Heteroplasmic sites in T. rathkei
Coverage across the T. rathkei mitochondrial genome was quite high, >300× across most of the genome in each sample, except at the very ends of the monomer sequence (Figure 2). There were no fixed sequence differences between the two individuals sequenced. However, eight heteroplasmic sites were identified (Figure 2, Table 1). Six of these heteroplasmic sites were detected with confidence in both individuals, and each allele occurred at a frequency of approximately 0.5 in all of these cases. Three of the six occurred toward the ends of the assembled monomer sequence (positions 62, 73, and 14107), and thus had relatively low coverage. The remaining three occurred in putative tRNA genes, and in the anticodon of each (positions 9346, 11785, and 12188). These three were supported by high coverage in both individuals; we also confirmed all three in additional unrelated wild-caught individuals using Sanger dye-terminator sequencing (Figure S1).
Figure 2  Sequencing depth across the monomeric unit of the mitochondrial genomes of Cylisticus convexus and each of the two Trachelipus rathkei samples. Vertical bars represent putative heteroplasmic sites detected by mapping reads to the assembled genomes. The blue portion of each bar represents the frequency of the reference allele in the sequencing reads, whereas the red portion represents the frequency of the alternate allele.
Table 1  Mitochondrial sequence heteroplasmies in Trachelipus rathkei
Pos.  Ref. Allele  Alt. Allele  Alt. Freq., M  Cov., M  Alt. Freq., F  Cov., F  Notes
62  T  A  0.45  252  0.39  111  Noncoding palindrome sequence
73  T  A  0.47  299  0.39  127  Noncoding palindrome sequence
9346*  G  A  0.53  1740  0.51  718  tRNA Leu1/Leu2 anticodon shared with C. convexus
10074  G  A  0  1737  0.47  657  Noncoding
11244  A  —  0.39  1726  0.04  756  cox3 frameshift, affecting amino acids 92 – 277; creates premature stop codon at position 106
11785*  G  C  0.58  1594  0.49  651  tRNA Arg/Gly anticodon shared with C. convexus; also causes cox3 substitution (E273Q)
12188*  A  G  0.41  1638  0.47  629  tRNA Ala/Val anticodon shared with C. convexus
14107  T  A  0.5  193  0.46  74  Noncoding palindrome sequence
Alt. freq., Cov. M, and Cov. F indicate the frequency of the alternative allele in the sequence reads, and the total coverage at that site, in the male and female samples, respectively. The — at site 11244 indicates that the alternate allele is a 1-bp deletion. *Heteroplasmic sites that were confirmed by Sanger sequencing. Finally, there were two additional putative heteroplasmic sites present at high frequencies in only one of the two individuals. One of these, unique to the female individual, was in a noncoding region (position 10074). The other was not a substitution, but a single base-pair deletion causing a frame shift within the cox3 gene (position 11244). Interestingly, the alternate allele at this position was detected in the female sample, but at a very low frequency (4%).
We were unable to identify the haplotype phase for most pairs of heteroplasmic sites, because they were too distantly spaced to be covered by single reads or read pairs in the Illumina data. However, for the polymorphisms at positions 62 and 73, we were able to identify reads covering both sites. In all cases, reads contained either both reference alleles or both alternate alleles (male sample: 133 reads with 62A and 73A, 119 reads with 62T and 73T; female sample: 68 reads with 62A and 73A, 40 reads with 62T and 73T; Figure S2).