International Society of communication and Development among universities www.europeansp.org Intersociety Energy Conversion Engineering Conference, ISSN: 0146-955

Genetic Diversity of Oak Curculio, Curculio glandium (Coleoptera: ) Among Geographical Populations in Southwestern Iran

Akram Ebrahimi1, Iraj Mehregan 1, Taher Nejad Sattari 1, Mostafa Assadi 2, Kambiz Larijani 3.

a Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran, 2. Research Institute of Forests and Rangelands, National Botanical Garden of Iran, Tehran, Iran, 3. Department of Chemistry, Science and Research Branch, Islamic Azad University, Tehran, Iran, * Corresponding author Islamic Azad University, Science and Research Branch, Hesarak, Daneshgah Blvd, Tehran, Iran.

Abstract

Persian oak (Quercus brantii Lindl.) is one of the most essential woody species of the Zagros forests in Iran. Among the species of that attack acorns, the most common is Curculio glandium Marsh. In this study, the variability of Curculio glandium populations in South Western forests of Iran was evaluated by amplified fragment length polymorphism (AFLP) technique. A total of 105 trees were sampled from 21 populations representing seven regions in the three provinces of Khuzestan (regions GOL, EMA, BAB, MOG and ALG), Chaharmahal and Bakhtiari (region MON) and Kohgiluyeh and Boyer-Ahmad (region DIS) of Iran. An analysis of molecular variance revealed that most genetic variation was confined within populations (88%), differences among populations account 5% and only 7% is attributed to variation among regions. Three regions of Khuzestan (EMA/ALG/MOG) have a similar genetic structure. This is also observed in the regions of MON and GOL. In addition, regions DIS and BAB have the same genetic structure. Two gene pools were identified in all regions, but further sampling is necessary to test for genetic variation and gene flow among these regions. Population genetic information can provide critical insights into range expansion and evolutionary potential to adapt to environmental changes, such as host shift and agricultural management changes.

© 2016 The Authors. Published by European Science publishing Ltd. Selection and peer-review under responsibility of the Organizing Committee of European Science publishing Ltd.

Keywords: AFLP, Curculio glandium, Diversity.

1. Introduction Downloaded from iecec.europeansp.org at 2:50 +0330 on Sunday October 3rd 2021 The genus Quercus L. (Fagaceae) is one of the most diversified groups of temperate trees with approximately 500 species distributed globally (Mehrnia et al. 2012). Zagros Oak forests are regarded as the largest in Iran with the coverage of ca. 40% (Sagheb-Talebi et al. 2004). These forests have sub-Mediterranean semiarid temperate climate, with dominant species of Quercus brantii Lindl. mixed with other deciduous, broad-leaved trees such as Pistacia atlantica Desf., Pistacia khinjuk Stocks., Pyrus spp., Amygdalus spp., Celtis spp. and Acer monspessulanum L. Q. brantii is mentioned as the most important tree species of the region with more than 50% coverage of the western forests of Iran (Heydari et al. 2013). The features such as genetic variation and high levels of phenotypic plasticity contribute to the success of the genus Quercus. (Kashani & Dodd 2002).

© 2016 The Authors. Published by European Science publishing Ltd. Selection and peer-review under responsibility of the Organizing Committee of European Science publishing Ltd. 2 Eicker et al. / Intersociety Energy Conversion Engineering Conference, ISSN: 0146-955 (2017)101_111

