Anim. Syst. Evol. Divers. Vol. 28, No. 1: 36-41, January 2012 http://dx.doi.org/10.5635/ASED.2012.28.1.036 Molecular Taxonomy of a Phantom Midge Species (Chaoborus flavicans) in Korea Haein An, Gila Jung, Chang-Bae Kim* Department of Green Life Science, Sangmyung University, Seoul 110-743, Korea ABSTRACT The larvae of Chaoborus are widely distributed in lakes, ponds, and reservoirs. These omnivorous Chaoborus larvae are crucial predators and play a role in structuring zooplankton communities, especially for small-sized prey. Larvae of Chaoborus are commonly known to produce predator-induced polyphenism in Daphnia sp. Nevertheless, their taxonomy and molecular phylogeny are very poorly understood. As a fundamental study for understanding the role of Chaoborus in predator-prey interactions in a freshwater ecosystem, the molecular identification and phylogenetic relationship of Chaoborus were analyzed in this study. A molecular comparison based on partial mitochondrial cytochrome oxidase I(COI) between species in Chaoborus was carried out for the identification of Chaoborus larvae collected from 2 localities in Korea. According to the results, the Chaoborus species examined here was identified as C. flavicans, which is a lake-dwelling species. Furthermore, partial mitochondrial genome including COI, COII, ATP6, ATP8, COIII, and ND3 were also newly sequenced from the species and concatenated 5 gene sequences excluding ATP8 with another 9 dipteran species were compared to examine phylogenetic relationships of C. flavicans. The results suggested that Chaoborus was more related to the Ceratopogonidae than to the Culicidae. Further analysis based on complete mitochondrial DNA sequences and nuclear gene sequences will provide a more robust validation of the phylogenetic relationships of Chaoborus within dipteran lineages. Keywords: phantom midge, Chaoborus flavicans, molecular identification, phylogenetic relationship, Korea INTRODUCTION several cryptic species have been suggested. Especially, 2 cryptic species in Chaoborus flavicanswere indicated accord- Chaoboridae, a family of Diptera, is commonly known as ing to its habitats, morphological characters, and mitochon- phantom midges. These are common midges with cosmopoli- drial cytochrome oxidase I(COI) sequences. Based on the tan distribution. Aquatic larvae of Chaoborus, a common morphological characters and 18S and 5.8S ribosomal DNA genus of the family, are widely distributed in lakes, ponds, sequences, it has been suggested that Chaoboridae is more and reservoirs. Omnivorous Chaoborus larvae are crucial closely related to the Culicidae than Ceratopogonidae in the predators in structuring zooplankton communities, especially culicomorphan Diptera(Miller et al., 1997; Sæther, 2000). In for the small-sized prey such as Daphnia, water flea. Larvae other molecular phylogenetic studies(Friedrich and Tautz, of Chaoborus are commonly known to produce predator- 1997; Cameron et al., 2007) conducted with the purpose of induced polyphenism in Daphniasp., which is a morphologi- testing the traditional hypotheses on relationships between cal defense for planktonic crustaceans by adaptive develop- families of the dipterans, the species in Chaoboridae were mental plasticity(Tollrian and Dodson, 1999; Simon et al., not included in analyses. 2011). Species in Chaoboridae have never known to inhabit Korea Despite the important ecological role of Chaoborus, their until recently, and even the family name did not appear in the taxonomy and phylogenetic relationships remain unresolved. Checklist of Insects from Korea(The Entomological Society According to Dupuis et al.