BMC Evolutionary Biology
BioMed Central
Open Access
Research article
A proteomic approach for studying insect phylogeny: CAPA
peptides of ancient insect taxa (Dictyoptera, Blattoptera) as a test
case
Steffen Roth1,3, Bastian Fromm1, Gerd Gäde2 and Reinhard Predel*1
Address: 1Institute of Zoology, University of Jena, Erbertstrasse 1, D-07743 Jena, Germany, 2Zoology Department, University of Cape Town,
Rondebosch 7701, South Africa and 3Institute of Biology, University of Bergen, Bergen N-5020, Norway
Email: Steffen Roth - steffen.roth@macnews.de; Bastian Fromm - B.Fromm@uni-jena.de; Gerd Gäde - gerd.Gade@uct.za;
Reinhard Predel* - reinhard.predel@uni-jena.de
* Corresponding author
Published: 3 March 2009
BMC Evolutionary Biology 2009, 9:50
doi:10.1186/1471-2148-9-50
Received: 6 October 2008
Accepted: 3 March 2009
This article is available from: http://www.biomedcentral.com/1471-2148/9/50
© 2009 Roth et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: 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 (Blaberidae, Blattellidae, Blattidae, Polyphagidae,
Cryptocercidae) and of the termite Mastotermes darwiniensis were chosen to test the above
hypothesis. The phylogenetic relationships within various groups of the taxon Dictyoptera (praying
mantids, termites and cockroaches) are still highly disputed.
Results: 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 61 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.
Conclusion: 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.
Background
Peptides are short proteins, whose power to resolve phylogenetic questions have already been recognized (e.g.
[1,2]). Peptide mass fingerprints support species recognition in many cases, particularly in organisms that exhibit
few morphological differences such as microorganisms
[3]. A specific group of peptides are the neuropeptides,
structurally diverse messenger molecules, which influence
a wide-range of physiological processes [4]. Due to their
role as ligands, which have to fit into the respective recep-
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BMC Evolutionary Biology 2009, 9:50
tors, 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 [5] 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 [6,7]. Other peptide families with multiple
forms such as allatostatins [8] 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 (Derst, Roth, Predel; unpublished).
Recent developments in mass spectrometric techniques [9],
however, have paved the way for a rapid identification of
mature neuropeptides from single insect specimens [1013], 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 [1416]. CAPA-genes are known from a number of holometabolous insects (e.g. Drosophila melanogaster: [17], Anopheles gambiae: [18], Apis mellifera: [19], Tribolium castaneum:
[20]). These genes encode for up to four peptides, which
belong to CAPA-periviscerokinins (PVKs) and CAPApyrokinins (PKs). Both groups of CAPA peptides bind to
different receptor types [21,22]. 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 (PSOs). Direct
mass spectrometric screening of these organs (see [10,23])
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.
http://www.biomedcentral.com/1471-2148/9/50
The taxon Dictyoptera includes praying mantids (Mantodea), termites (Isoptera), and cockroaches (Blattoptera)
(e.g. [24]), and members are among the oldest pterygote
insects known. Both morphological and molecular data
support a monophyly of Mantodea and Isoptera (see
[25]). 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 (e.g. [26-38]). 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
(analysis of morpho-anatomical data: [39,40]) and Gäde
et al. (analysis of adipokinetic hormones: [6]) placed
these wood-feeding cockroaches in the Polyphagidae.
Molecular data, however, suggest a sister-relationship
between termites and Cryptocercus [41-44], a historical
position [45] that is supported by Deitz et al. [46], and
Klass & Meier [47] based on morpho-anatomical data.
Inward et al. [44] 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. [44] 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 (see [44,47]). In cases
where these attempts result in conflicting hypotheses
about the placement and monophyly of different taxa,
additional characters (e.g. sequences of neuropeptides)
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 (AKH-1) and sulfakinins (SKs).