Among the species of insects that attack the acorns, the most common are of the family Curculionidae, particularly Curculio glandium (acorn ). This species attacks the acorn before release. Quercus species can be attacked by both small and large species of acorn and large seeds can feed on the larvae before they reach the embryo. The adult is 5-9 mm long, with an elongated oval body which varies from dark brown to yellow gray to black. The rostrum is long, thin and bent downward and can reach half its body length in males and slightly longer in females. The larva is white and legless with a yellowish brown cephalic capsule completely developed. The body is slightly curved, robust with a strong tegument (Oltean et al. 2014). One of the direct effects of this insect is the consumption of the embryo by the larvae or the damaging of the cotyledons to such an extent that the germination and the development of the seedling becomes impossible. Another of the direct effect is the premature abscission of the infested young acorns (Csóka & Hirka 2006). The weevils (Curculionoidea) constitute the most species-rich superfamily of . They consist of several thousand species in Europe and around 60,000 species have been described globally (Hundsdoerfer et al. 2009). The genus Curculio represents a large group of seed beetles made up of about 345 species distributed across Asia, Europe, Africa, and North America (Hughes & Vogler 2004). Evaluation of genetic diversity of oak trees and their pests is necessary for germplasm conversation and optimized forest management. Since the oaks are significant parts of temperate forests, they have been subject of several studies using different morphological and molecular markers (González-rodríguez & Oyama 2005; Birchenko 2008; Ballian et al. 2010; Crăciunesc et al. 2011; Lind & Gailing 2013; Lee et al. 2014; Yücedağ1 & Gailing 2013; Bakiş & Babaç 2014) but, with the exception of the study of Zhang et al. (2008) on genetic diversity of plum Curculio and Mynhardt (2006) on genetic diversity of pecan weevil, genetic diversity of genus Curculio has not been thoroughly investigated until now. Despite the economic impact of C. glandium, there is no information about its population genetic structure and gene flow patterns. For studying the genetic diversity of populations, DNA-based markers have several benefits than morphological markers as a result of lack of influence of the environment and high heredity (Duran et al. 2009). Amplified Fragment Length Polymorphism (AFLP) technique is a highly repeatable method that produces a large number of markers which makes it suitable for population genetic studies (Vos et al. 1995). AFLP technique produces a remarkably higher amount of markers in comparison to microsatellite markers (Mariette et al. 2002). Historically, Oaks have been an important source of fuel, fodder and building materials throughout their ranges. Other products include tannins and dyes. Oak bark and leaves were frequently used for tanning leather. The acorn flour can be used for the baking of cakes and bread. The widespread availability and previous use of acorns as food implies that the development of this crop for human consumption is possible. Furthermore, the fruits of this plant are used in a decoction or as powder for the treatment of acute diarrhea and inflammation and stomach ache in traditional medicine (Shikov et al. 2014). Considering the importance of the Persian Oak, we aimed at evaluating the genetic variation of C. glandium populations, the most common insect that attack acorns, in the three provinces (Khuzestan, Chaharmahal and Bakhtiari and Kohgiluyeh & Boyer-Ahmad) in SW Iran by AFLP fingerprint technique. Main text 2.1. Material and methods Downloaded from iecec.europeansp.org at 2:50 +0330 on Sunday October 3rd 2021 2.1.1. Oak Curculio populations This study was carried out on the materials of 21 populations collected from nine different regions in SW Iran (Table 1; Fig. 1). They were from provinces Chaharmahal and Bakhtiari (region MON), Khuzestan (regions ALG, GOL, MOG, BAB, and EMA) and Kohgiluyeh & Boyer-Ahmad (region DIS) (Fig. 1). Table 1 indicates the geographical coordinates and altitude corresponding to each region. Collection of acorns was done in three altitudes (low, middle and high) in each site during the autumn 2014. Each population was collected from at least five trees (individuals) with at least 50 m distance from each other. Larvae were collected from infested fruit and were delivered in 70-95% ethyl alcohol (EtOH) and were stored at -20ºC until needed. The larvae stage of C. glandium and its feeding on acorn is presented in Figure 2.

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Fig. 1. Map of Iran indicating locality of collected Specimens. Each point represents the region in which three populations are collected (further details in Table 1). Abbreviations: GOL, Golzar; EMA, Emamzadeh-Abdollah; BAB, Baloot-Boland; MOG, Mongasht; ALG, Alvar- Garmsiri; DIS, Dishmook; MON, Monj.

2.1.2. DNA Extraction Genomic DNA was extracted from each individual with a modified protocol of (Vogler et al. 1993) using a NucleoSpin® Tissue (Qiagen, Germany) following the manufacturer’s tissue protocol. The quality of extracted DNA was checked on 1% agarose gels. High quality DNA samples were stored at -20ºC until needed.