(2008), the monophyletic relation- of Korea and Korean Society of Applied Entomology, 1994). ships of species in Chaoborus were highly questionable, and Recently, Jeong(2010) reported Chaoborus flavicans from cc This is an Open Access article distributed under the terms of the Creative *To whom correspondence should be addressed Commons Attribution Non-Commercial License(http://creativecommons.org/ Tel: 82-2-2287-5288, Fax: 82-2-2287-0070 licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, E-mail: [email protected] and reproduction in any medium, provided the original work is properly cited. pISSN 2234-6953 Copyright The Korean Society of Systematic Zoology Molecular Taxonomy of Chaoborus flavicansin Korea the Sangchun reservoir in Gyeonggi-do, based mainly on based on the Kimura two-parameter model was inferred by mandible morphology and partial COI sequences. Therefore, PAUP4.0b10*(Swofford, 2003). Tree robustness was exam- their taxonomy and distribution are very poorly understood ined by bootstrap analysis using 1,000 replicates. in Korea. In this study, molecular identification and phylo- genetic relationships of Chaoborus were analyzed as a fun- PCR amplification and DNA sequencing of partial damental study for understanding the role of Chaoborus in mitochondrial genome predator-prey interactions in a freshwater ecosystem. For phylogenetic analysis, more than 4kb DNA including mitochondrial coding genes COI, COII, ATP6, ATP8, and COIII was amplified from a individual collected from the MATERIALS AND METHODS Sangchun reservoir by using PCR primers, CI-J-1632 and C3-J-5460(Table 1) by using the same PCR reaction compo- Collection and DNA extraction sition for the molecular identification and the following PCR Larvae of Chaoborus were collected by a Bongo net of 60 protocol: 92�C for 2min and 40 cycles of 92�C for 30 sec, cm mouth diameter and 300μm mesh size in the Sangchun 45�C for 30 sec, 68�C for 12min, followed by final exten- reservoir in Gyeonggi-do and the Ildae reservoir in Jeolla- sion, 68�C for 20min. For PCR amplification of genes inside nam-do. Collected specimens were preserved in 100% ethyl the long PCR fragment, several internal PCR primers were alcohol before storing at -20�C until DNA extraction. DNA designed(Table 1) and separate PCR reactions were execut- was extracted by using an AccuPrep Genomic DNA extrac- ed using these primers. ND3 was amplified by the PCR pri- tion kit(Bioneer, Korea). mers, C3-I-F and N4-I-R. PCR products were purified using the AccuPrep PCR Purification kit. Sequencing reactions Molecular identification were performed using BigDye Terminators kit 3.1, and run For molecular identification, the partial mitochondrial COI on an ABI 3730 Automated Sequencer. region was PCR amplified by employing primers COIF and COIR(Table 1), in a total volume of 20μL consisting of 2× Phylogenetic relationship TOPsimple DyeMIX-Tenuto(Enzynomics, Korea), ~100ng For comparison, another 9 dipteran species and Locusta template DNA, 2 pmol dNTPs, and 5 pmol of each primer. migratoria were used as an outgroup from which complete The PCR protocol consisted of initial denaturation at 94�C mitochondrial DNA sequences are known were retrieved for 3min, 35 cycles of 94�C for 30 sec, 45�C for 30 sec, and from the GenBank(Table 3). Based on the conserved(C)/vari- 72�C for 1min, followed by final extension, 72�C for 7min. able(V) site ratios and percentage of gaps and invariable sites PCR products were purified using the AccuPrep PCR Purifi- as an estimation of reliability of alignment(Lee et al., 2006), cation kit(Bioneer). Sequencing reactions were performed ATP8 did not show reliability(data not shown). ATP8 gene using the BigDye Terminators kit 3.1, and run on an ABI was commonly excluded from phylogenetic studies using 3730 Automated Sequencer(Applied Biosystems, USA). mitochondrial genome. Except ATP8 gene 5 genes were used Using Blastn search, COI sequences similar to the present for further phylogenetic analyses. After translating the DNA sequences were retrieved(Table 2) and multiply aligned by sequences, reading frames of DNA sequences were confirm- CLUSTAL W(Larkin et al., 2007) in the Geneious Pro 5.4.6 ed by concatenating