Supplementary Information - PCR amplification PCR amplification reactions for the partial mitochondrial cytochrome oxidase subunit I (COI), the
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1 Supplementary Information - PCR amplification PCR amplification reactions for the partial mitochondrial cytochrome oxidase subunit I (COI), the ribosomal 16S rdna gene and a fragment of the nuclear single locus recombination activating gene 1 (RAG1) spanning the highly variable 5`-end of the gene were performed in 20 µl reactions containing 2 µl 10x reaction buffer, mm MgCl 2, 0.2 mm dntps, 10 mm of forward and reverse primers, 0.5 U of AmpliTaq and ng template. Denaturation in 95 o C (10 mins) was followed by 35 cycles of a denaturation step of 94 o C (30 s), an annealing step of 50 o C, 54 o C or 57 o C for 16S, COI and RAG1 respectively for 30 s, an extension step of 72 o C (1 min) and a final extension of 72 o C (7 min). PCR products were quantified by agarose gel electrophoresis on a 1% TAE agarose gel using known standards. Amplicons were purified with AMPure XP (Agencourt) following manufacturer s recommendations and sequenced using the ABI Prism BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). 1
2 Table S1 Specimens used in this study; accession numbers of sequences and collection information. Details of the COI haplotypes are also given for the specimens of Neotrygon annotata, N. kuhlii and N. ningalooensis. Species Sample # COI 16s RAG1 Collection location Lat Long COI Haplotype Neotrygon annotata A11229 KC KC KC Australia, QLD, Torres Strait Hap35 Neotrygon annotata A11230 KC KC KC Australia, QLD, Torres Strait Hap35 Neotrygon annotata A11231 KC KC KC Australia, QLD, Torres Strait Hap37 Neotrygon cf annotata A2643 EU Australia, QLD, Torres Strait Hap36 Neotrygon cf annotata A2644 EU Indonesia, West Java, Muara Angke Hap36 Neotrygon cf annotata A2645 EU KC Indonesia, West Java, Muara Angke Hap36 Neotrygon cf annotata A2646 EU KC KC Indonesia, West Java, Muara Angke Hap36 Neotrygon cf annotata A2647 EU KC KC Indonesia, West Java, Muara Angke Hap36 Neotrygon kuhlii clade 1 A2584 EU KC KC Taiwan, Penghu Islands Hap1 Neotrygon kuhlii clade 1 A2585 EU KC Taiwan, Penghu Islands Hap3 Neotrygon kuhlii clade 1 A2586 EU KC Taiwan, Penghu Islands Hap1 Neotrygon kuhlii clade 1 A2587 KC KC Taiwan, Penghu Islands Hap1 Neotrygon kuhlii clade 1 A6218 GU KC KC Thailand, Gulf Coast Hap1 Neotrygon kuhlii clade 1 A6219 GU KC Thailand, Gulf Coast Hap1 Neotrygon kuhlii clade 1 A6276 GU KC KC Vietnam, Con Son Island fish market Hap2 Neotrygon kuhlii clade 1 A7817 HM KC Taiwan, Penghu Islands Hap1 Neotrygon kuhlii clade 1 A7818 KC Taiwan, Penghu Islands Neotrygon kuhlii clade 1 A7819 HM KC Taiwan, Penghu Islands Hap1 Neotrygon kuhlii clade 1 A7820 KC Taiwan, Penghu Islands Neotrygon kuhlii clade 1 BO409 KC KC KC Malaysia, Sarawak Hap2 Neotrygon kuhlii clade 2 A2575 EU KC KC Indonesia, West Java, Muara Angke Hap6 Neotrygon kuhlii clade 2 A2576 EU KC KC Indonesia, West Java, Muara Angke Hap4 Neotrygon kuhlii clade 2 A2577 EU KC Indonesia, West Java, Muara Angke Hap9 Neotrygon kuhlii clade 2 A2578 EU KC Indonesia, West Java, Muara Angke Hap7 Neotrygon kuhlii clade 2 A2579 EU KC KC Indonesia, West Java, Muara Angke Hap8 Neotrygon kuhlii clade 2 A6221 GU KC KC Thailand, Gulf Coast Hap5 Neotrygon kuhlii clade 2 A7737 GU KC Indonesia, West Java, Muara Angke Hap9 Neotrygon kuhlii clade 2 BO424 KC KC Malaysia, Sarawak Tanjung Manis Hap5 2
