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 (: )

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 ( cardamomum Maton) distributions showed population expansion in C. punctif- in India. The 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 Barcoding Castor enough to say that they are different species. The results Cardamom 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 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 . However, there are some difficulties that P. R. Shashank (&) 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 (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 , 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, 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 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, Madhya Pradesh, India 22.069N, Ricinus communis L. JX064416 This study 78.930E RHY Hyderabad, Andhra Pradesh, India 17.366N, Ricinus communis L. JX064418 This study 78.466E RHA1 Hassan, Karnataka, India 13.000N, Ricinus communis L. JX064419 This study 76.100E RHA2 Hassan, Karnataka, India 13.000N, Ricinus communis L. JX064420 This study 76.100E EMU Mudigere, Karnataka, India 13.130N, Elettaria cardamomum JX064422 This study 75.633E Maton EMU3 Mudigere, Karnataka, India 13.130N, Elettaria cardamomum JX064423 This study 75.633E Maton EMU2 Mudigere, Karnataka, India 13.130N, Elettaria cardamomum JX064424 This study 75.633E Maton EBA Bangalore, Karnataka, India 13.082N, Elettaria cardamomum JX064425 This study 77.570E Maton EBA1 Bangalore, Karnataka, India 13.082N, Elettaria cardamomum JX064426 This study 77.570E Maton EKE Idduki, Kerala, India 9.850N, Elettaria cardamomum JX064427 This study 76.940E Maton EKE1 Idduki, Kerala, India 9.850N, Elettaria cardamomum JX064428 This study 76.940E Maton ECO Kodagu, Karnataka, India 12.420N, Elettaria cardamomum JX064429 This study 75.739E Maton KC136065 Gumi, Gyeongsangbuk-do, South Korea 36.143N, – KC136065.1 NCBI 128.342E KC136063 Kohung, Jeollanam-do, South Korea 34.552N, – KC136063.1 NCBI 127.391E KC158239 Papua New Guinea 5.231S, – KC158239.1 NCBI 145.182E KC158238 Papua New Guinea 5.231S, – KC158238.1 NCBI 145.182E JX970237 Milne Bay, Louisiade, Papua New 10.65S, – JX970237.1 NCBI Guinea 152.80E JX970236 Milne Bay, Louisiade, Papua New 10.65S, – JX970236.1 NCBI Guinea 152.80E JX970235 Milne Bay, Louisiade, Papua New 10.67S, – JX970235.1 NCBI Guinea 152.80E JX970234 Milne Bay, Louisiade, Papua New 10.67S, – JX970234.1 NCBI Guinea 152.80E KC682499 China – – KC682499.1 NCBI

123 Appl Entomol Zool using sterile double distilled water. The entire exercise was distribution analyses were performed for all the sampling carried out over ice and the PCR tubes were immediately locations combined and each region separately to find the loaded into the thermal cycler (Eppendorf, Hamburg, evidence of past demographic expansions using DnaSP Germany) to prevent the amplification of non-specific 5.10.01 (Librado and Rozas 2009). products. The PCR reaction process was as follows: 94 °C Neutrality tests like Tajima’s D (1989), Fu and Li’s pre-denaturation, 94 °C denaturation for 1 min., annealing D (1993), and Fu’s FS(1997), were also calculated by at 52 °C for 30 s, and primer extension at 72 °C for 1 min DnaSP 5.10.01 (Librado and Rozas 2009) for investigating for 35 cycles of polymerization and a final primer exten- the historical population demographics and testing whether sion at 72 °C for 5 min. The amplification product was the sequences conformed to the expectations of neutrality. preserved in the refrigerator at 4 °C. A 5 ll aliquot of PCR We calculated pairwise FST using Arlequin 3.5 (Excoffier product were resolved on a 1 % agarose gel that contained and Lischer 2010), which accurately reflects patterns of 0.5 lg/ml ethidium bromide at 100 V for 30 min, and data genetic variation. Correspondingly, gene flow was esti- was acquired under a UV Tran-illuminator and photo- mated in Arlequin 3.5 (Excoffier and Lischer 2010). Two- graphed immediately for further interpretation using the level hierarchical analyses of molecular variance (AM- Gel-Doc system (BioRad, Hercules, CA). The remaining OVA) were conducted to evaluate possible population 45 ll of PCR product was purified using Qiaquick PCR genetic structure of Conogethes infesting castor and car- purification kits (Qiagen, Valencia, CA) following the damom using Arlequin 3.5 with 1,000 permutations. All manufacturer’s protocols. Purified PCR products are the sequences were verified using a GenBank BLAST sequenced in an automated sequencer (ABI Prism 3730; search and the Barcode of Life Database (BOLD) (Rat- Applied Biosystems, USA) at the specific commercial nasingham and Hebert 2007). facilities (SciGenome, India).

