EFTA Document EFTA01620819

New Phytologist Letters Fungal associations of basal vascular plants: reopening a closed book? Introduction The widely held hypothesis that Glomeromycota fungi alone formed the ancestral land plant—fungus symbiosis (Pirozynski & Da!pi, 1989; Selosse & Le Tacon, 1998; Wang & Qiu, 2006; Parniske, 2008) has recently been challenged by new lines of evidence from molecular, cytological, functional and palaeonto- logical studies. First, liverworts of the earliest divergent Glade, the Haplomitriopsida, form a mutualistic mycorrhiza-like relation- ship, whereby there is reciprocal exchange of plant carbon (C) for fungal nitrogen (N) and phosphorus (P), with members of the Mucoromycotina (Bidartondo et al, 2011; Field et 2014), a fungal lineage considered basal or sister to the Glomeromycota (James etal., 2006; Lin nal, 2014). Second, other basal plants, including complex and simple thalloid liverworts and homworts, enter into associations with both Mucoromycotina and Glomer- omycota fungi, sometimes simultaneously (Bidartondo et at, 2011; Desire, eta!, 2013). Third, dual partnerships involving fungi with affinities to Glomeromycota and Mucoromycotina have been reported in fossils of early vascular plants from the Devonian (Strullu-Derrien et al, 2014). Turning to the fungal associations of the extant representatives of the early diverging vascular plant lineages, the glomeromycete identity of fungi in ferns (Monilophyta) has never been questioned —a consensus borne out by cytology and limited DNA sequencing data (Wang & Qiu, 2006; Ogura-Tsujita etal., 2013). By contrast, the unusual cytology of fungal colonization in lycopods (Lycopodiophyra), highly reminiscent of the cytology reported in the Haplomitriopsida genus Treubia (Duckett et al, 2006), suggested unique fungal partnerships or ilycopodioid mycothallus interac- tions' (Duckett & Ligrone, 1992; Schmid & Oberwinlder, 1993) until a molecular study detected Glomeromycota in this group (Winther & Friedman, 2008), thus 'laying to rest over a century of speculations and uncertainty' surrounding their identity (Leake et al, 2008). However, Windier & Friedman's study, and a more recent investigation proposing a basidiomycete as the main symbiont in a member of the Lycopodiaceae (Horn eta!, 2013; but see rebuttal in Strullu-Derrien eta& 2014 criticizing their limited molecular and microscopical data), used methods that do not detect Mucoromycotina fungi. Therefore, it remains to be determined whether members of the Mucoromycotina related to the fungi known to enter into mutualism with basal liverworts (Field et al, 2014) also form associations with vascular plants. To test this possibility, we carried out molecular and microscopical analyses of the fungal associations of all the major lineages of lycopods and ferns. Materials and Methods Sampling sites were globally distributed (Supporting Information Table S1). At least one mature plant colony was collected from each site. Plants were processed for cytological and molecular analyses within 1 wk of collection by removing and cleaning roots with forceps and sterile water. Roots were prepared for scanning and transmission electron microscopy as previously described (Pressel et al, 2010; Desire end, 2013). Extraction and sequencing of genomic fungal DNA were performed using the method of Bidartondo et al (2011). In brief, the universal fungal 18S primer combination NS1 (White et 4, 1990) and EF3 (Smit et at, 1999) was used to amplify DNA which was cloned (TOPO TA; Invitrogen) and sequenced using an Applied Biosystems Genetic Analyser 3730 (Waltham, MA, USA). Between four and eight clones were sequenced for each sample and identified using NCBI BLAST (Altschul et al, 1997). Sequence editing and assembly were performed in Geneious v5.6 (http://www.geneious.com). The alignment algorithms of MUSCLE were used within MEGA v5.1 (Tamura et al, 2011), with reference sequences from GenBank. Using UCHIME (Edgar et al, 2011) within MOTHUR (http:// www.mothur.org), confirmed