High Regional Genetic Diversity and Lack of Host-Specificity in Ostrinia Nubilalis (Lepidoptera: Crambidae) As Revealed by Mtdna Variation
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Bulletin of Entomological Research (2016) 106, 512–521 doi:10.1017/S0007485316000195 © Cambridge University Press 2016 High regional genetic diversity and lack of host-specificity in Ostrinia nubilalis (Lepidoptera: Crambidae) as revealed by mtDNA variation M. Piwczyński1*, M. Pabijan2, A. Grzywacz1, W. Glinkowski1, P.K. Bereś3 and J. Buszko1 1Chair of Ecology and Biogeography, Nicolaus Copernicus University in Toruń, Lwowska 1, PL-87-100 Toruń, Poland: 2Department of Comparative Anatomy, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland: 3Institute of Plant Protection – National Research Institute, Regional Experimental Station, Langiewicza 28, 35-101 Rzeszów, Poland Abstract The European corn borer (Ostrinia nubilalis) infests a wide array of host plants and is considered one of the most serious pests of maize in Europe. Recent studies suggest that individuals feeding on maize in Europe should be referred to O. nubilalis (sensu nov.), while those infesting dicots as Ostrinia scapulalis (sensu nov.). We test if the clear genetic distinctiveness among individuals of O. nubilalis living on maize vs. di- cots is tracked by mitochondrial DNA (mtDNA). We used fragments of COI and COII genes of 32 individuals traditionally recognized as O. nubilalis collected on three host plants, maize, mugwort and hop, growing in different parts of Poland. In addition, we reconstructed the mtDNA phylogeny of Ostrinia species based on our data and sequences retrieved from GenBank to assess host and/or biogeographic patterns. We also compared haplotype variation found in Poland (east-central Europe) with other regions (Anatolia, Eastern Europe, Balkans, Far East, North America). Our study showed high mtDNA diversity of O. nubilalis in Poland in comparison with other regions and revealed rare haplotypes likely of Asian origin. We did not find distinct mtDNA haplotypes in larvae feeding on maize vs. dicotyledonous plants. Phylogenetic analyses showed an apparent lack of mtDNA divergence among puta- tively distinct lineages belonging to the O. nubilalis group as identical haplotypes are shared by Asian and European individuals. We argue that human-mediated disper- sal, hybridization and sporadic host jumps are likely responsible for the lack of a geo- graphic pattern in mtDNA variation. Keywords: COI, COII, European corn borer, genealogical sorting index (gsi), nucleotide diversity (π), Ostrinia scapulalis (Accepted 14 February 2016; First published online 28 March 2016) Introduction The European corn borer, Ostrinia nubilalis (Hübner) (Insecta: Lepidoptera: Crambidae), is naturally distributed in *Author for correspondence Europe, western Asia and north-west Africa and infests a Phone: +48 56 611 26 49 wide array of host plants Mutuura & Munroe (1970). North Fax: +48 56 611 44 43 American populations of this species are thought to have ori- E-mail: [email protected] ginated from accidental introductions in the early 20th century Genetic diversity of Ostrinia nubilalis 513 (Thompson & Parker, 1928). In Poland and other European 2008; Bourguet et al., 2014). A COII-based phylogeny showed countries, O. nubilalis is found mainly on mugwort that O. furnacalis (Guenée) (Asiatic corn borer) formed a sister (Artemisia vulgaris L.; Wocke, 1874), hop (Humulus lupulus L.; clade to the O. nubilalis group which included O. orientalis, Büttner, 1880; Romaniszyn & Schille, 1930), hemp (Cannabis O. scapulalis, O. narynensis and O. kurentzovi Mutuura & sativa L.; Romaniszyn & Schille, 1930) and maize (Zea mays Munroe (Hoshizaki et al., 2008). Most of these phylogenetic L.; Wocke, 1874). The European corn borer is one of the most and population studies sampled individuals from Asia and serious pests of maize in Europe. Extensive damage to crops to a lesser extent from Europe. For example, the majority of by O. nubilalis has been reported for Hungary, Italy, Spain, COII sequences available in GenBank for Ostrinia come from France, Germany and Poland. Meissle et al. (2010) found that samples collected in Japan and China. There are only a few se- in infested areas O. nubilalis occurs in a large proportion quences from Europe (e.g. Slovenia and France; Hoshizaki of maize fields, ranging from 20% (in Hungary) to 60% et al., 2008). In the case of COI, only sequences from Asian (in Spain), and estimated yield losses at between 5 and 30%. and North American specimens are available. Moreover, the In Poland O. nubilalis has caused serious damage to maize majority of studies used restriction enzymes to investigate since the 1950s (Kania, 1966). Currently, this pest is found haplotype variation of the COI gene (e.g., Martel et al., 2003; throughout the country (Bereś & Konefał, 2010). In regions Bourguet et al., 2014) and in consequence fewer sequences with intense maize cultivation (southern Poland) this species are available in GenBank than for COII. damages 50–80% and locally up to 100% of plants, causing a In this paper, we explore variation in