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Research Article
Identification and phylogenetic analysis in Pterorhinus chinensis (Aves, Passeriformes, Leiothrichidae) based on complete mitogenome
expand article infoGuirong Bai, Qingmiao Yuan, Qiang Guo, Yubao Duan
‡ Southwest Forestry University, Kunming, China
Open Access

Abstract

The Black-throated Laughingthrush (Pterorhinus chinensis) is a bird belonging to the order Passeriformes and the family Leiothrichidae, and is found in Cambodia, China, Laos, Myanmar, Thailand and Vietnam. Pterorhinus chinensis was once classified as belonging to the genus Garrulax. However, recent research has reclassified it in the genus Pterorhinus. In this study, we sequenced and characterized the complete mitogenome of P. chinensis. The complete mitochondrial genome of P. chinensis is 17,827 bp in length. It consists of 13 PCGs, 22 tRNAs, two rRNAs, and two control regions. All genes are coded on the H-strand, except for one PCG (nad6) and eight tRNAs. All PCGs are initiated with ATG and stopped by five types of stop codons. Our comparative analyses show irregular gene rearrangement between trnT and trnP genes with another similar control region emerging between trnE and trnF genes compared with the ancestral mitochondrial gene order, called “duplicate CR gene order”. The phylogenetic position of P. chinensis and phylogenetic relationships among members of Leiothrichidae are assessed based on complete mitogenomes. Phylogenetic relationships based on Bayesian inference and maximum likelihood methods showed that Garrulax and (Pterorhinus + Ianthocincla) formed a clade. Leiothrix and Liocichla also formed a clade. Our study provides support for the transfer of P. chinensis from Garrulax to Pterorhinus. Our results provide mitochondrial genome data to further understand the mitochondrial genome characteristics and taxonomic status of Leiothrichidae.

Key words

Black-throated Laughingthrush, duplicate control region, mitogenome, phylogeny, reclassification, taxonomy

Introduction

The mitochondrial genome (hereafter mitogenome) has proven very useful in phylogenetics and population genetics of avian taxa (Mindell et al. 1998; Jønsson et al. 2019). Inherited exclusively from the mother, the mitochondrial genome is highly conserved, with little to no recombination within the mitogenome (Koehn and Nei 1983; Lansman et al. 1983). Moreover, mitochondrial genomes are comparatively more conserved than nuclear genomes during transition events in the context of evolution, especially in birds (Rheindt and Edwards 2011). Such unique architecture, in terms of organization as well as evolutionary behavior, enables mitochondrial genomes to carry phylogenetic information more consistently than nuclear markers (Rheindt and Edwards 2011). Birds, however, are particularly noteworthy because their mitochondrial genomes are characterized by a gene order different from that found in the majority of vertebrates due to rearrangements near the control region (Desjardins and Morais 1990). Most of the mitochondrial DNA mutations are point mutations, with few insertions or deletions. Moreover, as mitochondrial gene evolution rates differ (Aquadro and Greenberg 1983; Cann et al. 1984), different genes in the mitochondrial genome can be used to address different issues in phylogenetics and population genetics (Wenink et al. 1994). Mitochondrial genes are more closely linked and easier to identify than nuclear genes. Because of these advantages, the mitogenome has been widely used in studies of vertebrate phylogeny. Accordingly, an increasing number of complete sequences of mitogenomes from birds has been determined, and their structural features have been studied (Haddrath and Baker 2001; Krzeminska et al. 2016; Master et al. 2016).

The babblers are a diverse group of oscine passerine birds. Fregin et al. determined five primary clades at the rank of family for the babbler assemblage: Sylviidae, Zosteropidae, Timaliidae, Pellorneidae and Leiothrichidae (Fregin et al. 2012). This taxonomy has been gradually recognized in recent taxonomic lists (Dickinson and Christidis 2014). However, some phylogenetic relationships and genus names remain controversial. Leiothrichidae represents the largest clade of babblers in terms of species diversity (Cibois et al. 2018). Distributed throughout Africa, most of southern Asia, and the Great Sunda region, Leiothrichidae are most diverse in the Sino-Himalayan and South-East Asian regions. Leiothrichidae are generally rather large birds which inhabit the understory of thickets in mountainous areas, with many species foraging mainly on the ground in a thrush-like manner (Cibois et al. 2018).