3 Neotrygon kuhlii clade 2 BO473 KC KC KC Malaysia, Sarawak Mukah Hap5 Neotrygon kuhlii clade 3 A6220 GU KC KC Thailand, Gulf Coast Hap10 Neotrygon kuhlii clade 3 BO423 KC KC Malaysia, Sarawak Tanjung Manis Hap10 Neotrygon kuhlii clade 4 P AB Japan, Okinawa, Ishigaki Is Hap11 Neotrygon kuhlii clade 5 A208 DQ KC KC Australia, QLD, Gulf of Carpentaria Hap14 Neotrygon kuhlii clade 5 A5649 KC KC Australia, QLD, Off Turtle Head Is Hap14 Neotrygon kuhlii clade 5 A5650 KC KC Australia, QLD, Torres Straits Hap14 Neotrygon kuhlii clade 5 A5960 KC KC Indonesia, Lombok, Tanjung Luar Hap16 Neotrygon kuhlii clade 5 A5961 KC KC Indonesia, Lombok, Tanjung Luar Hap16 Neotrygon kuhlii clade 5 A6198 KC KC Australia, WA, Shark Bay Hap12 Neotrygon kuhlii clade 5 A6199 GU KC Australia, WA, Shark Bay Hap15 Neotrygon kuhlii clade 5 A6849 KC KC KC Australia, QLD, off Weipa Hap13 Neotrygon kuhlii clade 5 A6850 KC KC Australia, QLD, off Weipa Hap14 Neotrygon kuhlii clade 5 A7794 HM Australia, QLD, off Weipa Hap14 Neotrygon kuhlii clade 6 A2571 EU KC Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 6 A2572 EU KC Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 6 A2573 EU Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 6 A2574 EU KC Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 6 A2580 EF KC KC Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 6 A2582 KC KC Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 6 A2583 EU KC Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 6 A5731 KC KC KC Indonesia, Central Java, Sadang Hap17 Neotrygon kuhlii clade 6 A5737 KC KC KC Indonesia, Central Java, Sadang Hap17 Neotrygon kuhlii clade 6 A5738 KC KC Indonesia, Central Java, Sadang Hap17 Neotrygon kuhlii clade 6 A5739 KC KC KC Indonesia, Central Java, Sadang Hap17 Neotrygon kuhlii clade 6 A7812 HM KC KC Indonesia, Bali, Kedonganan Hap17 Neotrygon kuhlii clade 7 A6222 GU KC Thailand, Andaman Coast Hap18 Neotrygon kuhlii clade 7 A6223 GU KC KC Thailand, Andaman Coast Hap20 Neotrygon kuhlii clade 7 A6224 GU KC Thailand, Andaman Coast Hap20 Neotrygon kuhlii clade 7 A6225 GU KC KC Thailand, Andaman Coast Hap19 Neotrygon kuhlii clade 7 A6226 GU KC KC Thailand, Andaman Coast Hap20 Neotrygon kuhlii clade 8 CHN 156 HM HM India, Kochi, Kerala Hap21 Neotrygon kuhlii clade KC KC KC Tanzania, Tanga Hap22 Neotrygon kuhlii clade 9 A6217 GU KC KC New Caledonia, Magenta Beach Hap24 3
4 Neotrygon kuhlii clade 9 A7791 HM KC KC Australia, NSW, off Yamba Hap25 Neotrygon kuhlii clade 9 A7792 HM KC Australia, NSW Hap25 Neotrygon kuhlii clade 9 A7793 HM KC Australia, NSW Hap25 Neotrygon kuhlii clade 9 A7808 HM KC KC Australia, QLD, Gladstone Hap28 Neotrygon kuhlii clade 9 A7809 HM KC Australia, QLD, Gladstone Hap25 Neotrygon kuhlii clade 9 A7810 HM KC Australia, QLD, Gladstone Hap26 Neotrygon kuhlii clade 9 A7811 KC Australia, QLD, Gladstone Neotrygon kuhlii clade 9 A7813 HM KC KC Australia, QLD, Moreton Bay Hap25 Neotrygon kuhlii clade 9 A7814 HM KC Australia, QLD, Moreton Bay Hap27 Neotrygon kuhlii clade 9 A7815 HM KC Australia, QLD, Moreton Bay Hap25 Neotrygon kuhlii clade 9 A7816 HM KC Australia, QLD, Moreton Bay Hap25 Neotrygon kuhlii clade 9 UGA008 KC Australia, QLD, Lizard Island Hap23 Neotrygon leylandi A2917 EU KC KC Australia, WA, Exmouth Neotrygon leylandi A2935 EU KC KC Australia, WA, Shark Bay Neotrygon leylandi A6235 KC KC Australia, WA, Coral Bay Neotrygon picta A5653 KC KC KC Australia, QLD, Torres Strait Neotrygon picta A5659 KC KC KC Australia, QLD, Torres Strait Neotrygon picta A5661 KC KC Australia, QLD, Torres Strait Neotrygon ningalooensis A6233 HQ KC KC Australia, WA, Coral Bay Hap34 Neotrygon ningalooensis A6234 KC KC KC Australia, WA, Coral Bay Hap34 Neotrygon ningalooensis A6205 GU KC KC Australia, WA, Shark Bay Hap32 Neotrygon ningalooensis A6215 GU KC Australia, WA, Shark Bay Hap30 Neotrygon ningalooensis A6216 GU KC Australia, WA, Shark Bay Hap29 Neotrygon ningalooensis A6204 GU Australia, WA, Shark Bay Hap32 Neotrygon ningalooensis A6206 GU Australia, WA, Shark Bay Hap31 Neotrygon ningalooensis A6207 GU Australia, WA, Shark Bay Hap31 Neotrygon ningalooensis A6212 GU Australia, WA, Shark Bay Hap33 Outgroups Dasyatis brevicaudata EU EU Dasyatis parvonigra A3755 EU KC KC Dasyatis ushiei BW-2176 EU KC KC Dasyatis zugei A3765 EU Dasyatis zugei A3766 EU KC Himantura astra A225 DQ KC
