High mycorrhizal specificity in the mycoheterotrophic Burmannia nepalensis and B. itoana (Burmanniaceae)

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Short communication

High mycorrhizal specificity in the mycoheterotrophicBurmannia nepalensis and B. itoana (Burmanniaceae)

Yuki Ogura-Tsujita a,*, Hidetaka Umata b, Tomohisa Yukawa c

aBotanical Gardens, Tohoku University, 12-2 Kawauchi, Aoba-ku, Sendai, Miyagi 980-0862, Japanb 5211 Kita-Takanabe, Takanabe-cho, Koyu-gun, Miyazaki 885-0002, JapancNational Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan

a r t i c l e i n f o

Article history:

Received 23 October 2012

Received in revised form

18 January 2013

Accepted 26 February 2013

Available online xxx

Keywords:

Arbuscular mycorrhiza

Burmannia liukiuensis

Glomeraceae

Glomus group A

Mycoheterotroph

* Corresponding author. Tel./fax: þ81 22 795E-mail address: [email protected] (Y.

Please cite this article in press as: Oguranepalensis and B. itoana (Burmanniaceae),

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a b s t r a c t

Mycorrhizal fungi of mycoheterotrophic Burmannia nepalensis and B. itoana were identified

by molecular identification methods based on fungal SSU nrDNA region. In B. nepalensis,

RFLP patterns and sequences from all root samples from 14 individuals were identical. A

single fungal sequence was also obtained from B. itoana roots from three individuals.

Phylogenetic analysis showed that the fungal sequences from these two species are

included in Glomeraceae (former Glomus group A). Our results indicate that the two Bur-

mannia species are associated with narrow phylogenetic ranges of arbuscular mycorrhizal

fungi.

ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Mycoheterotrophic plants (MHPs), which are achlorophyllous

and leafless, completely depend onmycorrhizal fungi for their

supply of carbon throughout their life cycle (Leake 1994).

Burmanniaceae, a monocotyledonous plant family, belongs to

the order Dioscoreales (Merckx et al. 2006). Most of the

members are achlorophyllous and mycoheterotrophic while

some species are chlorophyllous and autotrophic (Merckx

et al. 2010). Burmannia, the largest genus of the Burmannia-

ceae, is a pantropical genus comprising about 60 species, with

only a few species extending into temperate, humid areas of

East Asia (Leake 1994; Wu et al. 2010). Most species are

mycoheterotrophs and have been shown to be associatedwith

arbuscular mycorrhizal (AM) fungi by anatomical (van der Pijl

1934; Terashita and Kawakami 1991; Imhof 1999) and

6789.Ogura-Tsujita).

-Tsujita Y, et al., HighMycoscience (2013), htt

ycological Society of Jap

molecular investigations (Franke et al. 2006; Merckx and

Bidartondo 2008; Merckx et al. 2010; Suetsugu et al. 2012),

although only a few species were tested.

Burmannia nepalensis Hayata, often used as a synonymous

name B. liukiuensis, is a mycoheterotroph distributed in

Himalayas, Indochina, southern China, Taiwan, Philippines

and southern Japan (Wu et al. 2010). Burmannia itoana Makino

is distributed in southern China, Taiwan and southern Japan

(Wu et al. 2010), both of which are endangered species in Japan

(Environmental Agency of Japan 2000). Although Terashita

and Kawakami (1991) found the AM association of B. nepal-

ensis by anatomical observation, molecular identification has

never been performed in mycorrhizal fungi of both B. nepal-

ensis and B. itoana. Understanding their mycorrhizal

mycorrhizal specificity in the mycoheterotrophic Burmanniap://dx.doi.org/10.1016/j.myc.2013.02.004

an. Published by Elsevier B.V. All rights reserved.

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http://dx.doi.org/10.1016/j.myc.2013.02.004



my c o s c i e n c e x x x ( 2 0 1 3 ) 1e52

association is essential for establishing conservation strate-

gies of these endangered species.

