Eco-evolutionary

dynamics of microbial
communities in disturbed
freshwater ecosystems
Naíla Barbosa da Costa
Soutenance de thèse UdeM
25 nov. 2022
Mont Saint-Hilaire, the Gault reserve and lake Hertel (Google Earth)
 Thesis
structure

2
Chapter 1: introduction of agricultural
impacts in ecosystems

Thesis
structure

3
Chapter 1: introduction of agricultural Chapter 2: bacterioplankton ecological
impacts in ecosystems responses driven by pesticides contamination

BEFORE AFTER

Thesis
structure

4
Chapter 1: introduction of agricultural Chapter 2: bacterioplankton ecological
impacts in ecosystems responses driven by pesticides contamination

BEFORE AFTER

Thesis
structure

Chapter 3: changes in genes composition in

response to glyphosate based-herbicide (GBH)

5
BEFORE AFTER
Chapter 1: introduction of agricultural Chapter 2: bacterioplankton ecological
impacts in ecosystems responses driven by pesticides contamination

BEFORE AFTER

Thesis
structure

Chapter 4: evolutionary responses to Chapter 3: changes in genes composition in

GBH contamination response to glyphosate based-herbicide (GBH)

6
BEFORE AFTER BEFORE AFTER
 Agricultural land use
• Largest use of land on the planet (40% of its land surface)
• Major contribution to environmental changes

Cropland distribution across the world (USGS GFSAD30 Project)

7
The herbicide glyphosate
• GBHs are the most widely used herbicides
worldwide
• Use intensified after introduction of genetically
modified crops: Roundup Ready ® technology

8
 The herbicide glyphosate
Shikimate
pathway • GBHs are the most widely used herbicides
worldwide
• Use intensified after introduction of genetically
modified crops: Roundup Ready ® technology
Competitive
inhibitor
• Glyphosate targets EPSPS enzyme

Aromatic amino acids

9
 The herbicide glyphosate
Shikimate
pathway • GBHs are the most widely used herbicides
worldwide
• Use intensified after introduction of genetically
modified crops: Roundup Ready ® technology
Competitive
inhibitor
• Glyphosate targets EPSPS enzyme

• EPSPS variation in bacteria

• Sensitive (class I) and resistant (classes II, III
and IV) to glyphosate
• Genetically modified crops receive a transgene
from Agrobacterium
Aromatic amino acids
10
 Agricultural pollution
• Aquatic environments receive mixtures of agrochemicals (eg. fertilizers and
pesticides) mainly through leaching and runoff
• Reach non-target freshwater communities

11
Bacterioplankton
• If agrochemicals affect non-target
microorganisms they may lead to changes
in ecosystem functions

• They catalyze important reactions in

biogeochemical cycles

• Bacteria as interesting models to study

perturbations à fast responses time
• High adaptability, large mutation supply,
large population sizes
12
 Thesis general objective
• Investigate how agricultural contamination drive bacterial responses in
freshwater mesocosms at the level of:
• Communities (chapter 2)
• Genes (chapter 3)
• Populations/genomes (chapter 4)

Thesis
structure

13
Large Experimental Array of Ponds (LEAP) platform

Inflow
reser vo
Tank ir
s of ~
1000L
asin
tion b
Reten

14
LEAP in the Gault reserve © Vincent Fugère
Large Experimental Array of Ponds (LEAP) platform

Inflow
reser vo
Tank ir
s of ~
1000L
asin
Large scale 96-well
tion b

plate experiment: with

Reten

natural microbial
communities (plankton)
15
LEAP in the Gault reserve © Vincent Fugère
 LEAP: large scale outdoor tanks (mesocosms)
• Combine features of field and laboratory methodologies
• Allow to estimate causal effects of controlled variables under non-controlled
environmental conditions

Controlled addition of
contaminants

16
Timeline 3 months before All ponds filled
Days sampled for with lake water
DNA sequencing

Day 1: 1st sampling day (no pulse)

Day 6: pulse 1
Phase I

Day 34: pulse 2

Day 43
Day 44: pulse 3

Day 57: last sampling day

17
Timeline 3 months before All ponds filled Treatments in phase I (6 weeks)
Days sampled for with lake water A
Glyphosate (mg/L)
DNA sequencing

