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. 2024 Jul 10;14(7):e11687. doi: 10.1002/ece3.11687

Insights into phylogenetic positions and distribution patterns: Complete mitogenomes of two sympatric Asian horned toads in Boulenophrys (Anura: Megophryidae)

Hongmei Xiang 1, Qiang Zhou 1,2, Wei Li 3, Juan Shu 3, Zhirong Gu 4, Wansheng Jiang 1,2,✉
PMCID: PMC11237341  PMID: 38994208

Abstract

Boulenophrys sangzhiensis and Boulenophrys tuberogranulata, two narrow‐distributed toad species within the Megophryidae family in southern China, are experiencing population declines due to habitat loss and degradation. Despite their critical conservation status, the two species remain largely overlooked in public and scientific spheres. This study presented the first sequencing, assembly, and annotation of the complete mitogenomes of both species using next‐generation sequencing. The mitogenome of B. sangzhiensis was 16,950 bp, while that of B. tuberogranulata was 16,841 bp, each comprising 13 protein‐coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a noncoding control region (D‐loop). The gene content, nucleotide composition, and evolutionary rates of each mitogenome were analyzed. Both mitogenomes exhibited negative AT skew and GC skew with high A + T content. ATP8 exhibited the highest evolutionary rate, while COI had the lowest. A phylogenetic analysis based on 28 mitogenomes revealed two major clades of Megophryidae, supporting the classification of two subfamilies, Megophryinae and Leptobrachiinae. Within the subfamily Megophryinae, the genus Boulenophrys was divided into two species groups. Intriguingly, despite coexisting in Zhangjiajie City, B. sangzhiensis and B. tuberogranulata exhibited distinct origins from the two different species groups, underscoring the unique role of the coexisting area Zhangjiajie in driving their speciation and preserving their current populations. A parallel pattern was also identified in the Leptobrachiinae genus Leptobrachium within the same region. This study provided valuable data references and enhanced our understanding of the molecular characteristics of these threatened amphibian species.

Keywords: Asian horned toad, China, Megophryidae, mitochondrial genome, phylogenetic analysis

The mitogenomes of two endemic and threatened Asian horned toads, Boulenophrys sangzhiensis and Boulenophrys tuberogranulata, were sequenced and reported for the first time. A phylogenetic reconstruction based on all available mitogenomes within Megophryidae revealed a very special phylogenetic and distributional pattern of the two sympatric species relative to other congeners in Boulenophrys.

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1. INTRODUCTION

The toad family Megophryidae (Bonaparte, 1850) is an Asian endemic group that primarily distributed from eastern Pakistan to western China, and southward to the Philippines and the Greater Sunda Islands (Frost, 2023). Although the taxonomic validity of Megophryidae has been challenged for a long time in the last century (Zhou et al., 2023), it is now generally recognized as a monophyletic group and includes two subfamilies, Megophryinae and Leptobrachiinae (AmphibiaWeb, 2023; Frost, 2023). According to the latest statistics, the two subfamilies comprise 134 and 187 species, respectively (Frost, 2023), making Megophryidae one of the most diverse groups of amphibians in Asia as well as in the world.

The subfamily Megophryinae (Bonaparte, 1850), also known as Asian horned (or spadefoot) toads, basically reflects the distribution range and pattern of the whole Megophryidae family (Frost, 2023). Due to significant morphological diversity and similarity, especially the lack of easily recognizable generic diagnostic characters, debates regarding generic classifications within Megophryinae have persisted for decades. Several different generic hypotheses have been proposed from various studies, including five‐genus classifications (Chen et al., 2017; Delorme et al., 2006), seven‐genus classifications (Dubois et al., 2021; Fei & Ye, 2016; Lyu et al., 2021; Qi et al., 2021), or a single genus proposal encompassing all Megophryinae species together as a large group (Mahony et al., 2017). These specific generic classifications have attracted dedicated followings in later studies, being embraced or revised by databases over time. Among them, the proposal of a single large genus by Mahony et al. (2017) has been widely accepted in various studies (e.g., Gao et al., 2022; Liu et al., 2018; Tapley et al., 2021; Wang et al., 2019, 2020). However, a recent study has introduced a 10‐genus classification from an integrative taxonomic perspective (Lyu et al., 2023). This new classification is revealed by 10 well‐supported molecular phylogenetic clades and presented with combined morphological diagnoses for each genus, representing the most reliable and the latest taxonomic recognition of Megophryinae. These genera include Atympanophrys, Boulenophrys, Brachytarsophrys, Grillitschia, Jingophrys, Megophrys, Ophryophryne, Pelobatrachus, Sarawakiphrys, and Xenophrys. This 10‐genus classification of Megophryinae has been adopted shortly in mainstream databases such as Amphibian Species of the World (https://amphibiansoftheworld.amnh.org/) and AmphibiaChina (https://www.amphibiachina.org/), which is also the updated taxonomic system we used in this study.