In this study,mycorrhizal fungi of B. nepalensis and B. itoana

were identified with the small subunit (SSU) of nuclear ribo-

somal DNA sequences. In total, 36 roots of 14 individuals from

two sites of B. nepalensis and nine roots of three individuals

from one site of B. itoana were examined.

Materials of B. nepalensis (Fig. 1a) and B. itoana (Fig. 1b) were

sampled from Kagoshima Prefecture, Japan (Table 1).

Collected roots (ca. 1 cm long) were washed in water and kept

at �80 �C until use. Voucher specimens were deposited with

the Herbarium, National Museum of Nature and Science

(TNS).

DNA was extracted from root samples using the DNeasy

Plant Mini Kit (Qiagen, Valencia, CA, USA). Fungal SSU se-

quences were amplified with the primer NS31 (Simon et al.

1992) and AM1 (Helgason et al. 1998). All PCR products with

these primers were analyzed by restriction fragment length

polymorphism (RFLP) using the restriction enzymes HinfI and

MspI (Gardes and Bruns 1996) and representative samples for

Fig. 1 e Flowering plants of Burmannia nepalensis (a) and

B. itoana (b). Bars: 10 mm.

Please cite this article in press as: Ogura-Tsujita Y, et al., Highnepalensis and B. itoana (Burmanniaceae), Mycoscience (2013), htt

each plant were chosen and directly sequenced from the PCR

products (Table 1). To minimize PCR bias caused by primer

mismatch of the NS31/AM1 primers for Archaeosporaceae

and Paraglomaceae (Daniell et al. 2001; Lee et al. 2008), AML2, a

universal primer for all known AM fungal groups (Lee et al.

2008), was also used for PCR amplification with the primer

NS31. One representative root sample was chosen from four

individuals from two sites of B. nepalensis and one individual of

B. itoana, and in total, four roots of B. nepalensis and one root of

B. itoana were used for the NS31/AML2 amplification. PCR was

performed using the EX Taq PCR kit (Takara Bio, Shiga, Japan)

under the manufacturer’s protocols. Amplification conditions

were as follows: an initial denaturation at 94 �C for 5 min;

followed by 30 cycles of denaturing at 94 �C for 30 s, annealing

at 55 �C for 30 s, and extension at 72 �C for 30 s; with a final

extension at 72 �C for 7 min. The PCR products were purified

with ExoSAP-IT (USB Corporation, Cleveland, OH, USA) and

directly sequenced on an Applied Biosystems (Foster City, CA,

USA) 3100 genetic analyzer using BigDye v3.1 terminator cycle

sequencing ready reaction mix according to the manufac-

turer’s instructions. In addition, PCR products with the NS31/

AML2 primers were cloned. The cloning was performed using

the pGEM-T Vector System II (Promega, Madison, WI, USA)

and ten clones for each sample were used for sequencing

analysis. DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp)

accession numbers of the fungal sequences determined in this

study are shown in Table 1.

Fungal SSU sequences from B. nepalensis and B. itoana were

aligned manually against the data matrix in Schwarzott et al.

(2001) using MacClade v. 4.06 (Maddison and Maddison 2003).

The alignment length used for tree construction is 502 bp.

Phylogenetic analyses were conducted with PAUP* version

4.0b6 (Swofford 2001). Distance trees were obtained using the

neighbor-joining (NJ) method (Saitou and Nei 1987) with a

Kimura two-parameter correction (Kimura 1980). For assess-

ing the relative robustness for branches, the bootstrapmethod

(Felsenstein 1985) was used with 1000 replicates. Archaeospora

leptoticha (AJ006466) and Geosiphon pyriformis (Y15905) were

used as outgroups based on the results of phylogenetic anal-

ysis of Glomeromycota (Schwarzott et al. 2001). Fungal se-

quences from B. nepalensis and B. itoanawere analyzed using a

BLAST search (Altschul et al. 1997) against the NCBI sequence

database (National Center for Biotechnology Information,

GenBank) and MaarjAM database (Opik et al. 2010). Fungal

sequences from other plants in GenBank that were closely

matched to ours were added to the analysis.