The Black-throated Laughingthrush (Pterorhinus chinensis Scopoli, 1786) belongs to the order Passeriformes and the family Leiothrichidae. It is distributed in East Asia (China) and South and Southeast Asia (Cambodia, Laos, Myanmar, Thailand and Vietnam), and inhabits forest, shrubland and grassland (HBW-BirdLife 2022; IUCN 2023). This species is listed as Least Concern (LC) on the IUCN Red List, but the global population is decreasing (IUCN 2023). At present, the genus allocation of P. chinensis is controversial. It was once classified as Garrulax (Moyle et al. 2012). However, in a recent study, Cibois et al. (2018) collected the data of 102 species from 21 genera of Leiothrichidae, performed phylogenetic analysis and divergence time estimation based on molecular markers, and reclassified generic limits in combination with morphological characteristics. The results supported the transfer of 16 species from the original genus Garrulax to Pterorhinus, including the P. chinensis. Meanwhile, del Hoyo et al. (2016) and Cai et al. (2019) also support the classification of the Black-throated Laughingthrush into the genus Pterorhinus. In this study, we describe the complete mitogenome of P. chinensis and compare it with those of other species of Leiothrichidae. Our analysis of phylogenetic relationships confirms that P. chinensis should be classified as a member of Pterorhinus.

Material and methods

Samples and DNA extraction

The P. chinensis sample was collected in Dehong Dai and Jingpo Autonomous Prefecture (Yunnan, China) by the Dehong Prefecture Wildlife Shelter and Rescue Center and subsequently provided to the Department of Biodiversity Conservation, Southwest Forestry University. The tissue used in this study was preserved in absolute ethanol and stored at -20 °C until DNA extraction. Total DNA was extracted from muscle tissue of the bird using the TIANamp Genomic DNA Kit (DP304, TIANGEN, Beijing, China) according to the manufacturer’s instructions. DNA integrity was evaluated by 1% agarose gel electrophoresis, and DNA purity and concentration were measured on a NanoDrop 2000 (NanoDrop Technologies, Wilmington, DE, USA). Following Sangster and Luksenburg (2021), we verified the identity of our mitogenome sequence of P. chinensis with reference sequences of three commonly used markers in songbird systematics: NADH dehydrogenase subunit 2 (nad2, 1041 bp; n=1406, incl. 14 P. chinensis), part of cytochrome c oxidase subunit I (cox1, 696 bp; n=778, incl. two P. chinensis), and cytochrome b (cob, 1143 bp; n=939, incl. four P. chinensis). In each of these analyses, which were conducted with maximum likelihood analysis using a GTR+G+I model, our sequence of P. chinensis clustered with the reference sequences of P. chinensis, indicating that our sample was correctly identified.

Genome sequencing, assembly and annotation

The P. chinensis DNA library was sequenced by Shanghai Personal Biotechnology Co., Ltd (Shanghai, China) using an Illumina NovaSeq with 300 bp paired-ends. The mitogenome was sequenced by next-generation sequencing. Raw sequence data were deposited into the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) with the accession number MT457820. Assembly of the mitochondrial genome was completed using A5-miseq version 2.0 (Coil et al. 2014). The tRNA genes were verified using the MITOS WebServer (Bernt et al. 2013) (http://mitos2.bioinf.unileipzig.de/index.py) and tRNAscan-SE 2.0 (Lowe and Chan 2016) using the default settings for the vertebrate mitochondrial genetic code. The tRNA secondary structures were predicted by tRNAscan-SE. Protein-coding regions were identified using the open reading frame (ORF) finder (Pombert et al. 2004) on the NCBI website with settings for the vertebrate mitochondrial genetic code and translated into putative proteins using GenBank. Base compositions were calculated and relative synonymous codon usage (RSCU) values were analyzed with MEGA v.7.0 (Kumar et al. 2016). Composition skew was calculated using the formula “AT-skew = (A–T) / (A+T)” and “GC-skew = (G–C) / (G+C)” (Perna and Kocher 1995). A graphical map of the mitogenome was drawn using the CGView Server (http://stothard.afns.ualberta.ca/cgview_server/index.html) (Stothard and Wishart 2005).

