Monitoring Barcoding Network and RecordingMatthew ShepherdSenior Specialist, Soil Biodiversity, Natural England

Why monitor soils?

• Soil science has concentrated on agricultural systems, physical and chemical status.

• Learn from (semi) natural habitats– Lessons for managed ecosystems– Own interest

• A quarter of all biodiversity is found in the soil

Why monitor soils?

• Many monitoring efforts (eg. ECN, RSS) have focussed on chemical or physical parameters– yet soil biology does all the work!

• New advice from UK SIC, Defra SQuID project• CS2007 – more soil and soil biological parameters than ever

before• Try to be compatible, representative and affordable

Why monitor soils?

• CS survey in 1998 and 2007 measured soil biological parameters• Measured tRFLP, soil mesofauna

Why monitor soils?

• ~12.8 quadrillion soil invertebrates present in the top 8 cm of GB soils

• significant increase in total invertebrate catch in all Broad Habitats• except for agricultural areas on mineral soils• Due to increase in the catch of mites in 2007 samples• small reduction in the number of soil invertebrate broad taxa (0-

8cm) recorded• different seasonal conditions – more work needed• Needs linking with habitat and chemistry work

• Oribatid data – Thanks to Aidan Keith – now have loose locaitons – secret data!

LTMN Soils Method

• 1 habitat per NNR for soil assessment

• 22 so far of ~43 total• 8 broadleaved

woodlands• 5 heathlands• 6 calcareous

grasslands• 6 neutral grasslands• 2 dune grasslands• 2 blanket bogs• 4 raised bogs• 5 fens• 5 saltmarshes

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LTMN Soils Method

• NE help contribute to fieldwork and Macaulay Scientific Consulting do fieldwork and analysis.

• Use aerial photos and veg survey data to choose 5 ~similar points.

• Survey from Sept 16th to Oct 16th

• Use GPS to locate veg plot markers and lay out 20m by 20m soil plot to SW using compass

• Each contains 100 2m by 2m sub-plots

• Same 4 sampled for all plots – change next time.

LTMN Soils Method

• Take plot location photos• Sub-plot photos side and above• Vegetation survey• Soil auger assessment

LTMN Soils Method

LTMN Soils Method

• Cores taken – most bulked• Wrapped, labelled chilled and

sent to Scotland.• Different cores are letter coded:• C for “curface” (0-15)• A for “anderneath” (15-30)• Physico-chemical properties

• Bulk density• %C, %N• Loss On Ignition• pH• CEC and cations

LTMN Soils Method

• B for beasties (0-8 cm mesofauna)

• D for DNA (microbial community) – tRFLP, PLFA

• E for eelworms (nematodes)• F for fertiliser (N

mineralisation)

Baseline Results – Physico-chemical

0 10 20 30 40 50 60 70 80 90 1000

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

f(x) = − 0.3491330474727 ln(x) + 1.6603748327334R² = 0.974870383452913

0 - 15 cmLogarithmic (0 - 15 cm)15 - 30 cm

% Organic matter (LOI)

Dry

Bul

k D

ensi

ty g

cm

-3

0 10 20 30 40 50 60 70 80 90 1000

20406080

100120140

f(x) = 30.421973332908 ln(x) − 37.5060802352506R² = 0.69667033025645

Soil organic matter and cation exchange capacity

% Soil organic matter (loss on ignition)Cat

ion

Exch

ange

Cap

actit

y (m

Eq

100g

_1Baseline Results – Physico-chemical

0 10 20 30 40 50 60 70 80 90 1000

200

400

600

800

1000

1200

1400

1600

f(x) = 395.320529421108 ln(x) − 491.930103133124R² = 0.843366761182254

Soil organic matter and total soil PLFAs

% Soil Organic Matter (Loss on Ignition)

