Barcode Wales / Codbar Cymru: A complete DNA Barcode Dataset of a Nation’s Native Flowering

Plants: Creation, Applications and Public Engagement

Natasha de Vere, Tim Rich, Col Ford, Sarah Trinder, Charlie Long, Chris Moore, Danielle Satterthwaite, Helena Davies, Joe Moughan, Addie Griffith, Laura Jones, Joel Allainguillaume, Mike Wilkinson, Tatiana

Tatarinova, Hannah Garbett, Les Baillie, Jenny Hawkins

Barcode Wales: Cod Bar Cymru

• DNA barcode the native flowering plants and conifers of Wales

• Develop applications that utilise this research platform

Sample collection

• 1143 native flowering plants and conifers

• 455 genera, 95 families, 34 orders

• 4272 individuals sampled, 3637 herbarium, 635 freshly collected

• All specimens verified by taxonomic expert

• Herbarium vouchers and full collection details for all samples

DNA extraction, amplification and sequencing

• Qiagen kits, modified for herbarium material

• rbcL: 5 primer combinations

• matK 29 primer combinations

• Macrogen Europe for Sanger sequencing

Sequence editing and multiple alignment

• Sequencher 4.9. contig assembly and manual editing

• rbcL alignment: MUSCLE

• matK alignment: Transalign and Geneious Pro 5.4.4

• Sequences BOLD and Genbank

The workforce: research training

Analysis

• Interspecific and intraspecific divergence

• Species discrimination: BLASTn

Barcode Gap: min. interspecific p-distance > than max. intraspecific (CBOL Plant Working Group 2009)

• Test discrimination using GenBank data

• Discrimination at different spatial scales, using species distribution records

• Scripts written in Python

Recoverability

rbcL matK rbcL & matK

No. of spp. sequenced 1117 (98%) 1031 (90%) 1025 (90%)

No. of spp. with > 1 individual sequenced

1041 (91%) 814 (71%) 808 (71%)

Mean no. of individuals per spp.

3 2 2

Mode of individuals per spp. 3 3 3

Range of individuals per spp. 1 - 9 1 - 8 1 - 8

Total no. of individuals sequenced

3304 2419 2349

In total 5,723 barcode sequences obtained for the 1143 species

Fresh vs Herbarium

matK: Fresh = 5 primer combinations Herbarium = 29 primer combinations

Effect of herbarium specimen age

Spearman Rank Correlation: rbcL rho = 0.993*** matK rho = 0.986***

Intra and interspecific divergence

rbcL matK

No. of spp. showing intraspecific variation 66/1041 (6.3%)

136/814 (16.7%)

Mean intraspecific divergence: all individuals (SD) 0.0001 (0.0005)

0.0003 (0.0009)

Mean intraspecific divergence: theta(SD)

0.0001 (0.0006)

0.0004 (0.0011)

Mean coalescent depth (max. intraspecific) (SD) 0.0001 (0.0006)

0.0004 (0.0012)

Mean interspecific divergence (SD) 0.0063 (0.0069)

0.0174 (0.0231)

Using uncorrected p-distances

Relative discrimination

808 species

69

75

57

56

74

68

99

100

98

99

96

95

Recoverability and discrimination

1143 species

49

66

49

65

49

55

Testing discrimination

rbcL GenBank sequences Species % Genus % Family % Failed %

Sequences correctly identified (n = 1346)

57 93 99 1

Taxa correctly identified (n = 592)

58 94 100 0

matK GenBank sequences Species % Genus % Family % Failed %

Sequences correctly identified (n = 1380)

67 95 99 1

Taxa correctly identified (n = 533)

72 96 99 1

GenBank sequences queried against Barcode Wales database using BLASTn

rbcL discrimination

Scale n Mean discrimination % (SD)

10x10 km 253 72 (4)

2x2 km 1116 90 (9)

Species lists generated for each square, discrimination assessed by presence of a barcode gap

matK discrimination

Scale n Mean discrimination % (SD)

10x10 km 253 81 (3)

2x2 km 1116 93 (7)

Species lists generated for each square, discrimination assessed by presence of a barcode gap

rbcL & matK discrimination

Scale n Mean discrimination % (SD)

10x10 km 253 82 (3)

2x2 km 1116 93 (6)

Species lists generated for each square, discrimination assessed by presence of a barcode gap

DNA barcoding and drug discovery

• Collect wildflower honey from throughout UK

• Test antibacterial properties of honey against MRSA and Clostridium difficile

• DNA barcode honey

• Identify plant derived

phytochemicals

• New drug discovery

routes

Drug discovery – prelim results

• 150 honey samples

• Agar diffusion assay,

plates with MRSA,

activity present in

some samples

• Successfully amplified

rbcL from honey

Next:

• Identify cause of antimicrobial activity

• Next gen sequencing of honey samples

DNA barcoding and phylogenetics

ML tree for rbcL, RAxML (GTR+CAT) 1000 bootstraps, on the CIPRES supercomputer cluster

Good match with APGIII.

56% of species form monophyletic groups

44% with bootstrap support >70%

DNA barcoding and phylogenetic ecology

ML tree for rbcL, threatened species traced using Mesquite

DNA barcoding and art-science

DNA barcoding and community engagement

DNA barcoding and community engagement

Thank you!

• Funding from Welsh Government, National Botanic Garden of Wales, National Museum Wales, Countryside Council for Wales, Spirent Communications plc

• Sponsorship from the people of Wales

• www.gardenofwales.org.uk

• Science at the Garden of Wales on facebook