Thornhill et al. ME Supplementary Materials revision 08/2007 1

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1 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 1 Summary of clones obtained from estimates of Taq polymerase/bacterial cloning error in Symbiodinium type A3 (culture 77). The dominant clone from this culture was subjected to a second round of PCR and cloning. Comparisons are relative to the parental sequence. Sample Name Same as Dominant Sequence? GenBank Accession # A3.Direct - EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A No EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU074857

2 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 2 Summary of clones obtained from estimates of Taq polymerase/bacterial cloning error in Symbiodinium type C1 (culture 152). The dominant clone from this culture was subjected to a second round of PCR and cloning. Comparisons are relative to the parental sequence. Sample Name Same as Dominant Sequence? GenBank Accession # C1.Direct - EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C No EU C Yes EU C Yes EU C Yes EU C No EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C Yes EU C No EU C Yes EU C Yes EU C Yes EU C Yes EU C No EU C Yes EU C Yes EU074885

3 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 3 Summary of secondary structural disruptions and PCR-chimeras identified within the 5.8S-rDNA and ITS2 of bacterial clones originating from Symbiodinium type A3 (culture 77). A Y indicates that one or more disruptions were detected while a - denotes that no disruptions were identified. Sample Name Same as Dominant Sequence? GenBank Accession # 5.8S-rDNA Disruptions 1 ITS2 Disruptions 2 Suspected PCR Chimera A3.Direct - EU A Yes EU A Yes EU A Yes EU A No EU A No EU A Yes EU A No EU Y (124, 125) - - A Yes EU A Yes EU A No EU A Yes EU A Yes EU A Yes EU A No EU Y (110) - - A Yes EU A Yes EU A Yes EU A Yes EU A Yes EU A No EU Y (148) - A Yes EU A Yes EU Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 3 of the manuscript. 2 Numbers corresponds to the nucleotide position listed in the Supplementary Figure 3 alignment.

4 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 4 Summary of secondary structural disruptions and PCR-chimeras identified within the 5.8S-rDNA and ITS2 of bacterial clones originating from Symbiodinium type B1 (culture 13). A Y indicates that one or more disruptions were detected while a - denotes that no disruptions were identified. Sample Name Same as Dominant Sequence? GenBank Accession # 5.8S-rDNA Disruptions 1 ITS2 Disruptions 2 Suspected PCR Chimera B1.Direct - EU B No EU B No EU B Yes EU B No EU B No EU Y (21) - - B Yes EU B No EU Y (30) - B No EU Y (142, 151) - B Yes EU B Yes EU B Yes EU B Yes EU B No EU Y (73) - - B Yes EU B No EU B Yes EU B Yes EU B Yes EU B No EU B No EU Y (7) - - B No EU B No EU Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 3 of the manuscript. 2 Numbers corresponds to the nucleotide position listed in the alignment of Supplementary Figure 4.

5 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 5 Summary of secondary structural disruptions and PCR-chimeras identified within the 5.8S-rDNA and ITS2 of bacterial clones originating from Symbiodinium type C1 (culture 152). A Y indicates that one or more disruptions were detected while a - denotes that no disruptions were identified. Sample Name Same as Dominant Sequence? GenBank Accession # 5.8S-rDNA Disruptions 1 ITS2 Disruptions 2 Suspected PCR Chimera C1.Direct - EU C No EU Y (54, 60-68) - C Yes EU C No EU C No EU C Yes EU C No EU Y (95) - - C No EU C No EU C No EU C No EU Y (152) - C No EU C No EU Y (49, 123) - - C No EU C Yes EU C No EU C No EU C No EU Y (119) - - C No EU Y (54, 60-68) Y (C1/C1.1126) C Yes EU C No EU Y (26) - - C No EU C No EU Y (99) Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 3 of the manuscript. 2 Numbers corresponds to the nucleotide position listed in the alignment of Supplementary Figure 5. 3 Names correspond to the parent sequences of a suspected PCR-chimera. See Supplementary Figure 1.

