in previously published sequences match the regions of strong secondary structures determined within the sequences of w. argentea, s. margaritacea and m. fodiens. sequence composition and alignment among the mollusk taxa sampled for 18s, the cephalopoda, solenogastres and patellogastropoda show a combination of high gc content and elongated gene length. the variability within the selected taxa is measured with the disparity index, which has been shown to be more powerful than the commonly used χ test to assess substitution patterns. the compositional heterogeneity varies within the selected mollusk taxa and is not necessarily correlated with the sample size. for example, the disparity index within the caenogastropoda is remarkably low whereas heterobranchia is highly variable. the estimation of average maximum likelihood branch lengths required the recoding of ambiguously aligned and highly variable positions as binary presence/absence data from overall positions. the highest-ranking ml branch length values are found for cephalopoda, solenogastres and patellogastropoda. * brls and σ values were multiplied with for better readability. we used all sequences available for the mollusca, sipuncula and kamptozoa with a length over bp, supplemented by some other lophotrochozoa and priapulus caudatus. sequence composition of mollusca within selected taxa measured using the disparity index, compositional distance, gc content, gene length in base pairs and average branch lengths and their standard deviation. brls and σ values were multiplied with for better readability. we used all sequences available for the mollusca, sipuncula and kamptozoa above bp supplemented by several other lophotrochozoa and priapulus caudatus. this initially analyzed large dataset also guided the taxon selection for the phylogenetic analyses. the resulting taxa prank alignment comprises aligned positions and positions past trimming. the proportion of gaps or completely undetermined characters in the final alignment is %. the average disparity indices between the groups of sequences from this alignment show solenogastres distinct from all other mollusks except cephalopods and are depicted in the lower triangle of table additionally, we tested the homogeneity of substitution patterns calculating all pair-wise sequence comparisons in mega. significance with α≤ was tested with monte carlo replicates using the pair-wise deletion option. the substitution pattern of the three solenogastres sequences from this study are significantly different from all taxa except for cephalopoda, most patellogastropoda and some heterobranchs. * we considered homogeneity as significantly rejected with p- values ≤. the pairwise deletion option was used to compare all possible species pairs. anomalodesmata were treated separately and thus are excluded when showing results for the remaining heterodonta. phylogenetic analyses only the analysis using phase with the time heterogeneous model found the mollusca monophyletic, albeit with low support. we found full support for all class level taxa except bivalvia and confirmed all the well-established subgroups within the gastropoda, cephalopoda, and scaphopoda. polyplacophora and the single monoplacophoran representative together form the first branch of the molluskan tree. within bivalves, palaeoheterodonta and heterodonta form a robustly supported clade. pteriomorpha and protobranchia are outside this clade. protobranchia appears paraphyletic, with nuculoidea representing the sister group to pteriomorpha and with solemyoida included into a clade comprising scaphopoda, the aplacophoran clades, and cephalopoda. both bayesian and ml analyses revealed an identical internal branching pattern of +caudofoveata)+scaphopoda). the calculated ml bootstrap support values for this topology using different models and coding schemes are tabulated in figure all conducted analyses agree with large parts of the tree. the ml topologies corresponding to the three best fitting rna doublet models offer only minor topological differences as judged by eye and using the weighted robinson foulds distance accounting for node support values. rna doublet models of different complexity are not nested, thus we only determined the best fitting model within each class of substitution models using the akaike information criterion. the number of states describes the treatment of the possible ten non-watson-crick pairs, which account all together for 16% miss-matches in the taxa alignment. each model differs in the number of free parameters. the favored six state model is s6a. within the seven state models s7b and within the state models s were selected. we choose the s7b substitution model as a trade off between computational demand and complexity in the bayesian analyses. the phase software is designed to infer phylogenies from rrna genes with paired sites and is able to model compositional heterogeneity. the likelihood traces and most of the model parameters entered the stationary phase between and, generations. all five chains using the time heterogeneous model resulted in the identical branching pattern of the respective consensus tree and only minor differences in branch lengths. the consensus tree was calculated from a chain sampling mio generations to maximize the effective sample size for all estimated parameters. from a total of model parameters, six failed to extravagate the ess of and four parameters were only sparsely sampled. to further investigate the possible influence of exceptional sequence composition of solenogastres we modified the taxon sampling of gastropods in two analyses. all patellogastropoda and heterobranchia in the alignment were excluded and replaced by three fast evolving aeolidina. all gastropods were excluded but patellogastropoda. both experiments resulted in a regrouping of the above mentioned +caudofoveata)+scaphopoda) clade placing patellogastropoda between scaphopoda and caudofoveata or proposing aeolidina as sistergroup to solenogastres and cephalopoda. all trees and alignments are available from the authors. discussion gc rich 18s and the chimera problem amplification problems