Ryan J. Newton Sandra L. McLellan

Bacterial Assemblage Differences between

Urban-impacted Waterways & Oligotrophic Lake Michigan

BACKGROUND PRIMARY FINDINGS

CENTRAL QUESTION

THE RESULTS

Linwood

2 Mile 5 Mile

Bradford

McKinley

Kinnickinnic River

Menomonee River

Milwaukee River

N

Junction

3.5 Mile1 Mile

Doctor’sIn

Doctor’sMid

Doctor’sOut

Harbor wallsSampling site

How does urbanization interface with & impact aquatic microbial communities? Lake Michigan

Urban Estuary and Lake Michigan Communities are Distinct

Stormwater - A Unique Urban Bacterial Assemblage

to contact, email - newtonr@uwm.edu

ACKNOWLEDGEMENTS & REFERENCES

Within-Genus Habitat Specialization

OLIGOTYPING EXPLAINED1

www.uwm.edu/freshwater

Urbanization alters watershed ecosystem functioning through the movement, magnitude, and content of surface water runo�. However, there is still much to be learnt about the relationship between aquatic mi-crobial communities and urbanization. For example: What are the microbial biodiversity patterns associ-ated with urban areas? and Do urbanized waterways promote the maintenance of organisms that impact human health or well-being? With urban areas rapidly expanding and cities looking to become more sustainable, it will be important to integrate aquatic microbial ecology into the development of urban ecological theory and practice.

Here we aim to further our understanding of the bacterial connectivity between urban discharge and surface waters and the in�uence urbanized waterways have on community assemblages. We examine the prevalence of bacteria from urban stormwater in receiving water bodies and identify community assem-blage di�erences between a heavily urbanized estuary and a low urban-impacted oligotrophic lake.

We used massively-parallel sequencing (454 and illumina) of bacterial 16S rRNA genes (v6 region) ampli�ed from whole-community DNA extractions to characterize and compare the surface water bac-terial assemblages from stormwater, Milwaukee’s estuary, and nearshore Lake Michigan.

Stormwater area

Stormwater area 5,000 meters

Top left: Milwaukee estuary channel, looking toward Lake Michigan. Bottom left: Estuary discharge into Lake Michi-gan. Right: Map of sampling locations.

SAME14

acI-A

acI-B

Sediminibacterium

LD12

Limnohabitans

Polynucleobacter 1 10 100 1000 10000

110

100

1000

1000

0

Count in Lake Michigan Samples (abundance + 1)

Coun

t in

Urb

an E

stua

ry S

ampl

es

(abu

ndan

ce +

1)

-0.4 -0.2 0.0 0.2 0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

MDS1

MD

S2

Lake Michigan

Urban Estuary

Stormwater

NMDS plot of the bacterial taxonomic composition in samples from Lake Michigan (blue), the Milwaukee es-tuary (green), & Milwaukee stormwater outfalls (gold). Taxonomic a�liations for sequence reads were assigned with GAST5. Stress=0.03, dim. = 5

Minimum entropy decomposition2 was used to cluster se-quences into oligotypes/OTUs. LEfSe3 was then used to identify biomarkers (i.e. OTUs) that di�erentiated the urban stormwater environment from the other aquatic envi-ronments4. �e distribution of those biomarker OTUs are shown below in a boxplot (min, median, max, 1st, & 3rd quartiles indicated) & barplot (mean & std. dev. depicted).

20%

15%

10%

5%

0%

Between Enivronments

*

Urban EstuaryPreferred

Lake Michigan Preferred

4 2 2 4 6 8 10 121

**

*

*

*

*

*

AbundanceRatio

acI-AacI-BacI-C

acI-TH1acI-STL

AquilunaArcicella

AlgoriphagusFlavobacterium

FluviicolaSediminibacterium

LD12Sphingopyxis

HydrogenophagaLimnohabitans

PolynucleobacterRhodobacterLuteolibacter

Sample EnvironmentUrban EstuaryLake Michigan

Mann-Whitney U

68 ± 4037 ± 14

379*

Inverse Simpson Tail6

584 ± 463145 ± 85

370*

Mean & standard deviation are reported for diversity measures. Sequence data was grouped by closed-reference clustering. * indicates p<0.01

Right: Magnitude of a “habitat preference” for common freshwater genera/lineages. Habitat preference is deter-mined by the ratio of the mean relative abundance of each genus/lineage in the urban estuary samples vs. the Lake Michigan samples. Samples are normalized to max abundance for each sample transect. An asterisk indicates a signi�cant association with habitat (Mann-Whitney U test, p<0.01).

