Lecture on using genomics tools in Microbial Ecology

1. Genomics in Microbial Ecology
Dr. Ashish Malik
@ashish_a_malik
https://www.ashishmalik.co

2. Microbial ecology is the most important
and least developed area of ecology
Microbial ecology is the study of the interactions of
microorganisms with their environment, each other,
and plant and animal species.
Why are microbes so important
• They are ubiquitous - occupy the broadest range of
environments
• They drive all biogeochemical processes on earth
• They are essential for the existence of all animals and
plants
Global
Soil
Biodiversity
Atlas

3. Microbial ecology is the most important
and least developed area of ecology
Microbial ecology is the study of the interactions of
microorganisms with their environment, each other,
and plant and animal species.
Why least developed area of ecology
• Microbes are notoriously hard to cultivate
• Isolates might not be representative of their associated
natural populations
• They often exist as part of complex communities
• Lack of access to tools with sufficient depth of resolution
Global
Soil
Biodiversity
Atlas

4. Cultivation based methods
Genome sequencing of individual isolated microbial
strains from phylogenetically diverse lineages
Culturable
Unculturable
Large proportion of microorganisms
are unculturable with present methods

5. Ability to directly sequence DNA from
the environment permanently changed
microbial ecology

6. Classification systems
and the advent of DNA
sequencing technologies
Hug et al. 2016
Global Soil Biodiversity Atlas. 2016, European Commission.

7. Cultivation-independent methods
Allows survey of the resident microbiota
in a given community
Early examples:
• 16S rRNA gene clone library collections
• Group-specific fluorescence in situ hybridization
(FISH) Image à
Lukumbuzya et al. 2019

8. Marker gene sequencing
analyses
Gene amplicon sequencing:
16S rRNA, 18S rRNA, ITS
Description and quantitation of the phylogenetic
diversity of microbial communities
Linking these organisms to their functions and
ecological roles
Allows one to describe the microbial composition
and diversity based on the taxonomic groups
present in a community
Illustrations by A. Murat Eren (Meren)

9. Marker gene sequencing
analyses
Gene amplicon sequencing:
16S rRNA, 18S rRNA, ITS
Description and quantitation of the phylogenetic
diversity of microbial communities
Linking these organisms to their functions and
ecological roles
Allows one to describe the microbial composition
and diversity based on the taxonomic groups
present in a community
Bahram et al. 2020
ITS
16S rRNA

10. Shotgun metagenomics: sequencing of
microbial communities en masse
Functional analysis of collective microbial genomes
in a community
Who is there, and what are they doing?

11. Metagenomics: sequencing of microbial
communities en masse
Functional analysis of collective microbial genomes
in a community
Who is there, and what are they doing? −0.15 −0.10 −0.05 0.00 0.05 0.10
−0.10
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NMDS1
NMDS2
a)
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b)
Malik et al. 2017

12. Metagenomic assembly: reconstructing
genomes from metagenomes
Relate taxa to function and
identify new genes from species
Obtain hundreds to thousands of high-quality
microbial genomes to study individuals

13. Metagenomic assembly: reconstructing
genomes from metagenomes
Relate taxa to function and
identify new genes from species
Obtain hundreds to thousands of high-quality
microbial genomes to study individuals
Phylogenetic tree of 55 MAGs
Outer ring of coloured bars represents fold
enrichment (orange) or depletion (green) of
each MAG within drought-treated rhizosphere
compared with watered control rhizosphere.
Xu et al. 2021

14. http://merenlab.org/momics

16. Diversity of bacteria across habitats
Fig 1. Sample and geocluster locations. Map showing locations of each of the EMP samples used in this study (black dots) and the geoclustered samples
for each habitat (colored dots). Geoclusters were created from samples located within 110 km of each other.
https://doi.org/10.1371/journal.pone.0233872.g001
PLOS ONE Alpha-, beta-, and gamma-diversity of bacteria
Bacterial assemblages using 11,680 samples compiled by the Earth Microbiome Project

