OPOLE SCIENTIFIC SOCIETY
NATURE JOURNAL
No 44 – 2011: 73-91
COMPARATIVE STUDIES ON 12S AND 16S MITOCHONDRIAL rDNA SEQUENCES IN
PENTATOMOMORPHAN BUGS (HEMIPTERA: HETEROPTERA: PENTATOMOMORPHA)
JERZY A. LIS1, PAWEŁ LIS2, DARIUSZ J. ZIAJA3
1
Department of Biosystematics, Opole University, Oleska 22, 45-052 Opole, Poland;
e-mail: cydnus@uni.opole.pl, http://www.cydnidae.uni.opole.pl
2
Department of Genetics, Institute of Genetics and Microbiology, University of
Wrocław, S. Przybyszewskiego 63/77, 51-148 Wrocław, Poland;
e-mail: pawel.lis@microb.uni.wroc.pl
3
Department of Biosystematics, Opole University, Oleska 22, 45-052 Opole, Poland;
e-mail: d.ziaja@uni.opole.pl
ABSTRACT: Nucleotide sequences of the mitochondrial 12S and 16S ribosomal RNA
gene fragments (12S rDNA and 16S rDNA) of twenty species of Pentatomomorpha and
a single out-group species (i.e. Triatoma dimidiata, representing the infraorder
Cimicomorpha) have been determined. Their nucleotide composition, substitution
patterns and nucleotide divergence were investigated. To evaluate the usefulness of the
12S rDNA and 16S rDNA sequences for phylogenetic inference, the obtained data sets
were analyzed using different methods (MP, ME, NJ, Bayesian estimation). The average
A + T contents in studied species were as high as in other previously investigated
Heteroptera (above 75%), except for the Aradidae where the A + T content for 12S and
16S rDNA fragments were below 70%. The A/G and T/C transition biases found in both
rDNA fragments agreed with the observations suggesting that the transitional
substitutions occur more readily than transversional substitutions; however, the observed
A/T transversion frequency in the 16S rDNA of Pentatomomorpha was distinctly lower
(18.78%) than that found in cimicomorphan Anthocoridae (54.52%). Undirectional
nucleotide pair frequencies, in 12S rDNA and 16S rDNA fragments increase linearly
with an increase in taxonomic level indicating that the rate of nucleotide substitutions is
constant along studied taxonomic levels. Results of our studies suggested that the
nucleotide sequences of 12S and 16S rDNA fragments can be regarded as a good marker
for resolving the phylogeny of closely related genera. Moreover, it was found that the
Bayesian inference analysis of the combined 12S/16S rDNA dataset appears to be the
most appropriate method for phylogenetic reconstructions in Pentatomomorpha when the
mitochondrial ribosomal RNA genes are used.
74
KEY WORDS: Hemiptera, Heteroptera, Pentatomomorpha, mitochondrial DNA,
nucleotide sequences, nucleotide substitutions, nucleotide divergence, molecular
phylogeny, 12S rDNA, 16S rDNA, Bayesian estimation.
Introduction
Insect mitochondrial genome consists of 13 protein-coding genes (PCGs), 22 transfer
RNA genes and two ribosomal RNA genes (i.e., 12S rDNA and 16S rDNA); it contains
also a major non-coding region called the control region or A+T-rich region (Simon et
al. 1994, Boore 1999, Wolstenholme 1992, Li et al. 2009).
In recent years, different regions of the mtDNA (including the 12S rDNA and
16S rDNA subunits) have been regarded as good molecular markers for phylogenetic
inference (for a review, see, e.g., Simon et al. 2006, Vogler and Monaghan 2007, Bybee
et al. 2010).
The 12S rDNA, was initially regarded to be highly conserved and has mostly
been applied to understand the genetic diversity and phylogeny at higher taxonomic
levels, such as in phyla, classis and orders, whereas the 16S rDNA was very often used
in studies at the familial and generic level (for a review, see, e.g., Simon et al. 1994,
Gerber et al. 2001). However, in insects, these gene fragments have been proved
informative also in species-level studies (e.g., Kambhampati 1995, Shawn 1996,
Dopman et al. 2002, Saux et al. 2003, Yoshizawa and Johnson 2003, Cook et al. 2004,
Nagaraja et al. 2004, Mahendran et al. 2006, Liu et al. 2008), or even in intraspecific
investigations (Johnson et al. 2004).