Alpha Diversity Estimates Habitat Preference

Urban Estuary Lake Michigan GeneralistSpecialist - Urban EstuarySpecialist - Lake MIRare

Left: Bar plot of oligotype composition within common freshwater genera/clades. 6 of 18 examined genera/clades are illustrated. Not depicted: acI-C, acI-TH1, acSTL, Aquiluna, Arcicella, Algoriphagus, Flavobacterium, Fluviicola, Sphin-gopyxis, Hydrogenophaga, Rhodobacter, & Luteolibacter. Right: Oligotype habitat preference classi�cation results derived from the CLAM7 statistical approach, which implements a multinomial model for group classi�cation.

Important Points:• 80 of 351 oligotypes classi�ed as habitat specialists 51 for urban estuary and 29 for Lake Michigan

• Numerous urban estuary specialists from: Flavobacterium, Hydrogenophaga, Limnohabitans, & Rhodobacter

• Numerous Lake Michigan specialists from: acIA, acIB, acIC, acTH1, & Fluviicola

• 7 of 18 genera/lineages harbored oligotypes with ≥96% sequence identity, but opposite habitat specialization

4. For more info see Fisher et al., (2015) Elementa 3:000064

5. Huse et al., (2008) PLoS Genet. 4.6. Li et al., (2012) PLoS ONE 7(6):e32118.

1. Modi�ed from: Eren et al., (2013), Method. Ecol. Evol. 4:1111-2. Eren et al., (2015) ISME J. 9:968- 3. Segata et al., (2011) Genome Biol. 12

We would like to thank the crew of the R/V Neeskay and everyone in the McLellan lab who has contributed to sampling e�orts. We also thank Mitch Sogin’s group at the Marine Biological Laboratory for DNA sequening and data processing assistance and Jen Fisher for her work on the stormwater data.

DatasetGAACCTCTAAGAAAGGTTGAACCTCTAAGAAAGGTTGAACCACTAAGAAACGTTGAACCACTAAGAAACGTTGAACCTCTAAGAAAGGTTGAACCTCTAAGAAAGGTTGAACCACTAAGAAACGTTGAACCCCGGAGAAAGGTT

Sample 1

Sample 2

Entropy Analysis

GAACCTCTAAGAAAGGTT

1

Ent

ropy

OligotypingSample 1

0

Sample 2

OligotypesT A C

If further entropy

decomposition is needed

or

Stop

Repeat

• Urban stormwater systems contain a diverse & variable bacterial assemblage, but is an environment with its own identi�able bacterial community signature.

• Stormwater contributes signi�cantly to the organisms present in Milwaukee’s urban estuary bacterial assemblage (massive immigration - up to 20% of bacterial 16S rRNA genes).

• Milwaukee’s urban estuary community was distinct from that of Lake Michigan: 1. Estuary harbors a more diverse & variable community 2. Shifted numerical dominance among cosmopolitan freshwater lineages 3. Within-genus composition change - habitat specialization

Lake MI Estuary

% o

f bac

teria

l com

mun

ity

15%

10%

5%

0%Dry Rain

Estuary - Weather Scenarios

CSO

Dry = no rain in a 48 hr. period.CSO = sewage over�ow

-0.1

0.0

0.1

0.2

0.3

NM

DS

2

-0.2 -0.1 0.0 0.1 0.2MDS1

0.3

Lake Michigan

Oligotrophic lakes

Eutrophic lake

Urban Estuary

NMDS plot of oligo-type composition, for the 18 freshwater taxa mentioned above. Samples include three additional Wisconsin lakes (squares): Lake Mendota, Trout Lake, & Sparkling Lake.

SCHOOL OF FRESHWATER SCIENCES

What about other lakes?

• Further exploration - do urban areas select for genetic compositions or functional traits that di�er from un-impacted waters? or promote the maintenance of microbes that impact human health?

7. Chazdon et al., (2011) Ecology 92:1332-

Refs:

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