Single RFLP patterns for both HinfI andMSpI were obtained

from all root samples of B. nepalensis and SSU sequences from

all the individuals were also identical in the DNA analysiswith

the NS31/AM1 primers (Table 1). In the result of the NS31/

AML2 amplification, all detected Glomeromycota sequences

from B. nepalensis were 99% identical to those amplified using

the NS31/AM1 primers, except two samples included one

fungal sequence with 95 and 98% homology, respectively. In

B. itoana, a single RFLP pattern and identical single fungal

sequence were also obtained from all the root samples with

the NS31/AM1 primers. Furthermore, the sequences from the

NS31/AM1 primers were 99% identical to those from the NS31/

AML2 primers. These results indicate that a dominant fungal

partner of the two Burmannia species is limited within a single


http://www.ddbj.nig.ac.jp



Table 1 e Samples of Burmannia species used in this study and RFLP patterns of the fungal ITS fragments after digestionwithHinfI andMspI. The number of roots for each RFLP patterns is shown. Numerals in parentheses are the number of rootsamples used for sequencing analysis.

Taxa Locality No. ofindividuals

No. ofroots

RFLP Patterns Voucher Collectiondate

Accessionno.

A B

B. nepalensis Kirishima-shi, Kagoshima Prefecture, Japan 5 18 18 (6) 0 e 29 Sep. 2007 AB753043

Tarumizu-shi, Kagoshima Prefecture, Japan 9 18 18 (5) 0 TNS773863 5 Oct. 2007 e

B. itoana Yakushima Island, Kagoshima Prefecture, Japan 3 9 0 9 (4) TNS773862 27 Sep. 2007 AB753044

myc o s c i e n c e x x x ( 2 0 1 3 ) 1e5 3

phylotype, although these species may have some minor

mycorrhizal fungi.

Phylogenetic analysis of fungal SSU sequence showed that

the fungal sequences from the two Burmannia species are

Funneliformis

Funneliformis

Funneliformis

Glomus sp

Glomus m

Juniperus proc

Burmannia

Angiopteris l

Burmannia

Burmannia

Juniperus pro

Dictyostega or

Gymnosiphon

Dictyostega or

Burmannia n

Osumunda ja

Sciaphila tosa

Tacca planta

Uncultured G

Pyrus pyrifolia

Sciaphila led

Rhizophag

Rhizopha

Sclerocystis s

Sclerocy

Claroideoglomus

Claroideoglomus

Claroideoglomus lam

Claroideoglomus lu

Claroideoglomus

Claroideoglomus cla

Claroideoglomus etu

Archaeospora leptoticha (AJ006466)

Geosiphon pyriformis (Y15905)0.005 substitutions/site

100

100

100

100

99

100

76

84

9390

98

87

73

82

78

Fig. 2 e Phylogenetic placement of arbuscular mycorrhizal fung

on small subunit rDNA sequences. The sequences were amplifie

conducted using the neighbor-joining method with 1000 bootst

Classification of Glomeromycota follows Schußler and Walker (


included in Glomeraceae (former Glomus group A) (Fig. 2),

which has been reported as a mycorrhizal partner of Bur-

mannia species such as B. championii, B. cryptopetala (Suetsugu

et al. 2012) and B. hexaptera (Franke et al. 2006). Terashita

mosseae (Z14007)

geosporus (AJ245637)

coronatus (AJ276086)

