Bull. Natl. Mus. Nat. Sci., Ser. B, 46(3), pp. 119–127, August. 21, 2020

Herbarium Specimens Reveal the History and Distribution of
Seed-feeding Fly Infestation in Native Japanese Orchids
Yumi Yamashita1,2,*, Yuki Ogura-Tsujita3,4, Makoto Tokuda3,4 and Tomohisa Yukawa1
1

Tsukuba Botanical Garden, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
2
Graduate School of Symbiotic Systems Science and Technology, Fukushima University,
1 Kanayagawa, Fukushima 960–1296, Japan
3
Faculty of Agriculture, Saga University, 1 Honjyo, Saga 840–8502, Japan
4
The United Graduate School of Agricultural Sciences, Kagoshima University,
1–21–24 Korimoto, Kagoshima 890–8580, Japan*E-mail: yamashita@kahaku.go.jp(Received 11 May 2020; accepted 24 June 2020)

Abstract Fly infestation in the capsules of various wild orchids has become a serious issue forconservation in Japan in recent years. Herbivory caused by ﬂies is easily recognized through theobservation of coarctate pupa just beneath the pericarp or remnant puparium following adult emergence. We examined the capsules of herbarium specimens of ﬁve orchid species [Cephalanthera
erecta (Thunb.) Blume, C. falcata (Thunb.) Blume, C. longibracteata Blume, C. longifolia (L.)
Fritsch, and Cypripedium japonicum Thunb.] in eight herbaria. We found that 63 of 194 fruiting
specimens (32.5%) were damaged by seed-eating ﬂies. The morphology of the coarctate pupa,
puparium, and emergence holes were indicative of Japanagromyza tokunagai (Sasakawa). The
oldest infested capsules were collected in 1891. Since then, ﬂy-infested specimens have been collected throughout Japan, from Hokkaido Prefecture in the north to Kagoshima Prefecture in thesouth. We suggest that current-day severe infestation damage on orchids is not due to the population increase or geographic expansion of recently introduced ﬂies but rather to the population
increase in a native species that is widely distributed across Japan.
Keywords : Agromyzidae, herbarium, Orchidaceae, seed-feeding ﬂy, spatio-temporal analysis.

Introduction
Fly infestation of capsules or ovaries of wildorchid species presents a conservation issue inJapan (reviewed in Ogura-Tsujita et al., 2019).
The populations of many Japanese orchid species
have sharply declined in recent years; 67% of
these species are considered endangered by theJapanese Ministry of Environment (Ministry of
the Environment, 2020). No seeds are produced
when seed-feeding ﬂy larvae predate the immature capsules of orchids. The failure to reproduce© 2020 National Museum of Nature and Sciencesexually not only leads to population decline; it
can accelerate declines in genetic diversity,
which can lead to a more serious conservation
crisis. Our previous research and that of othershas determined that 50 taxa of 24 native Japaneseorchid genera can be infested by Japanagromyza
tokunagai (Sasakawa) (Diptera: Agromyzidae),as well as other dipteran species, and that damage from ﬂy infestation occurs across all ofJapan, i.e., from Hokkaido Prefecture to Okinawa
Prefecture (Ogura-Tsujita et al., 2019). Infestation by J. tokunagai on Japanese orchids became
obvious in the 1970s (Ogura-Tsujita et al., 2019),
but it remains unknown how these infestations

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Yumi Yamashita et al.

