Soil bacterial diversity in the Arctic is not fundamentally different from that found in other biomes

Haiyan Chu1, Noah Fierer2, Christian L. Lauber2, J. Gregory Caporaso2, Rob Knight2, Paul Grogan1

1Queen’s University, Canada, 2University of Colorado

1. “Everything is everywhere, but the environment selects” (Baas-Becking 1934) • Microbes are ubiquitous - small, abundant, fast reproduction and readily

transported -> dispersal limitation rare• Contemporary local environmental conditions determine whether individual

microbes are active/presentPrediction: Microbial distributions correlate with current local environmental

conditions

2. Historical factors also contribute (Martiny et al, 2006)• Dispersal limitation in the past (earlier evolutionary stage)• Local differences in environmental conditions over timePrediction: Geographic distance between sampling sites influences the distribution of

genotypes – the further apart, the more unlike are the microbial communities

Two main hypotheses:

What factors determine the spatial distributions of microbes?

What do we know about soil bacterial distributions?

What about arctic soil bacterial communities? – Is there evidence of spatial structuring, and is diversity relatively low?

•Technique limitations on taxonomic resolution and number of samples•Dispersal limitation can be important at very fine taxonomic levels•Soil pH is an important control in a variety of ecosystems from boreal to

tropical forests

Noatak

Toolik

Fairbanks

Taglu Island

Alexandra Fiord

Bear Island

Clifton Point

Kuujjarapik

Ramsay Lake

Torngat Mountains

Abisko

Artillery Lake

Bylot Island

Cambridge Bay

Cape Tanfield

Daring Lake

Dempster Highway

Kangerlussuaq

Kangirsuallujjuaq

Kangirsujjuaq

Lake Raudavatn

Kilpisjarvi village

Thule

Truelove lowland

Svalbard Island

Skallovaara

Laxardalur Valley

Zackenberg

Wolf Creek

AHDR: Arctic Human Development Report

Latitude: 55-79 degreesSoil pH: 4.3-7.6Soil C: 9-47%Soil N: 0.37-2.2%Soil C/N: 15-50

Our soil sampling sites across the Arctic

29 sites (6-5500 km)Within sites (0.02-0.1 km)Vegetation: dry heath onlySoil: Organic 2-5 cmSummer 2007, 2008

Soil DNA extraction

Sequences belonging to each site sorted using the barcodes

Pyrosequencing(of the pooled samples –

750,000 reads per run)

PCR amplification(16S rRNA gene sequences from each site

receiving a unique 12 bp barcode)

Bar-coded pyrosequencing

Bioinformatic analysis

AGCCTTAA …

GCTACCAT …

CGGATCAC …

CTCGATTC …

AGCCTTAA …

GCTACCAT …

CGGATCAC …

CTCGATTC…

AGCCTTAA …

GCTACCAT …

CGGATCAC …

CTCGATTC…

What were dominant bacterial phyla across the Arctic?

Dominant Phyla Rare phyla

* indicate sequences classified to the domain Bacteria, but not to a specific phylum.

0

5

10

15

20

25

30

35

Acidob

acter

ia

Alphap

roteo

bacte

ria

Actino

bacte

ria

Betapro

teoba

cteria

Proteo

bacte

ria*

Bacter

oidete

s

Gammap

roteo

bacte

ria

Verruc

omicr

obia

Gemmati

monad

etes

Deltap

roteo

bacte

riaTM7

Firmicu

tes

Cyano

bacte

riaChlo

roflex

iNitro

spira

OD1

Plancto

mycete

sOP10W

S3Bac

teria*

OP11BRC1

Rel

ativ

e ab

unda

nce

(%)

Does soil pH influence arctic soil bacterial community structure?

