<i>Stigmatodactylus sikokianus</i> (Orchidaceae) mainly acquires carbon from decaying litter through association with a specific clade of Serendipitaceae

Mycorrhizas usually engage in mutualism, which soil fungi contribute minerals to plants that turn supply photosynthetically fixed carbon (C) their fungal partners (Smith & Read, 2008). However, the functional status of such symbioses orchids remains somewhat controversial. Although all depend on symbiotic for essential nutrients (including C) during initial development (Dearnaley et al., 2016), most orchid species exhibit some level autotrophy at adult stage. As such, researchers have considered green are released from dependency heterotrophy C gain stage (e.g. Alexander Hadley, 1985). Indeed, radiocarbon tracer experiments demonstrated Goodyera repens engages a mutualistic symbiosis with its partner Ceratobasidium cornigerum (Cameron 2006, 2008), thereby indicating mycorrhizas can function as typical whose net flow is plant fungus. many obtain both photosynthesis and (mixotrophy or partial mycoheterotrophy) (Gebauer Meyer, 2003; Bidartondo 2004; Selosse 2004). Stable isotope analysis has identified fully mycoheterotrophic several highly enriched 13C 15N, reflecting incorporation fungi-derived organic nitrogen (N) symbionts Intriguingly, although mixotrophic either ectomycorrhizal (ECM) saprotrophic nonrhizoctonia (Merckx, 2013; Suetsugu 2019, 2021a), associated nonECM rhizoctonias, including Ceratobasidiaceae, Tulasnellaceae Serendipitaceae 2013). Therefore, rhizoctonias might not support demands exhibiting high degree mycoheterotrophy. recent studies also suggested mixotrophy may occur rhizoctonia-associated (Selosse Martos, 2014; Gebauer 2016; fungus still debated (Lallemand 2017; Jacquemyn 2021). Current evidence suggests less suited meet large demand than ECM (Schweiger 2018). Several reported shifts fungi, often precede evolution full mycoheterotrophy (Bidartondo given activities correlated temperature moisture (Martos 2009), least rhizoctonia 2014), warm conditions enable orchids. We note pure saprotrophs. The nutritional mode difficult understand detail because do produce conspicuous fruiting bodies be sampled isotopic 2014); furthermore, transition saprotrophy endophytism evolved multiple times (Veldre Weiß 2016). Consequently, it controversial whether otherwise detected roots endophytic 2014). In this respect, transit time (i.e. final utilization by plants, analogous diet age animals) estimated measuring concentration 14C derived nuclear bomb tests 1950s 1960s (Hyodo 2006; Hatté 2020; 2020). approach will help investigate levels litter-decaying endophytic, association receiving current-year photosynthates must result younger ages. study, we focused Stigmatodactylus sikokianus distributed temperate regions (Fig. 1). genus sometimes been classified (Furman Trappe, 1971; Campbell, retains chlorophyll stems small leaves. Based vitro isolation, mycobionts S. members Serendipitaceae, group (Yagame Yamato, Because grows decaying litter Cryptomeria japonica plantations without any adjacent trees, through an Serendipitaceae. Accordingly, investigated physiological ecology improve our understanding Specifically, were using high-throughput DNA sequencing, while stable (13C 15N) was used confirm sikokianus. addition, (Δ14C) test mainly litter-decaying, consequently, acquires 14C-enriched litter. All samples collected Kochi Prefecture, Japan 2 September 2017 (see Supporting Information Methods S1 detailed methodology). metabarcoding technique, community profiling revealed exclusively six operational taxonomic units (OTUs) belonging (Weiß OTUs (56 841 sequencing reads total) individuals. selected primers primarily designed Basidiomycota. they suitable studying diversity (Taylor McCormick, 2008; Waud possible other ascomycetous endophytes amplified due primer bias. present study belonged phylogenetic conducted