BACKGROUND
peptides are short proteins, whose power to resolve phylogenetic questions have already been recognized .
peptide mass fingerprints support species recognition in many cases, particularly in organisms that exhibit few morphological differences such as microorganisms  <cit> .
a specific group of peptides are the neuropeptides, structurally diverse messenger molecules, which influence a wide-range of physiological processes  <cit> .
due to their role as ligands, which have to fit into the respective receptors, neuropeptides are under considerable evolutionary constraint.
consequently, the regions of neuropeptide genes encoding for mature peptides may be highly conserved and suitable for the reconstruction of deep level phylogenetic relationships within higher taxa.
however, only few attempts have been made to use these substances for phylogenetic purposes.
gäde  <cit>  first introduced this approach for neuropeptides belonging to adipokinetic/hypertrehalosaemic hormones.
the few sequence variations of these hormones within insects, however, do not contain sufficient information for a detailed analysis of phylogenetic relationships, although grouping of certain taxa is possible  <cit> .
other peptide families with multiple forms such as allatostatins  <cit>  have both conserved and fast-evolving peptide sequences and are certainly more significant in this context but less extensively studied.
the conserved sequences may be suitable for the reconstruction of phylogenetic relationships within higher taxa and the fast-evolving sequences may be more suitable for the reconstruction of tip-level phylogenetic relationships within closely related taxa.
conducting a phylogenetic analysis of the genes encoding neuropeptides is not an easy task.
in most cases, only small portions of these genes have been highly conserved, specifically the regions encoding for mature peptides, which interact with their receptors.
thus, primers successfully used for the identification of neuropeptide genes in a certain insect species may fail to recognize the orthologous gene in a related species .
recent developments in mass spectrometric techniques  <cit> , however, have paved the way for a rapid identification of mature neuropeptides from single insect specimens  <cit> , thereby circumventing the genomic approach.
in the present study, tandem mass spectrometry was used for the first time to perform an extensive phylogenetic study on neuropeptides of insects, focusing on capa peptides of dictyoptera.
capa peptides were first identified from the american cockroach, periplaneta americana  <cit> .
capa-genes are known from a number of holometabolous insects .
these genes encode for up to four peptides, which belong to capa-periviscerokinins  and capa-pyrokinins .
both groups of capa peptides bind to different receptor types  <cit> .
besides their expression in a few interneurons, capa peptides are always part of the neuroendocrine system of the abdominal ventral nerve cord and are likely released into the haemolymph via abdominal perisympathetic organs .
direct mass spectrometric screening of these organs  allowed the unambiguous identification of the capa peptides from single specimens and cleared the way for a large-scale screening of these neuropeptides in the taxon dictyoptera.
the taxon dictyoptera includes praying mantids , termites , and cockroaches  , and members are among the oldest pterygote insects known.
both morphological and molecular data support a monophyly of mantodea and isoptera .
the relationships of mantodea, isoptera, and blattoptera, the monophyly of blattoptera and the relationships among several cockroach lineages are, however, a topic of conflicting conclusions .
in particular, the position of the genus cryptocercus within the blattoptera and its relationship with isoptera has been the focus of numerous phylogenetic studies.
grandcolas  and gäde et al.
placed these wood-feeding cockroaches in the polyphagidae.
molecular data, however, suggest a sister-relationship between termites and cryptocercus  <cit> , a historical position  <cit>  that is supported by deitz et al.
<cit> , and klass & meier  <cit>  based on morpho-anatomical data.
inward et al.
<cit>  presented convincing data to suggest that isoptera nest within blattoptera.
the monophyly of several cockroach taxa and subgroups of these taxa is, however, doubtful.
in a recent analysis of five gene loci, inward et al.
<cit>  found no support for the monophyly of the blattellidae and subordinated taxa within the blaberidae.
in some of these taxa, further data acquisition of conventional molecular and morphological characters and more species may provide sufficient information to resolve more precisely the phylogenetic relationship of certain taxa within the blattoptera .
in cases where these attempts result in conflicting hypotheses about the placement and monophyly of different taxa, additional characters  may be required to test the robustness of the different analyses.
to test the phylogenetic information of neuropeptides in general, we used a stepwise approach by analysing the topology and stability of the phylogenetic trees, starting with the capa peptide data set followed by repeated analyses with additional neuropeptide sequences, namely adipokinetic hormone  and sulfakinins .
the cladograms obtained from these peptide sequences confirmed that certain neuropeptide sequences of insects are able to complement molecular, biological and morphological data for the reconstruction of phylogenetic relationships.
