Host-ant specificity of endangered large blue butterflies (Phengaris spp., Lepidoptera: Lycaenidae) in Japan

Abstract

Large blue butterflies, Phengaris (Maculinea), are an important focus of endangered-species conservation in Eurasia. Later-instar Phengaris caterpillars live in Myrmica ant nests and exploit the ant colony’s resources, and they are specialized to specific host-ant species. For example, local extinction of P. arion in the U. K. is thought to have been due to the replacement of its host-ant species with a less-suitable congener, as a result of changes in habitat. In Japan, Myrmica kotokui hosts P. teleius and P. arionides caterpillars. We recently showed, however, that the morphological species M. kotokui actually comprises four genetic clades. Therefore, to determine to which group of ants the hosts of these two Japanese Phengaris species belong, we used mitochondrial COI-barcoding of M. kotokui specimens from colonies in the habitats of P. teleius and P. arionides to identify the ant clade actually parasitized by the caterpillars of each species. We found that these two butterfly species parasitize different ant clades within M. kotokui.

Full text

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Scientific RepoRts | 6:36364 | DOI: 10.1038/srep36364
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Host-ant specicity of endangered
large blue butteries (Phengaris
spp., Lepidoptera: Lycaenidae) in
Japan
Shouhei Ueda1,2, Takashi Komatsu3, Takao Itino1,4, Ryusuke Arai5 & Hironori Sakamoto6,7
Large blue butteries, Phengaris (Maculinea), are an important focus of endangered-species
conservation in Eurasia. Later-instar Phengaris caterpillars live in Myrmica ant nests and exploit the ant
colony’s resources, and they are specialized to specic host-ant species. For example, local extinction
of P. arion in the U. K. is thought to have been due to the replacement of its host-ant species with a less-
suitable congener, as a result of changes in habitat. In Japan, Myrmica kotokui hosts P. teleius and
P. arionides caterpillars. We recently showed, however, that the morphological species M. kotokui
actually comprises four genetic clades. Therefore, to determine to which group of ants the hosts of
these two Japanese Phengaris species belong, we used mitochondrial COI-barcoding of M. kotokui
specimens from colonies in the habitats of P. teleius and P. arionides to identify the ant clade actually
parasitized by the caterpillars of each species. We found that these two buttery species parasitize
dierent ant clades within M. kotokui.
Several orders of animals are found in ant nests. Some of them depend in some way on ants during their life cycle,
which are known as myrmecophiles1,2. In lepidopteran insects, more than half of Lycaenidae species have associa-
tions with ants that range from facultative association to obligate nest parasitism3. In order to communicate ants,
lycaenid caterpillars and pupae have some myrmecophilous organs, such as dorsal nectary organs, pore cupola
organs and tentacle organs, producing nectars and other substances, and organs for sound production3. By using
these myrmecophilous organs, lycaenids emit chemical and acoustic cues to manipulate their host ants.
Large blue Phengaris (Maculinea) butteries (Lepidoptera: Lycaenidae) are widely distributed in Europe and
Asia, and all known species (about 10) are considered to be obligately myrmecophilous. Phengaris butteries are
the best-known example of parasitic myrmecophily, and they exhibit a high degree of host-ant specicity4. Early
instar caterpillars feed on specic host plants (owers of Lamiaceae, Gentianaceae, or Rosaceae). When they
reach the fourth instar, they drop from their host plant to the ground and gain entry to a nest of Myrmica ants
(Myrmicinae) by using chemical mimicry to cause themselves to be recognized as ant larvae by worker ants, who
then carry them into their nest4–6. Ant nests are strongly protected by their ant inhabitants. erefore, if an organ-
ism can enter a nest without being attacked by the ants, the nest becomes a safe shelter against natural enemies1.
Once they gain entry into an ant nest, the caterpillars grow by exploiting the resources of the ant colony
(Fig.1). Phengaris uses two parasitic strategies: “predatory” caterpillars prey on the ant brood, and “cuckoo”
caterpillars are fed by the ants via regurgitation7,8. Phengaris teleius and P. arionides, which are widely distributed
in East Asia, including Japan, are predatory species. Caterpillars following both strategies gain more than 98%
of their biomass in the ant nest; thus, these buttery species are obligate parasites9. By the time the fourth-instar
1Department of Biology, Faculty of Science, Shinshu University, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan.
2Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai,
Osaka 599-8531, Japan. 3The institute of tropical agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku,
Fukuoka, Fukuoka 812-8581, Japan. 4Institute of Mountain Science, Shinshu University, Asahi 3-1-1, Matsumoto,
Nagano 390-8621, Japan. 5Department of Mountain and Environmental Science, Interdisciplinary Graduate School
of Science and Technology, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan. 6Brain
Science Institute, Tamagawa University, Tamagawagakuen 6-1-1, Machida, Tokyo 194-8610, Japan. 7Faculty of
Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan. Correspondence and requests for
materials should be addressed to S.U. (email: sueda@envi.osakafu-u.ac.jp)
Received: 01 July 2016
Accepted: 04 October 2016
Published: 03 November 2016
OPEN