began or how they came to be so widespread.
Evidence of ﬂy infestation in orchid capsules
is easily recognized by observing coarctate
pupae, which are typically found just below thepericarp, or puparia that are left after the emergence of adult ﬂies. Because these larval skins
persist, capsules on herbarium species may be asource of historical data on the timeline and distribution of Japanagromyza tokunagai infestations. It was recently suggested that the current
level of infestation on Japanese orchids is theresult of recently naturalized seed-feeding ﬂies
that originated in other geographic areas (Onuki,
2007; Suetsugu et al., 2018). Spatio-temporal
analysis using herbarium specimens may help totest this hypothesis. Here, we examined evidenceof ﬂy infestation in the capsules of herbarium
specimens and used collection information
including date, geographic area, and species, toshed light on the expansion of ﬂy infestation in
Japanese orchids.
Materials and Methods
We examined herbarium specimens of ﬁve
orchid species: Cephalanthera erecta (Thunb.)
Blume, C. falcata (Thunb.) Blume, C. longibracteata Blume, C. longifolia (L.) Fritsch, and Cypripedium japonicum Thunb. These species have beenpreviously investigated for seed-feeding ﬂy infestations (Ogura-Tsujita et al., 2019), and are widelydistributed in Japan with relatively large historical
population sizes. Therefore, they are suitable foruse in a spatio-temporal analysis of ﬂy infestation.
We examined specimens preserved in eight Japanese herbaria: Herbarium, Hokkaido University
Museum (SAPS); Herbarium of Sapporo Museum
Activity Center; Herbarium, Tohoku University
Botanical Garden (TUS); Herbarium, Institute of
University Materials, Fukushima University(FKSE); Herbarium, National Museum of Natureand Science (TNS); Makino Herbarium, Tokyo
Metropolitan University (MAK); Herbarium,
Kanagawa Prefectural Museum of Natural History (KPM); and Herbarium, Osaka Museum of
Natural History (OSA). We examined capsule-bearing specimens of all ﬁve species in these herbaria. Coarctate pupae and/or puparia were investigated using a stereomicroscope and the numberof ﬂy-infested specimens, the number of ﬂyinfested fruits per specimen and the number ofpupae and/or puparia per specimen were recorded.
Results and Discussion
1. Fly infestation detected from herbarium specimens
We examined a total of 194 fruiting specimensof all ﬁve orchid species across the eight herbaria.
Of these, 63 (32.5%) showed evidence of ﬂy
infestation (Table 1). Detailed information for ﬂyinfested specimen is provided in Appendix. Flyinfested specimens had remnant coarctate pupae
and/or puparia, as shown in Fig. 1. Coarctate
pupae were typically approximately 2 mm inlength and 1 mm in width, with characteristic anterior spiracles (Fig. 1A, B). Emergence holes in
capsules were typically 1–2 mm in diameter andtypically bore puparia fragments (Fig. 1C, D).
These characters are consistent with observationsof Japanagromyza tokunagai (Suga et al., 2018b;
Ogura-Tsujita et al., 2019). Japanagromyza tokunagai is the only known species in the genus todamage the seeds of Cephalanthera and Cypripedium (Ogura-Tsujita et al., 2019). Furthermore,
puparium morphology was examined (Fig. 1E, F)
and found to be highly similar to that of J. tokunagai. Speciﬁcally, the structure of the rows of
cuticular processes on the abdominal segments
was consistent with J. tokunagai, as were theshape of the anterior and posterior spiracles. As aresult of these observations, we identiﬁed theresidual puparia found on herbarium specimens as
belonging to J. tokunagai.
Previous ﬁeld studies have determined that
two species are primarily responsible for the
inhibition of seed production in wild orchids inJapan: Japanagromyza tokunagai and Chyliza
vittata Meigen (Diptera: Psilidae) (Ogura-Tsujita
et al., 2019). Japanagromyza tokunagai has beenreported to feed on 47 taxa of 22 orchid genera,
including the ﬁve species examined in this study.

Analysis of ﬂy infestation of orchids using herbarium specimens

121

Table 1. The number of ﬂy-infested (＋) and un-infested (−) fruiting specimens of Cephalanthera erecta,
C. falcata, C. longibracteata, C. longifolia and Cypripedium japonicum in each decade from 1881 to 2020
Collectionyear
1881–1890
1891–1900
1901–1910
1911–1920
1921–1930
1931–1940
1941–1950
1951–1960
1961–1970
1971–1980
1981–1990
1991–2000
2001–20102011–2020
Total
Infestation
rate for eachspecies