. W3347 (AJ301857)

acrocarpum (FR772325)

era symbiont (DQ085215)

itoana symbiont (AB753044)

ygodiifolia symbiont (AB594862)

hexaptera symbiont (EU417648)

hexaptera symbiont (DQ371688)

cera symbiont (DQ085210)

obanchoides symbiont (HM440258)

capitatus symbiont (JQ246045)

obanchoides symbiont (HM440261)

epalensis symbiont (AB753043)

ponica symbiont (AB594927)

ensis symbiont (AB556933)

ginea symbiont (EU417645)

lomus (FR693482)

symbiont (AB694992)

ermannii symbiont (EU417640)

us manihotis (Y17638)

gus irregularis (X58725)

inuosa (AJ133706)

stis coremioides (AJ249715)

sp. W3349 (AJ301856)

viscosum (Y17652)

ellosum (AJ276083)

teum (AJ276089)

sp. W3234 (AJ301855)

roideum (AJ276079)

nicatum (Y17639)

Glomeraceae

Claroideo-glomeraceae

i associated with Burmannia nepalensis and B. itoana based

d using the NS31/AM1 primers. Phylogenetic analysis was

rap replicates (values greater than 70% are shown).

2010) and Kruger et al. (2012).




my c o s c i e n c e x x x ( 2 0 1 3 ) 1e54

and Kawakami (1991) observed the root anatomy of B. nepal-

ensis and suggested that this species is associated with Glomus

fungi based on themorphological features of colonized fungal

hyphae and spores. Our results confirm this observation. The

fungal sequence from B. nepalensis shared 99% sequence

identity with those from mycoheterotrophic Sciaphila species

(AB556933), sequences of AM fungi virtual taxon VTX00166

(EU417645 and EU417640: Opik et al. 2010) and other Glomus

fungi (AB594927, FR693482 and AB694992) and these se-

quences formed a monophyletic group with 100% bootstrap

support (Fig. 2). The fungal sequence from B. itoana shared

96e98% sequence identity with those from several Burman-

niaceae species such as Dictyostega orobanchoides (HM440261;

VTX00178 and HM440258), Burmannia hexaptera (EU417648;

VTX00180 and DQ371688; VTX00181) and Gymnosiphon cap-

itatus (JQ246045), and also from other land plant species

(DQ085210; VTX00292, DQ085215; VTX00148, AB594862). These

sequences shared high homology (96e98%) with B. itoana

fungi.

Mycorrhizal specificity of MHPs in association with AM

fungi has been shown to be generally high (Bidartondo et al.

2002; Franke et al. 2006; Merckx and Bidartondo 2008; Yamato

et al. 2011ab), although the specificity level is somewhat vari-

able. Merckx et al. (2012) demonstrated that the specificity of

fungal partners in the mycoheterotrophic Burmanniaceae

varies from one to seven virtual taxa in Glomeraceae. In B.

championii and B. cryptopetala, four and three individuals

harbored eight and three distinct lineages of Glomeraceae,

respectively (Suetsugu et al. 2012). In contrast, only a single

type of funguswas found from otherMHPs associatedwith AM

fungi such as Arachnitis uniflora (Corsiaceae) (Bidartondo et al.

2002), Afrothismia (Thismiaceae) (Merckx and Bidartondo

2008). From all our results, it seems likely that B. nepalensis

and B. itoana are associated with narrow phylogenetic ranges,

possibly single clades of arbuscular mycorrhizal fungi. How-

ever, further work is required to confirm the hypothesis due to

a small sampling scale of this study.

MHPs in association with AM fungi are known to form

mycorrhizae in adjacent photosynthetic plants (Bidartondo

et al. 2002; Yamato et al. 2011b) and MHPs gain carbon from

neighboring autotrophs via AM fungi. Our sampling sites of B.

nepalensis and B. itoana are evergreen broad-leaved forests

dominated with Fagaceae (Castanopsis sieboldii, Quercus salicina

and Q. acuta) and Lauraceae trees (Machilus thunbergii, M.

japonica, Cinnamomum tenuifolium and Neolitsea sericea). The

Lauraceae plants and several Fagaceae species are known to

form arbuscular mycorrhiza (Zhao et al. 2001; Wang and Qiu

2006). The AM fungi from Burmannia species might be simul-

taneously mycorrhizal with neighboring AM plants. It is

crucial to identify autotrophic host plants that supply carbon

to MHPs via AM fungi for establishing conservation strategies

of these threatened Burmannia species.

