A total of 200 sequences of the SSU region of 18S rRNA were obtained from two samples of Pocillopora meandrina, one each from 5- and 10-meter depths. The GenBank accession numbers obtained from NCBI for the sequences from the SSU region were OQ581204–OQ581226 and OQ581434–OQ581457. The analysis of the sequencing data revealed the presence of multiple zooxanthellae clades under the Symbiodinium and Cladocopium genera in a single host (Fig. 2). While four clades (B, C, D, E) were identified under Symbiodinium, only one clade (C) was identified under Cladocopium. The maximum likelihood phylogenetic tree constructed from Symbiodinium using SSU 18S rRNA from the present study in comparison with the other studies on the same host is presented in Fig. 3. The percentage composition of the clades of both genera at 5 and 10 m depth are presented in Tables 1 and 2. No differences in clade composition were observed between 5 and 10 m depths (R = -0.064, P > 0.9). This was further confirmed by the non-multi-dimensional scaling tests (Fig. 4), which revealed no overall difference in clade communities at either depth.
Table 1
The percentage of Symbiodinium clades (B, C, D, and E) at 5 and 10 m depths).
Depth | B | C | D | E | Number of amplified sequences |
5 meters | 7.9 | 65.8 | 5.3 | 21.0 | 200 |
10 meters | 12.7 | 40.4 | 12.7 | 34.0 | 200 |
Table 2
The percentage of Cladocopium clades (B, C, D, and E) at 5 and 10 m depth).
Depth | B | C | D | E | Number of amplified sequences |
5 meters | 0 | 39 | 0 | 0 | 200 |
10 meters | 0 | 37 | 0 | 0 | 200 |
The PCR-amplified sequences exhibited a high base pair count (approximately 1075 bp) for the samples from 5 and 10 m depths. Analysis of the aligned sequences (those possessing > 99% similarity were selected using BLAST tools) revealed 145 out of 400 to be from the SSU region of the 18S rRNA of the zooxanthellae DNA extracted from Pocillopora meandrina. The pairwise genetic distances within the clades and between the clades calculated based on the maximum composite likelihood model were 0.001 and 0.043, respectively, for the lowest and highest pairwise distances for Pocillopora meandrina. Significant differences among the clades were observed from the pairwise distance data (R = 0.477, P = 0.0001; multivariate analysis of pairwise distance data - ANOSIM), implying that each clade is distinct. This was further confirmed by a cluster analysis of zooxanthellae clade data for the Symbiodinium and Cladocopium genera (Fig. 5).
The genetic diversity observed for each clade within the genera Symbiodinium and Cladocopium at 5 and 10 m is presented in Tables 3 and 4. At both depths, clade C, under both genera, demonstrated significant diversity (Tables 3 & 4). Among the Symbiodinium clades, clade C exhibited higher diversity than other clades (at 5 m, Hn = 25, Hd = 1, π = 0.0133; and at 10 m, Hn = 18, Hd = 0.9, π = 0.0125) (Table 3). Between depths, the Symbiodinium clades exhibited significant genetic differentiation (chi-square p-value = 0.0158, degrees of freedom = 203), while clade C of Cladocopium showed no significant genetic differentiation (chi-square p-value = 0.9997, degrees of freedom = 22).
Table 3
Genetic differentiation in Symbiodinium clades (5 and 10 m depths)
Clade with depth | n | Hn | H. diversity | π |
clade_c_5m | 25 | 25 | 1 | 0.0133 |
clad_E__5m | 8 | 3 | 0.7 | 0.0071 |
clad_B_5m | 3 | 1 | 0.1 | 0.001 |
clad_D_5m | 2 | 1 | 0.1 | 0.001 |
clad_c_10m | 19 | 18 | 0.9 | 0.0125 |
clad_D_10m | 3 | 1 | 0.1 | 0.001 |
clad_E_10m | 8 | 3 | 0.7 | 0.0071 |
* (Hn, is the number of haplotypes, Hd, is the haplotype diversity, π, is the nucleotide diversity)
Table 4
Cladocopium clades genetic differentiation (5 and 10 m depths).
Clade with depth | n | Hn | H. diversity | π |
clad_c_5m | 39 | 22 | 0.9770 | 0.01410 |
clade_c_10m | 37 | 20 | 0.97447 | 0.01290 |
* (Hn, is the number of haplotypes, Hd, is the haplotype diversity, π, is the nucleotide diversity)