representative Sebacinales sequences. Maximum-likelihood trees showed formed supported clade previously These Serendipita vermifera complex 2; Deshmukh Yagame known comprise orchids, Acianthus, Caladenia, Elythranthera, Eriochilus, Glossodia, Microtis, Pheladenia Prasophyllum (Deshmukh 2006). Along Stigmatodactylus, these belong tribe Diurideae, suggesting associations acquired secondarily instead partnerships common ancestors. It cannot provide sufficient resources mycoheterotrophy, whereas Sebacinaceae regularly amounts By contrast, results indicate sole prerequisite increasing (shown following two sections). Despite photosynthetic capability sikokianus, 13C. δ13C values (−23.3 ± 0.7‰; n = 6) significantly higher those only autotrophic reference (−33.8 0.8‰; 8; P < 0.001; Fig. 3; Table S1) but entangled rhizomes (−28.4 0.9‰; 6; 0.001). Notably, enrichment factor (10.6 1.4‰, slightly mean exploit (8.2 1.3‰, 94) (8.7 1.1‰, 24) 2009; Ogura-Tsujita Hynson Lee 2015; 2020), corroborating hypothesis nutrition associations. Such strong ability could compensate low production reduced leaf size inhabiting deeply shaded forest understoreys. contrast enrichment, 15N (2.5 0.6‰, 5) much lower (11.6 3.1‰, (Hynson 2013) similar (4.8 1.5‰, 42) relatively probably reflects mycobionts, generally 15N-enriched (Mayor 2009). patterns suggest nearly obtains uncertainties remained limitations approach, differences identities metabolism 2018; too provided depleted (Halbwachs 2013); therefore, responsible absent observed factors albino mutants so-called velutina (8.8 2019) Cypripedium debile (9.8 0.5‰), exploiting fungi. behave more like Litter-decaying well up (nearly) under favorable hot wet conditions. insufficient growth. Since Δ14C atmospheric CO2 globally elevated weapons testing early declined after ban treaty 1963 (Burchuladze, 1989), matter (such tissues) (Hatté al. (2020) wood-decaying (10- 40-yr-old longer mycoheterotrophs (0- 1.4-yr-old Both (23.8 4.6‰; 0.001) (27.0 7.7‰; exhibited Neolitsea sericea (10.0 2.9‰; 4) Pinellia ternata (10.9 4.2‰; 4), had assimilated bomb-derived C. between did differ significantly. Given 10 yr (Hobbie 2002), surrounding produced peak (c. 1964) 1989). 4.0 0.8 4.5 1.5 before sampling period, respectively. consistent feeders/consumers, 2002; Hyodo 2015). spp. directly assessed, owing microscopic habits atmosphere strongly rather latter would should noted provides Owing presence leaves, via diluted there no significant difference ∆14C litter, quantity likely negligible. Even though replacing greater supplies Roy, documents exciting exception. approaches included molecular barcoding, measurements, analysis) exploitation rhizoctonias. findings contradict previous notions always capable meeting Dearnaley 2020, 2021b). Our levels, especially tropical acquire partners, even contributes deeper interactions illustrates potential importance re-entry into thank Drs Erik Hobbie, Marc-André Selosse, Jun Matsubayashi anonymous reviewers constructive comments manuscript. Hisanori Takeuchi fieldwork Makoto Taniguchi, Takenori Yamamoto, Takako Shizuka, Takuto Shitara Michiko Ishida technical assistance. Editage (www.editage.com) English language editing. This work financially Mitsubishi Foundation (KS) JSPS KAKENHI (grant nos. 17H05016 16H02524 (IT). Joint Research Grant Environmental Isotope Study Institute Humanity Nature. KS planned research, materials wrote draft. KS, TFH HO laboratory carried out analyses. IT supervised experiments. TFH, revised manuscript approved version sequence data deposited Sequence Read Archive Data Bank (accession no. DRA011572). Further information found online section. natural abundance 13C, N concentrations each sample neighboring plants. Please note: Wiley Blackwell content functionality supplied authors. Any queries (other missing material) directed New Phytologist Central Office. publisher supporting content) corresponding author article.