Bull. Natl. Mus. Nat. Sci., Ser. B, 46(1), pp. 47–53, February 21, 2020

Floral Visitors of Critically Endangered Arisaema cucullatum (Araceae)
Endemic to Kinki Region of Japan
Satoshi Kakishima1, Masahiro Sueyoshi2 and Yudai Okuyama3,*
1

Center for Molecular Biodiversity and Research, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
2
Center for Biodiversity, Forestry and Forest Products Research Institute,
Forest Research and Management Organization, 1 Matsunosato, Tsukuba, Ibaraki 305–8687, Japan3
Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan*E-mail: yokuyama@kahaku.go.jp(Received 16 August 2019; accepted 25 December 2019)

Abstract Arisaema (Araceae) is one of the most species-rich plant groups in Japan, while 20 outof 64 Arisaema taxa are categorized as endangered species (CR or EN) in the Red List of Japan.
Although we need to comprehend their reproductive biology to conserve these species, the knowledge regarding their pollination systems is still limited. In this study, we examined the potential
pollinators of A. cucullatum, a critically endangered species endemic to Kinki Region of Japan. To
this end, we collected the insects trapped inside the spathes of A. cucullatum as well as the two
sympatric Arisaema species in the two native populations, and identiﬁed by both morphology andDNA barcoding based on mitochondrial cytochrome oxidase subunit I (COI). Sciaridae were themost abundant family in the trapped insects of A. cucullatum, and one sciarid species accounted
for 78.9% of the collected insects. Meanwhile, the insect assemblages trapped inside the spathes of
A. kishidae and A. yamatense that grow sympatrically with A. cucullatum were distinct from thoseof A. cucullatum. Accordingly, the difference in the pollinator assemblages may be important for
reproductive isolation among them, although further observation in different populations isneeded.
Key words : Arisaema, conservation biology, fungus gnats, Nara Prefecture, pollination, reproductive isolation, Sciaridae.

Introduction
Japanese Arisaema (Araceae) comprising 64
taxa is one of the most species-rich plant groups
in Japan (Iwatsuki et al., 2016; Murata et al.,2018). Moreover, 20 out of 64 taxa are categorized as endangered species (CR or EN) in the
Red List of Japan (Ministry of the Environment,
Japan, 2015). Even though in the increasing concern for the sustainability of these species, conservation measures for these plants have been
hardly performed. For example, although we© 2020 National Museum of Nature and Scienceneed to comprehend the reproductive biology ofthese endangered species to establish their conservation strategies, the knowledge regarding thepollination system of the species in Japan is stillbased on the pollinator records for only ＜10%of the diversity (5 spp.; Sasakawa, 1993, 1994a,
1994b; Nishizawa et al., 2005; Tanaka et al.,2013; Kakishima and Okuyama, 2018).
In this study, we examined ﬂoral visitors of anendangered species, Arisaema cucullatum M.
Hotta, which belongs to sect. Pistillata (Fig. 1A,
B). The sect. Pistillata is the largest group (54taxa) in Japanese Arisaema (Murata et al., 2018),
but the pollinators in this group were reported

Satoshi Kakishima et al.

48

Fig. 1. The three Arisaema species studied in the present study in its native habitat. A. A. cucullatum with maleinﬂorescence. B. A. cucullatum with female inﬂorescence. C. The trapped sciarid Leptosciarella sp. in the
spathe of A. cucullatum. D. A. kishidae with male inﬂorescence. E. A. yamatense with male inﬂorescence.

from only two species, A. serratum (Thunb.)
Scott (Sasakawa, 1993, 1994a, 1994b; Nishizawa
et al., 2005) and A. yamatense (Nakai) Nakai(Sasakawa, 1993). Most species of the sect. Pistillata in Japan are closely related to each otherand there are many similar species (Murata,
1995). Arisaema cucullatum is similar to A. seppikoense Kitam. in several vegetative characters
but distinguished by the incurved cucullate
spathe (Hotta, 1963) (Fig. 1A, B). The distribution of A. cucullatum had been originally known
in only Nara and Mie Prefectures in Kinki
Region of Japan (Hotta, 1963). A population in
Wakayama Prefecture next to Nara and Mie Pre-

fectures has recently been found (Naito et al.,2015). All the Red Lists of Japan and Nara,
Wakayama and Mie Prefectures categorize thisspecies as Critically Endangered (Wakayama
Prefecture, 2012; Ministry of the Environment,
Japan, 2015; Mie Prefecture, 2015; Nara Prefecture, 2016). In fact, the number of ﬂowering individuals in the Wakayama population was lessthan 10 in these years (Naito et al., 2015). In
addition, A. cucullatum does not vegetatively
reproduce through accessory buds so that pollinator-mediated reproduction is essential for thein-situ conservation of this species, although thepollinators have never been reported. Therefore,Pollination of Arisaema cucullatum