However, in Heteroptera, those two fragments of mtDNA have only been
sporadically used in phylogeographic and phylogenomic studies, i.e., 16S in Triatominae
of Reduviidae (Hypša et al. 2002, Silva de Paula et al. 2007) and in Pentatomoidea (Li et
al. 2006, Grazia et al. 2008), and 12S only in Triatominae of Reduviidae (Hypša et al.
2002).
Many studies have suggested that nucleotide composition and substitution
biases in mitochondrial DNA may affect the final results of phylogenomic analyses (e.g.,
Simon et al. 1994, 2006, Lockhart et al. 1994, Dowton and Austin 1995, 1997, Mindell
and Thacker 1996). Therefore, it appears important to define the suitability of the
nucleotide sequences for the phylogeny of certain insect taxa.
Such comparative studies on the characterization of the mitochondrial rDNA
sequences have almost been neglected in Heteroptera. The nucleotide composition,
substitution patterns and nucleotide divergence of the 16S rDNA sequences were
determined only in eight species of the family Anthocoridae (Muraji et al. 2000). In a
single paper (Hua et al. 2008) the complete (or nearly complete) mt-genomes of fifteen
heteropteran species were analyzed with respect to their genome organization and gene
rearrangement, but with no special attention to both ribosomal RNA genes.
Since no other heteropteran mitochondrial rDNA sequences have been
characterized yet, we decided to investigate the mitochondrial 12S and 16S rDNA
sequences of the heteropteran infraorder Pentatomomorpha, and provide the analyses of
75
their nucleotide composition, nucleotide divergence and base substitutions, as well as to
evaluate their phylogenetic utility.
Material and methods
The species used in this study are listed in Table 1. In total, 20 species representing 14
families and five superfamilies of the infraorder Pentatomomorpha, and a single outgroup species (i.e. Triatoma dimidiata, representing the infraorder Cimicomorpha) were
analyzed.
Table 1. Details of specimens of Pentatomoidea used in comparative and phylogenetic
analyses
species
PENTATOMOMORPHA
GenBank (NCBI) accesion numbers
12S
16S
Complete mt-genome
-
-
NC_012459.1
-
-
EU427344.1
-
-
EU427337.1
ARADOIDEA
ARADIDAE
1.
Neuroctenus parus Hsiao
COREOIDEA
ALYDIDAE
2.
Riptortus pedestris (Fabricius)
COREIDAE
3. Hydaropsis longirostris (Hsiao)
RHOPALIDAE
3.
Rhopalus latus (Jakovlev)
-
-
4.
Stictopleurus subviridis Hsiao
-
-
EU427333.1
[as Aeschyntelus notatus Hsiao]
NC_012888.1
-
-
EU427346.1
-
-
NC_012424.1
JQ234967
JQ234972
-
-
-
EU427339.1
JQ234968
JQ234973
-
-
-
NC_012457.1
[as Macroscytus subaeneus
(Dallas)]
LYGAEOIDEA
BERYTIDAE
5.
Yemmalysus parallelus Štusák
6.
Geocoris pallidipennis (A. Costa)
7.
Lygaeus equestris (Linnaeus)
8.
Malcus inconspicuous Štys
GEOCORIDAE
LYGAEIDAE
MALCIDAE
PENTATOMOIDEA
CYDNIDAE
9.
