A fine-scale spatial analysis of fungal communities on tropical tree bark unveils the epiphytic rhizosphere in orchids.

TL;DR: In this article, the authors conducted environmental metabarcoding of the ITS-2 region to understand the spatial structure of fungal communities of the bark of tropical trees, with a focus on epiphytic orchid mycorrhizal fungi.

Abstract: Approximately 10% of vascular plants are epiphytes and, even though this has long been ignored in past research, are able to interact with a variety of fungi, including mycorrhizal taxa. However, the structure of fungal communities on bark, as well as their relationship with epiphytic plants, is largely unknown. To fill this gap, we conducted environmental metabarcoding of the ITS-2 region to understand the spatial structure of fungal communities of the bark of tropical trees, with a focus on epiphytic orchid mycorrhizal fungi, and tested the influence of root proximity. For all guilds, including orchid mycorrhizal fungi, fungal communities were more similar when spatially close on bark (i.e. they displayed positive spatial autocorrelation). They also showed distance decay of similarity with respect to epiphytic roots, meaning that their composition on bark increasingly differed, compared to roots, with distance from roots. We first showed that all of the investigated fungal guilds exhibited spatial structure at very small scales. This spatial structure was influenced by the roots of epiphytic plants, suggesting the existence of an epiphytic rhizosphere. Finally, we showed that orchid mycorrhizal fungi were aggregated around them, possibly as a result of reciprocal influence between the mycorrhizal partners.

Summary

1. Introduction

• The authors hypothesized that (i) as described in soils, these communities have no random distribution on the bark .
• Due to the ability of many fungi to colonize plant roots, (ii) their distribution should be modulated by the distance to roots of vascular epiphytes.
• Particularly, (iii) communities of OMF should be aggregated around their orchid hosts.

2.1 Study area

• The elevation provides frequent fogs throughout the year, and the humidity is around 80%, even in the dry season.
• The climate of the region is humid subtropical mesothermic, with temperatures ranging from 17 to 23°C and annual rainfall averaging 1300 mm (Rolim & Ribeiro, 2001) .
• This forest is characterized by medium to large trees, and a high diversity of orchid species, the majority of which are epiphytic (Lana et al., 2018) .

2.2 Bark and root sampling

• Two trees belonging to Siparuna sp. (Siparunaceae; tree 1) and Himathanthus sucuuba (Apocynaceae; tree 2) were selected in February 2015 and February 2016 (95 m away from each other) respectively because they had epiphytic orchids growing on their lower trunk, namely Isochilus linearis and Epidendrum armeniacum.
• Bark was also collected under each root sample.
• All samples were frozen at -20°C within few hours in the nearby field laboratory of the Serra do Brigadeiro State Park headquarters for downstream molecular analyses.
• Two additional thin sections of orchid roots surrounding each sampled piece were collected to check for mycorrhizal fungal colonization on the following day under the microscope and all, without exception, displayed hyphal coils in at least one of each inspection section.

2.3 High-throughput sequencing of fungal communities

• Tagging system negative controls were performed at this step (Hornung et al., 2019; Zinger et al., 2019) , i.e., pairs of barcoded primers were intentionally omitted in the final sequencing to control for cross-contamination.
• Plate designs were randomized in order to avoid possible cross-contamination leading to misinterpretation in subsequent spatial analysis.
• After visualization on gel, the positive amplicons were purified with NucleoMag® NGS Clean-up and Size Select (Macherey-Nagel, GmbH & Co KG.), quantified by fluorescence with Qubit TM dsDNA High-Sensitivity (Invitrogen TM ), and pooled in equimolar ratios prior to library preparation and 2x250 bp paired-end sequencing on an Illumina MiSeq platform at Fasteris (Geneva, Switzerland).
• Three positive controls (mock community) and three negative controls (ultrapure water) were used per PCR trial , resulting in a total of 36 positive and 36 negative controls in total.