17. Alpha-, beta-, and gamma-
diversity of bacteria
varies across habitats
Fig 2. Alpha- and beta-diversity patterns. Alpha- and beta-diversity per habitat for all geoclusters used in study (A, C, and E) and per biome for soil
geoclusters (B, D, and F). (A and B) Boxplot of alpha-diversity (OTU richness). (C and D) Mean beta-diversity (distance from centroid) ± standard error.
For all bar and boxplots, letters above indicate significant differences among groups (Tukey test) where groups that share a letter are not significantly
different from each other. (E and F) NMDS of geoclusters.
whether these beta-diversity patterns might be driven by dispersal limitation [9,57] or spatial
variation in environmental conditions [6,45].
Fig 5. Geocluster accumulation curves. Geocluster accumulation curves (gamma-diversity) for mean OTU richness from a random sampling of
geoclusters (permutations = 999) with 95% confidence intervals drawn for each habitat.
https://doi.org/10.1371/journal.pone.0233872.g005
PLOS ONE Alpha-, beta-, and gamma-diversity of bacteria
Walters & Martiny 2020

18. Whole genome metagenomics on soils at
opposing ends of a landscape soil pH gradient
Malik et al. 2017
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16S
OTUs
a)
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annotated
functions
1 2 3 4
b)
pH

19. Whole genome metagenomics on soils at
opposing ends of a landscape soil pH gradient
Malik et al. 2017
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OTUs
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b)
pH
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NMDS2
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High pH indicator
Low pH indicator
Not significant
●
●
●
High pH indicator
Low pH indicator
Not significant
b)

20. Bacterial physiological adaptations to contrasting
edaphic conditions
Malik et al. 2017
0
10
20
30
40
50 Amino
Acids
and
Derivatives
Carbohydrates
Cell
Division
and
Cell
Cycle
Cell
Wall
and
Capsule
Clustering
based
subsystems
Cofactors,
Vits,
Pros.groups,
Pigments
DNA
Metabolism
Fatty
Acids,
Lipids,
and
Isoprenoids
Iron
acquisition
and
metabolism
Membrane
Transport
Metabolism
of
Aromatic
Compounds
Miscellaneous
Motility
and
Chemotaxis
Nitrogen
Metabolism
Nucleosides
and
Nucleotides
Phages,
Transpos.elems,
Plasmids
Phosphorus
Metabolism
Photosynthesis
Potassium
metabolism
Protein
Metabolism
Regulation
and
Cell
signaling
Respiration
RNA
Metabolism
Secondary
Metabolism
Stress
Response
Sulfur
Metabolism
Virulence,
Disease
and
Defense
freq
High pH indicator
Low pH indicator
P
)

21. Representation of a subsystem structure (Levels 1–3
classifications) – similar to KEGG pathways
Silva et al. 2016
K01647 Citrate Synthase [EC:2.3.3.1]