mitochondrial DNA direct loss in grain yield of 20–30%, and sometimes up to 40% (mtDNA) using fragments of COI and COII genes of indivi- (Lisowicz & Tekiela, 2004). duals traditionally recognized as O. nubilalis collected on Taxonomic revision of the genus Ostrinia by Mutuura & three of the most common host plants: maize, mugwort and Munroe (1970) placed the European corn borer within the hop. These study centers on East-Central Europe (different group of trilobed uncus species and the subgroup character- parts of Poland), a region in which various host plants of ized by males lacking a row of enlarged scales or a tuft of hair- O. nubilalis occur, but for which no information is available like scales on mid-tibia, i.e. the so-called ‘small mid-tibia’ to date. We test if the clear genetic distinctiveness among indi- phenotype. However, a number of studies have put into ques- viduals of O. nubilalis living on maize vs. dicots found at tion many traditional relationships within the genus since the nuclear microsatellite loci is also tracked by mtDNA. We sub- publication of Mutuura and Munroe’s monograph. Although sequently compare the mtDNA variation with results of other the division based on the shape of the uncus seems valid (Kim studies and contribute to unraveling the biogeographic history et al., 1999), the relationships within the trilobed group have and genetic variability of this serious pest in Europe. been strongly contested. For example, the mid-tibia morph- ology exhibits a simple Mendelian pattern of inheritance and extensive hybridization between species differing in this trait Material and methods has been shown (Frolov, 1981, 1984). Frolov et al. (2007) con- Sampling cluded that this group should be divided according to host type instead of mid-tibia morphology. They proposed that in- Thirty-two larvae of O. nubilalis were sampled from maize dividuals feeding on maize in Europe should be referred to as (19), mugwort (eight) and hop (five) from 12 localities in O. nubilalis (sensu nov.) while those infesting dicotyledonous Poland in 2012, 2013 and 2015 (fig. 1, Table S1). Plant material species such as mugwort, hemp and hop across Eurasia as was transported from the field to the laboratory where larvae Ostrinia scapulalis (sensu nov.) (Walker). Interestingly, signifi- were removed from stems, documented and immediately pre- cant divergence between O. nubilalis populations inhabiting served in 70 or 96% ethanol. Photographs of larvae and pre- maize and dicots, including prezygotic reproductive isolation, served material are available from the first author on request. was suggested much earlier (Judenko, 1938; Karpova, 1959; Frolov, 1984). Additionally, Frolov et al. (2007) synonymized DNA extraction, amplification and sequencing two Asian species, Ostrinia narynensis Mutuura & Munroe and Ostrinia orientalis Mutuura & Munroe, with O. scapulalis Total genomic DNA was isolated from approximately half (sensu nov.). According to the new division, O. scapulalis in- of each larvae using the DNeasy Blood & Tissue Kit (Qiagen, cluded all three mid-tibia morphotypes (small, medium and Valencia, CA, USA). A ca. 1.2 kb fragment of the COI gene was large), whereas O. nubilalis only encompassed individuals PCR-amplified using primers Ron and Pat (Dopman et al., with the small phenotype. Further studies based on microsat- 2005), whereas the entire COII region was PCR-amplified ellite variation corroborated this taxonomic treatment show- using primers O-tLEU and B-tLYS (Liu & Beckenbach, 1992; ing two genetic clusters corresponding to maize-derived Kim et al., 1999). Each 25 µl PCR was prepared using 1 × PCR – individuals and those infesting dicotyledons in Europe and buffer, 0.2 mM dNTPs, 0.2 µM of each primer, 3 4 mM MgCl2, Asia (Frolov et al., 2012; Bourguet et al., 2014). 1–2 U of Taq DNA polymerase (Qiagen) and 1 µl of DNA tem- Mitochondrial genes, especially cytochrome oxidase subu- plate. PCR cycles for the COI gene included initial denaturation nits I and II (COI and COII thereafter) are commonly used in at 94°C for 5 min, followed by 30 cycles comprising 45 s. phylogenetic, population and biogeographical studies of denaturation at 94°C, 45 s. annealing at 50°C, and 1:30 min. ex- Lepidoptera (e.g. Dopman et al., 2005; Hoshizaki et al., 2008; tension at 72°C. PCR cycles for the COII gene included initial Wiemers et al., 2010; Zhou et al., 2012). The COI fragment is denaturation at 94°C for 5 min., followed by 32 cycles com- a universal barcode for insects (Ratnasingham & Hebert, prising 30 s. denaturation at 94°C, 1 min. annealing at 48°C 2007), and is the most abundantly available marker in and 2 min. extension at 70°C. Both PCR cycling schemes GenBank for the majority of taxonomic groups (e.g. included final extension at 72°C for 10 min. Piwczyński et al., 2014). Both markers have been used for Each PCR product was electrophoresed in a 1% agarose gel population and phylogenetic studies of trilobed Ostrinia spe- and stained with ethidium bromide. No obvious polymorph- cies (Kim et al., 1999; Coates et al., 2004; Hoshizaki et al., ism (multiple bands from a single PCR product) was observed.