The genus Boulenophrys (Fei & Ye, 2016), which includes half of the recognized species within Megophryinae (67 out of 134 total species), is the largest genus in Megophryinae (Frost, 2023). China hosts the most diverse population of Megophryinae, housing over two‐thirds of the total cataloged species (Lyu et al., 2023). Unsurprisingly, species in Boulenophrys are predominantly found in southern China (61 species, see Figure 1), with a few extension westwards into Myanmar and southwards into northernmost Indochina, including Vietnam, Laos, and Thailand (Lyu et al., 2023). Navigating the intricate taxonomic history of Megophryinae, Boulenophrys has undergone several revisions despite being a relatively recent‐established genus (Fei & Ye, 2016). It was frequently considered as a subgenus or synonymous of Megophrys, Panophrys, or Xenophrys (e.g., Chen et al., 2017; Luo et al., 2021; Lyu et al., 2021; Mahony et al., 2017) until Dubois et al. (2021) recognized that the name Panophrys was preoccupied. They then reestablished Boulenophrys as the valid generic name following the International Code of Zoological Nomenclature. This recognition has been subsequently adopted by other studies (Lyu et al., 2023; Qi et al., 2021; Wang, Zeng et al., 2022) and databases (AmphibiaWeb, 2023; Frost, 2023).

FIGURE 1.

FIGURE 1

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The recorded distribution sites of Boulenophrys species in China (data from AmphibiaChina, 2024). The circles (in red show the species we collected in this study) on the map represent the type localities of Boulenophrys species distributed in China or near the national boundaries. The numbers 1–61 represent the species name as follow: (1) B. acuta, (2) B. anlongensis, (3) B. baishanzuensis, (4) B. baolongensis, (5) B. binchuanensis, (6) B. binlingensis, (7) B. boettgeri, (8) B. brachykolos, (9) B. caudoprocta, (10) B. cheni, (11) B. chishuiensis, (12) B. congjiangensis, (13) B. daiyunensis, (14) B. daoji, (15) B. daweimontis, (16) B. dongguanensis, (17) B. fanjingmontis, (18) B. fengshunensis, (19) B. hengshanensis, (20) B. hungtai, (21) B. insularis, (22) B. jiangi, (23) B. jingdongensis, (24) B. jinggangensis, (25) B. jiulianensis, (26) B. kuatunensis, (27) B. leishanensis, (28) B. liboensis, (29) B. lini, (30) B. lishuiensis, (31) B. lushuiensis, (32) B. minor, (33) B. mirabilis, (34) B. mufumontana, (35) B. nankunensis, (36) B. nanlingensis, (37) B. obesa, (38) B. ombrophila, (39) B. omeimontis, (40) B. palpebralespinosa, (41) B. puningensis, (42) B. qianbeiensis, (43) B. rubrimera, (44) B. sangzhiensis, (45) B. sanmingensis, (46) B. shimentaina, (47) B. shuichengensis, (48) B. shunhuangensis, (49) B. spinata, (50) B. tongboensis, (51) B. tuberogranulata, (52) B. wugongensis, (53) B. wuliangshanensis, (54) B. wushanensis, (55) B. xiangnanensis, (56) B. xianjuensis, (57) B. xuefengmontis, (58) B. yangmingensis, (59) B. yaoshanensis, (60) B. yingdeensis, (61) B. yunkaiensis.

Majority of species within Boulenophrys exhibit narrow distributions and small populations. For instance, Boulenophrys sangzhiensis (Jiang et al., 2008) is exclusively known from Mt. Tianping and Mt. Huping in northwestern Hunan Province, lived at elevations of 1300–1400 m a.s.l. (Fei & Ye, 2016; Lyu et al., 2023). Similarly, Boulenophrys tuberogranulata (Mo et al., 2010) is restricted to Tianzishan Nature Reserve and Mt. Tianping in northwestern Hunan Province, found at elevations ranging from 1000 to 1380 m a.s.l. (Lyu et al., 2023; Mo et al., 2010). Notably, B. sangzhiensis and B. tuberogranulata share overlapping distributions in Mt. Tianping. Despite this, they exhibit distinct morphological characteristics, suggesting a potentially early derivation from respective evolutionary lineages. Both species, however, face population declines due to threats such as habitat loss or degradation (Gao et al., 2022). The IUCN SSC Amphibian Specialist Group assessed their status in 2020, classifying B. sangzhiensis as Critically Endangered (CR) and B. tuberogranulata as Endangered (EN) on the IUCN Red List of Threatened Species (IUCN SSC Amphibian Specialist Group, 2020a, 2020b).