2.5 Fungal functional guilds

• OTUs found in at least one orchid root sample were considered as endophytes.
• Among them, those Basidiomycota belonging to Tulasnellaceae, Ceratobasidiaceae (Veldre et al., 2013) , Serendipitaceae (Weiß et al., 2016) , and Atractiellales (Kottke et al., 2010) were recognized as orchid mycorrhizal fungi (OMF) (Dearnaley et al., 2012) .
• Besides, trophic guilds were assigned to all OTUs using FunGuild (Zanne et al., 2019) : the authors chose to keep those which were either exclusively saprotrophs, symbiotrophs, plant pathogens, or lichenized fungi.
• For the remaining OTUs, guilds provided by FunGuild were validated based on the author's expertise.
• The OMF were kept in a separate category despite their saprotrophic and symbiotrophic ability (Dearnaley et al., 2012; Selosse & Martos, 2014) .

2.6 Statistical analyses

• As the similarities between samples are not independent of one another, coefficients of the binomial GLM were obtained using a leave-one-out Jackknife procedure as described in (Millar et al., 2011) .
• The significance of the distance decay of similarity was tested using a permutational Mantel test (Spearman method, 9999 permutations; Anderson et al., 2013) , while the significance of the distance decay of richness was assessed by ANOVA (F-test).

3.1 Roots and bark harbored distinct, but partially overlapping fungal communities

• Among the 31 OMF OTUs that were found, encompassing the four OMF families (see 2.5, Fig. 3 ), only five OTUs were shared between the two trees after rarefaction (Table S4 ) and only one OTU (Tulasnellaceae, TUL-1) when considering the roots only (Table S4 , S5).
• The sharing of OMF between grids was not statistically different to that of other fungi, meaning that the trees harbored different fungal communities overall.
• On grid 2, where two orchid species co-exist, OMF OTUs belonging to Ceratobasidiaceae (CER-1) and Serendipitaceae (SER-1) were shared between the two species when they were spatially close (Table S5 , Fig. S1 ).

3.2 All fungal communities were spatially structured

• Spatial autocorrelation of single OTUs showed that only OMF on grid 2 tend to be more frequently spatially clustered than other fungi (Table S6 ).
• OMF families showed vertical stratification on grid 2 that covered a greater height on the tree (1.7 m), whereas this pattern was not obvious on grid 1 (covering 0.7 m only; Fig. S9 ).

3.3 Epiphytic roots influenced all fungal communities

• The Jaccard similarity between roots and bark fungal compositions significantly decreased with increasing distance from the roots for the whole fungal community on both grids (Fig. 5 ).
• This was also observed for endophytes on both grids, for non-OMF symbiotrophs on grid 1 only, and for OMF, plant pathogens and saprotrophs on grid 2 only (Table 1, Fig. 5 ; see also Fig. S10 and Table S8 for details).
• The distance decay of bark fungal richness showed contrasting results with either non-significant or opposite results between grids (Fig. S12 -14, Table S9 ).
• By comparing the density distribution of OMF versus endophytes (distance from roots beyond which 80% of the occurrences of a given OTU are limited), the OMF were not statistically closer to roots than other endophytes (Wilcox tests, W = 370, p = 0.423 and W = 1142, p = 0.397 for grid 1 and 2, respectively).

4.1 Features of bark fungal communities compared to the soil's

• This thinness allowed us to exhaustively sample fungal communities at a given position.
• Whether these communities are spatially structured or are either homogeneously or randomly distributed remained an open question, which the authors investigated in this study.

4.5 Fungal communities could modulate epiphytic plant population dynamics

• Here, the OMF were more spatially clustered than any other fungi (Table S6 ), reflected in the vertical stratification on grid 2 (Fig. S9 ), which suggests that they could strongly constrain orchid seed germination.
• In soil, it has also been proposed that the patchiness of orchid individuals (Jacquemyn et al., 2007) could be due to that of their mycorrhizal partners (Jacquemyn et al., 2012) .

4.6 Conclusion and perspectives

• The authors observed a vertical niche differentiation for OMF communities, but not for other fungal guilds, probably because their sampling design was not appropriate to investigate such vertical gradients.
• Yet, a possible trend for lower vertical than horizontal structure was observed.