Tota

l Soi

l PLF

A c

onte

nt n

m g

-1Baseline Results – Biochemical

Baseline Results – Soil Function: C storage

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

140

f(x) = − 0.0386046856492471 x² + 4.32622221742989 x − 21.3420723217335R² = 0.619278454371842

f(x) = − 0.0417935316938523 x² + 4.62728130402153 x − 23.4024079966244R² = 0.624152989811183

0-15 cmPolynomial (0-15 cm)15-30 cmPolynomial (15-30 cm)

Field water content %

Carb

on s

tora

ge to

nnes

ha-

1

Baseline Results - Soil function: decomposition

0 10 20 30 40 50 60 70 800

5

10

15

20

25

30

35

40

f(x) = 4.97826664864687 ln(x) + 8.61792676524315R² = 0.689893058212357

f(x) = 0.270321059455835 x + 14.9948544668157R² = 0.588546519919854

% Cover of woody species

Soil

C:N

ratio

Baseline results - Soil organism communities: tRFLP

Baseline Results - Soil communities: tRFLP

Baseline results – interactions

-1 -0.5 0 0.5 1 1.5-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

bare ground

ferns

sedgesand rushes

littertrees

shrubsand climbers

ericaceae

bryophytesand lichens

grasses

Na

Fe

herbsVAM PLFAs

Gram +ve:-ve ratio

Gram -ve

Gram +vePLFAact

PLFA Fun:Bacratio

Fungal PLFA

Bac PLFAPLFA_total

tRFLP Shannon Diversity

tRFLP evenness

tRFLP richnessNO3-N min

NH4-N_min

Olsen P

Total PCEC

MnMg

K

Ca

Al

Exch_Acidity

C:N ratio

%C

%N

pH

LOI% waterBulk Density

PCA axis 1

PCA

axi

s 2

Baseline Results: Overall soil patterns (2011 data)

Baseline Results: implications for future work

• Size of change detectable varies site to site...– pH – ~0.4 pH units– ~20% change in bulk density– tRFLP - 7% change in evenness, 12% change in richness

• Soil physico-chemical properties change slowly• Soil biological properties may be more sensitive indicator...• Different habitats have distinct soil communities• Soil function – indicators and proxies – more measures needed?

• Continue with baseline – comparison over time• Include new analyses - earthworms, root biomass, genetic analysis• Develop new approaches

– Metabarcoding project – CEH & NHM – mesofauna– Earthworm DNA?– More multivariate analyses

• Write up – plan to present site by site data and full baseline report after 5 years

• Comparison with CS2007, CS1998 data – compare agricultural soils• Apply same methodology in experimental work, other monitoring

Future analyses and plans

Answering the big questions...

• Soil resistance and resilience to perturbations• Disturbance/fire at Thursley

• What are the soil communities in our priority habitats?• Clear microbial (and other?) communities

• How do these compare with other habitats?• Extend this method to other sites/experiments & compare

CS2007• Do soil characters/function lag or lead changes?• What will happen to soil carbon in seminatural habitats?• Trends in soil biodiversity – where are changes seen and why?

– Wait and see!

But...

• Most mesofauna samples are still not sorted and identified• Anyone interested – can borrow NE microscope• 5 samples - probably around 500-1000 beasts in total!

DNA metabarcoding?

We need a way to identify very large

numbers of invertebrate

specimens quickly and cheaply

Barcoding and Metabarcoding

• Alternative approach is metabarcoding.• Mitochondrial DNA passed down female line only – no

recombination during meiosis• Gradual change by mutations at “regular” rate• Differences and similarities should indicate timings of

divergence of species.• Similar story for ribosomal RNA• Sections of these are used as “barcodes” to characterise

