Metabarcoding of Mitochondrial COI Gene in Scrubwrens

We amplified 313 bp of the mitochondrial COI gene [39 ] using universal metazoan primers [40 (link), 41 (link)] with a 9 bp barcode which differed across samples in at least 4 bases. The following PCR conditions were used: initial denaturation at 94 °C for five minutes, denaturation at 94 °C for 30 s, annealing at 48 °C and extension at 72 °C for a minute each for 35 cycles. Final extension was carried out at 72 °C for five minutes. Samples were pooled at equal concentrations, cleaned using SureClean (Bioline Inc., London) following the manufacturer’s instructions, and sequenced on a MiSeq platform using a 300 bp paired-end run. Sequences were edited and assembled following Meier et al. [39 ] and samples with at least 10X coverage were used for further analyses. Based on these criteria, we were able to successfully amplify the COI gene fragment for 75 samples (Table S1).
In addition to the sequences generated in this study, we also included COI sequences for other scrubwrens from GenBank (Table S6). We performed multiple COI sequence alignments using MAFFT [42 (link)]. A phylogenetic haplotype network was constructed using the TCS method [43 (link)] in PopART 1.7 [44 ]. Pairwise net p-distances between species were computed in MEGA 7 [45 ].