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A ne-scale spatial analysis of fungal communities on
tropical tree bark unveils the epiphytic rhizosphere in
orchids
Remi Petrolli, Conrado Augusto Vieira, Marcin Jakalski, Melissa F Bocayuva,
Clément Vallé, Everaldo da Silva Cruz, Marc-andré Selosse, Florent Martos,
Maria Catarina M. Kasuya
To cite this version:
Remi Petrolli, Conrado Augusto Vieira, Marcin Jakalski, Melissa F Bocayuva, Clément Vallé, et al.. A
ne-scale spatial analysis of fungal communities on tropical tree bark unveils the epiphytic rhizosphere
in orchids. New Phytologist, Wiley, In press, �10.1111/nph.17459�. �hal-03279090�

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A fine-scale spatial analysis of fungal communities on tropical tree bark unveils the
1
epiphytic rhizosphere in orchids
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3
REMI PETROLLI
1*
, CONRADO AUGUSTO VIEIRA
1,2*
, MARCIN JAKALSKI
3
, MELISSA
4
F. BOCAYUVA
2
, CLEMENT VALLE
1
, EVERALDO DA SILVA CRUZ
2
, MARC-ANDRÉ
5
SELOSSE
1,2,3
§
, FLORENT MARTOS
1
§
, MARIA CATARINA M. KASUYA
2
§
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7
1
Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d’Histoire
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naturelle, CNRS, Sorbonne Université, EPHE, CP 39, 57 rue Cuvier, F-75005 Paris, France
9
2
Department of Microbiology, Viçosa Federal University (UFV), P. H. Rolfs street CEP:
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36570-900, Viçosa, Minas Gerais, Brazil
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3
University of Gdańsk, Faculty of Biology, ul. Wita Stwosza 59, 80-308 Gdańsk, Poland
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*
These authors contributed equally to this work.
13
§
These authors supervised equally this work.
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15
Rémi Petrolli (Corresponding author)
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Muséum National d’Histoire Naturelle
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UMR 7205, Institut de Systématique, Évolution et Biodiversité (ISYEB),
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12 rue Buffon, CP 39, 75005 Paris, France
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Email : remi.petrolli@mnhn.fr
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6162 words: 842 words (Introduction), 1916 words (M&M), 1146 words (Results) and 2258
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words (Discussion). 5 colored figures, 1 table, and 23 supplementary figures and tables.
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We declare no conflict of interest regarding this work.
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2
Abstract
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• Approximately 10% of vascular plants are epiphytes and, even though this has long
27
been ignored in past research, can interact with a variety of fungi, including mycorrhizal
28
ones. However, the structure of fungal communities on bark, as well as their relationship
29
with epiphytic plants, is largely unknown.
30
• To fill this gap, we conducted environmental metabarcoding of ITS-2 region to
31
understand the spatial structure of fungal communities of the bark of tropical trees, with
32
a focus on epiphytic orchid mycorrhizal fungi, and tested the influence of root
33
proximity.
34
• For all guilds, including orchid mycorrhizal fungi, fungal communities were more
35
similar when spatially closed on bark, i.e., displayed positive spatial autocorrelation.
36
They also showed distance decay of similarity from epiphytic roots, meaning that their
37
composition on bark increasingly differed, compared to roots, with distance from roots.
38
• We first showed that all the investigated fungal guilds presented a spatial structure at
39
very small scales. This spatial structure was influenced by the roots of epiphytic plants,
40
suggesting the existence of an epiphytic rhizosphere. Finally, we showed that orchid
41
mycorrhizal fungi were aggregated around them, possibly resulting from a reciprocal
42
influence between the mycorrhizal partners.
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Key words
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epiphytism; fungal guilds; metabarcoding; fungal spatial distribution; orchid mycorrhizal fungi;
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Tulasnellaceae
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3
1. Introduction
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Although globally distributed, microorganisms present a highly variable local richness and a
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spatial structure at every scale (from centimeters to thousands of kilometers), especially in soils
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(Green et al., 2004; Green & Bohannan, 2006). Much of the soil microbial biodiversity appears
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to be intrinsically linked with plants in the rhizosphere and controls their community structure
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by monitoring soil-root interactions (Bever et al., 2010). Reciprocally, soil microorganisms that
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develop nutritional and protective symbioses with roots are especially structured by host
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presence and diversity (Peay et al., 2013) such as the mycorrhizal fungi that associate with
