Biodiversity Data Journal 11: e101942 OO)
doi: 10.3897/BDJ.11.e101942 open access

Research Article

Mitochondrial genome analysis, phylogeny and
divergence time evaluation of Strix aluco (Aves,
Strigiformes, Strigidae)

Yeying Wangt, Haofeng Zhan*, Yu Zhang*, Zhengmin Long}, Xiaofei Yang

+ Guizhou Normal University, Guiyang, China
§ Guizhou University, Guiyang, China

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

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

Abstract
Background

Prior research has shown that the European peninsulas were the main sources of Siérix
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 Sérix 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-

© Wang Y et al. 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.

2 Wang Y et al

tree and Bl-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. a/uco.

Keywords

Strix aluco, phylogeny, divergence time, Pleistocene, climate oscillation, mountains uplift

Introduction

Strix aluco belongs to Strigidae (Strigiformes) and is a medium-sized owl (Grytsyshina et
al. 2016). S. aluco is a non-migratory and territorial nocturnal bird (Sunde et al. 2003, Dona
et al. 2016) with a wide distribution throughout mountainous broadleaf forest and mixed
forest in Eurasia; Israel is the southernmost country in the distribution area of S. aluco in
the Northern Hemisphere (Obuch 2011, Comay et al. 2022). It can feed on mammals, fish,
amphibians and even small birds such as sparrows (Obuch 2017) and voles are its most
preferred food (Karell et al. 2009, Solonen 2022). The IUCN listed this species as of "Least
Concern". The current population trend is stable and the estimated number of individuals
ranges from 1,000,000 to 2,999,999 (IUCN 2016: https:/Awww.iucnredlist.org/). In China, S.
aluco has been listed as a national class II protected animal.

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 (Yan et al. 2017, Sun et al. 2020).
Using them enables researchers to accurately infer phylogenetic relationships in birds
(Tuinen et al. 2000) and complete mitochondrial genomes generally achieve higher
accuracy than partial mitochondrial genomes (Haring et al. 2001, Harrison et al. 2004).
Through skeletal comparison, Strigidae has been divided into three subfamilies: Striginae
(13 genera), Surniinae (eight genera) and Asioninae (two genera) (Ford 1967). Previous
studies have defined the phylogenetic position of S. a/uco using a single gene or a
combination of multiple mitochondrial genes (Heidrich and Wink 1994, Fuchs et al. 2008,
Wood et al. 2017, Yu et al. 2021). Earlier research identified the monophyly of the
Strigiformes phylogeny through the cytochrome B (Cyt B) gene (Wink and Heidrich 2000).
In Strigiformes, the taxonomic relationship of subordinate branches of Strigidae has been
hotly debated (Salter et al. 2020). Phylogenetic relationships through the Cyt B gene also
showed that the order Strigiformes can be divided into two groups: (Tytonidae + Strigidae),
Tytonidae consisting of Tytoninae (containing Tyto) and Phodilinae (containing Phodilus);
and Strigidae can be divided into Striginae, Surniinae and Ninoxinae, amongst them,

Mitochondrial genome analysis, phylogeny and divergence time evaluation ... 3

Striginae can be subdivided into a clade of ((Bubo + Strix) + (Pulsatrigini + Asio)) +
(Psiloscops + Megascops) + Otus, with Surniinae consisting of two branches (with Surniini
and Aegolius), Surniini containing (Glaucidium + Athene) and Ninoxinae being mainly
composed of Ninox, possibly including Uroglaux and Sceloglaux (Wink et al. 2009, Wink
and Sauer-GUu 2021). In addition, a growing number of scholars have described a
framework for Strigiformes phylogeny. Zhang et al. (2016) completed the whole
mitochondrial genome sequencing of Asio flammeus and determined the paraphyletic
phylogenetic relationship amongst the three genera of Ofus, Ptilopsis and Asio; Kang et al.
(2018) completed the whole mitochondrial genome sequencing of Sifrix uralensis and
determined the inter-genus relationship of Ofus + (Asio + (Strix + Bubo)) by studying the
mitochondrial genome of Strigidae; Salter et al. (2020) combined morphological
characteristics and molecular biology, suggesting that a typical owl contains Striginae and
Surniinae; they further suggested that Athene, Otus, Asio, Megascops, Bubo and Strix are
paraphyletic, while Ninox and Glaucidium are polyphyletic; Koparde et al. (2018) showed
that the Striginae and Surniinae form a paraphyletic group in the South Asian subcontinent
population with Tytonidae as the out-group; their study showed that Strigidae and
Tytonidae diverged at about 42.5-47.7 Ma (mega-annum, million years); Uva et al. (2018)
Clarified the global distribution of Tytonidae and their time of divergence, their analysis
showing that Tytonidae and S. aluco split from a common ancestor dating back to about 45
Ma. Prior research has shown that Strix and Tyto diverged roughly about 40-50 Ma (Prum
et al. 2015). The phylogenetic relationship and timing of the divergence of Sfrix in China
remain unclear.