Data analysis Results

COI sequences in FASTA format were imported into the Morphological description of adult moths sequence alignment application of MEGA 5.05 (Tamura et al. 2011) software package and multiple sequence The moths of the CBR were pale yellowish with series of alignments were performed with the ClustalW (Jeanmou- black spots over the body (Fig. 1). Male moths had a dis- gin et al. 1998) algorithm using default parameters. The tinct black tuft of hair on tip of the abdominal segment. The Basic Local Alignment Search Tool (BLAST) (Altschul moths of the CBE were a straw yellow color with series of et al. 1990) was used to query the National Center for black spots over the body. The moths were larger in size. Biotechnology Information (NCBI) non-redundant nucle- Male moths had the anal tuft that was faded black with otide database and protein database with C. punctiferalis sparse hairs. The front of the head in both the CBR and COI sequence data in blastn and blastx searches, respec- CBE was covered with pale yellowish scales. The vertex tively. The sequence details were analyzed carefully and had dense, whitish-yellow scales. The antenna was filiform, base substitution mutations were identified manually. The brownish colored, covered with pale yellowish scales, and sequences were submitted to NCBI for GenBank Acces- between each flagellomere there were short cilia. The labial sions (Table 1). palpi were three segmented, upturned, and covered with Sequence divergences between individual moths feeding yellowish scales. The proboscis had black scales at the on castor and cardamom were calculated using the Kimura base. The patagium had a black spot on either side. The 2-Parameter distance model (Kimura 1980) and graphically tegula was covered with pale yellowish scales. displayed in a neighbor-joining (NJ) tree (Saitou and Nei The prothorax was marked by pale yellowish scales and 1987) by the program MEGA 5.05 (Tamura et al. 2011). had two black spots anterolaterally. The mesothorax had Tree robustness was evaluated by bootstrapping (Felsen- two black spots dorsolaterally. The mesoscutum was straw stein 1985) with 2,000 replicates with the Bombyx mori yellow and had a large round black spot dorsally. The COI sequence (GenBank # DQ415450: India) and Chilo metathorax was straw yellow and had three black spots, polychrysa COI sequence (GenBank # KC631647: India) one dorsal and the other two dorsolateral in both males and as outliers. females of the CBR. In the CBE, the prothorax had pale The number of haplotypes (h) and nucleotide (p) yellowish scales and two black spots anterolaterally. The diversities for the CBR and CBE were estimated using the mesothorax had two black spots dorsolaterally. The mes- software DnaSP 5.10.01 (Librado and Rozas 2009) and oscutum was straw yellow and had a large round black spot haplotype network constructed by NETWORKÒ 4.6.1.1. dorsally. The metathorax had only two spots, both of which software (Bandelt et al. 1999). Pairwise mismatch were dorsolateral. 123 Appl Entomol Zool

