Bull. Natl. Mus. Nat. Sci., Ser. B, 38(4), pp. 183–187, November 22, 2012

Pollination by Fungus Gnats in Mitella formosana (Saxifragaceae)
Yudai Okuyama
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
E-mail: yokuyama@kahaku.go.jp(Received 20 August 2012; accepted 26 September 2012)

Abstract The pollination system of Mitella formosana, the last Asimitellaria species (genusMitella sect. Asimitellaria, Saxifragaceae) in which the information regarding its ﬂower visitors
remained unreported, was studied in two study sites in Taiwan in April, 2011. Although the frequency of pollinator visits observed were very low (0.04 visits per hour per inﬂorescence on average in daytime), the principal pollinators of M. formosana were clariﬁed as the fungus gnats of thegenus Boletina and Coelosia, just similar as in the other Asimitellaria species in Japan. The apparent similarity of pollination systems of Asimitellaria species between Taiwan and Japan implies
the long-term maintenance and the evolutionary stability of the Mitella–fungus gnats pollination
mutualisms across Japan archipelago and Taiwan.
Key words : Asimitellaria, Boletina, Coelosia, Japan, pollination mutualism, Taiwan.

Introduction
A tremendous diversity of ﬂowering plants
today is reﬂected by the diversity of plant-pollinator interactions, and how the diversity of pollinator-associated ﬂoral traits has been shaped andhow it further facilitated the plant speciation has
been one of the major interests in plant evolutionary biology (Proctor et al., 1996; Fenster etal., 2004; Willmer, 2011). To address this issue,
it is very useful to have a model plant group inwhich variation in pollination systems and associated ﬂoral traits, as well as the phylogenetic
relationships therein are analyzed simultaneously.
Asimitellaria, a section of the genus Mitella(Saxifragaceae), is one of such the few plant lineages in which not only the phylogeny but also
its relationships with pollinators are comprehensively surveyed (Okuyama et al., 2004, 2005,2008, 2012). In Asimitellaria, almost all species
except M. doiana, which is presumably an obligate selfer, are known to be pollinated exclu-

sively by fungus gnats (Mycetophilidae)(Okuyama et al., 2004, 2008). Moreover, pollination by fungus gnats in Asimitellaria shows anotable variation, where some species are pollinated only by a long tongued fungus gnat species
Gnoriste mikado, while the other species are pollinated by short-tongued fungus gnats of genus
Boletina and Coelosia exclusively, or together
with Gnoriste mikado as a co-pollinator. Interestingly, in some populations, two to three Asimitellaria species co-occur, and the pollinator isolation mediated by the divergent pollinator fungus
gnats support their sympatric nature. Therefore,
to analyze the selection force behind the pollinator shifts in Asimitellaria should be useful tounderstand the mechanisms on pollinator-mediated isolation and speciation of ﬂowering plants.
The last Asimitellaria species without any
information of its relationships with pollinators isM. formosana, the only Asimitellaria species that
distributes outside Japan. Mitella formosana also
has a notable phylogenetic position in Asimitellaria, i.e., forming a well-supported clade with

184

Yudai Okuyama

Fig. 1. Coelosia sp. (Mycetophilidae) that visited on the M. formosana ﬂowers. A, Coelosia sp. nectaring on theM. formosana ﬂower. B, A microscopic image of Coelosia sp. collected on a M. formosana ﬂower. Notenumerous pollen grains are attached on the anterior side of the body. C, An electron microscopic image ofCoelosia sp. collected on a M. formosana ﬂower. The rugby-ball like morphology of pollen grains typical of
the pollen grains of Mitella is visible on the insectʼs surface.

M. japonica and M. yoshinagae (Fig. 2,Okuyama and Kato, 2009), which presumably
reﬂects the ancient ﬂoristic link between Taiwanand Japan. Okuyama et al. (2008) classiﬁed theﬂoral morphology of M. formosana as “saucershape”, implying this species might have rela-tionship with short-tongued fungus gnat speciesof the genus such as Boletina and Coelosia. To
test this prediction and to expand the knowledge
on pollination systems of Asimitellaria into thewhole lineage and geographic distribution, I conducted a survey on pollination biology of M. for-Pollination by Fungus Gnats in Mitella formosana (Saxifragaceae)

185Fig. 2. Phylogenetic distribution of pollination systems in Asimitellaria. Phylogenetic tree was redrawn from
Okuyama and Kato (2009).

mosana in April, 2011. The present study
revealed that M. formosana, a geographicallyisolated and the southernmost species of Asimitellaria, maintains a surprisingly similar pollination system to those of species in Japan.
Materials and Methods
Direct observation of pollinator visits to
ﬂowers of M. formosana were made at Lalashansite (N24°42′17.88″, E121°26′44.53″) during
1200–1700 on April 7 and 1600–1700 on April
10, and Szuyuanyakou site (N24°23′15.62″,E121°21′28.37″) during 0900–1700 on April 9and 1100–1200 on April 10. All insects ﬂew andlanded to the ﬂowers were collected and stored
dry separately to inspect their pollen loads undera KEYENCE VHX-2000 digital microscope(Keyence, Tokyo). In addition, pollinator visitswere further monitored with time-lapse photography using Optio W90 cameras (Pentax, Tokyo),each set in front of an individual of M. formosana, keeping ＞20 cm distance from the plants

and ＜5 m to each other. The time-intervals
between the shots were set to 2 min, as this is theminimum time-interval for camera battery to besustained overnight (＞12h). This setting is reasonable because the pollinator fungus gnats inother Asimitellaria species were known to stayon ﬂowers for ＞2 min, on average (Okuyama etal., 2004), and there is little discrepancy between
the ﬂower-visitation data from direct observationand time-lapse photography in Asimitellaria(Yudai Okuyama, personal observation; see also
the Results section). Five cameras were set eachin Lalashan site and Szuyuanyakou site for
13–46 h, focusing on 1–12 inﬂorescences in fullbloom (Table 1). Each insect individual ＞5 mmin body size photographed at nectaring on aﬂower was count as a single visit, and the insect
on the same ﬂower taken in the subsequent shot
was not count.
Results
Throughout the observation period, insect vis-186