22. Malik et al. 2017
C
Bact cyanide prod., tolerance mechs
Strep. pyogenes recombinatorial zone
mdtABCD multidrug resistance
mdtABCD multidrug resistance
Resistance to Vancomycin
Resistance to chromium compounds
Multidrug Resistance Efflux Pumps
Multidrug Resistance Efflux Pumps
MexE MexF OprN Multidrug Efflux System
Cobalt zinc cadmium resistance
Cobalt zinc cadmium resistance
Cobalt zinc cadmium resistance
Cobalt zinc cadmium resistance
Arsenic resistance
BlaR1 Regulatory Sensor transducer
Galactosylceramide, Sulfatide metabolism
Inorganic Sulfur Assimilation
Inorganic Sulfur Assimilation
Inorganic Sulfur Assimilation
Alkanesulfonates Utilization
Alkanesulfonate assimilation
Alkanesulfonate assimilation
SigmaB stress responce regulation
SigmaB stress responce regulation
Glutathione: Non redox reactions
Regulation of Oxidative Stress Response
Choline, Betaine Uptake, Betaine Biosynthesis
Choline, Betaine Uptake, Betaine Biosynthesis
Choline, Betaine Uptake, Betaine Biosynthesis
Heat shock dnaK gene cluster extended
Cold shock, CspA
family of proteins
Uptake of selenate, selenite
Tannin biosynthesis
Group II intron associated genes
Transcription
initiation, bacterial sigm
a
factors
ATP
dependent RNA
helicases, bacterial
RNA
processing, degradation, bacterial
Biogenesis
of cytochrom
e
c
oxidases
Form
ate
hydrogenase
Form
ate
hydrogenase
Form
ate
hydrogenase
Form
ate
hydrogenase
F
o
r
m
a
t
e
h
y
d
r
o
g
e
n
a
s
e
F
o
r
m
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t
e
h
y
d
r
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g
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n
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S
u
c
c
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d
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R
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1
R
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p
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d
r
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n
a
s
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s
1
R
e
s
p
i
r
a
t
o
r
y
C
o
m
p
l
e
x
I
N
i
F
e
h
y
d
r
o
g
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n
a
s
e
m
a
t
u
r
a
t
i
o
n
H
y
d
r
o
g
e
n
a
s
e
s
H
y
d
r
o
g
e
n
a
s
e
s
H
y
d
r
o
g
e
n
a
s
e
s
H
y
d
r
o
g
e
n
a
s
e
s
H
y
d
r
o
g
e
n
a
s
e
s
C
O
D
e
h
y
d
r
o
g
e
n
a
s
e
F
o
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m
a
t
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d
e
h
y
d
r
o
g
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n
a
s
e
V
T
y
p
e
A
T
P
s
y
n
t
h
a
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F
0
F
1
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y
p
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A
T
P
s
y
n
t
h
a
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e
F
0
F
1
t
y
p
e
A
T
P
s
y
n
t
h
a
s
e
T
e
r
m
in
a
l
c
y
t
o
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h
r
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e
d
u
b
iq
u
in
o
l
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id
a
s
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A
n
a
e
r
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b
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p
ir
a
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o
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d
u
c
t
a
s
e
s
T
e
r
m
in
a
l
c
y
t
o
c
h
r
o
m
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C
o
x
id
a
s
e
s
O
r
p
h
a
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g
u
la
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p
r
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in
s
O
r
p
h
a
n
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u
la
to
r
y
p
r
o
te
in
s
c
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M
P
s
ig
n
a
li
n
g
in
b
a
c
te
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ia
D
N
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b
in
d
in
g
r
e
g
u
la
to
r
y
p
r
o
te
in
s
,
s
tr
a
y
s
B
a
rA
U
v
rY
(S
ir
A
)
tw
o
c
o
m
p
o
n
e
n
t
re
g
u
la
to
r
y
s
y
s
te
m
S
tr
e
p
to
c
o
c
c
a
l
M
g
a
R
e
g
u
lo
n
C
o
a
g
u
la
ti
o
n
c
a
s
c
a
d
e
P
h
d
,D
o
c
,Y
d
c
E
,Y
d
c
D
to
x
in
a
n
ti
to
x
(p
rg
.c
e
ll
.d
e
a
th
)
T
o
x
in
a
n
ti
to
x
in
s
y
s
(n
o
t
R
e
lB
E
,M
a
z
E
F
)
T
o
x
in
a
n
ti
to
x
in
s
y
s
(n
o
t
R
e
lB
E
,M
a
z
E
F
)
P
ro
te
in
A
ce
ty
la
tio
n
,
D
e
a
ce
ty
la
tio
n
in
B
a
ct
G
3E
P
lo
op
G
T
P
as
es
m
et
al
lo
ce
nt
er
bi
os
yn
th
G
3E
P
lo
op
G
TP
as
es
m
et
al
lo
ce
nt
er
bi
os
yn
th
G
3E
P
lo
op
G
TP
as
es
m
et
al
lo
ce
nt
er
bi
os
yn
th
Pe
pti
dy
l
pr
oly
l
cis
tra
ns
iso
me
ra
se
