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Displacement of Torymus beneficus (Hymenoptera: Torymidae) by T. sinensis, an indigenous and introduced parasitoid of the chestnut gall wasp, Dryocosmus kuriphilus (Hymenoptera: Cynipidae), in Japanese chestnut fields: Possible involvement in hybridization
Biological Control 42 (2007) 148–154 www.elsevier.com/locate/ybcon
Displacement of Torymus beneficus (Hymenoptera: Torymidae) by T. sinensis, an indigenous and introduced parasitoid of the chestnut gall wasp, Dryocosmus kuriphilus (Hymenoptera: Cynipidae), in Japanese chestnut fields: Possible involvement in hybridization Kaori Yara a
a,*
, Terunori Sasawaki b, Yasuhisa Kunimi
c
National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan b Nagano Fruit-tree Experiment Station, Suzaka, Nagano 382-0071, Japan c Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8538, Japan Received 12 October 2006; accepted 25 April 2007 Available online 3 May 2007
Abstract Although there is empirical knowledge to suggest the displacement of Torymus beneficus (Hymenoptera: Torymidae) by Torymus sinensis, an indigenous and introduced parasitoid of the chestnut gall wasp Dryocosmus kuriphilus (Hymenoptera: Cynipidae), respectively, in Japanese chestnut fields, the underlying mechanisms are unclear. In this study, the displacement of the early-spring strain of T. beneficus by T. sinensis was surveyed in a chestnut field for nine successive years (n = 418), using two molecular markers, the internal transcribed spacer 2 of nuclear rDNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI). We also investigated whether or not hybridization between the parasitoids was involved as an important factor in the displacement. All individuals from 1993 to 1995 were of the early-spring strain of T. beneficus type for both ITS2 and COI. After 1996, individuals with the same types decreased and ones of the T. sinensis type for both ITS2 and COI increased. On the other hand, there was only one individual that had T. sinensis type for ITS2 and the early-spring strain of T. beneficus type for COI, suggesting that individuals are descendants of the F1 hybrid between the early-spring strain of T. beneficus females and T. sinensis males. These results indicate that hybridization between them was not closely related to displacement in the field. We have also found hybridization between T. sinensis and another type of T. beneficus: the late-spring strain; this is the first report to show their hybridization in chestnut fields. 2007 Elsevier Inc. All rights reserved. Keywords: Torymus sinensis; Torymus beneficus; rDNA internal transcribed spacer 2 (ITS2); Cytochrome oxidase subunit I (COI); Classical biological control; Non-target effect; Displacement; Hybridization; Dryocosmus kuriphilus
1. Introduction Torymus sinensis Kamijo is a parasitoid wasp introduced from China to Japan to control the chestnut gall wasp Dryocosmus kuriphilus Yasumatsu, an invasive and serious chestnut pest (concerning D. kuriphilus as a pest, see Mura-
*
Corresponding author. Fax: +81 29 838 8252. E-mail address: yara@affrc.go.jp (K. Yara).
1049-9644/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.04.017
kami, 1997; Moriya et al., 2002; Aebi et al., 2006). After a few small-scale trials, T. sinensis was initially released in 1982, and by 1999 had been released at 64 sites in Japan (Shirai et al., 1999). The introduced T. sinensis became ˆ take et al., 1984) and spread gradually (Morestablished (O iya et al., 1989). Simultaneously, the damage caused by D. kuriphilus (gall induction on buds followed by loss of production and ultimately death of the tree) fell precipitously (Moriya et al., 1989). This classical biological control strategy is regarded as one of the most famous and
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successful cases in Japan (Moriya et al., 1989; Shiga, 1999; Murakami et al., 2001). Torymus beneficus Yasumatsu et Kamijo is an indigenous parasitoid which also parasitizes D. kuriphilus (Yasumatsu and Kamijo, 1979). It is morphologically very similar to T. sinensis. Only the adult females of the two species can be discriminated empirically based on the slight difference in either the length of the ovipositor or the ratio ˆ take of the length of the ovipositor sheath to the thorax (O ˆ et al., 1984; Otake, 1987; Moriya et al., 1992); the males ˆ take et al., 1984). cannot be empirically discriminated (O Furthermore, T. beneficus has principally been divided into two types, provisionally designated in this paper as the early-spring and late-spring strains, based on the slight difˆ take, 1987; Murakaference in their emergence periods (O mi, 1988). Few of the ecological characteristics of the two T. beneficus types, as well as of T. sinensis, have been clarified. This is not only because morphological discrimination is difficult, but also because rearing methods under experimental conditions have not been fully developed. There has been interest in the interaction between T. sinensis and T. beneficus since T. sinensis was first released because it was feared that indigenous T. beneficus might compete with introduced T. sinensis and might adversely affect the outcome of the biological control. However, such apprehension disappeared while the damage caused by D. kuriphilus decreased. Instead, another apprehension— that the introduced T. sinensis might adversely affect the indigenous T. beneficus, i.e., a non-target effect—has appeared. According to field investigations based on morphological discrimination, T. sinensis increased while T. beneficus decreased after the release of T. sinensis (Moriya et al., 1992). These observations suggest that there has been displacement of indigenous T. beneficus by the introduced T. sinensis. Furthermore, it has been thought that the displacement may be closely related to hybridization between the Torymus species, since morphologically intermediate individuals between the two species have appeared in the field (Aoto and Murakami, 1992; Moriya et al., 1992). Moriya et al. (1992) reported that a hybrid (F1) between T. sinensis and T. beneficus could be obtained by artificial crossing, and that F1 females could be fertile. However, Moriya et al. (1992) also reported that some F1 females did show intermediate morphology between the parental species, but others had morphology similar to one or other of the parents. This makes it difficult to discriminate clearly between the species and their hybrids based on their morphological characteristics. It has been suggested that the results of previous field investigations based on morphological discrimination need to be reexamined by using other characteristics, for example, molecular markers. One isozyme marker, malic enzyme (EC 1.1.1.40) has been proposed as a means of discriminating between these Torymus species and their hybrids (Izawa et al., 1992,1996). Although malic enzyme cannot discriminate between T. beneficus (the late-spring strain) and T. sinensis due to
149
the same banding pattern (Izawa et al., 1996), the marker revealed that there were hybrids between T. sinensis and T. beneficus (early-spring strain) in the field (Izawa et al., 1996; Toda et al., 2000; Yara et al., 2000). However, little is known concerning hybridization as a causative factor of displacement. Variabilities in the cytochrome oxidase subunit I region (COI) of mtDNA (Yara, 2004) and in the internal transcribed spacer 2 of the rRNA coding region (ITS2) of nuclear DNA among these Torymus parasitoids are more effective molecular markers (Yara, 2006). One of the purposes of the present study is to substantiate the displacement of T. beneficus by T. sinensis using molecular markers. To achieve this aim, we selected a chestnut field where T. sinensis appeared not yet to have been distributed, and surveyed the frequency of the markers for nine successive years. We utilized two molecular markers: the internal transcribed spacer 2 of rRNA coding region (ITS2) and the cytochrome oxidase subunit I (COI). rRNA genes in nuclear DNA (nDNA) are recombining biparental markers that are able to reveal recent gene flow and hybridization. In contrast, COI in mitochondrial DNA (mtDNA) is maternally inherited. This makes it possible to examine the likelihood of hybridization as a factor for displacement. We also propose the use of PCR-RFLP of the COI region as a convenient method of determining mtDNA type. 2. Materials and methods 2.1. Sample collection and DNA extraction Samples were collected from a chestnut field in Obuse, Nagano Prefecture, Japan. T. sinensis had not been released in this field, whereas in May 1992, 720 females of T. sinensis were released in a chestnut orchard (Shirai et al., 1999), about 2 km distant from our research area. Both early-spring and late-spring strains of T. beneficus were originally present in the released orchard (Sasawaki and Yara, unpublished data). Every February from 1993 to 2001, several hundred withered galls of D. kuriphilus were collected from the chestnut field and kept in an instrument screen shelter, originally a meteorological screen designed to protect instruments from exposure to direct sunlight and precipitation while still allowing air to circulate freely, at the National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki Prefecture, Japan. From March to May, newly emerged adults of Torymus spp. from the withered galls were collected every day and stored at 70 C. Approximately 50 females were randomly selected from the emerged adults every year and were analyzed for both their nuclear and mitochondrial DNA type. In some years, no more than 50 females emerged, in which case all the females were used for analysis. DNA was extracted from single adult females using an Easy-DNA Kit (Invitrogen) according to the manufacturer’s instructions. One hundred microliters of DNA solution was obtained.
