Extended Data Fig. 7: Mitochondrial mutations in MarBTN.
(a) Neighbor joining tree built from variants called in all samples (170 SNVs) against the previously published M. arenaria reference mitogenome (excluding the repeated region). Bootstrap values in support of each clade are included on the preceding branch (bootstraps under 50 are not shown). The phylogenetic relationship generally reflects that built from genomic SNVs (that is, monophyletic MarBTN group with separate USA and PEI sub-lineages). The phylogeny within the USA sub-lineage deviates from that built from the nuclear genome, but only three SNVs are variable within the USA sub-lineage: one SNV unique to NYTC-C9 and two SNVs unique to MELC-A11. This causes the other samples to cluster more often with NYTC-C9 due to only one difference (versus two versus MELC-A11), but this relationship is still compatible with the USA branch structure from the nuclear phylogeny. (b) Observed SNVs (black) compared with expected counts estimated from nucleotide frequencies of the M. arenaria mitogenome and assuming equal mutation probability. This calculation was not collapsed to the usual 6 mutation types due to the imbalance of nucleotides in mitochondrial genomes (unequal frequencies of G/C and A/T). Likely somatic refers to SNVs found in a subset of BTN samples, while All USA and All PEI refer to SNVs found in all individuals from that sub-lineage, but not the other sub-lineage. (c) dN/dS ratios, where a ratio of 1 indicates neutrality, were calculated for mitochondrial SNVs found in healthy clams (n = 39), all BTN samples but not healthy clams (n = 13), and likely somatic mutations (n = 50). Error bars indicate 95% confidence intervals as estimated by dndscv and are quite large, due to the low number of mitochondrial SNVs. (d) Read depth across the mitochondrial genome for healthy clams (black), PEI MarBTN (red) and USA MarBTN (blue), normalized to mean depth outside D-loop. Bars above indicate the D-loop region (12,164–12,870 bp, black) and the region used to estimate duplicated region copy number (12,300–12,500 bp, gray), as shown in Fig. 3f. (e) Schematic (not to scale) of the control region of the M. arenaria control region in the previously published mitogenome with a single d-loop copy (top) versus the proposed mitochondrial genome with three d-loop copies and G-rich insertions (middle) with accompanying PCR results (bottom). Primer pair combinations are listed along top of gel and expected sizes are listed along bottom, molecular weights are in bp. Amplicon sizes from primers spanning the D-loop (67 with 62/71) support a single copy of the D-loop. However, we suspect this is a result of recombination and selection for the smaller product and loss of the G-rich insertions. Inverse PCR with outward-facing primers (65 with 72/72) indicates a tandem duplication allowing outward-facing primers to amplify. The inverse primers spanning the G-rich insertion (65 with 72) has a dim band at expected size, but two brighter bands at smaller sizes. PCR was run once.
