Biodiversity Data Journal :
Research Article
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Corresponding author: Yeying Wang (wangyeying0818@163.com), Xiaofei Yang (xfyang3@gzu.edu.cn)
Academic editor: Caio J. Carlos
Received: 14 Feb 2023 | Accepted: 11 Mar 2023 | Published: 20 Mar 2023
© 2023 Yeying Wang, Haofeng Zhan, Yu Zhang, Zhengmin Long, Xiaofei Yang
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wang Y, Zhan H, Zhang Y, Long Z, Yang X (2023) Mitochondrial genome analysis, phylogeny and divergence time evaluation of Strix aluco (Aves, Strigiformes, Strigidae). Biodiversity Data Journal 11: e101942. https://doi.org/10.3897/BDJ.11.e101942
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Background
Prior research has shown that the European peninsulas were the main sources of Strix aluco colonisation of Northern Europe during the late glacial period. However, the phylogenetic relationship and the divergence time between S. aluco from Leigong Mountain Nature Reserve, Guizhou Province, China and the Strigiformes from overseas remains unclear. The mitochondrial genome structure of birds is a covalent double-chain loop structure that is highly conserved and, thus, suitable for phylogenetic analysis. This study examined the phylogenetic relationship and divergence time of Strix using the whole mitochondrial genome of S. aluco.
New information
In this study, the complete mitochondrial genome of Strix aluco, with a total length of 18,632 bp, is reported for the first time. A total of 37 genes were found, including 22 tRNAs, two rRNAs, 13 protein-coding genes and two non-coding control regions. Certain species of Tytoninae were used as out-group and PhyloSuite software was applied to build the ML-tree and BI-tree of Strigiformes. Finally, the divergence time tree was constructed using BEAST 2.6.7 software and the age of Miosurnia diurna fossil-bearing sediments (6.0–9.5 Ma) was set as internal correction point. The common ancestor of Strix was confirmed to have diverged during the Pleistocene (2.58–0.01 Ma). The combined action of the dramatic uplift of the Qinling Mountains in the Middle Pleistocene and the climate oscillation of the Pleistocene caused Strix divergence between the northern and southern parts of mainland China. The isolation of glacial-interglacial rotation and glacier refuge was the main reason for the divergence of Strix uralensis and S. aluco from their common ancestor during this period. This study provides a reference for the evolutionary history of S. aluco.
Strix aluco, phylogeny, divergence time, Pleistocene, climate oscillation, mountains uplift
Strix aluco belongs to Strigidae (Strigiformes) and is a medium-sized owl (
Mitochondria are characterised by maternal inheritance, high conservation, multiple copies in each cell, low sequence recombination rate and high evolutionary rate; therefore, mitochondria are widely used in phylogenetic studies (
There are many reasons for the divergence of species, amongst which geological and climatic influences on species diversification cannot be ignored (
Divergence time analysis can provide a reference for the evolution process of species and provides a basis for further studies. To clarify the divergence time of species, it is necessary to obtain their gene sequence first; then, an appropriate evolutionary model needs to be selected and reliably calibrated, for example, by determining the age of fossils (
Part of the muscle tissue was extracted from the leg of one individual of S. aluco that died of an unknown cause in the Rescue Center of Leigong Mountain National Nature Reserve, Qiandongnan Prefecture, Guizhou Province, China (26° 49' 26.40" N, 104° 43' 33.60" E). The sample was stored in a refrigerated box with a built-in thermometer, the temperature was kept near freezing, until the sample was transported back to the laboratory for DNA extraction. To extract DNA, the standardised CTAB method was used (
The whole genome shotgun strategy was used to construct the library (
The concentration and purity of DNA extracted from the samples were assessed by Thermo Scientific NanoDrop 2000 (Thermo Scientific NanoDrop 2000, Thermo Fisher, Massachusetts, USA) and the integrity was assessed by agarose electrophoresis (Electrophoresis apparatus of Liuyi Company, Beijing, China) and Agilent 2100 Bioanalyzer (Agilent 2100 Bioanalyzer, Agilent Corporation, California, USA). The Covairs machine (Covairs machine of BRANSON Company in St. Louis, Missouri, USA) was used to break up and fragment DNA. The gene library was constructed according to the shotgun method described by
After DNA extraction, purification, library construction and sequencing, a raw image file was first obtained by sequencing. The raw data that can be read in FASTQ format were generated after the multi-step transformation, i.e. the offline data. Data transformation is automatically completed by the sequencing platform. According to the statistics of raw data, 7,947,240 reads (each sequence read is called one read) were obtained, the total number of bases was 1,192,086,000 bp, the percentage of fuzzy bases (uncertain bases) was 0.0016% and the GC content was 44.58%. The base recognition accuracy exceeding 99.00% accounted for 95.61% and the base recognition accuracy exceeding 99.90% accounted for 90.44%. The quality of off-machine data was tested through quality control and the software used is FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
