Journal of Genetics and Molecular Biology Vol. 19, No. 3, 158-167, September 1, 2008

Deciphering the Molecular Phylogenetics of Family and Inferring the Phylogeographical Relationships Using DNA Barcoding

Chandrasekhar N., Neetha N.V., Linda Koshy Vaidyan and Moinak Banerjee*

Rajiv Gandhi Centre for Biotechnology, Human Molecular Genetics Lab, Poojapura, Thiruvananthapuram, Kerala, India 695 014

Hyblaea puera (teak defoliator) is a pest of teak woodlands in India and other tropics. H. puera is a type genus that represents the fam- ily Hyblaeidae and superfamily Hyblaeoidea. The relationships between the superfamily Hyblaeoidea other smaller superfamilies like , , , , Torticoidea, and others are not well understood. This study provides substantial molecular evidence in supporting the morphological classification of Hyblaeidae fam- ily and its relationship with other superfamilies. As a molecular tool DNA barcoding has gained importance in species identification and taxonomic verification. Present case study on Hyblaea demonstrates the efficiency of the barcoding gene (folmer region) in discriminating global phylogeograph- ical variants among the Hyblaea species complex.

Key words: multigenic phylogeny, , Hyblaeidae, Molecular systematics, teak defoliator, DNA barcoding

Introduction ing ability demarcates the superfamilies of macrolepidoptera, like Mimallonoidea, is the second largest order , Bombycoidea, Noctuoidea, in the class Insecta comprising of but- Geometroidea, Axioidea, Calliduloidea, terflies, skippers and . So far 127 Hedyloidea, Hesperioidea and Papilionoidea families and 46 superfamilies have been from other Lepidoptera [1]. described. Around 98% of the species is an informal grouping of other moths and in the order Lepidoptera fall in Ditrysia families which are not included group. In Lepidoptera the Macrolepidoptera in the macrolepidoptera. It constitutes suborder constitutes about 60% of the paraphyletic assemblage of moths and but- total lepidopteran species. The better fly- terflies falling under , Pyralidae,

∗ Corresponding authors: Dr. Moinak Banerjee; Human Molecular Genetics Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram Kerala, India, 695 014 Tel: +91-471-234-5899, 234-8753 (O), 234-3367 (R) Fax: +91-471-234-8096 JGMB

