Morphological and genetic characteristics of Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae) infesting potato crops in Korea

Journal of Asia-Pacific Entomology 17 (2014) 281–286

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Morphological and genetic characteristics of Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae) infesting potato crops in Korea Rameswor Maharjan a, Hyun Woo Oh b, Chuleui Jung a,⁎ a b

Department of Bioresources Science, Graduate School, Andong National University, Andong 760-749, Republic of Korea Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea

a r t i c l e

i n f o

Article history: Received 21 November 2012 Revised 11 November 2013 Accepted 25 January 2014 Available online 3 February 2014 Keywords: Liriomyza huidobrensis Agromyzidae Molecular diagnostic Morphology Potato Damage

a b s t r a c t The pea leafminer fly, Liriomyza huidobrensis (Blanchard), was found infesting and damaging Korean potato crops during a 2012 field survey. Localized outbreaks of L. huidobrensis were observed in Miryang and Goryeong counties in the south of the Korean Peninsula. This paper describes the morphological and genetic characteristics of the Korean population of L. huidobrensis. The dorsal color pattern on the abdomen, and an array of microsetae on the thorax, were the main diagnostic characteristics of this species. On the basis of an analysis of a partial mitochondrial cytochrome c oxidase subunit 1 (COI) DNA sequence, we found that intraspecific variation in L. huidobrensis was negligible, and that the distance between L. huidobrensis and a closely related species, L. trifolii, was estimated to be 7.4–7.6 K2P. This is the first report of the infestation of field crops by the invasive leafminer L. huidobrensis in Korea. © 2014 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.

Introduction The Agromyzidae is one of the largest families in the order Diptera, with more than 2742 species in 27 genera worldwide (Spencer, 1990), and approximately 1165 species have been identified in the Palearctic region (Scheirs et al., 1999). Liriomyza Mik is a cosmopolitan group of more than 330 species, and many are economically important pests of field crops, ornamentals, and vegetables throughout the world (Spencer, 1973; Parrella, 1987; Kang, 1996; Cerny and Bartak, 2001). In Korea, eight species of Liriomyza are found (Suh and Kwon, 1998): L. congesta on Leguminosae (Anonymous, 1986); L. chinensis on Allium fistulosum (Choi et al., 1990); L. bryoniae on Raphanus sativus hortensis (Ahn et al., 1993); L. trifolii on Gerbera spp. (Ahn et al., 1994; Hong et al., 1996); L. bryoniae and L. yasumatsui on various vegetables (Choi and Woo, 1995); L. artemisiae on Sanguisorba officinalis, L. asterivora on Ixeris dentata, and L. pusilla on Callistephus chinensis (Suh and Kwon, 1998). Historically, Liriomyza species were classified as minor pests, but from the early 1980s the population increased rapidly, and some species, such as L. trifolii, L. huidobrensis, and L. sativae, have developed insecticide resistance (Trumble, 1981; Parrella et al., 1984; Macdonald, 1991). The larvae of this genus are polyphagous, and can cause both direct and indirect damage to ornamental and vegetable crops (Musgrave

⁎ Corresponding author. Tel.: +82 54 820 6191; fax: +82 54 820 6320. E-mail address: [email protected] (C. Jung).

et al., 1975; Minkenberg and Van Lenteren, 1986). Direct damage caused by larvae feeding on palisade parenchyma tissue can reduce the photosynthetic capacity of the plant by up to 62% (Johnson et al., 1983), and mines and infested leaves can result in a reduced yield (Chandler and Gilstrap, 1987). Indirect damage occurs when both adult males and females feed, and when females lay their eggs they may act as vectors for disease (Zitter and Tsai, 1977; Motteoni and Broadbent, 1988). Spencer (1990) noted that Liriomyza spp. have hosts across 14 plant families. L. huidobrensis is a highly polyphagous and invasive species that attacks a wide range of crops, and is now included in the latest A2 list of quarantine pests (EPPO, 2005). L. huidobrensis is known as the potato leafminer fly, and is the most serious leafminer species of potatoes worldwide; it also infests beet, spinach, peas, and beans. The species has become a serious worldwide pest due to human-mediated dispersal (Mikkenberg, 1988). There is significant potential for it to widen its distribution, as the Korean Peninsula is a hot spot of climate change, particularly global warming. However, until now, there has been no official report of L. huidobrensis infesting economic crops in Korea. During a 2011–2012 survey of potato pests in Korea, a new type of damage by leafminer flies was observed, and the leafminer fly species responsible was identified as L. huidobrensis. Here we report the morphological characteristics, as well as the DNA sequence information, of L. huidobrensis to assist in the identification of the species. We also describe the species' inter- and intraspecific DNA sequence variation, which may be useful in identifying possible sources of invasion.

