Jpn. J. Environ. Entomol. Zool. 31（1）：21－26（2020） 環動昆 第 31 巻 第 1 号：21－26（2020） Original Article

Development of microsatellite markers for the endangered butterfly Zizina emelina (de l’Orza, 1869) (: )

Daisuke Sato1), Shouhei Ueda1)*, Naoyuki Nakahama2), Ayako Izuno3), Yuji Isagi4), Masaya Yago5) and Norio Hirai1)

1） Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan 2） Institute of Natural and Environmental Sciences, University of Hyogo, 6 chome, Yayoigaoka, Sanda, Hyogo 669-1546, Japan 3）Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan 4）Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan 5）The University Museum, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

（Received: March 2, 2020; Accepted: April 1, 2020）

Abstract The lesser grass blue, Zizina emelina (de l’Orza, 1869) (Lepidoptera: Lycaenidae), is an endangered butterfly species in East Asia. In Japan and Korea, this species inhabits semi-natural grasslands with short vegetation, but available habitat is declining owing to the rapid loss of grasslands, including croplands and pastures, and natural riverbanks and seashores without concrete revetment, and the abandonment of regular grassland management. Here, we developed 14 microsatellite loci for Z. emelina based on de novo genome sequence data and observed that 13 loci exhibited polymorphisms in 51 individuals of this species. The number of alleles and the expected heterozygosity per locus were 1–10 and 0.06–0.85, respectively. A principal coordinate analysis based on this genetic information revealed genetic differentiation both among and within geographic populations of Z. emelina. The 13 microsatellite loci should be useful for future conservation genetic studies, including monitoring the genetic diversity and population structure of this species.

Keywords: conservation genetics, endangered butterfly species, genetic diversity, genetic structure, grassland butterfly

Introduction (Nakamura, 2011; Yago et al., 2016) has caused Z. emelina to be classified as "Endangered" in the fourth (latest) version of The lesser grass blue Zizina emelina (de l’Orza, 1869) is an the Japanese Red List (Ministry of the Environment in Japan, endangered butterfly species distributed in East Asia (Yago 2019). To develop a conservation strategy for an endangered et al., 2008). In Japan and Korea, this species inhabits species, it is important to understand its genetic diversity and semi-natural grasslands with short vegetation (Fukuda et al., population genetic structure, because a population with low 1984), a leguminous plant Lotus corniculatus on which the genetic diversity is at an increased risk of extinction due to larvae feed, and it used to be widely distributed in Honshu, inbreeding depression and because information of genotype Shikoku, and Kyushu islands. However, habitat loss due to the distribution would be effective to identify a conservation unit rapid loss of grasslands, including croplands and pastures, and (Frankhum et al., 2002). natural riverbanks and seashores without concrete revetment, Microsatellites are suitable genetic markers for analyzing the and the abandonment of regular grassland management genetic structure of a population (Rowe et al., 2017), but the

* Corresponding author: [email protected]