As endemic yet threatened amphibians, both B. sangzhiensis and B. tuberogranulata have received very limited public attentions and scientific concerns. Knowledges about these species in science have primarily been limited to initial morphological descriptions and distributions, with little engagement in phylogenetic studies, let alone investigations of their population structure and genetic diversity. Moreover, given the existence of over 60 species in Boulenophrys, constructing a robust phylogenetic tree using mitogenomes, for example, could significantly enhance our understanding of the phylogeny, trait evolution, and current distribution patterns within this group. However, the available mitogenome data remain quite limited. In this study, we utilized next‐generation sequencing (NGS) technology to sequence and assemble the complete mitogenomes of B. sangzhiensis and B. tuberogranulata for the first time. Our primary aim was to conduct a detailed analysis and description of these two complete mitogenomes to enhance the mitogenomic data of Boulenophrys. Then, we reconstructed a phylogenetic tree of Megophryidae based on all available mitogenomes in GenBank to scrutinize the current understanding of the phylogeny of this group. This study is expected to provide valuable data references for population genetics and conservation biology studies on these two threatened species. It will also contribute to the phylogenetic knowledge of Boulenophrys and other Asian horned toads in the future.

2. MATERIALS AND METHODS

2.1. Sample collection and sequencing

Permissions for the field survey in this study were obtained for scientific purposes from the local administrations, and the sample collections and experiment protocols were approved by the Biomedical Ethics Committee of Jishou University (No: JSDX‐2024‐0083) adhered to the relevant laws and guidelines of China. Following the “3R principle” (Reduction, Replacement, and Refinement) of animal ethics that required by National Ministry of Science and Technology (No. 398 [2006]), only one sample of each species was utilized. The sample of B. sangzhiensis was collected in May 2021 from its type locality within the National Nature Reserve of Badagongshan in Sangzhi County. The sample of B. tuberogranulata was collected in August 2022, also from its type locality within the Zhangjiajie National Forest Park in Wulingyuan District. The straight‐line distance between the two sample localities was only 60 km, and both are situated in Zhangjiajie City, Hunan Province, China. All animal collection and treatment procedures strictly followed the guidance outlined in the Code of Practice for the Housing and Care of Animals. The specimens were euthanized and preserved in 85% alcohol as voucher specimens, and then deposited at the molecular ecology laboratory in Zhangjiajie Campus, Jishou University (B. sangzhiensis, voucher no. JWS20210007; B. tuberogranulata, voucher no. JWS20221040). A small section of liver tissue from each sample was used for molecular analysis. Total DNA extraction was performed using the DNeasy Blood & Tissue Kit (Qiagen), and the DNA library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme). High‐throughput sequencing was conducted in paired‐end mode on the DNBSEQ‐T7 platform (Complete Genomics and MGI Tech), generating approximately 30 Gb of raw reads with a read length of 150 bp for each sample.

2.2. Sequence assembly, annotation, and analysis

The complete mitogenomes of B. sangzhiensis and B. tuberogranulata were assembled using NOVOPlasty 4.3 (Dierckxsens et al., 2016), with the mitogenome of Boulenophrys jingganggensis serving as a reference. The assembled sequences were then queried against the standard database of NCBI using the BLAST online program to identify highly similar sequences. 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 the MITOS Web server (Bernt et al., 2013). This annotation was cross‐verified by comparing it with the information available for congeners. Further annotation and gene map visualization were carried out using the web application GeSeq (Tillich et al., 2017). Codon usage and nucleotide frequencies of the PCGs were determined using MEGA X (Kumar et al., 2018). AT skew and GC skew were calculated using the formulas: AT skew = (A – T)/(A + T) and GC skew = (G – C)/(G + C) (Perna & Kocher, 1995). The relative synonymous codon usage (RSCU), nonsynonymous substitution rate (Ka), and synonymous substitution rate (Ks) of all PCGs were analyzed using DnaSP V6 (Rozas et al., 2017) to investigate potential signals of selective pressure.

2.3. Phylogenetic analysis

All available mitogenomes of Megophryidae species from GenBank were downloaded, and one representative sequence was selected for each species. A comprehensive dataset comprising a total of 28 Megophryidae species, including the newly sequenced B. sangzhiensis and B. tuberogranulata, was compiled for phylogenetic reconstruction. As an outgroup, Microhyla fissipes from the family Microhylidae was included. PhyloSuite (Zhang et al., 2020) was employed to extract each of the 13 PCGs from the 29 mitogenomes. Subsequently, individual PCG datasets were aligned using the MUSCLE module in MEGA X and manually checked for accuracy. Finally, these aligned and trimmed datasets were concatenated to create a combined PCGs dataset using PhyloSuite. Phylogenetic trees were reconstructed using both the maximum likelihood (ML) method with RAxML 8.0.2 (Stamatakis, 2006) and Bayesian inference (BI) method using MrBayes 3.2.7 (Ronquist & Huelsenbeck, 2003). The best‐fit partitioning scheme and nucleotide substitution models for ML and BI reconstructions were determined using PartitionFinder 2 (Lanfear et al., 2017). Statistical confidence was assessed by conducting a bootstrap test with 1000 replicates for ML trees. For BI trees, posterior probabilities were calculated under a simultaneous run of 1.0 × 107 million generations, with sampling every 1000 generations and discarding the initial 25% of generations as burn‐in.

