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DNA barcoding of sunn pest adult parasitoids using cytochrome c oxidase subunit I (COI)
Biochemical Systematics and Ecology 59 (2015) 70e77
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
DNA barcoding of sunn pest adult parasitoids using cytochrome c oxidase subunit I (COI)* Mehmet Duman a, Nurper Guz b, *, Erdal Sertkaya c a
Diyarbakir Plant Protection Research Station, Diyarbakir, Turkey Department of Plant Protection, Faculty of Agriculture, Insect Molecular Biology Laboratory, Ankara University, 06110, Diskapi, Ankara, Turkey c Department of Plant Protection, Faculty of Agriculture, Mustafa Kemal University, 31030, Hatay, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 September 2014 Accepted 2 January 2015 Available online
In this study, DNA barcoding was used in the identification of potential biological control agents of sunn pest adult parasitoid species, including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig). DNA analyses were assessed by sequencing cytochrome c oxidase subunit I (COI) gene. The obtained sequences were analyzed in terms of nucleotide composition, nucleotide pair frequency and haplotype diversity. Genetic divergence among haplotypes was estimated by constructing genetic distance matrix using DNA sequence variations, by Kimura 2-parameter model. Variable sites and average variations of the sequenced 603 base pair long DNA fragment were calculated. All COI barcodes were matched with reference sequences of expected species according to morphological identification. Neighbor-joining tree was drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. The evolutionary history inferred using the UPGMA method indicated two distinct mitochondrial haplotype lineages. The genetic variation between sunn pest adult parasitoids will be useful in sunn pest management, regulatory and environmental applications. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Adult parasitoids Cytochrome c oxidase subunit I DNA barcoding Sunn pest
1. Introduction The wide range of insecticides used to control insect pests has given rise to problems associated with the disruption of ecological balance, the useful insects, and human health. In addition to their high cost resistance has developed to various types of insecticides. Recent developments in genetics, systematic, population dynamics, and pesticide chemistry have led to inclusion of beneficial insects into Integrated Pest Management programs. The accurate identification of natural enemies and also the knowledge of the genetic variation in insect populations are critical for the success of control management strategies. Recently developed genetic markers make it possible to recognize a number of cryptic species and divergent evolutionary lineages. Moreover, they enable us to evaluate the levels of gene flow among populations in different geographic areas. Sunn pest is a major constraint to the production of wheat in several areas of the near and middle east, west and central Asia, North Africa, eastern and southern Europe (Brown, 1962; Critchley, 1998; Parker et al., 2002). Yield loss is commonly
*
Preliminary data of this study has been published as an abstract in Fifth Plant Protection Congress of Turkey. * Corresponding author. Tel.: þ90 312 596 1524; fax: þ90 312 318 7029. E-mail address: [email protected] (N. Guz).
http://dx.doi.org/10.1016/j.bse.2015.01.003 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77
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estimated at 20e30% in barley and 50e90% in wheat (Fatehi et al., 2009), and the damage can result in total crop loss without the use of proper control strategies (http://www.fao.org). Feeding activity by the sunn pest damages leaves, stems and grain heads. During feeding, the bug also injects saliva into the grain, causing protein denaturation due to the saliva's hydrolytic enzymes and this, in turn, greatly reduces the baking quality of the dough (Hosseininaveh et al., 2009). The current management strategy for this pest mainly relies on intensive use of insecticides which pose a risk to the balance of nature and resistance has developed to various types of insecticides (Critchley, 1998; Bandani et al., 2005; Sukhoruchenko and Dolzhenko, 2008). One component of IPM that is receiving increasing attention is the use of biological control agents in order to reduce reliance on pesticides. Several species of indigenous egg and adult parasitoids of sunn pest have already been identified (Abdulhai et al., 2007; Al-Izzi et al., 2007; Trissi et al., 2007). Identification of adult parasitoid species and their parasitization efficiency have been well studied (Memisoglu and Ozer, 1994; Islamoglu and Kornosor, 2003, 2007; Kececi et al., 2007; Gozuacik et al., 2010). Parasitization efficiency of adult parasitoids is usually determined using hatched adults however the aborted ones are ignored. Due to the great differences between the parasitized values it is difficult to estimate the actual parasitization efficiencies. Therefore, accurate identification of natural enemies becomes important in order to achieve a successful biological control program. Recently data acquired with molecular identification techniques provides vital information that must be obtained before a potential natural enemy is released to control a pest (Bigler et al., 2005). In this context, DNA barcoding is an efficient tool for many needs in biological control, such as linking unknown larvae with adults, associating males and females of the same species, identifying parasitoid species in their hosts and making rapid taxonomic identifications. This research attempts to provide information about the most common sunn pest adult parasitoid species including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig) using cytochrome oxidase I gene and also provide molecular markers that may be used for simple and rapid species identification. 2. Material and methods Information about the collection sites and geographical coordinates of different sunn pest adult parasitoids are listed in Table 1. The voucher specimens were deposited in Diyarbakir Plant Protection Research Station, Diyarbakir, Turkey. Parasitized sunn pest adults were brought to the laboratory and the parasitoid larvae were dissected under a microscope. Specimens were morphologically identified by Mikdat Doganlar with recent taxonomic keys. Both parasitoid adults and larvae were stored in 95% ethanol at 20 C until DNA extraction. DNA was extracted from adult parasitoids using Qiagen DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer's instructions. All DNA samples were electrophoresed in 0.7% agarose gel and visualized under UV transluminator. DNA concentrations were standardized to 50 ng/ml and stored at 20 C until PCR analysis. 603 bp COI fragment was amplified using universal HCO-LCO primer pairs (Folmer et al., 1994). PCR reactions were performed in total volumes of 50 ml by using 1 ml of DNA template and GoTaq Flexi DNA polymerase (Promega) according to manufacturer's instructions: 5x buffer, 10 mM of each dNTP, 10 mM of each primer and 1.25 u/ml DNA polymerase. PCR thermal cycling conditions consisted of an initial denaturation at 95 C for 2 min, followed by 30 cycles of 95 C for 30 s, 52 C for 45 s and 72 C for 1 min, followed by an extension at 72 C for 10 min. All the PCR products were visualized on 2% agarose gel to confirm the band corresponding to amplification product and purified with Wizard SV Gel and PCR Clean up System (Promega). The purified PCR fragments were cloned into PGEM T vector systems. Sequencing reactions were performed with DTCS Quick Start Kit (Beckman Coulter),
Table 1 Information about the collection sites, geographical coordinates and accession numbers of sunn pest adult parasitoids. Species
Specimens
Collection sites
Co-ordinates
Accession no.
Ectophasia crassipennis (F.) Elomyia lateralis (Meig) Eliozeta helluo (F.)