- 21 - Sato et al development of microsatellite markers has been extremely Scientific, Waltham) and sequenced by using the Ion PGM difficult in lepidopteran because the sequences of Sequencing 400 kit (Thermo Fisher Scientific, Waltham) and microsatellite flanking regions show high similarity owing to the Ion 318TM Chip v2 kit (Thermo Fisher Scientific, Waltham). the duplication or multiplication of microsatellite-containing The de novo sequencing produced 281,425 reads. Low-quality regions (Meglecz et al., 2004; Zhang, 2004). Recent advances bases (Phred quality < 20) were trimmed by using the in genome sequencing technologies, however, have made FASTX-Toolkit. Potential microsatellite loci (i.e. dinucleotides possible the development of microsatellite markers for and trinucleotides with at least eight and seven repeats, butterfly species such as Melitaea ambigua Ménétriés, 1859 respectively) were searched with MsatCommander v. 0.8.2 and Melitaea Protomedia Ménétriés, 1859 (Nakahama et al., software (Faircloth, 2008) according to the following criteria: 2015). amplification products comprising 70–150 bp; melting Sakamoto et al. (2015) investigated the spatial patterns of temperature between 57.0 and 62.0°C; GC content 35–65%. genetic variation in Z. emelina by reconstructing molecular PCR primers for amplifying these potential loci were designed phylogenies of mitochondrial (mtDNA) and nuclear (nrDNA) by using Primer 3 v. 4.0.0 software (Rozen and Skaletsky, gene sequences using specimens collected from Honshu, 2000). After screening, we obtained a total 29 candidate primer Shikoku, and Kyushu islands in mainland Japan. They pairs, and 14 of the 29 loci were effectively amplified by PCR constructed two haplotype networks based on mtDNA and with these primer pairs (Table 1). nrDNA sequences, each with six haplotypes, which revealed The adequacy of these 14 primer pairs as DNA markers was that the haplotype composition differed among populations. tested using 51 individual butterflies collected from four Although Sakamoto et al. (2015) demonstrated that six populations (Itami, Sumoto, Mima, and Kamogawa) in Japan haplotypes were detected from Honshu, Shikoku, and Kyushu (Table 2). To protect the butterflies, we determined minimum in Japan, and the proportional abundance of haplotypes were sampling and do not show the detailed collecting site locations. different among populations, to develop a conservation strategy, Multiplexes, each consisting of four loci, were designed by it is essential to analyze the fine-scale genetic structure and adding one of the following fluorescent dye-associated tags to diversity of Z. emelina using more sensitive genetic markers. each forward primer: FAM, 5’-GCCTCCCTCGCGCCA-3’; Nakahama et al. (2015) developed nine microsatellite markers VIC,5’-GCCTTGCCAGCCCGC-3’;NED,5’-CAGGACCAGG of short PCR product (70-190 bp) in an endangered butterfly, CTACCGTG-3’; or PET, 5’-CGGAGAGCCGAGAGGTG-3’ M. ambigua, based on its genome information. And Nakahama (Blackert et al., 2012; Table 1). The PCR products were and Isagi (2017) conducted the DNA fragment analysis with amplified in a total volume of 5 µl, consisting of 1.3 µl of these markers using M. ambigua specimens collected from the template DNA, 2.5 µl of 2× Type-it Multiplex PCR Master 1960s to the 2010s, and showed that negative correlations Mix in Type-it Microsatellite PCR Kit (QIAGEN, Hilden), 0.6 between the genotyping success probability and specimen age µl of a 0.06 µM solution of forward primer, and 0.6 µl of a for each of all microsatellite marker. Here, we used a 0.24 µM solution of reverse primer. Amplification was carried next-generation sequencing approach to develop microsatellite out with an initial denaturing step at 95°C for 5 min, followed markers of short PCR product for Z. emelina based on de novo by 25 cycles of denaturing at 95°C for 30 s, annealing at 60°C genome sequence data. for 90 s, and extension at 72°C for 30 s. After amplification of the targeted microsatellite loci, a second PCR amplification to Materials and methods attach the fluorescent dye was conducted in a total volume of 6.2 µl, consisting of the first PCR product, 0.6 µl of 2× Type-it Total DNA was extracted from leg muscles of a male Multiplex PCR Master Mix (QIAGEN, Hilden), and 0.6 µl of a individual collected in Itami City, Hyogo, Japan, on 8 May mixture of the four dye-labeled tails composed of the 2015, with a DNeasy Blood & Tissue Kit (Qiagen, Hilden), complementary base sequences of the FAM, VIC, NED and following the supplied protocol. A library for next-generation PET tags. Amplification was then carried out for 10 cycles of sequencing was prepared by using an Ion XpressTM Plus denaturing at 95°C for 30 s, annealing at 60°C for 90 s and Fragment Library Preparation Kit (Thermo Fisher Scientific, extension at 72°C for 30 s. The PCR products were detected on Waltham). The purified fragmented DNA was processed for an ABI Prism 3130 Analyzer (Applied Biosystems, Foster) emulsion polymerase chain reaction (emPCR) analysis by following the supplied protocol. Fragment lengths were using a PGMTM Template OT2 400 Kit (Thermo Fisher calculated using GeneMapper 4.1 software (Applied

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Table 1 Characteristics of 14 microsatellite loci for Zizina emelina, which were developed in this study.