Sequencing data contain low-quality reads with connectors, which will greatly interfere with subsequent analysis. To ensure the quality of subsequent information analysis, Fastp software (version 0.20.0) was used to remove sequencing connectors at the 3' end. Low-quality sequences (i.e. sequences with an average Q value of less than 20 and sequences with a sequence length shorter than 50 bp) were removed. The number of high-quality reads obtained was 7,611,984, accounting for 95.78% of the raw data and the number of bases of high-quality reads was 1,123,739,765 bp, accounting for 94.27% of the raw data (
A5-miseq v20150522 (
Through the above methods, the base compositions of the whole mitochondrial genome, protein-coding genes and rRNA genes were obtained. CGview visualisation software was used to draw the mitochondrial complete genome circle map (
Currently (until this study), in GenBank, there are 30 species with mitochondrial genomes greater than 10,000 bp, including 27 species of Strigidae and three species of Tytonidae. All taxonomic classifications of the species follow the current version of the IOC WORLD BIRD LIST (12.2) (http://dx.doi.org/10.14344/IOC.ML.12.2). The existing sequences of these thirty species were stored in a local folder using GenBank format. The registration number is shown in Table
Taxon |
GenBank accession |
Size (bp) |
Notes |
Reference |
Aegolius funereus |
17166 |
Partial |
Direct Submission |
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Asio flammeus |
18966 |
Complete |
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Asio otus |
17555 |
Complete |
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Athene brama |
16194 |
Partial |
Direct Submission |
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Athene noctua |
15776 |
Partial |
Direct Submission |
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Bubo blakistoni |
19379 |
Partial |
Direct Submission |
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Bubo bubo |
18956 |
Complete |
Direct Submission |
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Bubo flavipes |
19447 |
Partial |
Direct Submission |
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Bubo scandiacus |
18734 |
Complete |
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Ciccaba nigrolineata |
14875 |
Partial |
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Glaucidium brasilianum |
17717 |
Partial |
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Glaucidium brodiei brodiei |
17318 |
Complete |
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Glaucidium cuculoides |
17392 |
Complete |
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Ninox novaeseelandiae |
16223 |
Complete |
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Ninox scutulata |
16208 |
Complete |
Direct Submission |
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Ninox strenua |
16206 |
Complete |
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Otus bakkamoena |
17389 |
Complete |
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Otus lettia |
16951 |
Complete |
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Otus scops |
17413 |
Complete |
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Otus semitorques |
18834 |
Complete |
Direct Submission |
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Otus sunia |
17835 |
Complete |
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Sceloglaux albifacies |
15565 |
Partial |
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Strix aluco |
16490 |
Partial |
Direct Submission |
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Strix aluco |
18832 |
Complete, |
This study |
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Strix leptogrammica |
16307 |
Complete |
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Strix occidentalis |
19889 |
Complete |
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Strix uralensis |
18708 |
Complete |
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Strix varia |
18975 |
Complete |
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Out-group |
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Phodilus badius |
17086 |
Complete |
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Tyto alba |
16148 |
Partial |
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Tyto longimembris |
18466 |
Partial |
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The PhyloSuite software (downloaded from: https://github.com/dongzhang0725/PhyloSuite/releases) (
First, following the guided steps in the literature of
The result file of PartitionFinder 2.0 was selected, the ML method was completed in IQ-tree (
Miosurnia diurna fossils provide an approximate date of the origin of Surniinae and the age of the fossil-bearing sediments of the M. diurna is 6.0–9.5 Ma (
The total length of the mitochondrial genome sequence was 18,632 bp (GenBank entry number: OP850567). The results of genome annotation showed that the total number of genes was 39, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes, two OH genes and 0 OL genes. Amongst them were eight tRNA genes (trn-Q, trn-A, trn-N, trn-C, trn-Y, trn-P, trn-E and trn-S2) and the PCG gene nad6 on the main chain (J chain). The remaining 14 tRNA genes were trn-F, trn-V, trn-L2, trn-I, trn-M, trn-W, trn-D, trn-K, trn-G, trn-R, trn-H, trn-S1, trn-L1 and trn-T. Further, the two rRNA genes rrn-S and rrn-L were found and 12 PCGs genes encoding nad1, nad2, nad3, nad4, nad4L, nad5, atp6, atp8, cox1, cox2, cox3 and cytb on the secondary (N) chain were also found. There was no gene rearrangement (Fig.