E. crassipennis E. lateralis E. helluo Hap 1 Hap 2 Hap 3 Hap 4 Hap 5 Hap 6 Hap 7 Hap 8 Hap 9 P. subcoleoptrata Hap 10 Hap 11 Hap 12 Hap 13 Hap 14 Hap 15 Hap 16
Siverek, Sanliurfa, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey
37736033N 38154335N 38154335N 37736033N 37736033N 38154335N 38154335N 38154335N 38154335N 38154335N 38090808N 37736033N 37736033N 37717415N 37729358N 37717415N 37717415N 37717415N 37729358N 37717415N
KM233126 KM233125 KM233127 KM233128 KM233129 KM233130 KM233131 KM233132 KM233133 KM233134 KM233135 KM233136 KM233137 KM233138 KM233139 KM233140 KM233141 KM233142 KM233143 KM233144
Eliozeta helluo (F.)
Phasia subcoleoptrata (L.)
Phasia subcoleoptrata (L.)
39832819E (1835 m) 39823670E (802 m) 39823670E (802 m) 39832819E (1835 m) 39832819E (1835 m) 39823670E (802 m) 39823670E (802 m) 39823670E (802 m) 39823670E (802 m) 39823670E (802 m) 395021869E (778 m) 39832819E (1835 m) 39832819E (1835 m) 39834492E (1876 m) 39832369E (1846 m) 39834492E (1876 m) 39834492E (1876 m) 39834492E (1876 m) 39832369E (1846 m) 39834492E (1876 m)
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cleaned with Agencourt CleanSeq Kit (Agencourt Bioscience) and analyzed with the GenomeLab GeXP Genetic Analysis System (Beckman Coulter). Electropherograms of all sequences were assembled into contigs and proofread manually using the program Chromas, v.1.41. The DNA and deduced amino acid sequences were analyzed using the BLAST tool at the National Center for Biotechnology Information (NCBI) (www.ncbi.nih.gov/BLAST), the BOLD Identification System (http://www.boldsystems.org) and EXPASY (http://expasy.org). Sequence alignments were performed using the CLUSTALW v.1.82 software (Thompson et al., 1994). Conserved residues in the alignments were highlighted with Boxshade 3.21 (http://www.ch.embnet.org/software/ BOXform.html). All sequences have been deposited in the GenBank database. Models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the substitution pattern the best. For each model, AICc value (Akaike Information Criterion, corrected), Maximum Likelihood value (lnL), and the number of parameters (including branch lengths), are also calculated (Nei and Kumar, 2000). Analysis of variable parsimony informative sites, frequencies of transitional pairs, transversional pairs and R values, the average number of identical pairs, the number of transitions to the number of transversions for a pair of sequences were calculated using MEGA 6 software. The number of base substitutions per site from averaging over all sequence pairs and from averaging over all sequence pairs within each group is calculated using the Maximum Composite Likelihood model (Tamura et al., 2004). Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). The number of base substitutions per site from between sequences was conducted using the Maximum Composite Likelihood model (Tamura et al., 2004). Population differentiation and analysis of molecular variance (AMOVA) were conducted to identify number of haplotypes and fixation index (FST) using Arlequin 3.5.1.2 (Excoffier et al., 2005). Haplotype diversity was calculated using DnaSP vers. 3.5.1.2 (Excoffier and Lischer, 2010). Substitution pattern and rates were estimated under TamuraeNei model. Substitution model and rates of estimated transition/transversion bias (R) and estimates of evolutionary divergences between sequences were estimated under Kimura 2-parameter model. The evolutionary history of parasitoid haplotypes was inferred using the UPGMA method (Sneath and Sokal, 1973). Phylogenetic tree of adult parasitoids were constructed using the Neighbor-Joining (NJ) method (Saitou and Nei, 1987) implemented in the MEGA 6 program. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Estimates of Evolutionary Divergence between Sequences were conducted using the Maximum Composite Likelihood model (Tamura et al., 2004). The analysis involved 20 nucleotide sequences. Codon positions included were 1st þ 2nd þ 3rd þ Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 603 positions in the final dataset. Evolutionary analyses were conducted in MEGA 6 (Tamura et al., 2013).
3. Results and discussion Four adult parasitoids including E. helluo, P. subcoleoptrata, E. crassipennis and E. lateralis and sixteen parasitoid larvae were analyzed in terms of mtCOI gene sequences. The deduced sequences of all parasitoid species share >99% similarity to the same species of representative COI sequences in BOLD database. Among 20 specimens, 16 haplotypes were detected belonging to parasitoid larvae while E. helluo haplotypes from Diyarbakir province were identical (Hap3eHap4). All sequences were deposited under accessions KM233125eKM233144 in GenBank; GenBank accession numbers are detailed in Table 1. Conserved residues in the sequence alignments are highlighted with Boxshade 3.21 shown in Fig. 1. No insertions, deletions or stop codons were found indicating that all sequences correspond to a functional mitochondrial gene (Funk and Omland, 2003) and also indicating the absence of pseudogenes or nuclear copies of mitochondrial origin (NUMTs). Indels and nonsense/stop codons were not detected as well. The percentage of nucleotide composition at each codon position were found variable (Table 2). The mean frequency of COI sequences used in the analyses showed a bias of A þ T (T 38.7%, C 15.4%, A 30.9% and G 15.0%). The A þ T content at the third, second and first codon positions are 94.4%, 58%, and 56.3%, respectively (Table 2). First and second were less A þ T biased than the third codon position. The base frequencies of COI show a high adenine (A) and thymine (T) bias, with a mean rate of 69.6%, which is typical of insect mitochondrial sequences (Crozier and Crozier, 1993). The nucleotide G has the lowest (15%) and the A has the highest content (38.7%) in terms of first codon positions. Variation in base composition is important in the modeling of sequence evolution since the mtDNA loci verify the general observation from insects and especially Hymenopterans of a strong AeT bias (Crozier and Crozier, 1993; Dowton and Austin, 1995; Whitfield and Cameron, 1998). The mtDNA locus of sunn pest adult parasitoid species is AeT rich which agree with the cytochrome oxidase I data of Dowton and Austin (1995), showing a higher AeT content in parasitic wasps than in nonparasitic wasps. Maximum likelihood suitability of 24 nucleotide substitution models was analyzed. Assumed or estimated values of transition/transversion bias were also calculated for each model. For estimating ML values, a tree topology was automatically computed using MEGA 6 (Tamura et al., 2013). TamuraeNei model, with the lowest AICc score of 3397.361 was the best to describe the substitution pattern. The data set of COI alignment contains 603 nucleotide positions, of which 114 positions are variable and 72 are parsimony informative. The average number of identical pairs (ii) was 192 out of 603 base pairs. Transversional pairs (sv ¼ 18) were
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77
Fig. 1. Multiple alignment of COI nucleotide sequences from the following: E. lateralis (GenBank Accession: KM233125); P. subcoleoptrata (KM233137); E. crassipennis (KM233126); E. helluo (KM233127). The output for multiple sequence alignment is based on a Clustal W consensus sequence and sequences were shaded using the BoxShade program.