Size range Locus Repeat pattern Primer direction Primer sequence (5'-3') Ta (℃) Color labels * (bp) ZO70613 (GA)8 Forward TACGGATTTCCTCGTTCCTG 60 VIC 93-95 Reverse GGGAATAAAAAGTGGCTACGC 60 ZO61167 10 Forward GAAAGACAGGTAACGTGACGAA 59 NED 114-140 (TA) Reverse CCGTAGCCAATAACAACAACG 60 9 ZO7980 (GA) Forward TCATCCGTCCCACTGTGATA 60 PET 96-98 Reverse TTGCGCAATACACGGAAAC 61 5 ZO146891 (GAC) Forward TTAACAGGCTGAGACGACGA 60 FAM 96-102 Reverse AACCGGCATAAGAATCTCG 58

ZO70727 (TG)10 Forward TTCTCCAGTTGATGTGTGTGTG 60 VIC 80-104 Reverse ACTGAAGAGAGTGCGTGTCG 59 ZO126426 (CA)9 Forward CCCGGATAAACCTCCTTGC 62 NED 93-101 Reverse CGGTAAGTACACGAGGGTTCA 60 8 ZO29375 (CA) Forward TAGTATACACGCGCACATCG 58 PET 99-104 Reverse TAGCTTCGGCAGGGACAC 60 ZO91058 (TCG)5 Forward TGTTACGGGTACCAAACGAA 59 FAM 109

Reverse GCCTATGTCCTGCATTGGAC 60

ZO7123 (TAT)7 Forward GGTGTGATAGCGCCCATAGA 61 VIC 117-126 Reverse ACTGCCGACCATATTCACAA 59 ZO7311 (ATT)7 Forward GACGTGGTGTGATATCGTCC 59 PET 119-125 Reverse TGGTGTAGTGCGTAAGATAATGC 59 ZO112834 6 Forward GTTCCATCCGGAAACGAGTA 60 FAM 91-94 (ACG) Reverse AACGTTCGATTCATTGTCGTC 60

ZO41286 (GA)12 Forward GCGTTATACTGGTACAGTGAGAATAAA 59 VIC 90-100 Reverse TCTTATTACGGACGAGGCTGT 59 ZO77299 (ACG)7 Forward ACGTCAACAGGCCGAGAC 60 NED 92-107 Reverse TATCGTCATCGTCATCGTCA 59 ZO20168 (TTA)8 Forward AACACCTACTGTACCATATTCCTAGC 58 PET 105-123

Reverse CGTACTTAATACGTCCACTCTCC 57 Ta: annealing temperature; *: tag sequences of the color labels FAM: 5’- GCCTCCCTCGCGCCA -3’, VIC: 5’- GCCTTGCCAGCCCGC -3’, NED: 5’- CAGGACCAGGCTACCGTG -3’, PET: 5’- CGGAGAGCCGAGAGGTG -3’ (Blackert et al., 2012).

Biosystems, Foster). After the fragment lengths were measured, genetic diversity parameters were calculated by using the GenAlEx v. 6.5.0.1 software (Peakall and Smouse, 2006), and null allele frequencies were estimated by using MICRO-CHECKER software (Van Oosterhout et al., 2004). The Arlequin v. 3.5 software package (Excoffier and Lischer, 2010) was used to determine deviation from Hardy-Weinberg equilibrium (HWE) for a total of 56 pairwise combinations of the four populations and the 14 loci, and the linkage disequilibrium (LD) between loci for a total of 364 pairwise combinations of the 14 loci in

the four populations (i.e. 14 (14 – 1) / 2 × 4 populations). To

evaluate genetic differentiation among the four populations of Fig. 1 Principal coordinate analysis plot of Zizina emelina samples based on codominant genotypic distance. Axes Z. emelina, we performed a principal coordinate analysis 1 and 2 explain 25.37% and 13.93% of the variance, (PCoA) using the genetic data of each population with respectively. GenAlex version 6.41 software (Peakall and Smouse, 2006).

