Sarah stephenson csiro

LAND AND WATER

Sarah Stephenson| Research Project Officer

Metagenetic examinations of benthic eukaryl estuarine communitiesSMBE - Eukaryotic Omics 2013

Australian benthic eukaryl estuarine communities

Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson2 |

• Diverse and complex.

• 85% Australian’s live on the coast.

• Ecosystem services: Fishing, aquaculture, water purification.

• Multiple stressors: Mining, agriculture, urbanization, waste water treatment

plants, climate change.

Correlative and experimental studies:

Identify OTUs that are:

• Strongly correlated to the environmental variables of interest.

• Weakly correlated. Pick up subtle differences in biotic structure. • Whose presence is random across space and time, or which are derived from exogenous sources.

copper

sand (%)

salinity

turbidity

copper

sand (%)

salinity

turbidity

How can we link ecosystem function and structure to disturbance models?

Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson2

Ecological processes:RedundancyKeystoneIdiosyncratic

Limitations of using macrobenthic communities for environmental monitoring 25-40 relatively large

metazoan taxa

Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson4

Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson

What are we sequencing?• Complete analysis of the genomic constituents of a complex biological sample is

impractical for routine monitoring.

Chariton et al. (2010)Bik et al. (2012)

• Targeted DNA fragments• Ribosomal genes (rDNA) are required for translation (essential and universal). • 18S rDNA universal (many copies) in eukaryotes.• Sequences easily downloaded into sequence manipulation software.• Varying levels of taxonomic resolution. • Broad coverage. • Numerous approaches, with all DNA fragments having limitations.

Adaptor A Adaptor BBar-code Bar-codePrimer Primer

Forward Fusion primer Reverse Fusion primer

18S amplicon

5 |

AGGTTCTTCAATCGGACCCCATTCTTCAATCGGACC

TTCTTCTTCAATCGGACC

CTTCACTTCAATCGGACCz

PCR to produce a pool of 18S rDNA fragments

DNA is extracted & purified

Pooled samples are sequenced

Chemical separation: samples assigned a unique barcode and pooled prior to analysis.

Gasket A

Gasket B

Post analysis assignment of sequences into a priori defined samples.

Treatment A

Treatment B

Treatment B rep1

Treatment A rep1

Treatment A rep2

Treatment A rep3

Treatment A rep4

Treatment B rep2

Treatment B rep3

Treatment B rep4



South East Queensland Study

7 |

Australian population growth projections

Area contains 3.05 million people.Projection is 8.7 million by 2056.

South East Queensland Estuaries

• Aim: Use high throughput sequencing to examine the benthic composition along five estuaries of varying ecological integrity.

• Examine whether molecular biodiversity assessments can be discriminated among estuaries.

• Identify whether molecular derived biotic composition is correlated with nutrients and other physico-chemical variables.

• Utilise estuaries currently being monitored under the EHMP’s Healthy Waterways project.


No Biodiversity indicators

9 | Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson

www.healthywaterways.org


Estuary Score (2010)

Noosa B+

Maroochydore C

Pine C-

Currumbin C

Logan F

2010 catchment & estuary report card

C-

C

B+

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C


Experimental design

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site 1 site 2 site 3 site 4 site 5

