Use of genetics in species-level taxonomy

Pete Hollingsworth

Royal Botanic Garden Edinburgh

GCTTCACATTTTAACGCACATGACGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGATAATAC

GCTTCACACATTAACGCACATTTCGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGCCAATACGCCTCACACATTAACGCACATTTCGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGAGAAGAC

GCTTCATACATTAACGCACATGACGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGAGAATAC

The Problem.........• There are a lot of plant species........

• Species can be difficult to identify and delimit–Some plant groups are difficult–Some plant groups/geographical regions are

under-studied–Shortage of botanical experts–Some material is suboptimal.........................

ACGTGGGCTTGCATCGGGGAGTACATGCGTAACACTCGTTGCACGATCGATTGAAGCTACGTGCATGACGTGGGCTTGCATCGGGGAGTACATGCGTAACACTCGTTGCACGATCGATTGAAGCTACGTGCATGACGTAGGCTTGCATCGGGGAGTACATGCGTAACACTCGTTGCACGATCGATTGAAGCTACGTGCATGACGTGCACATGCATCATGGTGTACATGTGTAACACACGATGCAGGATCGGTCGAAGCTACGTGCATGACGTGCACATGCATCATGGTGTACATGTGTAACACACGATGCAGGATCGGTCGAAGCTACGTGCATGACGTGCACATGCATCATAGTGTACATGTGTAACACACGATGCAGGATCGGTCGAAGCTACGTGCATGACGTTTACATGCATCTGGGTGTACATGTGTAACTCCGATTGCACGAACGATCGAAGCTACGTGCATGACGTTTACATGCATCTGGGTGTACATGTGTAACTCCGATTGCACGAACGATCGAAGCTACGTGCATGACGTTTACATGCATCTGGGTGTACATGTGTATCTCCGATTGCACGAACGATCGAAGCTACGTGCATGACGTGCATATGCATCGGTGTGTCCAGCCGTTACACCCGTTGCATGATCAATCGAAGCTA CGTGCATGACGTGCATATGCATCGGTCTGTACAGCCGTTACATTCGTTGCATGATCAATCGAAGCTA CGTGCATGACGTGCATATGCCTCGGTGTGTACAGCCGTTACACCCGTTGCATGATCAATCGAAGCTA CGTGCATG

How can genetics help?

• Comparability

• Lack of environmental or developmental variation

• Automateability/scaleability

P. natansP. nodosus P. x schreberi

Tricophorum cespitosum(Cyperaceae)One taxon recognised in UK, but subtle differences in leaf sheath morphology suggested two taxa

Cryptic taxa

Hollingsworth & Swan, Watsonia 22, 235-242 (1999).

Ssp. cespitosum Nothossp foersteri Ssp. germanicum

Genetic markers support the recognition of two distinct taxa

Anacamptis robusta

Anacamptis fragrans

Type 3Type 1 & 2 Type 3 Chloroplast sequence

Axis 1 (50.8%)

Axi

s 2 (

5.0

%)

0.40.30.20.10.0-0.1-0.2-0.3-0.4-0.5

0.2

0.1

0.0

-0.1

-0.2

Legend

Hybrid zone G. urbanumPure G. rivalePure G. urbanum

Artificial F1Backcross G. rivaleHybrid zone F1Hybrid zone G. rivale

H34

H33

H32

H31

H30

H29

H27

H26

H25

H24

H23

H22

H21H20

H19

H18A

H17

H16

H15

H14

H13

H12

H11

H10

H9A

H8H7

H6A

H5A

H4

H3

H2

H1

PCO plot of AFLP data (203 markers)

H28

Pure rivale

Backcross rivale?

