Phylogeography of red muntjacs reveals three distinct mitochondrial lineages
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1 1 Phylogeography of red muntjacs reveals three distinct mitochondrial lineages Renata F. Martins, Jörns Fickel, Minh Le, Thanh van Nguyen, Ha M. Nguyen, Robert Timmins, Han Ming Gan, Jeffrine J. Rovie-Ryan, Dorina Lenz, Daniel W. Förster, Andreas Wilting Additional file 1 Table S1. Comparison of number of accepted red muntjac species and subspecies among three different recent publications. Number of recognized species range from 1 to 6 and number of recognized subspecies range from none to 10. Mattioli (2011)[10] Grubb and Groves (2011)[13] Timmins et al. (2016)[7] Species M. muntjak M. muntjak M. muntjak M. vaginalis M. vaginalis M. malabaricus M. aureus M. nigripes M. guongdongensis * Subspecies M. m. muntjak M. v. vaginalis not applicable M. m. annamensis M. v. curvostylis M. m. aureus M. m. curvostylis M. m. malabaricus M. m. mengalis M. m. montanus M. m. nigripes M. m. vaginalis M. m. yunnanensis * Identified as Incertae sedis, but referring to the different form described by Li J-x &Xu, 1996
2 2 Table S2. Complete dataset used for analyses, with information on origin, location and contact. Collection ID Sample name Name in Figures Genbank Source material Geographic origin Geographic origin MMU1 BAL1 KY nasal bone West Bali West Bali MMU2 JAV13 KY nasal bone West Java Java MMU3 THA2 KY nasal bone Thailand Thailand MMU6 IND5 KY nasal bone and tissue from India India MMU8 IND2 KY nasal bone India India MMU10 CHI3 KY nasal bone Yunnan 4837 MMU12 IND10 KY nasal bone East India East India MMU16 BOR2 KY nasal bone MMU17 LOM2 KY bone from MMU18 SRI1 KY nasal bone MMU20 CHI1 KY nasal bone MMU21 LSI1 KY Southeast Lombok East Sri Lanka Yunnan Lesser Sunda Islands Lombok Sri Lanka Lesser Sunda Islands MMU22 BOR7 KY nasal bone ZMA ZMA MMU24 SUM6 KY nasal bone Sumatra Sumatra 1950 MMU25 SUM1 KY nasal bone Sumatra Sumatra MMU27 JAV17 KY nasal bone and tissue from Year Java Java MMU28 JAV15 KY nasal bone Java Java MMU29 JAV5 KY nasal bone East Java Java MMU30 BOR5 KY antler drill MMU31 BOR6 KY antler drill 1902 Contact
3 MMU32 BOR4 KY antler drill MMU34 THA6 KY nasal bone Thailand Thailand MMU35 JAV6 KY nasal bone West Java Java MMU36 JAV11 KY nasal bone West Java Java MMU38 SUM4 KY nasal bone Sumatra Sumatra MMU40 SUM2 KY nasal bone Sumatra Sumatra MMU41 JAV14 KY nasal bone West Java Java MMU42 JAV1 KY nasal bone West Java Java MMU43 JAV16 KY nasal bone West Java Java MMU44 JAV3 KY nasal bone West Java Java MMU46 JAV8 KY nasal bone West Java Java MMU48 SUM5 KY tissue Sumatra Sumatra 1886 B3399 MMU49 JAV7 KY skin Java Java 1919 B3400 MMU50 JAV2 KY skin Java Java B3402 MMU51 JAV4 KY skin Java Java 1497 MMU53 SUM3 KY bone drill Sumatra Sumatra MMU59 LOM1 KY tissue Lombok Lombok MMU60 LOM3 KY tissue Lombok Lombok MMU62 IND6 KY tissue India India M122 MMU64 JAV9 KY skeleton Java Java Dr. Rainer Hutterer, Zoologisches Forschungsmuseum Alexander Koenig Bonn Dr. Irina Ruf, Senckenberg Forschungsinstitut Mammalogie Frankfurt Dr. Irina Ruf, Senckenberg Forschungsinstitut Mammalogie Frankfurt Dr. Irina Ruf, Senckenberg Forschungsinstitut Mammalogie Frankfurt M123 MMU65 NEP1 KY nasal bone Nepal Nepal 1838