and aligning 5 mitochondrial coding program(Biomatters, New Zealand). Neighbor-Joining tree genes by using CLUSTAL W in the Geneious Pro 5.4.6 pro- Table 1.List of PCR primers used in this study Target gene Primer Sequence(5′→3′) References COI-COIII CI-J-1632 TGATCAAATTTATAAT Kambhampati and Smith, 1995 C3-J-5460 TCAACAAAGTGTCAGTATCA Cameron et al., 2007 COI COIF GAAGCTAAAATTCAATGCACTAGTCT Dupuis et al., 2008 COIR CTTATTTTACTTCAGCAATAATTA Dupuis et al., 2008 COI C1F ACCTCCTTCTTTGACCCTGC In this study C1R GGACTACTCCTGTTAATCCTCC In this study COII C2F CTTAGGGTTAGCTGGAATGCC In this study C2R GGAAGAACAATACGATTATCTACATCT In this study COI-COIII C3IR AGGGGTCATGGGCTATAATCTACT In this study COIII-ND3 C3-I-F GGCATACGAATATATAGAAGCATC In this study N4-I-R TCAACCTGAGCGTTTACAGGCTGGG In this study CO, cytochrome oxidase. Anim. Syst. Evol. Divers. 28(1), 36-41 37 Haein An, Gila Jung, Chang-Bae Kim gram. 2004) under Akaike’s information criterion. The GTR++I++G For reconstucting the phylogenetic trees based on 5 mito- model was used to generate a Bayesian inference and a max- chondrial coding genes from dipteran species, a substitution imum likelihood(ML) tree. The Bayesian tree was obtained model was chosen using MrModeltest version 2.02(Nylander, with MRBAYES version 3.1.2(Ronquist and Huelsenbeck, 2003) with default options for the prior distribution in the Bayesian inferences. Metropolis-coupled Markov chain Table 2.List of COI nucleotide sequences of Chaoborus species Monte Carlo(MCMCMC) analyses were run with one cold using for phylogenetic analysis chain and 3 heated chains for 1,000,000 generations from 5 Species Location Accession no. mitochondrial coding genes, and sampled every 100 genera- C. flavicans Turkey DQ146242 tions. Two independent MCMCMC runs were performed C. flavicans Turkey DQ146264 and 2,500 trees were discarded as burn-in from the 5 mito- C. flavicans Sweden DQ146266 C. flavicans Sweden DQ146280 chondrial coding genes. The final trees near the optimum C. flavicans Sweden DQ146249 likelihood score were retained using the appropriate burn-in C. flavicans Sweden DQ146253 criterion. Trees from 5 mitochondrial coding genes were re- C. flavicans Indiana DQ146238 tained and used for calculation of posterior probabilities. A C. flavicans Indiana DQ146243 C. flavicans Newyork DQ146297 ML tree was constructed using PAUP4.0b10* by heuristic C. flavicans Newyork DQ146296 search with a truncated balanced realization algorithm. The C. cf. flavicans Alaska DQ146230 tree stability was examined by bootstrap analysis with 100 C. cf. flavicans Japan DQ146274 replicates. C. cf. flavicans Alaska DQ146233 C. cf. flavicans Alaska DQ146239 The sequence alignment is available upon request from C. cf. flavicans Alberta DQ146255 the corresponding author. C. cf. flavicans Alberta DQ146295 C. cf.flavicans Japan DQ146284 C. cf. flavicans Japan DQ146300 C. cf.flavicans Indiana DQ146271 RESULTS AND DISCUSSION C. cf.flavicans Indiana DQ146301 C. albatus Unknown AJ427614 Molecular identification C. americanus Alberta DQ146279 For molecular identification, 6 individual specimens from C. americanus British Columbia DQ146273 C. astictopus Unknown AJ427613 Sangchun reservoir in Gyeonggi-do and 3 from Ildae reservoir C. cooki Unknown AJ427616 in Jeollanam-do were used for generation of COI barcodes. C. crystallinus Finland DQ146256 In addition, 2 specimens from the National Institute for En- C. crystallinus Sweden DQ146305 vironmental Studies of Japan were also examined for com- C. obscuripes Unknown AJ427615 C. punctipennis Newyork DQ146236 parison. Sequences were deposited in GenBank(accession C. punctipennis Indiana DQ146283 nos: JQ277990-JQ278000). Based on the sequence compari- C. punctipennis Arkansas DQ146267 son of the COI gene, the specimens from the 2 Korean reser- C. pallidus Unknown AJ427622 voirs were almost identical(0.06%), while the specimens from C. trivitattus Unknown AJ427620 Anophele quadrimaculatus Unknown NC 000875 Korean and Japanese populations showed 18% differences in A. gambiae Unknown NC 002084 genetic distance which are close to values for putative cryptic species suggested by Dupuis et al.