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2.2. Typing of nuclear DNA (ITS2 of ribosomal RNA gene) To determine the ITS2 type of each female analyzed, fragment analysis followed by polymerase chain reaction (PCR) was conducted using the same method as described by Yara (2006). The fragment analysis showed the earlyspring strain of T. beneficus to have a 4/4 genotype (fragment length of 308 bp), most of T. sinensis a 0/0 genotype (312 bp), and the late-spring strain of T. beneficus and some T. sinensis individuals had a 2/2 genotype (310 bp) (Yara, 2006). Females with the 4/0 heteroallelic genotype were thus considered to be hybrids between the early-spring strain of T. beneficus and T. sinensis. Additionally, females with the 2/0 heteroallelic genotype were considered to be either T. sinensis or hybrids between the late-spring strain of T. beneficus and T. sinensis. 2.3. Typing of mitochondrial DNA (COI) Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was initially attempted to identify the polymorphisms in the mitochondrial DNA sequences of cytochrome oxidase subunit I (COI) among early- and late-spring strains of T. beneficus, and T. sinensis (Yara, 2004). Recognition sites for restriction endonucleases on the obtained COI sequences (Yara, 2004 and Accession Nos. AB070473–AB070504) were analyzed using Genetyx-MAC (Genetyx Corporation). It was concluded that endonucleases AluI and DraI should be able to discriminate clearly between the first half region of the COI sequences (518 bp) of T. sinensis, T. beneficus (early-spring strain) and T. beneficus (late-spring strain) (Table 1). PCR-RFLP was validated using specimens with each haplotype as obtained in Yara (2004). PCR for COI was performed in 25-ll reaction volumes using two primers, C1-J-1718 (5 0 -GGAGGATTTGGAAATTGATTAGTT CC-3 0 ) and C1-N-2191 (5 0 -CCCGGTAAAATTAAAAT ATAAACTTC-3 0 ) (Simon et al., 1994). The cycling conditions were as follows. Samples were preheated at 94 C for 1 min, followed by 35 cycles at 94 C for 30 s, 45 C for 30 s, and 72 C for 1 min. Four to 8 ll of the PCR products was incubated in a total reaction volume of 10 ll containTable 1 Expected restriction fragment size (bp) of partial COI region from Torymus species Species and strainsa
Endonuclease
TbE TbL Ts (haplotypeb 1–3, 6, 8–9, 11) Ts (haplotype 4, 7, 10) Ts (haplotype 5)
276 328 276 328 328
AluI
a
DraI 136 190 190 190 136
54 52 54
52
281 518 518 281 281
237
237 237
TbE: Torymus beneficus (early-spring strain), TbL: T. beneficus (latespring strain), Ts: T. sinensis. b Haplotypes of T. sinensis are referred to EMBL/GenBank/DDBJ.
ing 1.5 U restriction enzyme, AluI or DraI (Nippon Gene), and 1· reaction buffer for 2 h at 37 C. Restriction fragments (5 ll of reaction mixture) were electrophoresed in 2% agarose (AgaroseX, Nippon Gene) gel in 1· TAE buffer, then visualized by staining with ethidium bromide. PCR-RFLP banding patterns of samples for validation were consistent with the estimated patterns revealed by Genetyx (Fig. 1). Therefore, in this study, PCR-RFLP was conducted using the same method as described above to determine the mtDNA (COI) type for each Torymus sp. female. 3. Results 3.1. Annual change in frequency of nDNA (ITS2) type in Torymus parasitoids For three years from 1993 to 1995, all individuals analyzed proved to be the T. beneficus (early-spring strain) type, i.e., the 4/4 genotype as in Yara (2006) for ITS2 (Table 2). Thereafter, individuals that showed T. beneficus (early-spring strain) type decreased dramatically in number and were not detected in 1999 and 2001. In 1996, individuals of the T. sinensis type (0/0 genotype) first appeared and subsequently increased in number. After 1997, there were a few individuals with the 2/2 genotype, found in T. beneficus (late-spring strain) and some T. sinensis (Yara, 2006). There were also some individuals after 1996 with the 2/0 genotype, which were either hybrid F1 between T. beneficus (late-spring strain) and T. sinensis, or T. sinensis (Yara, 2006). There were no individuals with the 4/0 genotype, which would have been hybrid F1 between T. beneficus (early-spring strain) and T. sinensis. 3.2. Annual change in frequency of mtDNA (COI) type in Torymus parasitoids For three years from 1993 to 1995, all the individuals analyzed showed T. beneficus (early-spring strain) type for COI (Table 3). Thereafter, individuals which showed T. beneficus (early-spring strain) type decreased in number and were not detected in 2001. In 1996, individuals of the T. sinensis type first appeared and increased in number thereafter. There were a few individuals of the T. beneficus (late-spring strain) type after 1996. 3.3. Discrepancy between types of ITS2 and COI in Torymus parasitoid individuals In most individuals, there was no discrepancy between ITS2 and COI. Further, in analyzed Torymus parasitoids, the trend in frequency of ITS2 together with COI was similar to the trend of frequency of ITS2 alone; and the trend in frequency of ITS2 together with COI was also similar to the trend in frequency of COI alone (Fig. 2). That is, from 1993 to 1995, only individuals with T. beneficus (earlyspring strain) for both ITS2 and COI were detected; but
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Fig. 1. PCR-RFLP banding patterns (a: AluI, b: DraI) of the COI region on 2% agarose gel stained with ethidium bromide. M: 100 bp ladder size marker, 1: Torymus beneficus (early-spring strain), 2: T. beneficus (late-spring strain), 3: T. sinensis (haplotype of 1–3, 6, 8–9 or 11), 4: T. sinensis (haplotype of 4, 7 or 10), 5: T. sinensis (haplotype of 5). Haplotypes of T. sinensis are referred to EMBL/GenBank/DDBJ.