4 4 M505 MMU67 LSI2 KY nasal bone M537 MMU68 IND7 KY M1267 MMU72 THA4 KY M125 MMU74 THA1 KY nasal bone and tissue from Tissue in alcohol Belitung Island Belitung Island 1887 India India 1887 Thailand Thailand 1926 South Thailand Thailand MMU75 IND9 KY skin South India South India MMU81 IND3 KY skin India India MMU82 IND4 KY nasal bone India India MMU83 SRI2 KY nasal bone Sri Lanka Sri Lanka MMU86 THA5 KY nasal bone Thailand Thailand MMU87 IND8 KY nasal bone India India M M M M.4929 M.4916 M a M a 74035/ MMU90 JAV10 KY nasal bone West Java Java 1930 MMU91 THA3 KY MMU95 LSI3 KY Thailand Bangka Island Thailand 1989 Banka MMU100 JAV12 KY nasal bone Java Java MMU102 IND1 KY nasal bone India India MMU105 BOR3 KY MMU107 BOR1 KY C.2 CHI2 KY nasal bone Central Yunnan 1894 Douglas Yu, The Ecology, Conservation, and Environment Center RJT118 RJT118 LAO1 KY Laos Laos R. J. Timmins M2.3 M2.3 VIE3 KY M2.9 M2.9 VIE7 KY bone C. C. M2.14 M2.14 VIE2 KY bone Central Central
5 5 M3.8 M3.8 VIE11 KY bone E. M5.11 M5.11 VIE13 KY dry skin M6.5 M6.5 VIE8 KY bone M6.12 M6.12 VIE1 KY bone M6.17 M6.17 VIE4 KY bone x15 x15 VIE9 KY bone x17 x17 VIE12 KY bone x19 x19 VIE10 KY bone x20 x20 VIE6 KY bone x39 x39 VIE5 KY bone mm13 mm13 MAL1 KY mm20 mm20 MAL2 KY C. Central South C. C. C. C. C. Peninsular Peninsular Central South Peninsular Peninsular 2012 Monash University, Monash University, Table S3. Long-range PCR primer sequences and annealing temperatures used for bait development. Fragment 1 Fragment 2 Fragment 3 Position on ref. Sequence 5'-3' 2478 F: CGATTAAAGTCCTACGTGATCTGAG 6926 R: GTTATGATGTTGGCTTGAAACCAG 6506 F: GCTATYATRGGAGGATTTGTTCAC R: GATTAGGGCTGTTGTRGTAAATG F: TTACAAATCTTAACGCCTGAGACTTC 2548 R: TAGATAGAAACCGACCTGGATTACTC Annealing Temp. a 58 ⁰C 58 ⁰C 58 ⁰C a PCR was done with MyFi Mix (Bioline GmbH, Germany) with 1x MyFi Mix, 0.4 µm each primer and water to the final volume of 50 µl for each of the fragments separately, in a total of 35 amplification cycles.
6 6 Table S4. Sequencing results from all samples used in this study, with indication of sequencing platform, percentage of reads on target, average base coverage depth and percentage of reference genome covered with at least 3x coverage. Sample Sequencing Reads on Average read % genome Captured Name platform target depth covered 3x BAL1 yes PGM 53.65% JAV13 yes PGM 45.29% THA2 yes PGM 20.92% IND5 no Illumina 0.54% IND2 yes PGM 18.15% CHI3 no Illumina 0.06% IND10 yes PGM 46.65% BOR2 yes Illumina 2.21% LOM2 yes Illumina 36.22% SRI1 yes PGM 20.46% CHI1 yes PGM 4.30% LSI1 yes Illumina 45.26% BOR7 yes PGM 10.17% SUM6 yes PGM 36.05% SUM1 yes PGM 47.57% JAV17 yes PGM 71.62% JAV15 yes PGM 65.44% JAV5 yes PGM 55.88% BOR5 yes PGM 6.24% BOR6 yes PGM 31.17% BOR4 yes PGM 18.20% THA6 