Cephalauthera
erecta
＋

−

1

1
1

C. falcata

C. longibracteata

C. longifolia

Cypripedium
japonicum

＋

＋

＋

＋

−

1

1
1

1

2

1

3
3
2
4
3
1
1

1
1
3
2
2
1
2
4
3
1

21

20

2
1
2
8

27.6%

−
1
1
2
2
1
2
2
4
3
3
2
4
1
28

41.7%

−
3
2
2
2
1
7
1
9

2
4
1
1
4
4

1
1
1
1

1

7
11
9
5
2

1

22

61

3

2
3

26.5%

−

2
1
1
2
8
27.3%

Total
＋

−
4
5
5
5
4
13
3
17
7
19
18
13
13
5
131

2

1
1

0
4
5
0
1
4
5
11
7
2
6
8
5
5

10

13

63

1
1

1
1
3
1
1

1
1
5

43.5%

32.5%

Fig. 1. Fly infestation in capsules of herbarium specimens of Cephalanthera longibracteata. A, B. Pupa. C, D.
Puparium. E, F. Puparium dissected from a specimen. Arrowheads indicate a pupa (A) or a puparium (C). A,
B, E, and F were taken from a specimen collected by M. Majima s. n. in Hokkaido Prefecture, Chitose-shi, Mt.
Eniwadake in July 1906 (SAPS). C and D were taken from a specimen collected by S. Tanaka s. n. in Hokkaido Prefecture, Chitose-shi, Lake Shikotsuko on 1 October 1967 (SAPS). Scale bars ＝ 1 mm.

122Yumi Yamashita et al.

Table 2. The number of ﬂy-infested (＋) and un-infested (−) fruiting specimens collected in each district ofJapan in each decade from 1881 to 2020
Collection
year

KyusyuShikoku

Kinki

Hokuriku

Chubu

Kanto

TohokuHokkaido

＋

＋

＋

＋

＋

＋

−

＋

＋

1
1

3
1
1
3

1881–1890
1891–1900
1901–1910
1911–1920
1921–1930
1931–1940
1941–1950
1951–1960
1961–1970
1971–1980
1981–1990
1991–2000
2001–20102011–2020

1
1

Total

2

Infestation
rate foreach district

−

1
1
1
1
1

5

28.6%

1
1
1
1
1

5

−

−

−

1
1

1

1
2

5

50.0%

−

1

1
1
1
2

2
1
1

2
3

5

10

33.3%

1
1
2

4
1

1

3

1

1
1
1
5

37.5%

5

2

1
1
5

3
2
3
1
4
3

4
1
1
3

1
1
2
1
4

2
1

13

18

27

27.8%

40.0%

−

1

2

1
1
2
3
1
2
3
2

3

16

1
3
6
8
4
3
1
31

34.0%

−

Total
＋

−
4
5
5
5
4
13
3
17
7
19
18
13
13
5
131

3
2

2
1
1
2
5

1
2

2
1
4
4
8
2
3

0
4
5
0
1
4
5
11
7
2
6
8
5
5

35

63

1

9

20.5%

32.5%

Fig. 2. Number of ﬂy-infested (black) and un-infested (gray) fruiting specimens in Japan (A) and in Hokkaido
Prefecture (B) in each decade from 1881 to 2020.

Damage from this species has been recorded
from Fukushima Prefecture to Okinawa Prefecture (Ogura-Tsujita et al., 2019). Chyliza vittata
has been reported to damage the seeds ofCalanthe nipponica Makino, Epipactis helleborine (L.) Crantz, Gastrodia elata Blume, andNeottia furusei T.Yukawa & Yagame. Observa-

tions of damage by this species have beenreported from cool-temperate to subarctic regionsin Hokkaido and Nagano Prefectures (Kato et al.,2006; Suetsugu, 2013, 2016a, 2016b; Sueyoshi,
2013; Sugiura, 2016; Ogura-Tsujita et al., 2019;
Suetsugu et al., 2019). Pupal length is approximately 2 mm and 5 mm in J. tokunagai andAnalysis of ﬂy infestation of orchids using herbarium specimens

123

Fig. 3. Geographical distribution of ﬂy infestation of fruiting specimens in each prefecture of Japan. Prefecturesin which ﬂy infestation was detected are shown in dark hatch, and prefectures without detection are shown inpale hatch. The left- and right-hand ﬁgures in each prefecture denote the number of ﬂy-infested specimens andthe total number of fruiting specimens, respectively.