we surveyed the insects trapped inside thespathes of A. cucullatum, and compared theircomposition with those of two Arisaema species
belonging to sect. Pistillata, A. kishidae and A.
yamatense, growing sympatrically with A. cucullatum to examine if the potential pollinatorassemblages of A. cucullatum is distinct fromthese species (Fig. 1D, E).
Materials and Methods
Insect collection
We surveyed the insects trapped inside thespathes of Arisaema cucullatum, A. kishidae andA. yamatense in the two populations (Site 1 andSite 2) in Tenkawa Village, Nara Prefecture (precise localities are not shown for conservation).
The site 1 is located in a plantation forest of
Cryptomeria japonica and a deciduous broadleaved forest. The site 2 is located in a deciduous
broad-leaved forest along a road and is 2 kmaway from the site 1.
Most species of the sect. Pistillata make maleor female inﬂorescence every year depending on
the plant size (Schaffner, 1922; Maekawa, 1924;
Kinoshita, 1986; Takasu, 1987). The exit holes ofthe spathes found in only the male inﬂorescence.
A cotton plug was installed for preventing insects
going out from an exit hole (Kakishima andOkuyama, 2018). We collected insects inside 10
and four spathes and plugged the holes in the site
1 on 21 May 2019 and in the site 2 on 22 May
2019, respectively. Then, we collected thetrapped insects from both male and female inﬂorescence on 23 May 2019. Thus, the length of thetrap periods is two and one days in the site 1 and
2, respectively. The collected insects were stored
in 99.5% ethanol for identiﬁcation by morphology and DNA barcoding.
DNA extraction and sequencing
DNA barcoding was based on the sequences of
mitochondrial cytochrome oxidase subunit I(COI). The legs of the collected insects wereused for DNA extraction. Tissue was smashed in
20 µL of quick extraction buffer [16 mg/mL Che-49

lex-100 (Bio-Rad Laboratories, Hercules, CA)
and 1.25 mg/mL proteinase K (Wako, Osaka,
Japan) in distilled water], and then incubated at
55°C for 3 h and heat inactivated at 95°C for
10 min. A 0.5 µL supernatant of the solution wasthen used directly for polymerase chain reaction(PCR) using EmeraldAmp PCR Master Mix(TaKaRa, Shiga, Japan) or ExTaq DNA polymerase (TaKaRa, Shiga, Japan) with Ampdirect(Shimadzu, Kyoto, Japan), with the forward
primer LCO1490 (5′-GGTCAACAAATCATA
AAGATATTGG-3″) and the reverse primer
HCO2198 (5′-TAAACTTCAGGGTGACCAAA
AAATCA-3′) (Folmer et al., 1994). The PCR
was performed as follows: the initial denaturation at 98°C for 1 min, and 35 or 40 cycles of
98°C for 10 s, annealing at 55°C for 30 s, andextension at 72°C for 1 min. The ampliﬁed DNA
fragments were subjected to direct Sanger
sequencing using the Applied Biosystems 3500xl
Genetic Analyzer (Applied Biosystems, MA,
USA). Nucleotide sequences newly obtained inthis study are deposited in DDBJ under accession
numbers LC500531–LC500560.
Phylogenetic analysis and identiﬁcation
Species identiﬁcation of the collected insects
was based on both the morphology and thenucleotide sequences. Firstly we examined thecollected insects by a binocular microscope (SZ,
Olympus, Japan). Male genitalia of some fungus
gnats were removed from the body with sharpened tweezers for species identiﬁcation. They
were treated with 10% KOH solution at room
temperature overnight, and then observed undera stereoscopic microscope in 99.5% Ethanol.
When it were difﬁcult to identify some specimens by their morphology because of female or
broken bodies, we used DNA barcoding withuncorrected distance of 4% was used as thethreshold for differentiating species (Okuyama etal., 2018). The results of the DNA barcording
were also used for conﬁrming identiﬁcation
based on the morphology.
A phylogenetic analysis was conducted by
RAxML 8 (Stamatakis, 2014) using a maximum-

Satoshi Kakishima et al.