Phylogenetic analyses

At present, only a small number of complete mitogenomes of species of the Leiothrichidae are available from GenBank. Therefore, the phylogenetic position of P. chinensis within Leiothrichidae was determined by comparing the 13 PCGs identified in P. chinensis to those of 13 other species of Leiothrichidae from six genera: Pterorhinus, Ianthocincla, Garrulax, Trochalopteron, Liocichla and Leiothrix (Table 1). Alauda arvensis (GenBank Accession No. JQ322641) was used as an outgroup (Zhang et al. 2018a). Sequences of three species, Pterorhinus albogularis (NC_037464), Pterorhinus poecilorhynchus (NC_028082) and Trochalopteron milnei (NC_041141), were excluded because each of these represented a chimera with DNA of two different species (Sangster and Luksenburg 2021). A sequence of “Pterorhinus perspicillatus” (NC_026068) was misidentified on GenBank and was renamed Pterorhinus pectoralis, based on Sangster and Luksenburg (2021).

Table 1.

List of the 17 Leiothrichidae species and one outgroup used in this study with their GenBank accession numbers.

Family Genus Species GenBank No. References
Alaudidae Alauda Alauda arvensis Linnaeus, 1758 JQ322641 Qian et al. 2013
Leiothrichidae Pterorhinus Pterorhinus sannio Swinhoe, 1867 NC_028186 Unpublished
Pterorhinus courtoisi Ménégaux, 1923 NC_065197 Unpublished
Pterorhinus albogularis Gould, 1836 NC_037464 Unpublished
Pterorhinus lanceolatus Verreaux J, 1871 KR818090 Qi et al. 2016a
Pterorhinus poecilorhynchus Gould, 1863 NC_028082 Qi et al. 2016b
Pterorhinus pectoralis Gould, 1836 NC_026068 Unpublished
Pterorhinus chinensis Scopoli, 1786 MT457820 This study
Ianthocincla Ianthocincla maxima Verreaux, 1870 MZ129308 Unpublished
Ianthocincla ocellata Vigors, 1831 NC_027657 Zhou et al. 2016a
Ianthocincla cineracea Godwin-Austen, 1874 NC_024553 Xue et al. 2016
Garrulax Garrulax canorus Linnaeus, 1758 KT633399 Huang and Zeng 2016
Trochalopteron Trochalopteron elliotii Verreaux, 1870 NC_034373 Zhou et al. 2016b
Trochalopteron affine Blyth, 1843 NC_029402 Huang et al. 2016
Trochalopteron milnei David, 1874 NC_041141 Zhang et al. 2018b
Liocichla Liocichla omeiensis Riley, 1926 KU886092 Unpublished
Leiothrix Leiothrix lutea Scopoli, 1786 MN356265 Unpublished
Leiothrix argentauris Hodgson, 1837 HQ690245 Unpublished

The nucleotide sequences of the 13 PCGs of all 15 mitogenomes were concatenated and aligned using Clustal X in MEGA v.7.0 under the default parameters (Larkin et al. 2007). Phylogenetic analyses were performed by Bayesian inference (BI) and maximum likelihood (ML) methods. The Bayesian information criterion (BIC) in jModelTest v.0.1.1 was used to determine the optimal nucleotide substitution model, which was GTR+G+I (Santorum et al. 2014). The BI tree was produced using MrBayes v.3.2.1 with four Markov chains running simultaneously for 400,000 generations, sampling every 100 generations and discarding the first 25% as burn-in (Ronquist et al. 2012). The ML tree was produced using RAXMLGUI v.1.5b3 (Guindon and Gascuel 2003). A total of 1000 replicates were performed with the GTR+GAMMA substitution model. The resulting phylogenetic trees were visualized in FigTree v.1.2.2 (Rambaut and Drummond 2022).

