International Journal of Marine Science 2016, Vol.6, No.33, 1-7
2
USA) in accordance with the manufacturer’s protocol before direct sequencing. The sequences of the forward and
reverse strands were determined by using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, USA).
The similarity of the sequence was then verified using the Basic Local Alignment Search Tool (BLASTn).
Maximum parsimony (MP) analyses were conducted using MEGA 5 software (Tamura et al., 2011). The MP trees
were obtained using the Close-Neighbor-Interchange algorithm (Nei and Kumar, 2000) with search level 3 in
which the initial trees were obtained with the random addition of sequence (10 replicates). All positions
containing gaps and missing data were eliminated from the dataset (Complete Deletion option). The sequence
divergences were calculated using Kimura’s two-parameter distance bootstrapped using MEGA 5 with 1000
replications to calculate the standard deviation.
Table 1 Nucleotide sequences of the PCR primers for amplification of target regions.
Region
Primer sequence (5’ – 3’)
T
a
(°C)
References
cox
2-3
spacer
COX2for
GTACCWTCTTTDRGRRKDAAATGTGATGC
50
Zuccarello et al. 2006
COX3rev
GGATCTACWAGATGRAAWGGATGTC
ITS region
NS7 forward
GAGGCAATAACAGGTCTGTGATGC
56
White et al. 1990
ITS4
TCCTCCGCTTATTGATATGC
RuBisCo
spacer
pRBCf
TGTGGACCTCTACAAACAGC
52
Tan et al. 2013
pRBCr
CCCCATAGTTCCCAAT
Fig.1. Undescribed
Kappaphycus
sp. showing morphological differences from
K. alvarezii
,
K. striatus
,
K. malesianus
and
E.
denticulatum
