Introduction to HerbariaHerbaria

Professor Philip StevensonR l B t i G d KRoyal Botanic Gardens, Kew

Natural Resources Institute, University of Greenwich

The HerbariumThe Herbarium

• A collection of dried plants• A store of reference materialA store of reference material• A means of identification• An arbiter of correct names• A comprehensive data-bank• A comprehensive data-bank

“The essential working tool for systematics”

Role of the HerbariumRole of the Herbarium

• Identification• Taxonomic researchTaxonomic research• Data repatriation (including databasing)• IUCN Conservation ratings• Voucher specimens• Voucher specimens

– Ecological– DNA sequencing– Phytochemicaly

Fieldwork and the HerbariumFieldwork and the Herbarium

Fi ld Id tifi ti diffi lt• Field Identifications difficult– Tropical habitats very diverse– Family or genus level– Floras often not available (tropics especially)( p p y)– Floras bulky

E l i l fi ld k• Ecological fieldwork:– Plots, often sterile vouchers, shorter-term

• Taxonomic fieldwork:General collecting fertile material long term– General collecting, fertile material, long-term

Herbaria around the worldHerbaria around the world

• General or international herbaria• National and regionalNational and regional

– E.g., Forest Research Institute Malaysia (FRIM)F t H b i S k (SAR)Forest Herbarium, Sarawak (SAR)Forest Research Centre, Sabah (SAN)

• University herbaria• Local botanists = local contacts• Local botanists = local contacts• Index Herbariorum

H b i f d th ldHerbaria from around the world

Field to specimenField to specimen

SpecimenSpecimen

Determinavit slipp

Loose parts

Label

Barcode

Herbarium stamp

The Kew HerbariumThe Kew Herbarium• Approx 5 million specimens• Approx. 5 million specimens

• Comprehensive collections from all regionsComprehensive collections from all regions

Wing C - 1857 Wing A - 1903

The Kew herbarium contdThe Kew herbarium contd.

• Specimen arrangement– Phylogenetic according to Bentham and y g g

Hooker (1880)– Re-arrangement with the new wingRe arrangement with the new wing

• Mabberley’s Plant Book (new edition)

St ff ‘ t’• Staff ‘arrangement’– Regional teams– Systematic teams

Regional TeamsRegional Teams

• South-East Asia and Pacific• Drylands AfricaDrylands Africa• Africa Wet tropics• South America• Temperate• Temperate

• Name all material except systematic families• Sorts identification field guides and research• Sorts, identification, field guides, and research

Systematic TeamsSystematic Teams

M l i hi l (E h b )• Malpighiales (Euphorbs)• Myrtaceae (Eucaplypts etc.)y ( p yp )• Labiatae/Lamiaceae (Mints)• Rubiaceae (Coffee)• Rubiaceae (Coffee)• Leguminosae (Peas and beans)• Monocots

• Monographic and phylogenetic research

Digitisation progress

Nearly 50,000 legume specimens

Data repatriation of 4000 specimens f M i d

32 legume “species pages”

on-linefrom Mexico and Central America

Tephrosia vogeliip g

Plants reportedly used (%) in Malawi for pest control (n=167)

Some farmers report no activity from TephrosiaSome farmers report no activity from Tephrosia

Callosobruchus maculatus

Oviposition by fecund bruchid beetles on cowpea seeds treated withon cowpea seeds treated with

powdered plant @1% w/v after 48 h. eg

gsbe

r of e

n nu

mb

Mea

n

Rotenoids from Tephrosia vogelii leaves

HHO

OO

O OMeR

OO

O

OR

H

O

OMe

OMe O

OMe

OMeDeguelin R=H Tephrosin R = OH

Rotenone R=H 12α-hydroxyrotenone R = OH

OO

O OO

O

OHO

OMe

OMe O OMeOH

OMeSarcolobine Toxicarol

Is Is TT. . candidacandida effective? effective?

