The complete mitochondrial genome of aglaeactis castelnaudii (bourcier & mulsant, 1848) (apodiformes: trochilidae: aglaeactis) and phylogenetic analysis

Abstract In this study, we employed high-throughput sequencing data to assemble the mitochondrial genome (mitogenome) of the White-tufted Sunbeam (Aglaeactis castelnaudii). The total length of the mitogenome was found to be 16,872 base pairs (bp), containing 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region. The nucleotide composition was as follows: A 30.6%, T 24.0%, C 31.2%, and G 14.2%, resulting in a GC content of 45.4%. Phylogenetic analysis, utilizing the concatenation of the 13 mitochondrial PCGs, indicated a closer evolutionary relationship between the genus Aglaeactis and the genus Coeligena compared to other genera within the family Trochilidae investigated in this study. The mitogenome of A. castelnaudii not only contributes to species identification but also provides valuable insights for phylogenetic and conservation genetic analyses of A. castelnaudii.


Introduction
The White-tufted Sunbeam, scientifically known as Aglaeactis castelnaudii (Figure 1), belongs to the hummingbird family Trochilidae.A. castelnaudii inhabits a restricted and fragmented range, exclusively found in the Andes of Peru, Bolivia, and Northern Chile.It prefers damp montane forests and high-altitude shrublands in subtropical or tropical regions (Parker & O'Neill 1980).Playing a significant role in ecosystem regulation, A. castelnaudii primarily feeds on insects or flower nectar from blooming plants in its Andean habitat (Watts 2008).The International Union for Conservation of Nature (IUCN) describes this species as 'common but patchily distributed' within its habitat, although an accurate estimation of the overall population is currently lacking.As of 2017, the White-tufted Sunbeam is classified as 'Near Threatened' (NT) on the IUCN Red List, with its population size and trend assessed as 'Decreasing' (BirdLife International 2017).
The genus Aglaeactis comprises four species (Schuchmann 1985) and belongs to the Andean hummingbird clade (McGuire et al. 2014).All Aglaeactis hummingbirds inhabit the high Andes at elevations ranging from 2500 to 4300 m, where they have evolved several distinct adaptive phenotypes to survive in this challenging environment (Schuchmann 1990;Ocampo et al. 2020).Consequently, this group stands as a notable example of adaptive radiation in temperate and high latitudes.Remarkably, the genus Aglaeactis remains among the least researched hummingbird genera globally, with the mitogenome of this genus yet to undergo sequencing.In this study, we present and elucidate the mitogenome of A. castelnaudii for the first time.This study offers genetic data crucial for future studies into species identification, biodiversity surveillance, conservation genetics, and investigations into adaptive evolution.

Materials and methods
In this study, the mitogenome of A. castelnaudii was assembled utilizing publicly available high-throughput genomic data in NCBI, which was submitted by the Sam Noble Oklahoma Museum of Natural History.An individual muscle tissue sample from A. castelnaudii was collected in Peru, situated at coordinates 14.058 S latitude and 73.001 W longitude.This sample was preserved at the Sam Noble Oklahoma Museum of Natural History (https://samnoblemuseum.ou.edu/, contact person: Jessica McLaughlin, email: jfmclaughlin@ berkeley.edu)under the sample label ca169414.Genomic DNA was isolated from the muscle tissue and subjected to sequencing on an Illumina NovaSeq 6000 platform utilizing 150 bp paired-end runs (2 � 150).The genomic data supporting the findings of this study can be accessed at NCBI's Sequence Read Archive (SRA) via the following link: https:// www.ncbi.nlm.nih.gov/sra/, with the accession number SRR19461672.Data collection was conducted in compliance with the IUCN policies research involving species at risk of extinction (see Guidelines for appropriate uses of IUCN Red list data).Furthermore, we adhered to the regulations of Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.
We extracted the mitochondrial reads from the genomic data and proceeded to perform assembly using MitoZ v3.6 (Meng et al. 2019).After a manual examination, the resulting mitogenome was annotated and visually displayed using Proksee (Grant et al. 2023) (https://proksee.ca/).The nucleotide sequences of each PCG of A. castelnaudii and its related species were aligned using MUSCLE v3.8.31 (Edgar 2004).The best-fit substitution model for each alignment was determined using the ModelFinder model (Kalyaanamoorthy et al. 2017) integrated within IQ-TREE2 (Nguyen et al. 2015).Phylogenetic analysis using Maximum Likelihood (ML) of A. castelnaudii and other species of hummingbirds and one outgroup (Apus apus) was performed using IQ-TREE2 (Nguyen et al. 2015) based on the sequences from 13 mitochondrial protein-coding genes (PCGs).Individual gene alignments were combined to form a partitioned supermatrix encompassing 11,385 bp.This supermatrix was then employed to construct a ML tree with separate model partitions for each gene.The reliability of the branches within the tree was evaluated using UFBoot2 (Hoang et al. 2018) with 1,000 bootstrap replicates.

Results
The complete mitogenome of A. castelnaudii spans 16,872 bp (NCBI accession number: PP754509).The average read depth coverage for the mitogenome reached 902.9 � (Fig. S1).The mitogenome consists of 13 PCGs, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a single control region referred to as the D-loop (Figure 2).This genetic arrangement adheres to the typical structure of avian mitochondrial genes.The nucleotide composition of the genome is as follows: Adenine (A) 30.6%,Thymine (T) 24.0%, Cytosine  (C) 31.2%, and Guanine (G) 14.2%, demonstrating a slight bias toward AT content at 54.6%.Notably, among the PCGs, ND5 is the longest, spanning 1,815 bp, while ATP8 is the shortest at 168 bp.The two rRNA genes (12S rRNA and 16S rRNA) are 974 bp and 1,592 bp in length respectively, positioned between tRNA Phe and tRNA Leu , and separated by the tRNA Val gene.Furthermore, this genomic configuration encompasses 22 transfer RNAs (tRNAs) with lengths ranging from 67 to 80 bp.