1226-8615/$ – see front matter © 2014 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. http://dx.doi.org/10.1016/j.aspen.2014.01.013

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Materials and methods Field survey A sampling survey was conducted in the following potato-growing areas: Boseong (Jeollanam-do) in August 2011; and Kimje (Jeollabukdo), Goryeong (Gyeongsangnam-do), Miryang (Gyeongsangnam-do), Pyeongchang (Gangwon-do), Hongcheon (Gangwon-do), and Andong (Gyounsangbuk-do) in May and June 2012 (Table 1). The sampling sites were selected based on secondary data (the potato growing season). In total, 157 potato fields were surveyed. The specimens used in this study were from Miryang (Gyeongsangnam-do) (35°21′N, 128°44′E) and Goryeong (Gyeongsangnam-do) (35°42′N, 128°24′E). Miryang and Goryeong are located in the south of the Korean Peninsula. In addition, specimens of L. trifolii were obtained from Dongbu Ceres, Chungcheongnam-Do, Korea. Morphological characteristics The collected flies were identified based on Spencer (1973), Suh and Kwon (1998), and other sources. We looked for the following diagnostic morphological features of L. huidobrensis. On the head, the third antennal segment is slightly enlarged and usually darkened, and the frons is yellow, with generally more orange than pale lemon yellow, and the upper orbits are slightly darkened. The mesonotum is black, and the femurs are yellow, with black striations. On the abdomen, the male distiphallus consists of two distal bulbs, meeting only at their rims, and the anepisternum is yellow, with variable black patches across the lower three-quarters. Specimens for electron microscopy were dried naturally at room temperature and gold sputter-coated using a Hitachi HUS-5GB High Vacuum Evaporator (Tokyo, Japan), prior to being examined and photographed at 20 kV using a Hitachi S-520 SEM. Adult size and wing size, and thoracic microsetae locations and density, were measured using electron micrographs. Genetic characteristics DNA extraction, PCR, and sequencing DNA was extracted from most individuals using a DNeasy kit (Qiagen, Hilden, Germany) following the manufacturer's protocol, except that the final elution step was performed with 60 μL of distilled water instead of 200 μL of buffer. Specimen cuticles were recovered after DNA extraction, and then stored in ethanol. A 658-base pair (bp) segment of the barcode region was amplified using the primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al., 1994). PCRs were performed using a Maxime® PCR PreMix (iNtRON Biotechnology, Seongnam, Korea), with 2.0 pmol of each primer and 2–50 ng of template DNA in a 20-μL reaction. PCR thermocycling was carried out under the following conditions: 2 min at 95 °C; five cycles of 40 s at 94 °C; 40 s at 45 °C, 60 s at 72 °C; 35 cycles of 40 s at 94 °C; 40 s at 51 °C; 60 s at 72 °C; 5 min at 72 °C; held at 4 °C. PCR products were visualized on a 2% agarose gel stained with ethidium bromide, and

Table 1 Mean number of leafminer fly collection during the survey on potato fields in 2011 and 2012. Date

Locality

Number of surveyed field

Collection (mean [±SE]/sample/field)

Aug. 2011, May 2012

Bosang, JN Miryang, GN Goryeong, GN Andong, GB Kimje, JB Pyeongchang, GW Hongcheon, GW