Sehirus luctuosus Mulsant et Rey
10. Macroscytus gibbulus (Ellenrieder)
76
PLATASPIDAE
11. Coptosoma bifarium Montandon
-
-
NC_012449.1
12. Megacopta cribraria (Fabricius)
-
-
NC_015342.1
JQ234969
JQ234974
-
14. Antheminia lunulata (Goeze)
JQ234970
JQ234975
-
15. Carpocoris purpureipennis (De Geer)
SCUTELLERIDAE
13. Eurygaster testudinaria (Geoffroy)
PENTATOMIDAE
JQ234971
JQ234976
-
16. Halyomorpha halys (Stål)
-
-
NC_013272.1
17. Nezara viridula (Linnaeus)
-
-
NC_011755.1
-
-
EU427343.1
-
-
EU427335.1
-
-
NC_002609.1
PYRRHOCOROIDEA
LARGIDAE
18. Physopelta gutta (Burmeister)
PYRRHOCORIDAE
19. Dysdercus cingulatus (Fabricius)
CIMICOMORPHA
REDUVIOIDEA
REDUVIIDAE
20. Triatoma dimidiata (Latreille)
The partial mtDNA sequences (12S and 16S rDNA) of four pentatomoid and
one lygaeoid species were analyzed (see Table 2); their nucleotide sequence data were
submitted to GenBank (the accession numbers are listed in Table 1 and Table 2). All
other sequences used in our study were obtained directly from GenBank (Table 1). For a
question of proper identification of Macroscytus subaeneus, the only named species of
the family Cydnidae placed into GenBank database (see Lis J.A. and Lis B.2011).
Specimens of all studied species were collected directly in pure ethanol (for
collecting data, see Table 2). DNA extraction, purification and amplification were
performed at the Centre for Biodiversity Studies (Department of Biosystematics, Opole
University, Poland) using techniques described by Lis J.A. et al. (2011). All primer
sequences used for their PCR amplification are also listed in Lis J.A. et al. (2011). The
remains of the studied specimens were inserted in tubes with 96% ethanol and lodged in
a deep freezer (for the Opole University sample numbers, see Table 2). Sequencing was
conducted at the Health Care Centre GENOMED (Warsaw, Poland).
Sequence alignments were made with Clustal X (using default parameters) in
MEGA 4.0.2 software (Tamura et al. 2007, Kumar et al. 2008). The obtained sequences
were used in BLAST searches, which showed their high similarities to sequences of
other pentatomomorphan species (this procedure ensured our results were not
contaminants).
77
Table 2. Details of specimens of Lygaeoidea and Pentatomoidea used for comparative
and phylogenetic analyses (stored at the Center for Biodiversity Studies, Department of
Biosystematics, Opole University, Poland)
Superfamily/ family/ species
LYGAEOIDEA
LYGAEIDAE
1. Lygaeus equestris (Linnaeus)
PENTATOMOIDEA
CYDNIDAE
2. Sehirus luctuosus Mulsant et Rey
SCUTELLERIDAE
3. Eurygaster testudinaria (Geoffroy)
PENTATOMIDAE
4. Antheminia lunulata (Goeze)
5.
Carpocoris purpureipennis (De Geer)
subunit
Locality data
GenBank
(NCBI)
accesion
numbers
Opole
University
sample number
12S
16S
Štramberk, NE Moravia,
Czech Republic, 2010
JQ234967
JQ234972
ES2
12S
16S
Suchy Bór n. Opole,
Lower Silesia,
Poland, 2009
JQ234968
JQ234973
3P1
12S
16S
Łagów, WielkopolskoKujawska Lowland,
Poland, 2009
JQ234969
JQ234974
86ET
12S
16S
Szaniec n. Busko Zdrój,
Małopolska Upland,
Poland, 2010
Suchy Bór n. Opole,
Lower Silesia,
Poland, 2007
JQ234970
JQ234975
125C
JQ234971
JQ234976
122CP
12S
16S
The nucleotide composition, substitution patterns and nucleotide divergence
were calculated in MEGA 4.0.2 (Tamura et al. 2007, Kumar et al. 2008). The pattern of
nucleotide substitutions was estimated using Maximum Composite Likelihood (Tamura
et al. 2004). All positions containing gaps and missing data were eliminated from the
datasets (Complete-deletion option). The overall transition/transversion bias (R) was
calculated for each data set, where R = [A*G*k1 + T*C*k2]/[(A+G)*(T+C)] (Tamura et
al. 2004).
Phylogenetic analyses using maximum parsimony (MP), minimum evolution
(ME) and neighbor-joining (NJ) approaches were performed using MEGA 4.0.2
software (Tamura et al. 2007, Kumar et al. 2008).
