Characterization of the complete mitochondrial genome of Elanus caeruleus Desfontaines, 1789 (Accipitriformes: Accipitridae)

Abstract The complete mitochondrial genome of the Elanus caeruleus was sequenced via next-generation sequencing. The circular mitogenome is 18,898 bp in length, containing 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, a control region and a pseudo control region. The sequences (1210 bp) in the middle part of the two control regions are complete identical to each other. The gene order of the E. caeruleus mitogenome is identical to those of other Accipitridae species. The phylogenetic analysis indicated that E. caeruleus formed a basal lineage sister to other species within the Accipitridae family.

The black-winged kite, Elanus caeruleus (Desfontaines, 1789), is a small diurnal bird of prey belonging to the Accipitridae family. It has a very large distributed range encompassing Africa, India, southeastern Asia and southwestern Europe (Balbont ın et al. 2008). This species is listed as Least Concern (LC) on the International Union for the Conservation of Nature Red List of Threatened Species (BirdLife International 2019). However, in China, it has been classified as a secondclass key protected wild animal (National Forestry and Grassland Administration 2021). So far, the complete mitochondrial genomes have been reported for several species from the other subfamilies within the Accipitridae (for example, Choi et al. 2021), but not including species from the subfamily Elaninae. In this study, we sequenced the complete mitogenome of E. caeruleus and analyzed its phylogenetic position within the Accipitridae.
Muscle tissues of E. caeruleus was collected from a dead individual in Liancheng, Longyan City, Fujian Province, China (116.75 E,25.72 N) and preserved in 95% ethanol. The specimen was stored at À80 C in the laboratory at College of the Environment and Ecology, Xiamen University, Xiamen, China (https://cee.xmu.edu.cn/, Xiaoping zhou, xpzhou@xmu.edu.cn) under voucher number CEE2016Aves-EC-01. Genomic DNA was extracted using the EasyPure V R Genomic DNA Kit (TransGen Biotech Co., Ltd., Beijing) and then sequenced on the Illumina NovaSeq 6000 sequencing platform with a pairend sequencing protocol of 150 bp read length (PE150). FastP (Chen et al. 2018) was used to remove adaptors and low-quality reads. Clean pair-end reads were assembled into a complete mitogenome using GetOrganelle v1.7.5 (Jin et al. 2020) and total 21 different assemblies were generated. After annotated by MITOS2 (Donath et al. 2019), we found that the sequences of the 21 assemblies were different in the 3 0 end of the two control regions. Thus, we amplify the of the 3 0 end of the two control regions with two primer pairs (ARCR372F þ ARProR and ARCR372F þ AR12SR1, Zhou et al. 2014) and aligned the sequences of the PCR products with the 21 assemblies to identify the correct result.
All the PCGs use ATG as start codon except for ND3 using ATC and ND5 using ATA. The most common stop codon is TAA, while ND1, COX1 and ND6 end with AGG, ND2 ends with TAG, COX3 and ND4 end with a single T. Additionally, ND3 has one extra cytosine in the 174th nucleotide position, which is presumed not to be translated (Mindell et al. 1998).
The gene order of the E. caeruleus mitogenome is identical to those of other Accipitridae species. The CR (1795 bp) is located between tRNA Thr and tRNA Pro , whereas the WCR (1586 bp) is located between tRNA Glu and tRNA Phe . The sequences (1,210 bp) in the middle part of the CR and WCR are complete identical to each other, which contain several conserved boxes, such as F, E, D, C and bird similarity box, generally existing in the Avian mitogenome control regions (Zhou et al. 2014;Choi et al. 2021).
The concatenated nucleotide sequences of the 13 PCGs of the C. caeruleus mitogenome and 14 published mitogenomes from differ genus (one species per genera) within the Accipitridae were used for phylogenetic analysis, and Sagittarius serpentarius from Sagittariidae and Pandion haliaetus from Pandionidae were used as the outgroups. A Maximumlikelihood (ML) tree with 1000 bootstraps was constructed by IQ-TREE v1.6.2 (Trifinopoulos et al. 2016) using the substitution model GTR þ F þ I þ G4, which was selected by Modelfinder (Kalyaanamoorthy et al. 2017). The result (Figure 1) indicated that E. caeruleus formed a basal lineage sister to other species within the Accipitridae family, which was in consistent with previous molecular studies (Starikov and Wink 2020).

Ethical approval
This study was approved by the Ethics Committee for Animal Experimentation of the Xiamen University. The experiments were conducted in accordance with ethical guidelines.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Author contributions
HL performed the experiments, conducted data analyses, and wrote the first draft of the manuscript; XZ, WF and XC conceived and directed the experiments, helped for writing, and revised the manuscript. QL collected the samples and helped for lab work. All authors read and approved the final manuscript.