2.1.3. PCR-AFLP analysis For the amplified fragment length polymorphism (AFLP), the method of Scalone & Albach (2012) with the following modifications was employed. Total DNA was concurrently digested with 0.1 µl EcoRI and 0.1 µl MseI (10U/µl, Thermoscientific, Lithuania), and ligated with T4 DNA ligase (5U/µl, Thermoscientific, Lithuania) in a reaction buffer [10 mM tris-HCl (pH 7.5), 10 mM Mg- acetate and 50 mM K-acetate, 1 µl 0.5 mol NaCl and 0.25 µl bovin serum albumin (BSA, 2mg/ml, Thermoscientific, Lithuania) and 0.2 µl T4 DNA ligase for 3 hours at 37ºC. Two adaptors, one for the 0.5µl 5pmol/µl EcoRI (5’– CTCGTAGACTGCGTACC –3’; 3’– AATTGGTACGCAGTC - 5’) ends and one for the 0.5µl 5pmol/µl MseI (5’ – GACGATGAGTCCTGAG –3’; 3’ – TACTCAGGACTCAT - 5’) ends, were designed to avoid the reconstruction of the restriction sites. After incubation, enzymes were inactivated at 65ºC for 10 min. Then, the ligation product was diluted 1:3 for primary amplification. Primary amplification was done in a standard Polymerase Chain Reaction (PCR) cocktail containing 1.5mM MgCl2, 0.2 mM of each dNTP, 0.1 mM of Taq DNA polymerase, PCR Buffer, 1% Tween 20 (Ampliqon, Denmark), 0.4 µl 10pmol/µl preselective primers E01 (5’- GACTGCGTACCAATTCA- 3’) and M02 (5’- GATGAGTCCTGAGTAAC-3’), 6.7 µl DEPC H2O and 5 µl diluted product (1:3) of the digestion-ligation reaction. For the primers, the EcoRI primer sequence was identical to the adapter sequence, while the MseI primer had an

Downloaded from iecec.europeansp.org at 2:50 +0330 on Sunday October 3rd 2021 extra cytosine (“C”) as a selective nucleotide. The PCR reaction was carried out in a thermocycler (Labcycler, SENSQUEST, Germany) for 20 cycles using the following cycling parameters: 20s at 94ºC, 30s at 56ºC, 2min at 72ºC and 30min at 60ºC. The primary amplification product was then diluted 1:10 for selective amplification which involved the application of 4 different primer pair combinations. Selective amplification was done in a standard PCR reaction cocktail containing 2 mM MgCl2, 0.2 mM of each dNTP, 0.1 mM of Taq DNA polymerase, PCR Buffer, 1%Tween 20, 0.3 µl 10pmol/µl EcoRI primer and 0.4 µl 10pmol/µl MseI primer, 1.8 µl DEPC H2O and 5 µl diluted product of the preselective amplification. The three primer combinations (Table 2) for the selective PCR were E38 combined with M57; E45 with M54; E40 with M55. The PCR program had two cycles: 10 cycles of 20s at 94ºC, 30s at 65ºC and 2min at 72ºC, followed by 25 cycles of 20s at 94ºC, 30s at 56ºC and 2min at 72ºC. The products of selective amplification were resolved on 6 percent polyacrylamide native gel. Gels were stained with Gelstar (FMC Bioproducts, Rockland, ME, U.S.A.) following manufacturer’s protocol and recorded with a Kodak DC 120 digital camera.

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Table 1. Geographical and climatic details of 21 populations of Quercus brantii collected for this study. Abbreviations: Pop., population; Reg., Region; Prov., Province; Abr., Abbreviation; Alt., Altitude; Lat., Latitude; Long., Longitude.

Pop. Reg. Prov. Abr. Alt. Lat. Long.