Table 2 Changes in nDNA (ITS2) type of Torymus spp. emerging from 1993 to 2001 at chestnut fields in Obuse Type of nDNA ITS2a
Emergence year 1993
1994
1995
1996
1997
1998
1999
TbE (4/4) TbL or Ts (2/2) Ts (0/0) ‘‘Ts · TbL’’ or Ts (2/0) ‘‘Ts · TbE’’ (4/0) Untypedb
46
48
49
33 14 2
6 2 22 14
4 3 25 5
3 26 10
4
1
1
1
6
1
Total
50
49
50
50
50
38
2000
2001
4 7 6
3 33 11 3
39
17
50
a
TbE: Torymus beneficus (early-spring strain), TbL: T. beneficus (late-spring strain), Ts: T. sinensis. In parenthesis, ITS2 genotypes for each Torymus parasitoid (Yara, 2006) are denoted. b PCR product was not obtained.
Table 3 Changes in mtDNA (COI) type of Torymus spp. emerging from 1993 to 2001 in chestnut fields in Obuse Type of mtDNA COIa
Emergence year 1993
1994
1995
1996
1997
1998
1999
TbE TbL Ts Untypedb
46
48
50
4 2 31 1
4 4 9
1 49
1
8 3 38 1
1 1 37
4
32 3 11 4
Total
50
49
50
50
38
39
17
50
a b
50
2000
2001
TbE: Torymus beneficus (early-spring strain), TbL: T. beneficus (late-spring strain), Ts: T. sinensis. PCR product was not obtained.
such individuals decreased in number in 1996. Simultaneously, individuals with T. sinensis for both ITS2 and COI were first detected in 1996, and such individuals increased in number thereafter. In some individuals, on the other hand, there was a discrepancy between types of
ITS2 and COI. That is, one individual in 1996 showed both T. sinensis type for ITS2 and T. beneficus (late-spring strain) type for COI. There were also some individuals that had both the T. sinensis type for ITS2 and T. beneficus (late-spring strain) type for COI after 1996.
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100%
h
e
d
Frequency
80%
b
g
f
c
60% a
40%
20%
0%
93
94
95
96
97
98
99
00
01
Year Fig. 2. Annual changes in frequency of types of both nDNA ITS2 and mtDNA COI in Torymus parasitoids (T. beneficus (early-spring strain): TbE, T. beneficus (late-spring strain): TbL, T. sinensis: Ts) in chestnut plantations in Obuse from 1993 to 2001. a: 4/4 genotype for ITS2 and TbE type for COI, subsequently concluded to be TbE; b: 0/0 genotype for ITS2 and Ts type for COI, subsequently concluded to be Ts; c: 2/0 genotype for ITS2 and Ts type for COI, subsequently concluded to be ‘‘Ts or hybrid of ‘Ts · TbL’’’; d: 2/2 genotype for ITS2 and Ts type for COI, subsequently concluded to be ‘‘Ts or hybrid of ‘Ts · TbL’’’; e: 2/2 genotype for ITS2 and TbL type for COI, subsequently concluded to be ‘‘TbL or hybrid of ‘Ts · TbL’’’; f: 2/0 genotype for ITS2 and TbL type for COI, subsequently concluded to be hybrid of ‘‘Ts · TbL’’; g: 0/0 genotype for ITS2 and TbL type for COI, subsequently concluded to be hybrid of ‘‘Ts · TbL’’; h: 0/0 genotype for ITS2 and TbE type for COI, subsequently concluded to be hybrid of ‘‘Ts · TbE’’. For further explanations also see the text.
4. Discussion From 1993 to 1995, all the individuals analyzed showed T. beneficus (early-spring strain) type for both nDNA (ITS2) and mtDNA (COI) (Fig. 2). It therefore appears that originally there was only T. beneficus (early-spring strain) in the research field. After 1996, however, these individuals sharply decreased in number and they were not obtained in 1999 or 2001. Simultaneously, individuals of the T. sinensis type for both ITS2 and COI were first obtained in 1996 and increased thereafter. Thus, it is apparent that T. sinensis appeared in the field in 1996 and that T. sinensis had been dominant in years after this event. Furthermore, it is possible that the T. sinensis individuals that appeared in 1996 are descended from the T. sinensis that had been introduced in 1992 near the research field. These results make it clear that T. beneficus (early-spring strain) had been displaced by T. sinensis in the field. By using molecular markers, displacement by T. sinensis was substantiated genetically for the first time. One individual in 1999 had the T. sinensis type for ITS2 and the T. beneficus (early-spring strain) type for COI (h in Fig. 2), that is to say, there was a discrepancy between nDNA and mtDNA type in the individual. Therefore, this
individual is clearly a hybrid between T. sinensis and T. beneficus (early-spring strain). It is further apparent that hybridization had occurred between a female T. beneficus (early-spring strain) and a male T. sinensis, since its maternal mtDNA (COI) type was T. beneficus (early-spring strain). It is also apparent that the individual is a descendant of a hybrid F1 since its ITS2 type was not heteroallelic between T. sinensis and T. beneficus (early-spring strain) but was the homoallelic T. sinensis type (Yara, 2006). Of 418 individuals analyzed over 9 years, this individual is the only evidence that hybridization between T. sinensis and T. beneficus (early-spring strain) had occurred. Thus, it seems reasonable to suppose that hybridization had only rarely occurred between T. beneficus (early-spring strain), which had come dominant by 1995, and T. sinensis, which had appeared in 1996 and subsequently displaced T. beneficus. Yara et al. (2000) also reported that there were few F1 hybrids between T. beneficus (early-spring strain) and T. sinensis in other chestnut plantations where T. sinensis appeared to have become established, according to the results for malic enzyme marker. It is possible to state that the hybridization is not a main factor in displacement by T. sinensis, although there are other cases where hybridization plays a significant role in displacement and extinction (Rhymer and Simberloff, 1996).