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Table 2 Specimens of Zizina emelina for the microsatellite Results and discussion analysis. Collection All 14 loci were amplified, and 13 loci showed Voucher # Prefecture Location Date polymorphisms in 51 Z. emelina individuals (Table 1). The OA2 Hyogo Itami 2017.05.10 OA11 Itami 2017.06.16 number of alleles per locus (A) and the number of private OA18 Itami 2017.08.16 alleles per locus (PA) was 1–10 and 1–5, respectively (Table OA19 Itami 2017.08.16 2). Null alleles were detected in two locus–population OA24 Itami 2017.08.16 SK1 Itami 2017.05.10 combinations (ZO7311–Itami and ZO70727–Sumoto; P < SK5 Itami 2017.05.10 0.05). The observed heterozygosity was 0.06–1.00 (mean 0.29), SK6 Itami 2017.05.10 and the expected heterozygosity was 0.06–0.85 (mean 0.32) SK19 Itami 2017.06.16 SK29 Itami 2017.07.19 (Table 3). Significant deviations from HWE were detected in SK36 Itami 2017.08.16 the same two locus–population combinations (ZO7311–Itami SK42 Itami 2017.09.13 and ZO70727–Sumoto; P < 0.05). The null alleles might TC1 Itami 2017.06.16 DKN6 Itami 2017.09.13 plausibly explain the deviation from HWE (e.g. Dakin and DKN10 Itami 2017.09.23 Avise, 2004). Significant LD was detected in two pairs of loci TDR1 Itami 2017.08.25 (ZO61167–ZO126426 and ZO112834–ZO41286) in the Mima TDR3 Itami 2017.09.13 ATN12 Itami 2017.10.26 population, three pairs of loci (ZO61167–ZO20168, FR2 Itami 2017.07.19 ZO126426–ZO41286, and ZO126426–ZO77299) in the FG7 Itami 2017.07.19 Sumoto population, and one pair (ZO7123–ZO112834) in the 1058 Tokushima Mima 2012.07.31 1064 Mima 2012.07.31 Kamogawa population (P < 0.05). These pairs did not include 1065 Mima 2012.07.31 the ZO7311 and ZO70727 loci, where significant deviations 1066 Mima 2012.07.31 from HWE and null alleles were detected; thus, physical 1067 Mima 2012.07.31 1068 Mima 2012.07.31 linkage between these pairs of loci with significant LD may be 1069 Mima 2012.07.31 unlikely. The significant LD may have resulted from a recent 1070 Mima 2012.07.31 population bottleneck or high levels of inbreeding in the AW1 Hyogo Sumoto 2017.06.27 AW2 Sumoto 2017.06.27 population (e.g. Rowe et al., 2017). AW3 Sumoto 2017.06.27 The PCoA based on the 13 microsatellite loci differentiated AW4 Sumoto 2017.06.27 the Kamogawa population of eastern Japan from the other AW5 Sumoto 2017.06.27 AW6 Sumoto 2017.06.27 three populations, all in western Japan; among the western AW7 Sumoto 2017.06.27 Japan populations, the Sumoto population was differentiated AW8 Sumoto 2017.06.27 from the Itami and Mima populations (Table 2; Fig. 1), but AW9 Sumoto 2017.06.27 AW10 Sumoto 2017.06.27 no clear genetic differentiation between the Itami and Mima AW11 Sumoto 2017.06.27 populations could be detected (Fig. 1). Genetic polymorphism AW12 Sumoto 2017.06.27 among individuals within each population was respectively AW13 Sumoto 2017.06.27 AW14 Sumoto 2017.06.27 detected (Fig. 1). Thus, the PCoA based on the 13 AW15 Sumoto 2017.06.27 microsatellite loci revealed genetic differentiation both among AW16 Sumoto 2017.06.27 and within populations of Z. emelina. These results suggest K3 Chiba Kamogawa 2007.09.09 K4 Kamogawa 2007.09.09 that these microsatellite loci should be useful for future K5 Kamogawa 2007.09.09 conservation genetic studies, including for monitoring the K6 Kamogawa 2007.09.09 genetic diversity and structure of populations, in order to K8 Kamogawa 2007.09.09 K9 Kamogawa 2007.09.09 establish an effective conservation strategy for the endangered K10 Kamogawa 2007.09.09 butterfly Z. emelina. The microsatellite makers developed in our study were designed to amplify short PCR products (80–140 bp) (Table 1) because Nakahama and Isagi (2017) showed that short microsatellite markers in M. ambigua can be successfully used to investigate genetic diversity in older

