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

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 investigated intraspecific variation in E. guttigerus using mitochondrial NADH dehydrogenase subunit 2 (ND2), cytochrome oxidase subunit 1 (CO1), cytochrome b (Cytb), tRNA-Serine (tRNA(ser)), 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 estimated 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.

In general, species that are widely distributed have various geographical characteristics (Mayr 1942). These differences are classified into two major types: continuous and discontinuous variations. In the former, Bergmann’s rule is well known, while in the latter, geographic barriers such as the movement of continents, changes in sea level, or climate change prevent the transfer of genes among populations. In recent years, geographical variations have been found in many species through molecular phylogenetic analysis (e.g., Avise et al. 1979, Bernatchez et al. 1992, Brower 1994). Geographical variations are also seen in various animals in Japan, particularly in the Ryukyu Islands in the southwest of Japan (Ota 1998). Geographical variations are also seen in butterflies, dragonflies, fireflies and stinkbugs in populations in the Ryukyu Islands (Suzuki 1997, Kato and Yagi 2004, Kiyoshi 2008, Hosokawa et al. 2014).

Eysarcoris guttigerus (Thunberg) (Hemiptera: Pentatomidae) is distributed in East Asia and the Pacific region. In Japan, the species is found in grassy or composite weeds in the southern area of the Mainland and Ryukyu Islands (Ishikawa et al. 2012). The bug is also classified as a rice pest (Tomokuni et al. 1993). The species belonging to the genus Eysarcoris have two white spots on their scutellum (Ishikawa et al. 2012) with the exception of Ishigaki Island population, which has red spots instead (Tomokuni et al. 1993) (Fig. 1). This population also displays differences from other populations in the color of spots on the scutellum, suggesting that a large intraspecific variation may exist in this species. In this study, we investigated the geographic variation of E. guttigerus in the western mainland and Ryukyu Islands of Japan, using molecular analysis with mitochondrial DNA to obtain useful data on the intraspecific variation (e.g., Suzuki 1997, Kato and Yagi 2004, Kiyoshi 2008).

Fig. 1.

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

Materials and Methods

Insect Samples

Eysarcoris guttigerus were mainly distributed southern area of the Mainland of Japan (Ishikawa et al. 2012). So, we have collected this bug in southern Mainland and Ryukyu Islands. Samples of E. guttigerus were collected from 13 populations (Seven populations from the Mainland Area, five populations from Central Ryukyu Islands, and one population from South Ryukyu Islands including Ishigaki Island) in Japan (Fig. 2 and Table 1). We have collected several bugs in the same area. Many adults and nymphs were collected by sweeping with a net.

Fig. 2.

Map of the collection sites. The number indicates the site of collection (see Table 1).

Table 1.

Insect sample information

Species	Region	Locality	Map number	Date	Number of individuals	Accession number
						ND2	CO1(1)	CO1(2)	Cytb	tRNAser	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

As the outgroup species in phylogenetic analysis, Eysarcoris annamita Breddin was collected from Inzai shi, Chiba prefecture. These samples were stored at −20°C prior to DNA extraction. We used several samples from each population for the experiments.

DNA Extraction, PCR Amplification, and Sequencing DNA

The thorax was removed from each adult sample and homogenized in 180 μl of PBS using a pellet pestle in a 1.5-ml sample tube. DNA was then extracted using a DNeasy blood & tissue kit (QIAGEN Sciences, Germantown, MD) according to the manufacturer’s instructions. PCR reactions were performed in 10-μl reaction volumes and contained 0.05 μl of Takara Ex Taq (Takara Bio Inc., Shiga, Japan), 1 μl of 10× Ex Taq buffer (Takara Bio Inc.), 1 μl of dNTP mixture (Takara Bio Inc.), 0.5 μl of each 10 μM primer, 1–50 ng of DNA template, and sterile water. PCR was performed on a Thermal Cycler Dice (Takara Bio Inc.) by using an initial denaturing step at 94°C for 5 min followed by 25–35 cycles of 94°C for 30 s, 42–55°C for 30 s, 72°C for 30 s, and a final extension step of 72°C for 7 min. The primers listed in Table 2 were used to partially amplify the mitochondrial ND2, CO1, Cytb, tRNAser, ND1, and 16SrRNA genes. To confirm whether amplification was successful, 2 μl of the amplified product was electrophoresed on 1.5% agarose gel (1× Tris borate-EDTA), stained with ethidium bromide, and observed under a UV transilluminator. The PCR product was then purified using an ExoSAP-It (Usb) before sequencing by the direct sequencing method. A dye terminator-labeled cycle sequencing reaction was conducted with a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems Inc., Foster City, CA), with the analyses of the reaction products performed using an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems Inc.).

Table 2.

