Botanica Pacifica. A journal of plant science and conservation. 2018. 7(2): 31–39 DOI: 10.17581/bp.2018.07202 Diversity of fungal communities associated with mixotrophic pyroloids (Pyrola rotundifolia, P. media and Orthilia secunda) in their natural habitats Ekaterina­F.­Malysheva1*,­Vera­F.­Malysheva1, Elena­Yu.­Voronina2­&­Alexander­E.­Kovalenko1 Ekaterina­F.­Malysheva1* ABSTRACT e­mail:­[email protected] As it was shown, mixotrophic plants (MxP) strongly depend on their mycorrhi­ Vera­F.­Malysheva­1 zal fun gi for carbon, at least at the early stages of life cycle, and have rather high e­mail:­[email protected] spe­ci­ficity­for­mycobionts.­However,­the­diversity­of ­fungi­associated­with­MxP Elena­Yu.­Voronina­2 and their role in plant’s life are still poorly known, especially under natural condi­ e­mail:­[email protected] tions.­In­the­present­study,­the­diversity­of ­mycobionts­of ­the­three­mixotrophic­ py ro loid species (Pyrola rotundifolia, P. media and Orthilia secunda) was investigated Alexander­E.­Kovalenko­1 by se quencing nrITS­ from­ roots­ and­ rhizomes.­ At­ the­ same­ time,­ we­ studied­ e­mail:­[email protected] ec­to­my­corrhizal­fungal­communities­of ­neighboring­trees.­The­mycobiont­di­ versity slightly differed berween the three species, but they also shared similar 1 fungal­taxa.­The­species­of ­basidiomycete­genera­Tomentella, Piloderma, Russula ­Komarov­Botanical­Institute­ and Mycena­ were­dominant­fungal­partners­of ­the­studied­pyroloids.­The­plants­ of ­the­Russian­Academy­of ­Sciences,­ were also colonized by other ectomycorrhizal and saprotrophic basidiomycetes Saint­Petersburg,­Russia and ­as­co­my­cetes.­The­research­results­showed­MxP link to tree species by shared 2­Lomonosov­Moscow­State­University,­ my­co­bionts­and­partial­mycoheterotrophy­by­involvement­into­mycelial­network.­ Moscow, Russia Thirty­nine­fungal­taxa­(at­species­and­genera­level)­inhabiting­pyroloid­root­sys­ tem ­as­mycobionts­and­root­endophytes­were­detected.­Their­role­for­plant­per­ formance­requires­further­investigation. * corresponding author Keywords: Pyroleae, arbutoid mycorrhiza, mixotrophy, nrITS,­operational­taxonomic­ units, symbionts, endophytes Manuscript­received:­14.05.2018 РЕЗЮМЕ Review­completed:­03.07.2018 Accepted­for­publication:­11.07.2018 Малышева Е.Ф., Малышева В.Ф., Воронина Е.Ю., Коваленко А.Е. Published­online:­13.07.2018 Раз нообразие сообществ грибов, связанных с миксотрофными гру­ шан ко выми (Pyrola rotundifolia, P. media и Orthilia secunda) в их ес тест­ вен ных местообитаниях. Миксотрофные­растения­(MxP) ­сильно­зависят­ в ­углеродном­питании­от­микоризных­грибов,­по­крайней­мере,­на­ранних­ ста­диях­ развития,­ и­ обладают­ высокой­ специфичностью­ к­ ми­ко­бионтам.­ Однако ­еще­довольно­мало­известно­о­разнообразии­грибов,­свя­зан­ных­с­ MxP,­и­их­роли­в­жизни­растений,­особенно­в­естественных­эко­сис­темах.­В­ настоящей­работе­было­исследовано­разнообразие­микобионтов­трех­ви­ дов ­грушанковых­с­миксотрофным­типом­питания­(Pyrola rotundifolia, P. media и­ Orthilia secunda) ­ путем­ выделения­ ДНК­ и­ последующего­ сек­ве­нирования­ nrITS­из­корней­и­корневищ.