24 30 30 8 20 30 15

0 123.1 ± 22.54 3.5 ± 0.71 0 0 0 0

June 2012

bidirectionally sequenced using a BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster, CA, USA) using an ABI 3730XL capillary sequencer (Applied Biosystems Inc.). Contigs were assembled using CodonCode Aligner 3.5.6 (CodonCode Co., Dedham, MA, USA), and were subsequently aligned using the same software, or MEGA version 5 (Tamura et al., 2011). Sequence divergences were calculated using the Kimura 2-parameter (K2P) model (Kimura, 1980), and a neighbor-joining tree was generated by MEGA version 5. Results and discussion Field survey We collected 3698 flies in Miryang and 67 in Goryeong. No leafminer flies were observed in the other survey areas (Table 1). This is the first finding of a heavy infestation of L. huidobrensis on a field crop in Korea, although there have been some reports of infestations on greenhouse crops that have not been officially reported (SH Lee, HS Lee, personal communication). To date, eight species of Liriomyza have been reported in Korea (Suh and Kwon, 1998), with the dominant species being L. trifolii. IIE (1996) reported that L. huidobrensis is a serious pest of potato crops in Argentina, Brazil, Chile, Indonesia, Israel, Kenya, Malaysia, Mexico, and Peru, as well as in Central America and North Africa. Morphological characteristics All the collected leafminer flies were identified as L. huidobrensis, based on their morphology (Spencer, 1973; Spencer and Steyskal, 1986; Suh and Kwon, 1998) and DNA sequences (see below). L. huidobrensis has a darker body than other Liriomyza species, and the second visible tergite is divided by a yellow middle furrow (Fig. 1A). Liriomyza species have similar colors, and although the lateral and caudal margins of each tergal segment have been reported as being colored yellow in L. huidobrensis, neither drawings nor detailed descriptions have been provided (Sasakawa, 1961; Shiao, 2004). L. huidobrensis has medium-length microsetae (5–6 μm), at a density of 36–37 per 40 μm2 and local arrangement (Fig. 1B); both microsetae length and density are important in identifying the species (Zehnder et al., 1983). It is a medium-sized species, with a wing length of 1.7 mm in males and 2.3 mm in females (Fig. 3). The frons is yellow, and is approximately 1.5 times as wide as the eye. The gena and postgena are bright yellow, whereas the vertical angle, ocellar triangle, and occiput are dark brown to black. The antennae are yellow, with the 3rd segment rounded laterally, and the arista is dark brown and pubescent. There are four pairs of orbital bristle; the upper two pairs are directed upwards, the lower two pairs obviously inclinate. Both the inner and the outer vertical bristles are on a brown ground. Dorsocentral bristles are of the 1 + 3 type. The acrostichals are distributed in four irregular rows, and the halters are yellow. The costa extends to M1 + 2 near to the wing tip, and the proportions of the 2nd to the 4th costal sections are 5.2:1:1.3. It has brown squama with long dark fringes. Regarding the male terminalia, the surstylus has one stout spine and four to five sensory hairs on the posteroventral tip. The cerci are covered with long hairs. The phallus length is approximately 0.17 mm, and the distiphallus is paired and sac-shaped as seen from the ventral side. The mesophallus is well developed, with long membranous processes, and a membranous area divides the distiphallus from the mesophallus; the basiphallus is short but highly sclerotized (Fig. 1C). Regarding the female terminalia, the 9th sternite has four pairs of marginal setae, and the cerci have seven setae and six tactile sensilla. Genetic characteristics To confirm species identification, we conducted nondestructive DNA extraction and cytochrome c oxidase subunit 1 (COI) barcoding, using the similar species L. trifolii as a reference. PCR amplification and bidirectional sequencing generated 561–658-bp COI barcodes. DNA sequence

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Fig. 1. Diagnostic characteristics of Liriomyza species (L. huidobrensis; L. trifolii, L. sativae, L. bryoniae and L. chinensis). (A) Abdominal color patterns and abdominal tergites. (B) Local enlargement of thoracic microsetae. (C) Distiphallus of adult male, and (D) typical characteristics of mines. Drawings based on the observation by the first author.