sequences were not chimeric. Evo- lutionary models were tested in MEGA. Bayesian inference was carried out using MrBayes (Huelsenbeck & Ronquist, 2001) and FigTree v1.4 (http://tree.bio.ed.ac.uk) for visualization and edit- ing. Representative DNA sequences have been deposited in GenBank (KJ952212—KJ952241). Results Molecular and cytological analyses showed that both Mucoromy- cotina and Glomeromycota fungi associate with lycopods and ferns (Figs 1, 2). We examined samples from 20 lycopod and 18 fern species, and detected fungi in seven and 13 species, respectively (Table S1). Glomeromycota fungi were present in three lycopod species while Mucoromycotina were found in four. Fungal colonization was detected in only 17 of the 101 lycopod samples analysed. Diverse Mucoromycotina fungi colonized lycopods, sometimes occurring within the same species, and even the same plant, and six new Mucoromycotina clades were discovered (Fig. S2). Colonization rates in ferns were higher (33 out of 58 samples) and showed specificity to Glomeromycota (Fig. SI). Ferns exclusively contained members of the order Glomerales, with the exception of one Ophioglossum (Diversisporales), one Psi/arum (Archaeosporales), one Tmetipteris (Archaeosporales), and three 1394 New Phytologist (2015) 205: 1394-1398 wmv.newphytologisteom g) 2014 The Audio's New !biologist cD 2014 New Phrologist Trust EFTA01620819 Ncv. Phytulo,•kt Letters Forum 1395 0 o 11 Sens osaccharomyces pombe X51866 I C r nus ccmatus A Y665772 1 or sith 8oltychium duntlii 8536 Gloms erancum HM153420 1 with Selagoaria sedaginoides 8530 Uri , r Fonnerforma caledcnium 1r 1 7635.3 with Anogranyna leptophyle WR856-8 olEr with Glecheno mcrophyffe 8963-E ead ,_ t with Ophrogbssum yuktatum 8949.0 Gorrus .-acro:arpu- FR750376 I oat .it_ with Lycopodum cemuum 8964 2-8 WO Tmesiptens Warden 8369 with Ancgramma loptophyNa WR864-A Rr zepriagus .r!fariK:ces Ai30189 I o si V watt Setagmet.tu kraussona WR9I 1-C with Prune purpurascens A139-1 with Lycoredium comuum WR90743 I r with Nephrolopo hrsutute AU98b-4 L— with Xrphoptens ascensonensis Alan 3 inn with Salagmelfe kraussrana WR911,A with PsAatum radon A11.2 with Oplacglossum ythOatum 8956-D I Dwersopora (Ivaco FP686936 1 on I with Anogramnie hyptophytia WR865-8 with Pbsena porpurascens (gamotophyte) A181-2 Archaeospcea serenely, FR773150 1 ayth Psibium nut:turn .412-3 Paraglomus cauttum 4J276081 3 r -- Sphaorccreas pubescent 48755407 I—with Treutaa pygmaea 8356 0414230.1 r with Lycopodrella mwntiala Th6-2 o 9 i_ ,,,,, Rhoeoceros carohnsanus 8445 3 KC708396 1 with Anogramme itiptophylle WR86543 - o ' I I f —with Lycopodmille inundate ThEF4 with Phaeoceros carohnianus 8873 4 KC 708428 1 with Lyoopodiette inundate 77•86-4 .yeth Nothott3f0S vineenhanus 8843 6 KC 708395 I cycepodreile inundate Th8b-3 L WW1 LyttpotheAla inundate 8528 with Anogramma leplophyab JD92 Endogone podorrms 10322628 Endogone intrastate 90444 r Endorne fectifius MA59900-A I Endogone /Num.:corona $414206 r vath Lyvopodium ennotinurn WR266-C I- Froth Lycoporilum fastiglatum WR148 843 YAM PRasomEetatos coataceos 8797 4 KC708415 I Mortrerela mutdevancata AF157144 004 eloaAwoJawoo euRopAumioDniry Fig. 1 Representative fungal associates of basal vascular plants in a Bayesian full 18S nrDNA analysis. Both lycopods and ferns harbou rdivene Mucoromycotina and Glomeromycota fungi. Reference sequences from GenBan k are highlightedin grey.Analysiswasperformed using an HKY85 model (nit = 2)and invgamma rates. Four heated chains were run simultaneously with a chain length of 1.1 x 106. Anogramme (Mucoromycotina and Diversisporales) specimens; Anogramme was the only fern genus harbouring Mucoromycotina fungi. All samples analysed were sporophytes, with the exception of one fern gametophyte (Presents sp.), which contained Gigaspora- ceae fungi. This investigation added two new samples to the still limited database of Endogone fruiting body DNA sequences (including the first E. incresram) and supported the placement of Spharrortrar pubes-nu (Hirose etal., 2014) in