}
0 0.04 0.1 0.3 0.7 2.0 5.5 15
Day 1: 1st sampling day (no pulse)
Imidacloprid (μg/L)
Day 6: pulse 1
Replicated in
low and high
0 0.15 0.4 1.0 3.0 8.0 22 60 nutrient
Phase I
backgrounds
Glyphosate (mg/L) and imidacloprid (μg/L)

0/0 0.04/0.15 0.1/0.4 0.3/1.0 0.7/3.0 2.0/8.0 5.5/22 15/60

Day 34: pulse 2 B

Gradient
Microbial carbon
from environmentally relevant to toxic*
Pulse 1 Pulse 2

● ● ● ● ● ●
substrate use
Bacterioplankton concentrations
Day 43 ● ● ● ● ● ●

Day 44: pulse 3 (DNA and density)

Regulatory
Total nitrogen Acceptable Concentration in Canada (CCME):
● ● ● ●

- TotalGlyphosate
phosphorus 800 µg/L long-term exposure
● ● ● ● ●

- Glyphosate
Imidacloprid
27 mg/L short-term exposure
● ● ● ●

Day 57: last sampling day - Imidacloprid

Glyphosate
0.23 µg/L interim● ● ● ●

*For phytoplankton or zooplankton

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Day of experiment
18
Timeline 3 months before All ponds filled
Days sampled for with lake water
DNA sequencing Treatments in phase I vs. II
Day 1: 1st sampling day (no pulse)
Day 6: pulse 1
Phase I

Day 34: pulse 2

Day 44: pulse 3

Phase II

Day 57: last sampling day

19
Methodology by chapter
• Same experiment was used to answer questions of the 3 main chapters, with
variations in:
• The set of samples/treatments selected for each study
• Chap 2: all treatments Phase I
• Chap 3 & 4: glyphosate treatments Phase I and II

20
Methodology by chapter
• Same experiment was used to answer questions of the 3 main chapters, with
variations in:
• The set of samples/treatments selected for each study
• Chap 2: all treatments Phase I
• Chap 3 & 4: glyphosate treatments Phase I and II
• The methodological approach (sequencing technology + analyses)
• Chap 2: 16S rRNA gene sequencing, flow cytometry (bacterial density), and BiOLOG EcoPlates
(functional profiles)
• Chap 3 & 4: shotgun metagenomics, assembly, binning, Metagenome-Assembled Genomes (MAGs)
• Chap 3: genomes annotation (prediction of genes)
• Chap 4: inference of Single Nucleotide Variants (SNVs) within genomes

21
Objectives by chapter
• Chapter 2: investigate ecological responses of bacterioplankton to the
exposure to a GBH and the neonicotinoid insecticide imidacloprid at different
nutrient backgrounds

Thesis
structure

22
Objectives by chapter
• Chapter 2: investigate ecological responses of bacterioplankton to the
exposure to a GBH and the neonicotinoid insecticide imidacloprid at different
nutrient backgrounds
• Chapter 3: test if GBH doses cross-selected for antibiotic resistance genes
(ARGs)

Thesis
structure

23
Objectives by chapter
• Chapter 2: investigate ecological responses of bacterioplankton to the
exposure to a GBH and the neonicotinoid insecticide imidacloprid at different
nutrient backgrounds
• Chapter 3: test if GBH doses cross-selected for antibiotic resistance genes
(ARGs)
• Chapter 4: investigate how intraspecific diversity changes in response to GBH
contamination, connecting evolutionary dynamics to ecological responses

Thesis
structure

24
 Chapter 2
Resistance, resilience, and functional
redundancy of freshwater
bacterioplankton communities facing
a gradient of agricultural stressors in
a mesocosm experiment

Barbosa da Costa et al. 2021 in Mol Ecol

 Chapter 2: research motivation
Ecotoxicological assessments
• Laboratory assays in small scale
• Single targeted species
• Isolated stressors
Our research
• Large-scale array of mesocosms
• Effect on the whole community
• Combination of stressors
26
 Chapter 2: research questions and highlights
• How bacterioplankton is affected by one or multiple stressors?
• Bacterial density à slightly increased with addition of glyphosate-based herbicide (GBH)
• Alpha diversity à no significant time-dependent effect of any treatment
• Community composition à highest dose of GBH treatment changed taxonomic
composition, no effect of imidacloprid or nutrients