yes PGM 26.33% JAV6 yes PGM 6.35% JAV11 yes PGM 40.60% SUM4 yes PGM 44.44% SUM2 yes PGM 61.77% JAV14 yes PGM 30.97% JAV1 no Illumina 0.92% JAV16 yes PGM 30.47% JAV3 no Illumina 0.18% JAV8 yes Illumina 4.88% SUM5 no Illumina 0.05% JAV7 yes Illumina 3.88% JAV2 yes Illumina 9.26% JAV4 yes PGM 50.03% SUM3 yes PGM 24.66% LOM1 yes PGM 28.73% LOM3 yes PGM 33.09% IND6 yes PGM 50.73% JAV9 yes PGM 27.96% NEP1 yes PGM 59.64% LSI2 yes PGM 16.87% IND7 yes PGM 47.52% THA4 no Illumina 0.03% THA1 yes Illumina 14.59% IND9 yes PGM 33.76% IND3 yes Illumina 1.76% IND4 yes Illumina 8.41% SRI2 yes PGM 61.03% THA5 yes Illumina 29.52%
7 7 IND8 yes Illumina 64% JAV10 no Illumina 0.07% THA3 yes PGM 39.65% LSI3 yes Illumina 18.50% JAV12 yes Illumina 3.52% IND1 yes PGM 74.84% BOR3 yes Illumina 19.56% BOR1 yes Illumina 39.98% CHI2 no Illumina 0.19% LAO1 no Illumina 0.18% VIE3 no Illumina 0.04% VIE7 no Illumina 0.03% VIE2 no Illumina 0.07% VIE11 no Illumina 0.06% VIE13 no Illumina 0.08% VIE8 no Illumina 0.10% VIE1 no Illumina 0.03% VIE4 no Illumina 0.03% VIE9 no Illumina 0.12% VIE12 no Illumina 0.20% VIE10 no Illumina 0.07% VIE6 no Illumina 0.03% VIE5 no Illumina 0.06% MAL1 no Illumina 0.93% MAL2 no Illumina 0.58% Table S5: Number of Variable sites (V) and Parsimony informative sites (Pi) per coding gene, throughout the full mitogenomes obtained. ND1 ND2 Cox1 Cox2 Cox3 ND3 ND4L ND4 ND5 ND6 Cytb D-loop V Pi
8 8 SRI2 SRI1 * * BOR1 BOR2 BOR3 MAL1 MAL2 BOR7 SUM4 SUM5 SUM6 SUM3 LSI3 JAV17 JAV6 JAV5 JAV2 JAV4 JAV3 JAV1 BAL1 BOR5 BOR4 LOM3 LOM1 * LOM2 LSI1 BOR6 JAV15 JAV16 LSI2 JAV14 JAV13 * JAV12 JAV11 SUM2 JAV9 JAV10 SUM1 JAV7 JAV8 VIE1 VIE2 VIE12 THA1 VIE3 VIE4 THA6 THA4 THA5 THA3 CHI1 CHI2 CHI3 VIE13 THA2 VIE8 VIE7 VIE6 IND3 VIE5 IND4 IND5 IND2 IND6 IND7 IND8 IND10 NEP1 IND9 VIE9 VIE10 VIE11 LAO1 IND Figure S1. Best tree obtained with Maximum Likelihood analyses with RAxML. The same topology is recovered as with Bayesian inferences, with three distinct mitochondrial clades. All branches are supported with at least 90% bootstrap support, except when indicated otherwise with *.
9 9 IND1 LAO1 VIE1 VIE2 VIE12 THA1 THA6 THA3 THA4 THA5 VIE3 VIE4 THA2 CHI2 VIE13 CHI1 CHI3 VIE8 VIE6 VIE5 VIE11 VIE7 VIE9 VIE10 NEP1 IND10 IND9 IND2 IND6 IND4 IND3 IND5 IND8 IND7 BOR3 BOR1 JAV7 BOR2 SUM5 SUM4 MAL1 BOR7 MAL2 BOR6 LSI3 SUM3 SUM6 BOR5 BOR4 LOM1 LOM2 LOM3 LSI1 BAL1 JAV6 JAV1-5 Muntiacus reevesi JAV9 JAV10 JAV15 JAV16 SUM2 JAV13 JAV14 LSI2 JAV12 JAV11 JAV7 JAV8 SUM1 WG4 WG5 SRI2 SRI1 WG2 WG3 WG Figure S2. Best tree obtained with Maximum Likelihood analyses of the Cytochrome B gene of all red muntjac samples included in this study and five additional Genbank sequences from the Western Ghats. This tree was obtained with RAxML and shows similar topology as the trees with full mitogenome, although bootstrap values are generally lower. This result shows the relationship between Sri Lanka and Western Ghats populations, as they cluster together in the same clade.
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