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caterpillars pupate, the host-ant colonies have suered serious damage, yet the ants transport these parasites into
their nest in their own mandibles.
Because of the high specicity of parasitic Phengaris butteries toward their host plants and ants, both must
occur together for a habitat to be suitable for the butteries. As a result, these butteries are vulnerable to envi-
ronmental change, and all species of Phengaris are endangered worldwide10. Two Phengaris species, P. teleius and
P. arionides, are distributed in the Japanese archipelago, and geographic sub-species of P. teleius are classied as
“Near reatened” or “Critically Endangered”, and P. arionides is classied as “Near reatened” in the 4th (latest)
version of the Japanese red lists11. In addition, in March, 2016, the government of Japan’s Ministry of Environment
added a sub-species P. teleius kazamoto living in Chubu area of central Honshu to the list of “National Endangered
Species”, and prohibited the collecting and transferring of it.
To develop a conservation strategy for endangered Phengaris butteries, it is essential to identify their host-ant
species. In the United Kingdom, P. a r io n became extinct aer its host-ant species was replaced by unsuitable
congeners5,12. Before the 1980s, it was thought that Phengaris caterpillars could parasitize any Myrmica ant
species13, but in a comprehensive investigation of host specicity among eight Myrmica species and ve Phengaris
species, omas, et al.5 found a one-to-one association between each ant and buttery species. For example, they
found that the survival rate of P. a r io n caterpillars in nests of M. sabuleti and M. scabrinodis was on average 15%
and 2%, respectively5. us, the major host-ant species of P. a ri o n is M. sabuleti, and it is dicult for the caterpil-
lars to mature in a nest of M. scabrinois.
In past morphological studies, the host-ant species of Japanese Phengaris species was identied as Myrmica
kotokui2,14–16. However, Ueda, et al.17 showed that the species recognized as M. kotokui on the basis of morphology
actually consisted of four genetic clades. erefore, the host-ant specicity of Phengaris needs to be determined
not just at the species level but also at the genetic level. Moreover, Ueda, et al.18 showed that each cryptic clade
prefers a dierent habitat and nesting microhabitat. us, P. teleius, which inhabits grasslands, and P. arionides,
which lives in woodlands, might parasitize dierent ant clades within M. kotokui. To determine the true host ant
of P. teleius and P. arionides, we (1) investigated M. kotokui colonies in the habitats of P. teleius and P. arionides,
(2) used DNA barcoding to estimate the frequencies of the dierent ant clades in each habitat, and (3) then iden-
tied the ant clade that the caterpillars of each buttery species actually parasitized.
Results
The DNA clade of each of the 99 ant colonies collected from the six Phengaris habitats was identified by
neighbor-joining (NJ) analysis of 470-bp sequences of the mitochondrial COI gene (Fig. S1). We found that four
belonged to the L1 clade, 67 to the L2 clade, and 28 to the L3 clade (Table1). us, L2 was the dominant clade in
the P. teleius grassland habitats (86.2–100%), and in the woodland P. arionides habitat, all ant colonies belonged to
the L3 clade (Table1). ese habitat preferences of the ant clades are congruent with the ndings of Ueda, et al.18.
Next we identied the DNA clade of each ant colony parasitized by Phengaris caterpillars. e four ant col-
onies parasitized by P. teleius belonged to L2, and the three ant colonies parasitized by P. arionides belonged to
L3 (Table1). Although the sample size is too small for statistical testing, based on the habitat preferences of ants
and the parasitic frequency of Phengaris caterpillars, we tentatively conclude that P. teleius parasitizes L2 colonies
and P. arionides parasitizes L3 colonies under natural conditions. To determine the specicity of the Japanese
Figure 1. A Phengaris arionides caterpillar feeding on larvae belonging to the L3 clade of Myrmica kotokui
(Photo by T. Komatsu).