Before GBH After GBH 27

 Chapter 2: research questions and highlights
• How bacterioplankton is affected by one or multiple stressors?
• Bacterial density à slightly increased with addition of glyphosate-based herbicide (GBH)
• Alpha diversity à no significant time-dependent effect of any treatment
• Community composition à highest dose of GBH treatment changed taxonomic
composition, no effect of imidacloprid or nutrients
• Functional diversity (carbon substrate use) à has not been altered by GBH
besides changes in taxonomic composition: evidence of functional redundancy

Before GBH After GBH 28

lse 1 Pulse 2 Pulse 1 Pulse 2

Chapter 2: community resilience at phylum level sp2262

sp188 (
A Phylum
B sp1895
ASV sp2118
Proteobacteria sp2155
sp307 (
1.0

Pulse 1 Pulse 2 Pulse 1 Pulse 2

sp284 (

0.4
sp130 (
sp2262 (Alphaproteobacteria)
Cyanobacteria sp188 (Lhab-A4)
0.5

sp1895 (Azospirillum massiliensis)

Actinobacteria sp283 (

0.2
sp2118 (alfVI)
Proteobacteria
Treatment effect

Treatment effect
sp2155 (Agrobacterium) sp2111
sp307 (Nevskia ramosa)
sp284 (betI-A)
Bacteroidetes sp130 (betIV-A)
0.0

0.0
Cyanobacteria
Actinobacteria sp283 (betI-A)
sp2111 (Rhodobacter)

-0.2
-0.5

Bacteroidetes

-0.4
-1.0

0 10 20 30 40 0 10 20 30 40

Day of experiment Day of experiment

Pesticide treatment
Pesticide treatment
Glyphosate (0.3 mg/L) Imidacloprid (1 μg/L) Glyphosate (0.3 mg/L) and imidacloprid (1 μg/L)
Glyphosate (0.3 mg/L) Imidacloprid (1 μg/L) Glyphosate (0.3 mg/L) and imidacloprid (1 μg/L)
Glyphosate (15 mg/L) Imidacloprid (60 μg/L) Glyphosate (15 mg/L) and imidacloprid (60 μg/L)
Glyphosate (15 mg/L) Imidacloprid (60 μg/L) Glyphosate (15 mg/L) and imidacloprid (60 μg/L) 29
 Chapter 2: take-home
• GBH was the main driver of community changes
• Glyphosate treatments favored members of Proteobacteria, including Agrobacterium, known to
have a resistant target enzyme (EPSPS)
• Community resilience depends on phylogenetic depth
• Functional diversity remained stable besides changes in composition: evidence of
functional redundancy

30
 Chapter 2: take-home
• GBH was the main driver of community changes
• Glyphosate treatments favored members of Proteobacteria, including Agrobacterium, known to
have a resistant target enzyme (EPSPS)
• Community resilience depends on phylogenetic depth
• Functional diversity remained stable besides changes in composition: evidence of
functional redundancy
• It may be different in already impacted communities with lower richness

Lake Champlain during a bloom

(2016) © Inès Levade Lake Hertel in the Gault Nature Reserve (fall 2016) 31
BEFORE AFTER
Chapter 3
A glyphosate-based herbicide
cross-selects for antibiotic
resistance genes in
bacterioplankton communities
BEFORE AFTER
Barbosa da Costa et al. 2022 in mSystems
 Chapter 3: research motivation and question
• Detection of species with the sensitive EPSPS thriving in GBH treatments
• Could other genes be indirectly selected by glyphosate?

Penicillin
Glyphosate

Antibiotics

DIRECT SELECTION INDIRECT SELECTION

33
 Chapter 3: research motivation and question
• Evidences of ARGs cross-selection in laboratory (isolates) and in a soil field study

34
 Chapter 3: research motivation and question
• Evidences of ARGs cross-selection in laboratory (isolates) and in a soil field study

What about
freshwater?
35
log10(Unique ARGs per mi
0.10

Chapter 3: ARGs frequency within communities

10 20 30 40 50
Day of experiment
0.05

• ARG frequency increased in response to GBH pulses

0.00
Phase 0I − Pulse 1 Pulse 2 Phase II
• Highest GBH dose in Phase I and singleDaydose
10
in Phase II in all treatments
of experiment
20 30 40 50

B 3 Phase I − Pulse 1 Pulse 2 Phase II

log10(ARG reads per million reads +1)