Complete mitochondrial genome of S. aluco. The total length of the mitochondrial genome of S. aluco was 18,632bp. The genes located on the N strand or J strand are positioned inside or outside the circle. Contains two D-Loop regions. The GC Skew+ region contains more Guanine than Cytosine and the GC Skew- region contains more Cytosine than Guanine.
In this study, both the ML-tree and BI-tree showed the same tree topology with good support. The tree showed that Strigidae and Tytonidae are two distinct lineages under the owl shape. Athene noctua is a sister group of Athene brama; Glaucidium, Athene and Aegolius constitute the same group, Aegolius funereus is closely related to (G. cuculoides + G. brasilianum), but BI/ML (posterior probability/bootstrap) is 0.97/52, the phylogenetic relationship between them is Glaucidium brodiei brodiei + ((A. funereus + (G. cuculoides + G. brasilianum)) + (Athene noctua + A. brama). Ciccaba nigrolineata is nested in Strix. It shows that BI/ML is 1/100, S. aluco in this study is a sister group of S. uralensis, Strix aluco MN122823 + (Strix aluco OP850567 + Strix uralensis) had formed with S. aluco; Strix is a sister to Bubo clade and forms an Asio + (Strix + Bubo) monophyletic group with Asio and a higher monophyletic group with Otus + [Asio + ((Strix + Ciccaba nigrolineata) + Bubo)]. Additionally, Sceloglaux albifacies is nested in Ninox, BI/ML is 1/99; this monophyly emerged simultaneously with (Sceloglaux albifacies + Ninox) and the monophyly exhibited as dyadic taxa (Fig.
BI/ML-tree, Bayesian phylogenetic tree of 37 genes (24 rRNAs, 13PCGs) from 31 species of Strigiformes. The node labels are BI/ML posterior probability and bootstrap support value, respectively and the scale indicates the probability of nucleotide change within each branch length. The GenBank of the sequences has been indicated next to the species name. Branches of different subfamilies are distinguished by different colours, with Tytoninae (with Phodilus badius, Tyto longimembris and Tyto alba) being the out-group. The Strix aluco mitochondrial genome obtained by this sequencing has been marked by ★.
The divergence time tree, based on 37 genomes, shows that the time interval between Strigidae and Tytonidae from the common ancestor of Strigiformes was 8.05–12.75 Ma. However, in the out-group, Tyto alba, Tyto longimembris and Phodilus badius diverged from the common ancestor at about 4.23–6.69 Ma. The divergence of Strigidae began at about 6.32–10.01 Ma, the common ancestor of Ninoxinae and Striginae in Strigidae split into two species at 5.50–8.7 Ma and the earliest divergence of Surniinae occurred in Strigidae, Aegolius, Athene and Glaucidum occurred at about 6.0–9.5 Ma, Aegolius diverged from the common ancestor of Surniinae during 5.10–8.08 Ma, Athene and Glaucidium diverged completely into two species during 4.82–7.64 Ma.
The common ancestor of Strix and Bubo diverged completely during 3.49–5.53 Ma and, during 2.53–4.0 Ma, Strix began to gradually diverge into multiple species. In this study, the divergence time between S. aluco (OP850567) and S. aluco of Margaryan. A (MN122823) was found to be about 1.47–2.33 Ma. The divergence time between S. aluco and S. uralensis in China was about 1.28–2.02 Ma (Fig.