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Phengaris—Myrmica interaction more denitively, additional sampling is essential. During this study, however,
we decided not to collect more specimens because we judged that additional collections risked excessively deplet-
ing the populations of both butteries and ants.
Discussion
We showed that two Japanese Phengaris buttery species apparently parasitize the nests of dierent ant clades
within the M. kotokui morphological species. is nding raises the question, does this apparent specicity rep-
resent an adaptation on the part of the buttery, or did the dominance of L2 and L3 clades in grasslands and
woodlands, respectively, lead to this apparent one-to-one correspondence without adaptation? It is possible that
Japanese Phengaris—Myrmica interactions are an example of parasitic adaptation, because preliminary tests indi-
cate that P. teleius caterpillars and their host ants have some cuticular hydrocarbons (CHCs) in common (R. Seki,
personal communication). To conrm that the adaptation has occurred, in addition to a CHC analysis, acoustic
measurements should also be performed to compare the sounds produced by caterpillars and buttery pupae to
those of worker and queen ants, because both chemical and acoustical mimicry by P. r eb el i caterpillars of their
host Myrmica ants have been demonstrated6,19–22.
In this study, we found cryptic host-ant specicity in Phengaris butteries for the rst time. erefore, to
preserve these East Asian butteries, it is important to maintain their particular host ant clades. Do declines
in the number of host-ant colonies in appropriate buttery habitats drive the extinction of the butteries? We
investigated the ant species composition in the area of the most endangered population of P. teleius in habi-
tat E (Table1). We found that because the soil had acidified, become drier and swampy meadows, suitable
micro-habitat to L2 clade, decreased (Table1), probably as a result of changing agricultural practices, only Lasius
japonicus (Formicidae) and Myrmica jessensis, neither of which are suitable host ants, occurred beneath or near
Sanguisorba ocinalis (Rosaceae), the host plant of early-stage P. teleius caterpillars. Because all previous studies
showed that P. teleius and P. arionides in Japan parasitize the nest of M. kotokui, we determined that M. jessensis
may not be a suitable host ant species. ere were some reports that the caterpillars of the Japanese Phengaris spe-
cies parasitize the nest of Aphaenogaster japonica14,23, but the ant’s name was mistake for M. kotokui15. In this area,
we were able to ne only two ant nests of the L1 clade, and it was located at the edge of a forest and far from any
suitable host plants for the caterpillars. Given the suitable host ant of P. teleius is L2 clade, the caterpillars cannot
live in any of the ant nests in habitat E. e displacement of ant species in Phengaris habitats in the UK has been
shown to lead to the extinction of native populations of P. a r io n5,12. omas et al.11 showed that this high specic-
ity triggered the local extinction of P. arion. Myrmica scabrinodis prefers to nest in tall grass, whereas M. sabuleti
prefers areas where grass height is kept low by herbivory. When herbivores were excluded from the P. a ri o n habitat
and the grass became high, M. sabuleti replaced M. scabrinodis and, as a result, the P. a ri on population declined
sharply12,24. On the basis of this nding, in UK sanctuaries for P. ar i on , a suitable environment for the host ant was
produced by controlled burning and grazing. en, once M. sabuleti was re-established in the restoration sites,
the butteries were successfully re-introduced from Sweden5,12. is nding suggests the displacement of ant
species in Japan could cause P. teleius and P. arionides to become extinct. To save Japanese Phengaris butteries
from extinction, Phengaris populations and their habitats should be surveyed, interactions between the butteries
and ants should be investigated, and the anthropogenic impact on their habitats and hosts should be evaluated.
Methods
Parasitization rates of Japanese Phengaris on Myrmica kotokui. It is not necessary to acquire gov-
ernment permission to collect the ant samples in the concerned regions. However, we got approval to collect the
samples from the managers of each buttery sanctuary. We searched for M. kotokui nests in four P. teleius hab-
itats (A–E) separated from one another by more than 100 km, and in one P. arionides habitat (F). To protect the
butteries, we do not show the detailed collecting site locations here, but each habitat area has a large population
of butteries except for habitat E. e population size of the buttery may relate to colony density of M. kotokui
(Table1). e buttery population in the habitat (E) with the lowest colony density was much smaller than the
others. And the colony density of the ants may relate to soil moisture (Table1). In each P. teleius habitat A–E,
M. kotokui nested in the muddy soil of a moist grassland, and the swampy meadows decrease may lead to decreas-
ing the colony density of the ants (Table1). To determine whether P. teleius caterpillars were present in a nest, we
removed all soil to a depth of 0.5 m within a radius of 1.0 m of the nest entrance. We le all ants, including queen
ants, in the colony, except for some worker specimens removed for DNA analysis. In all, we investigated 29 ant
Phengaris
species Region Location
Soil
moisture
Colony density
of M. kotokui
No. of ant nests
examined
Ant clades of nests No. of nests
with Phengaris
Clades of parasitized ants
L1 L2 L3 L1 L2 L3
P. teleius Hokkaido A + + + + + 29 1 (3.4%) 25 (86.2%) 3 (10.3%) 3 0 3 0
Tohoku B+ + + + + + 14 0 (0%) 14 (100%) 0 (0%) 1 0 1 0
Tohoku C+ + + + + + 18 0 (0%) 18 (100%) 0 (0%) 0 0 0 0
Chubu D+ + + + 11 1 (2.8%) 10 (93.1%) 0 (0%) 0 0 0 0
Chubu E+ + 2 2 (0%) 0 (0%) 0 (0%) 0 0 0 0
P. arionides Chu-bu F + + + + + 25 0 (0%) 0 (0%) 25 (100%) 3 0 0 3
Table 1. Occurrence of ant clades within Myrmica kotokui in six Japanese Phengaris habitats (A–F) and
the clades of the ant colonies actually parasitized by caterpillars. Ant clades were identied by mitochondrial
COI barcoding.