F1s

Pure

urbanum

ACGTGGGCTTGCATCGGGGAGTACATGCGTAACACTCGTTGCACGATCGATTGAAGCTACGTGCATGACGTGGGCTTGCATCGGGGAGTACATGCGTAACACTCGTTGCACGATCGATTGAAGCTACGTGCATGACGTAGGCTTGCATCGGGGAGTACATGCGTAACACTCGTTGCACGATCGATTGAAGCTACGTGCATGACGTGCACATGCATCATGGTGTACATGTGTAACACACGATGCAGGATCGGTCGAAGCTACGTGCATGACGTGCACATGCATCATGGTGTACATGTGTAACACACGATGCAGGATCGGTCGAAGCTACGTGCATGACGTGCACATGCATCATAGTGTACATGTGTAACACACGATGCAGGATCGGTCGAAGCTACGTGCATGACGTTTACATGCATCTGGGTGTACATGTGTAACTCCGATTGCACGAACGATCGAAGCTACGTGCATGACGTTTACATGCATCTGGGTGTACATGTGTAACTCCGATTGCACGAACGATCGAAGCTACGTGCATGACGTTTACATGCATCTGGGTGTACATGTGTATCTCCGATTGCACGAACGATCGAAGCTACGTGCATGACGTGCATATGCATCGGTGTGTCCAGCCGTTACACCCGTTGCATGATCAATCGAAGCTA CGTGCATGACGTGCATATGCATCGGTGTGTACAGCCGTTACACCCGTTGCATGATCAATCGAAGCTA CGTGCATGACGTGCATATGCATCGGTGTGTACAGCCGTTACACCCGTTGCATGATCAATCGAAGCTA CGTGCATG

• Application of multiple techniques to solve taxonomic problems in difficult groups– Individual solutions for individual problems– Our capacity to do this is getting much better...

• Can we use massive scale DNA sequencing of biodiversity to create a unified database of life?

• DNA barcoding: sequence one (or a few) ‘standard’ short stretches of DNA– Individuals of a species should be more similar in DNA sequence, than they

are to individuals of other species

• Establish reference database of high quality sequences from well identified material (supported by voucher specimens)– Take some tissue, sequence it, find out what it is, and what is known about it

• Provide a mechanism for speeding up the process of characterising biodiversity– Identification of unknown specimens to known species– Assist in the discovery of new species

• Approach not novel…..scale and coordination is……

GCTTCACATTTTAACGCACATGACGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGATAATACGCTTCATACATTAACGCACATGACGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGAGAATACGCTTCACACATTAACGCACATTTCGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGCCAATACGCCTCACACATTAACGCACATTTCGTGTCACAGAACGAGTGCCTACGTCGAGCAATTAGAGAAGAC

DNA barcoding www.barcodinglife.org

This pioneering effort in DNA barcoding will set in motion the single most significant project in biology that I know today

Dan Janzen (2003)

“DNA barcoding generates information, not knowledge”

“…..the practice is counterproductive to furthering our understanding of life”

Ebach & Holdrege (2005) Nature 434, 697

“the noisome weed of DNA barcoding”Will et al. (2005) Syst. Biol. 54, 844–851

“an excursion into futility”Wheeler (2005) Cladistics 21, 405–407

What are these objections based on? • It won’t work in all groups• A sequence from a single locus is inadequate for describing new species• It is not as intellectually ‘worthwhile’ as detailed taxonomic research• It might draw attention away from traditional taxonomy/understanding organisms

Proposed solutions:Chase et al (2007): rpoC1 + rpoB + matK(Taxon 56, 295-299) rpoC1 + matK + trnH-psbA

Kim et al (2007): matK + atpF-H + psbK-I (unpublished) matK + atpF-H + trnH-psbA

Kress/Erickson (2007): rbcL + trnH-psbA(PLoS ONE 2, e508 )

Lahaye et al. (2008): matK (PNAS 105, 2923–2928Nature Precedings http://hdl.handle.net/10101/npre.2008.1896.1)

Plant working group (2007) matK + trnH-psbA + atpF-H (minutes of Taipei meeting)

Reviewed by Ledford (2008) Nature 451, 616; Pennisi (2007) Science 318, 190-191.