6 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 6 Summary of secondary structural disruptions and PCR-chimeras identified within the 5.8S-rDNA and ITS2 of bacterial clones originating from Symbiodinium type D1a (culture A001). A Y indicates that one or more disruptions were detected while a - denotes that no disruptions were identified. Sample Name Same as Dominant Sequence? GenBank Accession # 5.8S-rDNA Disruptions 1 ITS2 Disruptions 2 Suspected PCR Chimera D1a.Direct - EU D1a.2010 Yes EU D1a.2014 No EU Y (44-136) - - D1a.2017 No EU Y (48) - D1a.2019 Yes EU D1a.2023 No EU Y (48, 50, 93) - D1a.2025 Yes EU D1a.2027 No EU D1a.2028 No EU Y (44-136) Y (48) - D1a.2038 No EU D1a.2040 No EU Y (105) Y (48) - D1a.2042 No EU D1a.2050 No EU D1a.2052 No EU Y (150) - D1a.2056 Yes EU D1a.2062 No EU D1a.2064 No EU D1a.2066 No EU Y (121) - D1a.2068 No EU Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 3 of the manuscript. 2 Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 4 of the manuscript.

7 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 7 Summary of secondary structural disruptions and PCR-chimeras identified within the 5.8S-rDNA and ITS2 of bacterial clones originating from Symbiodinium type E2 (culture CCMP 421). A Y indicates that one or more disruptions were detected while a - denotes that no disruptions were identified. Same as Dominant Sequence? 5.8S-rDNA Disruptions 1 ITS2 Disruptions 2 Sample Name GenBank Accession # Suspected PCR Chimera 3 E2.Direct - EU E No EU Y (22) Y (49, 176, 195) Y (E2/E2.2094) E Yes EU E Yes EU E Yes EU E No EU Y (105) - - E No EU Y (157) - E No EU Y (51, 152, 176, 195) - E No EU Y (51, 152, 176, 195) - E No EU Y (49) - E No EU Y (49) - E No EU Y (49) Y (E2/E2.2094) E No EU E No EU Y (49) - E Yes EU E No EU E Yes EU E Yes EU E No EU E Yes EU E No EU Y (51, 152, 176, 190, 195) Y (E2/E2.2094) E No EU E No EU Y (119) - - E No EU Y (51, 152, 176, 195) - E No EU Y (51, 152, 176, 195) Y (E2/E2.2094) 1 Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 3 of the manuscript. 2 Numbers corresponds to the nucleotide position listed in the alignment of Supplementary Figure 6. 3 Names correspond to the parent sequences of a suspected PCR-chimera. See Supplementary Figure 1.

8 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Table 8 Summary of secondary structural/processing site disruptions and PCR-chimeras identified within the 5.8SrDNA and ITS2 of bacterial clones originating from Symbiodinium populations of Hawaiian Porites spp. corals (data from Apprill & Gates 2007). A Y indicates that one or more disruptions were detected while a - denotes that no disruptions were identified. Sample Name 5.8S Disruptions 1 ITS2 & Conserved Processing Site Disruptions 2 Suspected PCR Chimera 3 GenBank Accession # C15 AY KB1 DQ Y ( , 110, 114) Y (78, 98, 110, 119, , 123) - KB2 DQ Y ( , 110, 114, 123) Y (78, 98, 110, 121) - KB3 DQ Y ( , 114) Y (55, 78, 110, 121) - KB4 DQ Y ( , 114) Y (55, 78, 110, 121) - KB102C_5B DQ Y ( , 114) - Y (KB4/C15) KB102C_8B DQ Y ( , 114) Y (78, 110, 121) - KB119_16 DQ Y (94) Y ( ) - KB129_4 DQ KB135_42 DQ Y ( , 110, 114) Y (78, 98, 104, 110, 121) - KB135_63 DQ Y ( , 110, 114) Y (78) Y (KB1/C15) KB135_89 DQ Y ( , 114) Y (78, 110, 121) - KB162R_3 DQ Y ( , 110, 114) Y (78, 98, 110, 119, , 123) - KB162R_4 DQ Y ( , 110, 114) Y (78, 98, 110, 119, , 123, 182) - KB179A_18 DQ KB179B_21 DQ Y ( , 114, 128) Y (78, 110, 121) - KB179B_24 DQ Y (40, 154) - KB179B_7 DQ Y ( , 114) Y (55) Y (KB4/C15) KB184_1 DQ Y ( , 110, 114) Y (78, 98, 110, 119, , 123) - KB184_3 DQ Y ( , 110, 114) Y (18, 78, 98, 110, 119, , 123) - KB184L_1 DQ Y ( , 110, 114) Y (78, 98, 110, 119, , 123) - KB184RL_4 DQ Y ( , 110, 114, 123) Y (78, 98, 110, 121) - KB184RR_3 DQ Y ( , 110, 114, 123) Y (78, 98, 110, 121) - KB184RR_4 DQ Y ( , 110) Y (33, 78, 98, 110, 119, , 123) - 1 Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 3 of the manuscript. 2 Numbers corresponds to the nucleotide position listed in the alignment presented in Figure 5 of the manuscript. 3 Names correspond to the parent sequences of a suspected PCR-chimera. See Supplementary Figure 7. 8

