Intraspecific Variation of Eysarcoris Guttigerus (Hemiptera: Pentatomidae) in Japanese Southwest Population Based on Mitochondrial DNA

Journal of Insect Science (2016) 16(1): 7; 1–7 doi: 10.1093/jisesa/iev147 Research article

Intraspecific Variation of Eysarcoris guttigerus (Hemiptera: Pentatomidae) in Japanese Southwest Population Based on Mitochondrial DNA

Takuya Yamaji,1 Tadashi Ishikawa,3 and Masashi Nomura2

1Laboratory of Applied Entomology, Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan (yamata- [email protected]), 2Corresponding author, e-mail: [email protected], 3Laboratory of Insect Resources, Faculty of Agriculture, Tokyo University of Agriculture, Atsugi, Kanagawa, 243-0034 Japan ([email protected])

Subject Editor: Julie Urban

Received 16 July 2015; Accepted 19 November 2015

Abstract The white-spotted globular bug Eysarcoris guttigerus (Thunberg) (Hemiptera: Pentatomidae) is widely distributed in East Asia and the Pacific region. In Japan, the species is found in grassy or composite weeds in the western area of the main islands and Ryukyu Islands of Japan. One notable characteristic of the Eysarcoris genus is the two white spots on the scutellum. This is not the case with the Ishigaki Island population, however, which sports red spots instead of white, suggesting that intraspecific variation exists in the species. Therefore, we in- vestigated intraspecific variation in E. guttigerus using mitochondrial NADH dehydrogenase subunit 2 (ND2), cytochrome oxidase subunit 1 (CO1), cytochrome b (Cytb), tRNA-Serine (tRNAser), NADH dehydrogenase subunit 1 (ND1), and 16S ribosomal RNA (16SrRNA) genes from 13 populations of Japan. The obtained maximum likelihood phylogenetic tree was divided into three groups—Group 1: Mainland, Group 2: Central Ryukyu Islands (Okinawa-Amamioshima Islands), and Group 3: South Ryukyu Islands (Ishigaki Island). The Ishigaki population was significantly separated from the other populations with consistent differences in spot color. The es- timated period of divergence between the Ishigaki population and the other populations was consistent with the period of formation of the Kerama Gap in the Ryukyu arc. Thus, the process of formation of the Kerama Gap may have influenced the intraspecific variation of E. guttigerus.

Key words: Eysarcoris guttigerus, intraspeciﬁc variation, mitochondrial DNA, phylogenetics, Ryukyu Islands

In general, species that are widely distributed have various geo- belonging to the genus Eysarcoris have two white spots on their scu- graphical characteristics (Mayr 1942). These differences are classi- tellum (Ishikawa et al. 2012) with the exception of Ishigaki Island fied into two major types: continuous and discontinuous variations. population, which has red spots instead (Tomokuni et al. 1993) In the former, Bergmann’s rule is well known, while in the latter, (Fig. 1). This population also displays differences from other popu- geographic barriers such as the movement of continents, changes in lations in the color of spots on the scutellum, suggesting that a large sea level, or climate change prevent the transfer of genes among pop- intraspecific variation may exist in this species. In this study, we in- ulations. In recent years, geographical variations have been found in vestigated the geographic variation of E. guttigerus in the western many species through molecular phylogenetic analysis (e.g., Avise mainland and Ryukyu Islands of Japan, using molecular analysis et al. 1979, Bernatchez et al. 1992, Brower 1994). Geographical var- with mitochondrial DNA to obtain useful data on the intraspecific iations are also seen in various animals in Japan, particularly in the variation (e.g., Suzuki 1997, Kato and Yagi 2004, Kiyoshi 2008). Ryukyu Islands in the southwest of Japan (Ota 1998). Geographical variations are also seen in butterflies, dragonflies, fireflies and stink- Materials and Methods bugs in populations in the Ryukyu Islands (Suzuki 1997, Kato and Yagi 2004, Kiyoshi 2008, Hosokawa et al. 2014). Insect Samples Eysarcoris guttigerus (Thunberg) (Hemiptera: Pentatomidae) is Eysarcoris guttigerus were mainly distributed southern area of the distributed in East Asia and the Pacific region. In Japan, the species Mainland of Japan (Ishikawa et al. 2012). So, we have collected this is found in grassy or composite weeds in the southern area of the bug in southern Mainland and Ryukyu Islands. Samples of E. guttigerus Mainland and Ryukyu Islands (Ishikawa et al. 2012). The bug is were collected from 13 populations (Seven populations from the also classified as a rice pest (Tomokuni et al. 1993). The species Mainland Area, five populations from Central Ryukyu Islands, and one

VC The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2016, Vol. 16, No. 1

Fig. 1. Adults of E. guttigerus. (A) Kochi (spots are white). (B) Ishigaki (spots are red).