RESULTS
data acquisition and alignment
direct mass spectrometric analysis of abdominal pso preparations of single specimens  revealed complete sequences of capa peptides from  <dig> cockroach/termite species.
the species list covers major taxa of cockroaches  and the termite mastotermes darwiniensis.
from most species, three capa-periviscerokinins , and a single capa-pyrokinin  were sequenced.
cryptocercus and the blattellid cockroaches symploce pallens and loboptera decipiens express only two different pvks.
a fourth pvk  was found in the madagascan blaberidae and the table mountain cockroach aptera fusca  or pvk- <dig> , did not influence the topology of phylogenetic trees and were not included in the final alignments.
the average size of the pvks was  <dig> amino acids  and that of the pk was  <dig> aa.
sequences of the capa peptides were combined for each species and aligned .
with the inclusion of gaps and sequences of the outgroup species , the alignment resulted in  <dig> characters.
thirteen characters were constant,  <dig> variable characters were parsimony-uninformative, and  <dig> variable characters were parsimony-informative.
the sequence of pvk- <dig> was found to be highly conserved and did not contain phylogenetically informative substitutions.
sequence variation of capa peptides within and among populations
we did not observe a single sequence variation of capa peptides from males, females, and larvae within any of the cockroach populations investigated.
the psos of the american cockroach, p. americana, served as control in most mass spectrometric analyses , and there was a lack of variability of neuropeptides at the individual level.
we compared the capa peptides for a number of species  that had been raised in a culture for multiple generations with specimens collected in the field.
in addition, three south african populations of bantua robusta that were collected in the rainforest , fynbos , and karroo vegetation  were investigated but no sequence variations were found .
analysis of phylogenetic relationships by means of capa sequences
due to the high level of conservation in the sequences of pvk- <dig> as well as in the c-termini of the other capa peptides, only  <dig> amino acid positions contained phylogenetically informative characters.
it was intriguing to see that the maximum parsimony  analysis  obtained from these data was generally in agreement with recent molecular  <cit>  and morphological  <cit>  analyses, although the bootstrap values were relatively low.
significant support  was found for the monophyly of blaberoidea  and blattidae.
the cladograms also support sister-group relationships between blaberoidea and blattoidea, blattellidae and blaberidae, and blattidae and polyphagidae + cryptocercidae + mastotermes.
within the latter clade, the three polyphagid species  appear as a monophyletic group separated from an unsolved sister-group containing cryptocercus kyebangensis and mastotermes darwiniensis.
a bayesian consensus tree : phylogenetic relationships based on neuropeptide sequences represented by a bayesian majority rules consensus tree) yielded almost identical topologies with those that were obtained from maximum parsimony.
although the relationships within the blaberidae  were poorly resolved, the different clades comprised, with few exceptions, only members of specific subfamilies.
this was found for blaberinae , oxyhaloinae , panchlorinae , and perisphaeriinae .
in contrast, monophyly was not supported for some genera which are currently grouped in the perisphaeriinae .
the australian genus laxta and african genus derocalymma, both containing extremely flattened cockroaches which are adapted for living under bark, did not show close relationships with each other or with the remaining perisphaeriinae.
instead, derocalymma was found in a clade also containing gyna and the table mountain cockroach, aptera fusca.
blepharodera discoidalis, which was removed from the perisphaeriinae by grandcolas  <cit> , contained capa peptides typical of perisphaeriinae.
to test if the topology of the phylogenetic trees remains stable, the phylogenetic analysis was repeated with additional neuropeptide sequences, namely adipokinetic hormone  and sulfakinins  .
these peptides are stored in the corpora cardiaca, and mass fingerprints from these organs were sufficient for the correct assignment of the group-specific sequences in all cases.
the resulting cladograms confirmed the topology of the former analysis, and increased the bootstrap values : phylogenetic relationships based on peptide sequences represented by a bayesian majority rules consensus tree).
discussion
the current investigation represents the first comprehensive survey of neuropeptides of insects for entirely phylogenetic purposes.
although the introduction of novel characters is consistently requested to corroborate existing hypotheses on phylogenetic relationships in insects , such new character sets and methods have to compete with well-established methods.
in order for our methodological approach to be acceptable by systematists using established methods, we developed techniques that allowed us to sample sufficient taxa and perform the analysis quickly.
