Two Complete Mitochondrial Genomes of Leptobrachium (Anura: Megophryidae: Leptobrachiinae): Characteristics, Population Divergences, and Phylogenetic Implications

Abstract

The mustache toads Leptobrachium boringii and Leptobrachium liui are two attractive species in Megophryidae, in which adult males have mustache-like keratinized nuptial spines on their upper lip. However, both are under threat due to multiple factors, of which scientific studies are still very limited. In this study, two new complete mitochondrial genomes of L. boringii and L. liui were sequenced, assembled, and annotated based on next-generation sequencing. The mitogenome lengths of L. boringii and L. liui were found to be 17,100 and 17,501 bp, respectively, with both containing 13 protein coding genes, 23 tRNAs, 2 rRNAs, and 1 non-coding control region. Nucleotide diversity analyses indicate that atp8, atp6, and nad2 showed higher nucleotide diversity than cox1, cox3, and cytb. The intraspecific genetic distances among three different populations of L. boringii exceed 4%, and those between two populations of L. liui reach 7%. Phylogenetic relationships support their division into two subfamilies of Megophryidae (Leptobrachiinae and Megophryinae) as well as two species groups within Leptobrachium, corresponding to the number of keratinized nuptial spines (10–48 in the L. boringii species group vs. 2–6 in the L. liui species group). The two new mitogenomes reported in this study provide valuable data for future molecular evolutionary and conservation studies of the genus Leptobrachium and other Megophryidae toads.

1. Introduction

The toads of the family Megophryidae (Bonaparte, 1850) represent a group endemic to Asia and are found mainly distributed in tropical and subtropical forestry regions, ranging from northeast India, east Himalayas, eastward to South China, and southward to Southeast Asia [1]. For a long time, Megophryidae was known as Megophryinae, a subfamily within Pelobatidae (Bonaparte, 1850), until morphological studies showed that Megophryinae should be excluded from Pelobatidae based on skeletal structures; thus, it was suggested it be promoted to family level [2,3]. The validity and monophyly of Megophryidae has been supported by subsequent molecular phylogenetic studies [4,5]. At present, Megophryidae is widely recognized as an independent family that includes two subfamilies, the Leptobrachiinae and the Megophryinae, which comprise 179 and 129 species, respectively [6].

According to AmphibiaWeb (2023), Leptobrachium (Tschudi, 1838), the type genus of the subfamily Leptobrachiinae, includes 38 species of which 11 have been recorded in China, with 9 being endemic [7]. The toads in Leptobrachium prefer montane habitats in broad-leaf temperate forests at elevations of 700~1700 m, and they usually require slow-flowing streams with dense vegetation for breeding and forested terrestrial uplands to occupy outside the breeding season [7,8,9]. The taxonomy within the genus Leptobrachium is, however, debatable to some extent. It was originally divided into two subgenera, Vibrissaphora and Leptobrachium, according to whether or not adult males have keratinized nuptial spines [10,11]. There were also studies that further treated the spines in adult males as a basis for distinguishing Vibrissaphora and Leptobrachium as two distinct genera [7,12]. However, recent phylogenetic studies have proposed a single genus, Leptobrachium, including all species without subgeneric division, for the reason that Vibrissaphora species were not recovered as a single monophylum but dispersed within the lineage of Leptobrachium [8,13,14]. Leptobrachium species have a pair of large dark eyes with the upper half of the iris extraordinarily colored, such as sky-blue or green. Since these toads always hide in the daytime and come out at night, a study on their retina showed that the cell organization is principally adapted to enable sight at low light intensities [15]. This characteristic also seems to be useful for species identification and may bear phylogenetic signals; however, more data support for this speculation is still required [14]. Another unique aspect of the genus is its choice of cold season for breeding, whereas most frogs and toads breed in warmer months [16].

Leptobrachium boringii (Liu, 1945), known as the Emei mustache toad, is an attractive species in the genus. It was named after the mustache the males use as weapon for fighting during the breeding seasons, which is actually small keratinized nuptial spines growing on the upper lip [17]. The males are aggressive toward each other and outgrow the females to also aid in male–male combat, which is pretty rare in anurans [18]. Studies on reproductive behavior have indicated that males provide paternal care and larger males have higher mating success [19,20]. Both male combat and paternal care have been suggested to have adaptive significance in competing for nests, improving mating opportunities, and offspring survival [18,19,21]. Adult males of another species, L. liui (Pope, 1947), also have keratinized spines (mustache) on the upper lip but less in number than L. boringii (2–4 vs. 10–16) [22]. The distribution ranges of L. liui and L. boringii are basically separate but overlap at a few boundary areas (Figure 1). Leptobrachium liui is known as having two subspecies, L. liui liui and L. liui yaoshanensis, with two and four keratinized spines, respectively [22].