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 (examples given in Figures 1, 2) revealed complete sequences of CAPA peptides
from 61 cockroach/termite species. The species list covers
major taxa of cockroaches (Blattidae, Polyphagidae, CrypPage 2 of 12
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1146.61
Blaberus craniifer
CAPA-PK
3 PVKs
1835.88
3.8E+4
1094.59
1102.59
Signal Intensity
1090.60
1102.60
1.5E+4
1146.60
Bantua robusta
3 PVKs
300
Intensity, counts
tocercidae, Blaberidae, Blattellidae) and the termite Mastotermes darwiniensis. From most species, three CAPAperiviscerokinins (PVKs), and a single CAPA-pyrokinin
(PK) were sequenced. Cryptocercus and the blattellid cockroaches Symploce pallens and Loboptera decipiens express
only two different PVKs. A fourth PVK (designated PVK-4)
was found in the Madagascan Blaberidae and the Table
Mountain cockroach Aptera fusca (for sequences see [48].
These PVK-4 peptides, whose sequences suggest an internal gene duplication of PVK-1 (Elliptorhina, Gromphadorhina, Princisia) or PVK-2 (Aptera), did not influence
the topology of phylogenetic trees and were not included
in the final alignments. The average size of the PVKs was
11 amino acids (aa) and that of the PK was 17 aa.
Sequences of the CAPA peptides were combined for each
species and aligned (Table 1). With the inclusion of gaps
and sequences of the outgroup species (Locusta migratoria
and Drosophila melanogaster), the alignment resulted in 58
characters. Thirteen characters were constant, 12 variable
characters were parsimony-uninformative, and 33 variable characters were parsimony-informative. The sequence
y5
Blattella
PVK-1
[M+H] +: 1072.6
GSSGLIPMGRVa
558.4
[M+2H]2+
536.9
b2
145.1
0
100
b4 y3
b5
289.2 330.3
300
b5
402.3 402.3
y4
y6
671.5
461.3
500
y8
y7 841.6
y9
784.6
700
928.6
900
Mass/Char ge
2+
[M+2H]
CID spectrum
Figure
2 536.9(ESI-QTOF
([M+H]+:1072.6)
MS) of Blattella germanica PVK-1 at
CID spectrum (ESI-QTOF MS) of Blattella germanica
PVK-1 at [M+2H]2+ 536.9 ([M+H]+:1072.6). The y- and
b-type fragment ions are labelled. Fragments were analyzed
manually and the resulting sequence is given in the inset.
of PVK-2 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 (n > 400), and there
was a lack of variability of neuropeptides at the individual
level. We compared the CAPA peptides for a number of
species (Diploptera punctata, Loboptera decipiens, Blaberus
craniifer) 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 (Tsitsikamma), fynbos (Cape Town), and Karroo vegetation (Kamieskroon)
were investigated but no sequence variations were found
(data not shown).
CAPA-PK
1780.86
1000
1200
1400
1600
1800
2000
Mass/Char ge
Figure
Comparison
2000
craniifer
Da)
1
and
of Bantua
single
of MALDI-TOF
abdominal
robusta (=mass
PSO
peptide
spectra
preparations
hormone
(massof
fingerprint)
range
Blaberus
1000–
Comparison of MALDI-TOF mass spectra (mass
range 1000–2000 Da) of single abdominal PSO preparations of Blaberus craniifer and Bantua robusta (=
peptide hormone fingerprint). Only few abundant substances are detectable. Underlying sequences were used for
phylogenetic analyses.