Fig. 2. Map of the collection sites. The number indicates the site of collection (see Table 1). population from South Ryukyu Islands including Ishigaki Island) in was then extracted using a DNeasy blood & tissue kit (QIAGEN Japan (Fig. 2 and Table 1). We have collected several bugs in the same Sciences, Germantown, MD) according to the manufacturer’s area. Many adults and nymphs were collected by sweeping with a net. instructions. PCR reactions were performed in 10-ll reaction vol- As the outgroup species in phylogenetic analysis, Eysarcoris umes and contained 0.05 ll of Takara Ex Taq (Takara Bio Inc., annamita Breddin was collected from Inzai shi, Chiba prefecture. Shiga, Japan), 1 llof10 Ex Taq buffer (Takara Bio Inc.), 1 llof These samples were stored at 20C prior to DNA extraction. We dNTP mixture (Takara Bio Inc.), 0.5 ll of each 10 lM primer, used several samples from each population for the experiments. 1–50 ng of DNA template, and sterile water. PCR was performed on a Thermal Cycler Dice (Takara Bio Inc.) by using an initial denatur- DNA Extraction, PCR Amplification, and Sequencing DNA ing step at 94C for 5 min followed by 25–35 cycles of 94C for The thorax was removed from each adult sample and homogenized 30 s, 42–55C for 30 s, 72C for 30 s, and a final extension step of in 180 ll of PBS using a pellet pestle in a 1.5-ml sample tube. DNA 72C for 7 min. The primers listed in Table 2 were used to partially ora fIsc Science Insect of Journal

Table 1. Insect sample information 06 o.1,N.16 No. 16, Vol. 2016, ,

Species Region Locality Map Date Number Accession number number of ser individuals ND2 CO1(1) CO1(2) Cytb tRNA ND1 16SrRNA

E. guttigerus Mainland Mie, Shima shi, Isobe cho 1 26-VIII-14 1 LC027266 LC027112 LC027252 LC027575 LC027589 LC027603 LC027561 Kochi, Nagaoka gun, Motoyama cho 2 7-VIII-14 4 LC027260 LC027106 LC027246 LC027569 LC027583 LC027597 LC027555 Kochi, Kochi shi, Katsushima 3 8-VIII-14 2 LC027258 LC027104 LC027244 LC027567 LC027581 LC027595 LC027553 1 LC027258 LC027104 LC090795 LC027567 LC027581 LC027595 LC090811 1 LC027258 LC027104 LC027244 LC090798 LC027581 LC090804 LC090812 Kochi, Nankoku shi, Monobe 4 9-VIII-14 1 LC027262 LC027108 LC027248 LC027571 LC027585 LC027599 LC027557 1 LC027262 LC090790 LC027248 LC027571 LC027585 LC090803 LC027557 Yamaguchi, Shunan shi, Tokuyama 5 19-X-14 1 LC027268 LC027114 LC027254 LC027577 LC027591 LC027605 LC027563 Yamaguchi, Shunan shi, Otsushima 6 20-X-14 1 LC027264 LC027110 LC027250 LC027573 LC027587 LC027601 LC027559 Kagoshima, Aira shi, Kajiki cho 7 28-XI-14 2 LC027257 LC027103 LC027243 LC027566 LC027580 LC027594 LC027552 Central Ryukyu Islands Kagoshima, Oshima gun, Tatsugo cho 8 29-XI-14 2 LC027267 LC027113 LC027253 LC027576 LC027590 LC027604 LC027562 Kagoshima, Oshima gun, Setouchi cho 9 30-XI-14 2 LC027265 LC027111 LC027251 LC027574 LC027588 LC027602 LC027560 Kagoshima, Amami shi, Nazehirata cho 10 1-XII-14 1 LC027263 LC027109 LC027249 LC027572 LC027586 LC027600 LC027558 Okinawa, Kunigami gun, Kunigami son 11 11-VIII-14 2 LC027259 LC027105 LC027245 LC027568 LC027582 LC027596 LC027554 Okinawa, Naha shi, Syurisueyoshi cho 12 17-VIII-14 1 LC027261 LC027107 LC027247 LC027570 LC027584 LC027598 LC027556 1 LC090788 LC090789 LC090796 LC027570 LC027584 LC027598 LC090808 1 LC027261 LC027107 LC027247 LC027570 LC027584 LC027598 LC090809 South Ryukyu Islands Okinawa, Ishigaki shi, Tonoshiro 13 24-IX-14 1 LC091533 LC027102 LC027242 LC027565 LC027579 LC027593 LC027551 1 LC090785 LC027102 LC090791 LC090799 LC027579 LC090805 LC090808 1 LC090786 LC027102 LC090792 LC090800 LC027579 LC090806 LC027551 1 LC090787 LC027102 LC090793 LC090801 LC027579 LC090807 LC027551 E. annamita Mainland Chiba, Inzai shi, Iwato – 31-VIII-14 1 LC096061 LC096062 LC096063 LC096064 LC096067 LC096065 LC096066 3 4 Journal of Insect Science, 2016, Vol. 16, No. 1