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Table 2 Table of nucleotide composition at all sites and three codon positions. Specimen
All sites
Codon position 1
T
C
A
G
T-1
C-1
A-1
G-1
E. crassipennis E. helluo E. lateralis P. subcoleoptrata E. helluo (means of Hap 1e9) P. subcoleptrata (means of Hap 10e16) Average
38.6 38.0 38.5 39.5 38.1 39.5 38.7
15.1 16.1 14.4 15.1 15.8 15.0 15.4
31.0 31.2 31.3 30.5 31.3 30.3 30.9
15.3 14.8 15.8 14.9 14.8 15.1 15.0
27 26 27 28 26 28 27
15.4 16.4 14.4 14.9 16.3 14.6 15.5
28.4 29.4 28.9 29.4 29.9 28.8 29.3
29.4 27.9 29.4 27.9 27.6 28.6 28.2
Specimen
Codon position 2
E. crassipennis E. helluo E. lateralis P. subcoleoptrata E. helluo (means of Hap 1e9) P. subcoleptrata (means of Hap 10e16) Average
Codon position 3
T-2
C-2
A-2
G-2
T-3
C-3
A-3
G-3
43 43 43 43 43 43 43
25.9 25.9 25.9 25.9 25.9 25.8 25.9
14.9 14.9 14.9 14.9 15.0 14.9 15.0
15.9 15.9 15.9 15.9 16.0 15.9 15.9
46 44 45 47 45 47 46
4.0 6.0 3.0 4.5 5.2 4.8 4.9
49.8 49.3 50.2 47.3 48.9 47.3 48.4
0.5 0.5 2.0 1.0 9.0 0.8 0.9
found to be higher than transitional pairs (si ¼ 7). The number of transitions to the number of transversions for a pair of sequences (si/sv ¼ R) was 2.86 for the data set. The number of base substitutions per site from averaging over all sequence is 7.0% (SE ¼ 0.9%), whereas the mean genetic distance between Eliozeta helluo haplotypes and Phasia subcoleoptrata haplotypes was 1.3% and 0.9%, respectively (SE ¼ 0.3 and 0.2). Highest genetic distance was calculated between E. helluo-P. subcoleoptrata (0.327) and the lowest genetic distance was between E. helluo and E. crassipennis (0.012). The distance matrix for all analyzed species is presented in Table 3. Fixation index (FST) is one of the most widely-used measures of population differentiation for estimating and testing the magnitude of genetic divergence (Wright, 1943, 1951, 1965). FST, ranges from 0.0, indicates identical allele frequencies in a pair of populations (no differentiation), to 1, which indicates alternate fixation for a single unique allele in each population. The values between 0.0 and 1.0 show the magnitude of genetic differentiation among samples. In this study, overall FST of the Eliozeta helluo haplotypes was calculated as 0.036, while no significant pair-wise differences of Fst values were observed among Phasia subcoleoptrata haplotypes. A low genetic differentiation between E. helluo haplotypes among collection sites in Diyarbakir and Siverek, which might indicate that some combination of drift and reduced gene flow might be operating enough to generate significant genetic variation among regions. Maximum likelihood estimation of substitution pattern and rates of matrix were estimated under the Tamura-Nei (1993) model (Table 4). Substitution rates represent the probability of substitution (r) from one base (row) to another (column). Rates of different transitional substitutions are shown in bold and those of transversional substitutions are shown in italics in Table 4. Transitional substitution rates of A/G, T/C, C/T and G/A were 4.85%, 11.76%, 29.52% and 10.00%, respectively. Transversional rates are given in Table 4. The estimated transition/transversion bias (R) was 1.13 which is important not only to understand the patterns of DNA sequence evolution, but also for a reliable estimation of sequence distance and phylogeny reconstruction. The estimated transition to transversion ratio was higher than those observed in sunn pest egg parasitoid species (Guz et al., 2013). The nucleotide frequencies are 30.90% (A), 38.70% (T), 15.41% (C), and 14.99% (G). The evolutionary history of sunn pest adult parasitoid species and haplotypes was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and UPGMA method (Sneath and Sokal, 1973). The UPGMA method indicates that two distinct mitochondrial haplotype lineages within the sampled populations. Nine haplotypes (Hap 1e9) were recorded in specimens determined to be Eliozeta helluo and seven haplotypes (Hap 10e16) were recorded in specimens determined to be Phasia subcoleoptrata (Fig. 2a). The haplotype diversity was calculated as 0.992. The percentage of replicate NJ trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). Clustering patterns of the consensus tree (Fig. 2b), with the sum of branch length (1.398) pointed out a correlation between genetic distances. Sunn pest egg parasitoid species (Hymenoptara: Scelionidae) inferred by Guz et al. (2013) were chosen as out groups. According to the phylogenetic tree based on COI gene, Hymenopteran sunn pest parasitoids were separated in two as egg parasitoid species (Scelionidae) clustered together in one clade and adult parasitoid species (Tachinidae) clustered in the second clade. Adult parasitoid species were also separated in two including E. helluo, Ectophasia crassipennis and Elomyia lateralis in one clade and P. subcoleoptrata clustered in a separate clade. Genetic analyses of sunn pest adult parasitoid populations showed that the COI gene, known as the DNA barcode, successfully distinguishes genetic variation among parasitoid populations sampled from different regions and provide a valuable resource as a molecular marker. The analyzed partial COI gene region showed conserved and variable regions that provide information for identifications at species level and for revealing intraspecific variation among parasitoid populations. The data obtained from this study do not only provide molecular data to identify sunn pest adult parasitoid species but also will serve as a useful tool for detecting accurate parasitism rate of parasitoids which is important in biological control studies. It
1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
E. lateralis E. crassipennis E. helluo P. subcoleoptrata Hap 1 Hap 2 Hap 3 Hap 4 Hap 5 Hap 6 Hap 7 Hap 8 Hap 9 Hap 10 Hap 11 Hap 12 Hap 13 Hap 14 Hap 15 Hap 16
2 0.016
0.094 0.096 0.087 0.101 0.096 0.099 0.099 0.110 0.097 0.109 0.099 0.099 0.084 0.083 0.087 0.085 0.087 0.085 0.093
0.060 0.096 0.072 0.067 0.071 0.071 00.77 0.067 0.078 0.069 0.069 0.092 0.092 0.096 0.094 0.096 0.094 0.097
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0.015 0.011
0.014 0.017 0.018
0.016 0.012 0.004 0.018
0.015 0.012 0.004 0.018 0.003
0.016 0.012 0.004 0.018 0.004 0.004
0.016 0.012 0.004 0.018 0.004 0.004 0.000
0.017 0.013 0.005 0.019 0.005 0.005 0.005 0.005