Fig. 1 Moths of Conogethes punctiferalis breeding on castor and cardamom

In both the CBR and CBE, the forewings were a bright spots were placed between vein Rs to CuA2, of which a straw-color on the dorsal side and paler on the ventral side. spot placed between M1 and M2 was displaced inwardly. The costal margin had black yellow scales, and the apical The forewing length of male and female moths of the margin and anal margin were covered with pale yellow CBR was 9.79 ± 0.171 mm and, 10.61 ± 0.138 mm, fringed scales. Four black spots were present at the base of respectively. The width of the forewing in the case of the forewing, one at the base of costa, one below, one below CBR (males) was 4.40 ± 0.094 and for females was the discal cell and another one on the inner margin. A black 4.84 ± 0.086 mm, respectively (n = 25). The forewing spot was on the middle of the discal cell. A short black bar length of cardamom moths (males) was 11.37 ± 0.273 mm. was seen on the discocellular vein. A series of antemedial, In the case of female cardamom moths, the forewing length medial, post medial, and submarginal black spots were was 12.60 ± 0.242 mm. The width of the forewing in the present. In the antemedial series, three black spots were case of cardamom moths (males) was 5.22 ± 0.103 mm. situated outwardly oblique from the costa to the inner Whereas, the width of the forewing in females was margin. The medial series possessed four black spots 5.59 ± 0.072 mm in CBE (n = 25). The variation in length arising from the posterior angle of cell and inwardly obli- and width of the forewing between the castor and cardamom que toward the inner margin. The post medial series posses feeders was highly significant. ten spots, of which four were between the costa and M1, Concerning the male genitalia in both the CBR and outwardly oblique and almost banded together. The other CBE, the anterior margin of the tegumen was completely six spots deviated outwards between vein M1 and the inner sclerotized and the lateral arms were narrow. The uncus margin. Six spots were present in the submarginal series, was narrow, slender, ventrally curved, apically one-third which are moderately excurved between R4 and CuA2, of swollen, and evenly covered with the setae dorsally, and its which a spot placed between vein M1 and M2 is highly apical tip with two small, thin lobeless processes bearing a displaced inward. few bristles ventrally (looks like cattle egret, Bubulcus ibis The hind wings are paler than the forewings. A large head). The vinculum U shaped with the apex produced into black spot was present on the discocellular. An antimedial a conical point. The valve was broad at the apex and nar- series consisting of three spots placed between CuA2 and row at the base, somewhat broadly oval, with the saccular 2A was almost coalesced. In the post-medial series, seven margin weakly protruded. The sacculus was rather narrow, spots were present between vein Rs to 1A, of which spots sclerotized, and tapered with fringed hairs. The transtilla placed between M1 and M2, and CuA1 and CuA2 were was somewhat broad, with plate-like elements, weakly displaced inwardly. A submarginal series of six roundish sclerotized, each termen of the element was slightly

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Fig. 2 Male genitalia of Conogethes punctiferalis breeding on a castor and b cardamom. Lateral view of male genitalia of CBR and CBE (right vulva removed), c and d aedeagus of CBE and CBR, and e and f female genitalia of CBR and CBE, respectively

indented, and the two elements were closely approaching, sequence reads were trimmed based on base-call Phred but fused medially. The juxta was elongated and expanded quality parameter (Ewing et al. 1998) and used to generate basally. The saccus was short, tapered, and the anterior a 567-bp multiple sequence alignment. The analysis of margin was evenly rounded (Fig. 2a, b). However, the nucleotide composition showed a high A ? T content aedeagus in the CBR was very long, slender, and strongly (29.6 % A and 38.9 % T, averaged across the multiple curved near the base. The aedeagus in the CBE was smaller sequence alignment). The mean composition distance is than the CBR and strongly curved near the base (Fig. 2c, 0.019, which shows a low mean difference in base com- d). position per site. An annotation of 18 COI gene sequences In the female genitalia, the ovipositor of the CBR and showed 34 point mutations and hence, these are a substi- CBE was triangular and covered with a mixture of long and tution mutation type (Table 2). Out of 34, mutations at short setae. The apophysis anterioris was about as long as eight position changes, A to G or G to A, and 19 positions the length of the posterioris, and the anterioris was either involved C to T or T to C transitions. Transversion muta- thicker or thinner than the posterioris. The ostium was tions of T to G (336) and A to T (330, 339, 573) types were narrow, membranous, and funnel shaped. The ductus bursa also represented. The estimated ratio of transition to was narrow and very long in the CBR compared that of the transversion bias was (R) = 5.69 (Fig. 3). CBE being narrow and shorter. The corpus bursa was From 18 COI gene fragments (567 bp), eight haplotypes ovate, but irregular in shape and size (Fig. 2e, f). (six from CBR and two from CBE) were identified and the haplotype network was constructed and presented in Fig. 4. Sequence variation and genetic diversity The haplotype diversity (Hd), and nucleotide diversity (p) for each population of CBR and CBE are given in Table 3. Eighteen individuals of C. punctiferalis breeding on castor The haplotype frequency within the CBR and CBE col- (10 individuals) and cardamom (eight individuals) were lected from different locations was too low to delineate collected from six and four different localities, respectively them as different. (Table 1). The COI gene was amplified and sequenced Corrected (K2P) pairwise genetic distances among using the universal barcoding primer. The resulting individuals varied from 0.000 to 0.058. The highest genetic