(2008). According to the COI, cytochrome oxidase I. Table 3.List of species compared with Chaoborus for phylogeny reconstruction Order Suborder Family Species Accession no. Diptera Brachycera Calliphoridae Cochliomyia hominivorax NC_002660 Drosophilidae Drosophila sechellia NC_005780 Drosophilidae Drosophila simulans NC_005781 Muscidae Haematobia irritans irritans NC_007102 Nematocera Ceratopogonidae Culicoides arakawae NC_009809 Culicidae Anopheles darlingi NC_014275 Culicidae Anopheles gambiae NC_002084 Culicidae Aedes albopictus NC_006817 Culicidae Culex pipiens pipiens NC_015079 Orthoptera Caelifera Acrididae Locusta migratoria NC_001712 38 Anim. Syst. Evol. Divers. 28(1), 36-41 Molecular Taxonomy of Chaoborus flavicansin Korea Fig. 1. Neighbor-joining tree for Chaoborus species based on partial mitochondrial cytochrome oxidase I(COI) gene sequences. Values above the branches indicate ¤50% bootstrap support. Boxes include specimens examined in the study. neighbor-joining tree(Fig. 1), all the specimens examined in chondrial genome of C. flavicansincluding COI, tRNA-Leu, this study belonged to the Chaoborus flavicansgroup. Two COII, tRNA-Lys, tRNA-Asp, ATP8, ATP6, COIII, tRNA- subgroups were recognized in the C. flavicansgroup 2. Each Gly, ND3, tRNA-Ala. was 4446bp in length(Table 4). The of the Korean and Japanese populations was assigned to a overall AT content for six coding genes was 70.1%(T==39.0 separate subgroup. The 2 subgroups corresponded to the 2 %, C==17.7%, A==31.1%, G==12.3%) similar to that of dipter- cryptic species suggested by Dupuis et al.(2008). According an species. to Dupuis et al.(2008), 2 cryptic species have been recogniz- The order of protein coding genes and tRNA was identical ed by having different habitats(lake-dwelling and pond-dwel- to that reported from many of insect species. The initiation ling) and morphological characteristics, especially the man- and termination codons of the genes examined here were dible. Korean populations were included in the lake-dwell- identified using the open reading frame finder and by com- ing group and were well discriminated from the pond-dwell- parison with mitochondrial gene sequences of other dipteran ing group, including the Japanese population. Populations species. ATG and ATT were used as initiation codons(Table from the Palearctic and Nearctic were well recognized in the 4). In case of COI the initiation codon could not found be- lake-dwelling group, as indicated by Dupuis et al.(2008). cause of the truncation of start region of the gene. The usual TAA termination codon found for all genes examined. Organization of protein coding genes of C. flavicans The codon usage of C. flavicansfor six mitochondrial pro- Sequences of the partial mitochondrial genome were depo- tein coding genes and the relative synonymous codon usage sited in GenBank(accession no: JQ235548). The partial mito- values are given in Table 5. Most of values differed from the Anim. Syst. Evol. Divers. 28(1), 36-41 39 Haein An, Gila Jung, Chang-Bae Kim Table 4.Annotation and gene organization of Chaoborus flavicanspartial mitochondrial genome Overlapping Non-coding Gene Direction Position Size Initiation Termination region region CO I F 1-1381 12 1381 TAA tRNA-Leu F 1394-1461 8 68 CO II F 1470-2153 2 684 ATG(Met) TAA tRNA-Lys F 2156-2226 18 71 tRNA-Asp F 2245-2312 68 ATP8 F 2313-2474 7 162 ATT(Ile) TAA ATP6 F 2468-3136 22 669 ATG(Met) TAA CO III F 3159-3947 4 789 ATG(Met) TAA tRNA-Gly F 3952-4014 63 ND3 F 4015-4368 9 354 ATT(Ile) TAA tRNA-Ala F 4378-4446 69 Fig. 2.Maximum likelihood(ML) tree for the selected dipteran species based on 3,840bp of five concatenated mitochondrial gene sequences. Values above and below the