It was recently suggested (e.g., Li et al. 2006, Hahn 2007, Ronquist and Deans
2010, Rasmussen and Kellis 2011) these methods frequently make reconstruction errors
and can be not sufficiently accurate for systematic use. Therefore, in order to avoid the
possible effect of gene duplication and loss events on our inferences, the phylogenetic
trees of Pentatomomorpha were also calculated using the Bayesian estimation. Bayesian
inferences (BI) trees (inferred from separate and combined data set) were obtained with
MrBayes v. 3.2 (Ronquist et al. 2011) using the Markov Chain Monte Carlo technique
(MCMC).
We chose the evolutionary model GTR + I + Γ (nst=6), and each run lasted for
1,000,000 generations with a sampling frequency every 100th generation, giving 10,000
78
samples. MCMC sampling was used with a Metropolis coupling set to use four Markov
chains (three heated and one cold chain) with a starting temperature of 0.1 (default in
MrBayes v.3.2). Burning percentage was set to default, discarding the first 25% of the
samples from the cold chain. After 1,000,000 generations, the standard deviations of
split frequencies were <0.005 for 12S, 16S and combined 12S/16S analyses.
All trees for MP, ME and NJ were edited using MEGA 4.0.2 (op. cit.); all BI
trees were edited using FigTree 1.3.1 (Rambaut 2009).
Results and discussion
Nucleotide alignment. We obtained 376-base pair long sequences for the 3´ end of the
12S rDNA (Fig. 1) and 428-base pair long sequences for the 3´ end of the 16S rDNA
(Fig. 2).
In the 12S rDNA sequences – 122 conserved sites, 242 variable sites, 173 parsimonyinformative sites and 64 singleton sites were detected, while in 16S rDNA sequences –
161 conserved sites, 262 variable sites, 209 parsimony-informative sites and 53 singleton
sites were detected. Our results suggest that in Pentatomomorpha the 16S rDNA
sequences are a little more conserved than the 12S rDNA sequences (38.6% for
conserved sites in the 16S sequences; 33.8% for conserved sites in the 12S sequences).
These results are contrary to those regarding the 12s rDNA sequences highly conserved
in insects, and useful for understanding the genetic diversity and phylogeny only at
higher taxonomic levels. Additionally, when the variable sites of the 16S rDNA
sequences of our twenty species of Pentatomomorpha are compared to those of eight
species of the family Anthocoridae representing Cimicomorpha (Muraji et al. 2000), it
can be supposed that the 16S rDNA sequences are more conserved in Cimicomorpha
than they are in Pentatomomorpha (variable sites: 44.0% in Anthocoridae, 60.3% in
studied Pentatomomorpha).
Nucleotide composition. The average A + T contents in studied species of
Pentatomomorpha were 76.1% and 75.1%, for the 12S rDNA and16S rDNA,
respectively (Table 3 and 4). The similar high A + T content in the 16S rDNA was
observed also in other Heteroptera (Muraji et al. 2000; Hua et al. 2008; Lee et al. 2009;
Li, Liu et al. 2011; Li, Gao et al. 2011). Only in Neuroctenus parus (Aradidae) the A + T
content was distinctly lower than in other pentatomomorphan bugs (68.2% for12S
rDNA, 69.4% for 16S rDNA). Such low A + T content was noticed in Aradidae also by
Hua et al (2008). The fact of low A + T content may validate the isolated position of the
family Aradidae within this infraorder (this family is sometimes regarded as not
belonging to Pentatomomorpha, but constituting a separate infraorder within the
suborder Heteroptera (see Sweet 2006).
