Materials and Methods
Sample collection
A total of 263 T. viridipunctatus and T. luyeanus were captured during 2004 to 2006. The potential contact zone is around Sinchen, near the Taroko Gorge, and extends 2.5 kilometers to the south and north of the Liwu River (Figure 1). Collected samples were mapped with GPS (GARMIN GPS V). The geographic coordinates of all collected
individuals in the adjacent area of the Liwu River are N 24° 08’ to N 24°
10’, E 121° 36’ to E 121° 38’. Samples of T. viridipunctatus distributed adjacent to the potential contact zone were collected from Heren (11 individuals), while T. luyeanus samples are from Shoufen and Wanrong (25 individuals),
Collected samples were stored in 100% ethanol. DNA was extracted from tail tissues using a modified LiCl method (Gemmell & Akiyama 1996) and stored in a -20◦C freezer.
Mitochondrial DNA typing
Mitochondrial cytochrome b sequences were used to determine the maternal haplotype. Two sets of primers were designed by
species-specific sequences, to amplify a partial fragment from T.
viridipunctatus and T. luyeanus. The sequences of these two sets of
primers are listed: NL: 5’-TTG TAG AGT GAG TAT GGG GG-3’ and NH: 5’-TTG TTT TGA TAA ATG AGT GA-3’, for T. viridipuctatus and EL: 5’-TCG TAG AGT GGG TAT GAG GC-3’ and EH: 5’-TTG CTT TGA TAG GTG AAT AT-3’ for T. luyeanus. Polymerase chain reaction
(PCR) were conducted in a 10 μL reaction volume containing about 50 ng genomic DNA, 0.2 μM each primer, 0.625 mM dNTP, 1 X PCR buffer (10 mM Tris-HCl, pH 9.0; 50 mM KCl, 0.01% (w/v) gelatin, and 0.1%
Triton X-100), 0.4 U Taq DNA polymerase (Amersham Biosciences) and 1.5 mM MgCl2. The thermocycler conditions were set to 94 ◦C for 3 min, then 30 cycles of 94 ◦C for 30 s, 50 ◦C for 30 s and 72 ◦C for 30 s,
followed by 72 ◦C for 3 min (Lin 2004). Under such condition, each individual was expected to be amplified by only a single set of primers. If any individual was amplified by both sets of primers, the amplified
fragment would be sequenced, aligned, and compared to sequences of T.
viridipunctatus and T. luyeanus (Lin 2003).
Microsatellite genotyping
Thirteen microsatellite loci (Lin et al., 2006) were applied in detecting hybridization of the nuclear genome. PCR was conducted in a 10 μL reaction volume containing about 50 ng genomic DNA, 0.2 μM of each primer, 0.625 mM dNTP, 1 X PCR buffer (10 mM Tris-HCl, pH 9.0;
50 mM KCl, 0.01% (w/v) gelatin, and 0.1% Triton X-100), 0.4 U Taq DNA polymerase (Amersham Biosciences). The thermocycle conditions were set to 94 ◦C for 3 min, then 30 cycles of 94 ◦C for 30 s, 58 ◦C for 30 s and 72 ◦C for 30 s, followed by 72 ◦C for 3 min (Lin et al. 2006).
Analysis 1: The PCR products were electrophoresed on a
MegaBACE 1000 autosequencer (Amersham Biosciences) using ET-400 (Amersham Biosciences) as size standards. Sizing of alleles was analysed with the software genetic profiler 2.0 (Amersham Biosciences). We
calculated the observed and expected heterozygosities using the software
Cervus version 2.0 (Marshall et al. 1998). The conformance to Hardy-Weinberg expectations of each locus was calculated using
GENEPOP version 3.4 (Raymond & Rousset 1995). When corrected for multiple comparisons using the Bonferroni method.
RstCalc (Goodman 1999) and GENEPOP were used to calculate Rst
(Slatkin 1995) and Fst (Wright 1969) of T. viridipunctatus subpopulations, T. luyeanus subpopulations, and both sides of the river-bank populations.
In addition, calculations of correlation between genetic and geographic distance were performed through, Rst and geographic distance,
respectively.
Intraspecific and interspecific migration rates of T. viridipunctatus and T. luyeanus were calculated by BayesAss (Wilson and Rannala 2003).
Analysis 2: The Bayesian clustering method described by Pritchard et al. (2000) and implemented in the program STRUCTURE 2.1 were
applied to the genetic data. Using multilocus genotypes to assign
individuals to K number of groups. To choose the 100,000 burn-in length and to 1,000,000 iterations, several runs were assayed at each number of groups. Posterior probabilities of the number K were estimated assuming uniform prior values on K and comparing the Ln of the probability of the data for each one. The highest likelihood value is assumed to indicate the number of groups in sample data.
After the number of K groups was estimated and compared to the Ln of the probability of the data, the highest likelihood value was assumed to indicate the number of groups in the data pool. However, if the K values were similar, Kruskal-Wallis, Mann-Whitney-Wilcoxon, and likelihood
ratio tests were used to calculate the differences of likelihood, between the two K values. If there were no significant statistic differences between them, the K value with the more simple and realistic value in accordance with taxonomy of the genus Takydromus was chosen.
Analysis 3: Using STRUCTURE to assign individuals to the distinct K groups with (1) all 13 loci and (2) 6 loci which show high Rst values (Rst > 0.1). Integrating and comparing data from (1) and (2) helps us evaluate the results as well as evaluate the effect of homoplasy on loci with low Rst.
Determination of hybrids
Mitochondrial DNA and microsatellite data were used to determine individuals which may belong one of the four situations below (Figure 2):
(1) Pure species: Individuals with both mitochondrial DNA and microsatellite loci congruent with geographic distribution.
(2) Migrants: Individuals with both mitochondrial DNA and
microsatellite loci incongruent with geographic distribution were assigned to migrants.
(3) Hybrids: Individuals with mitochondrial DNA congruent with geographic distribution, but representing partial microsatellite
characteristics of the other species (>10%, the range of variance of Ln estimated by STRUCTURE).
(4) Hybrids or descendants of hybrids: Individuals with mitochondrial DNA incongruent, but microsatellite pattern congruent or partially congruent with geographic distribution. Descendants of hybrids may have experienced recent or ancestral female introgression unless
previously assigned to migrants.
