Variation in mycorrhizal communities and the level of mycoheterotrophy in grassland and Forest populations of <i>Neottia ovata</i> (Orchidaceae)

Read the free Plain Language Summary for this article on Journal blog. Orchid mycorrhiza is exclusively formed in highly diversified plant family Orchidaceae and represents a unique study system to address fundamental questions about evolutionary trajectory of trophic mode mycorrhizal associations (Jacquemyn & Merckx, 2019; Rasmussen et al., 2015; van der Heijden 2015). All orchid species are mycoheterotrophic at early stages development when their minute seeds achlorophyllous protocorms have an obligate demand fungal-derived C (Dearnaley 2016; 2013; Rasmussen, 1995). Although most adult orchids assumed become autotrophic (being capable performing photosynthesis), fully or partially continue depend throughout entire life cycle combination with photosynthesis, respectively (Merckx, Selosse Roy, 2009; Tĕšitel 2018). Partially usually show intermediate enrichment stable isotopes (13C, 15N 2H) between (Gebauer Gebauer Meyer, 2003; Hynson Jacquemyn 2021). Recent measurements 2H frequently used 13C isotope abundance suggested that partial mycoheterotrophy may be much more widespread among than previously acknowledged Schweiger, 2021; Schiebold Mycoheterotrophy has been hypothesized adaptation survival low-light habitats (Gomes, Bodegom, 2009) degree can variable light availability different (Bidartondo 2004; Preiss 2010; Schweiger 2019). Mycoheterotrophic often rely fungi compared relatives, suggesting shift from autotrophy accompanied by fungal symbionts 2022; Wang However, we still do not know ecological drivers behind these associations. The transition autotrophy, via full mycoheterotrophy, shifts partners, typical rhizoctonia (members three families Ceratobasidiaceae, Tulasnellaceae Serendipitaceae; Dearnaley 2012) non-rhizoctonia ectomycorrhizal wood/litter saprobes phyla Basidiomycota Ascomycota Motomura Ogura-Tsujita 2012, Yagame 2016). For instance, tribe Neottieae, one scarce lineages comprising broad range putatively autotrophic, (Feng Lallemand Zhou Jin, 2018), switch (e.g. Sebacinaceae, Russulaceae, Tuberaceae, Pyronemataceae) Girlanda 2005; Roy 2002; Suetsugu 2017; Těšitelová 2012). Yet, environmental conditions under which increase partner breadth occurs, remain unclear. Neottia ovata chlorophyllous terrestrial sister genus It therefore good investigate variation symbiotic level heterotrophy how they linked conditions. First, within Neottia, switches likely occurred Serendipitaceae Sebacinaceae towards end point (characterized N. nidus-avis) 2015, McKendrick Oja 2016), showing occur order Sebacinales (Weiß Second, despite its inconsistent across 2015), reported significantly enriched 2019), possibly indicating gain fungi. Third, several studies using molecular identification techniques shown mainly associated Serendipitaceae, while occasional were observed as well forest Vogt-Schilb 2020). These results suggest high plasticity increased levels particular (Selosse This associating multiple functional groups potentially places repeatedly (Figure 1). A detailed simultaneous investigation communities ecophysiology over contrasting shed factors influence variables. In study, examined whether specific microhabitats community composition relates changes mycoheterotrophy. Mycorrhizal investigated high-throughput sequencing six sites ranging open grassland shady forests Europe. set 13C, 15N, 18O signatures was measured, microhabitat (light intensity edaphic factors). Specifically, root (hereafter ‘root communities’) differ populations (#H1). We further expected determined soil influenced (#H2). Lastly, (represented