Mucoromycotina Group L (ram Desire et al, 2013). The cytology of fern—fungal associations hitherto undescribed is illustrated in Fig. 2. In Anogramma colonized by Mucoromycotina (Fig. 22,6), the exclusively intracellular fungus produces large hyphae, finer short-lived coils and vesicles (Fig. 2b). Fungal structures are surrounded by host plasma membrane and healthy host cytoplasm packed with mitochondria (Fig. 2a). Fungal associations in both the roots and gametophytes of Anima (Fig. 2c—g) comprise structures typical of Glomeromycota coloni- zation, including arbuscules, large vesicles and hyphal coils, which are intimately associated with the plant cell wall. Discussion This study demonstrates for the first time that the extant representatives of the earliest diverging clades of vascular plants, lycopods and ferns, form associations with both Mucoromycotina and Glomeromycota fungi. Lycopod sporophytes rely on a variety of strategies, entering into partnership with either Glomeromycota or Mucoromycotina, both or often neither. By contrast, all the ferns SD 2014 The Audio's New PhytologistO 2014 New Phytologist Titorc New Phoyfoght (2015) 205: 1394-1358 www.newphytologistcom EFTA01620820 1396 Forum Liners New Phytologist sampled associated exclusively with Glomeromycota, with the exception of the derived gen US Anogramma where dual partnerships were detected. Our discovery finally provides an explanation for the unusual colonization patterns reported before in some lycopods (Duckett & Ligrone, 1992; Schmid & Obenvinkler, 1993), consisting of an intracellular phase and extensive fungal prolifer- ation in gametophytic mucilage-filled intercellular spaces, as also reported in other Mucoromycotina-associated groups: the Haplo- miotriopsida liverwort genus Treubia (Duckett a al., 2006), several hornwort genera (Desire eta!, 2013), and the Devonian fossil plant Horneoplryton hstneri (Strullu-Derrien etnl., 2014). We hypothesize that the associations between Mucoromycotina fungi and vascular plants are mutualistic. Beyond microscopy, our main line of evidence is the recent demonstration of mutualism between Haplomitriopsida liverworts and Mucoromycotina fungi (Field a al., 2014) closely related to those now detected in vascular plants. Our observations demonstrate that intercellular fungal prolif- eration is a signature of Mucoromycotina colonization, and lend further support to the hypothesis that the early Devonian vascular plant Nothia, which also harboured inter- and intracellular fungi Fig. 2 Fungal colonization in ferns. (a, b) Transmission electron micrographs of Anogramma leptophyna colonized by Mucoromycotina fungi. Fungal colonization is largely confined to a zone where the tubers join the main root system and the lipid-filled tubers, as in hornworts and liverworts, are fungus-free. (a) Early stage in fungal colonization showing living farrowed) and collapsed (9 hyphae surrounded by healthy host cytoplasm packed with mitochondria (M). (b) Later stage of colonization showing a large hypha, clusters of collapsed short-lived hyphae and a vesicle farrowed). (c-g) Scanning electron micrographs of Ptisana purpurascern colonized by Glomeromycota fungi. (c) Fungal structures (indicated by arrows) in root inner cortex cells packed with amyloplasts. (d) Large vesicle and fine hyphal coil. (e) Hyphae tightly appressed to the inner walls of colonized cells (indicated by arrows). (f) Arbuscules. (g) Fungal entry is via the root hairs (indicated by arrows). Bars: (O 50 pm; (d-g) 20 pm. (Berbee & Taylor, 2007; Krings ttat, 2007a,b), formed associa- tions with Mucoromycotina fungi (Pressel etal., 2010; Strullu- Derrien et al, 2014). Nonetheless, where the fungi are exclusively intracellular (e.g. Anogramma), it is impossible to ascertain from cytology alone to which fungal group they belong, as both Glomeromycota and Mucoromycotina produce vesicles and hyphal coils. The short-lived fungal swellings or lumps typical of Mucoromycotina colonization in the Haplomitriopsida (Carafa et al, 