Table 2. Primers used in this study

Amplified region References

ND2 179F 50-AGCTAATAGGTTCATACCCTA-30 Hosokawa et al. (2014) 1452R 50-GTTCAATAGATAAAGTGGCTG-30 TM-J210 50-AATTAAGCTACTAGGTTCATACCC-30 Simon et al. (2006) TY-N1433 50-GGCTGAATTTTAGGCGATAAATTGTAAA-30 CO1(1) partial LCO1490 50-GGTCAACAAATCATAAAGATATTGG-30 Folmer et al. (1994) HCO2198 50TAAACTTCAGGGTGACCAAAAAATCA-30 CO1(2) partial C1-J-2183 50-CAACATTTATTTTGATTTTTTGG-30 Simon et al. (1994) TL-N-3014 50-TCCAATGCAACTAATCTGCCATATTA-30 Cytb partial,tRNAser,ND1 partial CB-J11335 50-CATATTCAACCAGAATGATA-30 Simon et al. (2006) N1-N12067 50-AATCGTTCTCCATTTGATTTTGC-30 16SrRNA partial LR-J12888 50-CCGGTTTGAACTCARATCATGTAA-30 Simon et al. (2006) LR-N13889 50-ATTTATTGTACCTTKTGTATCAG-30

Fig. 3. Linearized phylogenetic ML tree of E. guttigerus using nucleotide sequences of ND2, CO1, Cytb, tRNASer, ND1, and 16SrRNA genes. The total length of the nucleotide sequence is 3,143 bp. Bootstrap conﬁdence levels calculated based on 1,000 replications are shown near the nodes. Journal of Insect Science, 2016, Vol. 16, No. 16 5 amplify the mitochondrial ND2, CO1, Cytb, tRNAser, ND1, and Discussion 16SrRNA genes. To confirm whether amplification was successful, Molecular Phylogenetic Analysis 2 ll of the amplified product was electrophoresed on 1.5% agarose The Ishigaki population of E. guttigerus was significantly sepa- gel (1 Tris borate-EDTA), stained with ethidium bromide, rated from the other 12 populations. Moreover, the substitution and observed under a UV transilluminator. The PCR product was rates between the Ishigaki population and the other 12 popula- then purified using an ExoSAP-It (Usb) before sequencing by tions showed high values (2.0–2.6%). These results suggested the direct sequencing method. A dye terminator-labeled cycle that the Ishigaki population of E. guttigerus has been isolated sequencing reaction was conducted with a BigDye Terminator from the other populations for a long period of time. Our results version 3.1 cycle sequencing kit (Applied Biosystems Inc., Foster showed that the Ishigaki population is differentiated from the City, CA), with the analyses of the reaction products other populations not only in spot-color but also in molecular performed using an ABI PRISM 3130xl Genetic Analyzer (Applied characteristics. In recent studies, major genetic differences have Biosystems Inc.). been found in some insects between the southern Ryukyu Islands and central Ryukyu Islands (Suzuki 1997, Kato and Yagi 2004, Kiyoshi 2008). Our results were generally consistent with these Molecular Phylogenetic Analysis studies. Sequence alignment was performed using BioEdit (Hall 1999), However, our results show that the Yamaguchi–Kagoshima and sequences for each individual were deposited in DDBJ/ clade is closely related to the Okinawa population. The similar EMBL/GenBank under the accession numbers (Table 1). result was also found in a phylogeographic study of the plataspid Aligned nucleotide sites containing gaps were removed from the stinkbug (Hosokawa et al. 2014). The relationships among these dataset. populations need to be clarified by further molecular phylogenetic Phylogenetic analyses were performed using MEGA software analysis. ver. 6.06 for maximum likelihood (ML) method (Tamura et al. 2013). The best-fit nucleotide substitution model was selected using the Bayesian Information Criterion score with Find Best DNA Model (ML) in MEGA. For this analysis, the ML tree was Estimate of Divergence Time inferred using the selected Tamura 3-parameter model with Geographic variations have been found in many animals living in Gamma distribute (TN92þG) (Tamura 1992). Reliability of each the Ryukyu Islands (Ota 1998), and are thought to be related to the branch node was evaluated using the bootstrap test based on geological history of the Islands (Kizaki and