There are many reasons for the divergence of species, amongst which geological and
climatic influences on species diversification cannot be ignored (Claramunt and Cracraft
2015). The Cretacean-Tertiary extinction event was a mass extinction event in Earth's
history that occurred 65 Ma and wiped out most animals and plants at the time, including
the dinosaurs. It also wiped out the direct ancestors of tree-dwelling waterbirds on Earth
today with the few survivors evolving rapidly thereafter (Field et al. 2018). Bird ancestry
began to increase exponentially at the end of the Eocene, from an original 100 species to
the 10,000 species of today (Ksepka and Phillips 2015). Since the late Miocene, many
birds in the Palaearctic have migrated on a large scale and their changing ranges have led
to gene flows that have provided opportunities for the origin of various bird subfamilies
(Drovetski 2003, Holm and Svenning 2014). Climatic oscillation during the Quaternary
Period, especially throughout the Pleistocene (2.58-0.01 Ma), promoted the evolution of
species on a global scale (Hewitt 2000, Hewitt 2004, Lamb et al. 2019). Pleistocene glacial
gyre played a positive role in speciation (Zhao et al. 2013, Hung et al. 2014, Kozma et al.
2018, Mays et al. 2018). Brito (2005) studied 14 populations of S. a/uco in Western Europe
and found that these could be divided into three branches originating from three glacial
sanctuaries in the Iberian Peninsula and the Balkan Peninsula in Europe. This finding
supports the "glacier refuge hypothesis" that describes the origin of S. aluco in estern
Europe. However, the origin and divergence of S. aluco in mainland China remain a
mystery.

4 Wang Y et al

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
(Ho and Phillips 2009, Ho and Duchéne 2014). To clarify the phylogenetic position,
divergence time and reasons for divergence of S. aluco from China, this study sequenced
the complete mitochondrial genome of S. aluco and used it (combined with the
mitochondrial genome of other birds in Strigiformes) to reconstruct the phylogenetic tree of
Strigiformes. Fossil data are usually used to evaluate the divergence time of birds and the
divergence time of Surniinae fossils was used as the correction point to analyse the
divergence time of Strix. Possible reasons for its divergence are discussed in depth.

Materials and methods
Sample origin and DNA extraction

Part of the muscle tissue was extracted from the leg of one individual of S. a/uco 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 (Lutz et al. 2011).

Sequencing and assembly

The whole genome shotgun strategy was used to construct the library (Roe 2004). Next
generation sequencing technology was used for paired-end sequencing, based on the
Illumina NovaSeq sequencing platform (Illumina NovaSeq, Illumina Inc., San Diego,
California, USA).

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 Roe (2004). The Agilent 2100 Bioanalyzer was used to assess the size of the
library and fluorescence quantitative detection was used to assess the total concentration
of the library. The optimal amount of the library was selected and sequenced on the
Illumina NovaSeq sequencing platform. A single-stranded library was used as a template
for bridge PCR amplification and sequencing was performed during synthesis.

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

Mitochondrial genome analysis, phylogeny and divergence time evaluation ... 5

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:/Awww.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
(Chen et al. 2018).

