DNA Barcoding of Philippine Plants:

Systematics and DNA Barcoding of

Philippine Orchids

Genera
Species and varieties

152
1,142

10-15% of the Philippine flora

Orchids

(Schuiteman, undated)

040
074

Bulbophyllum cumingii
Bulbophyllum sp.
070

075

Bulbophyllum laxiflorum

Bulbophyllum sp. sect. Desmosanthes

035
Dendrochilum wenzelii
055

Dendrochilum anfractum

053

Dendrochilum cootesii

086

Dendrobium gerlandianum
087
088

Dendrobium sanderae

Dendrobium amethystoglossum

089

Eria sp.
046

Eria javanica

Miscellaneous
007

018

Aerides odorata
008

Phaelonopsis aphrodite

025

Ceratostylis retisquama

Ascocentrum aurantiacum
Subsp. philippinense

034

Trichotosia lagunensis

2001-2010
54 new species discovered!
under 25 genera
3 monotypic genera
Schuitemania
Santotomasia
Samarorchis

Orchids

New Plant Discoveries Per Year, 20012010

Source: Agoo 2011

New Species Per Family, 2001-2010

33
55
4
5
7
8
9
11

39
22

TOTAL = 192

Source: Agoo 2011

Morphology

Source: Agoo 2011

Anatomy

Palynology

DNA Barcoding : >100 Orchid Species

Robustness in PCR
Amplification and
Sequence Alignment
matK and trnH-psbA easiest to amplify
difficult in certain species (i.e. Paphiopedilum spp.;
Epidendroideae group for rbcL)

DNA Barcoding

Amplified Gene Length

rbcL, 774 bp
matK, 865 bp
trnH-psbA, 880 bp

DNA Barcoding

Ability to Identify or
Define Interspecific
Sequence Differences
Average divergence between sequences
highest in rbcL
matK
lowest trnH-psbA

DNA Barcoding

0.625
0.055
0.018

Ability to Identify or
Define Intraspecific
Sequence Differences
Divergence within species
least in rbcL
trnH-psbA
highest in matK

0.012
0.029
0.035

This indicates that any other differences in sequence compared to other species is due to
nucleic acid substitutions, and not merely insertion-deletion events, thus this makes rbcL
more effective in defining a species.

DNA Barcoding

Nucleotide Diversity or
Divergence

highest in rbcL 0.04532 to 0.74340

matK 0.00668 to .08724
lowest psbA 0.00082 to .03620
Nucleotide diversity (Jukes and Cantor), Pi (JC), is the average number of nucleotide
substitutions per site between two sequences where the correction has been performed in
each pairwise comparison.

DNA Barcoding

Reliability of the DNA Barcode Sequences

Investigated whether sequences can be used for species

identification (DNA Barcoding)
Misidentifications are due to wide overlap between intraand interspecific genetic variability
Tested whether DNA sequences yield an unambiguous
species level taxonomy based on assembled pairwise
distance threshold

Reliability of the DNA Barcode Sequences

Reliability of the DNA Barcode Sequences

Pairwise Distance Threshold:

Test for overlap between intra- with interspecific genetic variability

- plot uncorrected pairwise distances for conspecific sequences

- Also distances for interspecific , congeneric sequences
- for uniqueness of DNA barcode, test whether identical
sequences are shared by individuals from different species

Reliability of the DNA Barcode Sequences

rbcL
Relative Abundance

Intraspecific

6-7% gap

Relative Abundance

Pairwise Distance

matK

Pairwise Distance

NO OVERLAP J
Interspecific

trnH-psbA

overlap

rbcL
shortest gene
greatest ability of species differentiation
exhibits highest nucleotide diversity

matK
(for rbcL-difficult to amplify),
most easily amplified, & sequenced
effective in determining interspecific differences
high nucleotide diversity

DNA Barcoding

Taxonomic problems currently addressed

Taxonomic position of new endemic genera/new generic
records for the Philippines, i.e.

Oberonia-Liparis-Stichorkis
Eria complex: Mycaranthes, Pinalia, Trichotosia,
Ascidieria, Cylindrolobus, and Eria
Grammatophyllum and Sadokum

DNA Barcoding

Colocasia

Colocasia esculenta

Colocasia formosana

matK gene supports the infrageneric classification of Colocasia

C. affinis and C. esculenta belong to Section Tuberosae
C. gigantea belongs to Section Caulescentes
trnH-psbA is more diverse than matK gene

Cinnamomum

Cycas vespertilio

Cycas edentata

Herbarium Specimen
Accession Number

DNA Accession
Number

Living Collection
Accession Number

Botanical Research and Herbarium Management System

http://herbaria.plants.ox.ac.uk/bol/

Sequence

Chromatogram

Digital Images

NON COMMERCIAL
MATERIAL SUPPLY AGREEMENT FOR DNA
The recipient will
- will be used for scientific research, education, conservation
- will not be sold, distributed or used for profit or any other
commercial application
- share fairly and equitably the benefits arising from their use of
the Material
- will transfer the Material, to a bona fide third party such as
university or scientific institution for non-commercial use in
the
areas of scientific research, education, conservation

Acknowledgments

Commission on Higher Education, Philippines

De La Salle University
University Research Coordination Office
Center for Natural Science & Ecological Research

Philippine National Herbarium

E de Vogel, A Schuiteman (Kew), J Vermeulen
(NHN),
H Pedersen (Denmark), J Cootes