0.016 0.012 0.004 0.018 0.004 0.004 0.004 0.004 0.005
0.017 0.013 0.005 0.019 0.006 0.006 0.005 0.005 0.006 0.004
0.016 0.012 0.004 0.018 0.004 0.004 0.004 0.004 0.005 0.002 0.004
0.015 0.012 0.005 0.018 0.005 0.005 0.005 0.005 0.006 0.003 0.005 0.002
0.014 0.016 0.018 0.004 0.018 0.018 0.018 0.018 0.019 0.018 0.018 0.018 0.018
0.014 0.016 0.018 0.002 0.018 0.018 0.018 0.018 0.019 0.018 0.018 0.018 0.017 0.003
0.014 0.016 0.018 0.004 0.018 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.003 0.003
0.014 0.016 0.018 0.003 0.019 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.003 0.002 0.004
0.014 0.017 0.018 0.003 0.018 0.018 0.018 0.018 0.018 0.018 0.019 0.018 0.018 0.004 0.003 0.004 0.004
0.014 0.016 0.018 0.003 0.018 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.004 0.003 0.004 0.003 0.004
0.015 0.017 0.018 0.005 0.018 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.006 0.005 0.006 0.005 0.005 0.004
0.118 0.012 0.010 0.010 0.010 0.019 0.010 0.020 0.012 0.015 0.114 0.114 0.118 0.115 0.118 0.116 0.117
0.116 0.114 0.116 0.116 0.120 0.112 0.120 0.114 0.114 0.010 0.003 0.010 0.005 0.007 0.005 0.013
0.008 0.012 0.012 0.013 0.012 0.022 0.013 0.017 0.111 0.116 0.116 0.117 0.112 0.114 0.115
0.010 0.010 0.019 0.010 0.020 0.012 0.015 0.110 0.110 0.114 0.112 0.114 0.112 0.113
0.000 0.015 0.010 0.020 0.012 0.015 0.112 0.112 0.116 0.114 0.112 0.114 0.115
0.015 0.010 0.020 0.012 0.015 0.112 0.112 0.116 0.114 0.112 0.114 0.115
0.015 0.020 0.017 0.020 0.116 0.120 0.120 0.122 0.112 0.118 0.120
0.010 0.002 0.005 0.108 0.108 0.112 0.110 0.112 0.110 0.111
0.012 0.015 0.116 0.116 0.120 0.118 0.120 0.118 0.119
0.003 0.110 0.110 0.114 0.112 0.114 0.112 0.113
0.110 0.110 0.114 0.112 0.114 0.112 0.113
0.007 0.007 0.005 0.010 0.012 0.020
0.007 0.002 0.007 0.005 0.013
0.008 0.010 0.012 0.020
0.008 0.007 0.015
0.008 0.017
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77
Table 3 The number of base substitutions per site from between sequences is shown. Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). Analyses were conducted using the Maximum Composite Likelihood model. Codon positions included were 1st þ 2nd þ 3rd þ Noncoding. All ambiguous positions were removed for each sequence pair.
0.012
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M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77 Table 4 Each entry is the probability of substitution (r) from one base (row) to another base (column). Substitution pattern and rates were estimated under the TamuraeNei (1993) model (Tamura and Nei, 1993). Rates of different transitional substitutions are shown in bold and those of transversionsal substitutions are shown in italics. Relative values of instantaneous r should be considered when evaluating them. For simplicity, sum of r values is made equal to 100. The nucleotide frequencies are A ¼ 30.90%, T/U ¼ 38.70%, C ¼ 15.41%, and G ¼ 14.99%. For estimating ML values, a tree topology was automatically computed. The maximum Log likelihood for this computation was 1661.145.
A T/U C G
A
T/U
C
G
e 6.78 5.37 10.00
8.9 e 26.03 8.49
3.38 11.76 e 3.38
4.85 3.29 2.6 e
Fig. 2. Evolutionary relationships of taxa. The evolutionary history of species and haplotypes was inferred using the UPGMA (2A) and Neighbor-Joining (2B) methods. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.
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might be useful to address a broader sampling between geographically different populations in order to get better insight to the parasitoid population dynamics. Acknowledgment We are grateful to Mikdat Doganlar (Mustafa Kemal University) for the morphological identifications of sunn pest adult parasitoid species. References Abdulhai, M., Canhilal, R., El Bouhssini, M., Reid, W., Rihawi, F., 2007. Survey of sunn pest adult parasitoids in Syria. In: Parker, B.L., Skinner, M., El Bouhssini, M., Kumari, S.G. (Eds.), Sunn Pest Management: a Decade of Progress 1994-2004, pp. 315e318. Al-Izzi, M.A.J., Amin, A.M., Al-Assaid, H.S., 2007. Role of biocontrol agents in decreasing population of Sunn Pest in northern Iran. In: Parker, B.L., Skinner, M., EL- Bouhssini, M., Kumair, S.G. (Eds.), Sunn Pest Management, a Decade of Progress. 1994-2004, pp. 265e272. ICARDA, Aleppo, Syria, Arab Society for plant protection. Bandani, A.R., Alizadeh, M., Talebi, K., 2005. Toxicity of fenitrothion, an organophosphorus pesticides summer population of sunn pest, Eurygaster integriceps Put. (Hemiptera: Scutelleridae). Commun. Agric. Appl. Biol. Sci. 70, 775e777. Bigler, F., Bale, J.S., Cock, M.J.W., Dreyer, H., Greatrex, R., Kuhlmann, U., Loomans, A.J.M., van Lenteren, J.C., 2005. Guidelines on information requirements for import and release of invertebrate biological control agents in European countries. Biocontrol News Inf. 26, 115Ne123N. Brown, E.S., 1962. Notes on parasites of Pentatomidae and Scutelleridae (Hemiptera- Heteroptera) in Middle East countries, with observations on biological control. Bull. Entomol. Res. 53, 241e256. Critchley, B.R., 1998. Literature review of sunn pest Eurygaster integriceps Put. (Hemiptera, Scutelleridae). Crop Prot. 17, 271e287. Crozier, R.H., Crozier, Y.C., 1993. The mitochondrial genome of the honeybee Apis mellifera: complete sequence and genome organization. Genetics 133, 97e117. Dowton, M., Austin, A.D., 1995. Increased genetic diversity in mitochondrial genesis correlated with the evolution of parasitism in the Hymenoptera. J. Mol. Evol. 41, 958e965. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin version 3.01: an integrated software package for population genetics data analysis. Evol. Bioinform Online 1, 47e50. Excoffier, L., Lischer, H.E.L., 2010. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol. Ecol. Resour. 10, 564e567. Fatehi, F., Behamta, M.R., Zali, A.A., 2009. Evaluating the resistance to sunn pest (Eurygaster integriceps Put) and its relationship with high-molecular-weight glutenin subunit in wheat. Asian J. Plant Sci. 8, 82e85. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783e791. Funk, D.J., Omland, K.E., 2003. Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu Rev. Ecol. Syst. 34, 397e423. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Bio Biotechnol. 