Although most species in the Leptobrachium genus are only found very narrowly distributed in a few regions, L. boringii and L. liui are two exceptions that are found over a relatively large area. The current known populations of L. boringii are in Chongqing, Guangxi, Guizhou, Hunan, Yunnan, and Sichuan Provinces, and populations of L. liui are recorded in the Fujian, Zhejiang, Jiangxi, Hunan, Guangxi, and Guangdong Provinces of China (Figure 1; [7]). Although having relatively wide distribution, many populations of the two species are undergoing rapid decline due to over-catching, habitat loss, and degradation, among other factors [7,9]. The tadpoles of both L. boringii and L. liui take about four months to hatch and usually require three years to complete metamorphosis, which aggravates their chances of survival [22,23]. At present, L. boringii is classified as “Endangered” in the IUCN Red List of Threatened Species [24] and listed in the second class of National Key Protected Wild Animals of China [25], while L. liui was classified as “Least Concern” on the IUCN Red List of Threatened Species [26].

Speciation of amphibians has been driven by environmental factors and physiological adaptations during their short-term life cycle and long-term evolutionary history, which have shaped their current distribution and diversity patterns, showing similar characteristics or unique traits in morphology [22]. With more than 300 species in Megophryidae, a robust phylogenetic relationship reconstructed using mitogenomes would provide a basic framework to understand the phylogeny and trait evolution of this group. However, the available mitogenome data are still very limited. For instance, only 18 complete and 9 nearly complete mitogenomes of Megophryidae are available from NCBI to date. As for L. boringii, although it is relatively widely distributed, details of its population diversity and genetic differentiation are still largely unknown. There are two previous studies reporting the mitogenomes of L. boringii from two isolated populations: the Emei Mountains (EM) in Sichuan Province and Pengshui County (PS) in Chongqing Province [27,28]; however, these two mitogenomes were obtained using a traditional PCR-targeted sequencing method, and the sequences were incomplete. When it comes to L. liui, there was also a mitogenome reported from the population of the Jiulongshan National Nature Reserve (JLS) in Zhejiang Province. Similarly to L. boringii, however, it was only briefly described in the paper [29]. In this study, using next-generation sequencing (NGS) technology, we report two newly obtained complete mitogenomes of the genus Leptobrachium, one from L. boringii based on its easternmost population from the National Nature Reserve of Badagongshan in Sangzhi County (SZ) and the other from L. liui based on its northernmost population from the Zhangjiajie National Forest Park in Wulingyuan District (WLY). Both Sangzhi County and Wulingyuan District belong to Zhangjiajie City in Hunan Province and, interestingly, represent a boundary area of L. boringii and L. liu here (Figure 1). This study aims to: (1) analyze and describe the characteristics of the updated complete mitogenomes of L. boringii and L. liui in detail, (2) compare the genetic distances among the three representative populations of L. boringii and the two populations of L. liui, and (3) reconstruct the phylogenetic relationships of Megophryidae and present implications of this evolutionarily interesting group.

2. Materials and Methods

2.1. Sample Collection and Sequencing

The sample of L. boringii was collected in May 2021 from National Nature Reserve of Badagongshan, Sangzhi County, and the sample of L. liui was collected in September 2021 from Zhangjiajie National Forest Park, Wulingyuan District, both in Zhangjiajie City in Hunan Province, China. Permissions of the field survey for scientific purposes were approved by the local administration, and the collection of toads used in this study complied with the Wildlife Protection Law of China. According to the “3R principle” (Reduction, Replacement, and Refinement) of animal sampling, only one sample of each species was used in this study. All procedures of animal collection and treatment comply with the guidance of the Code of Practice for the Housing and Care of Animals. The specimens were euthanized and preserved in 95% alcohol as reference specimens, with a small liver sample used for molecular analysis. DNA extraction was conducted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), and a DNA library was then constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Nanjing, China). High-throughput sequencing was performed in paired-end mode on the DNBSEQ-T7 platform (Complete Genomics and MGI Tech, Shenzhen, China), generating approximately 30 Gb of raw reads of 150 bp read length.

2.2. Sequence Assembly, Annotation, and Analysis

The complete mitogenomes of L. boringii and L. liui were assembled by NOVOPlasty 4.3 [30] based on the raw reads produced through NGS, both using the cytb gene (1141 bp) as seed sequences, from the previous reported mitogenome of L. boringii (KJ630505). Then, the positions and directions of protein coding genes (PCGs), ribosomal RNA genes (rRNAs), transfer RNA genes (tRNAs), and the control region (D-loop) were annotated using MITOS Web Server [31]. The sites and secondary structure of tRNAs were further inferred with the help of ARWEN and Scan-SE [32]. Visualization and circularization of the complete mitogenome were performed through the online server GeSeq [33]. Other analyses, such as nucleotide composition, AT and GC skew, and the determination of sequence genetic distances under Kimura’s two-parameter (K2P) model [34], were conducted through MEGA X [35]. Relative synonymous codon usage (RSCU), nucleotide diversity (Pi), and the ratio of the nonsynonymous substitution rate (Ka) and the synonymous substitution rate (Ks) were all calculated through DnaSP 6 [36].