C. vittata, respectively; thus, the pupae and/or
puparia observed in this study are unlikely tobelong to C. vittata.
2. Geographical and temporal distribution of ﬂyinfested specimens
Infested capsules were found on specimens
collected across a wide range of time periods(Table 1, Fig. 2A). The earliest ﬂy-infested specimens, individuals of Cephalanthera erecta andC. longibracteata from two sites in Hokkaido,
were collected in 1891 (Appendix). Thereafter,
between 1–11 damaged specimens were found ineach decade, excluding the 1910s (Table 1, Fig.
2A). For the remaining three species, the ﬁrst
records of ﬂy infestation were in 1894 for C. lon-

gifolia, 1904 for Cypripedium japonicum, and1923 for C. falcata (Table 1, Appendix). The earliest evidence of ﬂy infestation throughout Japan
was that observed in Hokkaido in 1891, followedby records in the Kanto region in 1899, Shikoku
in 1904, Tohoku in 1906, and Kinki in 1950
(Table 2, Appendix).
Specimen data therefore suggest that seed-eating ﬂies spread throughout Japan from Hokkaido
Prefecture to Kagoshima Prefecture (Fig. 3,
Appendix). Fruiting specimens of the ﬁve species were available for 31 of the 47 Japanese prefectures covering most of the distribution rangeof these species (Fig. 3); infestations wereobserved in specimens collected from 25 prefectures. However, we note that J. tokunagai has

124

Yumi Yamashita et al.

been found feeding on the capsules and inﬂorescences of Cephalanthera erecta, C. falcata, Cymbidium goeringii (Rchb.f.) Rchb.f., C. kanran
Makino, Cypripedium macranthos Sw., andOreorchis coreana Finet on Jeju Island, South
Korea (Kim and Lim, 2019). Therefore, examination of specimens preserved in other Japanese
herbaria, as well as those in other countries, may
help to clarify the spread and extent of ﬂy infestation in orchids more accurately.
Although previous reports have suggested thatC. vittata was the only seed-feeding ﬂy in
Hokkaido (Nakamura et al., 2014; Ogura-Tsujita
et al., 2019), our results provide the ﬁrst evidence of the occurrence of J. tokunagai in
Hokkaido (Table 2, Fig. 3). Nine of the 44 fruiting specimens collected in Hokkaido were
infested by J. tokunagai (Fig. 3). However, ourresults suggest that ﬂy infestation was less severein Hokkaido relative to other areas. Compared tothe infestation rate for Japan as a whole (32.5%),
infestation in Hokkaido was relatively low, at
20.5% (Table 2). Furthermore, infestations were
only observed on specimens collected in 1891,
1894, 1904, 1906, 1953, 1967, 1968, and 1991,
although specimens of the ﬁve orchid species
were collected continuously over this period.
This indicates that J. tokunagai is relatively rarein Hokkaido Prefecture (Fig. 2B, Appendix).
3. Infestation rates
Our examination of herbarium specimens
revealed signiﬁcant damage resulting from ﬂyinfestation in all target species (Table 1). In Cypripedium japonicum, 43.5% (10 out of 23) offruiting specimens showed evidence of damage,whereas 90–92% of capsules in Ibaraki Prefecture and 83% of capsules in Yamanashi Prefecture were found to be damaged in a recent ﬁeld
survey (Suga et al., 2018c). In Cephalanthera
falcata, 41.7% (20 out of 48) of fruiting herbarium specimens showed evidence of infestation,
but recent ﬁeld surveys reported capsule damage
rates of 91% and 89% in Ibaraki Prefecture andthe Tokyo Metropolitan area, respectively (Sugaet al., 2018c). In C. erecta, 27.6% (8 out of 29)

