Sequences were cleaned and analysed using CodonCode Aligner Program V. 6.0.2 (CodonCode Corporation, Centerville, MA). ITS2 and COI sequences from
Anopheles specimens were submitted as queries to the National Center for Biotechnology Information's (NCBI) web-based Basic Local Alignment Search Tool (BLAST) [
19] against the nucleotide collection in Genbank under default parameters [max High-scoring Segment Pairs (HSP) 250, expect threshold 10, word size 28, optimized for highly similar hits, not specific to any organism]. The
Anopheles subject sequences from NCBI that formed HSP with the queries were identified.
Phylogenetic analyses of ITS2 and COI were employed to search for sister taxon relationships between isolates of
Anopheles from east Ethiopia and voucher specimens from
Anopheles with orthologous sequence data stored in NCBI.
Anopheles sequences from east Ethiopia and closest sequence hits in BLAST that had more than 85% sequence coverage were combined into datasets for COI and ITS2 separately. In some cases, there were multiple sequences from the same location and study. In these instances, only representative sequences were taken from those population sets. Alignments were created with MAFFT version 7 under default parameters [
18] and ragged ends were trimmed using Mesquite 3.51 [
20]. Phylogenetic relationships with the Ethiopian
Anopheles sequences and
Anopheles sequences from NCBI were inferred using RAxML [
21] which is based on a maximum likelihood (ML) approach. The GTRGAMMA option that uses GTR model of nucleotide substitution with gamma model of rate of heterogeneity was applied. Both 100 and 1000 replicates were completed with the strategy searching for the heuristically-best-scoring tree and bootstrap analysis in one run. Best scoring trees under ML with bootstrap values from RAxML were viewed and rooted under the outgroup criterion in FigTree [
22] for each locus. Outgroups were chosen based on availability of sequence data for each locus, overall coverage, and its use in previous phylogenetic analyses. For the COI analysis,
Anopheles implexus sequence was used as an outgroup based on sequence availability and use in similar analyses of
Anopheles species [
4]. For the ITS2, a different species,
Anopheles christyi, was used as an outgroup primarily because
An. implexus sequence was not available. Compatible
An. christyi ITS2 sequence was available and this species had been used in a similar analysis [
23].