Appl Entomol Zool DOI 10.1007/s13355-014-0248-0 ORIGINAL RESEARCH PAPER DNA barcoding reveals the occurrence of cryptic species in host-associated population of Conogethes punctiferalis (Lepidoptera: Crambidae) P. R. Shashank • A. K. Chakravarthy • B. R. Raju • K. R. M. Bhanu Received: 18 November 2013 / Accepted: 26 January 2014 Ó The Japanese Society of Applied Entomology and Zoology 2014 Abstract Conogethes punctiferalis (Gue´nee) is a critical differentiations among the two sampled populations, pest that commonly infests castor (Ricinus communis reflecting limited gene flow. Neutrality tests and mismatch Linnaeus) and cardamom (Elettaria cardamomum Maton) distributions showed population expansion in C. punctif- in India. The moths of both castor and cardamom appear to eralis, while the results of an analysis of molecular vari- be similar in wing pattern and color. However, the results ance (AMOVA) indicated the existence of significant of behavioral studies elicited a doubt that there may be genetic variation among the examined host races. Con- differences in terms of host specialization. In the present clusively, analysis using mitochondrial DNA showed an study, we conducted morphological studies and DNA amount of genetic divergence between the two host-asso- barcode analyses using cytochrome oxidase I gene, which ciated populations compatible with cryptic species rather unraveled the mystery of C. punctiferalis breeding on than host races. castor and cardamom. The differences in male aedeagus and female bursae were prominent, yet, not sufficient Keywords Conogethes Á Barcoding Á Castor Á enough to say that they are different species. The results Cardamom Á Cryptic species showed high haplotype diversity (0.817 ± 0.073) and nucleotide diversity (0.0285 ± 0.002) in C. punctiferalis. In addition, topologies of neighbor-joining trees indicate that Conogethes sp. breeding on castor belongs to C. Introduction punctiferalis while those on cardamom are of a separate clade. Further genetic analysis revealed significant genetic The accurate identification of insect species is one of the important aspects in entomological science. In most groups, traditional taxonomic research is based on mor- Electronic supplementary material The online version of this phological characters. It is not impossible to identify article (doi:10.1007/s13355-014-0248-0) contains supplementary material, which is available to authorized users. cryptic and polymorphic species through morphological taxonomy. However, there are some difficulties that P. R. Shashank (&) Á A. K. Chakravarthy depend on experts. For this purpose, molecular methods Department of Agricultural Entomology, Gandhi Krishi Vigyan have been found valuable in discriminating cryptic species Kendra, University of Agricultural Sciences, Bangalore 560065, India of insects (Jackson and Resh 1998; Pilgrim et al. 2002). e-mail: [email protected] Cryptic species are defined as two or more distinct species classified as a single nominal species because they are B. R. Raju morphologically indistinguishable (Bickford et al. 2007). Department of Crop Physiology, Gandhi Krishi Vigyan Kendra, University of Agricultural Sciences, Bangalore 560065, India One of the mechanisms thought to promote speciation in phytophagous insects is shifts to new hosts that lead to the K. R. M. Bhanu establishment of new species via an intermediate step of A Division of Pest Control (India) Pvt. Ltd., Bio-Control Research Laboratories, 36/2, Sriramanahalli. Nr. Rajankunte, host-race formation (Bush 1969; Dres and Mallet 2002). Dodballapur Road, Bangalore 561 203, India The occurrence of insect host races exhibit recently 123 Appl Entomol Zool evolved genetic differentiation with respect to host-plant Entomology, UAS, Bangalore) and transferred to plastic use (Dres and Mallet 2002). jars of dimensions 37 9 25 9 25 cm3 for rearing Conogethes punctiferalis (Gue´nee) is commonly refer- (25 ± 4 °C; 75 ± 5 % RH; 16:8 LD). The jars were cov- red to as yellow peach moth, cardamom stem borer, or ered with muslin cloth and the larvae were fed daily until castor shoot and fruit borer. Larvae of this moth are con- adult emergence. Male and female moths were pinned sidered polyphagous, attacking more than 120 wild and through the thorax using Bohemia Insect pinsÒ (No. 3). cultivated plants, including peach, chestnut, durian, citrus, Antennae and wings were stretched and each specimen was papaya, ginger, eggplant, and maize (Sekiguchi 1974). The labeled with information on the date of collection, locality, genus Conogethes Meyrick, 1884 (Lepidoptera: Crambi- latitude, longitude, elevation, name of collector, and host dae) is a large, taxonomically complex taxon of crambids on which it was collected. The specimens were dried in a