of fruiting specimens were infested, whereas
42% of capsules examined in Ibaraki Prefecture
were found to be damaged (Suga et al., 2018c).
In C. longifolia, 27.3% (3 out of 11) of fruiting
specimens were infested, whereas 64% of surveyed capsules in the ﬁeld in the Tokyo Metropolitan area were found to be infested (Suga etal., 2018c). In C. longibracteata, 26.5% (22 outof 83) of fruiting specimens were infested,
whereas 68% of surveyed capsules in Ibaraki
Prefecture were found to be infested (Suga et al.,2018c). These data highlight the fact that infestation rates in the ﬁeld may be much higher thanlevels reﬂected in herbarium specimens.
These diﬀerences in damage rates may be due
to under-estimation of the infestation rate in theherbarium specimens. In immature capsules,
J. tokunagai remains in the larval stage, and thus
may not be easily recognizable. Infestation istherefore only observed following coarctate
pupation. In a study from Chiba Prefecture, Sugaet al. (2018a) reported that most individuals of
J. tokunagai pupate in June or later in Cephalanthera falcata and Cypripedium japonicum capsules. However, several specimens in this study
were collected prior to June. It is therefore possiblethat capsules appeared visually intact even thoughlarvae were present; alternatively, coarctate pupae
or emergence holes may have been overlooked ifthey were located on the underside of the herbarium specimens, as our examination was nondestructive. Furthermore, ovaries or immature capsules that fell oﬀ the plant shortly after infestation
would have been overlooked in this study. Therefore, infestation rates from ﬁeld and herbarium surveys cannot be directly compared.
An alternative explanation for the discrepancies
in these rates is the possibility that the populationof J. tokunagai in Japan has increased in recent
years due to a decrease in its parasites. Several
parasitic wasps are known to prey on J. tokunagai
and thus could inﬂuence its population size (Kimand Lim, 2019; Matsuo et al., 2019; Suetsugu etal., 2019). Habitat change and other factors may
have reduced parasitic wasp populations, resulting
in an increase in the J. tokunagai population. A

Analysis of ﬂy infestation of orchids using herbarium specimens

125

third possible explanation is that specimen collection sites and ﬁeld observation locations diﬀered.
Collection details recorded on herbarium specimen labels indicate that specimens were largely
collected in natural ecosystems, whereas the ﬁeld
observation data were mostly collected in periurban ecosystems, such as secondary woodland,urban parks and gardens (Ogura-Tsujita et al.,2019). Therefore, observed diﬀerences in infestation rates between herbarium specimens and ﬁeld
surveys could be due to diﬀerent biological interactions present in the diﬀerent habitats.

Mami Yamazaki, Masayuki Maki, Takahide
Kurosawa, Atsushi Ebihara, Hidetoshi Kato,
Yukira Mochida, Norihisa Tanaka, Masashi
Yokogawa for allowing us to examine specimens.
We also are grateful to Mr. Tomoo Sobagaki fortaking beautiful pictures of coarctate pupae (Fig.
1E, F) of Japanagromyza tokunagai. This work is
supported partly by the National Museum of
Nature and Science research project entitled “Integrated Analysis of Natural History Collections forConservation of Highly Endangered Species” to Y.
Yamashita and T. Yukawa.

4. Conclusions
We assessed the temporal and geographic
expansion of seed-feeding ﬂy damage in native
Japanese orchids using herbarium specimens anddetermined that i) infestation by J. tokunagai isevident in all regions and on all species over an
extended period of time; ii) the record of infestation by J. tokunagai is more-or-less uninterrupted
from 1891 to the present day (i.e. over a 129-yeartimespan); iii) all ﬁve orchid species examined inthis study have been infested by J. tokunagai sinceat least 1923; and iv) nationwide damage has
occurred since the time of earliest collections.
Therefore, it is likely that J. tokunagai is not a
recent foreign or domestic inter-regional introduced invasive species, but rather a component of
the native Japanese fauna. This study highlights
the utility of herbarium specimens in documenting
and explaining patterns of insect herbivory. We
note that the specimens assessed in this study do
not reﬂect the entirety of Japan and that the number of specimens available for examination was
limited. We suggest that future surveys target geographic gaps in herbarium records to provide
greater understanding of the ecology of seed-feeding ﬂies in Japan. Finally, assessing the genetic
diversity of ﬂies collected from herbarium specimens may allow for an understanding of population and migration dynamics.