­Одновременно­изучался­со­с­тав­грибов,­фор­ мирующих­эктомикоризу­с­соседними­деревьями.­Обна­ру­женные­сообще­ ства­ микобионтов­ немного­ отличались­ у­ трех­ видов­ гру­шан­ковых,­ хотя­ были­выявлены­общие­симбионты.­Доминирующими­парт­не­ра­ми­в­арбу­ тоидной­микоризе­изученных­видов­растений­явились­ба­зи­дио­мицеты­из­ родов­Tomentella, Piloderma, Russula­и­Mycena. ­Растения­бы­ли­также­колонизи­ рованы ­другими­эктомикоризными­и­сапротрофны­ми­ба­зи­дио­мицетами­и­ аскомицетами.­Результаты­исследования­показали­связь­MxP­ с­древесными­ растениями­ в­ природе­ благодаря­ наличию­ общих­ ми­ко­бион­тов,­ с­ помо­ щью­ которых­ растения­ вовлекаются­в­ единую­ мице­ли­аль­ную­сеть.­Всего­ было­выявлено­39­грибных­таксонов­(видового­и­ро­до­вого­рангов),­обитаю­ щих­в­корневой­системе­грушанковых­в­качестве­ми­ко­бион­тов­и­корневых­ эндофитов.­Их­роль­в­жизни­растений­требует­даль­нейшего­изучения. Ключевые слова: Pyroleae,­арбутоидная­микориза,­миксотрофия,­nrITS, ­опе­ра­ци­ он­ные­таксономические­единицы,­симбионты,­эндофиты Phylogeny and physiology of pyroloid plants (members presence­ of ­ both­ leafless­ (potentially­ mycoheterotrophic­ of tribe Pyroleae) have attracted great interest from scien­ plants) and transitional to fully autotrophic forms in the tists­for­more­than­200­years­(Hynson­et­al.­2009,­2013).­ genus Pyrola L.­As­a­result­of ­recent­studies­based­on­mo­ One of the problems with the group’s taxonomy is the lecular­data­(Freudenstein­1999, Kron­et­al.­2002,­Liu­et­al.­ ©Botanical Garden-Institute FEB RAS. 2018 31 Malysheva et al. 2010),­pyroloids­have­come­to­be­treated­as­a­separate­tribe­ Such ­associations­have­important­environmental­con­se­quen­ (Pyroleae)­in­the­subfamily­Monotropoideae­of ­Ericaceae.­ ces: pyroloids growing under the forest canopy are provided One of the essential features of these plants is the pro­ with additional source of carbon in the form of ectomycor­ duction of a vast number of dust­like seeds (the phenom­ rhizal woody plants` photosynthetic pro ducts, but for their enon ­of ­mic­ro­spermy)­with­a­minimum­nutrient­supply.­It­ part ­serve­as­a­“depot”­of ­the­mycobiont,­which­is­essential­ is known that this trait (dust­like seeds) is also typical for when the tree layer is restored after dis tur ban ces caused, for many plants, from at least 12 different families including example,­by­clear­cutting­(Smith­&­Read­2008). orchids, and has emerg ed independently in different phy­ Until­the­present,­not­much­efforts­has­been­devoted­to­ logenetic­lines­(Erik­sson­&­Kainulainen­2011).­Because­of ­ studying the phenomenon of mixotrophy in pyroloids as limited carbon re serve such seeds evidently strongly depend compared­to­the­orchids­(Matsuda­et­al.­2012).­The­subject­ on the external carbon sources for their germination, and of study was and remains: a study of seed germination phy­ they­compensate­for­this­deficiency­by­symbiotic­connec­ sio logy; revealing the diversity of fungi forming mycorrhi­ tions­with­fungi. za with plants at different stages of ontogenesis as well as This ­nutritional­mode­was­termed­“initial­mycoheterotro­ the ­specificity­of ­these­symbionts­for­different­species­from­ phy”e ­(Leak ­1994,­Merckx­2013).