5 Sample grabsa b c d e

1. Noosa 3. Currumbin 4. Pine2. Maroochydore 5. Logan

EHMP sites sampled in 2010Physico-chemistry


Bioinformatics and statistical method

Pissodes castaneus AJ850007 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAAATAG GCG------- -T---TTTTG ACATCCTAAA GGCCCGCCGA CGG------- G--------- --GG------ ---------- ---------- Chrysomela scripta AF267436 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGTG ATTTGTCTGG TTAATTCCGA TAACGAACGA GATTCTAGCC TGCTAACTAG GCGT------ -T---TTTCG ACATCCCAAA GGCCCGCCGG TTG------- A--------- --GG------ ---------- ---------- Plegaderus cf. nitidus AY02833 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAAATAG GCGT------ TA---ATTCG ACATCTCAAA GGCCCGCCGG CCC------- G--------- --GC------ ---------- ---------- Rhynchophorus cruentatus AF389 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGC------ -T---TTTTG ACATCCTAAA GGCCCGCCGG CCG------- G--------- --CG------ ---------- ---------- Ocypus opthalmicus AJ810739 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGTG ATCTGTCAGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGT-----A CT----TTCG ACATCTCAAA GGTCG----- ---------- ---------- ---------- ---------- ---------- Galerucella nympheae AF267443 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGT------ -A---TTTCG ACATCCCAAA GGCCCGTCGG TAT------- G--------- --GA------ ---------- ---------- Microsporus sp. AF427599 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGTG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTTTGAGA ATTCAATCCG GTATCTATGA GGTGCGTGCA CGCGATCAGC TATGGGCTGA GAGAGCGCCG TCGGTGGCAT GGACCGTTGT Apotomus rufithorax AF012497 --TGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGATTG ATTTGTCTGC TTAATTGCGA TAACGAACGA GATTCTAGCC TGCTAAATAG GCGTT----- ACTTA--CCG GTATCTCGAA GATTCGCATC TCC------- --TGGTCGTT CAGCATGTTG TTCGTTGCAT GTGTGGGCGT Carabus nemoralis AF012507 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGT-----A CT---TTCCG GTATCTCGAA GGCCGACGTC TCT------- C--------- --GGGTTTCC GTTTG----- -----GCGCG Oregus aereus AF012500 GGTGGGTGGT GGTGCATG-C CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A CT---TACCG GTATCTCGAA GGCTCGCGTC TCT------- C--------- --GGTTCCCG CGT-----GT TGCGTTTGCG Lancetes varius AJ318684 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAAATAG GCGTT----A CT---TTCCG GCATCCCGAA GGCTCGCGTC ACC------- C--------- --GTTCGCC- ---------- -------GGT Hydraena iberica AJ810730 GGTGCGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGATCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTT----T CT-----CCG ACATCTCAAA GGCCA----- ---------- ---------- ---------- ---------- ---------- Trechus sp. nr. chalybeus AF00 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGACCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTT----A CT---TACCG GTATCCTGAA GGCTC--GTG TCT------- T--------- --TCGTATTC TGGCGTGCAT T-TGTCG--- Dalyat mirabilis AY926478 Cara ---GGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGTTAACTAG GCGTT----A CT---TACCG GTATCTCGAA GGCTCGCGTC TCT------- C--------- --GTATTCG- ---------- ---------- Diaprepes abbreviatus AY157729 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A AA---TTTTG ACATCTTAAA GGCCCGCCGG CCG------- A--------- --CGCGGTGT GTGGCGTGCG TGCGCGGCGC Pachnaeus litus AY157730 Curcu GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A AA---TTTTG ACATCTTAAA GGCCCGCCGG CCG------- A--------- --CGCGGTGT GTGGCGTGCG TGCGCGGCGC Meonis sp. AF398722 Harpalinae ----GGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A CAAAATACCG GTATCTTGAA GGCTCCGACA CTT------- G--------- --CTTTTTGA TTTCATTCAC TGCATTTCTT Moriosomus seticollis AF398721 ----GGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TACTAAATAG ACGTT----A CACT-TACCA GTATCTCGAA GGCTC--ACA TCT------- C--------- --TCGTTTAC TTT---TCAC TATATAA---

E137_AY922318_GenBank (0.0068)A71_6 (0.0000)A22_11 (0.0034)A99_4 (0.0060)

A108_3 (-0.0034)A29_9 (0.0034)

A116_3 (0.0068)A30_9 (0.0000)

A137_2 (0.0000)A74_5 (0.0000)

A15_13 (0.0034)A129_2 (0.0000)

A111_3 (0.0034)A35_9 (-0.0034)

A113_3 (0.0000)A82_5 (0.0000)A85_5 (0.0000)A86_4 (0.0000)A122_3 (0.0000)

A123_3 (-0.0034)A63_6 (0.0034)A12_14 (0.0034)

A40_8 (-0.0034)A139_2 (-0.0034)