A5-miseq v20150522 (Coil et al. 2015) and SPAdesv 3.9.0 (Bankevich et al. 2012) were
used for the de novo sequencing of high-quality next-generation sequencing data. To
construct contig and scaffold sequences, the sequences were extracted according to the
sequencing depth of de novo splicing sequences. The sequences with high sequencing
depth were compared with the NT (Nucleotide) library on NCBI by Blastn (BLAST
v2.2.31+) and the mitochondrial sequences of each splicing result were selected. To
integrate splicing results, the mitochondrial splicing results obtained by the different
software above were combined with reference sequences. Collinearity analysis was
performed using mummer v.3.1 software (Kurtz et al. 2004) to determine the position
relationship between contigs and fill gaps between contigs. The results were corrected by
using pilon v.1.18 software (Walker et al. 2014) to obtain the final mitochondrial sequence.
The complete mitochondrial genome sequence obtained by splicing was uploaded to the
MITOS web server (http://mitos2.bioinf.uni-leipzig.de/index.py) for functional annotation
(Bernt et al. 2013). RefSeq 81 Metazoa was selected as reference, the genetic code was
set to a second set of vertebrate codons and other parameters were set according to the
default parameters proposed by MITOS.

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 (Stothard and Wishart 2005).

Mitochondrial genome data collection in Strigiformes

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

6 Wang Y et al

these thirty species were stored in a local folder using GenBank format. The registration
number is shown in Table 1.

Construction of phylogenetic trees

The PhyloSuite software (downloaded from: hitps://github.com/dongzhang0725/
PhyloSuite/releases) (Zhang et al. 2020) was used to drag the 30 GenBank format files
(downloaded from NCBI) and the GenBank format files of S. aluco sequence (obtained by
the sequencing in this study) into the main interface.

First, following the guided steps in the literature of Zhang et al. (2020), a series of
standardised operations were conducted. Mitogenome sequence types were chosen;
meanwhile, the annotation error tRNA file was exported and modified comments were
uploaded to the ARWEN website (http://130.235.244.92/ARVWEN/). The site of the modified
comments is copied and pasted for modification after the box. After that, the corrected 13
protein-coding genes (PCGs) and 24 RNAs were extracted successfully. The second set of
two vertebrate mitochondrial codes is selected here and the extracted 13 PCGs and 24
RNAs are imported into MAFFT (PhyloSuite programme) for multiple sequence alignment.
The 37 gene files exported by MAFFT were selected and imported into ‘concatenate
sequence’ (PhyloSuite programme), using the '-auto' strategy and ‘normal’ alignment
mode. The concatenated completion file was selected and PartitionFinder 2.0 (PhyloSuite
programme) (Lanfear et al. 2017) was used to perform a greedy search, select the
'Nucleitide' mode and ‘branc-lengths' can be ‘linked’. Here, 'mrbayes' was chosen for
models and the model supported by MrBayes was calculated. 'AlCc' is the model selection
criterion recommended by PartitionFinder authors, the optimal partitioning strategy and
model selection was calculated, this place using a separate GTR+G model for each data
block automatically.

Table 1.

Mitochondrial genome sequences used in this study.

Taxon GenBank accession Size (bp) Notes Reference
Aegolius funereus MN122880 17166 Partial Direct Submission
Asio flammeus KP889214 18966 Complete Zhang et al. (2016)
Asio otus MG916810 17555 Complete Lee et al. (2018)
Athene brama KF961185 16194 Partial Direct Submission
Athene noctua MN122903 15776 Partial Direct Submission
Bubo blakistoni LC099106 19379 Partial Direct Submission
Bubo bubo MN206975 18956 Complete Direct Submission
Bubo flavipes LC099100 19447 Partial Direct Submission
Bubo scandiacus MG681084 18734 Complete Kang et al. (2018)
Ciccaba nigrolineata MN356178 14875 Partial Feng et al. (2020)
Glaucidium brasilianum MN356303 17717 Partial Feng et al. (2020)

Mitochondrial genome analysis, phylogeny and divergence time evaluation ... 7

Taxon GenBank accession Size (bp) Notes Reference
Glaucidium brodiei brodiei KP684122 17318 Complete Sun et al. (2016)
Glaucidium cuculoides KY092431 17392 Complete Liu et al. (2019)
Ninox novaeseelandiae AY309457 16223 Complete Harrison et al. (2004)
Ninox scutulata KT943750 16208 Complete Direct Submission
Ninox strenua KX529654 16206 Complete Sarker et al. (2016)
Otus bakkamoena KT340631 17389 Complete Park et al. (2019a)
Otus lettia MW364567 16951 Complete Yu et al. (2021)
Otus scops KT340630 17413 Complete Park et al. (2019b)
Otus semitorques LC541473 18834 Complete Direct Submission
Otus sunia MF346692 17835 Complete Zhou et al. (2019)
Sceloglaux albifacies KX098448 15565 Partial Wood et al. (2017)
Strix aluco MN122823 16490 Partial Direct Submission
Strix aluco OP850567 18832 Complete, This study