References

Acknowledgments
We would like to thank Drs. Kohtaroh Shutoh,

Kato, M., Tsuji, K. and Kawakita, A. 2006. Pollinator andstem- and corm-boring insects associated with mycoheterotrophic orchid Gastrodia elata. Annals of the
Entomological Society of America 99: 851–858.
Kim, I. K. and Lim, J. S. 2019. Seasonal occurrence ofJapanagromyza tokunagai (Sasakawa) (Diptera: Agromyzidae) on Cymbidium kanran Makino (Orchidaceae)
in Jeju Island, Republic of Korea and prevention of
infestation by the ﬂy. Acta Horticulturae 1262: 225–233.
Matsuo, K., Suga, M. and Ogura-Tsujita, Y. 2019. A new
host of Sphegigaster hamugurivora Ishii, 1953 (Hymenoptera: Pteromalidae). Japanese Journal of Systematic
Entomology 25: 43–44.
Ministry of the Environment. 2020. Ministry of the Environment Red List 2020. http://www.env.go.jp/press/
107905.html. (Accessed: 22 April 2020).
Nakamura, T., Saigusa, T. and Suwa, M. (eds.) 2014. Catalogue of the Insects of Japan. Volume 8. Diptera (Part2 Brachycera Schizophora). The Entomological Societyof Japan and Touka Shobo, Fukuoka (in Japanese).
Ogura-Tsujita, Y. Murata, M., Yamashita, Y. and Yukawa, T.
2019. Infestation of four Japanese orchids by ﬂies,
including Japanagromyza tokunagai. Japanese Journalof Conservation Ecology 24: 191–199 (in Japanese).
Onuki, K. 2007. An attempt to conserve the natural habitat of orchids in Inokashira Park. Orchid Sciences 16:
21–29 (in Japanese).
Suetsugu, K. 2013. Delayed autonomous self-pollinationin two Japanese varieties of Epipactis helleborine(Orchidaceae). Botanical Journal of the Linnean Society 173: 733–743.
Suetsugu, K. 2016a. Infestation of the mycoheterotrophic
orchid Yoania japonica by the two-winged ﬂy, Chyliza
vittata (Diptera: Psilidae). European Journal of Entomology 113: 393–396.
Suetsugu, K. 2016b. The autotrophic orchid Calanthe
nipponica is a potential host plant of Japanese popula-

126

Yumi Yamashita et al.

tions of the two-winged ﬂy, Chyliza vittata (Diptera:Psilidae). Entomological News 126: 231–236.
Suetsugu, K., Fukushima, S. and Sueyoshi, M. 2018. Substantial impact of seed-feeding ﬂy on seed productionof ﬁve endangered Japanese orchids. Ecology 99:
2871–2873.
Suetsugu, K., Kitamura, S. and Sueyoshi, M. 2019. Infestation of the orchid Cephalanthera spp. by Paralleloma
vittatum (Meigen, 1826) (Diptera: Scathophagidae) inJapan. Entomologycal Scinence 22: 121–125.
Sueyoshi, M. 2013. Orchidophaga gastrodiacola Kato,
2006, is Chyliza vittata Meigen, 1826 (Diptera: Psilidae): discussion on its taxonomy and biology. Zootaxa
3620: 473–480.
Suga, M., Fukushima, S., Yamashita, Y., Yukawa, T.,
Tokuda, M. and Ogura-Tsujita, Y. 2018a. Identiﬁcationof agromyzid ﬂies infesting six native orchid species in

Chiba Prefecture, Japan, and their damage assessment.
Japanese Journal of Entomology (New Series) 21: 167–174 (in Japanese).
Suga, M., Tokuda, M. and Ogura-Tsujita, Y. 2018b. Infestation of Japanagromyza tokunagai (Diptera: Agromyzidae) on native orchids in Kashibaru Marsh, Saga Prefecture. Saga Nature Study 23: 49–53 (in Japanese).
Suga, M., Yamashita, Y., Suetsugu, K., Yukawa, T.,
Tokuda, M. and Ogura-Tsujita, Y. 2018c. Identiﬁcationof ﬂies infesting wild orchid ﬂowers and fruits inJapan. Japanese Journal of Applied Entomology andZoology 62: 249–255 (in Japanese).
Sugiura, N. 2016. Mate-seeking and oviposition behaviorof Chyliza vittata (Diptera: Psilidae) infesting the leafless orchid Gastrodia elata. Entomological Science 19:
129–132.