50

Table 1. The number of the collected insects from three Arisaema speices
A. cucullatum
Site 1

A. kishidae

A. yamatense

Site 1Site 1Site 2

Order

FamilySpecies

Male

FemaleMale

Male

Female

Male

Total

DipteraCalliphoridae
Cecidomyidae
Ceratopogonidae
Chironomidae
Mycetophilidae

Melanomya nana
Cecidomyidae gen.
Ceratopogonidae gen.
Chironomidae gen.
Allodia pravdini
Allodia sp.
Mycetophila ocellus
Mycetophila ruﬁcollis
Mycomya fuscata
Mycomya neodentata
Corynoptera sp.
Ctenosciara insolita
Leptosciarella sp.

1
0
0
0
0
0
0
0
0
0
0
0
5
6

0
1
0
0
0
0
0
0
0
0
0
2
2
5

0
0
0
0
0
0
0
0
0
0
0
0
8
8

0
0
0
1
0
0
1
0
0
0
1
0
0
3

0
0
1
0
1
1
0
1
0
1
0
0
0
5

0
0
0
0
0
0
0
0
3
0
0
0
0
3

1
1
1
1
1
1
1
1
3
1
1
2
15
30

Sciaridae
Total
Table 2.
Sex

Site

Male

Site 1Site 2
Subtotal
Site 1

Female
Total

Pollinator visits to Arisaema cucullatum during 21–23 May 2019

Number of
inﬂorescence

Number of trapped
insects

Visits per
inﬂorescence

Visits per inﬂorescenceper day

9
4
13
1
14

6
8
14
3
17

0.67±1.12
2.00±2.83
1.08±1.80
3.00
1.21±1.81

0.33
2.00
0.85
1.50
0.89

likelihood (ML) method with a GTR＋G likelihood model for nucleotide substitutions. The ML
tree and bootstrap proportions (BPs) wereobtained by simultaneously running rapid bootstrapping with 1,000 iterations followed by asearch for the most likely tree.
Results
We found 12 and four ﬂowering individuals of
A. cucullatum in the site 1 and 2, respectively.
Among them, one female and nine male ﬂowering individuals in the site 1 and four male in thesite 2 were surveyed. In total, we collected 19
insect individuals from the spathes of A. cucullatum (Table 1; Fig. 1C). Two and three insect individuals were collected from the only female
plant individual in the site 1 on 21 and 23 May,
respectively. Six and eight insect individuals
from the male inﬂorescence were collected in thesite 1 and 2 on 23 May, respectively. On average,

1.21 insect individuals were found in the individual spathe of A. cucullatum on 23 May, andtherefore 0.89 insect individuals per day were
supposed to be trapped (Table 2).
Overall, 13 dipteran species were found in thisstudy (Table 1; Fig. 2). In A. cucullatum, Sciaridae was the most abundant ﬂower visitor (17 outof 19 individuals), in which two species, Leptosciarella (Leptosciarella) sp. and Ctenosciara
insolita (Sasakawa), were recorded. Leptosciarella sp. was found in both sites and from bothmale and female, accounting for 78.9% (15 individuals) of the collected insects. Other than
Sciaridae, one individual of Melanomya nana
(Meigen) (Calliphoridae) and one individual of
Cecidomyiidae were collected in A. cucullatum.
In contrast, Mycetophilidae was most abundantamong the insects in A. kishidae (ﬁve out of eightindividuals) and in A. yamatense (all three individuals). The species of the collected insects
were not overlapped among the three Arisaema

Pollination of Arisaema cucullatum

51

Fig. 2. A maximum likelihood tree of 25 individuals of fungus gnats (Sciaridae and Mycetophilidae) based on
617-bp of mitochondrial COI. Bootstrap supports are shown for nodes above the species-level. Each OTU
label represents the sample name followed by the Arisaema species from which the sample was collected, i.e.,
A. cucullatum (AC), A. kishidae (AK) and A. yamatense (AY).