Oshiro 1980, Kimura 1,000 replications. The number of base substitutions per 1996, Ota 1998, Osozawa et al. 2013). We calculated the diver- site between sequences was computed by pair-wise distance anal- gence time of E. guttigerus populations to investigate the relation- ysis. We have analyzed using several individuals from each ship between the geographic variations in E. guttigerus and the population. geological history of the Ryukyu Islands. The evolutionary rate in mtDNA was inferred from the combined data of seven arthropod species by Brower (1994), showing a pair-wise sequence divergence of 2.3% per million years. From the data of substitution rates Results (2.0–2.6%), the divergence time between Group 3 (Ishigaki popula- We determined the nucleotide sequences of ND2, CO1, Cytb, tion) and the other two groups was inferred to be 0.87–1.13 mil- tRNAser, ND1,and16SrRNA genes obtained from 13 populations lion years ago (Mya). From the substitution rate (1.0–1.2%) of E. guttigerus. The lengths of nucleotide sequences were ND2: between Groups 1 and 2, the divergence time was inferred to be 871 bp, CO1: 1,047 bp, Cytb: 204 bp, tRNAser: 104 bp, ND1: 0.44–0.52 Mya. Osozawa et al. (2012) have reported that the 222 bp, and 16SrRNA: 698–700 bp, with a total length of Ryukyu Islands existed on the continental edge 2.0 Mya. When the 3,146–3,148bp. Nucleotide sequence insertions were found in the rifting of the Okinawa Trough began 1.55 Mya, a rift valley also same position of the 16SrRNA gene from five populations (Shima, began to form in the back arc of the Ryukyu arc. The Tokara Gap, Nagaoka, Kochi, Nankoku, and Ishigaki). In addition, another Kerama Gap, and Yonaguni Gap were subsequently formed in the insertion was found in the same position as that of four popula- back arc of the Ryukyu arc. Over a long period of time, many tions (Shima, Nagaoka, Kochi, and Nankoku). Differences in islands formed in the area due to changes in sea level. The Tokara sequences among individuals within the same population were Gap and Kerama Gap are very deep, indicating that there were very small (0–0.3% exception Okinawa–Naha population probably never any land bridges between the respective islands in 0.1–1.0%). the glacial period (Ota 1998). Therefore, it can be assumed that In this way, we demonstrated the phylogenetic trees from DNA these gaps interfered with genetic relationships among many ani- sequencing data (Fig. 3). In the phylogenetic trees we obtained, 13 mals. The divergence time between Groups 2 and 3 is similar to the populations of E. guttigerus were divided into three major groups period during which the Kerama Gap formed. Thus, the formation (Group 1: Mainland, Group 2: Central Ryukyu Islands, Mainland process of the Kerama Gap may have influenced the geographic var- of Kagoshima and Yamaguchi, and Group 3: South Ryukyu iation of E. guttigerus. Islands), and the Group 3 was significantly separated from the other In conclusion, E. guttigerus has geographic variations in Japan. populations. These three groups were very reliably supported by Moreover, the Ishigaki population of E. guttigerus is significantly bootstrap values (85 and 99%). From the results of pair-wise dis- separated from other populations. We are interested in the relation- tance analysis, the substitution rates between the Group3 population ship between the Ishigaki population and populations in other areas and the other Groups populations showed very high values (2.0– of the South Ryukyu Islands. In the future, the investigation of pop- 2.6%) (Table 3). On the other hand, the substitution rates between ulations in other areas may be able to clarify the association between the Group 1 population and the Group 2 population were low (1.0– intraspecific variations of E. guttigerus and the geological history of 1.2%). the Ryukyu Islands. 6