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Table 3 Results of population genetics for 14 microsatellite loci in four populations of Zizina emelina. Itami (N=20) Mima (N =8) Sumoto (N =16) Kamogawa (N =7) Locus APA H o H e APA H o H e APA H o H e APA H o H e ZO70613 2 0 0.15 0.14 2 0 0.13 0.13 2 0 0.06 0.06 2 0 0.14 0.14 ZO61167 10 5 0.80 0.85 3 0 0.63 0.57 4 1 0.50 0.47 2 1 0.33 0.30 ZO7980 2 0 0.45 0.48 2 0 0.25 0.53 1 0 - - 2 0 0.43 0.36 ZO146891 1 0 - - 2 1 0.13 0.13 1 0 - - 1 0 - - ZO70727 5 1 0.60 0.67 4 1 0.63 0.58 2 0 0.19 0.50 6 5 0.57 0.68 ZO126426 3 1 0.55 0.61 2 0 0.25 0.40 2 0 0.50 0.51 4 2 0.86 0.69 ZO29375 3 0 0.40 0.50 4 1 0.75 0.72 1 0 - - 2 0 0.14 0.14 ZO9105810- - 10- - 10- - 10- - ZO7123 2 0 0.25 0.36 2 0 0.13 0.13 2 0 0.44 0.47 2 1 0.17 0.17 ZO7311 2 0 0.21 0.48 2 0 0.13 0.13 1 0 - - 1 0 - - ZO112834 2 0 0.25 0.30 2 0 0.25 0.40 2 0 0.13 0.12 2 0 0.43 0.54 ZO41286 2 1 0.50 0.49 2 0 0.38 0.53 2 0 0.56 0.51 2 1 0.14 0.36 ZO7729910- - 10- - 210.190.27210.290.26 ZO20168 4 0 0.65 0.72 2 0 0.25 0.23 3 0 0.69 0.51 4 2 1.00 0.82 N: number of samples; A: number of alleles, PA: private allele, Ho: observed heterozygosity, He: expected heterozygosity. specimens (from the 1960s onward). Therefore, the markers Meglecz, E., F. Petenian, E. Danchin, A. C. D’Acier, J. Y. developed here can potentially be used to evaluate temporal Rasplus and E. Faure (2004) High similarity between changes in genetic diversity in a local population over a period flanking regions of different microsatellites detected of more than 30 years. within each of two species of Lepidoptera: Parnassius apollo and Euphydryas aurinia. Mol. Ecol. 13: Acknowledgements 1693-1700. Ministry of the Environment, Japan (2019) The Red List of We are grateful to Dr. Y. Sakamoto, Mr. S. Morichi, Mr. S. Insects of Japan. https://www.env.go.jp/press/106383.html Minohara, and Dr. T. Koyama for providing the butterfly (accessed January 4, 2020). samples and advice on the experimental methods. Nakahama, N. and Y. Isagi (2017) Availability of short microsatellite markers from butterfly museums and References private specimens. Entomol. Sci. 20: 3–6. Nakahama, N., A. Izuno, K. Arima and Y. Isagi (2015) Blacket, M. J., C. Robin, R. T. Good, S. F. Lee and A. D. Development of microsatellite markers for two Miller (2012) Universal primers for fluorescent labelling endangered grassland butterflies, Melitaea ambigua and of PCR fragments–an efficient and cost–effective M. protomedia (Nymphalidae), using Ion Torrent approach to genotyping by fluorescence. Mol. Ecol. Res. next-generation sequencing. Conserv. Genet. Resour. 7: 12: 456–463. 525–527. Dakin, E. E. and J. C. Avise (2004) Microsatellite null alleles Nakamura, Y. (2011) Conservation of butterflies in Japan: in parentage analysis. Heredity 93: 504–509. status, actions and strategy. J. Conserv. 15: 5–22. Excoffier, L. and H. E. L. Lischer (2010) Arlequin suite ver. Peakall, R. O. D. and P. E. Smouse (2006) GENALEX 6: 3.5: a new series of programs to perform population genetic analysis in Excel. Population genetic software for genetics analyses under Linux and Windows. Mol. Ecol. teaching and research. Mol. Ecol. Notes 6: 288–295. Res. 10: 564–567. Rowe, G., M. Sweet and T. J. C. Beebee (2017) An Faircloth, B. C. (2008) MSATCOMMANDER: detection of Introduction to Molecular Ecology. Oxford University microsatellite repeat arrays and automated, locus-specific Press, New York. primer design. Mol. Ecol. Res. 8: 92–94. Rozen, S. and H. Skaletsky (2000) PRIMER3 on the WWW for Frankham, R., D. A. Briscoe and J. D. Ballou (2002) general users and for biologist programmers. Methods Introduction to Conservation Genetics. Cambridge Mol. Biol. 132: 365–386. University Press, Cambridge, UK. Sakamoto, Y., N. Hirai, T. Tanikawa, M. Yago and M. Ishii Fukuda, H., E. Hama, T. Kuzuya, A. Takahashi, M. Takahashi, (2015) Population genetic structure and Wolbachia B. Tanaka, H. Tanaka, M. Wakabayashi and Y. Watanabe infection in an endangered butterfly, Zizina emelina (1984) The Life Histories of Butterflies in Japan, Vol. 3. (Lepidoptera, Lycaenidae), in Japan. Bull. Entomol. Res. Hoikusha, Osaka, Japan (in Japanese). 105: 152–165.

