Table 2.

4X rule calculation for species delimitation.

Gene	Cox1		16s		12s
Clade	Nepal/Bangladesh	Sri Lanka/Malaysia	Nepal/Bangladesh	Sri Lanka/Malaysia	Nepal/Bangladesh	Thailand	Nepal/Bangladesh	Sri Lanka	Thailand	Sri Lanka
n	8	3	5	4	11	3	11	2	3	2
K	0.105		0.065		0.016		0.024		0.03
d	0.003	0.009	0.003	0.015	0.001	0	0.001	0.003	0	0.003
π = d*(n/n − 1)	0.0034	0.0135	0.0038	0.0227	0.0011	0.006	0.0011	0.006	0	0.006
θ = π/(1 − 4π/3)	0.0104	0.0428	0.0037	0.0233	0.0033	0.0184	0.0033	0.0184	0	0.0184
K/θ	2.451		2.7809		0.8676		1.3013		1.6267

Calculations used to delimit species under the 4X rule (Birky, 2013) for cox1, 16s and 12s show that under a conservative model the two S. spindale population represent a single species as all results for K/θ are below 4, with the exception of the 12s populations from Thailand and Sri Lanka. Where n = numbers of sequences; K = total pairwise differences between clades; d = pairwise differences within clades.