79
Table 3. Nucleotide content of the 12S rDNA sequences of 21 species used in this study
Taxon
Nucleotide frequencies
(in %)
Total
(bp)
T
C
A
G
Neuroctenus parus (Aradidae)
27.8
20.7
40.4
11.1
324
Riptortus pedestris (Alydidae)
30.9
16.4
44.8
7.9
330
Hydaropsis longirostris (Coreidae)
30.3
16.1
45.5
8.2
330
Rhopalus latus (Rhopalidae)
33.5
13.6
45.1
7.7
337
Stictopleurus subviridis (Rhopalidae)
31.1
14.8
44.9
9.2
325
Yemmalysus parallelus (Berytidae)
32.8
13.6
45.5
8.1
332
Geocoris pallidipennis (Geocoridae)
32.3
15.9
43.7
8.1
334
Lygaeus equestris (Lygaeidae)
31.8
14.7
45.3
8.3
327
Malcus inconspicuus (Malcidae)
32.7
14.5
43.6
9.1
330
Sehirus luctuosus (Cydnidae)
32.6
16.3
42.4
8.6
337
Macroscytus gibbulus (Cydnidae)
32.1
14.6
44.1
9.2
349
Coptosoma bifarium (Plataspidae)
33.5
15.7
41.5
9.2
337
Megacopta cribraria (Plataspidae)
29.9
17.2
44.1
8.8
331
Eurygaster testudinaria (Scutelleridae)
31.7
15.0
44.3
9.1
341
Antheminia lunulata (Pentatomidae)
33.3
14.6
44.3
7.7
336
Carpocoris purpureipennis (Pentatomidae)
33.1
13.9
44.4
8.6
338
Halyomoropha halys (Pentatomidae)
32.6
14.2
45.1
8.1
344
Nezara viridula (Pentatomidae)
32.9
14.2
45.1
7.8
346
Physopelta gutta (Largidae)
29.3
16.7
45.1
9.0
335
Dysdercus cingulatus (Pyrrhocoridae)
34.0
13.1
44.5
8.4
335
Average for Pentatomomorpha
Triatoma dimidiata (Reduviidae)
[Cimicomorpha]
Average for Heteroptera
31.9
15.3
44.2
8.6
334.9
31.5
20.1
38.3
10.2
324
31.9
15.5
43.9
8.7
334.4
80
Table 4. Nucleotide content of the 16S rDNA sequences of 21 species used in this study
Nucleotide frequencies
(in %)
Total
(bp)
Taxon
T
C
A
G
Neuroctenus parus (Aradidae)
27.4
19.7
42.0
10.9
402
Riptortus pedestris (Alydidae)
33.9
14.1
41.8
10.1
404
Hydaropsis longirostris (Coreidae)
32.4
16.2
42.4
9.0
401
Rhopalus latus (Rhopalidae)
33.4
16.0
41.0
9.6
407
Stictopleurus subviridis (Rhopalidae)
32.3
15.1
43.4
9.2
403
Yemmalysus parallelus (Berytidae)
35.4
12.7
41.6
10.2
401
Geocoris pallidipennis (Geocoridae)
34.5
14.5
41.8
9.3
400
Lygaeus equestris (Lygaeidae)
33.6
13.5
44.9
8.0
399
Malcus inconspicuus (Malcidae)
31.8
14.3
44.5
9.5
400
Sehirus luctuosus (Cydnidae)
35.8
13.9
42.3
8.0
411
Macroscytus gibbulus (Cydnidae)
32.0
16.1
42.7
9.3
410
Coptosoma bifarium (Plataspidae)
31.0
17.4
39.7
11.9
403
Megacopta cribraria (Plataspidae)
28.0
16.6
44.3
11.1
404
Eurygaster testudinaria (Scutelleridae)
32.2
16.7
42.3
8.8
407
Antheminia lunulata (Pentatomidae)
31.0
16.7
43.3
8.9
406
Carpocoris purpureipennis (Pentatomidae)
31.7
17.1
41.7
9.5
398
Halyomoropha halys (Pentatomidae)
33.9
15.5
41.8
8.8
407
Nezara viridula (Pentatomidae)
32.8
14.8
43.7
8.6
405
Physopelta gutta (Largidae)
32.0
15.8
43.3
9.0
400
Dysdercus cingulatus (Pyrrhocoridae)
36.4
13.9
42.3
7.4
404
Average for Pentatomomorpha
Triatoma dimidiata (Reduviidae)
[Cimicomorpha]
Average for Heteroptera
32.6
15.5
42.5
9.4
403.6
29.8
17.8
43.0
9.5
400
32.4
15.6
42.6
9.4
403.4
Nucleotide substitutions (Table 5 and 6). There were a total of 285 positions in the final
12S rDNA dataset, and 342 positions in the final 16S rDNA dataset. The nucleotide
frequencies are 0.438 (A), 0.309 (T/U), 0.159 (C), and 0.094 (G) in the 12S rDNA
sequences, and 0.396 (A), 0.324 (T/U), 0.173 (C), and 0.106 (G) in the 16S rDNA
sequences. The transition/transversion rate ratios are k1 = 1.965 (purines) and k2 = 2.278