Appendix. List of ﬂy-infested fruiting specimens of ﬁve Japanese native orchid species preserved in herbaria
Locality
Species

District

Prefecture

Detailed locality

Cephalanthera
erectaHokkaido
Kanto

Hokkaido
Saitama

Sapporo-shi
Hannou-shi,
Mt. Tenranzan
Ashigarakami-gun, Yamakitamachi
Shimoina-gun, Oshika-muraTahara-shi, Cape Irago
Kaizuka-shi, Baba
Izumi-shi,
Nabetani Pass
Iwade-shi, Sakaidani
Ishinomaki-shi, Kitazakai
Higashimatsushima-shi, JASDF
Matsushima Airbase
Sendai-shi, Taihaku-ku,
Akiu-machi
Iwanuma-shi,
Mt. Inokurayama
Iwanuma-shi,
Mt. Inokurayama
Fukushima-shi, Mizubayasi
Park
Fukushima-shi, Mizubayasi
Park
Iwaki-shi, Yamada-machi
Tsukuba-shi, Tsukuba University Campus
Niiza-shi, Nobitome, Heirinji
TempleFunabashi-shi, Tsuboi
Sagamihara-shi, Shiroyamamachi
Minamiashigara-shi, Mt.
Maruyama
Kanazawa-shi,
Mt. Utatsuyama

Kanagawa
Chubu
Kinki

Cephalanthera
falcata

TohokuNaganoAichi
Osaka

Wakayama
Miyagi

Fukushima

Kanto

Ibaraki
Saitama
Chiba
Kanagawa

Hokuriku

Ishikawa

Kinki

Mie
Hyogo
Ehime
Tokushima

Shikoku

Nabari-shi, Akame Valley
Ako-gun, Kamigori-cho
Shikokuchuou-shi, Doi-cho
Kaifu-gun, Mugi-cho

Collection
dateCollector andcollection
number

Voucher

1891-7-7
1939-6-5

Tokubuchi s. n.
T. Makino s. n.

SAPS
MAK193684

1999-7-5

Mashiko et al. s. n.

2006-9-25
1954-12-27
2003-7-6
1981-10-25

M. Maeshima s. n.
N. Satomi s. n.
C. Shimizu
2003-074
C. Shimizu s. n.

KPMNA0115518
MAK411161
TUS177757
OSA280824

1985-8-5
1993-10-25
1995-9-4

S. Yamashita 6452
Y. Sasaki 93-0689
Y. Ueno et al. 38193

OSA104880
TUS51304
TUS263153

1961-6-20

H. Ohashi 3271

TUS26214

1990-5-30

TUS151723

2009-7-19

T. Koga & T.
Kurosawa s. n.
T. Koga & T.
Kurosawa s. n.
T. Ando et al. 319

2010-6-21

T. Ando et al. 64

FKSE64337

1933-6-24
1980-7-10

J. Nozaki s. n.
Collector unknown

TUS342078
TNS01083089

1951-9-20

H. Ohashi s. n.

TUS50660

1991-10-17
2009-7-4

1969-6-22

H. Nakamura 1510
F. Sakai &K. Sakai 43558
E. Nomura
NMR-2236
T. Fukui s. n.

KPM428808
KPMNA0163598
KPMNA0297255
TUS60503

1950-8-19
1992-9-12
1950-8-8
1923-9-12

T. Kodama s. n.
S. Miyake 1261
T. Yamanaka 5408
A. Kimura s. n.

OSA
TNS665112
TUS409791
TUS232703

YearMonth-Day

1990-10-16

2015-7-19

OSA204582

TUS152354
FKSE64528

Analysis of ﬂy infestation of orchids using herbarium specimens

127

Appendix. List of ﬂy-infested fruiting specimens of ﬁve Japanese native orchid species preserved in herbaria (continued)
Locality

CollectiondateSpecies

District

Prefecture

Detailed locality

Cephalanthera
falcata

KyusyuNagasakiKagoshima

Tsushima-shi, Mine-machi
Satsuma-cho

1944-5-5
1960-5-19

Cephalanthera
longibracteata

Hokkaido

Hokkaido

Tokoro-gun, Saroma-cho, Mt.
Horoiwayama
Kamikawa-gun
Kitahiroshima-shi,
Mt. Sanbetsusan
Chitose-shi, Mt. Eniwadake