Number of unique phylotypes (richness):community-level diversity at a single level of taxonomic resolution

Faith’s index of phylogenetic diversity:an integrated index of the phylogenetic breadth across all taxonomic levels

Soil pH

R = 0.54P < 0.0001

300

400

500

600

700

4 5 6 7 8

Num

ber o

f uni

que

phyl

otyp

es(p

er 1

,000

seq

uenc

es)

R = 0.62P <0.0001

10

20

30

40

50

4 5 6 7 8

Phyl

ogen

etic

dive

rsity

(per

1,0

00 s

eque

nces

)

2

P < 0.0001

300

400

500

600

700

4 5 6 7 8

(per

1,0

00 s

eque

nces

)

R = 0.62P <0.0001

10

20

30

40

50

4 5 6 7 8

Soil pH

2

No sign. relationships with latitude, soil H2 O,C, N, DON, NH4 , NO3 , MBC, MBN, MBP

Unifrac distance between two communities =

How to compare communities among sites? UniFrac pairwise distances indicate differences in composition weighted according to phylogeny -> overall phylogenetic distance between each pair of communities

Unique branch lengthTotal branch length

B

A

Abi3

AF3Tru1

CT2CBKil Sva

DL2

CT1

H3

Abi2

DL

Art

Art

Kuu

Kuu

Abi

Abi

CT

Too

Too

Byl

BylTM AF

Ayl

RL

Ska

DHH1

FaiBI

KAV

AF1

KGRDL3

KG

WCTru2

Yam LRAF2

H4Too1

Abi1Lax

DL1

H2

Too2

Noa

Tag

Too3

CT2

AF1

Lax

KAV

AF2Abi2

Abi1

DH

Too1

LR

Abi3

BI

AF

FaiAF3TM

Ska

DL RL

KGR

H2

Yam

DL2

Kil

DL1

H3

H4

Tag

Sva

CT CB

WC

Noa

CT1

2D Stress: 0.09

DL3Ayl

Tru1

Too3H1

Tru2

Too2

KG

2D Stress: 0.07

4Soil pH

5 6 7 8

B

A

Abi3

AF3Tru1

CT2CBKil Sva

DL2

CT1

H3

Abi2

DL

Art

Art

Kuu

Kuu

Abi

Abi

CT

Too

Too

Byl

BylTM AF

Ayl

RL

Ska

DHH1

FaiBI

KAV

AF1

KGRDL3

KG

WCTru2

Yam LRAF2

H4Too1

Abi1Lax

DL1

H2

Too2

Noa

Tag

Too3

CT2

AF1

Lax

KAV

AF2Abi2

Abi1

DH

Too1

LR

Abi3

BI

AF

FaiAF3TM

Ska

DL RL

KGR

H2

Yam

DL2

Kil

DL1

H3

H4

Tag

Sva

CT CB

WC

Noa

CT1

2D Stress: 0.09

DL3Ayl

Tru1

Too3H1

Tru2

Too2

KG

2D Stress: 0.07

4Soil pH

5 6 7 8

The Arctic is no different: Soil pH is a strong determinantWeighted UniFrac distance (accounts for relative

abundances of taxa）

Unweighted UniFrac Distance(presence/absence of taxa）

Does the pattern of overall phylogenetic structure relate to soil pH?

Dispersal limitation (historical factors) seem to have little effect

Geographic distance (km)

0.0

0.1

0.2

0.3

0.4

0 1000 2000 3000 4000 5000 6000

Wei

ghte

d U

niFr

acdi

stan

ce

R 2 =0.0003

0.6

0.7

0.8

0.9

1.0

0 1000 2000 3000 4000 5000 6000Unw

eigh

ted

Uni

Frac

dist

ance

R 2 =0.0027

Does geographic proximity influence the phylogenetic structure of arctic soil bacterial communities?

Temperate coniferous forestTemperate deciduous forest

Temperate grasslandMediterranean shrubland

Arctic tundra

Tropical forest

Desert

Prairie

Boreal forest

Alpine tundra

Is the phylogenetic structure of soil bacterial communities in the Arctic different from that of other biomes?

Phylogenetic variability within arctic soils is similar to lower latitude soils

2D Stress 0.12

2D Stress 0.12

Unweighted UniFrac Distance

Weighted UniFrac Distance

Is the diversity of soil bacterial communities in the Arctic lower than in other biomes?