2.3. Phylogenetic Analysis

The sequences used for phylogenetic reconstruction consisted of L. boringii (SZ population) and L. liui (WLY population) sequences we obtained in this study and another 27 mitogenomes that were downloaded from NCBI, including 2 mitogenomes of L. boringii (EM and PS populations), 1 mitogenome of L. liui (JLS population), and 24 mitogenomes of other 21 representative species within Megophryidae. Microhyla fissipes in the Microhylidae family was used as the outgroup. Each of the 13 PCGs was extracted from the dataset of 30 mitogenomes and manually checked, and all PCGs were then aligned using the inbuilt MUSCLE module in MEGA X and concatenated to make a combined PCG dataset. To uncover the population status and phylogenetic position of L. boringii and L. liui, the phylogenetic analyses were reconstructed using both the maximum likelihood (ML) method conducted in RAxML 8.0.2 [37] and the Bayesian inference (BI) method via MrBayes 3.2.7 [38]. Partitioning scheme and nucleotide substitution models for ML and BI phylogenetic analyses were selected using PartitionFinder 2 [39] based on the Akaike information criterion (AIC). The statistical confidence was assessed through a bootstrap test with 1000 replicates in ML trees and posterior probability calculation of the BI trees that under simultaneously run for 1.0 × 10⁷ million generations, with sampling conducted every 1000 generations and discarding the initial 25% generations.

3. Results

3.1. Mitogenome Annotation and Nucleotide Composition

The assembled complete mitogenomes of L. boringii and L. liui were 17,100 and 17,501 bp in length, which were deposited in NCBI under accession numbers OP373724 and OP503540, respectively. The mitogenomes of both species had a typical gene organization, with 13 PCGs, 2 rRNAs, 23 tRNAs, and a D-loop region (

Table 1. Characteristics of the mitogenome of L. boringii (LB) and L. liui (LL).

Gene	Position		Length (bp)		Start Codon		Stop Codon		Anti Codon	Strand *	Intergenic Nucleotide #
	LB	LL	LB	LL	LB	LL	LB	LL			LB	LL
tRNAPhe	1–67	1–67	67	67					GAA	H
12S rRNA	68–1007	68–1004	940	937						H
tRNAVal	1004–1072	1001–1069	69	69					TAC	H	−4	−4
16S rRNA	1083–2662	1099–2661	1580	1563						H	10	29
tRNALeu	2661–2735	2660–2734	75	75					TAA	H	−2	−2
nad1	2736–3709	2735–3708	974	974	ATG	ATG	TA+	TA+		H
tRNAIle	3712–3782	3711–3781	71	71					CAT	H	2	2
tRNAGln	3782–3852	3781–3851	71	71					TTG	L	−1	−1
tRNAMet1	3852–3920	3851–3919	69	69					CAT	H	−1	−1
tRNAMet2	4029–4085	4019–4086	57	68					CAT	H	108	99
nad2	4096–5139	4087–5130	1044	1044	ATG	ATG	TAA	TAA		H	10
tRNAAla	5144–5213	5135–5204	70	70					TGC	L	4	4
tRNAAsn	5214–5286	5205–5277	73	73					GTT	L
NCR	5289–5316	5280–5307	28	28						H	2	2
tRNACys	5316–5379	5307–5370	64	64					GCA	L	−1	−1
tRNATyr	5380–5449	5371–5440	70	70					GTA	L
cox1	5451–7013	5442–7004	1563	1563	GTG	GTG	TAA	TAA		H	1	1
tRNASer	7011–7081	7002–7072	71	71					TGA	L	−3	−3
tRNAAsp	7086–7153	7077–7144	68	68					GTC	H	4	4
cox2	7154–7841	7145–7832	688	688	ATG	ATG	T++	T++		H
tRNALys	7842–7915	7833–7906	74	74					TTT	H
atp8	7916–8101	7907–8071	186	165	GTG	GTG	TAA	CAT		H
atp6	8092–8774	8083–8765	683	683	ATG	ATG	TA+	TA+		H	−10	11
cox3	8774–9557	8765–9548	784	784	ATG	ATG	T++	T++		H	−1	−1
tRNAGly	9558–9626	9549–9617	69	69					TCC	H
nad3	9627–9971	9618–9962	345	345	GTG	GTG	TAA	TAA		H
tRNAArg	9970–10,038	9961–10,029	69	69					TCG	H	−2	−2
nad4l	10,039–10,335	10,030–10,326	297	297	ATG	ATG	TAA	TAA		H
nad4	10,329–11,706	10,320–11,697	1378	1378	ATG	GTG	T++	T++		H	−7	−7
tRNAHis	11,707–11,775	11,698–11,766	69	69					GTG	H
tRNASer	11,776–11,842	11,767–11,833	67	67					GCT	H
tRNALeu	11,843–11,915	11,834–11,906	73	73					TAG	H
nad5	11,947–13,779	11,908–13,737	1833	1830	ATG	ATG	TAA	AGG		H	31	1
nad6	13,752–14,261	13,743–14,252	510	510	ATG	ATG	AGG	AGG		L	−28	5
tRNAGlu	14,262–14,330	14,253–14,321	69	69					TTC	L
cytb	14,335–15,475	14,326–15,466	1141	1141	ATG	ATG	T++	T++		H	4	4
tRNAThr	15,476–15,545	15,467–15,536	70	70					TGT	H
tRNAPro	15,548–15,616	15,539–15,607	69	69					TGG	L	2	2
tRNATrp	15,626–15,694	15,618–15,686	69	69					TCA	H	9	10
D-loop	15,695–17,100	15,687–17,501	1406	1815						H	0	0