Strix leptogrammica KC953095 16307 Complete Liu et al. (2014)
Strix occidentalis MF431746 19889 Complete Hanna et al. (2017)
Strix uralensis MG681081 18708 Complete Kang et al. (2018)
Strix varia MF431745 18975 Complete Hanna et al. (2017)
Out-group

Phodilus badius KF961183 17086 Complete Mahmood et al. (2014)
Tyto alba EU410491 16148 Partial Pratt et al. (2009)
Tyto longimembris KP893332 18466 Partial Xu et al. (2016)

The result file of PartitionFinder 2.0 was selected, the ML method was completed in I|Q-tree
(Minh et al. 2020) mode (PhyloSuite programme) and the BI method was completed in
MrBayes mode (PhyloSuite programme). Phodilus badius, Tyto alba and Tyto
longimembris were set as out-groups. In |Q-tree mode, the Edge-linked partition style was
employed for 10,000 replicates of ultrafast bootstrapping (Sterli et al. 2013, Hoang et al.
2018). In MrBayes mode, the result folder of PartitionFinder 2.0 was opened, out-groups
were set, parameters were defined as Partition Models and algebra run as two parallel
runs, four chains, for 2,000,000 generations (where it must be ensured that the average
standard deviation of split frequencies values remains below 0.01), sampling freq is one
sampling run for 1000 times and 25% of the initial samples were discarded as burn-in.

Divergence time evaluation

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 (Li et al. 2022), the origin
times of Surniinae were set to 6.0 Ma and 9.5 Ma, which included Aegolius, Athene, and
Glaucidium (Wink and Sauer-GUti 2021). The 'NEX' file obtained by concatenating 37 genes
using the ‘concatenate sequence’ programme function in PhyloSuite was imported into

8 Wang Y et al

BEAUti 2.6.7, (http:/www.beast2.org/), Hasegawa-Kishino-Yano model, with four gamma
categories, Strict clock with 1.0 clock rate and with a Yule process (speciation) prior. “
Aegolius funereus, Athene brama, Athene noctua, Glaucidium brasilianum, Glaucidium
brodiei brodiei and Glaucidium cuculoides” (sequence file name) was chosen and Prior
was added. Then, the “monophyletic” option was checked, the Mean set to 6.0/9.5 and
Sigma set to 0.1. A Markov Monte Carlo Chain Bayesian analysis with a chain length of
10,000,000 and with states recorded every 1000 iterations was run using BEAST 2.6.7.
Log files were assessed using TRACER 1.7.2 (http://tree.bio.ed.ac.uk/software/tracer/) to
ensure that posteriors were normally distributed and that all statistics had attained effective
sample sizes of > 200. If ESS < 200, optimisation was employed by adding 5,000,000
iterations (chain length) each time. A burn-in of 10% was discarded, the maximum clade
credibility tree was determined and mean heights were chosen using TreeAnnotator 2.6.7.
Finally, FigTree 1.4.4 was used to assess the divergence time. Finally, Adobe Illustrator
1.0.0.2 was used for visual editing (all figures are the same).

Results

Genome annotation

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 O
H genes and 0 O, 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-l, 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. 1). The specific annotation results of each gene are shown in Suppl.
material 1.

Phylogenetic analysis

In this study, both the ML-tree and Bl-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 G/aucidium 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. al/uco 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)].

Mitochondrial genome analysis, phylogeny and divergence time evaluation ... 2)

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. 2).

Wi cps

i tRNA

MH rRNA
Other

HB GC content

BB GC skew+

BB GC skew-

trnF(gaa) —
control region \ i) a
~ !
rl
trnE (ttc),
nad6
trnP(tgg),

PotRNA

7imnL2(taa)
control region,

_nad1l

jornltgat)
—trnQ(ttg)
NtrnM(cat)

trnT(tgt)~ .