in recent years, maldi-tof mass spectrometric analysis has been routinely used for studying the peptidome of the neuroendocrine system of insects  <cit> .
the power of modern mass spectrometry means that only a few specimens of insects as small as the red flour beetle tribolium castaneum are necessary to confirm the expression of more than  <dig> neuropeptides when genome information is available  <cit> .
in the present study, however, genome information was not available, and the homologous peptides of the different species had to be de-novo sequenced.
this approach posed a bioanalytical challenge and required a decision about the neuropeptide species to be included before extensive taxon sampling.
the decision to select capa peptides first  was made because these peptides fulfil certain criteria for a successful reconstruction of phylogenetic relationships.
first, these peptides occur at high concentrations in neurohaemal tissues , which are fairly easy to dissect, do not contain other neuropeptides at high concentrations and, thus, allow sequence elucidation from psos from a single specimen.
moreover, the detection of specific neuropeptide gene products, such as capa peptides, from defined neurohaemal organs usually excludes the alignment of peptides with sequence similarities that result from convergent evolution .
second, multiple members of related peptides encoded by single genes exist in insects.
if the number of these often closely related peptide paralogues differs between related species, alignments may become difficult.
hence, it is more convenient to use a peptide family that contains the same number of peptide forms in the taxa of interest.
in such a case, the storage organ as well as the conserved sequences of the peptide hormones can be used to assign the homologous peptides.
several peptide families were initially included in preliminary experiments; the capa peptides met the aforementioned criteria best and were thus used for this phylogenetic study.
since the sequence information from these peptides spans a length of  <dig> amino acids only, the resulting phylogenetic tree shows low posterior probabilities and low bootstrap levels.
in a subsequent and very rapid experimental approach, we used mass fingerprint data to include further neuropeptide sequences from relatively conserved peptides  in the phylogenetic analyses.
the resulting topology of the cladograms did not change but the bootstrap values increased considerably.
since the additional neuropeptides did not differ very much between closely related taxa or did not differ at all, bootstrap levels of higher taxa were higher than those within lower taxa.
this supports the hypothesis that, as a result of the decelerated co-evolution of neuropeptides and their receptors, neuropeptide sequences may be particularly suitable for the reconstruction of phylogenetic relationships within higher taxa.
the cladograms in figures  <dig> and  <dig> show a topology that is in general agreement with recent molecular  <cit>  and morphological phylogenetic analyses  <cit> , including the recent phylogenetic arrangement placing termites within the cockroaches.
questions arising from the current data are: how can we solve existing polytomies, how can we enhance bootstrap supports for existing clades, and how can we possibly extend the analysis to higher or lower taxa?
sampling more taxa and only analysing capa peptides, akhs and sulfakinins is unlikely to provide sufficient data to solve all of these questions.
a combination of well chosen taxa sampling  and other neuropeptides will be needed to solve the relationship among the major lineages of dictyoptera.
at a lower taxonomic level, however, a higher number of analyzed species in well-defined groups  may provide sufficient information to re-assess the generic composition of that group.
our data regarding the perisphaeriinae differ, in part, from the suggestions made by grandcolas  <cit> , who analyzed head morphology and genitalia.
the data do not support the removal of blepharodera from this subfamily , and do not verify a close relatedness of derocalymma and laxta with the other genera of perisphaeriinae.
indeed, we found six genera of perisphaeriinae with completely identical neuropeptide sequences  and these are exactly the genera which were placed in a single tribe  by roth  <cit> .
we did not test how the choice of outgroup and ingroup taxa affects tree topology but further taxon sampling seems to be essential in termites and blattellid cockroaches.
for the latter taxon, we have already obtained partial sequences from further species , which support the para- or polyphyletic origin of this group.
in most cockroach groups , however, even a more representative and comprehensive incorporation of further taxa is unlikely to provide novel insights into phylogenetic relationships.
in these cases, further peptide families have to be included for phylogenetic analyses.
in the present initial attempt, seven homologous neuropeptides of  <dig> species of blattoptera were tested.
from a single cockroach, p. americana, roughly  <dig> neuropeptides have been elucidated by biochemical methods in recent years.
today, most of these peptides can be identified by mass spectrometric techniques as described in this manuscript, which makes these peptides generally suitable for phylogenetic studies.
fast evolving neuropeptides such as fmrfamides  <cit>  can provide phylogenetic information at the generic level   <cit>  but are not suitable for studying the deep level relationships of higher taxa within an insect order because the homology of such peptide copies among far related taxa can be difficult to assess.