3. RESULTS

3.1. Mitogenome assembly, annotation, and nucleotide composition

The complete mitogenome of B. sangzhiensis was 16,950 bp, while that of B. tuberogranulata was 16,841 bp in length after assembly using NOVOPlasty (Figure 2). Blastn searches revealed that the most similar sequences in NCBI to be those from Boulenophrys and the best‐matched sequences showed the species identification was correct. Both mitogenomes exhibited the typical characteristics of animal mitogenomes, comprising 37 genes, including 13 PCGs, 22 tRNAs, two rRNAs, and a D‐loop control region (Table 1 and Figure 2). Most of these genes were encoded by the heavy strand (H‐strand), except for one PCG (ND6) and eight tRNAs, which were encoded on the light strand (L‐strand). The final complete mitogenomes, along with annotated information for both species, have been deposited in GenBank under accession numbers OQ830572 and OQ830573.

FIGURE 2.

FIGURE 2

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Gene map of the mitogenome of Boulenophrys sangzhiensis (a) and Boulenophrys tuberogranulata (b).

TABLE 1.

Characteristics of the mitogenome of Boulenophrys sangzhiensis (BS) and Boulenophrys tuberogranulata (BT).

Genes Position Length Start codon Stop codon Anticodon Strand Intergenic nucleotide
BS BT BS BT BS BT BS BT BS/BT BS/BT BS BT
tRNAPhe 1–70 1–71 70 71 GAA H 0 0
12S 71–995 72–996 925 925 H –4 –4
tRNAVal 992–1061 993–1063 70 71 TAC H 3 14
16S 1065–2658 1078–2671 1594 1594 H –2 –2
tRNALeu2 2657–2731 2670–2743 75 74 TAA H 0 0
ND1 2732–3705 2744–3717 974 974 ATA ATG TA(A) TA(A) H 3 3
tRNAIle 3709–3779 3721–3791 71 71 GAT H −1 –1
tRNAGln 3779–3850 3791–3862 72 72 TTG L –1 –1
tRNAMet 3850–3918 3862–3930 69 69 CAT H 0 0
ND2 3919–4959 3931–4971 1041 1041 ATG ATT TAG TAG H −2 −2
tRNATrp 4958–5026 4970–5038 69 69 TCA H 0 0
tRNAAla 5027–5095 5039–5107 69 69 TGC L 0 0
tRNAAsn 5096–5168 5108–5180 73 73 GTT L 2 2
OL 5171–5198 5183–5212 28 30 H −1 −1
tRNACys 5198–5260 5212–5274 63 63 GCA L 0 0
tRNATyr 5261–5327 5275–5341 67 67 GTA L 1 1
COI 5329–6885 5343–6905 1557 1563 TTG GTG AGA AGA H –5 −10
tRNASer2 6881–6951 6896–6966 71 71 TGA L 3 3
tRNAAsp 6955–7021 6970–7037 67 68 GTC H 0 0
COII 7022–7706 7038–7722 685 685 ATG ATG T(AA) T(AA) H 0 0
tRNALys 7707–7779 7723–7795 73 73 TTT H 1 1
ATP8 7781–7945 7797–7961 165 165 ATG ATG TAA TAA H −10 −10
ATP6 7936–8618 7952–8634 683 683 ATG ATG TA(A) TA(A) H −1 −1
COIII 8618–9402 8634–9418 785 785 ATG ATG TA(A) TA(A) H −1 −1
tRNAGly 9402–9470 9418–9486 69 69 TCC H 0 0
ND3 9471–9815 9487–9831 345 345 TTG ATG TAG TAG H −2 −2
tRNAArg 9814–9882 9830–9898 69 69 TCG H 3 3
ND4L 9886–10,182 9902–10,198 297 297 ATG ATG TAA TAA H −7 −7
ND4 10,176–11,553 10,192–11,572 1378 1381 ATG ATG T(AA) T(AA) H 6 0
tRNAHis 11,560–11,628 11,573–11,641 69 69 GTG H 6 0
tRNASer1 11,635–11,696 11,642–11,708 62 67 GCT H 0 0
tRNALeu1 11,697–11,769 11,709–11,781 73 73 TAG H 0 0
ND5 11,770–13,587 11,782–13,599 1818 1818 ATG ATG TAA TAA H 7 3
ND6 13,595–14,104 13,603–14,112 510 510 ATG ATG AGG AGA L 0 0
tRNAGlu 14,105–14,173 14,113–14,181 69 69 TTC L 2 2
Cytb 14,176–15,315 14,184–15,323 1140 1140 ATG ATG TAG TAG H –1 –1
tRNAThr 15,315–15,385 15,323–15,392 71 70 TGT H 0 1
tRNAPro 15,386–15,454 15,394–15,462 69 69 TGG L 0 –1
D‐loop 15,455–16,950 15,462–16,841 1496 1380 H 0 0
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A total of 38 and 44 bp overlapping sites were shared in 13 and 14 pairs of neighboring genes in B. sangzhiensis and B. tuberogranulata, respectively, with lengths ranging from 1 to 10 bp. Additionally, there were 37 and 32‐bp intergenic nucleotides (IGNs) dispersed across 11 and 10 locations in the two species, with lengths ranging from 1 to 7 bp and 1–14 bp, respectively. Both species featured a short noncoding region located between tRNAAsn and tRNACys, measuring 28 bp in B. sangzhiensis and 30 bp in B. tuberogranulata. The nucleotide compositions of B. sangzhiensis were 27.45% A, 30.77% T, 14.81% G, and 26.97% C, while those of B. tuberogranulata were 27.79% A, 30.91% T, 14.55% G, and 26.75% C (Table 2). Similar to other species in Megophryidae, all the A + T values accounted for more than half, indicating an A + T bias with greater A + T than G + C contents. For the H‐strand sequence of all examined Megophryidae species, both AT skew and GC skew were negative, indicating a predominant bias toward T and C base pairs.