5 Himantura walga EU KC KC Pastinachus atrus A2161 EU KC KC Pteroplatytrygon violacea HM HM HM Taeniura lymma UG0012 KC KC KC Taeniura lymma UG0713 KC KC KC Taeniurops meyeni A6227 GU KC KC Urogymnus asperrimus A4563 KC KC KC Rhynchobatus australiae A2380 EU KC Rhynchobatus australiae A2925 EU Carcharhinus leuca U62645 *All Axxxx numbers refer to corresponding Barcode of Life Database specimen numbers BW-Axxxx Table S2 Kimura 2-Parameter (K2P) corrected Neighbour Joining distance matrix with minimum and maximum values (in brackets) for the Neotrygon kuhlii mitochondrial COI clades 1 to 9. Clade ( ) ( ) 0.58 (0-0.93) ( ) 1.33 ( ) 0 (0-0) ( ) 2.54 ( ) 2.48 ( ) N/A ( ) 3.16 ( ) 3.10 ( ) 3.47 ( ) 0.53 (0-1.4) ( ) 2.61 ( ) 2.53 ( ) 3.17 ( ) 2.68 ( ) 0 (0-0) ( ) 2.35 ( ) 2.33 ( ) 3.31 ( ) 2.84 ( ) 1.69 ( ) 0.16 ( ) ( ) 2.56 ( ) 2.36 ( ) 3.17 ( ) 2.53 ( ) 1.41 ( ) 1.58 ( ) 1.56 ( ) ( ) 4.05 ( ) 4.00 ( ) 3.57 ( ) 4.53 ( ) 3.97 ( ) 4.07 ( ) 3.8 ( ) 0.3 ( ) 5
6 Table S3 K2P-corrected neighbour-joining distance matrix of Neotrygon species calculated from the COI alignment. Genetic distance is along the lower diagonal and standard error along the upper diagonal. N. annotata N. cf annotata N. ningalooensis N. cf ningalooensis N. leylandi N. picta N. kuhlii clade N. kuhlii clade N. kuhlii clade N. kuhlii clade N. kuhlii clade N. kuhlii clade N. kuhlii clade N. kuhlii clade N. kuhlii clade
7 Table S4 Mean divergence times and their 95% HPD confidence intervals based on fossil calibrations and published COI mutation rates (0.58% / My and 0.9% / My) calculated in the spotted eagle ray (A. narinari) from the isthmus of panama closure for nodes 1 to 10. Fossil Mutation rate 0.58% / My 0.9% / My Node Age (95% CI) Age (95% CI) Age (95% CI) ( ) 7.9 ( ) 11.6 ( ) ( ) 3.9 ( ) 9.0 ( ) ( ) 1.2 ( ) 7.3 ( ) ( ) 8.9 ( ) 5.8 ( ) ( ) 2.9 ( ) 1.9 ( ) ( ) 2.0 ( ) 1.4 ( ) ( ) 2.3 ( ) 1.5 ( ) ( ) 2.9 ( ) 1.9 ( ) ( ) 3.0 ( ) 2.0 ( ) ( ) 0.8 ( ) 0.5 ( ) Mutation rates from the present study COI = 0.229% per My 16s = % per My RAG1 = % per My 7
8 Figure S1 Substitution frequencies of transitions (cross) and transversions (triangle) for: a) the 1 st, b) 2 nd and c) 3 rd codon positions of the partial COI gene. Observed frequencies are calculated with the K2P genetic distance. a b a 8
9 Figure S2 Substitution frequencies of transitions (cross) and transversions (triangle) for: a) the 1 st, b) 2 nd and c) 3 rd codon positions of the partial 16s gene. Observed frequencies are calculated with the K2P genetic distance. a b c 9
10 Figure S3 Substitution frequencies of transitions (cross) and transversions (triangle) for: a) the 1 st, b) 2 nd and c) 3 rd codon positions of the partial RAG1 gene. Observed frequencies are calculated with the K2P genetic distance. a b c 10
11 Figure S4 Mismatch distributions for pairwise comparisons of Neotrygon kuhlii COI mitochondrial clades. Monomorphic clades 3, 4 and 6 were excluded from the calculation. The frequencies of expected and observed differences are given as a solid and dashed line, respectively. Figure: Ts-Tv_ratio C1 C7 Substitution frequencies of transitions (cross) and transversions (triangle) for the 1 st, 2 nd and 3 rd codon positions for the COI, 16s and RAG1 gene. Observed frequencies are calculated with the K2P genetic distance. τ = τ = C2 C8 τ = τ = C5 C9 τ = τ =
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