1 32°12' 54.3" 49°43'40.1" 850 2 32°11'9.3" 49°42'46.2" Golzar Khuzestan GOL 1350 3 32°9'16.4" 49°43'36.5 " 1600

4 Emamzadeh- 50°9' 54.1 " 1630 31°24' 46.1" 5 Abdollah 50°9' 46.6" Khuzestan EMA 1801 31°23' 38.6" 6 50°9' 21.9 " 2086 31°23' 2.1"

7 31°45'26.4" 50°8'47.1" 910 8 Baloot-Boland Khuzestan BAB 31°41'51.1" 50°18'25.7" 1540 9 31°39'43.5" 50°23'26.6 " 2030

10 31°47'54.9" 49°59'9.3 " 950 11 Mongasht Khuzestan MOG 31°46'30.1" 49°59'37.0" 1285 12 31°46'15.7" 50°0'23.7" 1621

13 32°50'24.7" 48°22'34.5" 650 14 Khuzestan ALG 32°49'45.4" 48°16'2.8" Alvar-Garmsiri 800 15 32°53'8.2" 48°15'53.0" 1000

16 31°14'29.1" 50°18'0.2" Kohgiluyeh and 700 17 Dishmook DIS 31°17'8.5"3131 50°16'51.8" Boyer-Ahmad 1435 18 °16'20.8" 50°22'12.1" 2000

19 31°30'27.1" 50°38'55.5" Chaharmahal 1550 20 Monj MON 31°29'45.9 " 50°365'3.8" and Bakhtiari 1830 21 31°30'10.3" 50°35'29.0" 2245

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Table 2 . Name and sequences of primer pairs that was used in selective-PCR reactions.

Reverse primer sequences Forward primer sequences Reverse Forward (non- labeled) (labeled) Row primer primer

5’-GATGAGTCCTGAGTAACGG- M-57 5’-HEX-GACTGCGTACCAATTCACT- E-38 1 3’ 3’ 5’-GATGAGTCCTGAGTAACCT-3’ M-54 5’-FAM-GACTGCGTACCAATTCATG- E-45 2 3’ 5’-GATGAGTCCTGAGTAACGA- M-55 5’-NED-GACTGCGTACCAATTCAGC- E-40 3 3’ 3’

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A B

Fig. 2. Larvae stage of Curculio glandium and his host. A, Larvae shape; B, the acorn that attacked by larvae.

2.1.4. Statistical data analysis Bands on the gel obtained by Gelstar were scored as present (1) or absent (0) in readable region of bands from 50-500 bp in length with Gene Marker v. 1.95. Each peak with a signal intensity of more than 1000 was selected and checked for its presence in each sample. The genetic diversity and structure of populations were calculated by using the computer software GenAlex v. 6.501 and Structure v. 2.3.4. respectively. Some of this work was done by SPSS v. 22 and Adobe Illustrator CS6.

2.2. Results Analysis of molecular variance (AMOVA) in GenAlex shows that 88% of the total variation is attributable to differences among individuals within the same population. 5% and 7% of the total overall variation was attributed to differences among populations and regions respectively (Table 3).

Table 3. Analysis of molecular variance (AMOVA) of genetic variation in Curculio glandium populations. Abbreviation: Df, Degrees of freedom; SS, Sum of Squares; MS, Mean Squares; Est.Var., Estandard Variation; %, Percent of genetic variation. Source df SS MS Est.Var. %

Among Regions 6 157.367 26.228 1.210 7% Among Populations 20 372.440 18.622 0.797 5% Within Populations 78 1141.842 14.639 14.639 88% Total 104 1671.649 16.645 100%

Downloaded from iecec.europeansp.org at 2:50 +0330 on Sunday October 3rd 2021 The results of evaluation of genetic structure revealed that two genotype were available in populations which is shown by two color (red and green). Calculation of percent allele means in different regions (Table 4) indicated that three region (EMA, MOG and ALG) of Khuzestan have similar genetic structure. This is also seen in regions of MON and GOL. Regions of DIS and BAB have a specific and separate genetic structure. For better understanding, see Fig.3. Figure 4 indicates dendrogram by AFLP data that populations mix together and are not separated from each other.

Table 4. Mean % of allele in Curculio glandium populations in studied regions. The descriptive statistics are presented in terms of the Mean ± SD (Standard deviation). Mean values with the same letters indicate homogeneous subsets for P ≤ 0.05 according to Duncan test.