TABLE 2.

Base composition (in percentages) of the mitogenomes of Boulenophrys sangzhiensis, Boulenophrys tuberogranulata, and other 26 species in Megophryidae.

Species Total length T(U)% C% A% G% A + T% AT skew GC skew Accession numbers
Boulenophrys sangzhiensis 16,950 30.77 26.97 27.45 14.81 58.21 −0.057 −0.291 OQ830572 a
Boulenophrys tuberogranulata 16,841 30.91 26.75 27.79 14.55 58.70 −0.053 −0.295 OQ830573 a
Boulenophrys jingganggensis 17,262 31.62 26.28 27.62 14.48 59.24 −0.067 −0.289 MT683772
Boulenophrys omeimontis 17,013 31.76 25.72 28.34 14.18 60.10 −0.057 −0.289 KP728257
Boulenophrys spinata 16,024 30.63 27.11 27.16 15.10 57.79 −0.060 −0.284 ON646614
Boulenophrys boettgeri 16,597 31.51 26.42 27.84 14.23 59.35 −0.062 −0.300 OR529440
Boulenophrys kuatunensis 17,921 32.07 25.86 28.10 13.97 60.17 −0.066 −0.298 OR522721
Boulenophrys baishanzuensis 17,040 31.51 26.64 27.24 14.61 58.75 −0.073 −0.292 OR063945
Brachytarsophrys carinense 15,271 29.80 27.61 27.46 15.13 57.26 −0.041 −0.292 JX564854
Leptobrachium boringii 17,097 31.53 25.51 27.69 15.27 59.22 −0.065 −0.251 OP373724
Leptobrachium liui 17,499 32.74 24.32 28.10 14.84 60.84 −0.076 −0.242 OP503540
Oreolalax major 17,786 32.63 24.31 28.75 14.31 61.37 −0.063 −0.259 MN803320
Oreolalax xiangchengensis 17,110 33.02 23.62 29.18 14.18 62.20 −0.062 −0.250 MH727696
Oreolalax jingdongensis 17,864 32.73 23.92 29.10 14.26 61.82 −0.059 −0.253 MF953479
Oreolalax omeimontis 17,675 32.59 24.96 28.46 13.99 61.05 −0.068 −0.282 MN803321
Oreolalax multipunctatus 17,358 32.96 24.20 28.53 14.31 61.49 −0.072 −0.257 MF966382
Oreolalax lichuanensis 17,702 32.17 24.86 28.00 14.98 60.17 −0.069 −0.248 KU096847
Oreolalax schmidti 18,481 32.80 24.48 28.31 14.41 61.11 −0.073 −0.259 MT773151
Oreolalax rhodostigmatus 18,676 32.39 24.93 28.03 14.66 60.42 −0.072 −0.259 MF770485
Leptobrachium ailaonicum 17,318 31.83 24.99 27.90 15.27 59.74 −0.066 −0.241 MZ394043
Leptobrachium leishanense 17,485 32.64 24.38 28.15 14.83 60.79 −0.074 −0.244 KU760082
Scutiger ningshanensis 17,265 32.68 24.25 29.11 13.96 61.79 −0.058 −0.269 KX619450
Scutiger liupanensis 16,888 32.25 24.90 28.32 14.53 60.57 −0.065 −0.263 KX352261
Leptolalax oshanensis 17,747 29.85 26.26 28.77 15.11 58.62 −0.018 −0.270 KC460337
Leptobrachella alpina 17,763 30.77 25.64 28.53 15.05 59.30 −0.038 −0.260 MW487804
Leptolalax pelodytoides 14,682 29.07 27.80 27.67 15.46 56.74 −0.025 −0.285 JX564874
Atympanophrys shapingensis 17,631 31.48 26.05 28.18 14.29 59.66 −0.055 −0.291 JX458090
Atympanophrys gigantica 18,259 32.11 25.19 28.37 14.33 60.48 −0.062 −0.275 MZ364157
Microhyla fissipes 16,723 31.01 25.48 28.93 14.58 59.94 −0.035 −0.272 MN046210
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a

The sequence obtained in this study.