spp.• COi – cytochrome oxidase 1 gene• Also 18SRNA• Prokaryotes- 16SRNA

Barcode Region for Animals

The Mitochondrial Genome

COI

TargetRegion

mosquito-COI: CGCGACAATGATTATTTTCAACTAACCATAAGGATATTGGAACATTATATTTTATTTTTGGAGCTTGAGCAGGAATAGTAGGAACTTCTCTAAGTATTTTAATTCGAGCAGAATTAGGACACCCTGGAGCCTTTATTGGTGATGATCAAATTTATAATGTTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGACTAGTCCCTCTAATACTAGGGGCCCCAGATATGGCTTTCCCTCGAATAAATAATATAAGATTTTGAATATTACCCCCCTCTTTAACTCTTCTAATTTCTAGAAGTATAGTAGAAAATGGAGCTGGAACAGGGTGAACTGTATATCCTCCTCTATCCTCAGGAATTGCTCATGCAGGAGCTTCAGTAGATTTAGCTATTTTTTCATTACATTTAGCAGGAATTTCTTCAATTTTAGGAGCAGTTAATTTTATTACAACAGTTATTAATATACGAGCACCAGGAATTACTCTTGACCGAATACCGTTATTCGTTTGATCTGTAGTAATTACAGCAGTATTATTATTACTTTCTTTACCAGTATTAGCTGGAGCTATTACTATACTTTTAACAGATCGAAACTTAAATACATCATTC

An actual mosquito barcode - a 650 letter word:

• If you can extract, and amplify barcodes from a community – cross ref with barcodes for known species

• Generate spp. List• Not quantitative – differential amplification• Problem – not enough spp. barcoded• Problem – extraction, amplification methods not well developed

MetabarcodingMass sequencing reduces time and cost

Uses CO1 barcode gene

Next-generation sequencers

Produce many parallel sequences

Limited in the length of sequence reads

Error can occur at amplification and sequencing stages – leads to noisy results

e.g. ‘454’ / Illumina

~120,000 reads in two 1/8 plates (~345 bp/sequence)

Step 1: Denoise

These are our molecular OTUs

~2000 reads

Step 2: Cluster similar sequences

Spider

Anopheles Mosquito

Fruit fly Emerald tree python

~2000 readsStep 3: Assign taxonomy

Output: species x site table

7 Communities

Spec

ies

Control

Swipe

Forage harvest

Disc plough

Agricultural plough

Forestry plough

Turf stripping

Control

Which treatments work?

Pitfall trap data

The best treatments are the most aggressive ones

StandardMetabarcoding (All arthropods)Standard (Spiders, carabids,

ants)

R2=0.76

Pitfall trap data

StandardMetabarcoding (All arthropods)Standard (Spiders, carabids,

ants)

Conclusion: metabarcoding produces useful information for restoration ecology.

• NE project to develop method – Dave Spurgeon, Rob Griffiths, Daniel Read at CEH

• 3 sites sampled along transects at differing proximites– Old spp. Rich chalk grassland– Improved grassland– Grassland managed to “revert” to chalk grassland

• 2 sets of mesofauna extracted– metabarcoding– morpho ID & spp. barcoding

• Metabarcoding – problems with primers – will COi work?• 18S RNA better – but good enough for spp?

• Morpho ID shows some differences• Communities (PCA) similarities are

– old <–> improved <–> reverting– I’m ID’ing samples for NHM – Alfried Vogler – lots of photos– Use for this project and put on BOLD– Challenge for NHM in small size– Must retain voucher specimen!

• Barcoding a specimen leaves a “permanent” legacy of an ID, and enables comparison to others (checking or defining)

• Link with location – a genetic NBN• Many specimens on current databases wrongly ID’ed• Some barcodes are of foreign material• Correct group-specific primers should help...• Better photos of lots of features.

Issues and Questions

• What’s stopping you barcoding things you ID?• Reagents?• Costs?• Lack of knowing where to go?• Would coordination help• Is there a role for NE? Museums? Universities? Biological Record

Centres?• Biodiversity groups and networks?

Soil Biodiversity Support Groups

• No soil biodiversity society for UK – SES in USA/Canada• Help is out there!• Facebook page• Blogs• Us lot!• What else?

– Record centres?

And Recording?