other peptide families with multiple members, such as tachykinin-related peptides, pyrokinins, and allatostatins  <cit>  are likely to be most suitable for the incorporation in phylogenetic analyses.
these peptide families are represented by more than  <dig> paralogues in p. americana.
previous experiments have already shown that members of the blattidae, which cannot be further separated from each other by the analysis of capa peptides, akhs, and sulfakinins, are clearly distinguishable if species-specific pyrokinin sequences are identified  <cit> .
these findings confirm that even short neuropeptide sequences of insects are suitable to complement molecular biological and morphological data for the reconstruction of phylogenetic relationships.
CONCLUSIONS
the phylogenetic relationships within the major lineages of cockroaches  and their relationship to termites  were reconstructed by using the first comprehensive survey of neuropeptides of insects for solely phylogenetic purposes.
the cladograms resulting from the analysis of peptide sequences of  <dig> blattoptera species show a topology which is in general agreement with recent molecular and morphological phylogenetic analyses and also confirm the grouping of isoptera within blattoptera.
regarding other hypotheses about cockroach phylogeny, our data support the monophyly of blaberoidea  and blattidae.
the cladograms also support sister-group relationships between blaberoidea and a monophylum of the remaining cockroaches , paraphyletic blattellidae and blaberidae, and blattidae and polyphagidae + cryptocercidae + mastotermes.
this study verified that sequences of several neuropeptide families can complement molecular biological and morphological data for the reconstruction of phylogenetic relationships.

 peptides are short proteins, whose power to resolve phylogenetic questions have already been recognized. peptide mass fingerprints support species recognition in many cases, particularly in organisms that exhibit few morphological differences such as microorganisms. a specific group of peptides are the neuropeptides, structurally diverse messenger molecules, which influence a wide-range of physiological processes. due to their role as ligands, which have to fit into the respective receptors, neuropeptides are under considerable evolutionary constraint. consequently, the regions of neuropeptide genes encoding for mature peptides may be highly conserved and suitable for the reconstruction of deep level phylogenetic relationships within higher taxa. however, only few attempts have been made to use these substances for phylogenetic purposes. gäde first introduced this approach for neuropeptides belonging to adipokinetic/hypertrehalosaemic hormones. the few sequence variations of these hormones within insects, however, do not contain sufficient information for a detailed analysis of phylogenetic relationships, although grouping of certain taxa is possible. other peptide families with multiple forms such as allatostatins have both conserved and fast-evolving peptide sequences and are certainly more significant in this context but less extensively studied. the conserved sequences may be suitable for the reconstruction of phylogenetic relationships within higher taxa and the fast-evolving sequences may be more suitable for the reconstruction of tip-level phylogenetic relationships within closely related taxa. conducting a phylogenetic analysis of the genes encoding neuropeptides is not an easy task. in most cases, only small portions of these genes have been highly conserved, specifically the regions encoding for mature peptides, which interact with their receptors. thus, primers successfully used for the identification of neuropeptide genes in a certain insect species may fail to recognize the orthologous gene in a related species. recent developments in mass spectrometric techniques, however, have paved the way for a rapid identification of mature neuropeptides from single insect specimens, thereby circumventing the genomic approach. in the present study, tandem mass spectrometry was used for the first time to perform an extensive phylogenetic study on neuropeptides of insects, focusing on capa peptides of dictyoptera. capa peptides were first identified from the american cockroach, periplaneta americana. capa-genes are known from a number of holometabolous insects. these genes encode for up to four peptides, which belong to capa-periviscerokinins and capa-pyrokinins. both groups of capa peptides bind to different receptor types. besides their expression in a few interneurons, capa peptides are always part of the neuroendocrine system of the abdominal ventral nerve cord and are likely released into the haemolymph via abdominal perisympathetic organs. direct mass spectrometric screening of these organs allowed the unambiguous identification of the capa peptides from single specimens and cleared the way for a large-scale screening of these neuropeptides in the taxon dictyoptera. the taxon dictyoptera includes praying mantids, termites, and cockroaches, and members are among the oldest pterygote insects known. both morphological and molecular data support a monophyly of mantodea and isoptera. the relationships of mantodea, isoptera, and blattoptera, the monophyly of blattoptera and the relationships among several cockroach lineages are, however, a topic of conflicting conclusions. in particular, the position of the genus cryptocercus within the blattoptera and its relationship with isoptera has been the focus of numerous phylogenetic studies. grandcolas and gäde et al. placed these wood-feeding