Discrimination of DNA barcodes.

As shown in table (blab la bla) each barcode has shown a higher interspecific distance
compared to their intraspecific distance. Wilcoxon two sample test confirmed that the
difference between inter- and intraspecific is statistically significant (p < 0.05; table something)
This result proves that all 3 barcodes have the ability to discriminate Piper species from one
another.

Among the barcodes tested, the mean interspecific divergences were highest in (insert values
here), followed by marker (insert values), marker(insert values) then marker (insert values) for
(species) and Species), respectively (table#). To confirm the difference among each pair of
barcode, Wilcoxon Signed Rank test was performed. For Species, the test revealed that
(MARKER) has the highest interspecific divergence, followed by (insert markers) and (marker)
(p<0.05) (table#), While for Species, Marker has the highest interspecific divergence, followed
by markers and marker (p<0.05)(table #) For the intraspecific divergence of species, the lowest
were for markers and marker (0), followed by ITS (values) (table #), while for species, all
markers obtained a value of zero (table #). Based from these results, ITS has highest
discriminatory power for both genera because it has the highest interspecific variation while
having a relatively low intraspecific variation

Resolution of (species) and (Species) species

For a barcode to be efficient, it should be able to identify species up to the specific

epithet. To estimate the reliability of species identification, BLAST method was applied. Results
indicated that for species, marker and marker correctly identified 100% of the samples up until
to their species level. For marker, and marker, they failed to blab la. Meanwhile, for species,
marker and marker failed to identify 3 samples up to their species level, but got 2 of the
samples’ genera identification correctly, whereas marker indentified the genus of one sample
correctly, but failed to identify up to its species level (insert figure). Individual trees using each
of the # of barcodes were also created for species (figure) ad species (figure) species. All #
barcodes created their respective species clades with BS=100% for both ITS (insert figure) and
matK(insert figure), BS=92% for rbcL (insert figure), and BS=99% for psbA-trnH (insert figure),
and BS=99% for both matK (insert figure) and rbcL (insert figure)

Universality

For Blumea, a total of 101 consensus sequences from ITS (number), matK (number), rbcL
(number) including outgroup _______ were obtained.A total of _____ consensus sequences
from ITS, (markers etc) were gathered (refer to appendix for genbank accession numbers).
Multiple sequence alignment revealed that rbcL has the longest mean length with 560 bp for
Blumea followed by ITS, matK. . ITS yielded the highest number of parsimony informative
characters for both genera. The utilization of ITS has been applied in plants because it is more
variable than the plastid genes.
 In assessing a DNA barcode, proper short sequence length should be observed to
facilitate easy DNA amplification and sequencing (Kress et al., 2005). The most efficient barcode
in terms of universality for Blumea are ITS, and psba. Relatively low amplification and
sequencing success rates, can be attributed to the inexistence of a universal primer pair for the
genes (Kress & Erickson, 2008; Yu et al., 2011).

Figure something. Efficiency of PCR amplification and sequencing success rtes of the 4
candidate barcodes for both Blumea and Ehretia representatives.