2003; Duckett et al, 2006) are unique to this group, the only land plant lineage to date known to associate exclusively with Mucoromycotina fungi (Field cal, 2014). Arbuscules, the signature of Glomeromycota colonization in angiosperms, are produced in some lycopod and fern—Glomeromycota associations (e.g. Ptisana, Angioptens, °mune& — Ogura-Tsujita et A, 2013) but are lacking in others (see Strullu-Derrien cal, 2014 and references therein), as is also often the case in liverworts and hornworts. The presence of Glomeromycota and Mucoromycotina fungi in lycopods and the predominance of Glomeromycota in the later diverging ferns fit the phylogenetic distribution of these fungi in New Ph )tologht (2015) 205: 1394-1398 www.newphytologist.com X) 2014 Tht Authors New Phywlogist €.> 2014 New Phytologist Trust EFTA01620821 New Phytologist Lams Forum 1397 other 'lower land plant groups. As such, dual partnerships are the norm in basal thalloid liverworts, while more derived clades have, like ferns, the specificity to Glomeromycota typical of later vascular plant lineages (Smith & Read, 2008). Together with the occurrence of multiple fungal associations in Devonian plants (Strullu-Derrien mat, 2014), this lends further weight to the notion of shifting symbiotic encounters between early land colonists and soil- dwelling fungi before the Glomeromycota became dominant. The presence of Mucoromycotina in Anogramma may be a recent reacquisition, on a par with Endogone forming ectomycorrhizas with pines (Walker, 1985), and probably relates to its unique life cycle among ferns — comprising short-lived sporophytes and aestivating tubers (Goebel, 1905). It is also possible that associa- tions with Mucoromycotina in lycopods and other plants represent recent acquisitions. However, this seems unlikely, given that the genes required for mycorrhiza formation in angiosperms are highly conserved across major plant lineages and that mycorrhizal genes from Mucoromycotina-associated Haplomitriopsida liverworts recovered the Glomeromycota mycorrhizal phenotype in a trans- formed mutant of the angiosperm Medicago minnow& (Wang mat, 2010). These finding', coupled with the occurrence of Mucoromycotina in extant basal groups of both nonvascular and vascular plants. as well as fossil plants (Strullu-Derrien et 2014), indicate that associations between Mucoromycotina and land plants are extremely ancient. During this investigation, we examined sporophytes only and it would be desirable now to study the cryptic nonphotosynthetic gametophytes of a range of lycopods and ferns, which are expected to be more heavily and consistently colonized by fungi (Read eta!, 2000; Ogura-Tsujita et al, 2013). Nevertheless, our discovery that lycopods enter into partnerships with both Mucoromycotina and Glomeromycota fungi opens a new chapter in understanding the origins and evolution of fungal symbioses in vascular plants. Functional studies into the nature of these associations, like those conducted by Field etal. (2014) on Haplomitriopsida—Mucoro- mycotina symbioses, are now needed. Acknowledgements M.i.B. and S.P. thank NERC for grants NEJI027193/1 and NE/ I025360/1. J.G.D. thanks the Leverhulme Trust for an Emeritus Fellowship. A Darwin Initiative Grant enabled S.P. and J.G.D. to collect fungal samples from Ascension Island. We thank Jim Trappe (Oregon State University) and Maria Martin (Royal Botanic Garden of Madrid) for fungal fruiting bodies, and Tatiana Solovieva (supported by the Society for Biology and Imperial College Undergraduate Research Opportunities Programme) for analysing Ascension Island samples. Our thanks go to the Editor and three anonymous referees for their comments. William R. Rim ington 11213*, Silvia Breese"), Jeffrey G. Duckett3 and Martin I. Bidartondola 'Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK; 2Jodrell Laboratory. Royal Botanic Gardens, ICew,TW9 3DS, UK; 3 Department of Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD, UK ("Author for correspondence: tel +44 (0)20 8332 5379; email [email