Supplementary Methods
SNP Genotyping & Method Validation
We developed species-specific diagnostic SNP assays to explore species assignment of
putative hybrid individuals (Appendix: Table 3). Eleven to fourteen individuals morphologically
and genetically identified as pure species from each of the three species were selected as
references for the diagnostic SNP marker development. We obtained sequence data for four
nuclear genes (SH3PX3, Zic1, Glyt, ENC1) based on the degenerate and nested PCR primers
and protocol described in Li et al. (2007). Successful amplification rates vary among species and
loci, ranging from five complete sequences to all fourteen. Selected PCR products were purified
using the Agencourt AMPure kit (Beckman-Coulter) and sequenced on the ABI 3730 DNA
Analyzer (Applied Biosystems, Inc.).
Four diagnostic SNPs (i.e. informative and fixed or monomorphic within species) were
selected in each of the four genes from consensus sequence alignments viewed in
SEQUENCHER version 4.8 (Gene Codes), totaling 16 SNP markers. Assays for the selected
SNPs were designed and genotyped by means of KASP by Design assays (KBioscience – LGC
Genomics). PCR amplification was conducted using nanofluidic array chips (BioMark 48x48
Dynamic Array, Fluidigm) and each individual was genotyped three times at each of 16 SNPs.
Each sample mixture consisted of 2.5 μl of each DNA sample (~20 ng/μl), 3 μl 2X KASPar
Reagent Mix (KBioscience – LGC Genomics), 0.3 μl 20X GT Sample Loading Reagent
(Fluidigm), and 0.2 μl of DNAse-free water per inlet. Assay pre-mix consisted of 2.5 μl 2X
Assay Loading Reagent (Fluidigm), 1.8 μl of DNAse-free water, and 0.7 μl of the KASPar Assay
primer mix. Thermal cycling conditions were as follows: thermal mix step consisting of 70°C
for 30 minutes followed by 25°C for 10 minutes, hot start step at 94°C for 15 minutes, followed
by 10 cycles of 94°C for 20 seconds and 64°C for 1 minute (-0.8°C per cycle), additional 26
cycles of 94°C for 20 seconds and 57°C for 1 minute, and a cooling step at 20°C for 30 seconds.
Endpoint genotype data were analyzed using the Fluidigm SNP Genotyping Analysis software
with the default confidence threshold. Heterozygous bases were manually verified by two
people.
In addition to using nuclear loci, a portion of the mitochondrial cytochrome b gene
(CYTB) of each sample was sequenced using the conserved animal primers, H15149 and
L14724 (Kocher et al. 1989; Irwin et al. 1991). Sequences were aligned in SEQUENCHER
version 4.8 (Gene Codes Corporation) and species assignment was done by creating a neighborjoining phylogenetic tree on Geneious Pro version 4.7 with the Tamura-Nei genetic distance
model. All sequences were submitted to GenBank (Submission ID: 1676420).
Literature Cited
Irwin DM, Kocher TD, Wilson AC (1991) Evolution of the Cytochrome-B gene of mammals.
Journal of Molecular Evolution, 32, 128-144.
Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC (1989)
Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing
with conserved primers. Proceedings of the National Academy of Sciences, 86, 61966200.
Li C, Ortí G, Zhang G, Lu G (2007) A practical approach to phylogenomics: the phylogeny of
ray-finned fish (Actinopterygii) as a case study. BMC Evolutionary Biology, 7, 44.