branches indicate ML bootstrap values and BI posterior probabilities, respectively. equilibrium frequency and the use of synonymous codons Sæther, 2000). These prior studies suggested that Ceratopo- was distorted. CUG(Leu), UCG(Ser), UAG(Termination), gonidae was diverged early and Chaoboridae and Culicidae AAG(Lys), UGG(Trp), AGG(Ser) were not used in C. fla- were more related. In this study, Ceratopogonidae and Chao- vicans. Since mtDNA of insects show a high bias against G boridae were more related to each other, however, this rela- and C, this could explain the lack of these codons. tionship received little statistical support. In this study, Chaoborus species from 2 reservoirs in Korea Phylogenetic relationship were identified as Chaoborus flavicansby mitochondrial COI The topology of resulting phylogeny based on ML and gene sequences. Phylogenetic trees based on 5 mitochondrial Bayesian interferences analyses recognized two clusters of coding genes by ML and Bayesian inferences showed Chao- the Diptera consisting of Brachycera and Nematocera with borus was more closely related to the Ceratopogonidae than high boostrap supports. In the Nematocera, Chaoboridae was to Culicidae, however, this relationship received little statis- more closely related to Ceratopogonidae than to Culicidae in tical support. Therefore, further analyses based on complete both analyses employed(Fig. 2). However, the present phy- mitochondrial DNA sequences and nuclear gene sequences logeny suggested different relationships between families in are needed for a more robust validation of the phylogenetic Nematocera from previous analyses(Miller et al., 1997; relationship of Chaoborus within dipteran lineages. 40 Anim. Syst. Evol. Divers. 28(1), 36-41 Molecular Taxonomy of Chaoborus flavicansin Korea Table 5.Chaoborus flavicanscodon usage of six protein coding region of partial mitochondrial genome Codon(aa) n(RSCU) Codon n(RSCU) Codon n(RSCU) Codon n(RSCU) UUU(F) 93(1.54) UCU(S) 39(2.79) UAU(Y) 26(1.16) UGU(C) 5(1.67) UUC(F) 28(0.46) UCC(S) 10(0.71) UAC(Y) 19(0.84) UGC(C) 1(0.33) UUA(L) 101(3.39) UCA(S) 32(2.29) UAA(*) 6(2) UGA(W) 43(2) UUG(L) 7(0.23) UCG(S) 0(0) UAG(*) 0(0) UGG(W) 0(0) CUU(L) 42(1.41) CCU(P) 33(2.06) CAU(H) 36(1.71) CGU(R) 7(1.17) CUC(L) 3(0.1) CCC(P) 16(1) CAC(H) 6(0.29) CGC(R) 1(0.17) CUA(L) 26(0.87) CCA(P) 13(0.81) CAA(Q) 29(1.87) CGA(R) 14(2.33) CUG(L) 0(0) CCG(P) 2(0.13) CAG(Q) 2(0.13) CGG(R) 2(0.33) AUU(I) 126(1.7) ACU(T) 39(1.7) AAU(N) 45(1.43) AGU(S) 9(0.64) AUC(I) 22(0.3) ACC(T) 17(0.74) AAC(N) 18(0.57) AGC(S) 2(0.14) AUA(M) 76(1.85) ACA(T) 35(1.52) AAA(K) 21(2) AGA(S) 20(1.43) AUG(M) 6(0.15) ACG(T) 1(0.04) AAG(K) 0(0) AGG(S) 0(0) GUU(V) 21(1.4) GCU(A) 26(1.53) GAU(D) 18(1.2) GGU(G) 7(0.35) GUC(V) 5(0.33) GCC(A) 14(0.82) GAC(D) 12(0.8) GGC(G) 1(0.05) GUA(V) 32(2.13) GCA(A) 26(1.53) GAA(E) 28(1.87) GGA(G) 51(2.58) GUG(V) 2(0.13) GCG(A) 2(0.12) GAG(E) 2(0.13) GGG(G) 20(1.01) A total of 1346 codons were analyzed. RSCU, relative synonymous codon usage; n, frequency of each codon. *Termination codons. ACKNOWLEDGMENTS Lee ES, Shin KS, Kim MS, Park H, Cho S, Kim CB, 2006. The mitochondrial genome of the smaller tea tortrix Adoxophyes honmai(Lepidoptera: Tortricidae). Gene, 373:52-57. This work was supported by the National Research Foun- Miller BR, Crabtree MB, Savage HM, 1997. Phylogenetic rela- dation of Korea(NRF) grant funded by the Korea govern- tionships of the Culicomorpha inferred from 18S and 5.8S ment(MEST)(No. 2009-0080737). We thank Dr. Shigeto ribosomal DNA sequences(Diptera: Nematocera). Insect Oda, the National Institute for Environmental Studies of Ja- Molecular Biology, 6:105-114. pan for sending us Japanese specimens. Nylander JAA, 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University, Uppsala. REFERENCES Ronquist F, Huelsenbeck JP, 2003. MrBayes 3: Bayesian phy- logenetic inference under mixed models. 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