protected]) References Altschul SF. Madden TL.SchafferAA, ZhangJH, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Met Renard, 25: 3389-3402. Barbee ML Taylor JW. 2007. Rhynie chert: a window into a lost world of complex plant-fungus interactions. New Phytologat 174: 475-479. Bidartondo MI. Read DJ. Trappe JM, Mercies V. Ligrone R. Duckett JG. 2011. The dawn of symbiosis between plants and fungi. Biology Letters 7: 574-577. Carats A. Duckett JG. Ligrone R. 2003. Subterranean gametophytic axes in the primitive liverwort Haplominium harbour a unique type of endophytic association with aseptare fungi. New Plytobgist 160: 185-197. Desire A, Duckett JG, Prersel S. Villarreal JC, Bidanondo MI. 2013. Fungal symbioses in hornworts: a chequered history. Proceeding, oily Rope! Swirly Series B-Biologintl.Seienen 280: 20130207. Duckett JG, Carafa A. Ligrone R. 2006. A highly differentiated glomeromnotean association with the mucilage-secreting. primitive antipodean liverwort Trembia (Treubiacen): clues to the origins of mycorrhizas. Americanfiturnai/Bozatur93: 797-813. Duckett JG, Ligrone FL 1992. A light and electron-microscope study of the fungal endophytes in the sporophyte and gametophyte of Lyeoporlisent ant:tune with observations on the gametophyte—sporophyce junction. Canadian Journal of Botany70: 58-72. Edgar RC. Haas BJ.Clemente JC, Quince C. Knight R. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatia 27: 2194-2200. Field KJ. Rimington WEL Bidartondo MI. Munson KE, Seeding DJ, Cameron DD. Duckett JG, Leaks JR. PresselS.2014. First evidence of muntalism between ancient plant lineages (Haplomitriopsida liverworts) and Mucoromycotina fungi and its response to simulated Palaeozoic changes in atmospheric CO2. New Plrologin 205: 743-756. Goebel K. 1905. aganografrby of plant, Pan IL Translated I. B. Balfour. Oxford. UK: Clarendon Press. Hirose D. Degawa Y, Yamamoto K, Yamada A. 2014. Sphaentereas pubnewu is a member of the Mucoromycotina closely related to fungi associated with liverworts and hornworts. *continue55: 221-226. Horn K. Franke T. Untersehet M, Schnieder M. Deaden. L 2013. Morphological and molecular analyses of fungal endophytes of achlorophyllous gametophytes of Diphasiastrumalpinum (Lycopodiaceae). Anwrintn Journal of Botany WO: 2158- 2174. HuelsenbeckJP.Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinfonnatin 17:754-755. James TY. Kauff F. Schoch CL Matheny PB. Hofiteuer V. Cox CJ, Celio G, Gueidan C, Faker E, Miadlikowska J et al. 2006. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature 443: 818-822. Krthp M. Taylor Tit Han H. Kerp H. Dottier N. Herman EJ. 2007a. An alternative mode of early land plant colonization by putative endomycorrhial fungi. Plant Signaling & Behavior 2: 125-126. Krinp M. Taylor TN. Han H. Keep H. Dottier N. Hermon EJ. 20076. Fungal endophytes in a 400-million-yr-old land plant: infection pathways. spatial distribution, and host responses. New Apologist 174:648-657. Leak. J Ft. Cameron DD, Beetling DJ. 2008. Fungal fidelity in the myco-Imerotroph-to-autotroph life cycle of Lycopodiaceae a case of parental nurture? New Phytologist 177: 572-576. Lin K. Umpens E, Zhang ZH, Nam S, Saunders DGO, Mu DS. Pang EL Cao HF, Ora HH, Lin T et AL 2014. Single nucleus gnome sequencing reveals high similarity among nuclei of an endomyconhiral fungus. PLoS Gerwtia 10: e1004078. Ogura-Tsujita Y. Sake& A. Ebihara A. Yukawa T. lmaichi R. 2013. Arboscular mycorrhiza formation in cordate gametophytes of two ferns. Angiopteris lygoaSif olio and Onnumia japonica. Journal of Plant Research 126: 41-50. 