TephrosiaTephrosiavogelii.

Controls bruchids

Tephrosia candidacandidaPromoted for soil

improvement (N2 fixing & green mulch)& green mulch)

and (assumed) pest control properties.

(growing at an International (growing at an International AgroforestryAgroforestry Centre)Centre)

Oh Oh –– its actually Tits actually T. . vogeliivogelii

TephrosiaTephrosiavogelii.

Controls bruchids

Tephrosia candidacandidaPromoted for soil

improvement (N2 fixing & green mulch) 2& green mulch)

and (assumed) pest control properties.

1 2

Plastid Ltrn region, ITS nuclear DNA sequences & morphology indicate both to be T. vogelii

Effect on C. maculatus of cowpea t t d ith t t t ftreated with acetone extracts of T. vogelii chemotypes after 48 h.

80

90

100

60

70

80

ects

(%

)

30

40

50 DeadSickHealthy

ber o

f ins

e

10

20

30

Num

b

02% 0.20% 0.02% 2% 0.20% 0.02% AcO

Chemotype 1 Chemotype 2 Control

Sick insects are alive but paralysed

LCLC--MS chromatograms of MS chromatograms of

PhilSAPP3 x 150 mm Phenomenex Luna C18(2), 95:0:5(0 min) 0:95:5(20 min) 0:95:5(25 min), water : MeOH : ACN +1% formic acidTvogelii herb specimen 34mg/ml

PhilSAPP22-Oct-2009

Fig 1a HPLC chromatogram of T. vogelii chemotype 1

T. vogeliiT. vogelii chemotypes 1 & 2chemotypes 1 & 2PhilSAPP3 x 150 mm Phenomenex Luna C18(2), 95:0:5(0 min) 0:95:5(20 min) 0:95:5(25 min), water : MeOH : ACN +1% formic acidTvogelii herb specimen 34mg/ml

PhilSAPP22-Oct-2009

PhilSAPP3 x 150 mm Phenomenex Luna C18(2), 95:0:5(0 min) 0:95:5(20 min) 0:95:5(25 min), water : MeOH : ACN +1% formic acidTvogelii herb specimen 34mg/ml