Results

Mitogenome organization

The mitochondrial genome of P. chinensis is a typical closed-circular and double-stranded DNA molecule of 17,827 bp in length (Fig. 1). It contained the 37 typical mitochondrial genes (13 PCGs, 22 tRNAs and 2 rRNAs) and 2 control regions. Most gene sequences were on the H-strand including 12 PCGs, 14 tRNAs, 2 rRNAs and 2 control regions; however, nad6 and 8 tRNAs (trnA, trnC, trnE, trnN, trnP, trnQ, trnS2, and trnY) were encoded on the L-strand (Fig. 1, Table 2). The mitochondrial gene order of P. chinensis corresponded to nad5/cob/trnT/CR1/trnP/nad6/trnE/CR2/trnF/12S. Thus, there were two similar control regions, and the rearrangement type was “duplicate CR”. The mitogenome contained 33 overlapping nucleotides that were 1–10 bp in length and located in 10 pairs of neighboring genes. A comparison of all genes revealed the longest overlap (10 bp) between atp8 and atp6. A total of 978 intergenic nucleotides were found in 22 locations, ranging in size from 1 to 311 bp. The longest intergenic spacer (311 bp) was located between trnT and CR1.

Table 2.

Annotation of the complete mitogenome of P. chinensis.

Gene Strand Start Stop Length (bp) Intergenic length Anticodon Start codon Stop codon
trnF H 1 70 70 -1 GAA
12S H 70 1056 987 -1
trnV H 1056 1125 70 7 TAC
16S H 1133 2722 1590 1
trnL2 H 2724 2798 75 14 TAA
nad1 H 2813 3790 978 8 ATG TAA
trnI H 3799 3872 74 5 GAT
trnQ L 3878 3948 71 1 TTG
trnM H 3948 4016 69 0 CAT
nad2 H 4017 5057 1041 -1 ATG TAA
trnW H 5057 5127 71 1 TCA
trnA L 5129 5197 69 10 TGC
trnN L 5208 5280 73 1 GTT
trnC L 5282 5347 66 -1 GCA
trnY L 5347 5417 71 1 GTA
cox1 H 5419 6969 1551 -9 ATG AGG
trnS2 L 6961 7033 73 4 TGA
trnD H 7038 7106 69 10 GTC
cox2 H 7117 7800 684 0 ATG TAA
trnK H 7801 7870 70 1 TTT
atp8 H 7872 8039 168 -10 ATG TAA
atp6 H 8030 8713 684 5 ATG TAA
cox3 H 8719 9502 784 0 ATG T(AA)
trnG H 9503 9571 69 0 TCC
nad3 H 9572 9922 351 -1 ATG TAA
trnR H 9922 9991 70 1 TCG
nad4l H 9993 10289 297 -7 ATG TAA
nad4 H 10283 11660 1378 0 ATG T(AA)
trnH H 11661 11730 70 0 GTG
trnS1 H 11731 11796 66 -1 GCT
trnL1 H 11796 11866 71 0 TAG
nad5 H 11867 13684 1818 8 ATG AGA
cob H 13693 14835 1143 3 ATG TAA
trnT H 14839 14907 69 311 TGT
CR1 H 15219 15882 664 126
trnP L 16009 16077 69 6 TGG
nad6 L 16084 16602 519 1 ATG TAG
trnE L 16604 16675 72 297 TTC
CR2 H 16973 17651 679 175
Figure 1. 

Gene map of the P. chinensis mitogenome.

The nucleotide composition of the complete mitogenome was as follows (Table 3): A = 29.45%, T = 23.26%, G = 14.48%, and C = 32.81%. The A + T content (52.71%) was substantially higher than that of G + C (47.29%). The overall AT-skew and GC-skew in the P. chinensis mitogenome were 0.12 and -0.39, respectively. The GC-skew, except for tRNAs, was slightly negative (-0.39 to -0.11), showing a higher occurrence of C than G. In contrast, the overall AT-skew, except for the 2 control regions, was slightly positive (0.01 to 0.24), showing a higher occurrence of A than T.

Table 3.

Composition and skew values for P. chinensis.