(pyrimidines) for 12S rDNA, and k1 = 1.6 (purines) and k2 = 2.077 (pyrimidines) for 16S
81
rDNA. The overall transition/transversion bias (R) is similar for both studied gene
fragments, i.e., 0.485 for 12S rDNA and 0.494 for 16S rDNA. Strong A/G and T/C
transition biases were found in both rDNA fragments (25.4% and 25.96%, respectively,
in 12S rDNA; 20.96% and 26.91%, respectively, in 16S rDNA) what agrees with
observations suggesting that the transitional substitutions occur more readily than
transversional substitutions (Brown et al. 1979; Kondo et al. 1993). However, our results
relating to the frequency of nucleotide substitutions in 16S rDNA fragments among
species of Pentatomomorpha stay in contradiction with the frequency among eight
species of Anthocoridae – see, Table 6 in the present study, and the Table 3 in Muraji et
al. (2000). Moreover, a very high A/T transversion frequency (54.52%) and a zero G/C
transversion frequency in the sequences of 16S rDNA among species of Anthocoridae
(Muraji et al. 2000) were not observed among species studied by us (transversion
frequency 18.78%, 7.28%, respectively). This may suggest diverse transitional and
transversional biases in the mitochondrial rDNA sequences in different infraorders of
Heteroptera.
Table 5. Frequency of nucleotide substitutions in the 12S rDNA sequences among
studied species of Pentatomomorpha. Each entry shows the probability of substitution
from one base (row) to another base (column) instantaneously. Rates of different
transitional substitutions are shown in bold and those of transversional substitutions are
shown in italics
A
T
C
G
A
-
7.52
3.87
4.49
T
10.64
-
8.82
2.29
C
10.64
17.14
-
2.29
G
20.91
7.52
3.87
-
Table 6. Frequency of nucleotide substitutions in the 16S rDNA sequences among
studied species of Pentatomomorpha. Each entry shows the probability of substitution
from one base (row) to another base (column) instantaneously. Rates of different
transitional substitutions are in bold and those of transversional substitutions are in
italics
A
T
C
G
A
-
8.45
4.51
4.43
T
10.33
-
9.37
2.77
C
10.33
17.54
-
2.77
G
16.53
8.45
4.51
-
Nucleotide divergence. The pairwise comparisons were made between sequences on
three different taxonomic levels (Table 7), i.e., between pairs of genera representing the
same family, between families (pairs of genera representing different families within the
82
same superfamily), and between superfamilies (pairs of genera representing different
superfamilies within the same suborder). Our studies showed that undirectional
nucleotide pair frequencies, in 12S rDNA and 16S rDNA fragments, increased linearly
with an increase in taxonomic level (from genera to superfamilies). These results stay in
accordance with results of study on the 16S rDNA in Anthocoridae (Muraji et al. 2000),
where also no plateau has been observed in the frequency of base substitutions within the
studied taxa.
Table 7. Percentage of nucleotide substitutions in the 12S rDNA and 16S rDNA
fragments among studied species of Pentatomomorpha
Pairwise comparisons between:
Genera
(within the same family)
Families
(within the same superfamily)
Superfamilies
(within the same infraorder)
12S rDNA
16S rDNA
mean
min.-max
mean
min.-max.