Choosing a plant (chloroplast) barcode

TROUBLE AT MILL

RBGE Strategic Review November 2009

rbcL & matK selected as the core plant barcode

Chloroplast barcode - will not discriminate between hybrids and their parents

A broadly applicable tool, rather than targeted at complex groups

• Species discrimination asymptotes at ca 70-75%Remaining species identified to “species groups”

• Adding >2 chloroplast loci on average leads to diminishing returns of discrimination

1 locus 2 locus 3 locus

80%

70%

60%

50%

40%

What is the likely discriminatory power of a plant barcode?

Predictions of plastid barcoding success

• High

• Recent radiation

• Agamosperms

• Heavily split micro-species

• Long-lived trees

• Poor

• Hybridisation and/or polyploidy frequency

• Species age

• Reproductive mode

• Taxonomic splitting

• Life-history

• Seed dispersal(angiosperms)

• Low

• More ancient divergence

• Sexuals

• Not-over split

• Herbs

• Good

Low High

LAND PLANTS• c. 400,000 species

VASCULAR PLANTS• > 350,000 species• c. 13,888 genera• c. 511 families

Using a plant

barcode

Applications of plant barcoding• Identification of CITES protected materials

– Ogden et al. (2008) Endang Species Res 9: 255–261

• Identification of plant material in diets– Jurado-Rivera et al. (2009) Proc R Soc Biol Sci SerB 276: 639-648

• Identification of fern gametophytes– Schneider et al. (2006) Molecular Ecology Resources 6: 989-991– Squirrell et al. unpublished

• Identification of invasive species– Van de Wiel et al. (2009) Molecular Ecology Resources 9: 1086-1091

• Identification of plants in horticultural trade– Pryer et al. (2010) Molecular Ecology Resources: online early

• Identification of herbal medicine components– Baker and Little, Scientific American online ‘Observations’ 18/4/2010

• Cryptic species discovery in orchids – Lahaye et al. (2008) PNAS 105: 2923-2928

• Species identification and discovery in liverworts– Hollingsworth et al. (2009) Molecular Ecology Resources 9: 439-457 and unpublished

Bryophyte identification is difficult and there is a shortage of experts

Develop system for rapid low-cost identification and species discovery

New species, Distinct ecology & morphology

H. borealisHerbertus

rbcLmatK+ trnH-psbA+ ITS

Aneura sp. nov.

Cryptothallus (=Aneura) mirabilis

Typical Aneura pinguis

Barcoding of Aneura pinguis in UK has led to one new species being described, and identification of another four candidate new species

Barcoding Aneura

Major Barcoding Projects• iBoL– 500K species, 5 million specimens by 2015– 100K species of plants

• Many research groups with active plant barcoding projects

• Tree-BoL: Barcoding all trees• Grass-BoL: Barcoding all grasses• Flora of Kruger National Park, • Flora of Area de Conservacion

Guanacaste Costa Rica• British liverworts and UK BAP mosses• Flora of Korea• Plant barcoding China• Various medicinal plant projects• ‘All plant genera’

• Where we were (1990-2010)– Individual taxon studies

• Small numbers of well-characterised genetic markers• Large numbers of poorly-characterised markers

• Where we are– High throughput coordinated sequencing of a small

number of DNA regions in lots of taxa (DNA barcoding)– Individual taxon studies using higher numbers of well

characterised markers

• Where we would like to be (2015 onwards)– High throughput sequencing of lots of well characterised

DNA regions in lots of taxa

• Establishing the reference database (field collections, herbarium vouchers, determinations) will be the rate limiting step– Important to commence this process now and establish “DNA

ready” herbaria for future improvements in sequencing technologies

• Vast quantities of DNA sequence data divorced morphological and ecological information will be meaningless– Integration with taxonomy and ecology, not replacement