Pr
ote
in
ch
ap
ero
ne
s
Un
ive
rsa
l
GT
Pas
es
tRN
A
ami
noa
cyla
tion
,
Trp
tRNA
amin
oacy
lation
,
Ser
tRNA
aminoac
ylation,
Ala
Translation
elongation
factors
bacterial
Ribosome
SSU
bacterial
Ribosome
LSU
bacterial
Ribos
ome
LSU
bacte
rial
Puta
tive
TldE
TldD
prot
eoly
tic
com
plex
Pro
teo
lysi
s
in
bac
teri
a,
ATP
dep
end
ent
Pro
tei
n
de
gra
da
tio
n
Am
ino
pe
pti
da
se
s
(E
C
3.4
.11
.
)
Di
pe
pt
id
as
es
(E
C
3.
4.
13
.
)
H
yp
er
os
m
ot
ic
po
ta
ss
iu
m
up
ta
ke
P
ot
as
si
um
ho
m
eo
st
as
is
P
o
ta
ss
iu
m
h
o
m
e
o
st
a
si
s
P
o
ta
s
s
iu
m
h
o
m
e
o
s
ta
s
is
P
o
ta
s
s
iu
m
h
o
m
e
o
s
ta
s
is
P
o
ta
s
s
iu
m
h
o
m
e
o
s
ta
s
is
P
ro
te
o
rh
o
d
o
p
s
in
P
h
o
s
p
h
a
te
m
e
ta
b
o
li
s
m
P
h
o
s
p
h
a
te
m
e
ta
b
o
li
s
m
H
ig
h
a
ff
in
.p
h
o
s
p
h
a
te
tr
n
s
p
r
tr
P
H
O
r
e
g
u
lo
n
P
u
p
ta
k
e
(
c
y
a
n
o
b
a
c
te
r
ia
)
r
1
t
li
k
e
s
tr
e
p
to
c
o
c
c
a
l
p
h
a
g
e
s
C
B
S
S
2
0
3
1
2
2
.
1
2
.
p
e
g
.
1
8
8
C
B
S
S
2
0
3
1
2
2
.
1
2
.
p
e
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.
1
8
8
H
y
d
a
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o
in
m
e
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a
b
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s
m
H
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d
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m
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t
a
b
o
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s
m
X
a
n
t
h
in
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M
e
t
a
b
o
li
s
m
in
B
a
c
t
P
u
r
in
e
U
t
il
iz
a
t
io
n
P
u
r
i
n
e
c
o
n
v
e
r
s
i
o
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s
P
u
r
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c
o
n
v
e
r
s
i
o
n
s
N
i
t
r
o
g
e
n
f
i
x
a
t
i
o
n
N
i
t
r
o
g
e
n
f
i
x
a
t
i
o
n
N
i
t
r
o
g
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n
f
i
x
a
t
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n
N
i
t
r
o
g
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n
f
i
x
a
t
i
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n
N
i
t
r
o
g
e
n
f
i
x
a
t
i
o
n
N
i
t
r
o
g
e
n
f
i
x
a
t
i
o
n
A
m
m
o
n
i
a
a
s
s
i
m
i
l
a
t
i
o
n
N
i
t
r
a
t
e
,
n
i
t
r
i
t
e
a
m
m
o
n
i
f
i
c
a
t
i
o
n
N
i
t
r
a
t
e
,
n
i
t
r
i
t
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a
m
m
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n
i
f
i
c
a
t
i
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n
N
i
t
r
a
t
e
,
n
i
t
r
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a
m
m
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n
i
f
i
c
a
t
i
o
n
N
i
t
r
a
t
e
,
n
i
t
r
i
t
e
a
m
m
o
n
i
f
i
c
a
t
i
o
n
N
i
t
r
a
t
e
,
n
i
t
r
i
t
e
a
m
m
o
n
i
f
i
c
a
t
i
o
n
F
l
a
g
e
l
l
a
r
m
o
t
i
l
i
t
y
F
l
a
g
e
l
l
a
r
m
o
t
i
l
i
t
y
Flagellar m
otility
Bacterial Chem
otaxis
Bacterial Chem
otaxis
Niacin
Choline
transport, m
etabolism
Iron
sulfur cluster assem
bly
Experim
ental COG2515
COG4319
COG3533
COG3533
COG3533
COG3533
COG0277
At1g01770
At1g26220 At1g32070
Broadly distributed proteins not in subsystems
ZZ gjo need homes
ZZ gjo need homes
Phenylacetyl CoA catabolic pathway (core)
Biphenyl Degradation
Catechol branch of beta ketoadipate pathway
Protocatechuate branch betaketoadipate pathway
Multi subunit cation antiporter
Multi subunit cation antiporter
Tricarboxylate transport system
Tricarboxylate transport system
Transport of Zinc
Transport of Zinc
Transport of Nickel, Cobalt
Ton, Tol transport systems
Transport of Manganese
Type VI secretion systems
General Secretion Pathway
General Secretion Pathway
Periplsm.bind.prot. Transport a Glucosides
Periplsm.bind.prot. Transport a Glucosides
Periplsm.bind.prot. Transport a Glucosides
ATP dependent efflux pump transporter Ybh
ABC transporter oligopeptide
ABC transporter branched chain amino acid
ABC transporter branched chain amino acid
ABC transporter branched chain amino acid
ABC transporter dipeptide
ABC transporter dipeptide
ABC transporter dipeptide
ABC transporter dipeptide
Transport of Iron
Iron acquisition in Vibrio
Heme, hemin uptake,use in GramNegatives
Campylobacter Iron Metabolism
Campylobacter Iron Metabolism
Encaps.protein DyP,ferritin like prot.oligos
Encaps.protein DyP,ferritin like prot.oligos
Glycerolipid, Glycerophospholipid Metabolism
Carotenoids
Carotenoids
Carotenoids
Fatty acid degradation regulons
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C
B
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a
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d
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s
Identity and abundances
of indicator genes for
low- and high-pH soils