3.2. Characteristics of PCGs and codon usage

The total length of the PCGs in B. sangzhiensis was 11,378 bp, and in B. tuberogranulata, it was 11,387 bp. The differences in length were attributed to 6 bp in COI and 3 bp in ND4, which were longer in B. tuberogranulata than in B. sangzhiensis; the lengths of other PCGs were identical in both species. Most PCGs in both species commenced with the ATG codon, except for ND1 using ATA, COI, and ND3 using TTG in B. sangzhiensis, and ND2 using ATT, COI using GTG in B. tuberogranulata. Generally, stop codon usage patterns were similar between the two species but varied among different genes. In general, B. sangzhiensis employed six types of stop codons, while B. tuberogranulata used five. Both species shared TAG as stop codons for ND2, CYTB, and ND3; TAA for ATP8, ND4L, and ND5; the incomplete TA(A) for ND1, ATP6, and COIII; the incomplete T(AA) for COII and ND4; and AGA for COI. However, AGA was used as stop codons for ND6 in B. tuberogranulata, while that in B. sangzhiensis was AGG (Table 1). Nucleotide composition analysis of the 13 PCGs revealed similar negative AT and GC skew patterns, except for the ND6 gene, which exhibited an extraordinary but positive GC skew (Table 3).

TABLE 3.

Nucleotide composition and skewness of different gene regions in the mitogenomes of Boulenophrys sangzhiensis (BS) and Boulenophrys tuberogranulata (BT).

Length (bp) T(U)% C% A% G% A + T% AT skew GC skew
BS BT BS BT BS BT BS BT BS BT BS BT BS BT BS BT
ND1 974 974 34.39 34.19 28.64 28.85 23.31 23.72 13.66 13.24 57.70 57.91 −0.192 −0.181 −0.354 −0.371
ND2 1041 1041 31.41 30.55 31.12 31.51 26.32 26.99 11.14 10.95 57.73 57.54 −0.088 −0.062 −0.473 −0.484
COI 1557 1563 31.47 32.05 25.43 24.63 24.98 25.27 18.11 18.04 56.45 57.33 −0.115 −0.118 −0.168 −0.154
COII 685 685 29.49 31.68 27.74 24.96 27.15 27.74 15.62 15.62 56.64 59.42 −0.041 −0.066 −0.279 −0.230
ATP8 165 165 35.76 30.91 25.45 30.30 28.48 29.70 10.30 9.09 64.24 60.61 −0.113 −0.020 −0.424 −0.538
ATP6 683 683 33.82 31.77 28.99 29.43 24.74 26.94 12.45 11.86 58.57 58.71 −0.155 −0.082 −0.399 −0.426
COIII 785 785 28.92 32.36 29.81 27.52 24.59 24.46 16.69 15.67 53.50 56.82 −0.081 −0.139 −0.282 −0.274
ND3 345 345 32.75 35.65 30.72 27.25 22.03 22.61 14.49 14.49 54.78 58.26 −0.196 −0.224 −0.359 −0.306
ND4L 297 297 35.69 34.01 26.60 29.29 24.24 22.56 13.47 14.14 59.93 56.57 −0.191 −0.202 −0.328 −0.349
ND4 1378 1381 34.40 33.74 27.79 28.24 24.53 24.98 13.28 13.03 58.93 58.73 −0.167 −0.149 −0.353 −0.368
ND5 1818 1818 33.55 33.83 27.34 27.67 26.90 26.73 12.21 11.77 60.45 60.56 −0.110 −0.117 −0.382 −0.403
ND6 510 510 38.63 37.84 10.00 11.18 21.18 19.80 30.20 31.18 59.80 57.65 −0.292 −0.313 0.502 0.472
CYTB 1140 1140 33.16 31.67 28.60 29.91 24.12 24.30 14.12 14.12 57.28 55.96 −0.158 −0.132 −0.339 −0.359
PCG‐all 11,378 11,387 32.95 32.95 27.37 27.35 24.93 25.17 14.76 14.54 57.87 58.11 −0.139 −0.134 −0.299 −0.306
PCGs–1st 3795 3798 24.96 24.48 24.72 24.77 26.94 27.52 23.37 23.24 51.90 52.00 0.038 0.058 −0.028 −0.032
PCGs–2nd 3795 3798 41.49 41.55 27.58 27.39 18.19 18.35 12.74 12.71 59.68 59.90 −0.390 −0.387 −0.368 −0.366
PCGs–3rd 3788 3791 32.39 32.80 29.80 29.90 29.64 29.63 8.17 7.67 62.03 62.44 −0.044 −0.051 −0.570 −0.592
12S‐rRNA 925 925 22.92 23.24 26.38 26.38 30.16 30.38 20.54 20.00 53.08 53.62 0.136 0.133 −0.124 −0.138
16S‐rRNA 1594 1594 25.97 26.10 22.58 23.59 33.94 33.25 17.50 17.06 59.91 59.35 0.133 0.121 −0.127 −0.160
rRNAs 2519 2519 24.85 25.05 23.98 24.61 32.55 32.20 18.62 18.14 57.40 57.24 0.134 0.125 −0.126 −0.151
tRNAs 1529 1525 28.17 28.91 19.87 19.40 30.65 30.40 21.31 21.29 58.82 59.31 0.042 0.025 0.035 0.046
D‐loop 1496 1380 34.49 33.16 22.75 25.40 30.36 28.48 12.39 12.97 64.86 61.63 −0.064 −0.076 −0.295 −0.324
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Codon usage patterns of PCGs were similar in both B. sangzhiensis and B. tuberogranulata (Figure 3). Codons UCU (Ser1), UCC (Ser1), CCC (Pro), GCC (Ala), CAA (Gln), and CGA (Arg) had the highest frequencies in both species. The RSCU analysis indicated that Leu was encoded by six synonymous codons, while other amino acids were encoded by fewer: Val, Ser1, Pro, Thr, Ala, Arg, and Gly were encoded by four codons, and the remaining amino acids (Phe, Ile, Met, Tyr, His, Gln, Asn, Lys, Asp, Glu, Cys, Trp, and Ser2) were encoded by two codons (Figure 3).