are a frequent phenomenon, even if using standard markers such as the 18s rrna gene. secondary structures and gc-rich sections as pronounced as for the solenogaster 18s rnas shown here, have not been reported earlier. may gc-rich sequences cause hampered pcr reactions in other taxa, too? based on our results, we assume this to be likely, but equally startling as the failure to obtain gene sequences is the danger to produce chimerical products. gc-rich sequences demand higher melting temperatures to be converted into and kept as single stranded dna molecules. under standard amplification conditions, sequence sections exclusively composed of gc residues will terminate elongation steps in pcr by forming highly stable stems or gc clamps causing amplification to be refractory. incompletely extended primers can anneal to heterologous 18s sequences and, despite of some degree of nucleotide mismatching, they will often be completed in the subsequent polymerization step resulting in chimeras. alternatively, compatible priming sequences from genes with lower gc contents are favored and successfully amplified. single stranded sequences with pronounced gc-stem regions also may lead the taq polymerase to skip the'locked' sections, which leads to shortened pcr products lacking the stem-loop regions. this potentially explains the lack of data for the gc-rich regions in the previously published solenogaster sequences, in contrast to the wirenia argentea, simrothiella margaritacea, and micromenia fodiens sequences described herein. this interpretation is corroborated by the severe amplification problems we experienced when using recombinant plasmids as templates under standard pcr conditions. the cloned wirenia argentea 18s inserts could not be amplified using conventional pcr protocols but resulted in blank agarose gels, although the number of templates accessible from clone amplification usually exceeds the gene copy number of genomic dna extractions by far. failure to amplify solenogaster 18s under such conditions demonstrates that clean starting material is just the first step. in addition to ours, a number of protocols for difficult or gc rich templates are published, supplemented by several commercial kits. contamination the solenogaster midgut is extremely voluminous and can hold undigested food that may provide considerable amounts of dna templates leading to non-target or chimerical amplification products. to avoid contamination with prey organisms, okusu and giribet suggested the use of gonad tissue, larval material or of species that are not predators of metazoan organisms. the first two options are feasible where material is available, but the third option can be misleading when the diet is unknown. a prey-predator relationship between helicoradomenia and non-cnidarian metazoan organisms, probably polychaetes, has been proposed earlier based on transmission electron microscopy data. starving animals in the laboratory for several days can reduce the amount of contaminating prey tissue considerably, but exogenous dna was amplified even after starvation times of up to three months when using standard pcr protocols. this may be due to residues of prey cnidocysts held back in the midgut epithelial cells. thus, we found isolation of total rna from starved specimens, followed by reverse transcription, to be most effective to avoid contamination. isolation of total rna followed by dnase digestion makes up to 95% of rrna available for rt-pcr and destroys contaminating non-target dna templates. due to the ubiquitous presence of rnases causing instability of rna outside of living cells, the presence of considerable amounts of exogenous rna within an isolate is highly unusual, but one always has to bear in mind the possibility of contamination with parasites, epibionts, and undigested prey tissue. phylogenetic inferences considering the pitfalls of phylogenetic analyses based on a single gene and taking into account well-established knowledge on mollusk relationships, a number of groupings in our tree are ambiguous. this concerns for example the clade composed of cephalopoda and solenogastres, where molecular inferences may reflect a shared bias in base composition rather than a sistergroup relationship. this assumption is corroborated by the two experiments testing the influence of taxon sampling by manipulating the gastropod dataset. in patellogastropoda and heterobranchia there are species with substitution patterns similar to solenogastres. for both taxa, the exclusion of species breaking up their long branches resulted in a placement next to species with a similar sequence composition. the branching pattern including all major gastropod lineages is robust in all analyses both within the gastropoda and the clade +caudofoveata)+scaphopoda). when we add the heterobranch taxa from the original analysis to the three extremely fast evolving aeolidina, the latter taxon is correctly placed inside the heterobranch radiation. within solenogastres our species selection aimed to reflect the taxon diversity by selecting representatives from two different orders, but the trimming of the alignment adapted to the broad taxon sampling diminished the phylogenetic signal at the species level. our new and more representative dataset of taxa covered for the first time all mollusk classes, but our analyses across the mollusca resulted in short internal branches combined with single long branching clades. this scenario is known to be critical in phylogenetic tree reconstructions. the structure aware and non-stationary models greatly improved the results by detecting "problematic" taxa and by resulting in increased node support for well established subgroups. thus we were able to recover nearly all mollusk classes, even though problematic fast evolving taxa, such as patellogastropoda, were included. in rejecting the major mollusk clade conchifera, however, our phylogenetic tree does not reflect traditional views. we detected a sister group of polyplacophora and monoplacophora as proposed by giribet et al. and wilson et al., but not with full support. both taxa show only minor differences in sequence