Analysis of phylogenetic relationships by means of CAPA
sequences
Due to the high level of conservation in the sequences of
PVK-2 as well as in the C-termini of the other CAPA peptides, only 33 amino acid positions contained phylogenetically informative characters. It was intriguing to see
that the Maximum Parsimony (MP) analysis (Figure 3)
obtained from these data was generally in agreement with
recent molecular [44] and morphological [47] analyses,
although the bootstrap values were relatively low. Significant support (bootstrapping, posterior probabilities of
Bayesian analysis) was found for the monophyly of Blaberoidea (Blattellidae + Blaberidae) and Blattidae. The
cladograms also support sister-group relationships
between Blaberoidea and Blattoidea, Blattellidae and Bla-
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Table 1: Sequences of CAPA peptides aligned with Clustal X
Species
Ergaula capucina
Polyphaga aegyptiaca
Blatta orientalis
Neostylopyga rhombifolia
Periplaneta americana
Periplaneta australasiae
Periplaneta brunnea
Periplaneta fuliginosa
Pseudoderopeltis bimaculata
Shelfordella lateralis
Deropeltis erythrocephala
Deropeltis atra
Deropeltis integerrima
Pseudoderopeltis flavescens
Pseudoderopeltis foveolata
Eurycotis floridana
Cryptocercus darwini
Cryptocercus kyebangensis
Mastotermes darwiniensis
Therea petiveriana
Gyna lurida
Gyna caffrorum
Aptera fusca
Blaberus craniifer
Blaberus giganteus
Eublaberus distanti
Eublaberus posticus
Eublaberus spec.
Blaptica dubia
Lucihormetica grossei
Lucihormetica subcincta
Lucihormetica verrucosa
Archimandrita tesselata
Panchlora spec.
Panchlora viridis
Cyrtotria poduriformis
Hostilia carinata
Perisphaeria aff. bicolor
Pilema dubia
Perisphaeria substylifera
Perisphaeria scabrella
Blepharodera discoidalis
Perisphaeria virescens
Bantua robusta
Diploptera punctata
Perisphaeria ruficornis
Elliptorhina spec.
Gromphadorhina portentosa
Gromphadorhina grandidieri
Princisia vanwaerenbeki
Rhyparobia maderae
Laxta spec.
Pycnoscelus surinamensis
Derocalymma cruralis
Derocalymma versicolor
Panesthia spec.
Blattella germanica
Loboptera decipiens
Supella dimidiata
CAPA-PVK1
CAPA-PVK2
CAPA-PVK3
CAPA-PK
GSS-GLISFPRTa
GTS-GLISFPRTa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
GAS-GLIPVMRNa
?????????????
?????????????
ASS-GLISMPRVa
GSS-GLISFPRNa
GST-GLIPFGRTa
GST-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPMGRTa
GSS-GLIPMGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRTa
GST-GLIPFGRPa
GST-GLISFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GSS-GLIPFGRTa
GST-GLIPFGRTa
GSP-GLIPFGRSa
GSSGGLITFGRTa
GSSGGLITFGRTa
GSS-GLISFPRVa
GSS-GLIPMGRVa
?????????????
GSS-GLIAMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISVPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLIPMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GSLTGLISMPRTa
GSLTGLISMPRTa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
--QLG-L-PFPRVa
--QVG-LIPFPRVa
GSSSG-LISMPRVa
GSSSG-LISMPRVa
GSSSG-LISMPRVa
GSSSG-LISMPRVa
GSSSG-LISMPRVa
GSSSG-LISMPRVa
GSSSG-LISMPRVa
GSSSG-LISMPRVa
GGSSG-LISMPRVa
GGSSG-LISMPRVa
GGSSG-LISMPRVa
GGSSG-LISMPRVa
GGSSG-LISMPRVa
GGSSG-LISVPRVa
G-SSG-LIAMPRVa
G-SSG-LIAMPRVa
S-SSG-LIPMPRVa
G-SSG-LISMTRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-IIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSGGMIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-LIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MISFPRTa
G-SSG-MISFPRTa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
SASG-SGESSGMWFGPRLa
SASGGAGESSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
GGGG-SGETSGMWFGPRLa
EGSG-SGETSGMWFGPRLa
GGSG-SGETSGMWFGPRLa
SASG-SGESSGMWFGPRLa
AGDT-SSEAKGMWFGPRLa
AGDT-SSEAKGMWFGPRLa
SGDT-SSQAKGMWFGPRLa
AGES-SNEAKGMWFGPRLa
AGES-SNEAKGMWFGPRLa
AGES-SNEAKGMWFGPRLa
AGES-SNEAKGMWFGPRLa
AGES-SNEAKGMWFGPRLa
GGES-SNEAKGMWFGPRLa
GGES-SNEAKGMWFGPRLa
GGES-SNEAKGMWFGPRLa
GGES-SNEAKGMWFGPRLa
EGAN-SNEAKGMWFGPRLa
GGET-GNDAKAMWFGPRLa
GGET-GSDAKAMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
SGET-SGEGNGMWFGPRLa
FGET-SGETKGMWFGPRLa
FGET-SGETKGMWFGPRLa
FGET-SGETKGMWFGPRLa
FGET-SGETKGMWFGPRLa
FGET-SGETKGMWFGPRLa
GGET-SGETKGMWFGPRLa
GGET-SGEGKGMWFGPRLa
DGDM-SGEGKGMWFGPRLa
TGDM-SGEGKGMWFGPRLa
GGET-SGEGKGMWFGPRLa
ESGG-SGEANGMWFGPRLa
GSGG-SGEANGMWFGPRLa
GGGS-SGETNGMWFGPRLa
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Table 1: Sequences of CAPA peptides aligned with Clustal X (Continued)
Supella longipalpa
Symploce pallens
Drosophila melanogaster
Locusta migratoria
GSS-GLIAMPRVa
?????????????