3, 294e299. Gozuacik, C., Kara, K., Karaca, V., Duman, M., Mutlu, C., Melan, K., 2010. Adult parasitoids of sunn pest, Eurygaster integriceps Put. (Het.: Scutelleridae) and their effectiveness in the Southeast Anatolia. J. Agric. Fac. HRU 14, 1e8 (in Turkish with English abstract). Guz, N., Kocak, E., Kilincer, N., 2013. Molecular phylogeny of Trissolcus species (Hymenoptera: Scelionidae). Biochem. Syst. Ecol. 48, 85e91. Hosseininaveh, V., Bandani, A.R., Hosseininaveh, F., 2009. Digestive proteolytic activity in the Sunn pest, Eurygaster integriceps. J. Insect Sci. 9, 70. Islamoglu, M., Kornosor, S., 2003. Investigations on the adult parasitoids (Diptera, Tachinidae) of the Sunn Pest in overwintering site and wheat fields in Gaziantep and Kilis. Plant Prot. Bull. 43, 99e110 (in Turkish with English abstract). Islamoglu, M., Kornosor, S., 2007. Investigations on the adult parasitoids (Diptera, Tachinidae) of the sunn pest in overwintering site and wheat fields in Kahramanmaras. Suleyman Demirel University J. Fac. Agric. 2, 53e61 (in Turkish with English abstract). Kececi, M., Teksam, I., Topuz, E., Oztop, A., 2007. Determination of adult parasitoid species (Dip.:Tachinidae) of Sunn Pests (Eurygaster integriceps Put.) (Het.: Scutelleridae) and their parasitoid ratios in Antalya and Burdur provinces. In: Second Plant Protection Congress of Turkey, p. 174 (in Turkish with English summary). Memisoglu, H., Ozer, M., 1994. Natural enemies and their activities of sunn pest (Eurygaster maura L., Hemiptera: Scutelleridae) in Ankara. In: The Third Turkish National Congress of Biological Control, pp. 175e186 (in Turkish with English summary). Nei, M., Kumar, S., 2000. Molecular Evolution and Phylogenetics. Oxford University Press, New York. Parker, B.L., Costa, S.D., Skinner, M., Bouhssini, M.E., 2002. Sampling sunn pest (Eurygaster integriceps Puton) in overwintering sites in northern Syria. Turk. J. Agr. For. 26, 109e117. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406e425. Sneath, P.H.A., Sokal, R.R., 1973. Numerical Taxonomy. Freeman, San Francisco. Sukhoruchenko, G.I., Dolzhenko, V.I., 2008. Problems of resistance development in arthropod pests of agricultural crops in Russia. EPPO Bull. 38, 119e126. Tamura, K., Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10, 512e526. Tamura, K., Nei, M., Kumar, S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. PNAS 101, 11030e11035. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725e2729. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673e4780. Trissi, A.N., El-Bouhssini, M., Ibrahem, J., Abdulhai, M., Reid, W., 2007. Survey of egg parasitoids of sunn pest in Northern Syria. In: Parker, B.L., Skinner, M., El-Bouhssini, M., Kumari, S.G. (Eds.), Sunn Pest Management: a Decade of Progress 1994e2004. Arab Society for Plant Protection, Beirut, Lebanon, pp. 309e314, 432 pp. Whitfield, J.B., Cameron, S.A., 1998. Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera. Mol. Biol. Evol. 15, 1728e1743. Wright, S., 1943. Isolation by distance. Genetics 28, 114e138. Wright, S., 1965. The interpretation of population structure by F-statistics with special regard to systems of mating. Evolution 19, 395e420. Wright, S., 1951. The genetical structure of populations. Ann. Eugen. 15, 323e353.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
DNA barcoding of sunn pest adult parasitoids using cytochrome c oxidase subunit I (COI)* Mehmet Duman a, Nurper Guz b, *, Erdal Sertkaya c a
Diyarbakir Plant Protection Research Station, Diyarbakir, Turkey Department of Plant Protection, Faculty of Agriculture, Insect Molecular Biology Laboratory, Ankara University, 06110, Diskapi, Ankara, Turkey c Department of Plant Protection, Faculty of Agriculture, Mustafa Kemal University, 31030, Hatay, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 September 2014 Accepted 2 January 2015 Available online
In this study, DNA barcoding was used in the identification of potential biological control agents of sunn pest adult parasitoid species, including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig). DNA analyses were assessed by sequencing cytochrome c oxidase subunit I (COI) gene. The obtained sequences were analyzed in terms of nucleotide composition, nucleotide pair frequency and haplotype diversity. Genetic divergence among haplotypes was estimated by constructing genetic distance matrix using DNA sequence variations, by Kimura 2-parameter model. Variable sites and average variations of the sequenced 603 base pair long DNA fragment were calculated. All COI barcodes were matched with reference sequences of expected species according to morphological identification. Neighbor-joining tree was drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. The evolutionary history inferred using the UPGMA method indicated two distinct mitochondrial haplotype lineages. The genetic variation between sunn pest adult parasitoids will be useful in sunn pest management, regulatory and environmental applications. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Adult parasitoids Cytochrome c oxidase subunit I DNA barcoding Sunn pest
1. Introduction The wide range of insecticides used to control insect pests has given rise to problems associated with the disruption of ecological balance, the useful insects, and human health. In addition to their high cost resistance has developed to various types of insecticides. Recent developments in genetics, systematic, population dynamics, and pesticide chemistry have led to inclusion of beneficial insects into Integrated Pest Management programs. The accurate identification of natural enemies and also the knowledge of the genetic variation in insect populations are critical for the success of control management strategies. Recently developed genetic markers make it possible to recognize a number of cryptic species and divergent evolutionary lineages. Moreover, they enable us to evaluate the levels of gene flow among populations in different geographic areas. Sunn pest is a major constraint to the production of wheat in several areas of the near and middle east, west and central Asia, North Africa, eastern and southern Europe (Brown, 1962; Critchley, 1998; Parker et al., 2002). Yield loss is commonly
*
Preliminary data of this study has been published as an abstract in Fifth Plant Protection Congress of Turkey. * Corresponding author. Tel.: þ90 312 596 1524; fax: þ90 312 318 7029. E-mail address: [email protected] (N. Guz).