* H and L indicate genes transcribed on the heavy and light strand, respectively. ^# Positive numbers correspond to the nucleotides separating adjacent genes; negative numbers indicate overlapping nucleotides.

; Figure 2). There was a total of 187 and 174 bp of intergenic nucleotides (IGNs) dispersed in 12 and 13 locations of two species, ranging from 1 to 108 bp and 1 to 99 bp in length, respectively (Table 1). Similar to other species in Megophryidae, the overall base composition of L. boringii was 31.5% T, 25.5% C, 15.3% G, and 27.7% A, while that of L. liui was 32.7% T, 24.3% C, 14.8% G, and 28.1% A (

Table 2. Base composition (in percentages) of the mitogenomes of three populations of L. boringii, two populations of L. liui, and another 21 species in Megophryidae.

Species	Total Length (bp)	T%	C%	G%	A%	A+T%	AT Skew	GC Skew	Accession Number
L. boringii (SZ)	17,100	31.5	25.5	15.3	27.7	59.2	−0.064	−0.250	OP373724 *
L. boringii (EM)	17,085	31.5	25.5	15.4	27.6	59.1	−0.066	−0.247	KJ630505
L. boringii (PS)	16,557	31.6	25.5	15.2	27.7	59.3	−0.066	−0.253	MH643882
L. liui (WLY)	17,501	32.7	24.3	14.8	28.1	60.8	−0.076	−0.243	OP503540 *
L. liui (JLS)	17,190	32.6	24.4	14.9	28.1	60.7	−0.074	−0.242	MW429348
Oreolalax major	17,786	32.6	24.3	14.3	28.7	61.3	−0.064	−0.259	MN803320
O. major	15,469	32.1	24.7	14.4	28.7	60.8	−0.056	−0.263	KU310894
O. major	17,431	32.4	24.5	14.4	28.8	61.2	−0.059	−0.260	KU127230
Oreolalax xiangchengensis	17,110	33.0	23.6	14.2	29.2	62.2	−0.061	−0.249	MH727696
Oreolalax jingdongensis	17,864	32.7	23.9	14.3	29.1	61.8	−0.058	−0.251	MF953479
Oreolalax omeimontis	17,675	32.6	25.0	14.0	28.5	61.1	−0.067	−0.282	MN803321
Oreolalax multipunctatus	17,358	32.0	24.2	14.3	28.5	60.5	−0.058	−0.257	MF966382
Oreolalax lichuanensis	17,702	32.2	24.9	15.0	28.0	60.2	−0.070	−0.248	KU096847
Oreolalax schmidti	18,481	32.8	24.5	14.4	28.3	61.1	−0.074	−0.260	MT773151
Oreolalax rhodostigmatus	18,676	32.4	24.9	14.7	28.0	60.4	−0.073	−0.258	MF770485
Leptobrachium ailaonicum	17,318	31.8	25.0	15.3	27.9	59.7	−0.065	−0.241	MZ394043
Leptobrachium leishanense	17,485	32.6	24.4	14.9	28.1	60.7	−0.074	−0.242	KU760082
Scutiger ningshanensis	17,265	32.8	24.2	14.0	29.1	61.9	−0.060	−0.267	KX619450
S. ningshanensis	16,799	32.6	24.3	14.3	28.8	61.4	−0.062	−0.259	KX352260
Scutiger liupanensis	16,890	32.2	24.9	14.5	28.4	60.6	−0.063	−0.264	KX352261
Leptobrachella oshanensis	17,747	29.9	26.3	15.2	28.8	58.7	−0.019	−0.267	KC460337
Leptobrachella alpina	17,763	30.8	25.6	15.1	28.5	59.3	−0.039	−0.258	MW487804
Leptobrachella pelodytoides	14,682	29.1	27.8	15.5	27.7	56.8	−0.025	−0.284	JX564874
Atympanophrys shapingensis	17,631	31.5	26.0	14.3	28.2	59.7	−0.055	−0.290	JX458090
Megophrys gigantica	18,259	32.1	25.2	14.3	28.4	60.5	−0.061	−0.276	MZ364157
Brachytarsophrys carinense	15,271	29.8	27.6	15.1	27.5	57.3	−0.040	−0.293	JX564854
Boulenophrys jingganggensis	17,263	31.6	26.3	14.5	27.6	59.2	−0.068	−0.289	MT683772
Boulenophrys pingjianggensis	17,866	32.0	25.9	14.3	27.8	59.8	−0.070	−0.289	KT601071
Boulenophrys omeimontis	17,013	31.8	25.7	14.2	28.3	60.1	−0.058	−0.288	KP728257

* the sequence obtained in this study.