0
10 20 30 40 50
0 10 20 30 40 50
Day of experiment
Day of experiment

Nutrient concentration
Nutrient concentration
high low high Pesticide
low treatment
Pesticide treatment Control Phase
Control II II
Phase Control
Control Phase
Phase I I Glyphosate
Glyphosate 0.3 mg/L 0.3 Glyphosate
mg/L Glyphosate 15 mg/L
15 mg/L

36
Chapter 3: ARGs in genomes predict species survival

• Species abundance in Phase II

−2

treatments depends on:

• Antibiotic resistance potential
−3

• EPSPS classification
MAG survival in Phase II (log10)

−4
• MAG abundance in Phase I
EPSPS putative classification
Resistant
Sensitive
Unclassified

−5

EPSPS putative classification

Resistant
−6
Sensitive
Unclassified

−7

0 1 2 3 4 5
MAG antibiotic resistance potential (RGI scrict hits)

37
Chapter 3: take-home
• Antibiotic efflux is the major mechanism of antibiotic resistance cross-selected
by GBH stress
• Corroborates with previous laboratory assays and soil metagenomic study
• GBH contamination may be an indirect pressure favoring ARGs in natural
communities
1 1

Glyphosate resistance might

Resistance mechanisms, as efflux pumps (1)
result of the cross-selection of
and target modification (2), might
additionally provide antibiotic resistance
ARGs whose mechanism is
based on efflux pumps (1)
Pesticides
Pesticidesapplied to fields
applied to fields
reach freshwater and
reach select
freshwater and select
resistant bacteria

glyphosate resistant
38
phenotypes
 Chapter 4
Genome-wide selective sweeps
rarely explain the ecological
success of bacterial populations
responding to a novel
environmental stress

Barbosa da Costa et al. 2022 in prep.

 Chapter 4: research motivation
• Stable ecotype model: evolutionary model of speciation
• Adaptive mutation (*) within a species spread through population after a periodic selection
• Assuming recombination is rare, the adaptive mutant and its clonal descendants replace the
rest of genetic diversity within the ecotype à genome-wide sweep

Mutation Consequence

Periodic
selection

Modified from Cohan & Perry (2007) in Current Biology

40
 Chapter 4: research motivation
• Stable ecotype model: evolutionary model of speciation
• Adaptive mutation (*) within a species spread through population after a periodic selection
• Assuming recombination is rare, the adaptive mutant and its clonal descendants replace the
rest of genetic diversity within the ecotype à genome-wide sweep
• Evidence of sweep in a freshwater lake
• In 9-year metagenomic time series 1 out of 30 bacterial populations went through a
genome-wide sweep (Bendall et al. 2016 ISME J)
• No selective pressure identified à not a selective sweep Mutation Consequence

Periodic
selection

Modified from Cohan & Perry (2007) in Current Biology

41
 Chapter 4: research questions
• Do GBH pulses purge diversity within species? How often do we observe
genome-wide sweeps in populations surviving to GBH contamination?
• Compared to Bendall et al. (2016) we have a much shorter time series (8-weeks) but a strong
selective pressure that promoted changes in the bacterial community and gene composition

42
Chapter 4: SNV profiling of 11 populations
• Intra-specific diversity changes à SNV frequency in genomes (SNVs/Mbp)
and the median MAF (minor allele frequency) after each pulse
• Six did not vary in intra-specific diversity after pulses 1, 2 or 3 (A-F)

A B C D E F
Prosthecobacter*a in pond C8 SYFN01c in pond D8 Roseococcusa in pond D8 Novosphingobiuma in pond D8 UBA2784a in pond D8 UBA2784a in pond C8
4 0.4

3 0.3

2 0.2

1 0.1
SNVs Mpb (log10)

0 0.0

Median MAF
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

G H I J K
Nevskia 1a in pond C8 Aquidulcibacterb in pond D8 Planktophila*b in pond D4 Allorhizobiuma in pond C8 Niveispirilluma in pond C8
4 0.4

3 0.3

Median MAF
2 0.2
SNVs/Mpb (log10)

1 0.1

0 0.0
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Pulse 43
Chapter 4: SNV profiling of 11 populations
• Intra-specific diversity changes à SNV frequency in genomes (SNVs/Mbp)
and the median MAF (minor allele frequency) after each pulse
• Six did not vary in intra-specific diversity after pulses 1, 2 or 3 (A-F)
• Three decreased (G-I) and two increased (J-K)
A B C D E F
Prosthecobacter*a in pond C8 SYFN01c in pond D8 Roseococcusa in pond D8 Novosphingobiuma in pond D8 UBA2784a in pond D8 UBA2784a in pond C8
4 0.4