-------------AA-GLFQFPRVa
GS-SGLISMPRVa
GS-SGLISMPRVa
AS--GLVAFPRVa
----GLLAFPRVa
62
1
77
1
52
0.72
95
52
62
0.94
1
7.0
G-SSG-MIPFPRVa
G-SSG-MIPFPRVa
GANMG-LYAFPRVa
TSS---LFPHPRLa
Drosophila melanogaster
Locusta migratoria
Cryptocercus darwini
Cryptocercus kyebangensis
Mastotermes darwiniensis
Therea petiveriana
Ergaula capucina
Polyphaga aegyptiaca
Blatta orientalis
Neostylopyga rhombifolia
Periplaneta americana
Periplaneta australasiae
Periplaneta brunnea
Periplaneta fuliginosa
Pseudoderopeltis bimaculata
Shelfordella lateralis
Deropeltis erythrocephala
Deropeltis atra
Deropeltis integerrima
Pseudoderopeltis flavescens
Pseudoderopeltis foveolata
Eurycotis floridana
Symploce pallens
Supella dimidiata
Supella longipalpa
Blattella germanica
Loboptera punctata
Panesthia sp.
Pycnoscelus surinamensis
Diploptera punctata
Perisphaeria ruficornis
Perisphaeria substylifera
Perisphaeria scabrella
Perisphaeria aff. bicolor
Pilema dubia
Cyrtotria poduriformis
Hostilia carinata
Blepharodera discoidalis
Perisphaeria virescens
Bantua robusta
Derocalymma cruralis
Derocalymma versicolor
Gyna lurida
Gyna caffrorum
Aptera fusca
Elliptorhina sp.
Gromphadorhina portentosa
Rhyparobia maderae
Gromphadorhina grandidieri
Princisia vanwaerenbeki
Laxta sp.
Panchlora sp.
Panchlora viridis
Blaberus craniifer
Blaberus giganteus
Eublaberus distanti
Eublaberus prosticus
Eublaberus sp.
Blaptica dubia
Lucihormetica grossei
Lucihormetica subcincta
Lucihormetica verrucosa
Archimandrita tesselata
GGGS-SGETNGMWFGPRLa
EGGS-SGEASGMWFGPRLa
TGPS---ASSGLWFGPRLa
DGGE---PAAPLWFGPRVa
“Cryptocercidae”
Isoptera
Polyphagidae
Blattidae
“Blattellidae”
Blattellidae
Panesthinae
Pycnoscelinae
Diplopterinae
“Perisphaerinae”
Gyninae
Epilamprinae
Blaberidae
Oxyhaloinae
“Perisphaerinae”
Panchlorinae
Blaberinae
Figure 3rulesrelationships
Phylogenetic
majority
consensus tree
of cockroaches based on CAPA peptide sequences represented by a maximum parsimony (MP) 50%
Phylogenetic relationships of cockroaches based on CAPA peptide sequences represented by a maximum parsimony (MP) 50% majority rules consensus tree. Numbers on the branches indicate bootstrap values (≥ 50) for MP. Italic
numbers on the nodes indicate posterior probability values (≥ 0.5) (proportion of the 18205 sampled trees that contain the
node). Tree length = 142, Consistency index (CI) = 0.768, Homoplasy Index (HI) = 0.232, Retention index (RI) = 0.907, Rescaled consistency index (RC) = 0.696.