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colonies in habitat A, 14 in habitat B, 18 in habitat C, 11 in habitat D and 2 in habitat E (Table1); we found cater-
pillars in three habitat A colonies and one caterpillar in a habitat B colony, but no caterpillars in habitats C and D
(Table1). us, the parasitization rate of P. teleius in ant nests was 5.4%, which is lower than rates reported this
species in Poland and France5,25. e lower parasitizing rate in Japan may indicate that the P. teleius population
is small, or it may be an underestimate, because in our survey we did not completely excavate the ant colonies.
In P. arionides habitat F, M. kotokui nested in decayed logs in a forest. We opened decayed wood from around
each nest to determine whether P. arionides caterpillars were present in the nest. We investigated 25 ant colonies
in habitat E, and found caterpillars in three of them (Table1). us, the parasitization rate of P. arionides in the
ant nests was 12.0%. is rate is higher than the P. teleius rate (5.4%), but we cannot compare it with rates in other
regions because, to our knowledge, this is the rst report of the parasitization rate of P. arionides in ant nests.
DNA barcoding of ants. During the nest survey, we collected 10 to 20 worker ants from each colony
for DNA barcoding and preserved them in 99.5% EtOH until the analysis. We deposited voucher specimens
at the Faculty of Science, Shinshu University, Matsumoto, Japan. We extracted DNA from the whole body of
each ant using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s proto-
cols. en we amplied the mitochondrial COI gene by polymerase chain reaction (PCR) using Takara Ex Taq
polymerase (Takara Bio, Shiga, Japan), and the primers MyrCOI-F1 (5′ -TA GGR TCR CCT GAT ATA GC-3′)
and MyrCOI-R1 (5′ -CC AGG TAY YAT TAA AAT ATA AAC TTC-3′ )18. e reaction was carried out for 30
cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 40 s. Aer amplication, the PCR products were puried with
ExoSap-IT reagent (USB, Cleveland, Ohio, USA). Both strands were sequenced with a BigDye Terminator v1.1
Cycle Sequencing Kit (ABI, Weiterstadt, Germany) on an ABI 3130 Genetic Analyzer.
e mitochondrial COI sequences were edited and aligned with SeqScape v. 2.5 soware (ABI, Weiterstadt,
Germany). We imported the obtained COI dataset into the COI dataset of Ueda et al.17, and then determined the
clade of the ants in each colony by a neighbor-joining NJ analysis, performed with MEGA6 soware26. Although
Ueda et al.17 used both COI and LwRh sequences to infer the molecular phylogeny, in this study we analyzed only
the COI sequences because the mutation rate of the LwRh gene is slow and it is possible to determine the clade by
using only the COI gene data. e GenBank accession numbers of the COI gene sequences are listed in Table S1.
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Acknowledgements
We thank T. Ando, S. Shimamoto, M. Hattori, K. Abe, K. Maruyama and T. Kobayashi for sampling support;
T. Itoh, T. Nakatani, Y. Hagiwara, S. Hiraga, M. Yago and S. Yamaguchi for providing information about sampling
sites; T. Akino and R. Seki for providing information about chemical mimicry; and H. Mohri for the initiation of
this research project.
Author Contributions
S.U., T.I. and H.S. conceived and designed the experiments. S.U., T.K., R.A. and H.S. performed the sampling and
experiments. S.U. and T.K. analyzed the DNA data. S.U. and T.I. contributed reagents/materials/analysis tools.
S.U., R.A. and H.S. wrote the paper and T.K. prepared Fig. 1. All authors reviewed the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Ueda, S. et al. Host-ant specicity of endangered large blue butteries (Phengaris spp.,
Lepidoptera: Lycaenidae) in Japan. Sci. Rep. 6, 36364; doi: 10.1038/srep36364 (2016).
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