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Since 1996, on the other hand, there were a few individuals that showed the T. sinensis type for ITS2 and the T. beneficus (late-spring strain) type for COI (g in Fig. 2). Consequently, it is apparent that these individuals are descendants of F1 hybrids between female T. beneficus (late-spring strain) and male T. sinensis, for reasons similar to those mentioned above. This is the first report in which hybridization between T. beneficus (late-spring strain) and T. sinensis has occurred in the field. Furthermore, it is noteworthy that hybridization between them had proceeded from F1 to their descendants. Individuals that are descendants of hybrids between female T. beneficus (late-spring strain) and male T. sinensis were first obtained in 1996. As mentioned above, it appears that there were originally only T. beneficus (early-spring strains) in the research field from 1993 to 1995. Torymus beneficus (both early-spring and late-spring strains) and T. sinensis are univoltine: they emerge, mate and oviposit in spring, and adults of the next generation emerge in the spring of the following year. Therefore, it is likely that, in a location other than the research field, at least before 1994, a female T. beneficus (late-spring strain) had crossed with a male T. sinensis. Thereafter, it is likely that the resultant F1 hybrids arrived at the research field and oviposited in the spring of 1995. It is also likely that descendants of the F1 hybrid arrived there. It is noteworthy that hybrids between T. beneficus (late-spring strain) and T. sinensis may show an expanding distribution. Yara (2004, 2006) showed that there was little genetic variation between T. sinensis and T. beneficus (both earlyand late-spring strain), concerning COI and ITS2 sequences, suggesting that these three have a very close relationship. This is also supported by the results in the present study that there were descendants of F1 hybrids between T. sinensis and T. beneficus (both the early- and late-spring strain) in the field, although their frequencies were very low. Further studies will be needed to revise their taxonomic positions. There were also other individuals whose nDNA and mtDNA type were incompatible (c, d and e in Fig. 2). However, such individuals cannot be identified as being hybrids or not between T. beneficus (late-spring strain) and T. sinensis, since the ITS2 marker cannot accurately discriminate T. beneficus (late-spring strain) from T. sinensis (Yara, 2006). Even if all such individuals were hybrids, it seems reasonable to suppose that hybridization between T. beneficus (late-spring strain) and T. sinensis is also not closely related to the displacement of T. beneficus (earlyspring strain) by T. sinensis, or to hybridization between T. beneficus (early-spring strain) and T. sinensis. Reasons for this are that no introgression of T. beneficus (earlyspring strain) genes was observed in such possible hybrids between T. beneficus (late-spring strain) and T. sinensis, and that individuals which had the T. sinensis type for both COI and ITS2 predominate after 1996. The displacement may be explained by another factor (or factors) associated with parasitization or competition.
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For example, Piao and Moriya (1992) reported the difference in (potential) reproductive ability between the two parasitoid species; the number of eggs laid per female of T. sinensis was about 2.7 times greater than that of T. beneficus (early-spring strain) under outdoor temperature conditions. Torymus sinensis, with its longer ovipositor, may excel T. beneficus (the early-spring strain), which has a shorter ovipositor, at efficiently exploiting large galls, as suggested in other parasitoid species of gall makers (Stone et al., 2002; Weis and Abrahamson, 1985; Price and Clancy, 1986). Furthermore, inter-specific larval competition between them for limited host resources in gall structures may be an important factor in the observed displacement. Molecular makers for identifying the Torymus species at the larval or egg stages would be useful for assessing the importance of these possible factors. Acknowledgments We wish to express our gratitude to Drs. T. Shimoda and S. Moriya (National Agricultural Research Center) for helpful comments and suggestions on the draft. We are indebted to Dr. Yano (Kinki University) for providing us with the samples. We appreciate the two anonymous reviewers’ comments that improved this paper immensely. This research was supported in part by Grants-in-Aid for Scientific Research (No. 19380037) from Japan Society for the promotion of Science. References Aebi, A., Scho¨nrogge, K., Melika, G., Alma, A., Bosio, G., Quacchia, A., Picciau, L., Abe, Y., Moriya, S., Yara, K., Seljak, G., Stone, G., 2006. Parasitoid recruitment to the globally invasive chestnut gall wasp Dryocosmus kuriphilus. In: Ozaki, K., Yukawa, J., Ohgushi, T., Price, P.W. (Eds.), Galling Arthropods and Their Associates Ecology and Evolution. Springer, Tokyo, pp. 103–121. Aoto, I., Murakami, Y., 1992. Dispersion of a Torymus (Syntomaspis) sinensis population in Fukuoka Prefecture (Hymenoptera: Torymidae). Proc. Assoc. Pl. Prot. Kyushu 38, 193–196 (in Japanese with English summary). Izawa, H., Osakabe, Mh., Moriya, S., 1992. Isozyme discrimination between an imported parasitoid wasp, Torymus sinensis Kamijo and its sibling species, T. beneficus Yasumatsu et Kamijo (Hymenoptera: Torymidae), attacking Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae). Jpn. J. Appl. Entomol. Zool. 36, 58–60 (in Japanese with English summary). Izawa, H., Osakabe, Mh., Moriya, S., Toda, S., 1996. Use of malic enzyme to detect hybrids between Torymus sinensis and T. beneficus (Hymenoptera: Cynipidae) attacking Dryocosmus kuriphilus (Hymenoptera: Cynipidae) and possibility of natural hybridization. Jpn. J. Appl. Entomol. Zool. 40, 205–208 (in Japanese with English summary). Moriya, S., Inoue, K., Mabuchi, M., 1989. The use of Torymus sinensis to control chestnut gall wasp, Dryocosmus kuriphilus, in Japan. FFTC Techn. Bull. 118, 1–12. Moriya, S., Inoue, K., Shiga, M., Mabuchi, M., 1992. Interspecific relationship between an introduced parasitoid, Torymus sinensis Kamijo, as a biological control agent of the chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu, and an endemic parasitoid, Torymus beneficus Yasumatsu et Kamijo. Acta Phytopathol. Entomol. Hungarica 27, 479–483.