FIGURE 3.

FIGURE 3

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Relative synonymous codon usage (RSCU) of the mitogenome of Boulenophrys sangzhiensis (a) and Boulenophrys tuberogranulata (b).

3.3. Characteristics of rRNAs, tRNAs, and the control region

Both B. sangzhiensis and B. tuberogranulata had two rRNA genes: the 12S rRNA located between tRNAPhe and tRNAVal with a length of 925 bp in both species, and the 16S rRNA located between tRNAVal and tRNALeu with lengths of 1594 bp in B. sangzhiensis and 1595 bp in B. tuberogranulata. Both mitogenomes contained 22 tRNAs, with total lengths of 1529 and 1525 bp for B. sangzhiensis, and B. tuberogranulata, respectively. Individual tRNA sizes ranged from 62 bp (tRNASer1) to 75 bp (tRNALeu2) in B. sangzhiensis and from 63 bp (tRNACys) to 74 bp (tRNALeu2) in B. tuberogranulata. Unlike the pattern of PCGs, both rRNAs and tRNAs had positive AT skew, and the tRNAs additionally exhibited positive GC skew, indicating a different base bias compared to PCGs. The noncoding control region, also known as the D‐loop, showed significant size variations, with a length of 1496 bp in B. sangzhiensis and 1380 bp in B. tuberogranulata. The D‐loop was located between tRNAPro and tRNAPhe in both species, and it exhibited similar A + T contents, AT skew, and GC skew patterns with PCGs rather than rRNAs and tRNAs (Table 3).

3.4. Positive selection and phylogenetic relationships

The Ka/Ks ratio was calculated to assess the evolutionary rates of each PCG (Figure 4). The highest Ka/Ks value was observed in ATP8 (0.246), while the lowest was in COI (0.037). Other PCGs such as ATP6, ND2, and ND4 exhibited relatively fast evolutionary rates, whereas COI, COIII, and CYTB showed relatively slow rates. However, all Ka/Ks ratios for the 13 PCGs were less than 1, indicating that they did not exhibit strong positive selection signals and possibly evolved under purifying selection.

FIGURE 4.

FIGURE 4

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The Ka/Ks of 13 PCGs among 28 species within Megophryidae.

Both ML and BI analyses generated similar tree topologies (Figure 5). The Megophryidae family could be divided into two major clades, corresponding to the subfamilies Leptobrachiinae (Clade I) and Megophryinae (Clade II). Clade I could be further divided into four well‐supported major groups: Leptobrachella, Scutiger, Oreolalax, and Leptobrachium. Clade II contained species from the genera Atympanophrys, Brachytarsophrys, and Boulenophrys. Each examined genus appeared as monophyletic, and the intergeneric relationships among the two clades were also well‐supported. The genus Boulenophrys could be further divided into two subgroups, with the two species sequenced in this study appearing in each of these subgroups. Boulenophrys sangzhiensis grouped with B. omeimontis and then clustered with B. spinata. Comparatively, B. tuberogranulata clustered with B. jinggangensis, B. boettgeri, B. kuatunensis, and B. baishanzuensis.

FIGURE 5.

FIGURE 5

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Phylogenetic relationships within Megophryidae derived from BI method based on 13 PCGs. The numbers on the branches represent the bootstrap values and posterior probabilities of ML/BI analyses. The number after species name is the GenBank accession number. Names in red show the phylogenetic positions of Boulenophrys sangzhiensis and Boulenophrys tuberogranulata that we sequenced in this study.

4. DISCUSSION

Mitochondrial genomes (or called mitogenomes) serve as valuable molecular markers and have found widespread applications in molecular biology and ecology. In vertebrates, especially, mitogenomes are extensively utilized due to their conservative structure, typically comprising 13 PCGs, 2 rRNAs, 22 tRNAs, and 1 noncoding control region (D‐loop), with a sequence length usually ranging from 16 to 17 kb. In comparison to the complex nuclear DNA, vertebrate mitogenomes possess unique characteristics, including maternal inheritance, a rapid evolutionary rate, a simple structure with conserved coding regions, low levels of recombination, and multiple copy numbers (Boore, 1999; Zardoya & Meyer, 1996). These characteristics make mitochondrial DNA as valuable markers for reconstructing phylogenetic relationships, revealing population genetic structures, estimating divergence times, testing selective pressures, and identifying species using mitochondrial barcoding genes (Jiang et al., 2023; Lan et al., 2023; Shu et al., 2021; Zhang et al., 2023).