http://dx.doi.org/10.1016/j.bse.2015.01.003 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77
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estimated at 20e30% in barley and 50e90% in wheat (Fatehi et al., 2009), and the damage can result in total crop loss without the use of proper control strategies (http://www.fao.org). Feeding activity by the sunn pest damages leaves, stems and grain heads. During feeding, the bug also injects saliva into the grain, causing protein denaturation due to the saliva's hydrolytic enzymes and this, in turn, greatly reduces the baking quality of the dough (Hosseininaveh et al., 2009). The current management strategy for this pest mainly relies on intensive use of insecticides which pose a risk to the balance of nature and resistance has developed to various types of insecticides (Critchley, 1998; Bandani et al., 2005; Sukhoruchenko and Dolzhenko, 2008). One component of IPM that is receiving increasing attention is the use of biological control agents in order to reduce reliance on pesticides. Several species of indigenous egg and adult parasitoids of sunn pest have already been identified (Abdulhai et al., 2007; Al-Izzi et al., 2007; Trissi et al., 2007). Identification of adult parasitoid species and their parasitization efficiency have been well studied (Memisoglu and Ozer, 1994; Islamoglu and Kornosor, 2003, 2007; Kececi et al., 2007; Gozuacik et al., 2010). Parasitization efficiency of adult parasitoids is usually determined using hatched adults however the aborted ones are ignored. Due to the great differences between the parasitized values it is difficult to estimate the actual parasitization efficiencies. Therefore, accurate identification of natural enemies becomes important in order to achieve a successful biological control program. Recently data acquired with molecular identification techniques provides vital information that must be obtained before a potential natural enemy is released to control a pest (Bigler et al., 2005). In this context, DNA barcoding is an efficient tool for many needs in biological control, such as linking unknown larvae with adults, associating males and females of the same species, identifying parasitoid species in their hosts and making rapid taxonomic identifications. This research attempts to provide information about the most common sunn pest adult parasitoid species including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig) using cytochrome oxidase I gene and also provide molecular markers that may be used for simple and rapid species identification. 2. Material and methods Information about the collection sites and geographical coordinates of different sunn pest adult parasitoids are listed in Table 1. The voucher specimens were deposited in Diyarbakir Plant Protection Research Station, Diyarbakir, Turkey. Parasitized sunn pest adults were brought to the laboratory and the parasitoid larvae were dissected under a microscope. Specimens were morphologically identified by Mikdat Doganlar with recent taxonomic keys. Both parasitoid adults and larvae were stored in 95% ethanol at 20 C until DNA extraction. DNA was extracted from adult parasitoids using Qiagen DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer's instructions. All DNA samples were electrophoresed in 0.7% agarose gel and visualized under UV transluminator. DNA concentrations were standardized to 50 ng/ml and stored at 20 C until PCR analysis. 603 bp COI fragment was amplified using universal HCO-LCO primer pairs (Folmer et al., 1994). PCR reactions were performed in total volumes of 50 ml by using 1 ml of DNA template and GoTaq Flexi DNA polymerase (Promega) according to manufacturer's instructions: 5x buffer, 10 mM of each dNTP, 10 mM of each primer and 1.25 u/ml DNA polymerase. PCR thermal cycling conditions consisted of an initial denaturation at 95 C for 2 min, followed by 30 cycles of 95 C for 30 s, 52 C for 45 s and 72 C for 1 min, followed by an extension at 72 C for 10 min. All the PCR products were visualized on 2% agarose gel to confirm the band corresponding to amplification product and purified with Wizard SV Gel and PCR Clean up System (Promega). The purified PCR fragments were cloned into PGEM T vector systems. Sequencing reactions were performed with DTCS Quick Start Kit (Beckman Coulter),
Table 1 Information about the collection sites, geographical coordinates and accession numbers of sunn pest adult parasitoids. Species
Specimens
Collection sites
Co-ordinates
Accession no.
Ectophasia crassipennis (F.) Elomyia lateralis (Meig) Eliozeta helluo (F.)
E. crassipennis E. lateralis E. helluo Hap 1 Hap 2 Hap 3 Hap 4 Hap 5 Hap 6 Hap 7 Hap 8 Hap 9 P. subcoleoptrata Hap 10 Hap 11 Hap 12 Hap 13 Hap 14 Hap 15 Hap 16
Siverek, Sanliurfa, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Ergani, Diyarbakir, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey Siverek, Sanliurfa, Turkey
37736033N 38154335N 38154335N 37736033N 37736033N 38154335N 38154335N 38154335N 38154335N 38154335N 38090808N 37736033N 37736033N 37717415N 37729358N 37717415N 37717415N 37717415N 37729358N 37717415N
KM233126 KM233125 KM233127 KM233128 KM233129 KM233130 KM233131 KM233132 KM233133 KM233134 KM233135 KM233136 KM233137 KM233138 KM233139 KM233140 KM233141 KM233142 KM233143 KM233144
Eliozeta helluo (F.)
Phasia subcoleoptrata (L.)
Phasia subcoleoptrata (L.)
39832819E (1835 m) 39823670E (802 m) 39823670E (802 m) 39832819E (1835 m) 39832819E (1835 m) 39823670E (802 m) 39823670E (802 m) 39823670E (802 m) 39823670E (802 m) 39823670E (802 m) 395021869E (778 m) 39832819E (1835 m) 39832819E (1835 m) 39834492E (1876 m) 39832369E (1846 m) 39834492E (1876 m) 39834492E (1876 m) 39834492E (1876 m) 39832369E (1846 m) 39834492E (1876 m)
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cleaned with Agencourt CleanSeq Kit (Agencourt Bioscience) and analyzed with the GenomeLab GeXP Genetic Analysis System (Beckman Coulter). Electropherograms of all sequences were assembled into contigs and proofread manually using the program Chromas, v.1.41. The DNA and deduced amino acid sequences were analyzed using the BLAST tool at the National Center for Biotechnology Information (NCBI) (www.ncbi.nih.gov/BLAST), the BOLD Identification System (http://www.boldsystems.org) and EXPASY (http://expasy.org). Sequence alignments were performed using the CLUSTALW v.1.82 software (Thompson et al., 1994). Conserved residues in the alignments were highlighted with Boxshade 3.21 (http://www.ch.embnet.org/software/ BOXform.html). All sequences have been deposited in the GenBank database. Models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the substitution pattern the best. For each model, AICc value (Akaike Information Criterion, corrected), Maximum Likelihood value (lnL), and the number of parameters (including branch lengths), are also calculated (Nei and Kumar, 2000). Analysis of variable parsimony informative sites, frequencies of transitional pairs, transversional pairs and R values, the average number of identical pairs, the number of transitions to the number of transversions for a pair of sequences were calculated using MEGA 6 software. The number of base substitutions per site from averaging over all sequence pairs and from averaging over all sequence pairs within each group is calculated using the Maximum Composite Likelihood model (Tamura et al., 2004). Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). The number of base substitutions per site from between sequences was conducted using the Maximum Composite Likelihood model (Tamura et al., 2004). Population differentiation and analysis of molecular variance (AMOVA) were conducted to identify number of haplotypes and fixation index (FST) using Arlequin 3.5.1.2 (Excoffier et al., 2005). Haplotype diversity was calculated using DnaSP vers. 3.5.1.2 (Excoffier and Lischer, 2010). Substitution pattern and rates were estimated under TamuraeNei model. Substitution model and rates of estimated transition/transversion bias (R) and estimates of evolutionary divergences between sequences were estimated under Kimura 2-parameter model. The evolutionary history of parasitoid haplotypes was inferred using the UPGMA method (Sneath and Sokal, 1973). Phylogenetic tree of adult parasitoids were constructed using the Neighbor-Joining (NJ) method (Saitou and Nei, 1987) implemented in the MEGA 6 program. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Estimates of Evolutionary Divergence between Sequences were conducted using the Maximum Composite Likelihood model (Tamura et al., 2004). The analysis involved 20 nucleotide sequences. Codon positions included were 1st þ 2nd þ 3rd þ Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 603 positions in the final dataset. Evolutionary analyses were conducted in MEGA 6 (Tamura et al., 2013).