­It­is­noteworthy­that­all­ Py ro leae; a study of changes in plant trophic strategies un­ species of Pyrola are initially mycoheterotrophic at the early der ­different­environmental­conditions.­Initially­it­was­sup­ stages of ontogeny but upon reaching full maturity they de­ posed,­and­then­there­was­evidence­(Hynson­&­Bruns­2009,­ mon strate practically the complete range of trophic strate­ Hashimoto­et­al.­2012)­that­all­pyroloids­(like­orchids)­are­ gies, from obligate autotrophy to full mycoheterotrophy very ­demanding­for­the­specific­composition­of ­the­ecto­ (e.g. ­achlorophyllous­Pyrola aphylla­Smith)­including­transi­ my co rrhizal fungi, but, unlike other representatives of Mo­ tio­nal ­forms.­The­phenomenon­of ­mixotrophy­(or­partial­ no tro poideae, not throughout the entire life cycle, but only my­co­heterotrophy)­is­inherent­in­such­transitional­forms.­It­ in­the­early­stages­of ­their­development.­However,­the­ac­ con sists in the possibility of simultaneous use of two inde­ cu­mulated­data­is­still­insufficient­to­talk­about­the­direct­ pen dent sources of carbon by adult green plants – one as de pen dence of mixotrophic plant on fungi, even for the a product of photosynthesis, and another coming through most­widespread­species­of ­pyroloids.­There­are­still­a­lot­ a ­mycorrhizal­association­with­fungi­(Tedersoo­et­al.­2007,­ of studies to be done in this direction, and the work presen­ Zim­mer­ et­ al.­ 2007,­ Selosse­ &­ Roy­ 2009,­ Matsuda­ et­ al.­ ted­herein­is­one­of ­them. 2012). ­The­mixotrophic­plants­were­shown­to­demonstrate­ The­present­research­addresses­the­study­of ­mycorrhi­ δ13С­and­δ15N values which are the average between these zal co lo nization of three pyroloid species (Pyrola rotundifolia in di ces typical for fully mycoheterotrophic and autotrophic L.,­P. media­.Sw­and­Orthilia secunda­(L.)­House)­widely­distri­ plants­(Hynson­et­al.­2009).­However,­the­δ13С ­value­in­mi­ bu ted in coniferous and mixed forests of the European part xo trophic pyroloids, as in orchids, may depend on the stage of ­Russia.­Data­on­their­mycorrhizas­are­limited­and­vary­ of development, type of habitat, lighting conditions and con si derably depending on the area of study and the type other ­factors­(Bidartondo­et­al.­2004,­Tedersoo­et­al.­2007,­ of ­plant­communities. Zimmer­et­al.­2007,­Johansson­et­al.­2015).­Moreover,­it­was­ Several­populations­of ­plants­growing­in­different­habi­ observed that the same Pyrola species occurring in different tats ­were­studied.­Identification­of ­fungal­mycobionts­using­ geographic­regions­significantly­vary­in­the­degree­of ­auto­­ mo­lecular­techniques­was­carried­out. and heterotrophy, and the causes of this variability have not yet­been­definitively­elucidated­(Tedersoo­et­al.­2007,­Zim­ MATERIAL AND METHODS mer­et­al.­2007,­Hynson­et­al.­2009). The­tribe­Pyroleae­contains­four­genera­(Pyrola, Orthil­ Study site ia­.,Raf ­Chimaphila Pursh and Moneses­.Salisb ­ex­Gray)­and­ A ­total­of ­67­pyroloid­plants­at­different­developmental­ about­40­species­of ­herbs­and­subshrubs­distributed­in­the­ stages ­from­16­populations­and­more­than­100­mycorrhi­ Northern­ Hemisphere­ that­ usually­