Yudai Okuyama
Table 1. The detailed information of time-lapse photography in the present study

Lalashan site
Camera 1
Camera 2
Camera 3
Camera 4
Camera 5
Szuyuanyakou site
Camera 6
Camera 7
Camera 8
Camera 9
Camera 10

No. ofinﬂorescence

Photographing time of day

Totaltime

No. ofshots

No. ofinsect visits*

113321247 April 7–1041 April 91356 April 7–1038 April 81414 April 7–0733 April 9
1236–1734 April 7, 0953–1141 April 81332 April 7–0202 April 846 h
20.5 h
41 h
7h
13 h

1380
615
1230
210
390

1 (1)
0 (0)
9 (7)
0 (0)
1 (1)

3612461849 April 8–1128 April 10
1834 April 8–1349 April 9
1628 April 9–1101 April 101851 April 8–1135 April 10
1838 April 8–1543 April 941 h
19 h
18.5 h40.5 h21 h1230
5705551215630

3 (0)
0 (0)
0 (0)
3 (0)
0 (0)

* The numbers in parentheses indicate the number of mycetophilid fungus gnats

its were rare, although certainly present. Except
for mordellid beetles continuously stayed andcrawled on M. formosana ﬂowers, only I had
observed were three visits of Coelosia sp.
(Mycetophilidae) and two visits of the other dipterans in Lalashan site, and a single visit ofBoletina sp. (Mycetophilidae) in Szuyuanyakousite. This very rare insect visits were also conﬁrmed by a time-lapse photography that shot
only 17 insect visits in a grand total of 118 h daytime (0.04 visits per hour per inﬂorescence onaverage), in which 9 of 17 were mycetophilid
fungus gnats and the other 8 were other dipterans(Table 1). The all four mycetophilid fungus gnats(3 Coelosia sp. and 1 Boletina sp.) directly collected on the ﬂowers were conﬁrmed to carry＞100 pollen grains on their body (Fig. 1),
whereas the other dipterans collected on theﬂowers of M. formosana had ＜10 pollen grains.
Discussion
The present research partly clariﬁed the pollination system of M. formosana that has neverbeen reported before. From the direct observation and time-lapse photography, the major
ﬂower visitors of M. formosana was conﬁrmed tobe the short-tongued fungus gnats of the genus
Boletina and Coelosia, just similar as in Asimitellaria in Japan. Because these fungus gnats collected on the M. formosana ﬂowers always had
numerous pollen grains (＞100) on their bodyenough to fertilize all ovules of the ﬂower, it
would be reasonable to conclude these fungus
gnats are the principal pollinators of M. formosana. The other insects, non-mycetophilid dipterans and mordellid beetles, visited on the ﬂowers
were never found to carry pollen loads enough tofertilize a large proportion of ovules of theﬂower, or they seldom move across the inﬂorescences (mordellid beetles). Therefore, they are
unlikely to contribute largely to the cross pollination of the species, although mordellid beetles
might partly contribute to self-pollination.
Although the present research is limited to ashort time-period in only one year, the following
characteristics of pollination system of M. formosana were clariﬁed. First, M. formosana has surprisingly similar pollination system to those of
Asimitellaria species in Japan that involves thesame mycetophilid genera such as Boletina andCoelosia. Second, the fungus gnats of the genus
Gnoriste are unlikely to be involved in the pollination of M. formosana, conﬁrming what isexpected from the ﬂoral morphology (Okuyamaet al., 2008) and the fact that there is no record ofcollection of the genus Gnoriste in Taiwan. The
apparent similarity of pollination systems of
Asimitellaria species between Taiwan (M. formosana) and Japan (M. pauciﬂora, M. japonica
Honshu group, and M. yoshinagae) implies thelong-term maintenance and the evolutionary stability of the Mitella–fungus gnats pollination
mutualisms across the Japan archipelago andPollination by Fungus Gnats in Mitella formosana (Saxifragaceae)

Taiwan.
It is also noteworthy that, from the present
study, the complete picture of the pattern andvariation of pollination systems in Asimitellaria
became available (Fig. 2). As is evident looking
at Fig. 2, the different pollination modes in theAsimitellaria are scattered across the phylogeny,
indicating the pollinator shifts occurred recurrently in the lineage. This suggests that, using thepresent information, the common ecological andphysiological mechanisms behind the pollinator
shifts in Asimitellaria could be identiﬁed, whichis the topic of forthcoming research.
Acknowledgments
The author thanks Ching-I Peng for thearrangement of the ﬁeld research in Taiwan andChien-Hua Lu and Chien-I Huang for their generous guide in the ﬁeld sites.
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