Fig 1.
Distribution of nucleotides within codons across six insect orders.
Nucleotide frequencies at each of the three codon positions in COI barcode sequence of species of six insect orders in addition to an Odonata sp. used for comparison.
Fig 2.
Distribution of aminoacids within the barcodes across six insect orders.
Amino acid frequencies in barcode sequences of species of six insect orders in addition to an Odonata sp. used for comparison. AAs are grouped vertically based on their biochemical properties.
Fig 3.
Predicted three-dimensional COI protein structure of Batrachedra amydraula.
A model barcode of the insect consensus AA sequence in the heme 515 vicinity. A displays the 16 AAs that probably affect functions of the two hemes 515 and 516. These AAs are threonine (T), sulfur (S), isoleucine (I), arginine (R), tyrosine (Y), isoleucine (I), valine (V), histidine (H), alanine (A), methionine (M), isoleucine (I), methionine (M), valine (V), isoleucine (I), glycine (G) and tryptophan (W). These AAs exist at positions 15, 18, 21, 22, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 109 and 110 of the COI polypeptide chain, respectively, following AA numbering of Pentinsaari et al. (2016). Four AAs sharing six bonds with hemes 515 and 516 are shown inside red circles. One extra bond also exists between S at position 18 and R at position 22. B indicates the occurrence of two bonds between H (position 45) and heme 515. C indicates the occurrence of two bonds of W (position 110); one with heme 515 and the other with heme 516. D indicates the absence of hydrogen bond with heme 515 at position 38 in the Camponotus maculatus barcode sequence due to substitution of tyrosine (Y) for phenyl alanine (F). As indicated in C, default position of W during 3D structure prediction is 126, while position 110 following protein model of Pentinsaari et al. (2016). Different types of atoms in the structure are indicated by different color.
Fig 4.
Alignment of the predicted three-dimensional structures of COI proteins of Batrachedra amydraula and Camponotus maculatus.
A indicates the occurrence of a hydrogen bond between tyrosine of the first insect at position 38 and Heme 515, while lack of bonding between phenyl alanine of the second insect at the same position. B indicates the side chain structures of the two AAs facing Heme 515. C indicates the alignments of the six helices and five loops of the two 3D structures.
Fig 5.
Phylogenetic tree of species of the six insect orders based on multiple AA sequence alignment in COI barcode.
The tree also indicates substitutions of the six varied AAs (at positions 15, 21, 38, 41, 42 and 57) within the distance of < 4Å from heme 515. Different colors are given to substitutions at different positions, while same colors for substitutions at the same position. AA sequence of Odonata was used for comparison.
Fig 6.
Nucleotide substitution matrix derived based on the COI barcode.
Maximum composite likelihood estimates of the pattern of nucleotide substitution to detect transition and transversion frequencies based on DNA sequence alignment in COI barcode of species in the six insect orders.
Fig 7.
Maximum Likelihood computations of codon-by-codon synonymous (s) and nonsynonymous (n) substitutions in COI protein.
Description of COI codons that are under positive selection based on the values of dN-dS in 30 species of six insect orders live in Saudi Arabia. Sequence of COI in order Odonata was used as a reference.