8i 2014 The Authors Nam Apologist (2015) 205: 1394-1398 New Phytologist €3 2014 New Phytologist Trust www.newphytologist.com EFTA01620822 1398 Forum Letters New Phytologist Pamiske NI. 2008. Arbuscular mycorthira: the mother of plant root endosymbioses. Nature Perinea Mierobielagy6: 763-775. Pircaynski KA. Dalpi Y. 1989. Geological history of the Glomaceae with particular reference to mycorrhisal symbiosis. Symbiosis 7: 1-36. Praise] S. Bidartondo Mi. Ligrone R. Duckett JG. 2010. Fungal symbioses in bryophytes: new insights in the Twenty First Century. Plgemaxa 9: 238-253. Read DJ. Duckett JG. Francis R, Ligrone R. Russell A. 2000. Symbiotic fungal associations in lower' land plants. Philosophical Transactions of the Royal Society of London. Serin 8-Biological Sciences 355: 815430. Schmid E, Oberwinkler F. 1993. Myer:whin-like interaction between the achlorophyllous gametophyte of Lyropodiron clamant L. and its lunged endophyte studied by light and eleaton-microscopy. New Phyrolegist 124: 69-81. Sdosse MA. Le Taeon F. 1998. The land flora: a phototroph-fungus partnership? Trends in Ecology and Errohetion 13: 15-20. Smit E. Leeflang P. Glandorf B. van Ebas JD, Weman K. 1999. Analysis of fungal diversity in the wheat rhisosphete by sequencing of cloned PC:R-amplificd genes encoding 185 rRNA and temperature gradient gel electrophoresis. Applied and Enriremmental slcicrobielogy65: 2614-2621. Smith SE. Read DJ. 2008. Myron-hind gm/Pink Cambridge. UK: Academic Press. StrulltrDerrien C. Kenrick P. Pressel S, Duckett JG, Ftioult J-P.Strunu D.C. 2014. Fungal associations in Harneophyran hymn from the Rhynie Chest (e. 407 million year old) closely resemble those in extant lower land plants: novel insights into ancestral plant—fungus symbioses. New Phyrologit 203: 964-979. Tamun K, Peterson D. Peterson N. &ether G, Nei M. Kumar S. 2011. MEGAS: Molecular evolutionary genetics analysis using maximum likelihood. evolutionary distance, and maximum parsimony methods. Molecular Biology and Eve anion 28: 2731-2739. Walker C 1985. Enekoneletaillua forming ectomycorrhizas with Phew canton& Transaction, *film British AfyrologiralSeeiety84: 353-355. Wang B, Qiu YL 2006. Phylogenetic distribution and evolution of mycorthirm in land plants. Myeerrhiza 16: 299-363. Wang B, Yeun LH. Xue JY. Liu Y, Ane JM. Qiu YL 2010. Presence of three mymrrhizal genes in the common ancestor of land plants suggests a key role of myconhiras in the colonisation of land by plants. New Phytologist 186: 514-525. White T, Bruns T, Lee S. Taylor). 1990. Amplification and direct sequencing of fungal ribosomal RNAgenes for phylogenetia. In:Innis M.Gelfand D.Sninskyl. White T. eds. PCR protocols: a guide to method and applications Orlando. FL USA: Anaemic Press. 315-322. Windier JL, Friedman WE. 2008. Arbuscular mycorthiad associations in Lycopodiacrae. New Physologia 177: 790-801. Supporting Information Additional supporting information may be found in the online version of this article. Fig. Si Glomeromycota associates of basal vascular plants in a Bayesian full 18S nrDNA analysis. Fig. S2 Mucoromycotina associates of basal vascular plants in a Bayesian full I 85 nrDNA analysis. Table SI Lycopod, fern and fungal fruiting body samples analysed with their origin and fungi detected Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Key words: ferns. Glomeromycota. lycopods. Mucoromycotina. myconhin. • lib CI About New Phytologist • New Phytologist is an electronic (online-only) journal owned by the New Phytologist Trust, a not-for-profit organization dedicated. to the promotion of plant science, facilitating projects from symposia to free access for our Tansley reviews. • Regular papers, Letters, Research reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication 'as ready' via Early View- our average time to decision Is <26 days. There are no page or colour charges and a PDF version will be provided for each article. • The journal is available online at Wiley Online Library. Visit www.newphytologist.com to search the articles and register for table of contents email alerts. • If you have any questions, do get in touch with Central Office ([email protected]) or, if it is more convenient, our USA Office (np-usaoffice@lancasteracuk) • For submission instructions, subscription and all the latest information visit www.nitsvphytologist.com New ',geologist (2015) 205: 1394-1398 www.newphytologistcom ID 2014 The Authors New PlgtologisttE5 2014 New Phytologist Trust EFTA01620823