SMBE satellite meeting, Davis 2013

1. 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

2. 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. !

4. EM fungi
Do not form a monophyletic group (Ascomycota,
Basidiomycota)
Assemble in diverse communities dominated by rare taxa.
Ascomycota
Basidiomycota
Other Fungi
Glomeromycota

5. 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
Treeage
Agerer, 2001

6. 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

7. Differences in root density select for different exploration
types (Peay et al, 2011)!
Is there zonation around isolated trees?!

8. 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!

9. - 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.!

10. 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)!

11. 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

12. 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
01234567 Circle
NumberofOTUsOld Young
01234567
Age
OTUs
-1.0 -0.5 0.0 0.5 1.0 1.5
-1.0-0.50.00.51.0
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

13. 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.deDeemy.de
Medium distance !

14. Non-EM fungi – pooled bags
http://www.etgonline.com
Inner Outer
2030405060
Circle
NumberofOTUs
*
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
-0.4-0.20.00.20.4
NMDS1
NMDS2
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4
-0.4-0.20.00.20.4
NMDS1
NMDS2
Stress =.27 ; ADONIS R2=.07, P<.05 Stress =.27 ; ADONIS R2=.07, P<.05
Circle Tree age

15. Non-EM fungi – individual bags
Inner Outer
2030405060
Circle
NumberofOTUs
*
2-Way ANOVA, F=5.29, P=.05
Higher richness in outer circles
www.mycolog.com
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
-0.50.00.5
NMDS1
NMDS2
-0.5 0.0 0.5 1.0
-0.50.00.5
NMDS1
NMDS2
Circle Tree age

16. 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
-1.0-0.50.00.51.0
NMDS1
NMDS2
Inner circle samples; Stress =.27
Computational pooling and rarefying yields no differences in
richness (23 OTUs) or community structure (read depth!).

17. 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.

18. 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.

19. 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’). !

20. 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. !

21. 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).

22. Bags
Roots
Soil
Sampling fungal communities !
Different sampling techniques recover different fungal
communities.!

23. 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