123 plEtmlZool Entomol Appl Table 2 Nucleotide differences amongst mitochondrial COI gene sequences of Conogethes punctiferalis breeding on castor and cardamom

Sr. Base no. in the Base in the consensus RBA RGU RPU1 RMP RMP1 RHY RGU1 RHA2 RPU RHA1 EMU EMU3 EMU2 EBA EBA1 EKE EKE1 ECO No. consensus sequence sequence

16 G A 2 120 T CCCCCCCC 3 172 C TTTTTTTT 4 177 A GGGGGGGG 5 183 G AAAAAAAA 6 186 C TTTTTTTT 7 198 T CCCCCCCC 8 201 C TTTTTTTT 9 204 T CCCCCCCC 10 229 T CCCCCCCC 11 244 C TTTTTTTT 12 261 C TTTTTTTT 13 282 A G 14 312 T CCCCCCCC 15 321 T CCCCCCCC 16 330 A TTTTTTTT 17 334 G AAAAAAAA 18 336 T G 19 339 A TTTTTTTT 20 353 T CCCCCCCC 21 360 T CCCCCCCC 22 373 T CCCCCCCC 23 378 G AA 24 387 T CCCCCCCC 25 411 C T 26 414 T CCCCCCCC 27 426 C TTTTTTTT 28 435 A G 29 445 T CCCCCCCC 30 514 C TTTTTTTT 31 533 G AAAAAAAA 33 528 G AAAAAAAA 34 573 A T Total 34 0 0 0 0 0 0 3 1 2 1 27 26 26 26 26 26 26 26

123 Substitutions 34 0 0 0 0 0 0 3 1 2 1 27 26 26 26 26 26 26 26

Unfilled boxes show the base similarity to the respective base in the consensus sequence RBA1 RHA1 individuals from castor, EMU ECO individuals from cardamom Appl Entomol Zool

Fig. 3 Neighbor-joining (NJ) analysis of COI DNA barcode sequences for Conogethes punctiferalis breeding on castor and cardamom. The phylogeny was constructed with 29 COI sequences using MEGA 5Ò software

distance of 0.058 was evident between the CBE from study and 11 sequences from GenBank and outliers Mudigere and the CBR from Gujarat. The distance among (Table 1) using MEGA 5.05. Based on a strict-consensus CBRs of Bangalore, Hassan, Hyderabad, Madhya Pradesh, NJ tree, two major clades (excluding two outliers) were Punjab, and Mudigere ranged between 0.00 and 0.012. This recognized and those clades distinguish C. evaxalis from shows that the genetic variation is less in the Conogethes other Conogethes spp. The phylogenetic tree had a total population breeding on castor in different locations. The branch length of 1.1774 base substitutions per site. In the pairwise genetic distance of populations breeding on car- Conogethes spp clade there are four subclades, one con- damom from Bangalore, Mudigere, Coorg, and Kerala, sisting of 100 % CBE and the other three of CBR, C. varies from 0.000 to 0.002, which is again negligible. This punctiferalis, and Conogethes spp (Fig. 3). CBE and CBR suggests that there is not much variation within the popu- were found in two different clades in our study. While lations of CBR in different locations. While, the maximum 100 % of CBE had constituted one separate clade, the pairwise genetic distance between CBR and CBE was clade consisting of CBR also included C. punctiferalis 0.058, which is high enough to categorize them as cryptic from China and South Korea, confirming the fact that CBR species (Additional file 1). is the actual C. punctiferalis. The clade consisting of CBR was not monophyletic, but complex, as it includes indi- Phylogenetic analysis viduals collected from geographically distant locations. This suggests that CBR and CBE differed from each other, The neighbor-joining tree (NJ tree) was constructed based as evident from the NJ tree where they formed two separate on 29 sequences including 18 sequences from the present clades.

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Population structure analysis (p \ 0.001), suggesting significant genetic variation among the populations. Pairwise estimates of FST (0.950) and The AMOVA revealed that 94.57 % of the genetic varia- gene flow (Nm = 0.01) between CBR and CBE suggests tion was among populations and 5.47 % was within pop- that the subpopulation in this species are highly ulations (Table 4). The average UST value was 0.896 differentiated.