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Hyblaeidae, , Tineidae, than the rate of substitution at silent sites in Gelechiidae, Lecithoceridae, Limacodidae mammalian genes. The focus of the current and many others. The current study was study is to decipher the systematic position intended to resolve the systematics of of H. puera using mitochondrial and nuclear Hyblaea and understand the genetic relation- genes in the order Lepidoptera. ships between the family Hyblaeidae with micro and macro lepidopterans. The current Materials and Methods study also demonstrates the resolving power of DNA barcoding at species level. Hyblaea puera (Cramer, 1777) is a type DNA Isolation and Polymerase Chain genus representing the family Hyblaeidae Reaction and superfamily Hyblaeoidea in order DNA extraction was done from whole Lepidoptera. Hyblaeidae consists of two larvae as per the protocol described by major genera Hyblaea and Erythrochrus Andrew and Gary (1995) [10]. The quality while the family comprises of 20 species and quantity of the DNA was checked spec- found throughout the new and old world trophotometrically by taking the absorbance tropics and subtropics. The current study ratios of 260/280 nm. The primer sequences rationalizes the morphological classifica- used for the amplification of Mt genes and tion with the sequence data for the family nuclear genes are tabulated in table 1. The Hyblaeidae. H. puera was first described template of 200 ng of total genomic DNA in 1794, and was originally included in was subjected to PCR reactions in 30 µL the family Noctuidae (Macrolepidoptera) volume. Each reaction consisted of 1 X Taq and recognized as a serious forest pest by buffer with 1.5 mM MgCl2, 1.2 U of Taq Hampson in 1894 [2]. In the same year polymerase (Genei), 0.25 mM of dNTPs Fletcher and Nye (1894) [3], placed the fam- (Amersham) and 10 pmols of primers per ily Hyblaeidae along with the superfamily reaction (Sigma). PCR cycling conditions Pyraloidea based on the morphology, subse- were as follows: 16S, 12S, COI (folmer quently the family became independent and region) gene were amplified with initial got its own superfamily status Hyblaeoidea. denaturation at 94°C for 3 mins followed by Till date the systematics of Hyblaeoidea has 35 cycles of cycle denaturation at 94°C for not been supported by molecular data. 30 secs, annealing at 55°C for 30 secs, exten- Mt genomes are renowned for mutation sion at 72°C for 1 min, final extension at 72° hot spots or adaptive substitutions which C for 4 mins and held at 4°C. In case of COI makes the genome more noteworthy, and and COII genes, amplification was done results in the heterogeneous evolutionary using COa and COb primers with an initial rates across genes [4, 5]. The average rate denaturation at 94°C for 5 mins followed of evolution of the mitochondrial genome is by 35 cycles of cycle denaturation at 94°C known to be 5-10 times higher than that of for 30 secs, annealing at 56.8°C for 1 min, nuclear genome, in case of mammals [6, 7, 8]. extension at 72°C for 1 min, final extension In Drosophila it has been shown that nuclear at 72°C for 4 mins and held at 4°C. COc, genes evolve faster than mammalian nuclear COd and COe were used for sequencing the genes and mitochondrial genes evolve faster full length gene. The nuclear gene 28S was than that of nuclear genes with high codon amplified using 28Sf and 28Sr primers with bias and at approximately the same rate as initial denaturation at 95°C for 3 mins, fol- nuclear genes with low bias [9]. The rates lowed by 45 cycles of denaturation at 95°C of substitution at silent sites in Drosophila for 30 secs, annealing at 60°C for 30 secs, nuclear genes are at least three times higher extension at 72°C for 1.5 mins, final exten-

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Table 1. Primer sequences used for PCR amplification inH. puera.

S.No. Primer name Sequence 1 COIf TACAATTTATCGCCTAAACTTCAGCC 2 COIr CCCGGTAAAATTAAAATATAAACTTC 3 COa CAACATTTATTTTGATTTTTTGG 4 COb GAGACCATTACTTGCTTTCAGTCATCT 5 COc TCCAATGCACTAATCTGCCATATTA 6 COd GGTCAAACAATTGAGTCTATTTGAAC 7 COe CCACAAATTTCTGAACATTGACCA 12 16Sf CGCCTGTTTATCAAAAACAT 13 16Sr CCGGTTGAACTCAGATCA 14 12Sf AAGAGCGACGGGCGATGTGT 15 12Sr AAACTAGGATTAGATACCCTATTAT 16 EF1 CACAT(CT)AACATTGTCGT(GC)AT(CT)GG 17 EF3 GCTGAGCG(CT)GA(AG)CGTGGTATCAC 18 EF4 CAT(AG)TTGTC(GT)CCGTGCCA(GT)CC 19 EF6 GC(CT)TCGTGGTGCAT(CT)TC(GC)AC 20 EF7 CA(AG)GACGTATACAAAATCGG 21 EF10 ACAGC(ACG)AC(GT)GT(CT)TG(CT)CTCAT(AG)TC 22 28Sf AGAGAGAGTTCAAGAGTACGTG 23 28Sr TTGGTCCGTGTTTCAAGACGGG sion at 72°C for 4 mins and held at 4°C. EFα (Promega) and plasmids were isolated from gene amplification was performed using EF1 the positive clones and sequenced using Big and EF10 primers with initial denaturation dye terminator sequencing kit ver 3.1 as per at 95°C for 3 mins, followed by 40 cycles of the manufacturer’s protocol. The sequencing denaturation at 95°C for 30 secs, annealing was repeated twice before being submitted to at 55°C for 30 secs, extension at 72°C for 2 NCBI GenBank. The accession numbers are mins, final extension at 72°C for 8 mins and as follows AY572232 - 16S ribosomal RNA held at 4°C. EF3, EF4, EF6 and EF7 primers gene, AY575214 - 12S ribosomal RNA gene, were used as internal sequencing primers. AY572235 - Cytochrome oxidase subunit I The PCR amplicons were separated using (COI) and Cytochrome oxidase subunit II 1.2% agarose gel in 0.5 X TBE buffer. The (COII) genes, AY847953 - Cytochrome oxi- gel was stained with ethidium bromide (0.5 dase subunit I (Folmer region), AY572233 – µg/mL) and visualized in Fluor-STM multi 28S ribosomal RNA gene and AY575215 - imager system (Bio-Rad) using Quantity elongation factor 1 alpha gene. One software module. The PCR products were subsequently Dataset cloned in pGEMT easy vector system The dataset consist of 11 families