beridae, and Blattidae and Polyphagidae + Cryptocercidae
+ Mastotermes. Within the latter clade, the three polyphagid species (Polyphaga aegyptiaca, Ergaula capucina,
Therea petiveriana) appear as a monophyletic group separated from an unsolved sister-group containing Cryptocercus kyebangensis and Mastotermes darwiniensis. A Bayesian
consensus tree (see additional file 1): 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 (members of 9 of 11 subfamilies were included in this study)
were poorly resolved, the different clades comprised, with
few exceptions, only members of specific subfamilies.
This was found for Blaberinae (Blaberus, Eublaberus, Lucihormetica, Archimandrita and Blaptica), Oxyhaloinae
(Madagascan genera Princisia, Elliptorhina, Gromphadorhina as well as Rhyparobia), Panchlorinae (Panchlora
species), and Perisphaeriinae (Southern African Cyrtotria,
Perisphaeria, Bantua, Hostilia, and Pilema). In contrast,
monophyly was not supported for some genera which are
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currently grouped in the Perisphaeriinae (see [49]). 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 [49], 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 (AKH-1) and sulfakinins (SKs) (see [50]). These
peptides are stored in the corpora cardiaca, and mass fin-
0.98
59
0.51
87
54
0.93
1
94
0.99
84
0.97
74
0.55
51
0.54
60
0.97
1
7.0
gerprints 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 (Figure 4 and see additional file 2): 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 (see
[51]), such new character sets and methods have to compete with well-established methods. In order for our
Drosophila melanogaster
Locusta migratoria
Mastotermes darwiniensis
Cryptocercus kyebangensis
Cryptocercus darwini
Therea petiveriana
Ergaula capucina
Polyphaga aegyptiaca
Blatta orientalis
Neostylopyga rhombifolia
Periplaneta americana
Periplaneta australasiae
Periplaneta brunnea
Periplaneta fuliginosa
Pseudoderopeltis bimaculata
Shelfordella lateralis
Deropeltis erythrocephala
Deropeltis atra
Deropeltis integerrima
Pseudoderopeltis flavescens
Pseudoderopeltis foveolata
Eurycotis floridana
Symploce pallens
Supella longipalpa
Supella dimidiata
Loboptera punctata
Blattella germanica
Panesthia sp.
Pycnoscelus surinamensis
Diploptera punctata
Perisphaeria ruficornis
Perisphaeria substylifera
Perisphaeria scabrella
Perisphaeria aff. bicolor
Pilema dubia
Cyrtotria poduriformis
Hostilia sp.
Blepharodera discoidalis
Perisphaeria virescens
Bantua robusta
Derocalymma cruralis
Derocalymma versicolor
Gyna lurida
Gyna caffrorum
Aptera fusca
Elliptorhina sp.
Gromphadorhina portentosa
Rhyparobia maderae
Gromphadorhina grandidieri
Princisia vanwaerenbeki
Laxta sp.
Blaberus craniifer
Blaberus giganteus
Eublaberus distanti
Eublaberus prosticus
Eublaberus sp.
Blaptica dubia
Lucihormetica grossei
Lucihormetica subcincta
Lucihormetica verrucosa
Archimandrita tesselata
Panchlora sp.