distributed throughout the Oriental, Australian and tropical hot air oven at 40 °C for 48 h and preserved in an insect Asian regions. In Japan, Koizumi (1960) identified and cabinet box (45 9 30 cm). named two different types of C. punctiferalis, as the Fruit Tree Type and Conifer Type from angiosperms and Pina- Morphology of adult moths ceae gymnosperms, respectively, for convenience. Most of the work on the genus Conogethes is in progress in Japan, For genitalia preparation, the abdomen of male and female China, and Australia, and the species have been barcoded moths were separated using a pair of micro scissors and (Armstrong 2010). placed in a test tube containing 10 % KOH solution. The Earlier attempts made on the taxonomic studies by abdomen was left in the solution overnight or until it was Chakravarthy (1985) (personal communication) in Karna- cleared. Genitalia were then transferred to a cavity block taka revealed differences in the populations of C. punctif- containing water and washed repeatedly to remove excess eralis. The Conogethes larvae reared on castor bean and KOH. Later, they were placed in glycerol on a slide for cardamom required two different mass-rearing techniques further dissection. Genitalia were pulled out from the (Chakravarthy et al. 1991). Visual observations showed abdomen under a stereo binocular microscope. After distinct differences in the size of an adult and in the feeding examination, the parts of the specimens were transferred to behavior of larvae infesting castor and cardamom. Cono- a microvial containing glycerol (98 % pure) and the vial gethes punctiferalis is considered as a major pest on the was pinned below the specimen. The terminologies of two crops that causes up to 63 % yield loss in castor and Klots (1965) were adopted to describe the genitalia. more than 20 % yield loss in cardamom (Kapadia 1996). Hampson (1896) and Inoue and Yamanaka (2006) classi- The differences in habitat of castor and cardamom and in fication will be used as the basis for identification of the morphological characteristics of C. punctiferalis moths specimens collected during the study. Photographs were infesting both crops elicit a doubt that the C. punctiferalis taken using a Leica DFC 425 mounted on a Leica M205C. bred on castor (CBR) and on cardamom (CBE) may be different host races or cryptic species. DNA isolation and polymerase chain reaction The cytochrome oxidase subunit I (COI) region of mtDNA is particularly useful for determining intra- and Genomic DNA was isolated from insect legs using a interspecific phylogenetic relationships at the genus and DNeasy animal tissue kit following the manufacturer’s species level (Caterino and Sperling 1999; Trewick 2000) instructions (Qiagen, Valencia, CA). The rest of the insect and within families (Logan 1999). Here, we show differ- body was used as the specimen voucher in the Biosyste- ences between the COI region of the mitochondrial gene of matics Laboratory, Division of Entomology, UAS, Ban- C. punctiferalis reared from castor and the COI region of galore. The DNA was quantified by analyzing it on 0.8 % C. punctiferalis reared from cardamom in India, along with agarose gel with diluted uncut k DNA as standard. The demographic analysis and morphological descriptions. universal barcode primer described by Folmer et al. (1994) (LCO-50-GGT CAA CAA ATC ATA AAG ATA TTG G-30; HCO-50-TAA ACT TCA GGG TGA CCA AAA Materials and methods AAT CA-30) specific to mitochondrial cytochrome oxidase I (COI) was used in the present study. The polymerase Sampling chain reaction (PCR) steps were carried out using sterile PCR tubes. The master mixture consisted of 25 ng of The larvae of the shoot and fruit borer were collected from genomic DNA, 10 pmol each of forward and reverse different castor and cardamom growing areas in India primers, 10X PCR buffer, 0.3 U Taq polymerase (Fer- (Table 1). The larvae collected from the fields were mentas GmBH, St. Leon-Rot, Germany), 2 mM dNTP, and brought to the laboratory (Department of Agricultural 2 mM MgCl2. The reaction volume was brought to 50 ll 123 Appl Entomol Zool Table 1 Details of the samples used in the analyses Specimens Collection sites Co-ordinates Hosts GenBank accession Reference ID no. RBA Bangalore, Karnataka, India 13.082N, Ricinus communis L. JX064410 This study 77.570E RGU Junagadh, Gujarat, India 21.520N, Ricinus communis L. JX064411 This study 70.470E RGU1 Junagadh, Gujarat, India 21.520N, Ricinus communis L. JX064412 This study 70.470E RPU Ludhiana, Punjab, India 30.909N, Ricinus communis L. JX064413 This study 75.633E RPU1 Ludhiana, Punjab, India 30.909N, Ricinus communis L. JX064414 This study 75.633E RMP Chhindwara, Madhya Pradesh, India 22.069N, Ricinus communis L. JX064415 This study 78.930E RMP1 Chhindwara,