Funding

eDNA - a tool for efficient biodiversity monitoring

from rare species to communities and back

Bernd Hänfling

eDNA barcoding and metabarcoding eDNA barcoding:

• Targeted detection • Species-specific primers • Standard or qPCR • Traditional Sequencing

=

Free-floating DNA (from sloughed skin cells faeces/urine, gametes, decaying matter) and

microscopic taxa

Water sampled & filtered (>3 x 15 ml -2L samples)

eDNA metabarcoding: • Screen whole community • (Several) conserved primers • PCR • Next generation sequencing

= Extract DNA

eDNA metabarcoding of lake fish

Aim: • To develop and test eDNA metabarcoding

methods to describe lake fish communities. Motivation: • Water Framework Directive (WFD) lake fish

monitoring. • Currently not carried out to the degree

required by the WFD due to methodological considerations.

• Cost and time • Ethics (gill nets are destructive) • Practicalities on large lakes

Phase I: • Sampling: January 2015

Phase II: • Sampling: September 2015/January2016

eDNA metabarcoding of lake fish

Phase I – sampling: • Three Cumbrian lakes

• Windermere • Derwent Water • Bassenthwaite Lake

• 2L water samples along boat transect at

2m, 5m & 20m depths, as well as shore

sampling.

• Filtered on 0.45 μm membrane filters.

• Two mitochondrial gene fragments (12S

and Cytochrome b) sequenced at CEH on

an Illumina MiSeq

eDNA detects lake fish presence

Previously (ever) recorded species: 16 eDNA metabarcoding (Jan-15): 14 (88%)

Gill netting survey 2014: 4 (25%)

Gill netting survey 2015: 5 (31%)

0

0.2

0.4

0.6

0.8

1

12S freq

cytb freq

Prop

ortio

n of

site

s pre

sent

Lake Windermere, January 2015 (65 samples)

Hänfling et al. 2016, Molecular Ecology

Prop

ortio

n of

site

s pre

sent

0

0.2

0.4

0.6

0.8

1

12S freq

cytb freq

Lake Windermere, January 2015 (65 samples)

Species not previously recorded 12S: 6 species Cytb: 3 species

eDNA detects lake fish presence

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2 4 6 8 10 12 14 16 18

January 2015

Long term rank (based on 30 years of established method surveys)

eDN

A ab

unda

nce

(site

occ

upan

cy)

eDNA reflects species abundance

Windermere both basins

r = - 0.77; P < 0.001

• Perch detected throughout lake

• Arctic charr predominantly North Basin

• Bream more common in South Basin

Spatial distribution of eDNA

Occupancy data demonstrate that species are not equally distributed across the lake e.g.

eDNA distribution reflects fish ecology

Oligotrophic association

Eutrophic association

No association

0

5

10

15

20

25

30

1945 1955 1965 1975 1985 1995 2005Year

Mea

n SR

P in

firs

t 4 w

eeks

(mg

m-3

)North basin

South basin

Hänfling et al. 2016, Molecular Ecology

Mesotrophic

Eutrophic-mesotrophic

eDNA distribution reflects fish ecology

• Large and common species found consistently across transects

• Small littoral species predominantly in shallower habitats

• Arctic charr was only found in midwater and bottom samples

Shore-west 5m

20m Midline

Shore-East

0

0.2

0.4

0.6

0.8

1

eDN

A ab

unda

nce

(12S

)

Windermere North Basin

Sampling in Phase 2

eDNA detection of rare species Vendace (Coregonus albula) in Cumbrian lakes

• Five offshore and one shore line sample in each lake

• Positive detection in two Derwent Water sites, but none in Bassenthwaite Lake (only three individuals caught since 2000)

• Is qPCR more sensitive compared to metabarcoding? Ongoing work with Glenn Rhodes (CEH)

PhD studentship available (UoH collaboration with CEH and SNH): “eDNA monitoring of conservation priority fish in UK freshwaters” Deadline for applications 3rd June 2016

Derwent Water

Bassenthwaite Lake

Summary

• eDNA metabarcoding is highly sensitive for fish species detection and outperforms established methods even in oligotrophic lakes

• Relative abundance estimates from eDNA and long term data correlate strongly.

• eDNA can tell us about species distribution on a small geographical scale and provide important ecological information

• Some differences between shore and offshore sampling… important for monitoring strategies

• Metabarcoding may just be as powerful as single species approaches for detecting rare species

• eDNA based metabarcoding has a huge potential for large scale biodiversity monitoring and to provide data to answer fundamental ecological questions

So long and thanks for all the fish help … UoH team: Dr. Lori Lawson-Handley PostDocs: Dr. Christoph Hahn Dr. Helen Kimbell Dr. Paul Nichols Dr. Harriet Johnson Dr. Hayley Watson Dr. Rose Wilcox Dr. Rob Donnelly PhD students: Joe Li Rosie Blackman Marco Benucci Lynsey Harper

CEH team: Dr. Ian Winfield Dr. Dan Read Dr. Anna Oliver Ben James Janice Fletcher

FBA and MEFGL Bangor for use of facilities!

EA, SEPA and NRW team: Dr. Kerry Walsh Dr. Graeme Peirson Dr. Willie Duncan Dr. Alistair Duguid Dr. Tristan Hatton Ellis