9 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure Legends Supplementary Figure 1: Putative PCR-chimeras detected in the bacterial cloned dataset of Symbiodinium type C1 (culture 152) and type E2 (culture CCMP 421). Suspected chimeric sequences include clones: A) C1_1178, B) E2_2074, C) E2_2122, D) E2_2130, and E) E2_2102. For each, an alignment of the parent sequences and putative chimera is shown. Alignments include only those nucleotide positions that differ; the number at the top of an alignment (read vertically downwards) designates site position on an alignment that including all clones from that culture. Dashes (-) indicate an insertion/deletion (indel) while an arrow ( ) denotes the break point between parent sequences in the putative chimera. Nucleotide positions in the chimera differing from either parent sequence are indicated in gray; these are likely a result of Taq polymerase error or failure to recover the actual parental sequence(s). Diagrams are presented below each alignment indicating the portions of the parent molecules contributing to each chimera. Supplementary Figure 2: Unrooted maximum parsimony phylogenies of ITS2 region sequence variants generated via PCR and bacterial cloning from two clonal Symbiodinium cultures: (A) culture 77 ( type A3; GenBank Accession Numbers EU EU074938), (B) culture 152 ( type C1; GenBank Accession Numbers EU EU074961). For each polytomy, numbers of point substitutions and/or indels are related to branch length. Indels were weighted as a single change regardless of length. Numbers of clones with identical sequences are indicated by circle size at each node; identical sequences recovered more than three times have the corresponding number of replicates (in parentheses) following clone name. Supplementary Figures 3 6: Secondary structural analyses and alignments of ITS2 sequences from clonal isolates of Symbiodinium types : A3 (culture 77, Supplemental Figure 3), B1 (culture 13, Supplemental Figure 4), C1 (culture 152, Supplemental Figure 5), and E2 (culture CCMP 421, Supplemental Figure 6). Boundaries of the helices are labeled and correspond to those above the nucleotide alignment. The sequence generated directly from the PCR product is used as the template against which the cloned sequences are compared (see Materials and Methods). A nucleotide (A, C, G, or U) in place of a dot (.) denotes a substitution at that position. Dashes (-) indicate an insertion/deletion (indel). Numbers listed under Mut. Pos. represent positions where mutational differences exist between the direct- and clone-derived sequences. For these, effects to secondary structural stability of ITS2 are inferred (i.e., Effect SS ): N = no influence, + = positive influence, - = negative influence, ~ = weakened interaction (G-U pairing). Arrows identify variable sequence positions on the secondary structure diagram. Characters depicted in gray represent a highly conserved rrna processing site. Supplementary Figure 7: Putative PCR-chimeras detected in the bacterial cloned dataset of Symbiodinium populations of Hawaiian Porites spp. corals (data from Apprill & Gates 2007). Suspected chimeric sequences include clones: A) KB102C_5B, B) KB135_63, C) KB179B_7. For each, an alignment of the parent sequences and putative chimera is shown. Alignments include only those nucleotide positions that differ; the number at the top of an alignment (read vertically downwards) designates site position on an alignment that including all clones from Apprill & Gates (2007). Dashes (-) indicate an insertion/deletion (indel) while an arrow ( ) denotes the break point between parent sequences in the putative chimera. Nucleotide positions 9

10 Thornhill et al. ME Supplementary Materials revision 08/ in the chimera differing from either parent sequence are indicated in gray; these are likely a result of Taq polymerase error or failure to recover the actual parental sequence(s). Diagrams are presented below each alignment indicating the portions of the parent molecules contributing to each chimera. 10

11 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure 1 11

12 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure 2 12

13 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure 3 13

14 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure 4 14

15 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure 5 15

16 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure 6 16

17 Thornhill et al. ME Supplementary Materials revision 08/ Supplementary Figure 7 17

Factors affecting PCR

Lec. 11 Dr. Ahmed K. Ali Factors affecting PCR The sequences of the primers are critical to the success of the experiment, as are the precise temperatures used in the heating and cooling stages of the