alignment, followed by phylogenetic analysis, indicated a deep fork between L. huidobrensis and L. trifolii, with a K2P distance of 7.4–7.6 (Fig. 2A & B). There was no intraspecific variation in L. huidobrensis (Table 2). The most genetically distant individuals were 10 and 13 (94.8), which were L. chinensis and L. bryoniae, respectively (Table 2). There was no sequence variation among the specimens collected from the field (Fig. 2A; Table 2), suggesting that the outbreak originated from a single source population. The genetic structure of the fieldcollected L. huidobrensis population was not distinguishable from that reported from China (Fig. 2B). The sequence data from the Miryang and Goryeong L. huidobrensis populations were deposited in GenBank, with the accession numbers KC136091 and KC136093. As the economic importance of Liriomyza spp. increases, detailed studies of intra- and interspecific differences are required that use the PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) technique (Scheffer et al., 2001; Kox et al., 2005), or multiplex PCR using newly designed primers (Nakamura et al., 2013). The present study is the first to report DNA barcoding data from the COI of L. huidobrensis. Furthermore, this study also contributes to the documentation of the biological diversity, insecticide resistance status, and evolutionary relationships of economically important Agromyzidae species.

We used the mitochondrial COI barcoding technique to separate the species (Folmer et al., 1994). The advantage of using molecular methods is that the genotypic characters are the basis for classification. Accordingly, any stage of an organism's life cycle can be used as a source of genetic material. Indeed, a study using the PCR-RFLP method was performed by Scheffer et al. (2001) in order to identify organisms at the species level. Other molecular studies involving the Agromyzidae family have also been reported (Scheffer, 2000; Scheffer and Wiegmann, 2000; Scheffer and Lewis, 2001; Scheffer et al., 2001; Kox et al., 2005). Known distribution L. huidobrensis is found throughout the world (Spencer, 1989; CABI, 2007). It is native to Central and South America (Blanchard, 1926; Argentina, Belize, Brazil [Minas Gerais, Sao Paulo], Chile, Colombia, Costa Rica, Dominican Republic, Ecuador, El Salvador, French Guiana, Guadeloupe, Guatemala, Honduras, Mexico, Nicaragua, Panama, and Peru). It is an invasive species elsewhere in the world, including the USA (California, Florida, Hawaii, Utah, and Virginia), Uruguay, Venezuela, and Europe (Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Rep., Denmark, Finland, France, Germany, Greece [Crete],

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Fig. 2. Neighbor-joining dendograms of mtDNA COI of leafminer flies based on Kimura's two-parameter distances. Intraspecific variation within the populations of L. huidobrensis and L. trifolii (A), and interspecific variation among closely related species (B). Bootstrap confidence limits are shown above the branches of clades supported in N80% of 1500 replicates. ( ) indicates collected location and accession numbers.

Hungary, Ireland, Italy [Sicily], Malta, the Netherlands, Norway, Poland, Portugal, Spain [Canary Islands], Sweden, Switzerland, and the United Kingdom [England, Northern Ireland, Scotland]). It has also dispersed to Africa (Kenya, Comoros, Mauritius, Morocco, Reunion, Seychelles, and South Africa) and Asia (China [Guizhou, Hebei, Peking, Qinghai, Sandong, Sichuan, Yunnan], Democratic People's Republic of Korea, Republic of Korea, India [Uttar Pradesh], Indonesia [Java, Sumatra], Israel, Jordan, Lebanon, Malaysia, Nepal, Singapore, Sir Lanka, Syria, Thailand, Turkey, Taiwan, and Vietnam). Diagnostics of important Liriomyza Mik 1. Head with frons -

Head with yellow frons L. huidobrensis Head with yellow frons and orbits L. trifolii Head with yellow frons and orbits L. sativae Head with bright yellow frons L. bryoniae Head largely yellow L. chinensis

2. Mesonotum - Mesonotum matt black L. huidobrensis - Mesonotum matt gray L. trifolii - Mesonotum matt black, outer vertical bristles on black ground L. sativae - Mesonotum shiny black, but with matt undertone L. bryoniae - Mesonotum grayish-black L. chinensis 3. Abdominal tergite division (Fig. 1A) -

Second visible tergite is divided L. huidobrensis Four divided tergites L. trifolii Second visible tergite is divided L. sativae Two divided tergites L. bryoniae No tergites divided L. chinensis

4. Thoracic microsetae (Fig. 1B) - Medium, length 5–6 μm L. huidobrensis - Long, length 8–9 μm L. trifolii - Medium, length 4–5 μm L. sativae

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285

Table 2 Estimation of genetic distance among the Liriomyza species after molecular analysis (mitochondrial COI barcoding).