The mitochondrial genome structure of birds is a covalent double-chain loop structure, with a total of 37 genes, including 22 tRNAs, two rRNAs, 13 PCGs and 1–2 non-coding control regions (D-loop). The nad6 and eight tRNA encoding genes (trnQ, trnA, trnN, trnC, trnY, trnS2, trnP and trnE) are located on the J chain (light chain). The remaining 14 tRNAs, two rRNAs, 12 protein-coding genes and 1–2 non-coding control regions are all located on the N chain (heavy chain) (
The BI and the ML tree have a consistent topology and each node has high posterior probability. The phylogenetic tree of Strigiformes obtained by the mitochondrial genome in this study is consistent with the phylogenetic tree obtained by
The Pleistocene began 2.58 million years ago (2.58 Ma). The Pleistocene (especially climate change) had a profound effect on the phylogeographic structure of existing populations (
By sequencing the complete mitochondrial genome of S. aluco and mapping its phylogenetic tree and divergence time tree, the phylogenetic relationship of Strigiformes (Tytoninae + Phodilinae) + (Striginae + Ninoxinae + Surniinae) has been summarised. Tytonidae, including Tytoninae (with Tyto) and Phodilinae (with Phodilus), are defined as the out-group; Strigidae comprises Striginae (with Asio, Bubo, Strix, Ciccaba and Otus) + Ninoxinae + Surniinae (with Athene, Aegolius and Glaucidium). The divergence time tree shows that the divergence time between S. aluco of China and S. aluco of other countries was about 1.47–2.33 Ma, suggesting that the common ancestor of S. aluco was separated by geographical isolation at the beginning of the Pleistocene. The divergence between S. aluco and S. uralensis in China was about 1.28–2.02 Ma. During this time, the rapid uplift of the Qinling Mountains led to the divergence of the ancestors of Strix on the north and south sides of the Chinese mainland. At the same time, because of climatic oscillations during the Pleistocene, the existing S. aluco population on the Qinghai-Tibet Plateau may have rapidly expanded in relatively warm shelters, such as Leigong Mountain to form the current distribution pattern.
The complete mitochondrial genome of Strix aluco has been uploaded to NCBI, GenBank accession number: OP850567.
The authors would like to thank the Rescue Center of Leigong Mountain National Nature Reserve, Qiandongnan Prefecture, Guizhou Province for providing the tissue slice of Strix aluco.
Guizhou Provincial Science and Technology Foundation, Grant/Award Number: Qiankehe LH [2020] 1Y080;
Project supported by the Joint Fund of the National Natural Science Foundation of China and the Karst Science Research Center of Guizhou Province, Grant/Award Number: U1812401;
Science and Technology Foundation of Guizhou Forestry Bureau (Qianlinkehe [2020] 09),
Guizhou Universtiy Dr. Scientific Research Fund (Guidarenjihe (2018) 07).
Yeying Wang and Haofeng Zhan contributed equally to this study. Conceptualisation: Yeying Wang, Haofeng Zhang, Yu Zhan, Zhengmin Long; Methodology: Yeying Wang, Haofeng Zhang; Software: Haofeng Zhang; Formal analysis: Haofeng Zhang; Investigation: Yeying Wang, Xiaofei Yang, Yu Zhan, Zhengmin Long; Resources: Yeying Wang, Yu Zhan, Zhengmin Long, Rescue Center of Leigong Mountain National Nature Reserve; Data curation: Haofeng Zhang; Writing - original draft: Yeying Wang, Haofeng Zhang; Writing - review & editing: Yu Zhan, Zhengmin Long, Xiaofei Yang; Visualisation: Haofeng Zhang, Yeying Wang; Supervision: Xiaofei Yang; Project administration: Xiaofei Yang; Funding acquisition: Xiaofei Yang, Yeying Wang.
The authors declare no competing or financial interests.
The genome annotation results showed that the total number of genes was 39, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes, two OH genes and 0 OL genes. Amongst them, eight tRNA genes (trn-Q, trn-A, trn-N, trn-C, trn-Y, trn-P, trn-E and trn-S2), one PCGs gene: nad6, are on the main chain (J chain); and the remaining 14 tRNA genes are trn-F, trn-V, trn-L2, trn-I, trn-M, trn-W, trn-D, trn-K, trn-G, trn-R, trn-H, trn-S1, trn-L1 and trn-T; Two rRNA genes: rrn-S, rrn-L;with 12 PCGs genes encoding: nad1, nad2, nad3, nad4, nad4L, nad5, atp6, atp8, cox1, cox2, cox3 and cytb on the secondary (N) chain.