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approximately 90% of the vascular land flora (Van Der Heijden et al., 2015; Brundrett &
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Tedersoo, 2018). Fungal metabarcoding studies in soils have shown that the mycorrhizal taxa
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are not randomly distributed, but exhibit spatial structure at rather fine scales, in temperate as
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in tropical systems (Anderson et al., 2014; Bahram et al., 2016; Coince et al., 2013; Pickles et
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al., 2010; Tedersoo et al., 2010; Zhang et al., 2017), i.e., a patchiness due to host distribution,
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but also other factors such as spore dispersal and community interactions (Hanson et al., 2012).
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However, the characterization of the underground distribution of soil fungi (mycorrhizal fungi,
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saprotrophs or pathogens) is complicated by the three-dimensional nature of soils, since
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differences may exist between soil horizons (Anderson et al., 2014; Bahram et al., 2015).
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Unlike soils, tree barks can be easily investigated as their multiple layers can be sampled and
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sequenced at once, especially on young trees where the bark is usually thin. Thus, young barks
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can be seen as virtually two-dimensional and are ideal systems for surveying the spatial
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distribution of fungal communities and mycorrhizal taxa around their epiphytic plant hosts.
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Indeed, ca. 10% of vascular plant species root on barks in the tropical wet forests around the
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globe (Zotz, 2016). These plants have long been considered as essentially non-mycorrhizal in
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4
such aerial substrates (Lehnert et al., 2017; Brundrett & Tedersoo, 2018; but see Rowe &
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Pringle, 2005) and their fungal partners have thus so far largely been ignored. However, there
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is now growing interest in the field of epiphytic fungal endophytes which could strongly
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influence the dynamics of epiphyte plant populations (Leroy et al., 2019). One symbiosis that
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regularly occurs in the epiphytic habitats is the orchid mycorrhiza (Martos et al., 2012; Herrera
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et al., 2018; Novotná et al., 2018). Epiphytic orchids, representing no less than 80% of this
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hyper-diverse plant family (with over 25 000 species (Givnish et al., 2015)), harbor typical
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hyphal coils within their root cortical cells, which are formed by the same families but different
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species of saprotrophic basidiomycetes (Dearnaley et al., 2012; Martos et al., 2012; Xing et al.,
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2019) compared to soil. The fungi are also required for germination of the minute, nutrient-
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poor orchid seeds (Smith & Read, 2008). It was therefore hypothesized that the distribution of
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orchids must be constrained by that of their mycorrhizal fungi (McCormick & Jacquemyn,
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2014; McCormick et al., 2018)
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The distribution of orchid mycorrhizal fungi (OMF) has been investigated in soils (Jacquemyn
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et al., 2014, 2017; McCormick & Jacquemyn, 2014; McCormick et al., 2016, 2018; Voyron et
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al., 2017), but only marginally on barks (Kartzinel et al., 2013), perhaps because most studies
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focus on temperate and Mediterranean ecosystems where orchids are strictly terrestrial. For
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example, two recent studies (Waud et al., 2016b,a) showed a decline in abundance and
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similarity composition of OMF with distance from adult orchids, which likely explains the
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patchy distribution of grassland orchids (Jacquemyn et al., 2007, 2014). Still in grassland
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habitats, Voyron et al., (2017) found that communities of OMF are more similar in nearby soil,
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i.e., display spatial autocorrelation (Hanson et al., 2012). As for the epiphytic environment,
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very little is known on the spatial distribution of mycorrhizal fungi on bark [but see (Izuddin et
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al., 2019) for a first approach]. Similarly, the evolution of their community structure by distance
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