23. Bacterial physiological adaptations to contrasting
edaphic conditions
Malik et al. 2017

24. Hydrogen metabolism is widespread and active
members of soil microbial communities
Bay et al. 2021
Abundance of each gene in the metagenomic short reads
of four Australian depth profiles and eight global sites

25. Hydrogen metabolism is widespread and active
members of soil microbial communities
Bay et al. 2021
Metabolic potential of 757 MAGs:
number of genomes in each phylum that encode the gene of interest

26. Phylogenetic tree showing
sequence diversity and
taxonomic distribution of
Group 1 & 2 [NiFe] hydrogenase
The tree shows sequences from soil
MAGs and NCBI reference genomes
Bay et al. 2021

27. Diversity of bacteria across habitats
Microbial traits across a precipitation gradient
Loma Ridge Global Change Experiment, Southern California
Microbial functional response to drought
Lower decomposition
under drought?

28. Impact of drought on microbial traits and decomposition
Litter
bags
Grass
Shrub
Precipitation
Ambient Reduced
Increasing stress
Decreasing
resources
Ranking: rate of
litter decomposition
1 2
3 4

29. Impact of drought on microbial traits and decomposition
Grass T4
T2 T3
T1 T4
T0

30. Malik et al., in prep.
Taxonomic Functional
Metagenomics-derived, MG-RAST (read-based)
T: 0.26 ***
V: 0.20 ***
VxT: 0.13 ***
PxT: 0.05 **
VxPxT: 0.04 *
P: 0.03 ***
VxP: 0.02 *
T: 0.35 ***
V: 0.17 ***
VxT: 0.09 ***
VxPxT: 0.04 *
PxT: 0.04 *
P: 0.03 **
VxP: 0.02 *
PERMANOVA
Microbial community succession during decomposition

31. Microbial community succession during decomposition
Malik et al., in prep.
Metagenomics-derived, MG-RAST (read-based)
Taxonomic diversity Fungi vs bacteria
T: ***
V: ***
VxT: ***
VxP: *
T: ***
V: *
VxT: **
ANOVA

32. Metagenomics-derived:
Subsystems classification
Change with drought
Drought-related functional changes during decomposition

33. Metagenomics-derived: reads based using Subsystems classification
Drought-related functional changes during decomposition
z score
MetaSPAdes assembly configs: gene-calling using KOFAM Normalized gene
domain numbers

34. Drought-related functional changes during decomposition
T: ***
V: ***
VxT: **
VxP: *
Normalized
CAZy gene
domain numbers
z score
Carbohydrate-Active enZYmes Database

35. Population Community Ecosystem
Multi-scale integrated ecology approach
Sustainable
land use
Climate
change
resilience
Peatland
restoration
Microbial
Biogeochemistry
Lab

36. Microbial
Biogeochemistry
Lab
ashish.malik@abdn.ac.uk
@ashish_a_malik ashishmalik.co