3 0.3

2 0.2

1 0.1
SNVs Mpb (log10)

0 0.0

Median MAF
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

G H I J K
Nevskia 1a in pond C8 Aquidulcibacterb in pond D8 Planktophila*b in pond D4 Allorhizobiuma in pond C8 Niveispirilluma in pond C8
4 0.4

3 0.3

Median MAF
2 0.2
SNVs/Mpb (log10)

1 0.1

0 0.0
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Pulse 44
Chapter 4: SNV profiling of 11 populations
• Intra-specific diversity changes à SNV frequency in genomes (SNVs/Mbp)
and the median MAF (minor allele frequency) after each pulse
• Six did not vary in intra-specific diversity after pulses 1, 2 or 3 (A-F)
• Three decreased (G-I) and two increased (J-K): only two are glyphosate-sensitive
A B C D E F
Prosthecobacter*a in pond C8 SYFN01c in pond D8 Roseococcusa in pond D8 Novosphingobiuma in pond D8 UBA2784a in pond D8 UBA2784a in pond C8
4 0.4

3 0.3

2 0.2

1 0.1
SNVs Mpb (log10)

0 0.0

Median MAF
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

G H I J K
Nevskia 1a in pond C8 Aquidulcibacterb in pond D8 Planktophila*b in pond D4 Allorhizobiuma in pond C8 Niveispirilluma in pond C8
4 0.4

3 0.3

Median MAF
2 0.2
SNVs/Mpb (log10)

1 0.1

0 0.0
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Pulse 45
Chapter 4: genome-wide sweep in one population
• Among the two potentially glyphosate-sensitive
species (EPSPS classification and no ARG):
• A genome-wide sweep may have occurred in
Aquidulcibacter, a species favored by GBH treatment
(PRC species score)

• Planktophila is present after pulses but does not

thrive (low PRC species score) and alleles do not
show tendency to get fixed

46
Pulse
 Chapter 4: take home
• Ecological changes over short timescales are not always accompanied with
directional evolutionary changes such as genome-wide sweeps
• Different dynamics in glyphosate-resistant or sensitive species
• Sweep detected in the only sensitive population positively affected by GBH
• It remains to be confirmed if the ecotype selected within this population was resistant to glyphosate
(i.e. check for acquired mutations in the EPSPS gene or other resistance pathway)

Most individuals Were sensitive

are sensitive individuals purged?

3rd GBH
pulse Resistant
mutation?

Modified from Cohan & Perry (2007) in Current Biology

47
 Thesis highlights
• High concentrations of glyphosate shifted
bacterial community taxonomic - but not
functional - composition and cross-selected
for ARGs

48
 Thesis highlights
• High concentrations of glyphosate shifted
bacterial community taxonomic - but not
functional - composition and cross-selected
for ARGs

• ARGs may be useful for bacterial survival in

face of strong glyphosate stress

49
 Thesis highlights
• High concentrations of glyphosate shifted
bacterial community taxonomic - but not
functional - composition and cross-selected
for ARGs

• ARGs may be useful for bacterial survival in

face of strong glyphosate stress

• Selective genome-wide sweep may be a

process underlying adaptation to herbicide
contamination in glyphosate-sensitive species
50
 Thesis highlights
• High concentrations of glyphosate shifted
bacterial community taxonomic - but not
functional - composition and cross-selected
for ARGs

• ARGs may be useful for bacterial survival in

face of strong glyphosate stress

• Selective genome-wide sweep may be a

process underlying adaptation to herbicide
contamination in glyphosate-sensitive species
51
 Thesis contributions

l
g e ta
an e n
ch onm

Co
s

m
vi r

m
un
En
• Complexity of bacterial responses to

i ti e
s
agrochemical contamination while integrating
ecology and evolution with ecotoxicology

• Ecology, genetics and evolution à explain

interconnected processes that drive bacterial
responses to environmental changes Ge
s
n om e
es en
G

52
 Thanks!

Acknowledgment:
Shapiro & Fussmann labs
The LEAP team
GRIL & QCBS networks
Funding support:
LiberEro
CRC & CFI
FRQNT & NSERC
EcoLac
UdeM & McGill