Infestation of the mycoheterotrophic orchid Yoania japonica by the two-winged fl y , Chyliza vittata ( Diptera : Psilidae )

Chyliza vittata is known to utilize leaves, stems and underground parts of several leafy and leafl ess orchids. Compared to the well-recorded feeding habits of C. vittata in Europe, its feeding habits in Japan are poorly studied. Thus, further records of its host plants and the habits of its larvae in Japan are likely to reveal the similarities and differences in its feeding habits in Europe and Japan. The current study reports C. vittata feeding on the stems of the mycoheterotrophic orchid Yoania japonica in central Japan. This study also showed that in spite of the small size of Yoania its reproductive success is not severely reduced when infested with C. vittata, whereas the robust stems of Gastrodia elata, which is its main host plant in Japan, are thought to be a defence against infestation by C. vittata.

The genus Chyliza includes mainly medium-sized fl ies, which can be identifi ed by a combination of the following characteristics: head not deltoid in profi le, face concave in profi le, antenna normal, occiput not swollen, cell cup clearly shorter than cell bm, swelling on anatergite, 6 distinct scutellar bristles, postcoxal bridge well developed, and male genitalia with well-developed surstyli (Iwasa, 1989;Freidberg & Shatalkin, 2008;Bygebjerg et al., 2011).In common with all members of Psilidae, the larvae of the genus Chyliza are phytophagous (Steyskal, 1987;Iwasa, 1989), feeding on various organs and tissues of plants belonging to at least 10 different families, both angiosperm and gymnosperm (reviewed by Sueyoshi, 2013).For example, the larvae of Chyliza extenuata induce the formation of galls on the stems of holoparasitic plants belonging to the genus Orobanche (Giard, 1900;Chandler, 1975) and the larvae of Chyliza annulipes occur under the bark of Picea and Pinus sp.(Lyneborg, 1987).
Although seven species of Chyliza fl ies are reported in Japan, ecological data is only available for two species.Larvae of C. splendida feed on galls induced by the ampelopsis fruit midge Asphondylia baca Monzen (Cecidomyiidae) in the buds of Weigela the study site (Association of Wildlife Research & Envision Conservation Offi ce, 2012).Only three stems were collected and the underground structures of the plants were not excavated in order to minimize any negative effects on the Y. japonica population investigated.The stem samples were placed in plastic containers fi lled with vermiculite and incubated at 15°C under wet conditions for ca. 1 month before the temperature was reduced to 5°C during the period from October to the following March when they were removed from the incubator and inspected.ily be distinguished from the males because their wings were usually held erect and half-open (Sugiura, in press).The female fl ies were found on various plant structures and were observed walking between them.In addition, the females were also observed laying eggs on the plant surface, often gradually changing their abdominal position in order to place the eggs on different parts of the fl ower, including the inner portions of the fl ower, the outer surface of the sepals and petals, or the pedicels.Food-searching/ feeding behaviour was not recorded for either the males or females, although it was apparent that the plants on which the females laid eggs were used as an encounter site for the males, as is often the case with G. elata (Sugiura, in press).Indeed, males were sometimes observed grasping the females with swollen abdomens after oviposition, during which time copulation occurred (Fig. 1).When the site was revisited during the fruiting season (September), the puparia of C. vittata were found just beneath the epidermis of the basal stem (Fig. 1).Even the heavily infested stems of Y. japonica were still capable of producing well-developed fruit capsules with numerous viable seeds (Fig. 1).Adult C. vittata fl ies emerged from the pupae in the rotten stems collected the following March, indicating that C. vittata overwinters in Y. japonica as a puparium.

DISCUSSION
The present study clearly demonstrates that the mycoheterotrophic orchid Y. japonica is one of the host plants of the larvae of C. vittata.Furthermore, as in G. elata (Sugiura, in press), male C. vittata fl ies exhibited mate seeking behaviour on fl owering plants of Y. japonica to which females were attracted to lay eggs, which resulted in copulation.These facts indicate that Y. japonica can be used as a mating site for C. vittata.
Until recently, the only documented host of C. vittata in Japan was G. elata, of which both the buds and stems can be infested (Kato et al., 2006;Sueyoshi, 2013).However, European populations of C. vittata are known to be leaf or stem miners and root borers of at least fi ve genera of European orchids.For example, it is reported that the larvae of C. vittata can mine the leaf sheaths and stems of Cephalanthera damasonium, Epipactis helleborine and Epipactis purpurata and pupate in their roots (Pitkin et al., 2012).In addition, the larva of C. vittata also tunnel in the tubers of Neottia nidus-avis, and are associated with three other genera, including Dactylorhiza, Himantoglossum and Orchis, although the species of these plants were not identifi ed (de Meijere, 1940;Hering, 1957).With the exception of G. elata, all of the hosts utilized by C. vittata have relatively short stems of less than 50 cm in length.In contrast, the stems of G. elata are robust, growing to a height of approximately 1 m, and sometimes as high as 2 m.Such long fl ower stems are unusual amongst mycoheterotrophic plants and it is suggested by Kato et al. (2006) that the long stems of G. elata protect them from infestation by C. vittata.
Although the association between G. elata and Japanese populations of C. vittata appears to be exceptional compared to those recorded in Europe it is suggested that further research is needed to identify other host plants in Japan in order to determine whether Kato et al.'s (2006) hypothesis is well supported (Sueyoshi, 2013).A recent study by Suetsugu (2013a) indicates that Japanese populations of C. vittata utilize other species of hosts, in particular they oviposit in the fl owers of E. helleborine var.papillosa.Taken together with the results of the current study, these fi ndings suggest that the association between C. vittata and G. elata is not specifi c, even in Japan.In addition, even though the stems of Y. japonica are much shorter than those of G. elata, usually being less than 30 cm (Suetsugu & Yagame, 2014), it seems that infestation by C. vittata does not severely reduce the reproductive success of Y. japonica.The infested stems of Y. japonica were still capable of producing mature fruit capsules with numerous viable seeds, indicating that the larvae of C. vittata mainly consume the pedicels and stems, but not the developing ovaries.Such observations indicate that the robust fl ower stems of G. elata may not be a counter-adaptation to herbivory by C. vittata, but may have evolved for another reason, such as increasing pollinator attraction.Further research on the selection of host plants by C. vittata in Japan is required to gain a fuller understanding of the similarities and differences between their feeding habits in two geographically disjunct regions: Europe and Japan.
males and seven females of C. vittata were observed visiting fl owers of Y. japonica at the study site.The females could eas-study site, which was approximately 20 m by 40 m in area, contained about 30 fl owering plants of Y. japonica.Observations during the 20-h study period were made by walking around the study area or sitting near fl owering plants and monitoring the intrafl oral behaviour of insect visitors.Floral visitors were carefully observed, with some being captured immediately after they left infl orescences for detailed identifi cation.The study site was revisited in early September 2012, when most of the Y. japonica fruit had matured, to investigate the stems of the Y. japonica plants that C. vittata females laid eggs on during the fl owering season.Great care was taken when collecting the samples because Y. japonica is designated nearly threatened or endangered in 26 of the 47 prefectures in Japan, including Nagano Prefecture, the location of

Fig. 1 .
Fig. 1.A -fl owering plant of Yoania japonica f. lutea; B -a copulating pair of Chyliza vittata on a fl ower of Y. japonica; C -fruiting plant of Y. japonica whose stem is infested with Chyliza vittata; D -magnifi cation of the enclosed parts of C. Puparia in the stem are indicated by arrows.