), indicating an A+T bias with greater A+T than G+C content (59.2% vs. 40.8% and 60.8% vs. 39.1%). Additionally, both the AT and GC skew were negative for the mitogenomes of all Megophryidae species, reflecting a general bias toward T and C base pairs (Table 2).

3.2. Characteristics of rRNAs, tRNAs, and the Control Region

In both L. boringii and L. liui, there were 23 tRNAs interspersed in the whole mitogenome, ranging from 64 to 75 bp, with tRNA^Cys being the shortest and tRNA^Leu the longest. Similarly to other congeners in the genus Leptobrachium, there was also a tandem duplication of tRNA^Met that was separated by long IGNs (intergenic nucleotides, 108 bp in L. boringii and 99 bp in L. liui). There were two rRNAs with a total length of 2520 bp in L. boringii and 2500 bp in L. liui. The 16S rRNA was located between tRNA^Val and tRNA^Leu with a length of 1580 bp in L. boringii and 1563 bp in L. liui, whereas the 12S rRNA was located between tRNA^Phe and tRNA^Val with 940 bp in L. boringii and 937 bp in L. liui. The non-coding domain of D-loop was located between tRNA^Trp and tRNA^Phe, with a length of 1406 bp in L. boringii and 1815 bp in L. liui (Table 1). For both L. boringii and L. liui, most genes were encoded on the heavy (H) strand, except for the nad6 and eight tRNA genes (tRNA^Gln, tRNA^Ala, tRNA^Asn, tRNA^Cys, tRNA^Tyr, tRNA^Ser, tRNA^Glu, and tRNA^Pro) encoded on the light (L) strand.

Among all the 13 PCGs, the shortest was the atp8 gene, being only 186 bp in L. boringii and 165 bp in L. liui, while the longest was nad5 at 1833 bp in L. boringii and 1830 bp in L. liui. Three PCGs (cox1, atp8, nad3) started with GTG, while the other ten PCGs used ATG as the initiation codon in L. boringii; however, in L. liui, by comparison, there was one more PCG (nad4) starting with GTG, and the remaining nine PCGs start with ATG. The termination codon usage in L. boringii and L. liui was diverse: complete codons were found to be used in seven PCGs (TAA for nad2, cox1, nad3, nad4l, and nad5; TGA for atp8; AGG for nad6) and incomplete codons by the six other PCGs (TA+ for nad1 and atp6; T++ for cox2, cox3, nd4, and cytb) in L. boringii, while in L. liui, a difference appeared in the usage of complete termination codons (TAA for nad2, cox1, nad3, and nad4l; CAT for atp8; AGG for nad5 and nad6). Nucleotide composition analysis of the 13 PCGs revealed that they shared similar patterns, except for the nad6 gene, which had unusual base proportions of T (35.1%), C (12.7%), G (33.7%), and A (18.4%) in L. boringii and T (35.5%), C (12.9%), G (32.9%), and A (18.6%) in L. liui, respectively; thus, the nad6 gene presents an extraordinary but positive GC skew (

Table 3. Nucleotide composition and skewness of different regions in the mitogenomes of L. boringii (LB) and L. liui (LL).