cockroaches in the polyphagidae. molecular data, however, suggest a sister-relationship between termites and cryptocercus, a historical position that is supported by deitz et al., and klass & meier based on morpho-anatomical data. inward et al. presented convincing data to suggest that isoptera nest within blattoptera. the monophyly of several cockroach taxa and subgroups of these taxa is, however, doubtful. in a recent analysis of five gene loci, inward et al. found no support for the monophyly of the blattellidae and subordinated taxa within the blaberidae. in some of these taxa, further data acquisition of conventional molecular and morphological characters and more species may provide sufficient information to resolve more precisely the phylogenetic relationship of certain taxa within the blattoptera. in cases where these attempts result in conflicting hypotheses about the placement and monophyly of different taxa, additional characters may be required to test the robustness of the different analyses. to test the phylogenetic information of neuropeptides in general, we used a stepwise approach by analysing the topology and stability of the phylogenetic trees, starting with the capa peptide data set followed by repeated analyses with additional neuropeptide sequences, namely adipokinetic hormone and sulfakinins. the cladograms obtained from these peptide sequences confirmed that certain neuropeptide sequences of insects are able to complement molecular, biological and morphological data for the reconstruction of phylogenetic relationships.  data acquisition and alignment direct mass spectrometric analysis of abdominal pso preparations of single specimens revealed complete sequences of capa peptides from cockroach/termite species. the species list covers major taxa of cockroaches and the termite mastotermes darwiniensis. from most species, three capa-periviscerokinins, and a single capa-pyrokinin were sequenced. cryptocercus and the blattellid cockroaches symploce pallens and loboptera decipiens express only two different pvks. a fourth pvk was found in the madagascan blaberidae and the table mountain cockroach aptera fusca or pvk-, did not influence the topology of phylogenetic trees and were not included in the final alignments. the average size of the pvks was amino acids and that of the pk was aa. sequences of the capa peptides were combined for each species and aligned. with the inclusion of gaps and sequences of the outgroup species, the alignment resulted in characters. thirteen characters were constant, variable characters were parsimony-uninformative, and variable characters were parsimony-informative. the sequence of pvk- was found to be highly conserved and did not contain phylogenetically informative substitutions. sequence variation of capa peptides within and among populations we did not observe a single sequence variation of capa peptides from males, females, and larvae within any of the cockroach populations investigated. the psos of the american cockroach, p. americana, served as control in most mass spectrometric analyses, and there was a lack of variability of neuropeptides at the individual level. we compared the capa peptides for a number of species that had been raised in a culture for multiple generations with specimens collected in the field. in addition, three south african populations of bantua robusta that were collected in the rainforest, fynbos, and karroo vegetation were investigated but no sequence variations were found. analysis of phylogenetic relationships by means of capa sequences due to the high level of conservation in the sequences of pvk- as well as in the c-termini of the other capa peptides, only amino acid positions contained phylogenetically informative characters. it was intriguing to see that the maximum parsimony analysis obtained from these data was generally in agreement with recent molecular and morphological analyses, although the bootstrap values were relatively low. significant support was found for the monophyly of blaberoidea and blattidae. the cladograms also support sister-group relationships between blaberoidea and blattoidea, blattellidae and blaberidae, and blattidae and polyphagidae + cryptocercidae + mastotermes. within the latter clade, the three polyphagid species appear as a monophyletic group separated from an unsolved sister-group containing cryptocercus kyebangensis and mastotermes darwiniensis. a bayesian consensus tree: phylogenetic relationships based on neuropeptide sequences represented by a bayesian majority rules consensus tree) yielded almost identical topologies with those that were obtained from maximum parsimony. although the relationships within the blaberidae were poorly resolved, the different clades comprised, with few exceptions, only members of specific subfamilies. this was found for blaberinae, oxyhaloinae, panchlorinae, and perisphaeriinae. in contrast, monophyly was not supported for some genera which are currently grouped in the perisphaeriinae. the australian genus laxta and african genus derocalymma, both containing extremely flattened cockroaches which are adapted for living under bark, did not show close relationships with each other or with the remaining perisphaeriinae. instead, derocalymma was found in a clade also containing gyna and the table mountain cockroach, aptera fusca. blepharodera discoidalis, which was removed from the perisphaeriinae by grandcolas, contained capa peptides typical of perisphaeriinae. to test if the topology of the phylogenetic trees remains stable, the phylogenetic analysis was repeated with additional