Region Size (bp) A% C% T% G% A+T% G+C% AT-skew GC-skew
mtDNA 17,827 29.45 32.81 23.26 14.48 52.71 47.29 0.12 -0.39
PCGs 11,369 27.47 33.73 24.13 14.67 51.60 48.40 0.06 -0.39
tRNAs 1546 28.96 20.71 28.18 22.15 57.14 42.89 0.01 0.03
rRNAs 2577 33.18 25.89 20.36 20.57 53.54 46.46 0.24 -0.11
CRs 1343 20.86 31.55 30.27 17.32 51.13 48.87 -0.18 -0.29

Protein-coding genes

The total length of all PCGs in the mitogenome of P. chinensis was 11,369 bp and this accounted for 63.77% of the entire P. chinensis mitogenome. The A + T content in PCGs was 51.60% (Table 3). The gene with the highest number of base pairs was nad5 (1818 bp), and the gene with the lowest number was atp8 (168 bp). In addition, atp8 and atp6 shared 10 nucleotides; atp6 and cox3 had an interval of 5 nucleotides; nad4L and nad4 shared 7 nucleotides; and nad5 and cob had an interval of 8 nucleotides (Table 2). The initiation codon used for all PCGs was ATG for methionine. The predominantly used stop codons used were TAA (nad1, nad2, cox2, atp8, atp6, nad3, nad4l and cob). Whereas nad6 ended with TAG, cox1 ended with AGG, and nad5 ended with AGA. Incomplete stop codons (T**) were detected for cox3, and nad4 in P. chinensis.

RSCU values for the P. chinensis mitogenome for the third position are shown in Table 4. The total number of codons in PCGs was 5942. Codons encoding Trp were rare, while those encoding Pro were most frequent (Fig. 2). The codons Glu (GAA), Gln (CAA), and Lys (AAA) were mainly composed of T or A + T, while Gly (GGA), Leu1 (CUA) and Val (GUA) had a high G + C content (Fig. 3).

Table 4.

Codon number and relative synonymous codon usage (RSCU) of P. chinensis mitochondrial protein-coding genes (PCGs).

Codon Count RSCU Codon Count RSCU Codon Count RSCU Codon Count RSCU
UUU(F) 56 0.53 UCU(S) 93 1.08 UAU(Y) 61 0.69 UGU(C) 28 0.61
UUC(F) 154 1.47 UCC(S) 123 1.42 UAC(Y) 116 1.31 UGC(C) 64 1.39
UUA(L) 72 0.69 UCA(S) 93 1.08 UAA(*) 87 1.21 UGA(*) 78 1.08
UUG(L) 31 0.30 UCG(S) 31 0.36 UAG(*) 51 0.71 UGG(W) 30 1.00
CUU(L) 126 1.21 CCU(P) 281 1.57 CAU(H) 167 0.96 CGU(R) 44 0.74
CUC(L) 149 1.43 CCC(P) 209 1.17 CAC(H) 181 1.04 CGC(R) 71 1.20
CUA(L) 181 1.74 CCA(P) 173 0.97 CAA(Q) 173 1.42 CGA(R) 57 0.96
CUG(L) 65 0.63 CCG(P) 52 0.29 CAG(Q) 71 0.58 CGG(R) 39 0.66
AUU(I) 103 0.86 ACU(T) 172 1.43 AAU(N) 141 0.81 AGU(S) 53 0.61
AUC(I) 156 1.30 ACC(T) 143 1.19 AAC(N) 208 1.19 AGC(S) 126 1.46
AUA(I) 102 0.85 ACA(T) 136 1.13 AAA(K) 158 1.33 AGA(R) 80 1.35
AUG(M) 74 1.00 ACG(T) 30 0.25 AAG(K) 80 0.67 AGG(R) 64 1.08
GUU(V) 47 0.98 GCU(A) 62 0.90 GAU(D) 46 0.73 GGU(G) 38 0.76
GUC(V) 53 1.11 GCC(A) 102 1.48 GAC(D) 80 1.27 GGC(G) 64 1.27
GUA(V) 63 1.32 GCA(A) 87 1.27 GAA(E) 76 1.31 GGA(G) 72 1.43
GUG(V) 28 0.59 GCG(A) 24 0.35 GAG(E) 40 0.69 GGG(G) 27 0.54
Figure 2. 