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Region Red allele (%) Green allele (%) Golzar (GOL) 68.60 ± 34.96 c 31.40 ± 34.96 a Emamzadeh-Abdollah (EMA) 42.00 ± 41.95 abc 58.00 ± 41.95 abc Baloot-Boland (BAB) 7.20 ± 12.78 a 92.80 ± 12.78 c Mongasht (MOG) 23.00 ± 32.55 ab 77.00 ± 32.55 bc Alvar-Garmsiri (ALG) 33.67 ± 30.01 abc 66.33 ± 30.01 abc Dishmook (DIS) 5.60 ± 5.37 a 94.40 ± 5.37 c Monj (MON) 62.53 ± 33.91 bc 37.47 ± 33.91 ab Downloaded from iecec.europeansp.org at 2:50 +0330 on Sunday October 3rd 2021 8 Eicker et al. / Intersociety Energy Conversion Engineering Conference, ISSN: 0146-955 (2017)101_111

Fig. 3. Correlation between genetic structures of Curculio glandium populations in studied regions. Abbreviations: GOL, Golzar; EMA, Emamzadeh-Abdollah; BAB, Baloot-Boland; MOG, Mongasht; ALG, Alvar-Garmsiri; DIS, Dishmook; MON, Monj.

2.3. Discussion This is the first study on AFLP markers in Oak curculio populations. In this study, the AFLP was highly informative as a molecular marker in the evaluation of genetic diversity and structure of C. glandium populations. Analysis of variance shows that most of the total variation is attributable to differences among individuals within the same population and a little percent of the total overall variation was attributed to differences among populations and regions. This result is similar to that of Zhang et al. (2008) and Mynhardt (2006) on genetic diversity of plum Curculio and pecan weevil respectively. In addition, results of evaluation of genetic structure indicated that two gene pools were available in the populations and percent allele means in different regions is different from each other. In dendrogram by AFLP, populations are mixed together and not separated from each other. Conversely, weevil populations do not usually show separate and specified genetic foundations. Gene flow is a critical factor in species evolution that is effective on size population, hybridization and local adaptation (Gerber et al. 2014). The pattern of diversity and population genetic structure are determined by the interaction of different factors, some of which are related to life history traits and ecological features of species, evolutionary history, and natural disturbances. Therefore, the balance achieved by a population among these factors determines its special genetic structure. The genetic structure can be associated with geographical region and climatic conditions (Neophytou et al. 2010), hybridization (Curtu et al. 2014), breaking of primary reproductive barriers (Valencia-Cuevas et al., 2014), altitude, historical movements and evolution, (Zhang et al. 2013; Ohsawa et al. 2007; Wang et al. 2014). Oak curculio is a key pest of numerous tree fruits in Zagros forests. Knowledge of the genetic diversity and structure as well as the distribution status of this pest is essential for their management. This study would enhance our Downloaded from iecec.europeansp.org at 2:50 +0330 on Sunday October 3rd 2021 phylogeographical information concerning the pest from a molecular view and could provide an ecological and economic benefit for fruit production in Oak forests. In future studies, increase in sample size and application of other molecular markers to enlarge the informative sites for thorough comprehension of the evolution of Oak curculio populations should be considered.

3. Conclossion In spite of the fact that this investigation is done in a part of Zagros forests, analyses revealed the some molecular differentiations in some populations of oak Curculio in Iran. The pattern of diversity is different depending on the type of marker and analysis that was performed. Further molecular investigations in this case would be necessary to test for historical evolutionary forces that shape species population diversity and structure. Population genetic information can provide critical insights into range expansion and evolutionary potential to adapt to environmental changes, such as host shift and agricultural management changes. Eicker et al. / Intersociety Energy Conversion Engineering Conference, ISSN: 0146-955 (2017)101_111 9

Figure 4. Dendrogram of Curculio glandium populations based on AFLP marker (letters and numbers in left side show region and population and latter number show individuals of populations). Abbreviations: GOL, Golzar; EMA, Emamzadeh-Abdollah; BAB, Baloot-Boland; MOG, Mongasht; ALG, Alvar-Garmsiri; DIS, Dishmook; MON, Monj.

Acknowledgments This article is extracted from Akram Ebrahimi Ph.D thesis. We want to finally thank Islamic Azad University- Tehran Science and Research Branch for providing the facilities necessary to carry out the work.

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