neuropeptide sequences, namely adipokinetic hormone and sulfakinins. these peptides are stored in the corpora cardiaca, and mass fingerprints from these organs were sufficient for the correct assignment of the group-specific sequences in all cases. the resulting cladograms confirmed the topology of the former analysis, and increased the bootstrap values: phylogenetic relationships based on peptide sequences represented by a bayesian majority rules consensus tree). discussion the current investigation represents the first comprehensive survey of neuropeptides of insects for entirely phylogenetic purposes. although the introduction of novel characters is consistently requested to corroborate existing hypotheses on phylogenetic relationships in insects, such new character sets and methods have to compete with well-established methods. in order for our methodological approach to be acceptable by systematists using established methods, we developed techniques that allowed us to sample sufficient taxa and perform the analysis quickly. in recent years, maldi-tof mass spectrometric analysis has been routinely used for studying the peptidome of the neuroendocrine system of insects. the power of modern mass spectrometry means that only a few specimens of insects as small as the red flour beetle tribolium castaneum are necessary to confirm the expression of more than neuropeptides when genome information is available. in the present study, however, genome information was not available, and the homologous peptides of the different species had to be de-novo sequenced. this approach posed a bioanalytical challenge and required a decision about the neuropeptide species to be included before extensive taxon sampling. the decision to select capa peptides first was made because these peptides fulfil certain criteria for a successful reconstruction of phylogenetic relationships. first, these peptides occur at high concentrations in neurohaemal tissues, which are fairly easy to dissect, do not contain other neuropeptides at high concentrations and, thus, allow sequence elucidation from psos from a single specimen. moreover, the detection of specific neuropeptide gene products, such as capa peptides, from defined neurohaemal organs usually excludes the alignment of peptides with sequence similarities that result from convergent evolution. second, multiple members of related peptides encoded by single genes exist in insects. if the number of these often closely related peptide paralogues differs between related species, alignments may become difficult. hence, it is more convenient to use a peptide family that contains the same number of peptide forms in the taxa of interest. in such a case, the storage organ as well as the conserved sequences of the peptide hormones can be used to assign the homologous peptides. several peptide families were initially included in preliminary experiments; the capa peptides met the aforementioned criteria best and were thus used for this phylogenetic study. since the sequence information from these peptides spans a length of amino acids only, the resulting phylogenetic tree shows low posterior probabilities and low bootstrap levels. in a subsequent and very rapid experimental approach, we used mass fingerprint data to include further neuropeptide sequences from relatively conserved peptides in the phylogenetic analyses. the resulting topology of the cladograms did not change but the bootstrap values increased considerably. since the additional neuropeptides did not differ very much between closely related taxa or did not differ at all, bootstrap levels of higher taxa were higher than those within lower taxa. this supports the hypothesis that, as a result of the decelerated co-evolution of neuropeptides and their receptors, neuropeptide sequences may be particularly suitable for the reconstruction of phylogenetic relationships within higher taxa. the cladograms in figures and show a topology that is in general agreement with recent molecular and morphological phylogenetic analyses, including the recent phylogenetic arrangement placing termites within the cockroaches. questions arising from the current data are: how can we solve existing polytomies, how can we enhance bootstrap supports for existing clades, and how can we possibly extend the analysis to higher or lower taxa? sampling more taxa and only analysing capa peptides, akhs and sulfakinins is unlikely to provide sufficient data to solve all of these questions. a combination of well chosen taxa sampling and other neuropeptides will be needed to solve the relationship among the major lineages of dictyoptera. at a lower taxonomic level, however, a higher number of analyzed species in well-defined groups may provide sufficient information to re-assess the generic composition of that group. our data regarding the perisphaeriinae differ, in part, from the suggestions made by grandcolas, who analyzed head morphology and genitalia. the data do not support the removal of blepharodera from this subfamily, and do not verify a close relatedness of derocalymma and laxta with the other genera of perisphaeriinae. indeed, we found six genera of perisphaeriinae with completely identical neuropeptide sequences and these are exactly the genera which were placed in a single tribe by roth. we did not test how the choice of outgroup and ingroup taxa affects tree topology but further taxon sampling seems to be essential in termites and blattellid cockroaches. for the latter taxon, we have already obtained partial sequences from further species, which support the para- or polyphyletic origin of this