	Length (bp)		T%		C%		G%		A%		A+T%		AT Skew		GC Skew
	LB	LL	LB	LL	LB	LL	LB	LL	LB	LL	LB	LL	LB	LL	LB	LL
nad1	974	974	34.7	35.3	24.8	23.8	14.8	14.5	25.7	26.4	60.4	61.7	−0.149	−0.145	−0.253	−0.244
nad2	1044	1044	32.3	32.6	29.7	29.4	12.1	12.2	26.0	25.9	58.3	58.4	−0.108	−0.115	−0.421	−0.415
cox1	1563	1563	31.6	33.1	24.7	22.5	18.0	17.8	25.7	26.6	57.3	59.7	−0.103	−0.110	−0.157	−0.117
cox2	688	688	31.4	32.6	25.4	24.3	15.7	15.4	27.5	27.8	58.9	60.3	−0.065	−0.080	−0.236	−0.223
atp8	186	165	38.7	43.6	24.2	20.6	10.2	9.7	26.9	26.1	65.6	69.7	−0.180	−0.252	−0.407	−0.360
atp6	683	683	35.7	36.0	26.2	25.3	13.6	12.0	24.5	26.6	60.2	62.7	−0.186	−0.150	−0.317	−0.357
cox3	784	784	33.9	33.8	25.1	25.8	16.7	16.8	24.2	23.6	58.1	57.4	−0.167	−0.178	−0.201	−0.210
nad3	345	345	38.3	39.1	25.2	23.8	15.4	14.2	21.2	22.9	59.5	62.0	−0.287	−0.262	−0.241	−0.252
nad4l	297	297	35.4	37.4	27.9	25.9	12.8	12.8	23.9	23.9	59.3	61.3	−0.194	−0.220	−0.371	−0.339
nad4	1378	1378	33.4	35.2	28.4	26.1	12.7	12.3	25.5	26.4	58.9	61.6	−0.134	−0.143	−0.382	−0.357
nad5	1833	1830	33.8	34.8	26.0	25.0	13.1	12.9	27.0	27.4	60.8	62.1	−0.112	−0.119	−0.330	−0.319
nad6	510	510	35.1	35.5	12.7	12.9	33.7	32.9	18.4	18.6	53.5	54.1	−0.312	−0.312	0.453	0.436
cytb	1141	1141	34.1	35.1	27.0	26.1	14.7	13.7	24.2	25.1	58.3	60.2	−0.170	−0.167	−0.295	−0.313
PCGs	11,426	11,402	33.7	34.7	25.8	24.6	15.3	14.9	25.2	25.8	58.9	60.5	−0.144	−0.148	−0.255	−0.246
PCGs–1st	3809	3801	33.9	35.0	27.0	26.0	14.9	14.3	24.2	24.6	58.1	59.6	−0.167	−0.174	−0.289	−0.290
PCGs–2nd	3809	3801	34.7	35.4	26.0	24.9	14.2	14.3	25.2	25.4	59.9	60.8	−0.159	−0.164	−0.294	−0.270
PCGs–3rd	3808	3800	32.6	33.7	24.3	22.9	16.8	16.1	26.2	27.3	58.8	61.0	−0.109	−0.105	−0.182	−0.174
12S rRNA	940	937	25.0	25.5	23.7	23.5	19.9	19.9	31.4	31.2	56.4	56.7	0.113	0.100	−0.087	−0.084
16S rRNA	1580	1563	27.0	27.2	21.8	21.8	17.5	17.1	33.7	34.0	60.7	61.2	0.110	0.111	−0.109	−0.120
rRNAs	2520	2500	26.3	26.6	22.5	22.4	18.2	18.1	33.0	32.9	59.3	59.5	0.113	0.107	−0.106	−0.106
tRNAs	1536	1536	28.6	28.6	20.4	20.8	22.1	21.7	28.8	29.0	57.4	57.6	0.003	0.007	0.040	0.021
D-loop	1406	1815	35.1	38.8	21.2	19.4	13.8	12.8	29.9	29.0	65.0	67.8	−0.080	−0.145	−0.211	−0.205
Mitogenome	17,100	17,501	31.5	32.7	25.5	24.3	15.3	14.8	27.7	28.1	59.2	60.8	−0.064	−0.076	−0.250	−0.242

). There were several distinct but overlapping sites found in the mitogenomes of the two species: in L. boringii, these sites ranged from 1 to 28 bp and overlapped at 10 positions, occupying a total length of 50 bp, with the longest overlap (28 bp) located between the nad5 and nad6 genes; in L. liui, the overlapping sites were found dispersed in nine positions that range from 1 to 7 bp and a total length of 22 bp, with the longest (7 bp) lying between nad4l and nad4.

3.3. Codon Usage and Genetic Distances

The codon usage of the PCGs showed an extremely similar pattern that was shared between L. boringii and L. liui (Figure 3). The UCU (Ser1), CGA (Arg), UUA (Leu), and CAA (Gln) had the highest frequencies in both L. boringii and L. liui. RSCU analysis of both species indicated that six amino acids (Val, Pro, Thr, Ala, Arg, and Gly) were encoded by four synonymous codons, with the exceptions being Leu and Ser, encoded by six codons, and all others (Phe, Ile, Met, Tyr, His, Gln, Asn, Lys, Asp, Glu, Cys, and Trp) were encoded by two codons (Figure 3). Ka was generally less than Ks, and the average values between species pairs within 21 Megophryidae species ranged from 0 to 0.686 for Ka and from 0 to 6.2922 for Ks (Figure 4A). The Ka/Ks ratios of all the 13 PCGs were less than 1, with the highest Ka/Ks ratio (0.675) in atp8 (Figure 4B). None of the PCGs showed Ka/Ks ≥ 1, which indicates a generally negative or purifying selection. The atp8, atp6, and nad2 genes were found to have relatively fast evolutionary rates, whereas the cox1, cox3, and cytb genes have a relatively slow evolutionary rate.