Table 3. Estimates of evolutionary divergence between sequences

123456789101112131415161718192021222324252627282930

1 Shima1 2 Nagaoka1 0.002 3 Nagaoka2 0.002 0.000 4 Nagaoka3 0.002 0.000 0.000 5 Nagaoka4 0.002 0.000 0.000 0.000 6 Kochi1 0.002 0.000 0.000 0.000 0.000 7 Kochi2 0.002 0.001 0.001 0.001 0.001 0.001 8 Kochi3 0.002 0.001 0.001 0.001 0.001 0.001 0.001 9 Kochi4 0.002 0.000 0.000 0.000 0.000 0.000 0.001 0.001 10 Nankoku1 0.002 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 11 Nankoku2 0.002 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.001 0.001 12 Tokuyama1 0.011 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010 13 Otsushima1 0.011 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.000 14 Aira1 0.011 0.010 0.010 0.010 0.010 0.010 0.011 0.011 0.010 0.010 0.010 0.000 0.000 15 Aira2 0.011 0.010 0.010 0.010 0.010 0.010 0.011 0.011 0.010 0.010 0.010 0.000 0.000 0.001 16 Tatsugo1 0.012 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.009 0.009 0.009 0.008 17 Tatsugo2 0.012 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.009 0.009 0.010 0.009 0.001 18 Setouchi1 0.011 0.010 0.010 0.010 0.010 0.010 0.011 0.011 0.011 0.011 0.011 0.009 0.009 0.009 0.009 0.000 0.000 19 Setouchi2 0.012 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.009 0.009 0.010 0.009 0.001 0.001 0.000 20 Amami1 0.012 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.009 0.009 0.010 0.009 0.001 0.000 0.000 0.001 21 Kunigami1 0.011 0.010 0.010 0.010 0.010 0.010 0.011 0.011 0.011 0.010 0.011 0.004 0.004 0.005 0.004 0.009 0.010 0.010 0.010 0.010 22 Kunigami2 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.005 0.005 0.005 0.004 0.010 0.010 0.010 0.010 0.010 0.000

23 Naha1 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.005 0.005 0.005 0.004 0.010 0.010 0.010 0.010 0.010 0.000 0.001 Science Insect of Journal 24 Naha2 0.012 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.011 0.009 0.009 0.010 0.009 0.001 0.001 0.000 0.001 0.001 0.010 0.010 0.010 25 Naha3 0.011 0.011 0.011 0.011 0.011 0.011 0.012 0.012 0.011 0.011 0.011 0.005 0.005 0.005 0.005 0.010 0.010 0.010 0.010 0.010 0.001 0.001 0.001 0.010 26 Ishigaki1 0.022 0.022 0.022 0.022 0.022 0.022 0.022 0.022 0.022 0.022 0.022 0.026 0.026 0.026 0.026 0.024 0.024 0.024 0.024 0.024 0.024 0.024 0.024 0.024 0.024 27 Ishigaki2 0.021 0.021 0.021 0.021 0.021 0.021 0.021 0.021 0.021 0.021 0.021 0.024 0.024 0.024 0.024 0.023 0.022 0.022 0.023 0.022 0.023 0.023 0.023 0.023 0.024 0.003 28 Ishigaki3 0.021 0.020 0.020 0.020 0.020 0.020 0.021 0.021 0.021 0.021 0.021 0.024 0.024 0.024 0.024 0.022 0.022 0.022 0.022 0.022 0.023 0.023 0.023 0.022 0.023 0.003 0.001 29 Ishigaki4 0.021 0.020 0.020 0.020 0.020 0.020 0.021 0.021 0.021 0.021 0.021 0.024 0.024 0.024 0.024 0.022 0.022 0.022 0.022 0.022 0.023 0.023 0.023 0.022 0.023 0.003 0.001 0.000 30 E. annamita 0.087 0.087 0.087 0.087 0.087 0.087 0.087 0.087 0.087 0.087 0.087 0.089 0.089 0.090 0.090 0.089 0.089 0.089 0.089 0.089 0.088 0.088 0.089 0.089 0.088 0.088 0.087 0.088 0.088

The numbers of base substitutions per site between sequences are shown. 06 o.1,N.1 No. 16, Vol. 2016, , Journal of Insect Science, 2016, Vol. 16, No. 16 7

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