A83_5 (0.0034)A146_2 (0.0000)

A64_6 (0.0034)A8_18 (-0.0034)

A100_4 (0.0000)A135_2 (0.0068)

A96_4 (0.0051)A1_3756 (0.0000)

A101_4 (0.0069)A97_4 (0.0000)

A102_4 (0.0069)A103_4 (0.0034)A38_9 (0.0034)A104_4 (0.0034)A37_9 (0.0034)A105_4 (0.0034)A117_3 (0.0034)A106_3 (0.0000)A98_4 (0.0000)

A140_2 (0.0000)A6_22 (0.0000)

A107_3 (0.0069)A109_3 (0.0069)A10_16 (0.0069)A110_3 (0.0069)A112_3 (0.0069)A114_3 (0.0069)A115_3 (0.0069)A118_3 (0.0069)A119_3 (0.0069)A11_15 (0.0069)A120_3 (0.0034)A138_2 (0.0034)A121_3 (0.0069)A124_3 (0.0069)A125_3 (0.0034)A27_10 (0.0034)A126_3 (0.0034)A18_12 (0.0034)A127_3 (0.0069)A128_3 (0.0034)A84_5 (0.0034)A130_2 (0.0069)A131_2 (0.0069)A132_2 (0.0034)

A133_2 (0.0068)A2_67 (0.0000)A47_7 (0.0043)A134_2 (0.0034)A80_5 (0.0034)A136_2 (0.0051)

A41_8 (0.0205)A95_4 (0.0000)A13_14 (0.0069)A141_2 (0.0069)A142_2 (0.0069)A143_2 (0.0069)A144_2 (0.0069)A145_2 (0.0069)A14_14 (0.0069)A16_12 (0.0069)A17_12 (0.0069)A19_11 (0.0069)A20_11 (0.0034)A28_10 (0.0034)A21_11 (0.0069)A23_11 (0.0069)A24_10 (0.0069)A25_10 (0.0069)A26_10 (0.0069)A31_9 (0.0069)A32_9 (0.0069)A33_9 (0.0069)A34_9 (0.0069)A36_9 (0.0034)A45_7 (0.0034)A39_9 (0.0069)A3_34 (0.0069)A42_8 (0.0069)A43_8 (0.0069)A44_8 (0.0069)A46_7 (0.0069)A48_7 (0.0069)A49_7 (0.0069)A4_23 (0.0069)A50_7 (0.0069)A51_7 (0.0069)A52_7 (0.0069)A53_6 (0.0069)A54_6 (0.0069)A55_6 (0.0069)A56_6 (0.0069)A57_6 (0.0069)A58_6 (0.0069)A59_6 (0.0069)A5_23 (0.0069)A60_6 (0.0069)A61_6 (0.0069)A62_6 (0.0069)A65_6 (0.0069)A66_6 (0.0069)A67_6 (0.0069)A68_6 (0.0069)A69_6 (0.0069)A70_6 (0.0069)A72_6 (0.0069)A73_5 (0.0069)A75_5 (0.0069)A76_5 (0.0069)A77_5 (0.0069)A78_5 (0.0069)A79_5 (0.0069)A7_22 (0.0069)A81_5 (0.0069)A87_4 (0.0069)A88_4 (0.0069)A89_4 (0.0069)A90_4 (0.0069)A91_4 (0.0069)A92_4 (0.0069)A93_4 (0.0069)A94_4 (0.0069)A9_17 (0.0069)

CSIRO APDP

nMds, PERMANOVA,dbRDA, INDVAL (indicator analysis).

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De-noise & chimera clean up

Blast NCBI

Morgan et al., in review


Results

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Phylum

• >1 million sequences produced, 731,992 retained producing 2,966 OTUs• All control sequences recovered with no artificial inflation of OTUs • Richness was significantly greater in Logan than the other locations • Only 1 OTU, Naviculale (diatom) observed in all 125 samples• Diatoms are the most widely distributed taxa (>80% of the samples)


Results

Chlorophyta and nutrients?

