Francisco et al.

| 17

CHAPTER IV

RESULT AND DISCUSSION

Among the 27 samples obtained from Cagayan, only 20 samples had been

successfully sequenced because the cheliped of 3 samples were lost and the other samples

did not matched to CO1 sequences, possibly due to contamination. Using the DNA

barcoding approach, all of the 20 samples were identified to the species level. For each

species, a CO1 consensus sequence was generated to align with the sequences of

brachyurans obtained from GenBank. Figure 2 shows the phylogenetic tree of CO1

sequences from crab species samples. GenBank sequences of Portunus reticulatus and

Portunus armatus were used as outgroup. There were two species of Scylla identified,

namely Scylla serrata and Scylla olivacea.

BUGUEY 1
88
BUGUEY 9
50
APARRI 1

82 APARRI 6
SANTA ANA 1
61
BUGUEY 3
SANTA ANA 2
23
76 KC200562 Scylla serrata

99 BUGUEY 10
45 SANTA ANA 4
83
APARRI 2
APARRI 4

74 99
BUGUEY 13
80 BUGUEY 7

BUGUEY 2
BUGUEY 4
99
KC200563 Scylla olivacea
16
63 BUGUEY 16
71 BUGUEY 12

62 SANTA ANA 3

23 BUGUEY 14
69 APARRI 5
HM750949 Atergatis floridus
96 HM751078.1 Zosimus aeneus
EF661877 Portunus armatus
99 EF661976 Portunus reticulatus

0.1

Figure 3 Phylogenetic tree of CO1 sequence of market sold crabs obtained from different market of
Cagayan province (Buguey, Sta Ana, and Aparri) using neighbor-joining tree (kimura 2- parameter).
Portunus armatus and Portunus reticulatus served as outgroup. Sequence of all the samples were also
compared to 2 known Poisonous crabs, namely, Atergatis floridus and Zosimus aeneus.
 Francisco et al. | 18

624 BUGUEY 1
89 BUGUEY 9
50 APARRI 1
624
APARRI 6
81
SANTA ANA 1
59 BUGUEY 3
SANTA ANA 2
22
76 KC200562 Scylla serrata

99 BUGUEY 10
45 SANTA ANA 4
83
APARRI 2
APARRI 4

75 99 BUGUEY 13
80 BUGUEY 7
BUGUEY 2
BUGUEY 4
99
KC200563 Scylla olivacea
16
63 BUGUEY 16
71 BUGUEY 12

62 SANTA ANA 3

22 BUGUEY 14
68 APARRI 5
HM750949 Atergatis floridus
96 HM751078 Zosimus aeneus
|EF661877 Portunus armatus
99 EF661976 Portunus reticulatus

Figure 4 Topology of Phylogenetic tree of CO1 sequence of market sold crabs obtained from different
market of Cagayan province (Buguey, Sta Ana, and Aparri) using neighbor-joining tree (kimura 2-
parameter).

Ten of the samples clearly matched with the known Scylla serrata GenBank

sequences and 7 samples matched with the known Scylla olivacea Genbank sequences. The

two species of Scylla can differ to each other by identifying the colors of the cheliped.
 Francisco et al. | 19

Scylla serrata samples have a light green and light yellow to red color of pincers (Figure

4) while the Sylla olivacea samples have a dark green and red color of pincers.

A B

C D
Figure 5 Scylla serrata sample (A) frontal view (B) ventral view and Scylla olivacea (C) frontal view (D) ventral
view.

The Samples BU7, BU13 and AP4 are morphologically similar to Scylla serrata

and have little morphological differences. Their differences can slightly be seen in the color

of their pincers. AP4 has a purple and yellow color of pincer, BU13 has yellow and dark

red, and BU7 has light red and dark green color of pincer. These 3 samples were identified

as cryptic species by having the mean genetic distance ranged from 9.7 to 17.6 percent.
 Francisco et al. | 20

C
Figure 6 samples AP4 (A), BU7 (B) and BU13 (C).left picture (frontal view), right picture (ventral view)

Approximately, 600 base pair of the CO1 gene was successfully amplified from 20

out of 27 samples. The average nucleotide composition for all sequences were Adenine=

715.0 %, Thymine= 993.1%, Cytosine= 531.1 %, Guanine= 460.9 %. The intra specific

genetic distance ranged from 0.002 to 0.156 for Scylla serrata, from 0.006 to 0.141 for

Scylla olivacea. The 3 suspected cryptic species sample has the intra specific genetic

distance ranged from 0.004 to 1.26 between Scylla serrata and from 0.011-0.129 between

the Scylla olivacea.

 Francisco et al. | 21

Sequences of 20 samples were also compared to the 2 known species of poisonous

crabs in the Philippines namely Atergatis floridus and Zosimus aeneus. Fortunately, none

of the samples matched or grouped with the known poisonous crab GenBank sequences.

The mean genetic distance (Kimura 2-parameter model) of 20 samples from two toxic crabs

ranged from 20.5 to 64.3 percent, which means that the samples are not related to the toxic

crabs.

The classification for Scylla has been notorious. Estampador (1949) set Scylla to

four taxa (three species and one subspecies) comprising Scylla serrata, Scylla oceanica,

Scylla tranquebarica, and S. serrata var. paramamosain, while Stephenson and Campbell

(1960) proposed only one species (S. serrata) for this genus. Keenan and others (1998)

revised the taxonomy of genus Scylla using both morphometric and genetic approaches,

and separated this genus into four distinct species: S. serrata, S. olivacea, S. tranquebarica

and Scylla paramamosain. DNA barcoding is a new way of identification that can lead to

species discovery by flagging cryptic species. Worldwide campaign regarding the DNA

barcoding of all crustacean species is necessary. Building regional database of all

crustaceans can bring closer understanding to humans how wide is the diversity of

crustacean (Radulovici et al, 2009). Practical applications of DNA barcoding of

crustaceans comprises the detection of invasive species, authenticating of substituted

processed seafood and estimation of stock size of harvested species based on larval

abundance (Costa et al, 2007).