Turkish Journal of Zoology Turk J Zool (2020) 44: 146-155 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1908-9

DNA barcoding of black cherry cerasi (Fabricus, 1775) (: ) populations collected from Prunus avium and Prunus cerasus

1, 2 Burcu İNAL *, İrfan KANDEMİR  1 Directorate of Plant Protection Central Research Institute, Ankara, Turkey 2 Department of Biology, Faculty of Science, Ankara University, Ankara, Turkey

Received: 06.08.2019 Accepted/Published Online: 28.01.2020 Final Version: 04.03.2020

Abstract: Sequences of the mitochondrial cytochrome oxidase I (COI) gene of one Greek and 25 Turkish (Fabricus) (Hemiptera: Aphididae) populations collected from Prunus avium and Prunus cerasus were analyzed. The partial coding region of COI studied is 605 bp for all the populations, from which 565 nucleotides were conserved, 40 were variable, 37 were singleton, and 3 were parsimony-informative. Four haplotypes were identified based on nucleotide substitutions and the mean of intraspecific divergence was calculated to be 0.3%. Phylogenetic trees were constructed using maximum likelihood, neighbor joining, and minimum evolution. Myzus persicae (Sulzer) and Myzus borealis Ossiannilson were included as outgroups. The population ofM. cerasi from Isparta diverged from the rest of the groups and formed a clade (Haplotype B) with Myzus borealis. The rest of the haplotype diversity included Haplotype A and Haplotype C with individuals characterized as Myzus cerasi pruniavium and Haplotype D with Myzus cerasi cerasi. M. cerasi diverged into two subspecies and it must be reevaluated whether this pest is monophagous or oligophagous in terms of plant type dependence. Further studies will be required for a complete barcoding and host-specific status of black cherry aphid.

Key words: DNA barcoding, COI, black cherry aphid, nucleotide diversity

1. Introduction Hille Ris Lambers (1976) listed synonyms. In addition More than 4,000 species of have been described, to M. cerasi sensu stricto (Fabricius, 1775), three subspecies some 250 of which are pests of crops and ornamental plants. have also been described: M. cerasi pruniavium Börner, In Turkey, a total of 466 species and 12 subspecies belonging 1926; M. cerasi veronica (Walker, 1848); and M. cerasi to 141 genera and 13 tribes are listed. The family Aphididae umefolii Shinji, 1941 (Dahl, 1968; Müller, 1986; Gruppe, comprises the highest number of species (457), followed 1988). Polyacrylamide gel electrophoresis (PAGE) was by Adelgidae (6) and Phylloxeridae (3), respectively used for the identification of the subspecies ofM. cerasi (Görür et al., 2012). Myzus cerasi causes the leaves to and their esterases differed from each other. The results curl and also transmits several nonpersistent viruses of indicated that Myzus cerasi pruniavium and Myzus cerasi nonhost plants including bean yellow mosaic virus, celery sensu stricto are subspecies that may be on the way to mosaic virus, onion yellow dwarf virus, and potato developing into two different species (Gruppe, 1988). virus Y (Blackman and Eastop, 1984; Bokx and Piron, DNA barcoding technology has been widely used 1990). It is distributed throughout the Palearctic zone for identifying aphid species through BLAST sequence and is now almost cosmopolitan. Most populations of comparisons and has also taken part in pest management black cherry aphid alternate hosts and have a sexual and plant quarantine (Komazaki et al., 2010; Lee et al., stage in the life cycle. Host alternation is between cherry 2010; Shufran and Puterka, 2011; Chen et al., 2012; (Prunus cerasus and Prunus avium) as the primary Rebijith et al., 2013; Coeur d’Acier et al., 2014; Rakauskas host and bedstraws (Galium), eyebrights (Euphrasia), et al., 2014; Chen et al., 2016; Zhu et al., 2017). Based on and speedwell (Veronica spp.) as the secondary hosts. the partial sequences of the mitochondrial cytochrome Forms on Prunus cerasus and Prunus avium are sometimes oxidase (COI) gene, M. cerasi samples form two major considered as different subspecies. clades corresponding to two host-specific black cherry Fabricius (1775) first described M. cerasi, as Aphis aphid taxa. The range of genetic divergence (0.0%–0.5%) cerasi, from specimens collected in Germany. Eastop and between the two clades of M. cerasi appears to be of * Correspondence: [email protected] 146