Percent Identity 1

Divergence

1

2

3

4

5

6

7

8

9

10

11

12

13

100.0 100.0 100.0

93.0

92.8

89.2

89.2

89.3

45.5

45.5

45.4

44.9 48.0

1

L._huidobrensis_(Pyeongchang).seq

100.0 100.0

93.0

92.8

89.2

89.2

89.3

45.5

45.5

45.4

44.9 48.0

2

L._huidobrensis_(JN570505).seq

100.0

93.0

92.8

89.2

89.2

89.3

45.5

45.5

45.4

44.9 48.0

3

L._huidobrensis_Miryang_(KC136091).seq

93.0

92.8

89.2

89.2

89.3

45.5

45.5

45.4

44.9 48.0

4

L._huidobrensis_Goryeong_(KC136093).seq

99.8

89.2

89.2

89.3

46.1

46.1

46.4

46.1 48.0

5

L._trifolii_(KC136096).seq

89.0

89.0

89.2

46.0

46.0

46.4

46.0 48.0

6

L._trifolii_(KC136098).seq

99.7

99.8

46.8

46.8

47.2

46.5 48.6

7

L._sativae_(HQ333260).seq

99.8

46.8

46.8

47.4

46.8 48.6

8

L._sativae_(EU219613).seq

46.9

46.9

47.2

46.6 48.6

9

L._sativae_Suwon.seq

100.0

86.7

86.2 39.5

10

L._chinensis_(AB721342).seq

86.7

86.2 39.5

11

L._chinensis_(AB721344).seq

100.0 39.0

12

L._bryoniae_(AB721335).seq

39.0

13

L._bryoniae_(AB721337).seq

14

Cameraria ohridella.seq

2

0.0

3

0.0

0.0

4

0.0

0.0

0.0

5

7.4

7.4

7.4

7.4

6

7.6

7.6

7.6

7.6

0.2

7

11.7

11.7

11.7

11.7

11.7

11.9

8

11.7

11.7

11.7

11.7

11.7

11.9

0.3

9

11.5

11.5

11.5

11.5

11.5

11.7

0.2

0.2

10

100.4 100.4 100.4 100.4

98.0

98.7

95.5

95.3

94.8

11

100.4 100.4 100.4 100.4

98.0

98.7

95.5

95.3

94.8

0.0

12

102.2 102.2 102.2 102.2

97.9

97.9

94.8

94.2

94.8

14.7

14.7

13

103.9 103.9 103.9 103.9

98.5

99.2

97.3

96.1

96.6

15.4

15.4

14

90.3

90.3

90.3

90.3

89.9

89.9

87.5

87.5

87.5

1

2

3

4

5

6

7

8

9

0.0

14

135.7 135.7 139.2 139.2 10

- Medium, length 4–5 μm L. bryoniae - Longest, length 9–10 μm L. chinensis 5. Wing length and Cu1A (a and b sections) (Fig. 3) - Vein Cu1A, a 2–2.5 times length of b section, wing length 1.7–2.3 mm. L. huidobrensis - Vein Cu1A, a 3–4 times length of b section, wing length 1.2–1.9 mm L. trifolii - Vein Cu1A, a 3–4 times length of b section, wing length 1.3–1.7 mm L. sativae - Vein Cu1A, a 2 times length of b section, wing length 1.7–2.1 mm L. bryoniae

11

12

13

14

- Vein Cu1A, a 3–4 times length of b section, wing length 1.3–2.0 mm L. chinensis 6. Male distiphallus (Fig. 1C) - Two distal bulbs, meeting only at their rims L. huidobrensis - One distal bulb, with marked constriction between lower and upper halves L. trifolii - One distal bulb, with a slight constriction between lower and upper halves L. sativae - Two distal bulbs, bulb rims circular L. bryoniae - Aedeagus with distiphallus enlarged L. chinensis