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Appendix 1 - List of accession numbers used in the dataset for family level study. Families GENES Code or 12S 16S 28S COI COII EF Orders α Dip Diptera NC_001709 NC_001709 AF191294 NC_001709 NC_001709 NM_206593 Pie Pieridae DQ150058 DQ150095 AY521784 AY954565 AF044024 DQ082828 Pap Papilionidae AY351418 AF095450 DQ406739 DQ270142 DQ270139 AY804454 Lyc Lycaenidae NC_007976 NC_007976 AY954532 NC_007976 NC_007976 AY954622 Tor Tortricidae NC_008141 DQ073916 AJ844025 NC_008141 NC_008141 DQ232886 Geo Geometridae AF232885 AJ505592 DQ178927 AJ870409 AF064521 DQ018899 Noc Noctuidae AF232884 AF173062 AF178905 AJ420370 AB158623 DQ192234 Bom Bombycidae NC_003395 NC_003395 M 3 1 3 2 0 NC_003395 NC_003395 NM_001046653 Sat Saturniidae NC_004622 NC_004622 AF423922 NC_004622 NC_004622 AY301297 Hyb Hyblaeidae AY575214 AY572232 AY572233 AY847953 AY572235 AY575215 Pyr Pyralidae AJ560801 DQ150095 AY062912 DQ630742 AY320504 A F 4 2 3 8 1 1 Cra Crambidae NC_003368 NC_003368 DQ406739 NC_003368 NC_003368 A F 1 7 3 3 9 2 spanning 7 superfamilies along with a dip- ) [11]. All teran outgroup, were considered for analyses the members from the family Hyblaeidae (Appendix-1). Both mitochondrial genes were selected for the analysis irrespective and nuclear genes were used to resolve of its geographical locations. Barcoding the family level clade. 16S, 12S, COI and records show around 25 H. puera sequences COII Mt genes were selected along with from different tropical regions, majority of the two nuclear genes (EFα and 28S). The the accessions are from Papua New Guinea dataset consisted of three families from the and Costa Rica. A well documented record Papilionoidea superfamily namely Pieridae of four Hyblaea species including H. puera (PIE), Papilionidae (PAP) and Lycaenidae were found in the BOLD database of which (LYC), two families Bombycidae H. constellata, H. amboinae species were (BOM) and Saturniidae (SAT) represents found in tropics and H. firmamentum is a Bombycoidea superfamily and two fami- new species recently reported from Taiwan. lies representing superfamily Pyraloidea namely, Pyralidae (PYR) and Crambidae Statistical Analyses (CRA). One representative from family Nucleotide data sets were obtained from Tortricidae (TOR), Geometridae (GEO), GenBank and were aligned using Clustal-X Noctuidae (NOC) and Hyblaeidae (HYB) multiple alignment software [12]. The site were included in the study. The Standard properties was analysed using MEGA v 3.1 barcoding gene region (folmer region) near [13]. Nucleotide sequence divergence was the 5’ terminus of the COI gene was selected calculated using Kimura-2 parameter model for distinguishing the phylogeographical by Kimura (1980). Neighbour joining (NJ) ecotypes of Hyblaea species. The dataset for [14] was performed using Bionumerics v this study was obtained from the Barcode 2.0 (Applied Maths). The strength of the of Life Data System (BOLD website tree was checked using 1000 bootstraps.