Panchlora viridis
Isoptera
“Cryptocercidae”
Polyphagidae
Blattidae
“Blattellidae”
Blattellidae
Panesthinae
Pycnoscelinae
Diplopterinae
“Perisphaerinae”
Gyninae
Epilamprinae
Blaberidae
Oxyhaloinae
“Perisphaerinae”
Blaberinae
Panchlorinae
Figure
Phylogenetic
mum
parsimony
4
relationships
(MP) 50% of
majority
cockroaches
rules consensus
based on CAPA
tree peptides, AKH-1 and sulfakinin sequences represented by a maxiPhylogenetic relationships of cockroaches based on CAPA peptides, AKH-1 and sulfakinin sequences represented by a maximum parsimony (MP) 50% majority rules consensus tree. Numbers on the branches indicate bootstrap values (≥ 50) for MP. Italic numbers on the nodes indicate posterior probability values (≥ 0.5) (proportion of the 20206
sampled trees that contain the node). Tree length = 181, Consistency index (CI) = 0.796, Homoplasy Index (HI) = 0.204,
Retention index (RI) = 0.917, Rescaled consistency index (RC) = 0.729.
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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 [11-13,51-54]. 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 60 neuropeptides when genome information is available [20]. 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 (see [48]) 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
(abdominal PSOs), 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 (homoplasy). 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 50 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 (AKH-1
and sulfakinins) 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.
http://www.biomedcentral.com/1471-2148/9/50
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 3 and 4 show a topology that is
in general agreement with recent molecular [44] and morphological phylogenetic analyses [47], 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 (including the outgroup
taxa) 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 (e.g. Perisphaeriinae) 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 [49], who analyzed head morphology and
genitalia. The data do not support the removal of Blepharodera from this subfamily (see also [55]), 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 (Perisphaeria, Blepharodera, Pilema,
Hostilia, Bantua, Cyrtotria) and these are exactly the genera
which were placed in a single tribe (Perisphaeriini) by
Roth [56].
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 (unpublished
data), which support the para- or polyphyletic origin of
this group. In most cockroach groups (e.g. Blaberidae),
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 61 species of Blattoptera were
tested.
From a single cockroach, P. americana, roughly 80 neuropeptides have been elucidated by biochemical methods
in recent years. Today, most of these peptides can be iden-
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tified by mass spectrometric techniques as described in
this manuscript, which makes these peptides generally
suitable for phylogenetic studies. Fast evolving neuropeptides such as FMRFamides [57] can provide phylogenetic
information at the generic level (see Figure 5) [58] 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 [59] are likely to be most suitable for the
incorporation in phylogenetic analyses. These peptide
families are represented by more than 30 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 [23].
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.
Conclusion
The phylogenetic relationships within the major lineages
of cockroaches (Blaberidae, Blattellidae, Blattidae, Polyphagidae, Cryptocercidae) and their relationship to termites (Isoptera) 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 61 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 (Blattellidae + Blaberidae) and Blattidae. The cladograms also support sister-group relationships between
Blaberoidea and a monophylum of the remaining cockroaches (including Isoptera), 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.
Methods
(a) Insects
In total, 61 species of Dictyoptera, representing the five
cockroach taxa Polyphagidae, Cryptocercidae, Blattidae,
Blattellidae, Blaberidae, and the termite Mastotermes darwiniensis were analyzed. Locusta migratoria (Orthoptera)
and Drosophila melanogaster (Diptera) were used as outgroup species; the CAPA peptides of these species were
identified by Predel & Gäde [60], Clynen et al. [61] and
http://www.biomedcentral.com/1471-2148/9/50
Kean et al. [17]. The names and places of collection (or
sources of cockroach/termite cultures) of all species examined in this study, as well as the SWISSPROT accession
numbers for peptide sequences are given in additional file
3. For most of the species, a mass fingerprint which represented about 40 peptide hormones was obtained from the
major hormone release sites (corpora cardiaca, thoracic
and abdominal perisympathetic organs). The respective
fingerprints are typical of very closely related species (see
Figure 5) and may be species-specific (see [62,63]).
Remains of the insects as well as the fingerprint data can
be obtained from the corresponding author.