The overall genetic distances of PCGs between two of the three populations of L. boringii were 4.82% (EM and PS), 4.22% (EM and SZ), and 4.19% (PS and SZ). While looking at each of the PCGs, atp8 had the highest genetic variance, followed by cox2, cox1, and nad2, and cox3 had the lowest genetic variance among the three populations (Figure 5A). As for L. liui, the overall genetic distance of PCGs between the two populations (JLS and WLY) was 7.09%, in which atp8 and nad4l had the highest and the lowest genetic variances, respectively (Figure 5A). In addition, when using the sliding window analysis along the concatenated PCG dataset, we found that the nucleotide diversity (Pi) was also highly variable among different gene sequences, ranging from 0.198 (cox1) to 0.352 (atp8) of 13 PCGs within the 21 species. Generally, the atp8, atp6, and nad2 genes had relatively high nucleotide diversity, while the cox1, cox2, and cox3 genes had comparatively low nucleotide diversity, no matter the examined sequences, which were grouped from (1) three populations of L. boringii and two populations of L. liui (Figure 5B), (2) four subgroups of Clade II (Figure 5C), or two major clades of Megophryidae (Figure 5D).

3.4. Phylogenetic Analysis

The phylogenetic trees based on both ML and BI methods showed highly consistent topologies, with strong support values in most branches (Figure 6). The genus Leptobrachium was divided into two subgroups. L. boringii was recovered into the sister group of L. ailaonicum, and the three samples of L. boringii formed a monophyly, while the PS population branched first and the EM population and SZ population followed (obtained in this study). The two populations (WLY and JLS) of L. liui formed a monophylum and were recovered as the sister species of L. leishanese. According to the phylogenetic tree, the Megophryidae can be divided into two major clades corresponding to two subfamilies. Clade I (the subfamily Megophryinae) contains species of the following four genera: Atympanophrys, Megophrys, Brachytarsophrys, and Boulenophrys. Clade II (the subfamily Leptobrachiinae) can be further divided into four well-supported major groups in the following order: Leptobrachella (Group A) first, followed by Scutiger (Group B), then the sister groups Oreolalax (Group C) and Leptobrachium (Group D) (Figure 6).

4. Discussion

Mitochondrial DNA serves as an efficient molecular marker that has been widely applied in evolution-related studies for a variety of species [40]. Compared with relatively abundant and complex nuclear DNA, the mitochondrial genome has many favorable features, such as simple structure with conserved coding regions, low levels of recombination, multiple copy number, rapid evolutionary rate, and maternal inheritance [40,41]. Therefore, mitochondrial DNA has played a valuable role in reconstructing phylogenetic relationships, revealing population genetic structures, estimating divergence times, identifying relatedness between recently diverged species, etc. [29,42,43,44,45].

The traditional method for obtaining a mitogenome is the PCR-targeted method based on chain termination sequencing [46], which has greatly promoted the development of relevant studies. Although the PCR-targeted method is simple to operate, it is time-consuming and primer-dependent, and the mitogenomes combined based on different DNA fragments were usually uncomplete [27,28]. In recent years, the advent of next-generation sequencing (NGS) technology has revolutionized biological studies for producing thousands or even millions of DNA reads within a short period [47,48]. With the ever-increasing throughput and ever-decreasing costs with the development of NGS, more and more complete mitochondrial genomes have been reported and used as essential sources and optimal molecular markers for studies on evolution, phylogenetics, population genetics, and biological conservation [29,43,48,49,50,51]. Although previous studies [27,28] have already reported the mitogenomes of L. boringii based on the traditional PCR-targeted method, these mitogenomes were not as complete as what we assembled based on NGS in this study. Nevertheless, the gene organization, base composition pattern, and transcriptional direction were almost the same among the three mitogenomes of L. boringii, and they were also similar to those of other congeners [29,52], including L. liui that we sequenced and assembled in this study (Table 1, Table 2 and Table 3, Figure 2).

As a result of this study, we report and describe the characteristics of two newly sequenced mitogenomes of L. boringii and L. liui in more detail. For instance, codon usage bias is a phenomenon in which specific codons are used more frequently than other synonymous codons by certain organisms during the translation of genes to proteins. With rapid progress in whole-genome sequencing, analysis of codon usage bias at the genome level, rather than for a single gene or a set of genes, has been increasing [42]. The results of RSCU analyses indicate that in both L. boringii and L. liui, codons with A or T at the third position are always overused compared with other synonymous codons (Figure 3). The biased usage of AT nucleotides is also reflected in the form of codon usage, where the highly frequently used codons, UCU (Ser1), CGA (Arg), UUA (Leu), and CAA (Gln), all end with A or T, which might also partly contribute to the higher A+T content.

The positive selection analyses based on the PCGs of mitogenome can shed light on the question of whether natural selection affected the functional characteristics of the mitochondrion [53]. The ratio of Ka and Ks is a popular proxy to detect adaptive evolution from viruses to humans [54]. It is considered that Ka/Ks > 1 indicates positive selection, Ka/Ks = 1 neutrality, and Ka/Ks < 1 negative or purifying selection [55]. The Ka/Ks value from each of the PCGs within Megophryidae was less than 1 (Figure 4), which indicates that the overall evolution pattern of the mitogenomes of Megophryidae tend to be conservative in maintaining the functions of regularly generated proteins [56]. Interestingly, the Ka/Ks of each of the PCGs indicates that the evolutionary rates are relatively fast for atp8, atp6, and nad2 and relatively slow for cox1, cox3, and cytb (Figure 4). This pattern was also revealed in the nucleotide diversity analyses based on different PCGs or from different examined groups under sliding windows (Figure 5).