1953-10-25

Chitose-shi, Lake Shikotsuko
Tomakomai-shi

1967-10-1
1904-10-17

Kamaishi-shi, Kasshi-cho
Nishimurayama-gun,
Mt. Asahidake
Mt. Iidesan
Onuma-gun, Aizumisato-machi
Aizuwakamatsu-shi,
Mt. Otoyama
Koriyama-shi, Tamura-machi
Nishishirakawa-gun,Kashi-onsen
Ashikaga-shi, Oiwa-cho
Nerima-ku, Shakujii Pond
Suginami-ku, Omiya-Hachiman
ShrineHachioji-shi, Nakayama

Kanagawa

Sagamihara-shi, Mt. Shotosan

1985-10-10

Chubu

NaganoKitasaku-gun, Karuizawa-machi
Chiisagata-gun, Wada Pass

1936-7-22
1995-9-28

Hokuriku

Niigata

Hokkaido

Hokkaido

Kanto

Kanagawa

Nagaoka-shi, Oguni-machi
Minamiuonuma-gun, Yuzawamachi
Sapporo-shi, Minami-ku,
Ishiyama
Odawara-shi, Oniyanagi

Shikoku
Hokkaido

Kochi
Hokkaido

TohokuMiyagi

Kanto

Saitama

Chubu

Tokyo Met.
Chiba
Kanagawa
Shizuoka

Shikoku

TokushimaTohokuIwate
Yamagata

TohokuYamagata
Fukushima

Kanto

Cephalanthera
longifolia

Cypripedium
japonicum

Tochigi
Tokyo Met.

Kochi

YearMonth-Day

1891-8-11
1991-7-28

Collector andcollection
numberH. Marui s. n.
S. Sugaya & K.
Sohma s. n.
T. Misumi s. n.

Voucher
TNS708860
TUS26207
SAPS
SAPS
TUS75925

1994-8-23
1960-8-15

K. Miyabe s. n.
Y. Tateishi et al.
15131
M. Majimas. n.
S. Tanaka s. n.
K. Miyabe & J.
Hanzawa s. n.
H. Nakamura s. n.
M. Mizushima s. n.

1906-8-10
1967-10-1
1953-7-13

T. Makino s. n.
T. Saito s. n.
T. Suzuki s. n.

MAK237948
FKSE40230
TUS346926

1950-8-17
1967-8-9

H. Saze s. n.
K. Sohma 4446

FKSE416
TUS

1903-8-31
1951-11-7
1899-6-25

S. Aiba s. n.
Y. Yambe s. n.
T. Makino s. n.

TNS54274
KPM45176
MAK193757

2011-12-15

MAK404736

1982-10-2
1963-10-20

F. Sakai & K. Sakais. n.
M. Akiyama et al. s.
n.
K. Shirai s. n.
H. Nakamuras. n.
S. Iwano 18317
Collector unknown

1894-7-2

K. Miyabe s. n.

SAPS023726

2014-4-27

K. Ito OD30366

Kami-shi, Kahoku-cho, Arase
Tomakomai-shi, Hokkaido
Univ. Tomakomai Experimental
ForestKatta-gun, Shichikasyukumachi
Hanno-shi, Agano

1954-5-12
1968-9-25

T. Yamanaka s. n.
Nishikawa s. n.

KPMNA0297913
TUS402587
SAPS025932

1977-8-21

R. Suzuki s. n.

TNS374938

1953-6-7

TNS01063947

Hachioji-shi, Asakawa
Inbanuma Pond
Yotsukaido-shi
Yokohama-shi, Asahi-ku,
Kamikawai-cho
Fujinomiya-shi

1953-6-14
1947-6-22
2015-7-28
1952-8-9

T. Kawasaki
s. n.
T. Yano s. n.
B. Makino s. n.
Y. Yamashita 735
N. Deguchi s. n.

FKSE94275

Miyoshi-shi, Mt. Tsurugisan
Takaoka-gun, Ochi-cho,
Mt. Yokokurayama

1904-8-12
1934-8-22

Y. Yamashita et al.
1225
J. Nikai s. n.
G. Koidzumis. n.

1906-7-?

2016-10-23

SAPS
SAPS
SAPS
MAK451585
MAK336564

KPM72653
TNS66016
MAK452812
TUS146204
TNS791967

MAK341275
MAK299987
FKSE87328
KPM80272

TNS049109
OSA17392