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Yano, E., Matsuo, K., Shiyomi, M., Andow, D.A. (Eds.), Biological Invasions of Ecosystem by Pests and Beneficial Organisms. NIAES, Tsukuba, pp. 175–188. Shirai, Y., Adachi, I., Moriya, S., 1999. Records of release of Torymus sinensis Kamijo (Hymenoptera: Torymidae), an introduced biological control agent of chestnut gall wasp in Japan. Bull. Natl. Inst. Fruit Tree Sci. 33, 163–178 (in Japanese with English summary). Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651–701. Stone, G.N., Scho¨nrogge, K., Atkinson, R.J., Bellido, D., Pujade-Villar, J., 2002. The population biology of oak gall wasps (Hymenoptera: Cynipidae). Annu. Rev. Entomol. 47, 633–668. Toda, S., Miyazaki, M., Osakabe, Mh., Komazaki, S., 2000. Occurrence and hybridization of two parasitoid wasps, Torymus sinensis Kamijo and T. beneficus Yasumatsu et Kamijo (Hymenoptera: Torymidae) in the Oki islands. Appl. Entomol. Zool. 35, 151–154. Weis, A.E., Abrahamson, W.G., 1985. Potential selective pressures by parasitoids on a plant-herbivore interaction. Ecology 66, 1261–1269. Yasumatsu, K., Kamijo, K., 1979. Chalcidoid parasites of Dryocosmus kuriphilus Yasumatsu (Cynipidae) in japan, with description of five new species (Hymenoptera). Esakia 14, 93–111. Yara, K., Yano, E., Sasawaki, T., Shiga, M., 2000. Detection of hybrids between introduced Torymus sinensis and native T. beneficus (Hymenoptera: Torymidae) in central Japan, using malic enzyme. Appl. Entomol. Zool. 35, 201–206. Yara, K., 2004. Relationship between the introduced and indigenous parasitoids Torymus sinensis and T. beneficus (Hymenoptera: Torymidae) as inferred from mt-DNA (COI) sequences. Appl. Entomol. Zool. 39, 427–433. Yara, K., 2006. Identification of Torymus sinensis and T. beneficus (Hymenoptera: Torymidae), introduced and indigenous parasitoids of the chestnut gall wasp Dryocosmus kuriphilus (Hymenoptera: Cynipidae), using the ribosomal ITS2 region. Biol. Control 36, 15–21.
Displacement of Torymus beneficus (Hymenoptera: Torymidae) by T. sinensis, an indigenous and introduced parasitoid of the chestnut gall wasp, Dryocosmus kuriphilus (Hymenoptera: Cynipidae), in Japanese chestnut fields: Possible involvement in hybridization Kaori Yara a
a,*
, Terunori Sasawaki b, Yasuhisa Kunimi
c
National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan b Nagano Fruit-tree Experiment Station, Suzaka, Nagano 382-0071, Japan c Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8538, Japan Received 12 October 2006; accepted 25 April 2007 Available online 3 May 2007
Abstract Although there is empirical knowledge to suggest the displacement of Torymus beneficus (Hymenoptera: Torymidae) by Torymus sinensis, an indigenous and introduced parasitoid of the chestnut gall wasp Dryocosmus kuriphilus (Hymenoptera: Cynipidae), respectively, in Japanese chestnut fields, the underlying mechanisms are unclear. In this study, the displacement of the early-spring strain of T. beneficus by T. sinensis was surveyed in a chestnut field for nine successive years (n = 418), using two molecular markers, the internal transcribed spacer 2 of nuclear rDNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI). We also investigated whether or not hybridization between the parasitoids was involved as an important factor in the displacement. All individuals from 1993 to 1995 were of the early-spring strain of T. beneficus type for both ITS2 and COI. After 1996, individuals with the same types decreased and ones of the T. sinensis type for both ITS2 and COI increased. On the other hand, there was only one individual that had T. sinensis type for ITS2 and the early-spring strain of T. beneficus type for COI, suggesting that individuals are descendants of the F1 hybrid between the early-spring strain of T. beneficus females and T. sinensis males. These results indicate that hybridization between them was not closely related to displacement in the field. We have also found hybridization between T. sinensis and another type of T. beneficus: the late-spring strain; this is the first report to show their hybridization in chestnut fields. 2007 Elsevier Inc. All rights reserved. Keywords: Torymus sinensis; Torymus beneficus; rDNA internal transcribed spacer 2 (ITS2); Cytochrome oxidase subunit I (COI); Classical biological control; Non-target effect; Displacement; Hybridization; Dryocosmus kuriphilus
1. Introduction Torymus sinensis Kamijo is a parasitoid wasp introduced from China to Japan to control the chestnut gall wasp Dryocosmus kuriphilus Yasumatsu, an invasive and serious chestnut pest (concerning D. kuriphilus as a pest, see Mura-
*
Corresponding author. Fax: +81 29 838 8252. E-mail address: yara@affrc.go.jp (K. Yara).
1049-9644/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.04.017
kami, 1997; Moriya et al., 2002; Aebi et al., 2006). After a few small-scale trials, T. sinensis was initially released in 1982, and by 1999 had been released at 64 sites in Japan (Shirai et al., 1999). The introduced T. sinensis became ˆ take et al., 1984) and spread gradually (Morestablished (O iya et al., 1989). Simultaneously, the damage caused by D. kuriphilus (gall induction on buds followed by loss of production and ultimately death of the tree) fell precipitously (Moriya et al., 1989). This classical biological control strategy is regarded as one of the most famous and
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successful cases in Japan (Moriya et al., 1989; Shiga, 1999; Murakami et al., 2001). Torymus beneficus Yasumatsu et Kamijo is an indigenous parasitoid which also parasitizes D. kuriphilus (Yasumatsu and Kamijo, 1979). It is morphologically very similar to T. sinensis. Only the adult females of the two species can be discriminated empirically based on the slight difference in either the length of the ovipositor or the ratio ˆ take of the length of the ovipositor sheath to the thorax (O ˆ et al., 1984; Otake, 1987; Moriya et al., 1992); the males ˆ take et al., 1984). cannot be empirically discriminated (O Furthermore, T. beneficus has principally been divided into two types, provisionally designated in this paper as the early-spring and late-spring strains, based on the slight difˆ take, 1987; Murakaference in their emergence periods (O mi, 1988). Few of the ecological characteristics of the two T. beneficus types, as well as of T. sinensis, have been clarified. This is not only because morphological discrimination is difficult, but also because rearing methods under experimental conditions have not been fully developed. There has been interest in the interaction between T. sinensis and T. beneficus since T. sinensis was first released because it was feared that indigenous T. beneficus might compete with introduced T. sinensis and might adversely affect the outcome of the biological control. However, such apprehension disappeared while the damage caused by D. kuriphilus decreased. Instead, another apprehension— that the introduced T. sinensis might adversely affect the indigenous T. beneficus, i.e., a