~askop = Strix aluco
complete mitochondrial genomics a

Length: 18,632 bp = 5kop -

cob—

—nad2

trnW(tca)
~LtrnA(tgc)
—trnN(gtt)
\ trnC(gca)
trnY(gta)

nad5—-
~“cox1

trnL1(tag)—_
trnS1(gct)~
trnH(gtg

—trnS2(tga)
\trnD(gtc)

\cox2
nad4“ —trnk(ttt)
nad4 : am latps
trnR(tcg)_ “7. ~_ ——=, 2 atp6

nad3 trnG(tcc)©O%?

Figure 1. EES

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.

Divergence time evaluation

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, 7yto 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

10 Wang Y et al

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.

0.97/52 Aegolius funereus MN122880
j 1/100 Glaucidium brasilianum MN356303
1/100 Glaucidium cuculoides KY 092431
1/100 1/100 Athene brama KF961185
Athene noctua MN122903
Glaucidium brodiei brodiei KP684122
1/100 Asio flammeus KP889214
Asio otus MG916810
1/100 Bubo blakistoni LC099106
1/100 1/100 Bubo flavipes LC099100
1/100 Bubo bubo MN206975
: Bubo scandiacus MG68 1084
1/100 1/100 1/100 Ciccaba nigrolineata MN356178
1/100 Strix varia varia MF431745 Strigidae
j Strix occidentalis caurina MF431746
1/100 1/100 | 49 Strix aluco MN 122823
1/100 1/95 Strix aluco OP850567 *®
< Strix uralensis MG681081
Strix leptogrammica KC953095
1/100 Otus bakkamoena KT340631
1/81 1/100 Otus semitorques pryeri LC541473
1/100 Otus lettia MW364567
1/100 Otus scops KT340630
Otus sunia MF346692
1/99 Ninox novaeseelandiae AY 309457
oA 1/100 Sceloglaux albifacies KX098448
1/100 Ninox strenua KX529654
Ninox scutulata KT943750
Phodilus badius KF961183 .
1/100 Tyto longimembris KP893332 Tytonidae (Out-group)
Tyto alba EU410491 i

Tree seale: 0.1

Figure 2. EESl

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 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 (IMN122823)
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. 3).

Discussion

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) (Wolstenholme 1992, Boore 1999), which is consistent with the
complete mitochondrial genome structure of all birds (Hanna et al. 2017). The complete
mitochondrial genome sequence of S. a/uco obtained in this study was circular, with a total
length of 18,632 bp and a GC content of 46.76%. Its composition was as follows: the ration
of Adenine bases to the total base column (A%) was 29.57%; the ratio of the Guanine base

Mitochondrial genome analysis, phylogeny and divergence time evaluation ... 11

to the total base column (G%) was 14.09%; the ratio of the Cytosine base to the total base
column (C%) was 32.67%; the ratio of Thymidine to the total base column (T%) was
23.66%. The start codon of all 13 PCGs was ATG and the transcription stop codons were
AGG, TAG and TAA. The content of A+T (53.23%) was higher than that of G+C (46.76%),
which is consistent with the AT tendency of base bias in vertebrate mitochondrial genomes
(Broughton et al. 2001, Ma et al. 2015). This result is consistent with the mitochondrial
genome of other owls in Strigidae (Kang et al. 2018, Sun et al. 2020).

TIME SCALE (Ma)
[8.05, 12.75]

) Common Ancestor of Strigiformes

Aegolius finereus MN122880
510.808 PROS « Athene brama KF961185
4.82, 7.64] —— « Athene noctua MN122903
aris « Glaucidium brasilianum MN356303
Glaucidium cuculoides KY 092431
« Glaucidium brodiei brodiei KP684122
BB Midevolution 192,304) Asio flammeus KP889214
—— Asio otus MG916810

Bubo blakistoni LC099106
Bubo flavipes LC099100
Bubo bubo MN206975
Bubo scandiacus MG68 1084
Ciccaba nigrolineata MN356178
Strix varia varia MF431745
Strix occidentalis caurina MF43 1746
4.63, 7.33] 2.53, 4.0] Strix aluco MN122823

{1.47,2.33 ee ears CYPR
138 2.03) Strix aluco OP850567 *

[6.0, 9.50] (3.52, 5.57]

0.96, 1.52
14.03,6.39] | 65 94 4.65)