Acknowledgments

This study was funded by a Grant-in-Aid from the Ministry of

Education, Sports, Culture, Science and Technology of Japan

(nos. 21370038 and 24370040). YO is a Research Fellow of the

Japan Society for the Promotion of Science.


r e f e r e n c e s

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic AcidsResearch 25: 3389e3402.

Bidartondo MI, Redecker D, Hijri I, Wiemken A, Bruns TD,Domınguez L, Sersic A, Leake JR, Read DJ, 2002. Epiparasiticplants specialized on arbuscular mycorrhizal fungi. Nature419: 389e392.

Daniell TJ, Husband R, Young JPW, 2001. Molecular diversity ofarbuscular mycorrhizal fungi colonising arable crops. FEMSMicrobiology Ecology 36: 203e209.

Environmental Agency of Japan, 2000. Threatened wildlife of Japan.Red data book, 2nd edn (in Japanese). Japan Wildlife ResearchCenter, Tokyo.

Felsenstein J, 1985. Confidence limits on phylogenies: anapproach using the bootstrap. Evolution 39: 783e791.

Franke T, Beenken L, Doring M, Kocyan A, Agerer R, 2006.Arbuscular mycorrhizal fungi of the Glomus-group A lineage(Glomerales; Glomeromycota) detected in myco-heterotrophicplants from tropical Africa. Mycological Progress 5: 24e31.

Gardes M, Bruns TD, 1996. ITS-RFLP matching for identification offungi. In: Clapp JP (ed), Species diagnostics protocols: PCR andother nucleic acid methods, vol. 50. Humana Press, Incorporated,Totowa, New Jersey, pp 177e186.

Helgason T, Daniell TJ, Husband R, Fitter AH, Young JP, 1998.Ploughing up the wood-wide web? Nature 394: 431.

Imhof S, 1999. Subterranean structures and mycorrhiza of theachlorophyllous Burmannia tenella (Burmanniaceae). CanadianJournal of Botany 77: 637e643.

Kimura M, 1980. A simple method for estimating evolutionaryrates of base substitutions through comparative studies ofnucleotide sequences. Journal of Molecular Evolution 16:111e120.

Kruger M, Kruger C, Walker C, Stockinger H, Schußler A, 2012.Phylogenetic reference data for systematics andphylotaxonomy of arbuscular mycorrhizal fungi from phylumto species level. New Phytologist 193: 970e984.

Leake JR, 1994. Tansley review no. 69. The biology ofmycoheterotrophic (‘saprophytic’) plants. New Phytologist 127:171e216.

Lee J, Lee S, Young JPW, 2008. Improved PCR primers for thedetection and identification of arbuscular mycorrhizal fungi.FEMS Microbiology Ecology 65: 339e349.

Maddison DR, Maddison WP, 2003. MacClade: analysis of phylogenyand character evolution, version 4.06. Sinauer Associates,Sunderland.

Merckx V, Schols P, Maas-Van de Kamer H, Maas P, Huysmans S,Smets E, 2006. Phylogeny and evolution of Burmanniaceae(Dioscoreales) based on nuclear and mitochondrial data.American Journal of Botany 93: 1684e1698.

MerckxV,BidartondoMI,2008.Breakdownanddelayedcospeciationin the arbuscularmycorrhizalmutualism. Proceedings of the RoyalSociety Series B, Biological Sciences 275: 1029e1035.