Codon distribution of the P. chinensis mitogenome. Numbers on the Y-axis refer to the total number of codons and codon families are provided on the X-axis.

Figure 3. 

The relative synonymous codon usage (RSCU) of the P. chinensis mitogenome. Codons are shown on the X-axis and RSCU values are shown on the Y-axis.

Ribosomal and transfer RNA genes

The small 12S rRNA was 987 bp, with an A + T content of 50.80%; whereas the large 16S rRNA of P. chinensis was 1590 bp in length, with an A + T content of 54.40%. The two rRNAs were located between trnF and the trnL2, and isolated by trnV (Fig. 1 and Table 2).

The total length of tRNA ranges for P. chinensis was 1546 bp, and this accounted for 8.70% of the total mitogenome. The average length of tRNA was 70 bp, the longest was trnL2 (75 bp), and the shortest was trnC and trnS1 (66 bp) (Table 2). All tRNAs in the mitogenome of P. chinensis had the canonical cloverleaf structure with slight variation in sequence length of the stem regions of the main arms. The A + T content of the 22 tRNAs was 57.14%. The mismatched base pairs G-U, A-C, U-U, A-A, and C-C were found in all P. chinensis tRNAs except for trnR, trnI, trnK, trnF, trnW, and trnV (Fig. 4).

Figure 4. 

Secondary structures of the 22 transfer RNA genes of P. chinensis.

Non-coding sequencing

The mitogenome of P. chinensis contained two similar control regions (CR1, CR2), which were 664bp and 679bp in length, respectively. They were located between trnT and trnF genes, separated by trnP, nad6, and trnE genes (Fig. 1). The base composition of CR1 was 20.93% A, 30.72% T, 17.32% G, and 31.02% C. The A + T content (51.65%) was higher than the G + C content (48.34%). The base composition of CR2 was 20.76% A, 30.49% T, 17.08% G, and 31.66% C. The A + T content (51.25%) was higher than the G + C content (48.74%).

Phylogenetic analyses

Phylogenetic analysis based on concatenated alignments of 13 PCGs of 15 species was carried out. The topology of the phylogenies reconstructed by BI and ML analyses was identical (Fig. 5). To gain insight into the phylogenetic interrelationships within Leiothrichidae, we obtained the concatenated nucleotide sequences of 13 PCGs from 14 species of Leiothrichidae, including five Pterorhinus, three Ianthocincla, one Garrulax, two Trochalopteron, two Leiothrix and one Liocichla.

Figure 5. 

Phylogenetic relationships of Leiothrichidae species determined using concatenated nucleotide sequences of 13 PCGs. Both BI and ML analyses produced identical tree topologies. Values at nodes are BI posterior probabilities and ML bootstrap values, respectively.

Within Leiothrichidae, we found that the phylogenetic relationships among the six genera were: Leiothrix and Liocichla were sister to the other four genera. Garrulax and the clade (Pterorhinus + Ianthocincla) formed a clade (posterior probability 1.0, 97% bootstrap support), and Leiothrix and Liocichla were sister taxa (posterior probability 1.0, 100% bootstrap support).

In addition, P. sannio was sister to P. chinensis (posterior probability 1.0, 94% bootstrap support). The (P. chinensis + P. sannio) and ((P. lanceolatus + P. pectoralis) + P. courtoisi) formed a well-supported clade (posterior probability 1.0, 100% bootstrap support). Meanwhile, our results showed that P. chinensis was distantly related to Garrulax.