non-target effect—has appeared. According to field investigations based on morphological discrimination, T. sinensis increased while T. beneficus decreased after the release of T. sinensis (Moriya et al., 1992). These observations suggest that there has been displacement of indigenous T. beneficus by the introduced T. sinensis. Furthermore, it has been thought that the displacement may be closely related to hybridization between the Torymus species, since morphologically intermediate individuals between the two species have appeared in the field (Aoto and Murakami, 1992; Moriya et al., 1992). Moriya et al. (1992) reported that a hybrid (F1) between T. sinensis and T. beneficus could be obtained by artificial crossing, and that F1 females could be fertile. However, Moriya et al. (1992) also reported that some F1 females did show intermediate morphology between the parental species, but others had morphology similar to one or other of the parents. This makes it difficult to discriminate clearly between the species and their hybrids based on their morphological characteristics. It has been suggested that the results of previous field investigations based on morphological discrimination need to be reexamined by using other characteristics, for example, molecular markers. One isozyme marker, malic enzyme (EC 1.1.1.40) has been proposed as a means of discriminating between these Torymus species and their hybrids (Izawa et al., 1992,1996). Although malic enzyme cannot discriminate between T. beneficus (the late-spring strain) and T. sinensis due to
149
the same banding pattern (Izawa et al., 1996), the marker revealed that there were hybrids between T. sinensis and T. beneficus (early-spring strain) in the field (Izawa et al., 1996; Toda et al., 2000; Yara et al., 2000). However, little is known concerning hybridization as a causative factor of displacement. Variabilities in the cytochrome oxidase subunit I region (COI) of mtDNA (Yara, 2004) and in the internal transcribed spacer 2 of the rRNA coding region (ITS2) of nuclear DNA among these Torymus parasitoids are more effective molecular markers (Yara, 2006). One of the purposes of the present study is to substantiate the displacement of T. beneficus by T. sinensis using molecular markers. To achieve this aim, we selected a chestnut field where T. sinensis appeared not yet to have been distributed, and surveyed the frequency of the markers for nine successive years. We utilized two molecular markers: the internal transcribed spacer 2 of rRNA coding region (ITS2) and the cytochrome oxidase subunit I (COI). rRNA genes in nuclear DNA (nDNA) are recombining biparental markers that are able to reveal recent gene flow and hybridization. In contrast, COI in mitochondrial DNA (mtDNA) is maternally inherited. This makes it possible to examine the likelihood of hybridization as a factor for displacement. We also propose the use of PCR-RFLP of the COI region as a convenient method of determining mtDNA type. 2. Materials and methods 2.1. Sample collection and DNA extraction Samples were collected from a chestnut field in Obuse, Nagano Prefecture, Japan. T. sinensis had not been released in this field, whereas in May 1992, 720 females of T. sinensis were released in a chestnut orchard (Shirai et al., 1999), about 2 km distant from our research area. Both early-spring and late-spring strains of T. beneficus were originally present in the released orchard (Sasawaki and Yara, unpublished data). Every February from 1993 to 2001, several hundred withered galls of D. kuriphilus were collected from the chestnut field and kept in an instrument screen shelter, originally a meteorological screen designed to protect instruments from exposure to direct sunlight and precipitation while still allowing air to circulate freely, at the National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki Prefecture, Japan. From March to May, newly emerged adults of Torymus spp. from the withered galls were collected every day and stored at 70 C. Approximately 50 females were randomly selected from the emerged adults every year and were analyzed for both their nuclear and mitochondrial DNA type. In some years, no more than 50 females emerged, in which case all the females were used for analysis. DNA was extracted from single adult females using an Easy-DNA Kit (Invitrogen) according to the manufacturer’s instructions. One hundred microliters of DNA solution was obtained.
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2.2. Typing of nuclear DNA (ITS2 of ribosomal RNA gene) To determine the ITS2 type of each female analyzed, fragment analysis followed by polymerase chain reaction (PCR) was conducted using the same method as described by Yara (2006). The fragment analysis showed the earlyspring strain of T. beneficus to have a 4/4 genotype (fragment length of 308 bp), most of T. sinensis a 0/0 genotype (312 bp), and the late-spring strain of T. beneficus and some T. sinensis individuals had a 2/2 genotype (310 bp) (Yara, 2006). Females with the 4/0 heteroallelic genotype were thus considered to be hybrids between the early-spring strain of T. beneficus and T. sinensis. Additionally, females with the 2/0 heteroallelic genotype were considered to be either T. sinensis or hybrids between the late-spring strain of T. beneficus and T. sinensis. 2.3. Typing of mitochondrial DNA (COI) Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was initially attempted to identify the polymorphisms in the mitochondrial DNA sequences of cytochrome oxidase subunit I (COI) among early- and late-spring strains of T. beneficus, and T. sinensis (Yara, 2004). Recognition sites for restriction endonucleases on the obtained COI sequences (Yara, 2004 and Accession Nos. AB070473–AB070504) were analyzed using Genetyx-MAC (Genetyx Corporation). It was concluded that endonucleases AluI and DraI should be able to discriminate clearly between the first half region of the COI sequences (518 bp) of T. sinensis, T. beneficus (early-spring strain) and T. beneficus (late-spring strain) (Table 1). PCR-RFLP was validated using specimens with each haplotype as obtained in Yara (2004). PCR for COI was performed in 25-ll reaction volumes using two primers, C1-J-1718 (5 0 -GGAGGATTTGGAAATTGATTAGTT CC-3 0 ) and C1-N-2191 (5 0 -CCCGGTAAAATTAAAAT ATAAACTTC-3 0 ) (Simon et al., 1994). The cycling conditions were as follows. Samples were preheated at 94 C for 1 min, followed by 35 cycles at 94 C for 30 s, 45 C for 30 s, and 72 C for 1 min. Four to 8 ll of the PCR products was incubated in a total reaction volume of 10 ll containTable 1 Expected restriction fragment size (bp) of partial COI region from Torymus species Species and strainsa
Endonuclease
TbE TbL Ts (haplotypeb 1–3, 6, 8–9, 11) Ts (haplotype 4, 7, 10) Ts (haplotype 5)
276 328 276 328 328
AluI
a
DraI 136 190 190 190 136
54 52 54
52
281 518 518 281 281
237
237 237
TbE: Torymus beneficus (early-spring strain), TbL: T. beneficus (latespring strain), Ts: T. sinensis. b Haplotypes of T. sinensis are referred to EMBL/GenBank/DDBJ.