(b) Mass spectrometry
The dissection of the neurohaemal organs (abdominal
perisympathetic organs, corpora cardiaca) as well as the
sample preparation for MALDI-TOF MS (matrix-assisted
laser desorption ionization time-of-flight mass spectrometry) and ESI-QTOF MS (electrospray ionization time-offlight mass spectrometry) were performed as previously
described [11,57]. MALDI-TOF MS: Mass spectra were
obtained using an ABI 4700 proteomics analyzer (Applied
Biosystems, Framingham, MA). To determine the
sequences of the peptides, tandem MS experiments with a
CID (collision induced dissociation) acceleration of 1 kV
were performed. An unambiguous assignment of internal
Leu/Ile was achieved by means of CID under high gas
pressure that revealed unique patterns for the side chains
of Leu and Ile (see [64]). Samples with CAPA peptides that
contained Lys/Gln ambiguities were analysed again after
dissolving the respective abdominal PSO preparations in
acetic anhydride (2:1 methanol/acetic anhydride) which
results in rapid acetylation of the ε-amino group of Lys.
ESI-QTOF MS: In a few cases, data obtained from MALDITOF MS did not contain sufficient information to reveal
the complete sequences of CAPA peptides. To fill the
respective sequence gaps, nanoelectrospray mass spectra
were acquired in the positive-ion mode using the API
Qstar Pulsar (Applied Biosystems, Applera Deutschland
GmbH, Darmstadt, Germany) fitted with a Protana
(Odense, Denmark) nanoelectrospray source. Samples
were purified using a homemade spin column and analyzed as described in Predel et al. [57].
(c) Sequence alignments and phylogenetic analysis
Homologous peptides were aligned using the Clustal ×
program package separately (parameter setting: gap penalty = 1; Protein Weight Matrix = BLOSUM), in contrast to
aligning the whole data set simultaneously. There was no
variability in the alignment results. Assignment of homologous gene products was facilitated due to their storage in
specific neurohaemal organs and very similar C-terminal
sequences. Phylogenetic analyses of peptides were performed under maximum-parsimony (MP) and Bayesian
inference (BI) using PAUP4.0b10 [65] and MrBayes 3.1.2
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tPSO-3
900
1060
1641.61
1589.49
1641.62
1589.51
1423.48
1316.52
1660.78
1439.62
1154.60
Eublaberus spec.
1401.56
1316.67
1220
1588.61
1140.56
1091.66
1106.59
1058.63
1004.61
1031.63
1033.60
1095.59
2693
1439.50
1095.48 1091.46
1112.48
1123.50
1140.48
1154.49
B.giganteus
1031.53
1033.51
1058.53
Signal Intensity
1316.47
1379.45
1439.45
1095.37 1091.42
1123.39
1140.36
1154.38
1033.38
1004.40
1031.41
4146
1058.40
5187
1004.53
B.craniifer
1380
1540
1700
Mass/Char ge
2004)
MALDI-TOF
species,
Figure 5representing
mass spectra
FMRFamide
(neuropeptide
related mass
peptides
fingerprints)
which accumulate
from single
in thoracic
the neurohaemal
PSO preparations
organs ofofinsects
three (see
Blaberus/Eublaberus
Predel et al.
MALDI-TOF mass spectra (neuropeptide mass fingerprints) from single thoracic PSO preparations of three
Blaberus/Eublaberus species, representing FMRFamide related peptides which accumulate in the neurohaemal
organs of insects (see Predel et al. 2004). The selected species were not distinguishable by screening the CAPA peptides
from abdominal PSOs. All ion signals different from those of B. craniifer are marked. Such fingerprint data exist from all neurohaemal organs of all cockroach species investigated.
Page 9 of 12
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[66], respectively. In the MP analysis, the heuristic search
option with the tree-bisection-reconnection (TBR) branch
swapping and 100 stepwise random additions of taxa was
used. Gaps corresponding to missing data of few peptides
were treated as missing characters, all other gaps as 21st
amino acid. Levels of branch support were assessed using
bootstrap resampling [67] with 1000 replicates to evaluate the reliability of the inferred topology. In the MP analysis, we tested the different data sets, i.e. CAPA peptides,
adipokinetic hormone and sulfakinins, both separately
and simultaneously following the total evidence
approach. Because the topology of trees was similar
(results not shown), we only present the results for our
main data set (CAPA peptides) and overall data set. We
tested the consistency by calculating the consistency index
(CI), retention index (RI), and homoplasy index (HI) (see
Figure 3 and 4).