composition, which might mislead analyses of class level relationships. the failure to detect monophyletic bivalvia probably best demonstrates the limits of 18s analyses in mollusca using contemporary methods. bivalvia are known to show a number of exceptional morphological characters as well as several unusual structural features in the mitochondrial genome. thus, despite not obviously fast evolving, here the alignment methods may have failed to identify homologous positions correctly. alternatively, the phylogenetic signal in bivalves may be largely eroded. the close relationship between scaphopoda and protobranchia is worth mentioning because it supports the so-called diasoma hypothesis, recently rejected by a number of molecular and morphological analyses, but here again proposing a sistergroup relationship between bivalvia and scaphopoda, across all analyses we recovered a previously proposed clade composed of cycliophora and kamptozoa within the outgroup. sequence divergence in mollusca elevated substitution rates are known to be gene specific but also characteristic for certain lineages across different genes. high substitution rates in the generally well-conserved 18s gene thus may point to fast evolving taxa. to allow for general conclusions across the mollusca, we inferred substitution rates for all published molluscan 18s sequences longer than bp. the solenogaster 18s sequences obtained in this study are among the fastest evolving 18s sequences within the mollusca. solenogastres bear a number of assumed ancestral mollusk features, but substitution rates in the 18s gene are nearly doubled in our selected species compared to other presumably "basal" mollusks, such as caudofoveata and polyplacophora. if the assumed plesiomorphic morphological characters in solenogastres are in fact conserved ancestral features, then molecular and morphological rates of evolution are unlinked, at least for the 18s gene. similar cases of possibly ancestral morphology combined with exceptionally high substitution rates as shown in table are nautilus, the "living fossil" cephalopods, and patellogastropoda, the true limpets. a number of factors, for example functionality of proteins and rnas, generation time, metabolic rate, population size, and life histories, are thought to influence substitution rates.  we show that the solenogaster 18s gene has an exceptional base composition, resulting in a number of technical difficulties. amplification problems are a common phenomenon but can be overcome by combining known methods in a new framework and by employing alternative strategies. we suggest to include assays with modified pcr methods and to creatively vary pcr conditions if amplification fails or if it leads to'peculiar' results. we show that the practical amplification issues of 18s are conquerable, whereas the future of mollusk class level phylogeny appears not to lie in this gene. the incorporation of additional data, such as structural information to identify homologous positions between sequences, is a promising approach to improve alignments and phylogenetic analyses. within the mollusca, where the sequence composition is highly variable between clades, both alignment methods and models of evolution used in phylogenetic analyses at date still remain the main bottlenecks in tracing deep phylogeny. thus, multigene analyses are more effective to resolve such ancient splits, as recently demonstrated. 
our phylogenetic tree based on species, including representatives of all mollusk classes, shows limited resolution at the class level but illustrates the pitfalls of artificial groupings formed due to shared biased sequence composition. for these gc-rich regions we calculated strong secondary structures. we report here the first authentic 18s genes of three solenogastres species, each possessing a unique sequence composition with regions conspicuously rich in guanine and cytosine. the 18s rrna gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. detailed phylogenetic analyses were conducted using rna specific models that account for compensatory substitutions in stem regions. indication for contamination of previously published solenogastres sequences is presented. the extreme morphological diversity of mollusks is mirrored in the molecular 18s data and shows elevated substitution rates mainly in three higher taxa: true limpets, cephalopoda and solenogastres. the mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations.
 the 18s rrna gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. the mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations. although an enormous amount of 18s data is available for higher mollusks, data on some early branching lineages are still limited. despite of some partial success in obtaining these data from solenogastres, by some regarded to be the most "basal" mollusks, this taxon still remained problematic due to contamination with food organisms and general amplification difficulties.  we report here the first authentic 18s genes of three solenogastres species, each possessing a unique sequence composition with regions conspicuously rich in guanine and cytosine. for these gc-rich regions we calculated strong secondary structures. the observed high intra-molecular forces hamper standard amplification and appear to increase formation of chimerical sequences caused by contaminating foreign dnas from potential prey organisms. in our analyses, contamination was avoided by using rna as a template. indication for contamination of previously published solenogastres sequences is presented. detailed phylogenetic analyses were conducted using rna specific models that account for compensatory substitutions in stem regions.  the extreme morphological diversity of mollusks is mirrored in the molecular 18s data and shows elevated substitution rates mainly in three higher taxa: true limpets, cephalopoda and solenogastres. our phylogenetic tree based on species, including representatives of all mollusk classes, shows limited resolution at the class level but illustrates the pitfalls of artificial groupings formed due to shared biased sequence composition. 