This work is licensed under a Creative Commons Attribution 4.0 International License. İNAL and KANDEMİR / Turk J Zool intraspecific level. Hence, black cherry aphids inhabiting 2.2. DNA extraction sour and sweet cherries are still be considered as a single Total genomic DNA was extracted from each individual species (Rakauskas et al., 2014). aphid using the DNeasy Blood & Tissue Kit (QIAGEN) No clear study, except that of Rakauskas et al. (2014), protocol. DNA quality was measured using a NanoDrop has been fulfilled with respect to phylogenetic analysis of 2000 spectrophotometer (Thermo Scientific) according to M. cerasi. Here, we analyzed partial sequences of the COI two ratios of absorbance: 260/280 nm and 260/230 nm. gene of M. cerasi populations based on DNA barcoding. 2.3. COI amplification and sequencing We sequenced 605 nucleotide sequences from one Greek After DNA extraction from a single aphid, partial sequences of and 25 Turkish populations to estimate nucleotide the COI gene was amplified with either CO-I forward primer diversity and genetic intraspecific divergence. The aim 5’- TCTTCTCTTTACATTTAGCAGGAAT-3’ or CO-I of this research is to identify the haplotype diversity and reverse primer 5’-AATAGATGAATTAGCAAGAATTA-3’ genetic divergences between the populations of M. cerasi (Turcinaviciene et al., 2006). PCR amplification was and to evaluate the accuracy of DNA barcoding in pest performed in a thermal cycler (Techne TC-5000) in 50 µL management strategies. volumes containing 4 µL of genomic DNA, 2 µL of each primer (0.4 µM), 10 µL of 5X buffer, Promega Flexi Go Taq 2. Materials and methods (1X), 1 µL of dNTP mix (0.2 mM), 3 µL of 25 mM MgCl2, 2.1. Aphid sampling 0.25 U of Gotaq polymerase (5 U/µL), 4 µL of bovine

Both apterous and pterous viviparae of black cherry aphid serum albumin (10 µg/µL), and ddH2O to 23.75 µL. The adults were collected mainly from Prunus avium, but also thermal cycling parameters were as follows: denaturizing from Prunus cerasus during May and June in 2013–2015 at 95 °C for 10 min (1 cycle); denaturizing at 95 °C for 30 (Figure 1). A total of 120 aphid samples belonging to 25 s, annealing at 49 °C for 30 s, and extension at 72 °C for populations from different climates in Turkey (Figure 2; 30 s (37 cycles in total); and a final extension for 5 min Table 1) and one population from Greece were used for the (1 cycle) (Rakauskas et al., 2014). The amplified products molecular analyses. Aphid specimens were taken from the were tested by electrophoresis on 2.0% agarose gel and shoots of host plants using a soft brush and were put into visualized in a gel documentation system (UVP). an Eppendorf tube containing 95% alcohol. Aphid slide urified PCR mounting and identification were conducted according products were sequenced in both directions by auto- to Hille Ris Lambers (1950) and Blackman and Eastop mated Sanger sequencing (2006). All aphid samples were preserved in 95% ethanol The PCR products were bidirectionally sequenced and kept at –20 °C before taxonomic identification and by automated Sanger sequencing at GENOKS (Genetic DNA extraction. Diagnosis Center, Ankara, Turkey). After that, forward

Figure 1. Black cherry aphid colonies on the terminal growth of host plant (Denizli, Honaz).