ing 1.5 U restriction enzyme, AluI or DraI (Nippon Gene), and 1· reaction buffer for 2 h at 37 C. Restriction fragments (5 ll of reaction mixture) were electrophoresed in 2% agarose (AgaroseX, Nippon Gene) gel in 1· TAE buffer, then visualized by staining with ethidium bromide. PCR-RFLP banding patterns of samples for validation were consistent with the estimated patterns revealed by Genetyx (Fig. 1). Therefore, in this study, PCR-RFLP was conducted using the same method as described above to determine the mtDNA (COI) type for each Torymus sp. female. 3. Results 3.1. Annual change in frequency of nDNA (ITS2) type in Torymus parasitoids For three years from 1993 to 1995, all individuals analyzed proved to be the T. beneficus (early-spring strain) type, i.e., the 4/4 genotype as in Yara (2006) for ITS2 (Table 2). Thereafter, individuals that showed T. beneficus (early-spring strain) type decreased dramatically in number and were not detected in 1999 and 2001. In 1996, individuals of the T. sinensis type (0/0 genotype) first appeared and subsequently increased in number. After 1997, there were a few individuals with the 2/2 genotype, found in T. beneficus (late-spring strain) and some T. sinensis (Yara, 2006). There were also some individuals after 1996 with the 2/0 genotype, which were either hybrid F1 between T. beneficus (late-spring strain) and T. sinensis, or T. sinensis (Yara, 2006). There were no individuals with the 4/0 genotype, which would have been hybrid F1 between T. beneficus (early-spring strain) and T. sinensis. 3.2. Annual change in frequency of mtDNA (COI) type in Torymus parasitoids For three years from 1993 to 1995, all the individuals analyzed showed T. beneficus (early-spring strain) type for COI (Table 3). Thereafter, individuals which showed T. beneficus (early-spring strain) type decreased in number and were not detected in 2001. In 1996, individuals of the T. sinensis type first appeared and increased in number thereafter. There were a few individuals of the T. beneficus (late-spring strain) type after 1996. 3.3. Discrepancy between types of ITS2 and COI in Torymus parasitoid individuals In most individuals, there was no discrepancy between ITS2 and COI. Further, in analyzed Torymus parasitoids, the trend in frequency of ITS2 together with COI was similar to the trend of frequency of ITS2 alone; and the trend in frequency of ITS2 together with COI was also similar to the trend in frequency of COI alone (Fig. 2). That is, from 1993 to 1995, only individuals with T. beneficus (earlyspring strain) for both ITS2 and COI were detected; but
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Fig. 1. PCR-RFLP banding patterns (a: AluI, b: DraI) of the COI region on 2% agarose gel stained with ethidium bromide. M: 100 bp ladder size marker, 1: Torymus beneficus (early-spring strain), 2: T. beneficus (late-spring strain), 3: T. sinensis (haplotype of 1–3, 6, 8–9 or 11), 4: T. sinensis (haplotype of 4, 7 or 10), 5: T. sinensis (haplotype of 5). Haplotypes of T. sinensis are referred to EMBL/GenBank/DDBJ.
Table 2 Changes in nDNA (ITS2) type of Torymus spp. emerging from 1993 to 2001 at chestnut fields in Obuse Type of nDNA ITS2a
Emergence year 1993
1994
1995
1996
1997
1998
1999
TbE (4/4) TbL or Ts (2/2) Ts (0/0) ‘‘Ts · TbL’’ or Ts (2/0) ‘‘Ts · TbE’’ (4/0) Untypedb
46
48
49
33 14 2
6 2 22 14
4 3 25 5
3 26 10
4
1
1
1
6
1
Total
50
49
50
50
50
38
2000
2001
4 7 6
3 33 11 3
39
17
50
a
TbE: Torymus beneficus (early-spring strain), TbL: T. beneficus (late-spring strain), Ts: T. sinensis. In parenthesis, ITS2 genotypes for each Torymus parasitoid (Yara, 2006) are denoted. b PCR product was not obtained.
Table 3 Changes in mtDNA (COI) type of Torymus spp. emerging from 1993 to 2001 in chestnut fields in Obuse Type of mtDNA COIa
Emergence year 1993
1994
1995
1996
1997
1998
1999
TbE TbL Ts Untypedb
46
48
50
4 2 31 1
4 4 9
1 49
1
8 3 38 1
1 1 37
4
32 3 11 4
Total
50
49
50
50
38
39
17
50
a b
50
2000
2001
TbE: Torymus beneficus (early-spring strain), TbL: T. beneficus (late-spring strain), Ts: T. sinensis. PCR product was not obtained.
such individuals decreased in number in 1996. Simultaneously, individuals with T. sinensis for both ITS2 and COI were first detected in 1996, and such individuals increased in number thereafter. In some individuals, on the other hand, there was a discrepancy between types of
ITS2 and COI. That is, one individual in 1996 showed both T. sinensis type for ITS2 and T. beneficus (late-spring strain) type for COI. There were also some individuals that had both the T. sinensis type for ITS2 and T. beneficus (late-spring strain) type for COI after 1996.
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100%
h
e
d
Frequency
80%
b
g
f
c
60% a
40%
20%
0%
93
94
95
96
97
98
99
00
01
Year Fig. 2. Annual changes in frequency of types of both nDNA ITS2 and mtDNA COI in Torymus parasitoids (T. beneficus (early-spring strain): TbE, T. beneficus (late-spring strain): TbL, T. sinensis: Ts) in chestnut plantations in Obuse from 1993 to 2001. a: 4/4 genotype for ITS2 and TbE type for COI, subsequently concluded to be TbE; b: 0/0 genotype for ITS2 and Ts type for COI, subsequently concluded to be Ts; c: 2/0 genotype for ITS2 and Ts type for COI, subsequently concluded to be ‘‘Ts or hybrid of ‘Ts · TbL’’’; d: 2/2 genotype for ITS2 and Ts type for COI, subsequently concluded to be ‘‘Ts or hybrid of ‘Ts · TbL’’’; e: 2/2 genotype for ITS2 and TbL type for COI, subsequently concluded to be ‘‘TbL or hybrid of ‘Ts · TbL’’’; f: 2/0 genotype for ITS2 and TbL type for COI, subsequently concluded to be hybrid of ‘‘Ts · TbL’’; g: 0/0 genotype for ITS2 and TbL type for COI, subsequently concluded to be hybrid of ‘‘Ts · TbL’’; h: 0/0 genotype for ITS2 and TbE type for COI, subsequently concluded to be hybrid of ‘‘Ts · TbE’’. For further explanations also see the text.
4. Discussion From 1993 to 1995, all the individuals analyzed showed T. beneficus (early-spring strain) type for both nDNA (ITS2) and mtDNA (COI) (Fig. 2). It therefore appears that originally there was only T. beneficus (early-spring strain) in the research field. After 1996, however, these individuals sharply decreased in number and they were not obtained in 1999 or 2001. Simultaneously, individuals of the T. sinensis type for both ITS2 and COI were first obtained in 1996 and increased thereafter. Thus, it is apparent that T. sinensis appeared in the field in 1996 and that T. sinensis had been dominant in years after this event. Furthermore, it is possible that the T. sinensis individuals that appeared in 1996 are descended from the T. sinensis that had been introduced in 1992 near the research field. These results make it clear that T. beneficus (early-spring strain) had been displaced by T. sinensis in the field. By using molecular markers, displacement by T. sinensis was substantiated genetically for the first time. One individual in 1999 had the T. sinensis type for ITS2 and the T. beneficus (early-spring strain) type for COI (h in Fig. 2), that is to say, there was a discrepancy between nDNA and mtDNA type in the individual. Therefore, this
individual is clearly a hybrid between T. sinensis and T. beneficus (early-spring strain). It is further apparent that hybridization had occurred between a female T. beneficus (early-spring strain) and a male T. sinensis, since its maternal mtDNA (COI) type was T. beneficus (early-spring strain). It is also apparent that the individual is a descendant of a hybrid F1 since its ITS2 type was not heteroallelic between T. sinensis and T. beneficus (early-spring strain) but was the homoallelic T. sinensis type (Yara, 2006). Of 418 individuals analyzed over 9 years, this individual is the only evidence that hybridization between T. sinensis and T. beneficus (early-spring strain) had occurred. Thus, it seems reasonable to suppose that hybridization had only rarely occurred between T. beneficus (early-spring strain), which had come dominant by 1995, and T. sinensis, which had appeared in 1996 and subsequently displaced T. beneficus. Yara et al. (2000) also reported that there were few F1 hybrids between T. beneficus (early-spring strain) and T. sinensis in other chestnut plantations where T. sinensis appeared to have become established, according to the results for malic enzyme marker. It is possible to state that the hybridization is not a main factor in displacement by T. sinensis, although there are other cases where hybridization plays a significant role in displacement and extinction (Rhymer and Simberloff, 1996).