species.
Discussion
The principal ﬂower visitors of three Arisaema
species studied in the present study, namely A.
cucullatum, A. kishidae and A. yamatense, were
fungus gnats (Mycetophilidae or Sciaridae) as in
the cases reported previously in other Arisaema
species (Sasakawa, 1993, 1994a, 1994b; Vogel
and Martens, 2000; Nishizawa et al., 2005; Barriault et al., 2009, 2010; Tanaka et al., 2013;
Kakishima and Okuyama, 2018). Other than thetwo sciarid species, only a few insect individuals
were found to visit to the inﬂorescence in A.
cucullatum (Table 1). This result suggests that A.
cucullatum may rely on speciﬁc groups of sciarids for pollination. Leptosciarella sp. was thedominant species trapped in A. cucullatum,accounting for 78.9% of the collected insects(Table 1). In addition, although the morphologyof A. cucullatum is different between male andfemale as in the case of other Arisaema species(Murata, 1986), i.e. female individuals have longer pseudostems (Fig. 1A, B), Leptosciarella sp.
was found in the inﬂorescences of both sexes.
Taken together, Leptosciarella sp. is likely tofunction as the most important pollinator of A.
cucullatum in these study sites. Another sciarid
species trapped in the inﬂorescences of A. cucullatum is Ctenosciara insolita (Table 1), which
was also reported as the ﬂower visitor of A. serratum in Toyama Prefecture [Sasakawa (1994b),
as Phytosciara (Dolichosciara) insolita].
Although the species name Arisaema serratum
was sometimes applied to several morphologicaltypes or taxa (A. serratum sensu lato) dependingon the literatures (Murata, 1995), it is obviousthat A. cucullatum is distinct from A. serratum
sensu lato (Ohashi and Murata, 1980). Therefore,
C. insolita was found to have relationships withmultiple Arisaema species in two distant locations, although the mechanism underlying therelationships was unknown. A blow ﬂy, Melanomya nana, is newly recorded from the inﬂorescence of Arisaema. The body size of Melanomya

52

Satoshi Kakishima et al.

nana is larger than a hole of the spathe of A.
cucullatum, which would not allow the ﬂy to go
out from the hole. Therefore, M. nana does notseem to be an effective pollinator.
Meanwhile, the principal ﬂower visitors of A.
kishidae and A. yamatense were mycetophilids(Table 1). Three individuals of Mycomya fuscata(Mycetophilidae) were collected from A.
yamatense (Table 1). The result is congruent with
the independent record of Mycomia ornata from
A. yamatense in Kyoto Prefecture (Sasakawa,1993), suggesting that the genus Mycomia mightplay important roles as the pollinators of A.
yamatense. Mycomia neodentata collected fromA. kishidae was different from Mycomya species
collected from A. yamatense. Both M. fuscataand M. neodentata are newly recorded in Japan.
Other mycetophilids collected from A. kishidae
also includes rare or new records in Japan. Allodia pravdini is new to the Japanese fauna of fungus gnats and Mycetophila ocellus is the third
record in Japan since Okada (1939, 1940).
Judging from the fact that most species of thesect. Pistillata are endemic and closely related toeach other, it is most likely that the diversiﬁcation of this group has centered in the JapaneseArchipelago (Murata, 1995; Murata and Kawahara, 1995). In fact, a recent phylogenetic study
using chloroplast DNA sequences revealed that
there is only very small genetic variation in thisgroup, including A. cucullatum (Ohi-Toma et al.,2016). Therefore, it is of special interest tounderstand how these diverse species establish
reproductive isolation among them in order toelucidate the mechanisms underlying the remarkable speciation. Although the number of the collected insects was not sufﬁcient, the current
study supported the view that the mechanisms ofreproductive isolation among sympatric, closelyrelated Arisaema species involve pollinator difference, based on the observation that the three
Arisaema species do not have common pollinatorspecies in the two native populations. Further
observation in different populations would beneeded to conﬁrm if this view on pollinator-isolation in Japanese Arisaema is appropriate. Apartfrom pollinator isolation, the difference in theﬂowering season may also play an important role
in reproductive isolation (Murata et al., 2018). In
fact, Arisaema cucullatum was at the early ﬂowering stage but A. kishidae was at the late stage
in the study period.
In the present study, we could only ﬁnd 16
ﬂowering individuals of A. cucullatum in the twostudy sites, conﬁrming that A. cucullatum isindeed critically endangered. So it is crucial toidentify the environmental factors for their successful reproduction. To this end, the observationthat the number of trapped pollinators per day
was higher in the site 2 (2.00 individuals per day)than the site 1 (0.33 individuals per day) (Table2) might provide some hints, although both study
sites are common in that they were located onhumid slopes along a river in a limestone area.
The present observation also implies that A.
cucullatum may rely on speciﬁc taxonomic
groups of Sciaridae for their pollination (Table1). If this view is correct, we need to elucidate
the environmental factors suitable not only forthe plantsʼ growth but also pollinatorsʼ abundance
for making conservation strategy.
Acknowledgments
We thank S. Serizawa and T. Matsumoto for
information of the study sites and Y. Suzuki forsupporting DNA barcoding of the pollinators. We
also thank the Ministry of the Environment Government of Japan for permission to collect A.
cucullatum. This study was supported by the
JSPS KAKENHI Grant Number JP15H05604,
JP19H03292 (to Y.O.) and JP17K15182 (toS.K.), and Ichimura Foundation for New Technology.
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