Fig. 3. Comparison of the wing patterns of the female and male of L. huidobrensis and L. trifolii. Wing structure in L. huidobrensis, wing with last section of Cu1A, 2–2.5 times length of penultimate section, wing length 1.7–2.3 mm. In case of L. trifolii, section a of wing vein Cu1A much longer relative to section b than in L. huidobrensis. Wing with last section of Cu1A, 1, 3–4 times length of penultimate section, wing length 1.2–1.9 mm.

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7. Leaf mining nature (Fig. 1D) - Irregular serpentine mines, tends to be restricted by veins, and tendency is to mine towards the base of the leaf L. huidobrensis - Tightly coiled and almost blotch-like mines, and tendency is to mine away from the leaf stem L. trifolii - Loose, irregular serpentine mines L. sativae - Loose, irregular serpentine mines L. bryoniae - Mines initiate from one part of the leaf base and are concentrated on one side of the leaf L. chinensis Acknowledgments We thank Dr Doo-Sang Park, KRIBB, for the molecular work; Dr Heungsik Lee, QIA, for diagnostic advice; and Dr Ju-il Kim, HARC, RDA, for assistance with the field collection. RM was supported by the BK-21 program, ANU. The molecular study was made possible due to an RDA research fund (project number 906999), Korea. References Ahn, S.B., Lee, S.H., Choi, J.Y., Han, M.J., Choi, K.M., 1993. Investigation on the insect pests of new profitable crops in Korea. Ann. Rep. Agr. Sci. Ins. RDA 815–838. Ahn, S.B., Lee, S.H., Choi, J.Y., Han, M.J., Choi, K.M., 1994. Investigation on the insect pests of new profitable crops in Korea. Ann. Rep. Agr. Sci. Ins. RDA 881–913. Anonymous, 1986. A List of Plant Diseases, Insect Pests, and Weeds in Korea, 2nd ed. Kor. Soc. Plant Prot (633 pp.). Blanchard, E., 1926. A dipterous leaf-miner on Cinernria, new to science. Rev. Soc. Entomol. Argent. 1, 10–11. CABI, 2007. Crop Protection Compendium, 2007 edition ©. CAB International, Wallingford, UK. Cerny, M.M.V., Bartak, M., 2001. Agromyzidae. Catalog of the Diptera of the Australasian and Oceania Regions, 105. Folia Fac. Sci. Nat. Univ. Masaryk 349–364. Chandler, L.D., Gilstrap, F.E., 1987. Seasonal fluctuation and age structure of Liriomyza trifolii (Diptera: Agromyzidae) larval populations on bell peppers. J. Econ. Entomol. 80, 102–106. Choi, H.T., Woo, K.S., 1995. Four unrecorded species of the family Agromyzidae from Korea. Kor. J. Appl. Entomol. 34, 414. Choi, K.M., Han, S.C., Lee, M.H., Cho, W.S., Ahn, S.B., Lee, S.H., 1990. Color illustrations of ecology and control of vegetable insect pests. (224 pp. (In Korean)). EPPO, 2005. Diagonistics. EPPO Bulletin, 35. European and Mediterranean Plant Protection Organization 271–273. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome coxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Hong, K.J., Han, M.J., Kim, I.S., Ahn, S.B., Lee, M.H., 1996. Damage by American serptine leafminer, Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) and its host plants. RDA J. Agric. Sci. 38, 539–544. IIE (International Institute of Entomology), 1996. Distribution maps of plant pests. Series A: Map No. 568. CAB International, Wallingford, UK 3 p. Johnson, M.W., Welter, C., Toscano, N.C., Ting, I.P., Trumble, J.T., 1983. Reduction of tomato leaflet photosynthesis rates by mining activity of Liriomyza sativae (Diptera: Agromyzidae). J. Econ. Entomol. 76, 1061–1063. Kang, L., 1996. Ecology and Sustainable Control of Serpentine Leafminers. Science press, Beijing, China (254 pp.). Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120.

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