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Table 2. Site properties of four mitochondrial and two nuclear gene sequences within order Lepidoptera. Sites in % Families 12s 16s 28s COI COII EF-α Variable site BOM/HYB 46.7 17.0 63.1 19.0 24.5 56.8 PAP/HYB 60.1 69.5 80.5 25.2 30.6 60.7 GEO/HYB 6.9 47.3 22.8 11.4 14.5 9.6 TOR/HYB 41.8 48.5 54.3 14.4 12.9 51.9 NOC/HYB 6.0 46.9 24.5 12.0 16.9 8.3 PYR/HYB 45.5 10.2 61.1 18.3 23.3 16.9 ALL 72.7 81.1 97.6 39.3 47.5 82.4 Conserved sites BOM/HYB 50.8 73.5 29.1 80.9 75.4 39.4 PAP/HYB 39.0 23.6 15.0 74.7 69.3 35.6 GEO/HYB 90.2 36.6 66.5 88.5 85.0 86.6 TOR/HYB 51.6 38.8 36.4 85.5 86.6 38.4 NOC/HYB 91.0 39.6 65.0 87.9 82.6 87.9 PYR/HYB 52.4 80.0 30.3 81.6 76.6 79.3 ALL 27.2 12.8 0.9 60.6 52.4 15.4 Parsimony-informative sites ALL - - - 24.0 28.6 59.2 Two-fold degenerate sites ALL - - - 10.9 14.1 2.1 Four- fold degenerate sites ALL - - - 4.0 9.2 3.0

Principal component analyses (PCA) was showed more parsimony informative sites implemented by STATISTICA-6.0. when compared with CO complex (24% and 28.6%). Results Phylogenetic Analyses The combined dendrogram for the Mt Site Properties and nuclear genes showed two major clus- Site analysis on both mitochondrial ters which bifurcate from the dipteran out- and nuclear genes at the family level showed group. Majority of the macrolepidopterans, that COI and COII had maximum conserved cluster together, except Bombycoidea super- regions among lepidopterans (Table 2). In family. Interestingly Bombycoidea clusters general, compared to protein-coding genes with microlepidopteran subgroup which (COI, COII and EFα) the ribosomal genes comprises of Hyblaeoidea and Pyraloidea (12S, 16S and 28S) showed more variable with very high bootstrap values (Figure 1). sites. Among the six loci, the 28S gene Noctuoidea and Hyblaeoidea superfami- showed 97.6% variable site followed by EFα lies were distantly placed in two different (82.4%), 16S (81.1%) and 12S (72.7%). clusters based on their genetic distances, Among protein-coding genes EFα (59.2%) disproving Hampson’s morphological

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Fig. 1. Neighbour joining tree based on mitochondrial and nuclear genes showing the families and super families with bootstrap values highlighted on nodes. classification. It was further observed that clearly proves that the discrimination power the H. puera clusters with Pyralidae and of barcoding gene can interpret a taxon up Crambidae family members supporting the to the species level and also can differentiate Fletcher and Nye (1984) reclassification. the phylogeographical variants. The clade also confirms the superfamily sta- tus of Hyblaeoidea. Spatial Analysis The phylogenetic tree was constructed PCA explores similarity relationships using NJ method with K2P parameter for in Euclidean space and has the advantage the Hyblaea species. H. puera forms two of permitting genetically intermediate taxa distinct clusters comprising of old and to remain spatially intermediate, rather than new world teak moths. The NJ tree clearly fixing them to cluster into a pseudogroup illustrates that among the four Hyblaea spe- as in hierarchical methods [15]. In the pres- cies, H. puera belongs to the primitive lin- ent case a similarity matrix was constructed eage. Phylogeographical assemblage of H. using Bionumerics-2.0 by treating every puera from different geographical locations position in the alignment as a separate char- showed clearly that H. puera from Indian acter and ambiguous nucleotides as missing sub-continent (Kerala, Nilambur) clustered character (Appendix 2). The PCA analysis with H. puera complex from Papua New based on the combined similarity matrices Guinea which belong to old world trop- generated based on four mitochondrial and ics (Figure 2). This case study on Hyblaea two nuclear loci confirms that H. puera is spatially closer to the superfamily Pyraloidea

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Fig. 2. Neighbour joining tree based on the barcoding region of Hyblaea species.

