DOI: 10.5061/dryad.x69p8czf7
title: An unusual amino acid substitution within hummingbird cytochrome c oxidase alters a key proton-conducting channel

description: Hummingbirds in flight exhibit the highest metabolic rate of all
 vertebrates. The bioenergetic requirements associated with sustained
 hovering flight raise the possibility of unique amino acid substitutions
 that would enhance aerobic metabolism. Here, we have identified a
 non-conservative substitution within the mitochondria-encoded cytochrome c
 oxidase subunit I (COI) that is fixed within hummingbirds, yet exceedingly
 rare among other vertebrates. This unusual change is also rare among
 metazoans, but can be identified in several clades with diverse life
 histories. We performed atomistic molecular dynamics simulations using
 bovine and hummingbird COI models, thereby bypassing experimental
 limitations imposed by the inability to modify mtDNA in a site-specific
 manner. Intriguingly, our findings suggest that COI amino acid position
 153 (bovine numbering system) provides control over the hydration and
 activity of a key proton channel in COX. We discuss potential phenotypic
 outcomes linked to this intriguing alteration encoded by the hummingbird
 mitochondrial genome.
description: Figure 1A, Figure 1B, and Table S1:
 Mitochondria-encoded protein sequences were downloaded from
 RefSeq (release 92) and bird sequences were extracted. Sequences from Bos
 taurus were also included. Polypeptides sequences were arrayed and
 concatenated [Figure 1A and Table S1 and Figure 1B
 (Accession_by_Species_Aves)_RefSeq_JAN7_2019.xlsx] [Figure 1A and Table S1
 and Figure 1B (Sequences_by_Species_Aves)_RefSeq_JAN7_2019.xlsx].
 Duplicates or egregiously misaligned concatenate sequences were removed.
 Alignments were performed using T-coffee in regressive mode and taking
 advantage of the MAFFT G-INS-i strategy [Figure 1A and Table S1 and Figure
 1B (Aves_mtProt_NoDups_Clean_TCOFF_G)_RefSeq_JAN7_2019.fasta]. FastTreeMP
 was subsequently used to generate the NWK tree file [Figure 1A and Table
 S1 and Figure 1B [(Aves_mtProt_NoDups_Clean_TCOFF_G)_RefSeq_JAN7_2019.nwk]
 for downstream analysis by PAGAN and our custom-made scripts (available on
 GitHub at https://github.com/corydunnlab/hummingbird) [Figure 1A and Table
 S1 and Figure 1B (Aves_mtProt_NoDups_Clean_TCOFF_G_PAGAN).anctree; Figure
 1A and Table S1 and Figure 1B
 (Aves_mtProt_NoDups_Clean_TCOFF_G_PAGAN).fas]. Figure
 1C and Table S2: Mitochondria-encoded protein sequences
 were downloaded from RefSeq (release 92) and bird COI sequences were
 extracted. The COI sequence from Bos taurus was subsequently included.
 Sequences were aligned by MAFFT using the G-INS-i strategy [Figure 1C and
 Table S2
 (Aves_COX1_extracted_from_RefSeq_JAN7_2019_w_bovine_MAFFT_G-INS-i).fasta]. Table S3:  In BOLD Systems (February 12, 2020), the query "Apodiformes" was used. FASTA entries were downloaded and aligned by MAFFT using the L-INS-i strategy. Samples unaligned or uninformative (no character at selected positions) were removed [Table S3 (Apodiformes_bold_MAFFT_L-INS-i_correct_translation).fasta]. Samples without a specific species assignment were not processed further. Table S4:  In BOLD Systems (February 12, 2020), the query "Aves" was used. FASTA entries were aligned using MAFFT under the 'auto' setting [Table S4 (Aves_BOLD_MAFFT_auto).fasta]. Table S5: Mitochondria-encoded protein sequences were downloaded from RefSeq (release 92) and Chordata sequences were extracted. FASTA entries were aligned using MAFFT under the 'auto' setting [Table S5 (Chordata_COX1_MAFFT_auto).fasta], with subsequent analysis removing unaligned or uninformative sequences. Next, BOLD samples were sought for the relevant genera (February 28, 2019) using the terms "Ambystoma", "Serrivomer", "Pangio", and "Polypterus". Downloaded FASTA entries were aligned using MAFFT under the 'auto' setting: [Table S5 [Ambystoma_BOLDSYSTEMS_MAFFT_auto].fasta; Table S5 [Serrivomer_BOLDSYSTEMS_MAFFT_auto].fasta,; Table S5 [Pangio_BOLDSYSTEMS_MAFFT_auto].fasta; Table S5 [Polypterus_BOLDSYSTEMS_MAFFT_auto].fasta]. Subsequent analysis removed unaligned or uninformative samples. Table S6:  Mitochondria-encoded protein sequences were downloaded from RefSeq (release 92), and metazoan COI protein sequences were extracted and aligned by MAFFT under the 'auto' setting [Table S6 (Metazoan_COI_MAFFT_auto).fasta]. For BOLD analysis, FASTA sequences were sought for each of the relevant genera (February 29, 2020). Files were merged and limited to those entries obtained by the COI-5P barcode primers. Sequences were then aligned by MAFFT using the FFT-NS-2 option [Table S6 (BOLD_combined_COI-5P_MAFFT_FFT_NS_2).fasta]. Unlike other analyses performed with BOLD, assignment to specific species was not required to be counted for Table S6. Translation of these divergent entries was accomplished with the help of the gappyout function of trimal. Table S7:  BOLD samples were obtained for bee families using the queries "Andrenidae", "Apidae", "Colletidae", "Halictidae", "Megachilidae", and "Melittidae" (April 4, 2019). FASTA entries were aligned using MAFFT under the 'auto' setting: [Table S7 (Andrenidae_BOLD_COI-5P_MAFFT_auto).fasta; Table S7 (Apidae_BOLD_COI-5P_MAFFT_auto).fasta; Table S7 (Colletidae_BOLD_COI-5P_MAFFT_auto).fasta; Table S7 (Halictidae_BOLD_COI-5P_MAFFT_auto).fasta; Table S7 (Megachilidae_BOLD_COI-5P_MAFFT_auto).fasta; Table S7 (Melittidae_BOLD_COI-5P_MAFFT_auto).fasta]. Table S8:  BOLD samples were obtained for hoverfly genera using the queries "Anasimyia", "Chasmomma", "Eristalinus", "Eristalis", "Helophilus", "Lejops", "Mallota", "Mesembrius", "Palpada", "Parhelophilus", "Phytomia", and "Senaspis" (April 4, 2019). FASTA entries were aligned using MAFFT under the 'auto' setting: [Table S8 (Anasimyia_BOLD_MAFFT_auto).fasta; Table S8 (Chasmomma_BOLD_MAFFT_auto).fasta; Table S8 (Eristalinus_BOLD_MAFFT_auto).fasta; Table S8 (Eristalis_BOLD_MAFFT_auto).fasta; Table S8 (Helophilus_BOLD_MAFFT_auto).fasta; Table S8 (Lejops_BOLD_MAFFT_auto).fasta; Table S8 (Mallota_BOLD_MAFFT_auto).fasta; Table S8 (Mesembrius_BOLD_MAFFT_auto).fasta; Table S8 (Palpada_BOLD_MAFFT_auto).fasta; Table S8 (Parhelophilus_BOLD_MAFFT_auto).fasta; Table S8 (Phytomia_BOLD_MAFFT_auto).fasta; Table S8 (Senaspis_BOLD_MAFFT_auto).fasta].<br>