Download Systematics and DNA Barcoding of Philippine

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