Fig. 4 Haplotype network (Median-Joining network) of COI DNA barcode sequences for Conogethes punctiferalis breeding on castor and cardamom constructed by NETWORKÒ software

Table 3 Descriptive statistics of polymorphisms found in a 587-bp portion of the COI gene from Conogethes punctiferalis bred on castor and cardamom Index Castor Cardamom Both n 10 8 18 Hn 6 2 8 Hd 0.844 ± 0.103 0.250 ± 0.180 0.817 ± 0.073 p 0.00473 ± 0.00130 0.00043 ± 0.00031 0.0285 ± 0.002 k 2.77778 0.25000 16.77124 S Number of segregating sites 10 1 37 Total number of mutations 10 1 39 Total number of synonymous changes 10 1 37 Total number of replacement changes 0 0 2 Theta S 0.00609 0.00066 0.01906 Theta p 0.00476 0.00043 0.02970 n number of sequences, # of haplotypes (Hn), Hd Haplotype diversity, p pi nucleotide diversity, k average number of nucleotide differences, genetic distances among haplotypes, and theta values based on the number of polymorphic sites (S), and the mean number of pairwise differences

Table 4 Analysis of molecular Source of df Sum of Variance Percentage U Statistic variance (AMOVA) for the COI variation squares components variation sequences of Conogethes punctiferalis on castor and Among 1 129.181 14.43877 Va 94.53 – cardamom populations Within 16 13.375 0.83594 Vb 5.47 0.896 (p \ 0.001) populations Total 17 142.556 15.27471 Fixation index 0.94527

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Table 5 Results of the neutrality tests calculated for castor and cardamom populations Populations Tajima’s D Fu and Li’s D Fu and Li’s F Fu’s Fs

Castor -0.94931NS -0.39729NS -0.60024NS -1.002 Cardamom -1.05482NS -1.12639NS -1.20353NS -0.182 All populations 1.95217NS (0.10 [ p [ 0.05) 0.83703NS 1.34805NS 5.455 NS not significant, p [ 0.10

Demographic inference and neutrality tests

Demographic history changes were analyzed for CBR and CBE populations through neutrality tests and mismatch distribution. None of the neutrality tests (D value of Taj- ima, D and F values of Fu and Li, and Fs value of Fu) showed significant difference within and between CBR and CBE populations. For CBR and CBE, Tajima’s D values were non-significantly positive when both populations pooled in one group, but significantly negative when ana- lyzed separately (Table 5). The same results were seen in all other three neutrality tests. The mismatch analysis yielded a unimodal distribution of pairwise differences for CBE (Fig. 5a) compared to the multimodal distribution of CBR samples (Fig. 5b) and for the pooled samples (Fig. 5c), further elucidating the demographic history of C. punctiferalis. The observed mismatch distributions did not deviate from the expected values in CBE based on the sudden expansion model. The mismatch distribution analysis did not indicate a rapid demographic expansion, where the results were bimodal curves for CBR. The mismatch distribution test did not indicate a rapid demographic expansion, where the results were multimodal curves for the total C. punctiferalis population.

Sequence verification using GenBank and BOLD

The sequences obtained were blast searched (on December 10, 2011) in the NCBI Genbank using the BLAST program. But there were no hits related to the Conogethes species, instead, sequences from the CBR group showed a maximum identity (91 % identity: Accession no. JQ550378) with the COI gene of Phostria cyrisalis (Hampson), a Crambid moth. The CBE group showed maximum identity (91 % identity (Accession no. JQ578070.1) with the COI gene of Rhecto- craspeda periusalis (Walker), another Crambid moth. The sequences of CBR, when searched using the Barcode of Life Database (BOLD) specimen identification tool (on 12th January, 2012), showed a maximum identity with C. punc- tiferalis. But there were no hits in the BOLD specimen Fig. 5 Mismatch distributions of pairwise nucleotide differences for identification tool when searched with sequences of Cono- Conogethes punctiferalis breeding on a castor, b cardamom and gethes breeding on cardamom. c castor and cardamom