Merckx V, Stockel M, Fleischmann A, Bruns TD, Gebauer G, 2010.15N and 13C natural abundance of two mycoheterotrophic anda putative partially mycoheterotrophic species associatedwith arbuscular mycorrhizal fungi. New Phytologist 188:590e596.

Merckx V, Janssens SB, Hynson NA, Specht CD, Bruns TD,Smets EF, 2012. Mycoheterotrophic interactions are notlimited to a narrow phylogenetic range of arbuscularmycorrhizal fungi. Molecular Ecology 21: 1524e1532.

Opik M, Vanatoa A, Vanatoa E, Moora M, Davison J, Kalwij JM,Reier U, Zobel M, 2010. The online database MaarjAM revealsglobal and ecosystemic distribution patterns in arbuscular




myc o s c i e n c e x x x ( 2 0 1 3 ) 1e5 5

mycorrhizal fungi (Glomeromycota). New Phytologist 188:223e241.

Saitou N, Nei M, 1987. The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. MolecularBiology and Evolution 4: 406e425.

Schußler A, Walker C, 2010. The Glomeromycota: a species list withnew families and genera. The Royal Botanic Garden/BotanischeStaatssammlung Munich/Oregon State University, Edinburgh& Kew, UK/Munich, Germany/Oregon, USA. http://www.amf-phylogeny.com.

Schwarzott D, Walker C, Schußler A, 2001. Glomus, the largestgenus of the arbuscular mycorrhizal fungi (Glomales), isnonmonophyletic. Molecular Phylogenetics and Evolution 21:190e197.

Simon L, Lalonde M, Bruns TD, 1992. Specific amplification of 18Sfungal ribosomal genes from vesicularearbuscularendomycorrhizal fungi colonizing roots. Applied andEnvironmental Microbiology 58: 291e295.

Suetsugu K, Kawakita A, Kato M, 2012. Evidence for specificity toGlomus group Ab in two Asian mycoheterotrophic Burmanniaspecies. Plant Species Biology. http://dx.doi.org/10.1111/j.1442-1984.2012.00387.x.

Swofford DL, 2001. PAUP*: phylogenetic analysis using parsimony(*and other methods), version 4.0b10. Sinauer, Sunderland,Massachusetts.


Terashita T, Kawakami Y, 1991. An endomycorrhizal fungus ofBurmannia liukiuensis. Transactions of the Mycological Society ofJapan 32: 207e215.

van der Pijl L, 1934. Die mycorrhiza von Burmannia undEpirrhizanthes und die fortpflanzung ihres endophyten. Recueildes Travaux Botaniques Neerlandais 31: 761e779.

Wang B, Qiu YL, 2006. Phylogenetic distribution and evolution ofmycorrhizas in land plants. Mycorrhiza 16: 299e363.

Wu D, Zhang D, Saunders RMK, 2010. Burmanniaceae. In: Wu ZY,Raven PH, Hong DY (eds), Flora of China, vol. 23. Science Press,Beijing, pp 121e124.

Yamato M, Yagame T, Iwase K, 2011a. Arbuscular mycorrhizalfungi in roots of non-photosynthetic plants, Sciaphilajaponica and Sciaphila tosaensis (Triuridaceae). Mycoscience52: 217e223.

Yamato M, Yagame T, Shimomura N, Iwase K, Takahashi H,Ogura-Tsujita Y, Yukawa T, 2011b. Specific arbuscularmycorrhizal fungi associated with non-photosyntheticPetrosavia sakuraii (Petrosaviaceae). Mycorrhiza 21:631e639.

Zhao ZW, Xia YM, Qin XZ, Li XW, Cheng LZ, Sha T, Wang GH,2001. Arbuscular mycorrhizal status of plants and the sporedensity of arbuscular mycorrhizal fungi in the tropical rainforest of Xishuangbanna, southwest China. Mycorrhiza 11:159e162.


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High mycorrhizal specificity in the mycoheterotrophic Burmannia nepalensis and B. itoana (Burmanniaceae)