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2D Stress: 0.13

full dataResemblance: S7 Jaccard

LocationNoosaMaroochydoorePineCurrumbinLogan

2D Stress: 0.13

Compositional comparisons among estuaries

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Estuary Score (2010)Noosa B+Maroochydore CPine C-Currumbin CLogan F

nMDS ordination plot of 18S rDNA derived benthic assemblages

PERMANOVA identified that all estuaries contained unique compositions


-60 -40 -20 0 20 40

dbRDA1 (29.5% of fitted, 21.8% of total variation)

-40

-20

0

20

40

dbR

DA

2 (2

1%

of f

itted

, 15 .

6% o

f tot

al v

aria

tion) Noosa

Maroochydoore

Pine

Currumbin

Logan

Depth

Chlorophyll-a

Conductivity

Secchi depth

Ammonia

N (organic)

N (oxidised)

N (total)Dissolved oxygen

pHP (reactive)

P (total)

TemperatureTurbidity

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Relationships between biotic composition and the physico-chemical properties of the overlying waters (distance-based linear modelling)


Bacillario

phyta

Alveolata

Amoebozoa

Chlorophyta

stramenopile

s

Unknown

Chytridiomyco

ta (Fungi)

Foraminife

ra

Porifera

Basidiomyco

ta(fungi)

Cercozoa

Gastopoda

Gastrotri

cha

Glomeromycota(Fu

ngi)

Arthropoda

Coleoptera

Blastocla

diomycota (F

ungi)

Centrohelio

zoa

Cnidaria (p

arasitic)

dinoflagellate

Flabellin

ea

Mucoromyco

tina (Fugi)

Oligoch

aeta

Rotifera

Sphagnum

Telonemida

Bacillario

phyta

Foraminife

ra

Ascomyco

ta (Fungi)

Cnidaria (p

arasitic)

Harpactico

ida

Platyhelm

inthes0

2

4

6

8

10

12

OTUs present in >80% of Logan samples and absent from the NoosaOTUs present in >80% of Noosa samples and absent from the Logan

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Where to from here?South East Queensland• Develop and test predictive models which clarify the

relationships between OTUs and environmental variables.

• 2012 Estuaries were re-sampled for biota and greater number of environmental parameters and sites were examined.

• Biosentinals: Identify key taxa which characterise and discriminate environments e.g. Kinorhyncha, Diatoms.

• Develop species specific PCR.• Integrate ecotoxicology.

• Integrate the structural and functional attributes of the systems.

-

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Metagenetics: Are we meeting the needs ecologists?

Challenges• Unknown eukaryotes.• Presence / absence only.• 18S copy number.• OTU confidence: Only as good as

database e.g. SILVA, NCBI.• Extracellular DNA• How many replicates? Rare taxa.

Solutions• Metagenomic and deeper sequencing.• Target ITS for Fungi.• Complimentary target CO1 for

eukaryotes.• Biological/ Technical replicates.• ‘Mock community’ positive control.• Microcosm and lab manipulation studies.

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CSIRO LAND AND WATER

CSIRO LAND AND WATER

Holly Bik, SMBE and UC Davis

Anthony Chariton

Andy Steven

CLW Lucas Heights and CSIRO North Ryde

CLW field crew Dutton Park

Mathew Morgan

Chris Hardy and Leon Court

Australian Genome Research Facility

The traditional owners of Jagera, Turrbal and Kallangur, SEQ

Acknowledgements

CSIRO Land and WaterSarah StephensonResearch Project Officert +61 2 9E [email protected]

www.csiro.au/loremw www.csiro.au

Thank you

CSIRO LAND AND WATER LUCAS HEIGHTS

http://www.csiro.au/lorem


Results

• >1 million sequences produced, 731,992 retained producing 2,966 OTUs

• All control sequences recovered with no artificial inflation of OTUs

• Richness was significantly greater in Logan than the other locations

• Only one OTU, Naviculale (diatom) was observed in all 125 samples

• Diatoms were the most widely distributed taxa (>80% of the samples)

Streptophyta?

Chlorophyta and nutrients?

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Ascomycota and nutrients?

Sarah stephenson csiro

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