signatures) related (#H3). total 30 individuals sampled (forest sites: BE, VP VS; KA, ZI KL) North-East Bavaria (Germany) Netherlands June 2021 (Table Within each site, five 1 m2 plots containing randomly selected soon vegetative parts could identified. plot, sensor (silicon photodiode BPW 21, Infineon, Germany) installed beside targeted individual height approximately 15 cm record irradiance till sample collection (2 weeks month later) data logger (HOBO H8; ONSET, USA). Following calibration procedure (Preiss 2010), received during measuring period calculated (Appendix S1). collected leaf, samples plot budding flowering stage. One two pieces leaf (n = 30) reference plants 90) measurements. Reference always growing close spatial proximity identical respective individuals. individual, least (3–5 cm) chosen DNA extraction microscopic observation pelotons. topsoil cores taken 2.5-cm-diameter auger depth 5 below litter layer mixed homogenized bulk-soil nutrient analysis. preserved cooling box immediately after stored fridge (4°C) laboratory until processing. Root surface sterilized 1% sodium hypochlorite rinsed sterile distilled water. Total sections (1 mm thickness) extracted CTAB method (Doyle, 1987). bulk (0.25 g) DNeasy PowerSoil Pro Kit (QIAGEN). forward primer fITS7 (5′-GTGARTCATCGAATCTTTG-3′; Ihrmark reverse ITS4 (5′-TCCTCCGCTTATTGATATGC-3′; White 1990) amplify nuclear ITS2 region both samples. According our pilot S1; Table S1), pair amplified wider ITS3 ITS4OF specifically targeting (Taylor McCormick, 2008). Fungi forming captured sets primers Since due variability (Li Waud 2014), designed new detect fungi: Tul1F (5′-CGTYGGATCCCTYGGC-3′) Tul2F (5′-TGGATCCCTTGGCACGTC-3′) positioning 5.8S match ITS4Tul2 (5′-TTCTTTTCCTCCGCTGAWTA-3′; 2015) based sequence alignment S2). Thus, general newly pair. PCRs duplicated template reduce PCR stochasticity diversity detected (Alberdi After checking amplification products gel electrophoresis, duplicates amplicons pooled first step attached Nextera indexes (Illumina) second according manufacturer's guidelines. pool sequenced generate 300-bp paired-end reads Illumina MiSeq platform (BaseClear). demultiplexing, paired merged low-quality sequences (error rate >0.5) filtered out VSEARCH (Rognes Merged separated subsequently trimmed CUTADAPT 1.0 (Martin, 2011). Chimeric UCHIME chimera detection program (de novo algorithm) (Edgar quality filtering removal, operational taxonomic units (OTUs) clustered 97% similarity threshold VSEARCH. Global singletons removed because accuracy estimates (Ihrmark 2012; 2014). remaining OTUs assigned identities best USEARCH (Edgar, 2010) Unite dataset (utax_reference_dataset_10.05.2021.fasta, https://unite.ut.ee/repository.php) annotation resources. Finally, manually screened possible summarized literature 2021) previous quality-filtered generated 3342 (1,514,336 sequences) 60 samples, 543 (693,154 sequences—45.8% all putative belonging S3; Appendix Because did yield considerably pair, only retained analyses. material oven-dried 105°C, ground fine powder ball mill (Retsch Schwingmühle MM2) desiccator before abundances C, N, H, O N concentrations. conducted BayCEER Keylab Isotope Biogeochemistry. Relative natural analysis carbon (13C/12C) nitrogen (15N/14N) concentrations simultaneously EA-IRMS coupling combining elemental analyser (EA IsoLink CN; Thermo Fisher Scientific) continuous flow ratio mass spectrometer (delta V advantage; Scientific). hydrogen (2H/1H) oxygen (18O/16O) measured separately TC-IRMS coupling, links thermal conversion through pyrolysis unit (HTO; HEKAtech) Device ConFlo IV open-split interface (Thermo H abundance, four times row measures being neglected analysed target together batches