For BI, we analysed the CAPA peptides and complete data
set separately by using the fixed rates model test as default
in MrBayes. Model free analysis of the peptide data set,
however, did not change the topology of the trees (results
not shown). A Markov Chain Monte Carlo (MCMC) sampling was run for 1 × 106 generations and trees were saved
every 100 generations (with the first 1000 trees being discarded as "burn-in"). Gaps and missing characters were
treated as missing data. Posterior probabilities with values
greater than 49% are presented.
Authors' contributions
The strategy of the paper was mainly developed and coordinated by RP and to some degree by SR. RP and SR have
written the manuscript. SR, BF and RP carried out insect
dissection, sample preparation, and mass spectrometry;
RP was responsible for species identification. BF and SR
generated the phylogenetic analysis. Parts of the present
study are incorporated within BF's diploma thesis. GG
participated in the design and coordination of the study
and helped to draft and improve the manuscript. All
authors read and approved the final manuscript.
Additional file 2
Bayesian analysis of CAPA peptides, AKH-1 and sulfakinin sequences.
Phylogenetic relationships of cockroaches based on CAPA peptides, AKH1, and sulfakinin sequences represented by a Bayesian majority rules consensus tree. Numbers on the nodes indicate posterior probability values (≥
0.49) (proportion of the 20206 sampled trees that contain the node).
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712148-9-50-S2.pdf]
Additional file 3
Additional information about studied species and accession numbers
of peptides. Information about the species used in this study, including
collecting sites/source, accession numbers of CAPA-peptides, AKH-1, sulfakinin-1 to UniProt. The sequence of sulfakinin-2 (Uni-Prot P67802)
was identical in all species, except Loboptera decipiens, Symploce pallens and Blattella germanica (sequences not elucidated). Bold accession
numbers correspond to sequences identified in this study. For Drosophila
melanogaster peptides, the Gene-bank accession numbers are given.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712148-9-50-S3.doc]
Acknowledgements
We acknowledge the assistance of Stefan Richter (Rostock, Germany), Olaf
Bininda-Emonds (Jena, Germany), Dorit Liebers (Stralsund, Germany),
Annett Kocum (Hiddensee, Germany), and Christian Derst (Berlin, Germany) during the phylogenetic analyses. We thank Christian Fischer (Göttingen, Germany) for helpful comments on an earlier version of the
manuscript, and Susanne Neupert (Jena, Germany) as well as William K.
Russell (College Station, Texas) for assistance in mass spectrometric analyses. We also thank Horst Bohn (Munich, Germany) for identification of
some species of Blattellidae and Blattidae. The authors are grateful to S.
Kambhampati (Manhattan, Kansas, USA), Yung Chul Park (Seoul, South
Korea), Rüdiger Plarre (Berlin, Germany), Andreas Brune (Marburg, Germany), and Roland Dusi (frunol delicia, Delitzsch, Germany) for supporting
the study with living or frozen specimens (see Appendix 1). We are grateful
to Cathy Jenks (Bergen, Norway) for correcting the English language. The
work was supported by grants from the Deutsche Forschungsgemeinschaft
(Predel 595/6-1,2,4), the NRF (Pretoria, RSA; gun number 2053806 and
FA2007021300002), and the Rotary Club Rondebosch (RSA).
References
Additional material
Additional file 1
Bayesian analysis of CAPA peptides sequences. Phylogenetic relationships of cockroaches based on CAPA peptides sequences represented by a
Bayesian majority rules consensus tree. Numbers on the nodes indicate
posterior probability values (≥ 0.49) (proportion of the 18205 sampled
trees that contain the node).
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712148-9-50-S1.pdf]
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