0

100

200

300

400

500

600

700

800

Arctic

tundra

Tropical

forest

Temperate

coniferous

forest

Temperate

deciduous

forest

Desert Prairie Temperate

grassland

Mediterran.

shrubland

Boreal

forest

Alpine

tundra

Num

ber o

f uni

que

phyl

otyp

es(p

er 1

,000

seq

uenc

es)

A

35 34679

101217

17

0

10

20

30

40

50

60

Arctic

tundra

Tropical

forest

Temperate

coniferous

forest

Temperate

deciduous

forest

Desert Prairie Temperate

grassland

Mediterran.

shrubland

Boreal

forest

Alpine

tundra

Phyl

ogen

etic

dive

rsity

(per

1,0

00 s

eque

nces

)

B

173534

679

10

1217

0

100

200

300

400

500

600

700

800

Arctic

tundra

Tropical

forest

Temperate

coniferous

forest

Temperate

deciduous

forest

Desert Prairie Temperate

grassland

Mediterran.

shrubland

Boreal

forest

Alpine

tundra

Num

ber o

f uni

que

phyl

otyp

es(p

er 1

,000

seq

uenc

es)

35 34679

101217

17

0

10

20

30

40

50

60

Arctictundra

Tropicalforest

Temperateconiferous

forest

Temperatedeciduous

forest

Desert Prairie Temperategrassland

Mediterran.shrubland

Borealforest

Alpinetundra

Phyl

ogen

etic

dive

rsity

(per

1,0

00 s

eque

nces

) 173534

679

10

1217

(The digits above each column indicate the number of sampled sites)

Conclusions on patterns of soil bacterial community structure and their controls

Local environmental conditions (soil pH) are far more important than dispersal limitation (historical factor) in the Arctic - Soil fungal patterns similar???

Bacterial community composition, richness and phylogenetic diversity are NO less variable in the Arctic than in more temperate biomes- contrasts with latitudinal species richness gradient of macroecology1. Biome definitions are not useful for predicting variability in soil bacterial communities

2. The controls on bacterial distributions are fundamentally different from those observed for macro-organisms

We sincerely thank: The many research colleagues who collected soil samples across the

Arctic including friends from the North Slave Metis Association Linda Cameron and several undergraduate students for help with soil

processing and lab analyses Virginia Walker and Kate Buckeridge for their comments IPY CiCAT (Greg Henry et al) NSERC NSF Ontario Government Early Researcher Award program

Reference: Chu H, Fierer N, Lauber C, Caporaso JG, Knight R, Grogan P. 2010. Environmental Microbiology, 12(11) 2998-3006.

~108,000 bacterial sequences, ~2100 per site

• What taxa/lineages are present?• How are the communities structured?

– Endemism (richness and uniqueness)– Phylogenetic diversity

• Does variation in community structure correlate with environmental characteristics?

• How different are the communities among sites?– Compositional plus phylogenetic variation

Thank you for your attention

We sincerely thank the many research colleagues who collected soil samples across the Arctic for us. We also thank Linda Cameron and several undergraduate students for help with soil processing and lab analyses, and Virginia Walker and Kate Buckeridge for their comments.

Faith’s index of phylogenetic

diversity

(Faith. 1992. Biological Conservation)

Total Tree Length = 38Red Length = 34Blue Length = 19

Red Phylodiversity = 34/38 = 89.5%

Blue Phylodiversity = 19/38 = 50.0%

0

20

40

60

80

100

All soils 4.0-4.5 4.5-5.0 5.0-5.5 5.5-6.0 >6.0

Rel

ativ

e ab

unda

nce

(%)

VerrucomicrobiaGemmatimonadetesDelta-proteobacteriaGamma-proteobacteriaBeta-proteobacteriaAlpha-proteobacteria

Others

Bacteroidetes

ActinobacteriaAcidobacteria

Soil pH

How did the phylum distributions relate to soil pH?