group. in most cockroach groups, however, even a more representative and comprehensive incorporation of further taxa is unlikely to provide novel insights into phylogenetic relationships. in these cases, further peptide families have to be included for phylogenetic analyses. in the present initial attempt, seven homologous neuropeptides of species of blattoptera were tested. from a single cockroach, p. americana, roughly neuropeptides have been elucidated by biochemical methods in recent years. today, most of these peptides can be identified by mass spectrometric techniques as described in this manuscript, which makes these peptides generally suitable for phylogenetic studies. fast evolving neuropeptides such as fmrfamides can provide phylogenetic information at the generic level but are not suitable for studying the deep level relationships of higher taxa within an insect order because the homology of such peptide copies among far related taxa can be difficult to assess. other peptide families with multiple members, such as tachykinin-related peptides, pyrokinins, and allatostatins are likely to be most suitable for the incorporation in phylogenetic analyses. these peptide families are represented by more than paralogues in p. americana. previous experiments have already shown that members of the blattidae, which cannot be further separated from each other by the analysis of capa peptides, akhs, and sulfakinins, are clearly distinguishable if species-specific pyrokinin sequences are identified. these findings confirm that even short neuropeptide sequences of insects are suitable to complement molecular biological and morphological data for the reconstruction of phylogenetic relationships.  the phylogenetic relationships within the major lineages of cockroaches and their relationship to termites were reconstructed by using the first comprehensive survey of neuropeptides of insects for solely phylogenetic purposes. the cladograms resulting from the analysis of peptide sequences of blattoptera species show a topology which is in general agreement with recent molecular and morphological phylogenetic analyses and also confirm the grouping of isoptera within blattoptera. regarding other hypotheses about cockroach phylogeny, our data support the monophyly of blaberoidea and blattidae. the cladograms also support sister-group relationships between blaberoidea and a monophylum of the remaining cockroaches, paraphyletic blattellidae and blaberidae, and blattidae and polyphagidae + cryptocercidae + mastotermes. this study verified that sequences of several neuropeptide families can complement molecular biological and morphological data for the reconstruction of phylogenetic relationships. 
this study represents the first comprehensive survey of neuropeptides of insects for solely phylogenetic purposes and concludes that sequences of short neuropeptides are suitable to complement molecular biological and morphological data for the reconstruction of phylogenetic relationships. neuropeptide ligands have to fit exactly into their respective receptors and thus the evolution of the coding regions of their genes is constrained and may be strongly conserved. capa peptides of major lineages of cockroaches and of the termite mastotermes darwiniensis were chosen to test the above hypothesis. tandem mass spectrometry of neuropeptides from perisympathetic organs was used to obtain sequence data of capa peptides from single specimens; the data were analysed by maximum parsimony and bayesian interference. adipokinetic hormones and sulfakinins, were included, the general topology of the cladogram did not change but bootstrap values increased considerably. the resulting cladograms, taking species into account, show a topology which is in general agreement with recent molecular and morphological phylogenetic analyses, including the recent phylogenetic arrangement placing termites within the cockroaches. the phylogenetic relationships within various groups of the taxon dictyoptera are still highly disputed. as such, they may be suitable for the reconstruction of phylogenetic relationships within higher taxa.
 neuropeptide ligands have to fit exactly into their respective receptors and thus the evolution of the coding regions of their genes is constrained and may be strongly conserved. as such, they may be suitable for the reconstruction of phylogenetic relationships within higher taxa. capa peptides of major lineages of cockroaches and of the termite mastotermes darwiniensis were chosen to test the above hypothesis. the phylogenetic relationships within various groups of the taxon dictyoptera are still highly disputed.  tandem mass spectrometry of neuropeptides from perisympathetic organs was used to obtain sequence data of capa peptides from single specimens; the data were analysed by maximum parsimony and bayesian interference. the resulting cladograms, taking species into account, show a topology which is in general agreement with recent molecular and morphological phylogenetic analyses, including the recent phylogenetic arrangement placing termites within the cockroaches. when sequence data sets from other neuropeptides, viz. adipokinetic hormones and sulfakinins, were included, the general topology of the cladogram did not change but bootstrap values increased considerably.  this study represents the first comprehensive survey of neuropeptides of insects for solely phylogenetic purposes and concludes that sequences of short neuropeptides are suitable to complement molecular biological and morphological data for the reconstruction of phylogenetic relationships. 