assess potential transpiration effect relative caused differences stomata regulation non-orchid plants. Transpiration affects tissues (Cernusak da Silveira Lobo Sternberg, 1989; Ziegler, 1989). excluded if similarly distributed signature Measured (δ values) following equation: δ13C, δ15N, δ2H δ18O (Rsample/Rstandard − 1) × 1000 [‰], where Rsample Rstandard ratios heavy standard. To enable comparisons sampling sites, (ε) normalize δ values equation ε δS δREF, single value δREF mean same Gebauer, references available S4. rarefied fewest reads. Unless stated otherwise, ‘picante’ r package (Kembel alpha (α-diversity) differed habitats, OTU richness (SR) phylogenetic α-diversity, Faith's (1992) PD. phylogeny reconstructed Maximum Likelihood (see details analyses S1) calculation Analysis variance (ANOVA) Kruskal–Wallis rank-sum tests evaluate SR PD respectively. If null hypothesis rejected, post hoc Tukey HSD Dunn's test performed analyse pairwise differences. (#H1) α-diversity fitting linear models ‘Habitat’ fixed ‘Site’ random ‘lme4’ (Bates structure habitat types (β-diversity), Bray–Curtis (Bray Curtis, 1957) UniFrac dissimilarity metrics (Lozupone Knight, 2005) presence–absence information OTUs. index widely assessing ecology (Anderson 2006), distance considers relatedness fraction distances shared Variation visualized non-metric multidimensional scaling (NMDS) ‘vegan’ (Oksanen homogeneity variances function ‘betadisper’ ‘vegan’, PERMANOVA (Anderson, 2001) ‘adonis2’ sites. nested ‘strata Site’ specified constrain permutations addition, ran indicator identify characteristic given site ‘indval’ ‘labdsv’ (Roberts, Indicator preferentially community, reflects importance (Dufrêne Legendre, 1997). (#H2) soil, (SR PD) regressions. Mantel 10,000 examine Furthermore, corresponding PERMANOVA. recruited roots (MiSeq reads) site. composition, distance-based redundancy (db-RDA) ‘vegan’. Explanatory variables tested db-RDA model organic content (SOC), extractable (including nitrate concentration—NO3− ammonium—NH4+), P, pH moisture properties proportion explained controlling type vs. grassland), covariate. Edaphic fit ‘ordiR2step’ significance assessed ‘anova.test’ explore relationship relatives (N. nidus-avis, camtschatea accuminata), download ITS (Chen NCBI GenBank database. Those downloaded aligned reconstruct maximum likelihood trees RAxML Geneious Prime v. 2019. (KJ188478, KJ188509 KJ188545) al. (2015) also included reconstruction, Serendipitaceace outgroup sequences. compositions (ε13C, ε15N, ε18O, ε2H N) leaves models. ‘plot’ plot-wise scheme. Differences ANOVA tests. Linear effect. ε13C ε2H, individually communities, present regardless S1A,B), presence dependent. Apart members comprised (sites VS), Inocybaceae VS) Thelephoraceae whereas KA ZI) Ceratobasidiaceae KL S1A,B). Besides these, other occasionally <1% including Hymenogastraceae, Cortinariaceae, Pyronemataceae, Pezizaceae, Psathyrellaceae Tricholomataceae preference mapping 2; S5). Each (except species. had (BE—Sebacinaceae; VP—Inocybaceae, VS—Sebacinaceae, Thelephoraceae, Inocybaceae, Atheliaceae Russulaceae), (KA—Tulasnellaceae Ceratobasidiaceae; KL—Ceratobasidiaceae). S6), VS higher 3A,B; S6). Significant found (SR: R2 0.424, p < 0.001; PD: 0.324, P S7). (Bray–Curtis metric: 0.305, pseudo-F 3.164, 0.217, 2.065, 0.001) 3C,D) 0.142, 4.463, 0.179, 5.884, 0.001). Bulk (416 OTUs) (253 Similar varied substantially S1C,D) Tables S6 Soil 0.444, 3.669, 0.473, 4.131, 0.110, 3.321, 0.208, 7.100, There significant positive correlation indices S4). revealed (R 0.504, strong 4A) 0.086, 0.703, 0.866). (pseudo-F 1.721, 0.001, R2adj 0.134) 4B; S8). NO3−, SOC, (RDA1: F 3.767, S9). remained (partial db-RDA, 0.064, 1.6, 0.002). Despite