Five dominant phyla accounted for >85% in each of the soils.

Did the relative abundance of the major phyla correlate with soil pH?

Soil pH

Acidobacteriar = -0.816, P < 0.001

0

10

20

30

40

50

60

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%)

Alphaproteobacteriar = 0.749, P < 0.001

0

10

20

30

40

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%)

Actinobacteriar = 0.371, P = 0.014

0

5

10

15

20

25

30

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%)

Betaproteobacteriar = 0.479, P = 0.001

0

4

8

12

16

20

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%) Soil pH

Bacteroidetesr = 0.329, P = 0.031

0

3

6

9

12

15

4 5 6 7 8R

elat

ive

abun

danc

e (%

)

Acidobacteriar = -0.816, P < 0.001

0

10

20

30

40

50

60

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%)

Alphaproteobacteriar = 0.749, P < 0.001

0

10

20

30

40

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%)

Actinobacteriar = 0.371, P = 0.014

0

5

10

15

20

25

30

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%)

Betaproteobacteria

r = 0.479, P = 0.001

0

4

8

12

16

20

4 5 6 7 8

Rel

ativ

e ab

unda

nce

(%)

Bacteroidetes

r = 0.329, P = 0.0310

3

6

9

12

15

4 5 6 7 8R

elat

ive

abun

danc

e (%

)

Did bacterial diversity correlate to other environmental variables?

• Bacterial diversity strongly correlated to soil pH, and inversely correlated soil C/N ratio,

• No significant correlations with other environmental variables.

R2 Lat.Soil

moist.Soil pH Soil C Soil N

Soil C/N DOC DON NH4+ NO3-

Avail. P MBC MBN MBP

Richness 0.01 0.03 0.54 0.08 0.01 0.22 0.00 0.10 0.03 0.01 0.05 0.04 0.00 0.00

PD 0.04 0.01 0.63 0.10 0.01 0.25 0.00 0.11 0.04 0.02 0.03 0.07 0.00 0.01

P<0.0001 P<0.01

Soil microbes are diverse, ubiquitous and abundant……yet our understanding of their spatial distribution patterns is quite limited, and we don’t know if biome definitions are useful for microbes.

A biome is a large ecosystem where the community of certain plants and animals live best because they are adapted to particular range of climate and soil conditions.

Biomes of North America

http://www.omicsonline.com/Archiv eJCSB/2009/February/01/JCSB2.7

4.pdf

In 2006: $10,000  = 100 bacteria identified in 

each of 30 samples

In 2010:$10,000 = 10,000 bacteria identified in 

each of 100 samples

Advances in DNA sequencing technologies (e.g. pyrosequencing) 

have revolutionized the field of microbial ecology.

DNA extracted from samples

AGCCTTAA…

GCTACCAT…

CGGATCAC…

CTCGATTC…

AGCCTTAA…

GCTACCAT…

CGGATCAC…

CTCGATTC…

AGCCTTAA…

GCTACCAT…

CGGATCAC…

CTCGATTC…

Sequences belonging to each sample identified 

using the barcodes

Pyrosequencing

of the ssu

rRNA

gene from the 

pooled sample (>750,000 reads per run)

PCR amplification of the 16S rRNA

gene with 

each sample receiving a unique 12 bp

error‐

correcting barcode

Barcoded

pyrosequencing

The problem: How do you interpret vast amounts of sequence/data?

First 9 of 330,000 sequences

What types of taxa/lineages are present? (taxonomic 

description)

What is the diversity? (alpha diversity)

How are taxa/lineages shared among samples? (beta diversity, 

community divergence)

Faith’s index of phylogenetic

diversity

(Faith. 1992. Biological Conservation)

Total Tree Length = 38Red Length = 34Blue Length = 19

Red Phylodiversity = 34/38 = 89.5%

Blue Phylodiversity = 19/38 = 50.0%

Lozupone

and Knight. 2005. Appl. Environ. Microbiol.

UniFrac

Distance = Fraction of the total branch lengths unique to any 

particular environment or sample