PhilSAPP22-Oct-2009

3 0e+2

3.5e+2

Tvogelii herb specimen 1 3: Diode Array Range: 4.337e+219.40

deguelin3 0e+2

3.5e+2

Tvogelii herb specimen 1 3: Diode Array Range: 4.337e+219.40

deguelin3 0e+2

3.5e+2

Tvogelii herb specimen 1 3: Diode Array Range: 4.337e+219.40

deguelin1

AU

1.5e+2

2.0e+2

2.5e+2

3.0e+212.03

12.62

tephrosi rotenone

AU

1.5e+2

2.0e+2

2.5e+2

3.0e+212.03

12.62

tephrosi rotenone

AU

1.5e+2

2.0e+2

2.5e+2

3.0e+212.03

12.62

tephrosinrotenone

11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

5.0e+1

1.0e+218.75

15.12 20.35

Tcandida BI19309 3: Diode Array Range: 6.261e+120.68

tephrosin toxicarolsarcolobin

e11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

5.0e+1

1.0e+218.75

15.12 20.35

Tcandida BI19309 3: Diode Array Range: 6.261e+120.68

tephrosin toxicarolsarcolobin

e11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

5.0e+1

1.0e+218.75

15.12 20.35

Tcandida BI19309 3: Diode Array Range: 6.261e+120.68

toxicarolsarcolobine

4.0e+1

5.0e+1

g

12 00

Fig 1b HPLC chromatogram of T. vogeliichemotype 2 Obovatin 5-methylether

4.0e+1

5.0e+1

g

12 00

Obovatin 5-methylether4.0e+1

5.0e+1

g

12 00

Obovatin 5-methylether2 OO

AU

1.0e+1

2.0e+1

3.0e+1 12.00 12.58

19.93

Z-tephrostachin

4 & 5

31 & 2 6

AU

1.0e+1

2.0e+1

3.0e+1 12.00 12.58

19.93

Z-tephrostachin

4 & 5

31 & 2 6

AU

1.0e+1

2.0e+1

3.0e+1 12.00 12.58

19.93

Z-tephrostachin 4 & 5

31 & 2 6OOMe

Time11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

0.0 15.1213.40 20.37

Time11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

0.0 15.1213.40 20.37

Time11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

0.0 15.1213.40 20.37

Compound IDs based on 700MHz NMR and Orbitrap HR-EI MS

Flavanones and flavones from T. vogelii chemotype 2 (inactive)

OO O

OH

OMeO

O

O

OMe

O

OOMe

OMe2

OO

O

R

H

OOMe OOMe

Obovatin 5-methylether Z-tephrostachin3O

OMe

OMeR

Deguelin R=H Tephrosin R = OH

OO

Tephrosin R = OH

OO

*two of 6 newO

OHOMe

Yukovanol 5 methylether * T h l *O O

two of 6 new flavonoid aglycones

Yukovanol 5-methylether Tephrovogelone*Stevenson et al., Stevenson et al., PhytochemistryPhytochemistry (submitted)(submitted)

Mildbraediodendron excelsum

Herbarium sheet:Herbarium sheet:Specimen collected by Johannes Mildbraed in 1928

Living specimen:Grown from seed collectedi 1996 Mt K Cin 1996, Mt. Kupe, Cameroon

LC-UV Analysis of Mildbraediodendron excelsum

AULiving specimen

AUAUMildbraed 10643

2 4 6 8 10 12 14 t (min)ANALYTES: 50% aq. MeOH

LC chromatogram (total scan PDA) extracts of leaflet material

MILDBRAEDIN: a flavonol tetraglycoside fromMildbraediodendron excelsum

OH

OHO

OH

O OHCH3

α-RhaI

7

OOHO

O

OOHO

CH3

O1

1

2

31

3

OOH

OHCH2

OHCH3

HOHOCH3HO 6 α-RhaII

β-Gal

CH2OO

HO

HO

HO 1α-RhaIII

Main phenolic component of:(1) Herbarium leaf fragment (1928)(2) Li i i

HO

(2) Living specimen

Veitch et al. (2005) Tetrahedron Lett. 46, 8595

Flavonol pentaglycosides of Cordyla haraka

265348100

m/z = 1033LC UV

90

1001033MS

50Abu

ndan

ce

70

80

200 300 400λ (nm)

0

Rel

ativ

e

m/z = 104760

70

ndan

ce

1461461461621460 5 10 15 20 25 30 35

t (min)

0

Ion current chromatogram 40

50

Rel

ativ

e A

bun -146-146-146-162-146

20

30 741

433

m/z

10887595287

0

Electrospray mass spectrum

200 400 600 800 1000 m/z0

D. Du PuyCordyla haraka

Distribution of flavonol pentaglycosides in Cordyla s.l.

AmburanaMildbraediodendronCordyla C f i

M. excelsum

CordylaAldina

C. africanaC. densifloraC. harakaC. madagascariensis

Dupuya harakaDupuya madagascariensis

C. pinnataC. richardiiC. somalensis

O

OH

O

CH3HO7

CONCLUSIONS:

O

OOHO

O

O OH

OHO

CH3

O

OO

HO

HO

1

1

23 1

3

α RhaII

α-RhaI7

1

α-RhaIVC. haraka allied withC. pinnata & C. richardii

N t f t f f

OOH

OOH

OHCH2

OOHCH3

HOHOCH3HO

1 2

6

1 α-RhaIIβ-Gal

No support for transfer ofC. haraka to Dupuya

OO

HO

HO1

α-RhaIII

R = OH, Cordylasin BR = H, Cordylasin A

Veitch, Kite & Lewis (2008) Phytochemistry 69, 2329