The previously known mitogenomes of L. boringii and L. liui from other populations [27,28,29], and two newly sequenced mitogenomes in this study, together, provided us with an excellent opportunity to examine the intraspecific and interspecific genetic distances among the species of Leptobrachium. The intraspecific genetic distances of L. boringii were generally greater than 4%, which means the three examined populations are highly differentiated. This trend was also revealed in other studies. For instance, Yang et al. [57] found that L. boringii in the Sichuan and Hunan Provinces had significant genetic differentiation with relatively poor gene flow, and the genetic distance between them was 4.7% based on 1097 bp of cytb genes. Interestingly, our study further showed that the differentiation pattern of L. boringii is not straightforward along the current geographic distances. The PS population (in Chongqing Province) was between the EM (in Sichuan Province) and SZ population (in Hunan Province) from the geographic point of view; however, it divided first and allowed the remaining far-distanced EM and SZ populations to group together in the phylogenetic tree (Figure 6). This suggests the phylogeographic pattern of L. boringii among its ranges is shaped by multiple factors. Comparatively, the intraspecific genetic distance between the two populations of L. liui was even greater (7.09%) and almost reached that of the interspecific genetic distance between L. liui and L. leishanensis (7.85%). Although we could identify that the sample we sequenced was a subspecies (L. liui yaoshanensis) due to its four keratinized spines [22], we were unable to identify whether or not the JLS population was from another subspecies (L. liui liui) because no morphological traits were mentioned in the literature [29] and, additionally, whether the JLS population was in the boundary area of two subspecies. Even so, our results show that the intraspecific genetic distance of L. liui is substantially high; thus, further taxonomic studies are of worth. The interspecific genetic distance between L. boringii and L. liui was relatively high, as they were found in two different subgroups of Leptobrachium (Figure 5), which is consistent with another study based on 2 mitochondrial DNA markers (cytb and nad4) and 11 microsatellite loci [58].

While using 27 mitogenome sequences available from NCBI and 2 newly obtained in this study as the ingroups, with Microhyla fissipes in the Microhylidae family as the outgroup, a phylogenetic tree within Megophryidae based on mitogenomes was able to be reconstructed in this study (Figure 5). The family Megophryidae was divided into two clades (Clade I and II) corresponding to two subfamilies, Megophryinae and Leptobrachiinae, respectively. Although the species involved here are still limited, the division of two subfamilies is supported. Leptobrachiinae was divided into four groups corresponding to four genera and well supported based on the values: the genus Leptobrachella divided first and then Scutiger, followed by the sister group of Leptobrachium and Oreolalax. However, this is not consistent with a previous study based on three mitochondrial and nine nuclear genes, which showed that Leptobrachella divided first, followed by Leptobrachium, leaving Scutiger and Oreolalax to form a sister group [5]. This is a reminder that the intergeneric phylogeny within Leptobrachiinae requires further study for verification. When it comes to the phylogenetic relationships within Leptobrachium, our study shows that two lineages can be distinguished: one with L. boringii and L. ailaonicum, and the other one formed by L. liui and L. leishanense. The relationship of the above two lineages is consistent with the traditional distinction of two species groups according to morphological characteristics, especially the number of keratinized spines (10–48 in L. boringii species group vs. 2–6 in L. liui species group, [22]). However, this relationship is not well supported by previous studies using relatively limited mitochondrial DNA sequences. For instance, Rao et al. [8] and Matsui et al. [14] revealed a (L. ailaonicum, (L. boringii, (L. liui, L. leishanense))) relationship based on 16S rRNA, nad4, and cytb (a total of 2566 bp), and 12S rRNA, tRNA^Val, and 16S rRNA (a total of 2009 bp), respectively. Chen et al. [59] recovered a (L. boringii, (L. ailaonicum, (L. liui, L. leishanense))) relationship based on 1914 bp of 12S rRNA, tRNA^Val, and 16S rRNA sequences. Although the phylogenetic relationships would change along with the differences in the DNA markers used, it has been generally recognized that the use of a complete mitogenome is superior to individual genes in uncovering evolutionary relationships [60,61]. However, it has to be noted that the bootstrap value of the node of L. ailaonicum and L. boringii in our study was still not so supportive (only 70), which could also be treated and collapsed as an unresolved polytomy with a (L. ailaonicum, L. boringii (L. liui, L. leishanense)) relationship. In a word, the whole picture of the evolutionary history within the groups of Megophryidae is still as presented until more and more mitogenome data, such as for the two species reported in this study, become available in the future.