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Van Oosterhout, C., W. F. Hutchinson, D. P. M. Wills and P. Yago, M., N. Hirai and U. Jinbo (2016) The Red List of Shipley (2004) MICRO-CHECKER: software for Butterflies in Prefectures, Japan –Fourth Edition (2015)–. identifying and correcting genotyping errors in In “Decline and Conservation of Butterflies and Moths in microsatellite data. Mol. Ecol. Notes 4: 535–538. Japan Ⅶ” (Yago, M., N. Hirai and U. Jinbo, eds), pp. Yago, M., N. Hirai, M. Kondo, T. Tanikawa, M. Ishii, M. Wang, 88–351, Lepidopterological Society of Japan, Tokyo (in M. Williams and R. Ueshima (2008) Molecular Japanese with English summary). systematics and biogeography of the Zizina Zhang, D. X. (2004). Lepidopteran microsatellite DNA: (Lepidoptera: Lycaenidae). Zootaxa 1746: 15-38. redundant but promising. Trends Ecol. Evol. 19: 507–509.

絶滅危惧種シルビアシジミ（チョウ目:シジミチョウ科）のマイクロサテライトマー カーの開発

佐藤大輔 1)・上田昇平 1)*・中濵直之 2)・伊津野彩子 3)・井鷺裕司 4)・矢後勝也 5)・平井規央 1)

1) 大阪府立大学大学院生命環境科学研究科 2) 兵庫県立大学自然・環境科学研究所 3) 国立研究開発法人森林研究・整備機構森林総合研究所 4) 京都大学大学院農学研究科 5) 東京大学総合研究博物館

シルビアシジミ Zizina emelina (de l’Orza, 1869) （チョウ目：シジミチョウ科）は，東アジアに分布する絶滅 危惧種である．日本と韓国において本種は草丈の低い半自然草原に生息するが，本種が利用可能な生息地である 農耕地，牧草地や，コンクリートで被覆されていない自然の河川と海岸の堤防などの草原の喪失や，これらの草 原の管理放棄により減少しているとされる．本研究では，本種のゲノム情報から 14 遺伝子座のマイクロサテラ イトマーカーを開発した．それらを用いて本種 51 個体のフラグメント解析を実施した結果，13 遺伝子座で多型 を確認した．アリル数，ヘテロ接合度の期待値はそれぞれ 1-10，0.06-0.85 であった．これらの遺伝情報を用い た主座標分析では，本種の地域集団間および集団内の遺伝的差異を検出することができた．開発した 13 個のマ イクロサテライトマーカーは，本種の遺伝的多様性と遺伝的構造の評価を含む，保全遺伝学的研究に有用である と考えられる．

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