Fungi at a small scale: spatial zonation of fungal assemblages around single trees!

Sara Branco1, Tom Bruns1, Ian Singleton2 1University of California, Berkeley

2Newcastle University

Nhu Nguyen

Resources!

How are fungal assemblages maintained?!

Space! Time!

Ectomycorrhizal (EM) fungi

Soil fungi symbiotically associated with plant roots; provide water and nutrients and receive carbohydrates in return. !

EM fungi

Do not form a monophyletic group (Ascomycota, Basidiomycota)

Assemble in diverse communities dominated by rare taxa.

Ascomycota Basidiomycota

Other Fungi

Glomeromycota

Succession - early stage fungi colonize young trees as well as the outer edge of root systems.!

Spatial zonation in EM assemblages!

EM fungal exploration types!Deacon&Flemming (1992)

Root density; soil properties

Tree

age

Agerer, 2001

Trade off between dispersal and competition at the mycelial level – different root densities select for different exploration types.

Long range exploration

Short range exploration

Low root density Ideal; cords needed to find new roots

Unable to colonize new roots

High root density Wasteful strategy Root colonization at low cost

Differences in root density select for different exploration types (Peay et al, 2011)!

Is there zonation around isolated trees?!

Is there a zonation pattern in EM fungal assemblages around single trees? How does it change with tree age?!

Pinus muricata single trees at Point Reyes National Seashore, CA!

- Lower EM diversity at the drip line, with long distance taxa dominating; canopy should host higher diversity, with short exploration types.!

Modified from Peay et al, 2011

- Lower EM fungal diversity associated with younger trees; long distance taxa dominating.!

Expectations:

Drip line and inner canopy host different EM taxa due to differences in root density and soil properties.!

Sampled fungi associated with 20 isolated trees (10 old and 10 young):!

Buried 8 bags per tree (4 under the drip line and 4 1/3 to drip line) for 2-month periods over one year (today I will focus on the first two months).!

Ingrowth bags - filled with acid-washed sand; target actively growing fungi and enrich for mycorrhizal fungi (Wallander et al., 2001)!

DNA was extracted from sand and the ITS region pyrosequenced.

Data was rarified. Presence/absence and read abundance analyses yielded the same results.

QIIME (Caporaso et al, 2010), ITS1 (Nilsson et al, 2010), OTUs defined at 95% similarity.

OTUs were identified by blast – UNITE and Pt Reyes databases; OTUs separated into known EM and non-EM.

Bags were amplified separately and PCR products either pooled by inner or outer circle or sequenced individually (6 trees).

20 trees

6 trees

Results !

368 fungal OTUs; 70 mycorrhizal

EM fungi – pooled bags

No differences in communities across circle or age.

EM fungi – individual bags

More OTUs in old trees and inner circles; different communities in old and young trees.

Inner Outer

01

23

45

67

CircleN

umbe

r of O

TUs

Old Young

01

23

45

67

Age

OTUs

-1.0 -0.5 0.0 0.5 1.0 1.5

-1.0

-0.5

0.0

0.5

1.0

NMDS1

NMDS2

Stress =.1 ; ADONIS R2=.06, P<.05 2-Way ANOVA, F=5.82, P<.05 2-Way ANOVA, F=6.48, P<.05

* *

Fusarium Mortierella Cladosporium

Tomentella Pseudotomentella Amphinema

Top non-EM Top EM

EM fungi exploration types (http://www.deemy.de/, Hobbie & Agerer, 2010)

Long distance!Short distance !

No significant difference in exploration type across circle or tree age.

David Genney Deemy.de Deemy.de

Medium distance !

Non-EM fungi – pooled bags

http://www.etgonline.com

Inner Outer

2030

4050

60

Circle

Num

ber o

f OTU

s

*

2-Way ANOVA, F=5.29, P=.05

Higher richness on outer circles

Different composition across circle and tree age

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4

-0.4

-0.2

0.0

0.2

0.4

NMDS1

NMDS2

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4

-0.4

-0.2

0.0

0.2

0.4

NMDS1

NMDS2

Stress =.27 ; ADONIS R2=.07, P<.05 Stress =.27 ; ADONIS R2=.07, P<.05

Circle Tree age

Non-EM fungi – individual bags

Inner Outer

2030

4050

60

Circle

Num

ber o

f OTU

s

*

2-Way ANOVA, F=5.29, P=.05

Higher richness in outer circles

ww

w.m

ycol

og.c

om

Different composition across circle and tree age

Stress =.27 ; ADONIS R2=.07, P<.05 Stress =.27 ; ADONIS R2=.07, P<.05

-0.5 0.0 0.5 1.0

-0.5

0.0

0.5

NMDS1

NMDS2

-0.5 0.0 0.5 1.0

-0.5

0.0

0.5

NMDS1

NMDS2

Circle Tree age

Comparing pooled and individual bags (6 trees)

More fungi recovered from individual bags (140 vs 90; P<.05).

Fungal communities recovered from individuals bags differed from pooled bags.

-1.0 -0.5 0.0 0.5 1.0

-1.0

-0.5

0.0

0.5

1.0

NMDS1

NMDS2

Inner circle samples; Stress =.27

Computational pooling and rarefying yields no differences in richness (23 OTUs) or community structure (read depth!).

Root density and soil chemistry

Soil pH and C:N differ across circle and tree age - not significantly correlated with fungal diversity.

Very few roots…

No differences in root density across inner and outer circle or across old and young trees.

Root biomass from soil cores collected where bags were buried.

Is there a zonation pattern in fungal assemblages around single trees? !

EM fungi

Some differentiation across tree canopy, but no evidence for space partitioning by exploration type. Higher richness in inner circle only with individual samples.!

Yes, there is small scale partitioning in fungi. However the patterns can be weak and scale dependent.!

No evidence for predicted compositional zonation; trees seem to be old enough to host mature fungal communities (Inocybe, Lactarius, Russula).

Very few roots overall. Finding mature communities suggests root density is not a driver in this system.

Is there a zonation pattern in fungal assemblages around single trees? !Non-EM fungi

Unexpected highly diverse and distinct assemblages across circles and tree age.!

Soil chemistry does not explain this pattern. Moisture can be the main driver (fog and ‘drip line’). !

Conclusions !

Ingrowth bags do not target EM fungi, but provide wide overview of soil fungi. Active species only – non EM fungi growing inside or along inner side of bags?

Exploration types are not the basis of small scale space partitioning for EM fungi and root density is not an important driver for fungal diversity in this single tree setting. !

EM and non EM fungi behave very differently at a small scale. !

Sampling fungal communities !

Are ingrowth bags a good way to sample EM fungi?

Compare fungal diversity using different sampling techniques:

ingrowth bags (actively growing fungi), soil (all fungi), root tips (EM fungi associated with pine trees), and bioassays (spore bank fungi – able to colonize host de novo).

ITS pyrosequences from two pine forests (CA and NC).

Bags Roots Soil

Sampling fungal communities !

Different sampling techniques recover different fungal communities.!

Acknowledgements

Bruns and Taylor labs

Dimensions of Biodiversity, NSF

Pt Reyes National Seashore

Kabir Peay

Marie Curie Actions

Holly Edes

SMBE travel award

William Moore