14.8
9.6-20.9
15.9
11.4-19.9
18.4
10.0-24.7
19.8
14.7-24.4
26.3
18.3-31.8
25.4
19.0-28.3
Phylogenetic inference. In our studies, we found that the 12S and 16S rDNA sequences
of pentatomomorphan bugs contained nucleotide composition and substitution biases,
which may restrict their phylogenetic utility. To assess the usefulness of these rDNA
fragments for phylogenetic inference, the obtained data sets (Figs 1 & 2) were analyzed
using several widely accepted methods, i.e., maximum parsimony (MP), minimum
evolution (ME) and neighbor-joining (NJ) analyses (Figs 3-4). Moreover, in order to
avoid the possible effect of gene duplication and loss events on our inferences, the
phylogenetic trees of Pentatomomorpha were also calculated using the Bayesian
estimation (Fig. 5). In general, the nucleotide sequences of 12S and 16S rDNA
fragments seem to be a good marker to resolve the phylogeny of pentatomomorphan
taxa, e.g., even closely related genera (Figs 3-5), what was also confirmed for the 16S
rDNA in Anthocoridae (Muraji et al. 2000). The taxa in various families and
superfamilies always formed distinct clades regardless the estimation method (Figs 3-5).
As the most important, only the topology of the phylogenetic tree obtained from the
Bayesian inference analysis of the combined 12S and 16S rDNA dataset agreed
completely with conventional classification (Table 1); the topologies of other trees only
partially correspond to it. Moreover, our results are very similar to the results of the
studies on complete mitochondrial genomes in Pentatomomorpha (Hua et al. 2008).
Conclusions
This was the first study to examine the nucleotide composition, substitution patterns,
nucleotide divergence of the 12S and the 16S mitochondrial rDNA fragments, and their
usefulness for phylogenetic inference in pentatomomorphan bugs. Our results suggest
that in Pentatomomorpha:
(1) the 16S rDNA sequences are more conserved than the 12S rDNA sequences;
83
(2) the average A + T contents in studied species are as high as in other previously
investigated Heteroptera (above 75%), except for the Aradidae where the A + T
content for 12S and 16S rDNA fragments are below 70%;
(3) the A/G and T/C transition biases found in both rDNA fragments agree with the
observations suggesting that the transitional substitutions occur more readily
than transversional substitutions;
(4) the A/T transversion frequency in the 16S rDNA is distinctly lower (18.78%)
than that found in the cimicomorphan Anthocoridae (54.52%);
(5) the undirectional nucleotide pair frequencies, in 12S rDNA and 16S rDNA
fragments increase linearly with an increase in taxonomic level;
(6) the nucleotide sequences of 12S and 16S rDNA fragments can be regarded as a
good marker to resolve the phylogeny of closely related genera;
(7) the Bayesian inference analysis of the combined 12S/16S rDNA dataset appears
to be the most appropriate method for phylogenetic reconstructions when the
mitochondrial ribosomal RNA genes are used.
Acknowledgements
We thank M. Biskupek (Department of Biosystematics, Opole University) for her
technical assistance during our studies. This project was supported by the Opole
University grants (1/KBI/10-S and 1/KBI/11-S).
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Figures
Fig. 1. Nucleotide sequences of 12S rDNA in species used in the study. Nucleotide identical to the reference, Neuroctenus parus, are
indicated by a dot. Dashes indicate deletions
88
Fig. 2. Nucleotide sequences of 16S rDNA in species used in the study. Nucleotide identical to the reference, Neuroctenus parus, are
indicated by a dot. Dashes indicate deletions
89
Fig. 3. Phylogenetic trees for 12S rDNA sequences of 21 species used in this study
generated by: A – neighbor-joining (NJ) analysis; B – minimum evolution (ME) analysis;
C-E – maximum parsimony (MP) analysis (C-D – two most parsimonious trees, E –
consensus tree). The percentage of replicate trees (A-D) and parsimonious trees (E) in
which the associated taxa clustered together are shown next to the branches
90
Fig. 4. Phylogenetic trees for 16S rDNA sequences of 21 species used in this study generated by: A – neighbor-joining (NJ) analysis; B –
minimum evolution (ME) analysis; C – maximum parsimony (MP) analysis. The percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (500 replicates) are shown next to the branches
91
Fig. 5. Phylogenetic trees obtained from the Bayesian inference analyses of the 12S rDNA
sequences (A), 16S rDNA sequences (B), and combined 12S/16S rDNA dataset (C) of 21 species
used in this study. The Bayesian posterior probabilities are indicated at each node