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Figure 2. Sampling locations of Myzus cerasi in different geographical regions of Turkey (Google Earth). and reverse chromatograms were assembled and aligned marker that determines genetic distances among aphid using Clustal X 2.1. The nucleotide sequences were species (Komazaki et al., 2010; Lee et al., 2011; Shufran examined with the NCBI BLAST tool to compare them and Puterka, 2011; Rebijith et al., 2013; Coeur d’Acier et al., with the other aphid species. 2014; Rakauskas, 2014; Chen et al., 2016; Zhu et al., 2017). 2.4. Phylogenetic analyses Here, based on a DNA barcoding approach, 67 analyzed All the incomplete sequences with ambiguous nucleotides nucleotide sequences of M. cerasi were compared with were checked or removed, and ultimately 67 COI sequences database data by the NCBI BLAST tool. The coding region were obtained from 26 Myzus cerasi populations. In addition of the COI gene was 605 bp, which included 40 variable to the 67 sequences of Myzus cerasi, ten sequences were sites, 565 conserved sites, 37 singletons, and 3 parsimony- selected from the NCBI. The construction of the haplotype informative sites (219th, 300th, and 321st nucleotides). network by Rakauskas et al. (2014) showed that five of the The sequences in all M. cerasi populations had an average COI sequences were different (KF754337-HAP1- M. cerasi nucleotide composition of 41.3% T, 33.9% A, 12.8% C, cerasi, KF754302-HAP2- M. cerasi cerasi, KF754349- and 12.1% G. Commonly, the majority of the nucleotide HAP3- M. cerasi pruniavium, KF754312-HAP4- M. cerasi composition in aphids is composed of adenine and thymine pruniavium, KF754315-HAP4- M. cerasi pruniavium, (Rebijith, 2013; Rakauskas et al., 2014; Zhu et al., 2017). and KF754350-HAP5- M. borealis, KF754348, KF754343, To select the best-fit nucleotide substitution model KF754334, AB506741). Best model tests were performed to for 77 aligned sequences, model testing was used in get the most reliable branch lengths and bootstrap support constructing phylogenetic trees. The mean intraspecific values. Maximum likelihood (ML), neighbor joining (NJ), genetic divergence was estimated to be 0.3% using and minimum evolution (ME) trees were constructed by the Tamura 3-parameter model (Tamura et al., 2011). MEGA 7 based on the aligned sequences. Myzus persicae Aphids have the lowest intraspecific distance among the and Myzus borealis were included as outgroups for the groups despite a large shift in biological traits, phylogenetic analyses. The Tamura 3-parameter model host preference, and insecticide susceptibility (Komazaki (T92+G) of base substitution (Tamura et al., 2011) was et al., 2010; Coeur d’Acier et al., 2014). As reported selected in pairwise distance calculations for ML. The by Guillemaud et al. (2003) and Sanchez et al. (2013), Kimura 2-parameter (K2P) model (Kimura, 1980) was migration in relation to host plant alteration reduces the used for NJ and ME trees. genetic differentiation in cyclically parthenogenic aphids. The mode of reproduction is another factor influencing 3. Results and discussion genetic diversity. Aphids reproducing by parthenogenesis The mitochondrial cytochrome oxidase c subunit 1 (COI) have low genetic diversity on local and worldwide scales gene is considered to be the most suitable molecular (Carletto et al., 2009; Chen et al., 2013).