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Since 1996, on the other hand, there were a few individuals that showed the T. sinensis type for ITS2 and the T. beneficus (late-spring strain) type for COI (g in Fig. 2). Consequently, it is apparent that these individuals are descendants of F1 hybrids between female T. beneficus (late-spring strain) and male T. sinensis, for reasons similar to those mentioned above. This is the first report in which hybridization between T. beneficus (late-spring strain) and T. sinensis has occurred in the field. Furthermore, it is noteworthy that hybridization between them had proceeded from F1 to their descendants. Individuals that are descendants of hybrids between female T. beneficus (late-spring strain) and male T. sinensis were first obtained in 1996. As mentioned above, it appears that there were originally only T. beneficus (early-spring strains) in the research field from 1993 to 1995. Torymus beneficus (both early-spring and late-spring strains) and T. sinensis are univoltine: they emerge, mate and oviposit in spring, and adults of the next generation emerge in the spring of the following year. Therefore, it is likely that, in a location other than the research field, at least before 1994, a female T. beneficus (late-spring strain) had crossed with a male T. sinensis. Thereafter, it is likely that the resultant F1 hybrids arrived at the research field and oviposited in the spring of 1995. It is also likely that descendants of the F1 hybrid arrived there. It is noteworthy that hybrids between T. beneficus (late-spring strain) and T. sinensis may show an expanding distribution. Yara (2004, 2006) showed that there was little genetic variation between T. sinensis and T. beneficus (both earlyand late-spring strain), concerning COI and ITS2 sequences, suggesting that these three have a very close relationship. This is also supported by the results in the present study that there were descendants of F1 hybrids between T. sinensis and T. beneficus (both the early- and late-spring strain) in the field, although their frequencies were very low. Further studies will be needed to revise their taxonomic positions. There were also other individuals whose nDNA and mtDNA type were incompatible (c, d and e in Fig. 2). However, such individuals cannot be identified as being hybrids or not between T. beneficus (late-spring strain) and T. sinensis, since the ITS2 marker cannot accurately discriminate T. beneficus (late-spring strain) from T. sinensis (Yara, 2006). Even if all such individuals were hybrids, it seems reasonable to suppose that hybridization between T. beneficus (late-spring strain) and T. sinensis is also not closely related to the displacement of T. beneficus (earlyspring strain) by T. sinensis, or to hybridization between T. beneficus (early-spring strain) and T. sinensis. Reasons for this are that no introgression of T. beneficus (earlyspring strain) genes was observed in such possible hybrids between T. beneficus (late-spring strain) and T. sinensis, and that individuals which had the T. sinensis type for both COI and ITS2 predominate after 1996. The displacement may be explained by another factor (or factors) associated with parasitization or competition.
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For example, Piao and Moriya (1992) reported the difference in (potential) reproductive ability between the two parasitoid species; the number of eggs laid per female of T. sinensis was about 2.7 times greater than that of T. beneficus (early-spring strain) under outdoor temperature conditions. Torymus sinensis, with its longer ovipositor, may excel T. beneficus (the early-spring strain), which has a shorter ovipositor, at efficiently exploiting large galls, as suggested in other parasitoid species of gall makers (Stone et al., 2002; Weis and Abrahamson, 1985; Price and Clancy, 1986). Furthermore, inter-specific larval competition between them for limited host resources in gall structures may be an important factor in the observed displacement. Molecular makers for identifying the Torymus species at the larval or egg stages would be useful for assessing the importance of these possible factors. Acknowledgments We wish to express our gratitude to Drs. T. Shimoda and S. Moriya (National Agricultural Research Center) for helpful comments and suggestions on the draft. We are indebted to Dr. Yano (Kinki University) for providing us with the samples. We appreciate the two anonymous reviewers’ comments that improved this paper immensely. This research was supported in part by Grants-in-Aid for Scientific Research (No. 19380037) from Japan Society for the promotion of Science. References Aebi, A., Scho¨nrogge, K., Melika, G., Alma, A., Bosio, G., Quacchia, A., Picciau, L., Abe, Y., Moriya, S., Yara, K., Seljak, G., Stone, G., 2006. Parasitoid recruitment to the globally invasive chestnut gall wasp Dryocosmus kuriphilus. In: Ozaki, K., Yukawa, J., Ohgushi, T., Price, P.W. (Eds.), Galling Arthropods and Their Associates Ecology and Evolution. Springer, Tokyo, pp. 103–121. Aoto, I., Murakami, Y., 1992. Dispersion of a Torymus (Syntomaspis) sinensis population in Fukuoka Prefecture (Hymenoptera: Torymidae). Proc. Assoc. Pl. Prot. Kyushu 38, 193–196 (in Japanese with English summary). Izawa, H., Osakabe, Mh., Moriya, S., 1992. Isozyme discrimination between an imported parasitoid wasp, Torymus sinensis Kamijo and its sibling species, T. beneficus Yasumatsu et Kamijo (Hymenoptera: Torymidae), attacking Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae). Jpn. J. Appl. Entomol. Zool. 36, 58–60 (in Japanese with English summary). Izawa, H., Osakabe, Mh., Moriya, S., Toda, S., 1996. Use of malic enzyme to detect hybrids between Torymus sinensis and T. beneficus (Hymenoptera: Cynipidae) attacking Dryocosmus kuriphilus (Hymenoptera: Cynipidae) and possibility of natural hybridization. Jpn. J. Appl. Entomol. Zool. 40, 205–208 (in Japanese with English summary). Moriya, S., Inoue, K., Mabuchi, M., 1989. The use of Torymus sinensis to control chestnut gall wasp, Dryocosmus kuriphilus, in Japan. FFTC Techn. Bull. 118, 1–12. Moriya, S., Inoue, K., Shiga, M., Mabuchi, M., 1992. Interspecific relationship between an introduced parasitoid, Torymus sinensis Kamijo, as a biological control agent of the chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu, and an endemic parasitoid, Torymus beneficus Yasumatsu et Kamijo. Acta Phytopathol. Entomol. Hungarica 27, 479–483.
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