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Discussion detecting signals of population growth (Yuan et al. 2010). Fu’s Fs is significantly negative in CBR and CBE popula- Mitochondrial DNA has been used for elucidating genetic tions, suggesting the population expansion in both popula- variation and speciation of complex insect species (Brower tions. In our study, a higher FST value indicated a lower 1999; Kruse and Sperling 2001; Savolainen et al. 2005; level of gene flow (Nm) and higher genetic differentiation Sperling and Hickey 1995). There are several lines of among populations. The results of two-level AMOVA show evidence that show that the differences in the mtDNA that significant genetic variation exists among the examined influence the speciation process in the closely-related populations. These results provide a second line of support cryptic species in crambids and other families of Lepi- to a conclusion that the CBE is a different species. doptera (Hajibabaei et al. 2006; Hebert et al. 2004; Huemer The results of mitochondrial cytochrome oxidase I and Hebert 2011; Nagoshi et al. 2011; Nieukerken et al. (mtCOI) sequences of CBR when searched using the 2012; Vaglia et al. 2008; Wilson et al. 2010). Previous BOLD specimen identification tool showed 100 % identity studies from our laboratory showed that morphologically with C. punctiferalis. On the basis of these results as well dissimilar Conogethes larvae reared on castor and carda- as with the NJ tree, it is most likely that the Conogethes sp. mom required two different mass-rearing techniques breeding on castor is C. punctiferalis. However, there were (Chakravarthy et al. 1991). In the present study, an attempt no hits in the BOLD specimen identification tool when was made to use DNA barcoding for understanding the searched with sequences of CBE. This indicated that there variation between CBR and CBE. The morphological dif- were no sequences related to CBE that were previously ferences between CBR and CBE, which were described in deposited in BOLD, or that CBE is a new species. the results of the present study, were not sufficient to dif- In conclusion, the molecular evidence along with the ferentiate these two populations as different species. For morphology offered here seems to confirm the presence of confirming the species status, the study DNA for 18 indi- cryptic species within C. punctiferalis breeding on castor viduals of CBR and CBE were obtained from different and cardamom. Though further works are required to form locations. This is the first attempt from India in which the a conclusive understanding in this regard, our findings Conogethes species has been barcoded. suggest the apparent existence of two species pairs, where The mtDNA analysis revealed that C. punctiferalis there is evidence of genetic divergence by[5 %. This is an populations breeding on cardamom represent a distinct interesting case and warrants further studies. Results from lineage from those breeding on castor (Fig. 3). Our results our study strengthen information about the Conogethes demonstrate a clear differentiation of a cryptic lineage species complex and help in developing appropriate inte- (CBE), which can be viewed as a host-plant-based cryptic grated pest management strategies for these insect pests. species on the basis of high (5.47 %) sequence divergence (Table 4) because genetic divergence of more than 5 % Acknowledgments We thank Dr. Uday Kumar and Dr. M.S. She- suggests the likely occurrence of a new species (Hebert shshayee, Department of Crop Physiology, University of Agricultural Sciences, Bangalore, India for providing laboratory facilities to con- et al. 2004). The species complex is a model for duct this work, and the valuable comments by Dr. Hiroshi Honda, speciation of Lepidoptera and provides a basis for com- Graduate School of life and Environmental Sciences, Tsukuba, Japan. parison with our observations from C. punctiferalis (Coates We profoundly thank Dr. C. A. Viraktamath, for facilitating and et al. 2005; Kim et al. 1999; Ohno et al. 2006). Similar guiding in morphological work; Mr. H. M. Yeshwanth, for helping in photography of moths; Dr. N.E. Thygaraj and Mr. M.V. Nataraj, results were observed in the Maruca vitrata complex Department of Entomology, University of Agricultural Sciences, (Margam et al. 2011). Armstrong (2010) compared DNA Bangalore, India, who helped in the collection of samples. This work barcoding of different populations of Conogethes and was supported by the Department of Science and Technology (DST), revealed that Australian and Asian specimens form sepa- New Delhi, by awarding the INSPIRE fellowship. rate clades that are divergent by *6 %, and their data successfully distinguished C. punctiferalis and C. pluto. Different neutrality tests were chosen for the demo- References graphic history analysis, and significantly negative values of neutrality statistics can be indicative of background Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic selection, but are also consistent with either population local alignment search tool. J Mol Biol 215:403–441 Armstrong K (2010) DNA barcoding: a new module in New subdivision or expansion. Fu and Li’s D and F are more Zealand’s plant biosecurity diagnostic toolbox. 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