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Table 1. Analyzed samples of black cherry aphid populations Additionally, the average transition (ti)/transversion with collection date and coordinates. bias based on the point or base substitution was R = 1.69 (Table 2). Our estimate of the transition/transversion ratio Population Collection date Coordinates is similar to the finding of Rakauskas et al. (2014) for partial COI sequences of M. cerasi from European countries. 40°44′11.05″N Amasya 2013 28.05.2013 Four haplotypes were determined as a result of 35°57′21.05″E phylogenetic dendrograms generated by different methods 37°49′12.68″N Isparta/Eğirdir 22.05.2014 such as neighbor joining, maximum likelihood, and 30°52′28.10″E minimum evolution trees (Figures 3, 4, and 5). A large part 38°31′28.76″N Sultandağı 04.06.2013 of the M. cerasi populations are included in Haplotype A. 31°15′03.87″E Remarkably, the Isparta populations were clustered with 39°57′44.47″N Batıkent 02.06.2014 Myzus borealis, diverging from all the groups (Figures 3, 32°43′54.48″E 4, and 5). These conclusions are concordant with the DNA 38°31′19.98″N Sultandağı 2015 01.06.2015 31°14′47.65″E sequence analyses of M. cerasi that occurs as paraphyletic 38°25′31.40″N to M. borealis and differs by 0.2%–0.5% of the COI Manisa/Salihli 1 20.05.2015 28°6′56.34″E sequences (Rakauskas et al., 2014). 38°25′25.43″N The mitochondrial genome has many advantages such as Manisa/Salihli 2 20.05.2015 28°6′35.21″E the absence of introns, limited exposure to recombination, 37°2′25.26″N high number of copies in all cells, haploid characteristics, Konya/Hadim 1 25.05.2015 32°29′47.33″E and maternal inheritance compared to the nuclear genome. 37°2′33.32″N In addition to these advantages, the evolution of the COI Konya/Hadim 2 25.05.2015 32°38′10.97″E gene has allowed for the separation of close species and 40°6′32.71″N has taken place at a rate that could produce intraspecific Bursa/Kestel 1 14.05.2015 29°21′55.79″E variation that is related to geographical structure (Keskin 40°7′52.32″N Bursa/Kestel 2 14.05.2015 and Atar, 2013). However, it can be expected to find low 29°19′54.42″E levels of COI sequence divergence among different aphid 38°24′40.36″N İzmir/Kemalpaşa 1 21.05.2015 species. Hence, specific molecular markers should be 27°32′57.88″E chosen as standard DNA barcodes due to the low level of 38°26′32.17″N İzmir/Kemalpaşa 2 21.05.2015 genetic variation among species (Chen et al., 2012). 27°25′5.41″E In this study, while the majority of the populations 38°26′32.28″N İzmir/Kemalpaşa 3 21.05.2015 belonged to Haplotypes A and C with the subspecies M. ′ ″ 27°25 5.45 E cerasi pruniavium (KF754349-HAP3, KF754312-HAP4, 40°12′41.15″N Ankara/Güdül 1 15.06.2015 KF754315-HAP4), Kars and Kayseri formed Haplotype 32°12′28.12″E D with M. cerasi cerasi (KF754337-HAP1, KF754302- 40°12′40.36″N Ankara/Güdül 2 15.06.2015 HAP2) (Figures 3, 4, and 5). Thus, it is considered that 32°14′27.31″E Myzus cerasi has two subspecies in Turkey. Likewise, 37°43′27.55″N Denizli/Honaz 1 22.05.2015 29°15′41.80″E Myzus cerasi samples collected mainly from Prunus avium 37°46′8.26″N and Prunus cerasus in Europe formed two major clades Denizli/Honaz 2 22.05.2015 29°15′18.65″E referring to two host-specific subspecies and the average 37°46′19.49″N genetic divergence of COI sequences between two clades Denizli/Honaz 3 22.05.2015 29°17′11.15″E was reported to be 0.2% (Rakauskas et al., 2014). However, 40°41′57.55″N contrary to the opinion on host specificity, oligophagous Amasya 1 02.06.2015 35°57′16.56″E or polyphagous feeding patterns of black cherry aphid 40°41′9.82″N should be reassessed if we take into consideration that all Amasya 2 02.06.2015 35°57′24.55″E the M. cerasi populations, except Kars, were collected from 40°44′35.62″N the same host (Prunus avium) and the same variety. As a Amasya 3 02.06.2015 35°57′34.88″E matter of fact, the reasons for the monophagy, oligophagy, 40°5′10.41″N Ankara/Kalecik 13.05.2015 or polyphagy of numerous aphids can be physiological and 33°27′1.21″E phylogeographic (Blackman and Eastop, 2006). 40°19′53.75″N Kars/Sarıkamış 18.05.2015 In conclusion, the first study based on mitochondrial 42°34′40.04″E COI sequence analysis of M. cerasi populations collected 38°22′56.01″N Kayseri/Develi 25.05.2015 from different geographical regions in Turkey shows four 35°28′16.07″E different haplotypes. Different phylogenetic analyses