respond 4C). Rhizoctonia fungi, especially those exhibited relatively preferably recruits even though dominant community. Our showed accuminata camtschatea) Conversely, placed basal At BE) 2H, but S10). Notably, negative (BE, S10), might underestimated heterotrophic indicated enrichment. concentration 5; S11). no consistent, difference ε15N (KA (BE VP). VS. (R2 0.436, 0.149, 0.05) such 0.838) 0.137, 0.177) S12). While greatly 0.002, 0.823) 0.014, 0.633) S13). line 2020), predominantly recruiting wide site-dependent 2)—this common rhizoctonia-associated 2022). already tightly support (#H1), grasslands 3; Figure Moreover, correlated 4A; Figures S3 S4) 4B), supporting general, 5), confirming usual trend 2018; S11 S12) S13), rejecting third parallel conceivable (Yagame Such supported associates leafy leafless displaying degrees endpoint 2018) specializes Previous (Těšitelová confirmed current 2). disagreement findings separation nidus-avis closely camtschatae) indicate predisposition opened door stage, broad-scale inferences (Wang predicted ‘waiting room hypothesis’ Ectomycorrhizal tend access resources soil-dwelling explain distinct ectomycorrhizal-dominant Here, would leaves. variations 5). Considering predominant association (Figures 2 3) non-negligible host 4C), speculate contribute little nutritional budget studied take major role external supply hint stage waiting 2022), partners later (as endophytes) probably yet achieved. Light negatively amount compensate reduced photosynthetic exposed (Gonneau 2014; factor Cephalanthera associate Ophrys insectifera (Schweiger agreement reports (Onipchenko 2023; 2019) orchids, Cypripedium calceolus Goodyera repens (Liebel Epipactis palustris (Lallemand Particularly, E. reside genera consisting Neottieae. considered suitable nutrition inconspicuous find intensity. expectation met Several hamper understanding nutrition. include Onipchenko 1989), plants' alternative strategies adaption stoichiometry Van Soudzilovskaia, Detailed elaborations S1. Nonetheless, exact influencing awaits future situ ex manipulated. comprehensive investigations dual supplementary habitats. forests. cannot rule possibility enrichments masked rates individuals, contribution minimal play Vincent S. F. T. Deyi Wang, Gerhard Hans experiment. Franziska Zahn, HVDH contributed selection Jacquemyn, Zahn Johanna Lorenz processed input Sofia I. Gomes. wrote manuscript draft. authors commented approved final version manuscript. work China Scholarship Council (Grant No. 201804910634) Ecology Fund Royal Academy Arts Sciences (KNAWWF/807/19039) DW, Senoir Fellowship 2020 Center International Excellence Alexander von Humboldt VSFTM. like acknowledge Dunea Duin Water (the Netherlands) Regierung Oberfranken Mittelfranken authorization collect thank PhD student Izai Kikuchi Naturalis Biodiversity helping collection. Carina Bauer Petra Eckert (BayCEER Laboratory Biogeochemistry) skilful technical assistance. Master Christina Marburger University Bayreuth helpful discussions declare conflict interest. Additional Supporting Information will online section article. deposited BioProject accession number PRJNA952620 database (https://www.ncbi.nlm.nih.gov/bioproject/). S1: Methodological results. Fungal S2: S3: Alpha soil. S4: regression S5: Phylogenetic relatives. efficiency commonly pairs capturing Sequence list design. Taxonomic assignments Stable plants, type, properties. larger 0.5 P-value. S6: ANOVA, S7: Habitat S8: S9: Results dbRDA model. S10: Model effect, example, lmer (ε13C ~ Category + [1|Plot], BE). S11: S12: (1|Site)). S13: Test Please note: publisher responsible functionality any supplied authors. Any queries (other missing content) should directed author