CBIOMES e-meeting (Dec 10th, 2025)

Jesse McNichol, St. Francis Xavier University (StFX)

Implications of Prokaryotic Microdiversity for Ecosystem Functional Resilience - an Empirical Approach

NASA PACE

Julia Kadel, Unsplash

Biological data contains nested diversity

What is the right level for understanding ecosystem function?

https://simonscmap.com/catalog/datasets/GRUMP

Ecologically-relevant annotations aggregate complex ASV data into sensible groupings

Bacterioplankton
Phytoplankton
- Prochlorococcus (ecotype)

McNichol, Williams, et al., 2025

Biological data contains nested diversity

What is the right level for understanding ecosystem function?

One strategy: aggregate to broad "guild" level

But what about "microdiversity"?

A reservoir of diversity that can buffer environmental change, maintaining ecosystem function?
Neutral genetic variation?

In many cases, we don't know...

Ecosystem

Metagenomics

Work thus far in literature:

If metagenomes show different organisms have similar metabolic pathways → ecosystem resilience
My view: maybe, but this is "potential", not measured resilience
Ignores other aspects of microbial niches (T optimum, for e.g.)

But what about "microdiversity"?

A "model ecosystem" in Cape Breton

Me aboard Dr. Bruce Hatcher's research vessel Exocet in 2024

Stratified basin with anoxic, sulfidic waters ~ 17 m (historically ~1000 μM H₂S)
Temperature and oxygen gradients very steep at redoxcline, system has recently overturned in winter 2024 (?) causing a fish kill
With Katherine Rutherford (StFX) we are using metabarcoding, metagenomics to understand taxa present, dynamics, functional potential

W. Whycocomagh Bay

A "model ecosystem" in Cape Breton

A "model ecosystem" to ask questions about the link between diversity and function

Lots of genetic diversity, but relationship between diversity & function poorly known (some exceptions, e.g. Prochlorococcus)

A "model ecosystem" to ask questions about the link between diversity and function

...it is unclear and controversial how multiple disturbances affect microbial community stability and what consequences this has for ecosystem functions."

In this ecosystem, sulfur oxidation is an "ecosystem service"
"Microbial firewall"
Prediction: stable stratification means limited functional diversity
If disturbance (e.g. water overturning) occurs → limited resilience of function
Goal: experiments to generate empirical data to test this prediction

How resilient are microbial ecosystem services to disturbance?

W. Whycocomagh Bay

Does high genetic "microdiversity" observed in natural microbial ecosystems confer functional resilience of H₂S detoxification during deep water mixing, or will rapid changes in environmental conditions slow or stop this critical ecosystem service?"

A simple question

A simple question ... but how to answer it?

Vincent and Vardi, 2023

A "middle way" for understanding microdiversity

Pure cultures

Pros: Highly controlled

Cons: Selects for "lab rats", reduced diversity vs. environment

Environmental 'omics

Pros: Observe everything

Cons: Static measurement, limited functional information

Vincent and Vardi, 2023

A "middle way" for understanding microdiversity

Short-term incubation experiments of seawater

Pro: Generate empirical data for testing hypotheses

Con: The longer you incubate, the less the composition resembles the natural community. Need very short-term incubations, and single-cell activity measurements

McNichol, Dyksma et al., 2022

A "middle way" for understanding microdiversity

rRNA-FISH & FACS + ¹⁴CO₂ =

taxon-specific C-fixation
response to different chemical conditions in short-term incubations

How we plan to do the work

[H₂S]

[O₂]

depth

EUX ~3°C

RXL ~10°C

SRF ~22°C

Field sampling

DNA

-3DMB

-MAGs

-FL-16S

Lab processing

OOI

(function,

microdiversity)

OOI-specific measurements:

-Growth rate

-Respiration

-Resilience

Field incubations

FISH probes

(broad group level,

microdiverse subcluster level)

Katherine Rutherford (StFX)

Bruce Hatcher (CBU)

3DMB=3-domain metabarcoding, FL-16S=full-length 16S (PacBio)

SRF=surface, RXL=redoxcline, EUX=Euxinic (sulfide-rich waters)

Street map = meta'omics data from Whycocomagh (which organisms are involved in sulfur cycling)
Allows us to design FISH probes for particular organisms (including microdiversity within clade)
FISH probes allows us to derive:
- Cell size, biovolume, morphological variability
- In situ growth rates
- Metabolic rates during incubations

How we plan to do the work

How we plan to do the work: in situ growth rates

FISH probes + FODC method → OOI growth rate (including microdiverse subclusters)

Could be compared with MAG estimates (gRodon)

How we plan to do the work: incubations

[H₂S]

[O₂]

depth

EUX ~3°C

RXL ~10°C

SRF ~22°C

Simulate upwelling to surface by subjecting RXL or SRF communities to increased [H₂S], temp, [O₂] (or combinations of all) by mixing at different ratios and incubating at controlled temperatures for ~6-12 h. Response (compared to control) measured by:

Single-cell respiration rates
Bulk H₂S oxidation
Bulk community respiration w/ spot optodes

in situ conditions (control)

"disturbance" conditions

How we plan to do the work: organism-specific respiration

FISH probes + Redox Sensor Green → OOI respiration rate changes after short-term incubations

Munson-McGee et al., 2022

Possible outcomes (null)

Cell-specific respiration rate (RSG)

Killed

NIC

RXL @ RXL

NIC=No incubation control + RSG | RXL @ RXL=redoxcline water incubated at in situ conditions

RXL @ SRF=redoxcline water incubated at surface conditions (increased O₂, temp)

RXL @ SRF

Possible outcomes (limited resilience)

Cell-specific respiration rate (RSG)

Killed

NIC

RXL @ RXL

NIC=No incubation control + RSG | RXL @ RXL=redoxcline water incubated at in situ conditions

RXL @ SRF=redoxcline water incubated at surface conditions (increased O₂, temp)

RXL @ SRF

Possible outcomes (more activity with upwelling)

Cell-specific respiration rate (RSG)

Killed

NIC

RXL @ RXL

NIC=No incubation control + RSG | RXL @ RXL=redoxcline water incubated at in situ conditions

RXL @ SRF=redoxcline water incubated at surface conditions (increased O₂, temp)

RXL @ SRF

Possible outcomes (incubations not working)

Cell-specific respiration rate (RSG)

Killed

NIC

RXL @ RXL

NIC=No incubation control + RSG | RXL @ RXL=redoxcline water incubated at in situ conditions

RXL @ SRF=redoxcline water incubated at surface conditions (increased O₂, temp)

RXL @ SRF

Next steps

[H₂S]

[O₂]

depth

EUX ~3°C

RXL ~10°C

SRF ~22°C

in situ conditions (control)

"disturbance" conditions

Winter 2026:

Identify targets from bioinformatics, optimize FISH probes

Spring / summer 2026:

Apply incubation approach with FISH probes targeting organisms of interest

What knowledge we can gain?

An inventory of sulfur oxidation potential in system from metagenomics (Illumina + Nanopore)
Spatiotemporal diversity patterns (stochastic? predictable?)
In situ growth and respiration rates (FODC, RSG)
Resilience to realistic disturbances across diversity
"Traits" we can use to model future disturbance

STAY TUNED over the next 2-3 years!

Does high genetic "microdiversity" observed in natural microbial ecosystems confer functional resilience of H₂S detoxification during deep water mixing, or will rapid changes in environmental conditions slow or stop this critical ecosystem service?"

Much to learn!

Questions?

Thanks to:

Current and former CBIOMES collaborators (Fuhrman, Follows, Levine, Zakem labs)
Dr. Bruce Hatcher (CBU)
Katherine Rutherford (StFX)
All of you for listening!

stfxmicroeco.ca | jmcnicho@stfx.ca

https://slides.com/jcmcnch/cbiomes-e-seminar-2025/

Summary of incubation conditions

Ecosystem maps from eDNA

Ecosystem DNA

A Map

Metabarcoding

Vincent and Vardi, 2023

Ecosystem maps from eDNA → tackling big questions

Ecosystem maps can be cheaply generated from a "quiver" of DIY methods that I like to use

Kit-free, non-toxic DNA extraction with linear acrylamide as co-precipitant, allows for recovery of very small amounts of DNA for PCR (~10 mL seawater*)
Cheap, simple protocol for removal of PCR inhibitors with linear acrylamide (for difficult samples, Zymo 1-step is cheap & works)
Protocols for decontamination of reusable plasticware for molecular protocols (reduce waste, guarantee quality)
1-step PCR reaction for library prep in lab (advantages = speed, lower cost, flexibility)

*Other methods for as little as 1 µL exist

A story about ecosystem maps + meta'omics + models

Zakem, McNichol, Weissman, Raut et al., 2025

Figure 1: Overview of sampling and data processing workflow. Oxygen and sulfide depth profiles shown in blue and orange, respectively. SRF=surface, RXL=redoxcline, EUX=euxinic. 3DMB=3-domain metabarcoding, MAGs = metagenome assembled genomes, FL-16S = full-length 16S. OOI = organism of interest. FISH = fluorescence in-situ hybridzation.

[H₂S]

[O₂]

depth

EUX ~3°C

RXL ~10°C

SRF ~22°C

DNA

-3DMB

-MAGs

-FL-16S

OOI

(function,

microdiversity)

(broad group level,

microdiverse subcluster level)

FISH probes

OOI-specific measurements:

-Growth rate

-Respiration

-Resilience

Field sampling

Lab processing

Field incubations

Vincent and Vardi, 2023

Coastal systems are flexible: experiment across scales, depending on question

GRUMP 2.0 = a story about absolute units

Slide credits: Dr. Lexi Jones-Kellett (aejk@alum.mit.edu)

GRUMP 2.0 = a story about absolute units

Slide credits: Dr. Lexi Jones-Kellett (aejk@alum.mit.edu)

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