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Data Domain info 201.00 202.00 202.00 605.00 Total 1.00 30.00 42.00 73.00 GG 0.00 0.00 0.00 0.00 GA 0.00 0.00 0.00 0.00 GC 0.00 0.00 0.00 0.00 GT 0.00 0.00 0.00 0.00 AG 94.00 36.00 205.00 75.00 AA 0.00 0.00 0.00 0.00 AC 0.00 0.00 0.00 0.00 AT . 0.00 0.00 0.00 0.00 CG M. cerasi 0.00 0.00 0.00 0.00 CA 6.00 49.00 22.00 77.00 CC 1.00 0.00 1.00 0.00 CT 0.00 0.00 0.00 0.00 TG 0.00 0.00 0.00 0.00 TA 0.00 0.00 0.00 0.00 TC 99.00 87.00 63.00 249.00 TT 1.63 nc 3.00 1.69 R 1.00 0.00 0.00 1.00 sv 1.00 0.00 1.00 0.00 si 199.00 202.00 202.00 603.00 ii Transition and transversion ratio in cytochrome oxidase subunit I sequences of of I sequences subunit oxidase in cytochrome ratio transversion and Transition Avg Domain 3rd 2nd 1 1st All frequencies are averages (rounded) taxa.all over (rounded) averages are frequencies All Pairs Identical = ii Pairs = Transitional si Pairs sv = Transversional R = si/sv Table 2. Table

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Figure 3. A neighbor joining tree using cytochrome c oxidase subunit 1 sequences from Myzus cerasi populations.

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Figure 4. A maximum likelihood tree using cytochrome c oxidase subunit 1 sequences from Myzus cerasi populations.

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Figure 5. A minimum evolution tree using cytochrome c oxidase subunit 1 sequences from Myzus cerasi populations.

153 İNAL and KANDEMİR / Turk J Zool indicate that most of the populations are in the same group morphologically similar aphid species and some species with Myzus cerasi pruniavium, while populations collected groups could not be distinguished by mitochondrial gene at Kars and Kayseri clade with the haplotype of Myzus sequences (Chen et al., 2012). Although COI barcoding is cerasi cerasi. Although it is considered that M. cerasi has a useful identification tool for aphids in pest management two different subspecies in Turkey, these findings should be (Coeur d’Acier et al., 2014), detailed research using supported by morphometric analysis. different molecular markers such as microsatellites or Populations from Isparta particularly diverged from the nuclear genomes should be applied to evaluate the current rest of the studied populations, forming a different clade status of the Isparta population and the molecular basis of with Myzus borealis. Considering that Isparta produces the host specificity of the aphid species. 15% of the cherries in Turkey, it can be concluded that mutations resulting after insecticide applications in this Acknowledgments region or the biogeographic factors of Isparta province We would like to offer special thanks to Dr. Işıl Özdemir might lead to this separation. Some genetic studies of aphids (Directorate of Plant Protection Central Research Institute, based on microsatellite and mitochondrial DNA suggest Ankara) for identifying the aphid samples. We would that insecticide selection pressure, landscape features, and also like to thank Amalia N. Kati (Aristotle University of the suitable habitats surrounding the aphid populations Thessaloniki) for kindly providing the aphid samples from can influence genetic diversity (Dong et al., 2018). It should Greece. Sincere thanks to Dr. Gülten Yazıcı for her helpful also be pointed out that DNA barcodes may fail to identify comments on the manuscript.

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