Appendix 2 – Similarity values in percentage for the individual families obtained from the combined mitochondrial and nuclear genes.

DIP PIE PAP LYC TOR GEO NOC BOM SAT HYB PYR CRA

DIP 100 73.71 72.95 63.59 66.86 76.69 76.08 70.43 75.61 79.47 75.91 78.81 PIE 73.71 100 78.57 71.51 70.98 76.85 77.59 68.11 79.79 77.57 78.82 76.63 PAP 72.95 78.57 100 72.81 74.11 79.06 82.98 68.12 77.37 79.07 77.99 78.03 LYC 63.59 71.51 72.81 100 84.59 69.05 74.23 65.73 69.31 70.6 71.24 68.45 TOR 66.86 70.98 74.11 84.59 100 66.83 73.49 66.99 69.81 68.41 69.42 69.13 GEO 76.69 76.85 79.06 69.05 66.83 100 82.79 70.27 77.29 84.02 79.06 80.81 NOC 76.08 77.59 82.98 74.23 73.49 82.79 100 70.97 78.87 84.17 79.62 82.58 BOM 70.43 68.11 68.12 65.73 66.99 70.27 70.97 100 77.08 75.81 72.34 76.02 SAT 75.61 79.79 77.37 69.31 69.81 77.29 78.87 77.08 100 81.35 83.93 81.47 HYB 79.47 77.57 79.07 70.6 68.41 84.02 84.17 75.81 81.35 100 84.09 86.53 PYR 75.91 78.82 77.99 71.24 69.42 79.06 79.62 72.34 83.93 84.09 99.89 85.27 CRA 78.81 76.63 78.03 68.45 69.13 80.81 82.58 76.02 81.47 86.53 85.27 100

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Fig. 3. Principal components analysis based on similarity matrix generated by both Mitochondrial and Nuclear genes. in order Lepidoptera (Figure 3). Multigene phylogenetic studies inte- grated with the morphological characters in Discussion phylum has been demonstrated by Giribet et al. [18]. In a recent study Most often the phylogenetic studies using the multigenic approach of nuclear are either based on individual gene or few and mitochondrial genes the phylogeny and genes with similar evolutionary rate or phylogeography of ice crawlers have been entire mitochondrial genome. It has been resolved [19]. There are extensive reports on observed that phylogeny based on complete the use of COI, COII and 16S genes for spe- Mt genome can always produce exceptions cies, genus, and subfamily level studies in in the placement of few species in the clade Lepidoptera [20, 21, 22, 23, 24 & 25]. The [16]. Whereas phylogeny based on single EFα gene has the ability to resolve phylo- gene can also lead to erroneous interpre- genetic information for subfamily or lower tation of species tree because of variable levels [26, 27]. The nuclear ribosomal gene mutations associated with speciation. It has 28S was employed for studying family level been reported that evolutionary rate of cyto- phylogenies in order Lepidoptera [28, 29 chrome complex genes is three times more &30]. However, the ultimate challenge is to rapid than the 12SrRNA and 16SrRNA genes use precise combination of genes to address in eukaryotes [17]. In the current study the any phylogenetic relationship. Based on multigenic approach is demonstrated by this multigenic approach of selecting genes considering genes from variable mutational with variable mutation rate our data strongly rate, in understanding the systematics of H. supports the clustering of Hyblaeoidea in puera. This multigenic approach was based proximity to Pyraloidea within the micro- on four Mt genes and two nuclear genes to lepidopteran cluster. The study disproves the balance the mutational bias. Hampson’s morphological classification of

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