1.79, 2.84
© Approach to the present

-(6.32, 10.01] 3.49, 5.53] TERE

[I.61, 2.55]

NOW (2.20, 3.48] Strix uralensis MG68 1081
Strix leptogrammica KC953095
(1.63,258] oe bakkamoena K’ eae? 4 Die
‘s a ts semitorques pryeri LC.
pee 5:50, 8.70 ~ Lean Otus lettia MW364567

Sm ASP) (0.01, 0.02)" Otus scops KT340630
Ontus sunia MF346692
Ninox novaeseelandiae AY 309457
Sceloglaux albifacies (X098448
Ninox strenua KX529654
Ninox scutulata KT943750
Phodilus baditis KF961183
« Tyto albaEU410491
Tyto longimembris KP893332

2.0

(4.23, 6.69]

Figure 3. EES

Divergence time tree. Through the divergence time tree obtained by BEAST 2.6.7, based on
the Bayesian method, the node horizontal bar indicates that the posterior probability of this
age interval is 95% and the divergence time has been marked at the node.

Phylogenetic analysis of S. aluco

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 Li et al. (2022) through
morphology. Wink et al. (2009) compared Surniini (with Surnia, Gl/aucidium and
Taenioglaux), Athenini (with Athene) and Aegolini (with Aegolius) under Surniinae, in their
recent study, Surniini (with Surnia and G/aucidium) are monophyletic and cluster as a sister
to Aegolius and they found that Jyto alba probably originated in Australia. They also
believe that many owls that do not migrate will form new species in different places (Wink
and Sauer-GU 2021). Both Salter et al. (2020) and Wink and Sauer-Gu (2021) agree on the
phylogenetic relationship of (Glaucidium+Athene) + Aegolius and we agree that G. brodiei
does not form an evolutionary clade with other Glaucidium. According to the genome

12 Wang Y et al

analysis of Strigidae birds in Madagascar, Strix is most closely related to Bubo, followed by
Otus (Fuchs et al. 2008). The phylogenetic relationship of Strigidae forms a phylogenetic
relationship of Surniinae (with Surniini and Aegolius) + [Ninox + (Otus + (Asio + (Strix +
Bubo)))] in this study. The conclusion of Oftus + (Asio + (Strix + Bubo)) is consistent with
the conclusion of Kang et al. (2018). In the phylogenetic tree constructed by Yu et al.
(2021), C. nigrolineata was also nested in Strix. S. albifacies has been extinct on the island
of New Zealand and when Wood et al. (2017) extracted its mitochondrial genome from
museum specimens, they suggested changing its name to Ninox albifacies because it has
the same morphological structure and phylogenetic position as Ninox, in our phylogenetic
tree, with relatively high posterior probability and bootstrap supporting this point. S. aluco O
P850567 forms a sister group with S. uralensis, which was uploaded to GenBank by Kang
et al. (2018). However, S. aluco uploaded with Margaryan. A forms a monophyly of Stnx
aluco MN122823 + (Strix aluco OP850567 + Strix uralensis). Compared with the
mitochondrial genome of S. a/uco obtained by Margaryan. A, S. aluco in China is more
closely related to S. uralensis MG681081, which came from northeast China. Extant
Eurasian birds communicated through woodland corridors during the Pleistocene
interglacial. Combined with divergence-time tree analysis, such communication may have
existed in the common ancestor of S. aluco IMN122823 and S. aluco OP850567 (Voelker
2010). The likely reason is that, at the beginning of the Pleistocene, the common ancestor
of S. aluco MN122823 and S. aluco OP850567 had already been geographically isolated.
The isolation of the Pleistocene refugium led to the divergence of the whole genome of the
common ancestor of the forest owl both in China and internationally. Foreign studies have
shown that the Quaternary Period is characterised by a series of glacial-interglacial cycles
(Woodruff 2010), with the ancestors of modern species seeking refuge in suitable
environments. The existing species on the Qinghai-Tibet Plateau may be the result of rapid
population expansion in relatively warm refugia during the Pleistocene glaciation and
interglacial period, which formed the current distribution pattern and genetic diversity (Gao
et al. 2015). Leigong Mountain in Guizhou Province just played the role of a refugium for S.
aluco during the Pleistocene glaciation period. Mitochondrial phylogeographic studies
(Brito 2005) showed that the origin of S. aluco in Western Europe supports the "glacial
refuge hypothesis"; further, the species survived in three allopatric refugees in Italy, the
Iberian Peninsula and the Balkans, becoming the main source of S. a/uco in Europe during
the late glacial period. DNA barcoding technology also showed that the geographical
barrier of the Strait of Gibraltar played an extremely important role in the phylogenetic
history of S. aluco (Dona et al. 2016).