3. Results and discussion Four adult parasitoids including E. helluo, P. subcoleoptrata, E. crassipennis and E. lateralis and sixteen parasitoid larvae were analyzed in terms of mtCOI gene sequences. The deduced sequences of all parasitoid species share >99% similarity to the same species of representative COI sequences in BOLD database. Among 20 specimens, 16 haplotypes were detected belonging to parasitoid larvae while E. helluo haplotypes from Diyarbakir province were identical (Hap3eHap4). All sequences were deposited under accessions KM233125eKM233144 in GenBank; GenBank accession numbers are detailed in Table 1. Conserved residues in the sequence alignments are highlighted with Boxshade 3.21 shown in Fig. 1. No insertions, deletions or stop codons were found indicating that all sequences correspond to a functional mitochondrial gene (Funk and Omland, 2003) and also indicating the absence of pseudogenes or nuclear copies of mitochondrial origin (NUMTs). Indels and nonsense/stop codons were not detected as well. The percentage of nucleotide composition at each codon position were found variable (Table 2). The mean frequency of COI sequences used in the analyses showed a bias of A þ T (T 38.7%, C 15.4%, A 30.9% and G 15.0%). The A þ T content at the third, second and first codon positions are 94.4%, 58%, and 56.3%, respectively (Table 2). First and second were less A þ T biased than the third codon position. The base frequencies of COI show a high adenine (A) and thymine (T) bias, with a mean rate of 69.6%, which is typical of insect mitochondrial sequences (Crozier and Crozier, 1993). The nucleotide G has the lowest (15%) and the A has the highest content (38.7%) in terms of first codon positions. Variation in base composition is important in the modeling of sequence evolution since the mtDNA loci verify the general observation from insects and especially Hymenopterans of a strong AeT bias (Crozier and Crozier, 1993; Dowton and Austin, 1995; Whitfield and Cameron, 1998). The mtDNA locus of sunn pest adult parasitoid species is AeT rich which agree with the cytochrome oxidase I data of Dowton and Austin (1995), showing a higher AeT content in parasitic wasps than in nonparasitic wasps. Maximum likelihood suitability of 24 nucleotide substitution models was analyzed. Assumed or estimated values of transition/transversion bias were also calculated for each model. For estimating ML values, a tree topology was automatically computed using MEGA 6 (Tamura et al., 2013). TamuraeNei model, with the lowest AICc score of 3397.361 was the best to describe the substitution pattern. The data set of COI alignment contains 603 nucleotide positions, of which 114 positions are variable and 72 are parsimony informative. The average number of identical pairs (ii) was 192 out of 603 base pairs. Transversional pairs (sv ¼ 18) were
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77
Fig. 1. Multiple alignment of COI nucleotide sequences from the following: E. lateralis (GenBank Accession: KM233125); P. subcoleoptrata (KM233137); E. crassipennis (KM233126); E. helluo (KM233127). The output for multiple sequence alignment is based on a Clustal W consensus sequence and sequences were shaded using the BoxShade program.
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Table 2 Table of nucleotide composition at all sites and three codon positions. Specimen
All sites
Codon position 1
T
C
A
G
T-1
C-1
A-1
G-1
E. crassipennis E. helluo E. lateralis P. subcoleoptrata E. helluo (means of Hap 1e9) P. subcoleptrata (means of Hap 10e16) Average
38.6 38.0 38.5 39.5 38.1 39.5 38.7
15.1 16.1 14.4 15.1 15.8 15.0 15.4
31.0 31.2 31.3 30.5 31.3 30.3 30.9
15.3 14.8 15.8 14.9 14.8 15.1 15.0
27 26 27 28 26 28 27
15.4 16.4 14.4 14.9 16.3 14.6 15.5
28.4 29.4 28.9 29.4 29.9 28.8 29.3
29.4 27.9 29.4 27.9 27.6 28.6 28.2
Specimen
Codon position 2
E. crassipennis E. helluo E. lateralis P. subcoleoptrata E. helluo (means of Hap 1e9) P. subcoleptrata (means of Hap 10e16) Average
Codon position 3
T-2
C-2
A-2
G-2
T-3
C-3
A-3
G-3
43 43 43 43 43 43 43
25.9 25.9 25.9 25.9 25.9 25.8 25.9
14.9 14.9 14.9 14.9 15.0 14.9 15.0
15.9 15.9 15.9 15.9 16.0 15.9 15.9
46 44 45 47 45 47 46
4.0 6.0 3.0 4.5 5.2 4.8 4.9
49.8 49.3 50.2 47.3 48.9 47.3 48.4
0.5 0.5 2.0 1.0 9.0 0.8 0.9
found to be higher than transitional pairs (si ¼ 7). The number of transitions to the number of transversions for a pair of sequences (si/sv ¼ R) was 2.86 for the data set. The number of base substitutions per site from averaging over all sequence is 7.0% (SE ¼ 0.9%), whereas the mean genetic distance between Eliozeta helluo haplotypes and Phasia subcoleoptrata haplotypes was 1.3% and 0.9%, respectively (SE ¼ 0.3 and 0.2). Highest genetic distance was calculated between E. helluo-P. subcoleoptrata (0.327) and the lowest genetic distance was between E. helluo and E. crassipennis (0.012). The distance matrix for all analyzed species is presented in Table 3. Fixation index (FST) is one of the most widely-used measures of population differentiation for estimating and testing the magnitude of genetic divergence (Wright, 1943, 1951, 1965). FST, ranges from 0.0, indicates identical allele frequencies in a pair of populations (no differentiation), to 1, which indicates alternate fixation for a single unique allele in each population. The values between 0.0 and 1.0 show the magnitude of genetic differentiation among samples. In this study, overall FST of the Eliozeta helluo haplotypes was calculated as 0.036, while no significant pair-wise differences of Fst values were observed among Phasia subcoleoptrata haplotypes. A low genetic differentiation between E. helluo haplotypes among collection sites in Diyarbakir and Siverek, which might indicate that some combination of drift and reduced gene flow might be operating enough to generate significant genetic variation among regions. Maximum likelihood estimation of substitution pattern and rates of matrix were estimated under the Tamura-Nei (1993) model (Table 4). Substitution rates represent the probability of substitution (r) from one base (row) to another (column). Rates of different transitional substitutions are shown in bold and those of transversional substitutions are shown in italics in Table 4. Transitional substitution rates of A/G, T/C, C/T and G/A were 4.85%, 11.76%, 29.52% and 10.00%, respectively. Transversional rates are given in Table 4. The estimated transition/transversion bias (R) was 1.13 which is important not only to understand the