Our estimate of ν of mcy is 0.018, which is lower than the maximum sequence divergence (0.050) of mcy genes.
The maximum sequence divergence of mcyD, mcyG, and mcyJ in the whole dataset was 3.6%, 5.1%, and 2.9%, that in group A was 3.6%, 1.5%, and 2.0%, and that in group B was 0.7%, 2.0%, and 2.0%, respectively.
Maximum sequence divergences within each of the B, C, D and E clades reached 2.22, 0, 0, 2.41%; respectively, for the 16S rDNA gene (Table S2), whereas they reached 8.60, 2.10, 2.08, 8.77%; respectively for the COI gene (Table S2, S3, S4, S5).
Specifications of the full and stripped ITS sequence alignments are given in Table 2. Maximum pairwise sequence divergence was 13% for the full and 5% for the stripped alignment.
Excluding the Kunjin virus, the maximum nucleotide sequence divergence exhibited in lineage 1 (21.5%), was between the Indian isolate G 167919 and CAR isolate ArB 310.
The maximum pairwise sequence divergence within each MOTU, as well as the distance to the nearest neighbouring sequence belonging to a different MOTU, was calculated for all sequences of at least 500 bp using the Barcode Gap Analysis function in BOLD3 using the Kimura-2-parameter (K2P) distance model [ 49].
Direct support for the hypothesis of reproductive continuity within clades also comes from detailed studies on Rosemary Island [ 23], all from Clade D3, where nuclear microsatellite DNA provides clear evidence for gene flow between specimens that key to four of the described morphospecies, with a maximum percentage sequence divergence of COI of 4.4% (mean 1.24 ± SD 0.78).
We built a MSA for each data protein structure comprising the most similar (A maximum of 40% sequence divergence) protein sequences isolated from eukaryotic organisms.
However, we detected marked sequence divergence (maximum = 5.98%) among the 20 specimens, and this variation fell into three groups separated by more than 2.98% versus a maximum within-group divergence of 0.96% (Fig. 3).
However, the maximum fragment length and sequence divergence between homeoloci where HRM remains useful for SNP or mutation detection is unknown and further experiments are required.
A study of the mtDNA of these species found a maximum of approximately 7% sequence divergence at the cytochrome b gene among these species, which, based on a rough 1% per million years calibration for fishes [ 31], would suggest initial divergences in the upper Miocene [ 32].