Divergence time evaluation of S. aluco

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 (Lamb et al. 2019). On the Qinghai-Tibet Plateau, the impact of mountain uplift
on the formation of modern species (< 2.0 Ma) is limited and researchers have suggested
that climate fluctuations played a key role in the formation of species during the Middle
Pleistocene (Renner 2016, Wang et al. 2018). During the Quaternary Period and
Pleistocene (1.60—2.70 Ma), there were severe climate shocks (Lisiecki and Raymo 2005),

Mitochondrial genome analysis, phylogeny and divergence time evaluation ... 13

which positively promoted the formation of species (Rull 2008, Rull 2011, Rull 2015).
Climatic fluctuations during this period, especially during the ice age, affected the
distribution of forests in the Northern Hemisphere and the evolution of forest living species
(Song et al. 2021). This series of climate fluctuations in the Pleistocene promoted species
variation, which has led to species differentiation (Leonard et al. 2015). Glaciation has
played an important role in influencing the population size, species and community genetic
structure of today's species (Hewitt 2000, Hewitt 2004, Svendsen et al. 2004). The glacial-
interglacial gyrations of the same period also affected the distribution of species (Zhao et
al. 2013, Hung et al. 2014, Kozma et al. 2018, Mays et al. 2018). Glacial-interglacial cycles
led to periodic shifts in glacial refuges for Pleistocene birds (Nadachowska-Bizyska et al.
2015) and the isolation of glacier refugia led to the divergence of the whole genome of
species, thus forming different species (Provost et al. 2022). This is likely also the reason
why the common ancestor of S. aluco MN122823 and S. aluco OP850567 (this study)
diverged at 1.47—2.33 Ma. Genetic divergence of the same lineage because of the isolation
of refugees leads to divergence of lineages. Various kinds of species generally begin to
migrate to the best habitat during warm climate periods (Claramunt and Cracraft 2015); in
particular, species adapted to low altitudes in the early stage of climate change will move to
high altitudes at this time, resulting in the reproductive isolation of species in the two
separated places (Wiens 2004). During the Pleistocene-Holocene (1.10—0.60 Ma), the
Qinghai-Tibet Plateau experienced three stages of rapid uplift, with the formation of
mountains, the climate changing from moist and warm to dry and cold and the retreat of
forests to the edge of the plateau (Wang et a/. 2008). Thus, the forest landscape became
what it is today. The Quaternary Period climate shock led to the initial formation of the
existing forest and mountain distribution pattern in the Northern Hemisphere. Birds began
to distribute widely after leaving the glacier refuge at the end of the glaciation and initially
formed the existing distribution pattern (Pujolar et al. 2022). The S. uralensis may have
moved north at this time and thus diverged from S. aluco. In addition, the rapid uplift of the
Qinling Mountains from the end of the Early Pleistocene to the Middle Pleistocene may
have formed the Qinling Mountains as a barrier to north-south bird communication (Li et al.
2019). The rapid uplift of the Qinling Mountains prevented communication between S.
aluco and the common ancestor of S. uralensis, which was originally distributed on the
north and south sides.

Conclusions

By sequencing the complete mitochondrial genome of S. a/uco 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. a/luco 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.

14 Wang Y etal

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.

Data ability

The complete mitochondrial genome of Strix aluco has been uploaded to NCBI, GenBank
accession number: OP850567.

Acknowledgements

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.

Funding program

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).

Author contributions

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.

Mitochondrial genome analysis, phylogeny and divergence time evaluation ... 15

Conflicts of interest

The authors declare no competing or financial interests.

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Supplementary material

Suppl. material 1: Analysis of mitochondrial genome feature EE

Authors: Yeying Wang, Haofeng Zhan

Data type: Table

Brief description: 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 O, genes and 0 O,
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-l, 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.

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