Primers used in this study

Amplified region		References
ND2
179F	5′-AGCTAATAGGTTCATACCCTA-3′	Hosokawa et al. (2014)
1452R	5′-GTTCAATAGATAAAGTGGCTG-3′
TM-J210	5′-AATTAAGCTACTAGGTTCATACCC-3′	Simon et al. (2006)
TY-N1433	5′-GGCTGAATTTTAGGCGATAAATTGTAAA-3′
CO1(1) partial
LCO1490	5′-GGTCAACAAATCATAAAGATATTGG-3′	Folmer et al. (1994)
HCO2198	5′TAAACTTCAGGGTGACCAAAAAATCA-3′
CO1(2) partial
C1-J-2183	5′-CAACATTTATTTTGATTTTTTGG-3′	Simon et al. (1994)
TL-N-3014	5′-TCCAATGCAACTAATCTGCCATATTA-3′
Cytb partial,tRNAser,ND1 partial
CB-J11335	5′-CATATTCAACCAGAATGATA-3′	Simon et al. (2006)
N1-N12067	5′-AATCGTTCTCCATTTGATTTTGC-3′
16SrRNA partial
LR-J12888	5′-CCGGTTTGAACTCARATCATGTAA-3′	Simon et al. (2006)
LR-N13889	5′-ATTTATTGTACCTTKTGTATCAG-3′

Molecular Phylogenetic Analysis

Sequence alignment was performed using BioEdit (Hall 1999), and sequences for each individual were deposited in DDBJ/EMBL/GenBank under the accession numbers (Table 1). Aligned nucleotide sites containing gaps were removed from the dataset.

Phylogenetic analyses were performed using MEGA software 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 inferred using the selected Tamura 3-parameter model with Gamma distribute (TN92+G) (Tamura 1992). Reliability of each branch node was evaluated using the bootstrap test based on 1,000 replications. The number of base substitutions per site between sequences was computed by pair-wise distance analysis. We have analyzed using several individuals from each population.

Results

We determined the nucleotide sequences of ND2, CO1, Cytb, tRNA, ND1, and 16SrRNA genes obtained from 13 populations of E. guttigerus. The lengths of nucleotide sequences were ND2: 871 bp, CO1: 1,047 bp, Cytb: 204 bp, tRNA: 104 bp, ND1: 222 bp, and 16SrRNA: 698–700 bp, with a total length of 3,146–3,148bp. Nucleotide sequence insertions were found in the same position of the 16SrRNA gene from five populations (Shima, Nagaoka, Kochi, Nankoku, and Ishigaki). In addition, another insertion was found in the same position as that of four populations (Shima, Nagaoka, Kochi, and Nankoku). Differences in sequences among individuals within the same population were very small (0–0.3% exception Okinawa–Naha population 0.1–1.0%).

In this way, we demonstrated the phylogenetic trees from DNA sequencing data (Fig. 3). In the phylogenetic trees we obtained, 13 populations of E. guttigerus were divided into three major groups (Group 1: Mainland, Group 2: Central Ryukyu Islands, Mainland of Kagoshima and Yamaguchi, and Group 3: South Ryukyu Islands), and the Group 3 was significantly separated from the other populations. These three groups were very reliably supported by bootstrap values (85 and 99%). From the results of pair-wise distance analysis, the substitution rates between the Group3 population and the other Groups populations showed very high values (2.0–2.6%) (Table 3). On the other hand, the substitution rates between the Group 1 population and the Group 2 population were low (1.0–1.2%).

Fig. 3.

Linearized phylogenetic ML tree of E. guttigerus using nucleotide sequences of ND2, CO1, Cytb, tRNA, 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.

Table 3.

Estimates of evolutionary divergence between sequences

		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	28	29	30
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
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.

Discussion

The Ishigaki population of E. guttigerus was significantly separated from the other 12 populations. Moreover, the substitution rates between the Ishigaki population and the other 12 populations showed high values (2.0–2.6%). These results suggested that the Ishigaki population of E. guttigerus has been isolated from the other populations for a long period of time. Our results showed that the Ishigaki population is differentiated from the other populations not only in spot-color but also in molecular characteristics. In recent studies, major genetic differences have 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 studies.

However, our results show that the Yamaguchi–Kagoshima clade is closely related to the Okinawa population. The similar result was also found in a phylogeographic study of the plataspid stinkbug (Hosokawa et al. 2014). The relationships among these populations need to be clarified by further molecular phylogenetic analysis.

Estimate of Divergence Time

Geographic variations have been found in many animals living in the Ryukyu Islands (Ota 1998), and are thought to be related to the geological history of the Islands (Kizaki and Oshiro 1980, Kimura 1996, Ota 1998, Osozawa et al. 2013). We calculated the divergence time of E. guttigerus populations to investigate the relationship between the geographic variations in E. guttigerus and the 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 (2.0–2.6%), the divergence time between Group 3 (Ishigaki population) and the other two groups was inferred to be ∼0.87–1.13 million years ago (Mya). From the substitution rate (1.0–1.2%) between Groups 1 and 2, the divergence time was inferred to be ∼0.44–0.52 Mya. Osozawa et al. (2012) have reported that the Ryukyu Islands existed on the continental edge 2.0 Mya. When the rifting of the Okinawa Trough began 1.55 Mya, a rift valley also began to form in the back arc of the Ryukyu arc. The Tokara Gap, Kerama Gap, and Yonaguni Gap were subsequently formed in the back arc of the Ryukyu arc. Over a long period of time, many islands formed in the area due to changes in sea level. The Tokara Gap and Kerama Gap are very deep, indicating that there were probably never any land bridges between the respective islands in the glacial period (Ota 1998). Therefore, it can be assumed that these gaps interfered with genetic relationships among many animals. The divergence time between Groups 2 and 3 is similar to the period during which the Kerama Gap formed. Thus, the formation process of the Kerama Gap may have influenced the geographic variation of E. guttigerus.

In conclusion, E. guttigerus has geographic variations in Japan. Moreover, the Ishigaki population of E. guttigerus is significantly separated from other populations. We are interested in the relationship between the Ishigaki population and populations in other areas of the South Ryukyu Islands. In the future, the investigation of populations in other areas may be able to clarify the association between intraspecific variations of E. guttigerus and the geological history of the Ryukyu Islands.