patterns of DNA sequence evolution, but also for a reliable estimation of sequence distance and phylogeny reconstruction. The estimated transition to transversion ratio was higher than those observed in sunn pest egg parasitoid species (Guz et al., 2013). The nucleotide frequencies are 30.90% (A), 38.70% (T), 15.41% (C), and 14.99% (G). The evolutionary history of sunn pest adult parasitoid species and haplotypes was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and UPGMA method (Sneath and Sokal, 1973). The UPGMA method indicates that two distinct mitochondrial haplotype lineages within the sampled populations. Nine haplotypes (Hap 1e9) were recorded in specimens determined to be Eliozeta helluo and seven haplotypes (Hap 10e16) were recorded in specimens determined to be Phasia subcoleoptrata (Fig. 2a). The haplotype diversity was calculated as 0.992. The percentage of replicate NJ trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). Clustering patterns of the consensus tree (Fig. 2b), with the sum of branch length (1.398) pointed out a correlation between genetic distances. Sunn pest egg parasitoid species (Hymenoptara: Scelionidae) inferred by Guz et al. (2013) were chosen as out groups. According to the phylogenetic tree based on COI gene, Hymenopteran sunn pest parasitoids were separated in two as egg parasitoid species (Scelionidae) clustered together in one clade and adult parasitoid species (Tachinidae) clustered in the second clade. Adult parasitoid species were also separated in two including E. helluo, Ectophasia crassipennis and Elomyia lateralis in one clade and P. subcoleoptrata clustered in a separate clade. Genetic analyses of sunn pest adult parasitoid populations showed that the COI gene, known as the DNA barcode, successfully distinguishes genetic variation among parasitoid populations sampled from different regions and provide a valuable resource as a molecular marker. The analyzed partial COI gene region showed conserved and variable regions that provide information for identifications at species level and for revealing intraspecific variation among parasitoid populations. The data obtained from this study do not only provide molecular data to identify sunn pest adult parasitoid species but also will serve as a useful tool for detecting accurate parasitism rate of parasitoids which is important in biological control studies. It
1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
E. lateralis E. crassipennis E. helluo P. subcoleoptrata Hap 1 Hap 2 Hap 3 Hap 4 Hap 5 Hap 6 Hap 7 Hap 8 Hap 9 Hap 10 Hap 11 Hap 12 Hap 13 Hap 14 Hap 15 Hap 16
2 0.016
0.094 0.096 0.087 0.101 0.096 0.099 0.099 0.110 0.097 0.109 0.099 0.099 0.084 0.083 0.087 0.085 0.087 0.085 0.093
0.060 0.096 0.072 0.067 0.071 0.071 00.77 0.067 0.078 0.069 0.069 0.092 0.092 0.096 0.094 0.096 0.094 0.097
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0.015 0.011
0.014 0.017 0.018
0.016 0.012 0.004 0.018
0.015 0.012 0.004 0.018 0.003
0.016 0.012 0.004 0.018 0.004 0.004
0.016 0.012 0.004 0.018 0.004 0.004 0.000
0.017 0.013 0.005 0.019 0.005 0.005 0.005 0.005
0.016 0.012 0.004 0.018 0.004 0.004 0.004 0.004 0.005
0.017 0.013 0.005 0.019 0.006 0.006 0.005 0.005 0.006 0.004
0.016 0.012 0.004 0.018 0.004 0.004 0.004 0.004 0.005 0.002 0.004
0.015 0.012 0.005 0.018 0.005 0.005 0.005 0.005 0.006 0.003 0.005 0.002
0.014 0.016 0.018 0.004 0.018 0.018 0.018 0.018 0.019 0.018 0.018 0.018 0.018
0.014 0.016 0.018 0.002 0.018 0.018 0.018 0.018 0.019 0.018 0.018 0.018 0.017 0.003
0.014 0.016 0.018 0.004 0.018 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.003 0.003
0.014 0.016 0.018 0.003 0.019 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.003 0.002 0.004
0.014 0.017 0.018 0.003 0.018 0.018 0.018 0.018 0.018 0.018 0.019 0.018 0.018 0.004 0.003 0.004 0.004
0.014 0.016 0.018 0.003 0.018 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.004 0.003 0.004 0.003 0.004
0.015 0.017 0.018 0.005 0.018 0.018 0.018 0.018 0.019 0.018 0.019 0.018 0.018 0.006 0.005 0.006 0.005 0.005 0.004
0.118 0.012 0.010 0.010 0.010 0.019 0.010 0.020 0.012 0.015 0.114 0.114 0.118 0.115 0.118 0.116 0.117
0.116 0.114 0.116 0.116 0.120 0.112 0.120 0.114 0.114 0.010 0.003 0.010 0.005 0.007 0.005 0.013
0.008 0.012 0.012 0.013 0.012 0.022 0.013 0.017 0.111 0.116 0.116 0.117 0.112 0.114 0.115
0.010 0.010 0.019 0.010 0.020 0.012 0.015 0.110 0.110 0.114 0.112 0.114 0.112 0.113
0.000 0.015 0.010 0.020 0.012 0.015 0.112 0.112 0.116 0.114 0.112 0.114 0.115
0.015 0.010 0.020 0.012 0.015 0.112 0.112 0.116 0.114 0.112 0.114 0.115
0.015 0.020 0.017 0.020 0.116 0.120 0.120 0.122 0.112 0.118 0.120
0.010 0.002 0.005 0.108 0.108 0.112 0.110 0.112 0.110 0.111
0.012 0.015 0.116 0.116 0.120 0.118 0.120 0.118 0.119
0.003 0.110 0.110 0.114 0.112 0.114 0.112 0.113
0.110 0.110 0.114 0.112 0.114 0.112 0.113
0.007 0.007 0.005 0.010 0.012 0.020
0.007 0.002 0.007 0.005 0.013
0.008 0.010 0.012 0.020
0.008 0.007 0.015
0.008 0.017
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77
Table 3 The number of base substitutions per site from between sequences is shown. Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). Analyses were conducted using the Maximum Composite Likelihood model. Codon positions included were 1st þ 2nd þ 3rd þ Noncoding. All ambiguous positions were removed for each sequence pair.
0.012
75
76
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77 Table 4 Each entry is the probability of substitution (r) from one base (row) to another base (column). Substitution pattern and rates were estimated under the TamuraeNei (1993) model (Tamura and Nei, 1993). Rates of different transitional substitutions are shown in bold and those of transversionsal substitutions are shown in italics. Relative values of instantaneous r should be considered when evaluating them. For simplicity, sum of r values is made equal to 100. The nucleotide frequencies are A ¼ 30.90%, T/U ¼ 38.70%, C ¼ 15.41%, and G ¼ 14.99%. For estimating ML values, a tree topology was automatically computed. The maximum Log likelihood for this computation was 1661.145.
A T/U C G
A
T/U
C
G
e 6.78 5.37 10.00
8.9 e 26.03 8.49
3.38 11.76 e 3.38
4.85 3.29 2.6 e
Fig. 2. Evolutionary relationships of taxa. The evolutionary history of species and haplotypes was inferred using the UPGMA (2A) and Neighbor-Joining (2B) methods. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.
M. Duman et al. / Biochemical Systematics and Ecology 59 (2015) 70e77
77
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