Discussion

Mitogenome characteristics

In this study, the complete mitochondrial genome of P. chinensis was characterized for the first time. As in other species of Leiothrichidae, the mitogenome of P. chinensis consisted of 13 PCGs, two rRNAs, 22 tRNAs, and two control regions (Qian et al. 2013; Huan et al. 2016). Except for eight tRNAs (trnA, trnC, trnE, trnN, trnP, trnQ, trnS2 and trnY) and the nad6 gene, all the genes were coded on the H-strand, similar to that in most other vertebrates (Huan et al. 2016). Our study documents gene rearrangement in P. chinensis. Gibb et al. (2007) proposed the following nomenclature for avian mitochondrial gene orders: (1) Ancestral gene order; (2) remnant CR2 gene order; (3) duplicate CR gene order; and (4) duplicate tRNAThr-CR gene order. The mitogenome of P. chinensis includes two similar control regions and therefore we suggest that this represents a duplicate CR gene order. As in some other Passeriformes species, the two control regions (CR1 and CR2) were positioned between the trnT and trnF genes, and were separated by trnP, nad6, and trnE, with length of 664 and 679 bp, respectively (Huan et al. 2016). There were also intergenic spacers and overlaps between genes, as is seen in other birds (Dong et al. 2018).

The A + T content in the complete mitochondrial genome of P. chinensis was 52.71%, in line with the typical base bias of vertebrates (Huang and Zeng 2016). All PCGs of the mitogenome of P. chinensis were initiated with ATG, similar to those of most Passeriformes species (Huan et al. 2016). However, they were terminated with five types of stop codons, including TAA (nad1, nad2, cox2, atp8, atp6, nad3, nad4l, and cob), AGG (cox1), AGA (nad5), TAG (nad6), and incomplete stop codons T** (cox3 and nad4), as in as other species of Leiothrichidae (Qian et al. 2013; Huan et al. 2016). For the incomplete stop codons, the missing nucleotides may be the result of post-transcriptional polyadenylation, which is common in animal mitogenomes and could produce functional stop codons by polycistronic transcription cleavages and polyadenylation mechanisms (Ojala et al. 1981). The anticodons of all tRNAs in the mitogenome of P. chinensis were identical to those observed in most vertebrates (Sun et al. 2020). Furthermore, mismatched base pairs were identified in the stems of 22 different tRNAs, most of which are G-U pairs, which can form a weak bond in tRNAs and non-canonical pairs in tRNA secondary structures (Gutell et al. 2002). rRNAs includes 12S rRNA and 16S rRNA, with lengths of 987 bp and 1590 bp, respectively. As in most vertebrates, these rRNA genes were located between trnF and trnL2, and isolated by trnV (Lu et al. 2013).

Phylogenetic analyses

Mitochondrial sequences are widely used to infer phylogenetic relationships among vertebrate species (Anderson et al. 1981; Miya et al. 2001), including birds (e.g., Jønsson et al. 2019). In this study, we explored the phylogenetic relationship among members of Leiothrichidae based on 13 PCGs. Our study supports the phylogenetic relationships of ((((Pterorhinus + Ianthocincla) + Garrulax) + Trochalopteron) + (Leiothrix + Liocichla)). These results partially agree with the topologies inferred by Cibois et al. (2018) and Cai et al. (2019). The latter two agree with our findings that (1) Garrulax is sister to (Pterorhinus + Ianthocincla) and that (2) Leiothrix and Liocichla are more closely related to each other than either is to Garrulax, Pterorhinus or Ianthocincla. However, our results differ from those of Cibois et al. (2018) and Cai et al. (2019) in placing Trochalopteron closer to Garrulax, Pterorhinus or Ianthocincla than to Leiothrix and Liocichla, whereas Cibois et al. (2018) and Cai et al. (2019) inferred a closer relationship of Trochalopteron to Leiothrix and Liocichla than to Garrulax, Pterorhinus or Ianthocincla.

Our analyses support the reallocation of P. chinensis from the genus Garrulax to Pterorhinus, consistent with previous studies (Cibois et al. 2018; Cai et al. 2019). Currently, published mitochondrial genome data of species of Leiothrichidae are scarce. In order to better understand the phylogenetic relationships among Leiothrichidae, additional sequences of mitochondrial genomes are warranted.

Acknowledgments

We thank the reviewers for their valuable comments and suggestions to improve the manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the Science and Technology Project of Yunnan (202301BD070001082 and 202101AT070040) and Guizhou Provincial Science and Technology Foundation (Grant No. Qiankehe jichu [2020] 1Y080).

Author contributions

Methodology: QY. Software: GB, QG. Writing - original draft: QY, GB. Writing - review and editing: YD.

Data availability

All of the data that support the findings of this study are available in the main text.

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