To the best of our knowledge, this study represents the first assembly of the mitogenomes for two horned toads, B. sangzhiensis and B. tuberogranulata. The exploration of similarities and differences in gene orders, genetic structures, base compositions, evolutionary features, and codon usage offers valuable molecular insights into the taxonomic and phylogenetic characteristics of closely related species (Sun et al., 2022). Overall, the mitogenomes of the two species exhibited similarities in terms of size, organization (Figure 2 and Table 1), nucleotide composition of PCGs, rRNAs, tRNAs, control region, and codon usage of PCGs (Figures 3 and 4 and Table 3). The most notable difference between the two mitogenomes was observed in the length of the D‐loop (1496 bp vs. 1380 bp), contributing significantly to the total length variations (16,950 bp vs. 16,841 bp). Despite this distinction, the two newly obtained mitogenomes exhibited patterns similar to those of other Boulenophrys species reported previously (Liu et al., 2016; Wu et al., 2024), such as negative AT skew and GC skew, along with high A + T content. The AT skew and GC skew generally reflect base bias, which can vary among different groups. For instance, the species we previously detected in Salamandridae had a positive AT skew (Wang, Lan et al., 2022), whereas all the Megophryidae species in this study displayed a negative AT skew, consistent with the findings of Zhou et al. (2023). Furthermore, while the mitogenomes of species in Ranoidea exhibited gene rearrangements frequently (Igawa et al., 2008), no any rearrangements were identified among all the Megophryidae species examined in this study.

By utilizing the mitogenomes of 26 species available from NCBI and the two newly obtained in this study as ingroups, with M. fissipes from the Microhylidae family as the outgroup, we successfully reconstructed a phylogenetic tree within Megophryidae based on the 13 PCGs of 29 species (Figure 5). The phylogenetic analysis revealed that Megophryidae could be divided into two well‐supported clades, supporting the classification of the two subfamilies, Megophryinae and Leptobrachiinae. The intergeneric relationships among the two subfamilies were consistent with our previous study (Zhou et al., 2023), with the monophyly of each examined genus also receiving support. Based on the two most recent phylogenetic studies of Boulenophrys, the genus can be further divided into three well‐supported species groups: the B. minor group, the B. omeimontis group, and the B. boettgeri group (Lyu et al., 2023; Qi et al., 2021). In this study, two species groups were identified and supported, although no sequences of the B. minor group were included. Specifically, B. sangzhiensis, B. omeimontis, and B. spinata formed the B. omeimontis group, while B. tuberogranulata, B. jinggangensis, B. boettgeri, B. kuatunensis, and B. baishanzuensis grouped in the B. boettgeri group. These phylogenetic relationships within Boulenophrys, as elucidated here using mitogenomes, were largely consistent with previous studies that employed more species but fewer molecular markers (Lyu et al., 2023; Qi et al., 2021).

Interestingly, despite B. sangzhiensis and B. tuberogranulata being sympatric and coexisting in Zhangjiajie City, Hunan Province, this study revealed that they belong to two distinct phylogenetic clades (Figure 5). Boulenophrys sangzhiensis, along with other species in the B. omeimontis species group, mainly displayed a southwestern distribution pattern. In contrast, B. tuberogranulata and other species in the B. boettgeri species group were primarily distributed in the southeastern area of China. It appears that Zhangjiajie City serves as the boundary area between the two clades of Boulenophrys, especially considering that the type localities of B. sangzhiensis and B. tuberogranulata completely overlapped in Mt. Tianping in Sangzhi County. A similar pattern was observed in mustache toads of Leptobrachium we studied before, where L. boringii and L. liui were also collected in the same region of Zhangjiajie City but occupied very different phylogenetic lineages that showed a parallel southwestern and southeastern convergence (Zhou et al., 2023). Notably, another species, B. caudoprocta, was also found in Mt. Tianping in the same area (Figure 1). Although the phylogenetic position of B. caudoprocta was not assessed in this study, a previous investigation indicated that the three species found in Mt. Tianping did not form any sister group relationships and they were also morphologically distinct (Lyu et al., 2023). This distribution pattern in Boulenophrys is unique, with three species coexisting in the same mountain but having distinct phylogenetic histories. This pattern highlights the special role of the coexisting area, the Zhangjiajie City in driving the speciation of these species. Similar coexistence of species in nearby areas was also observed in other species of Boulenophrys. For instance, the type localities of B. boettgeri, B. ombrophila and B. kuatunensis were all in Kuatun village (Fujian Province, China), B. yangmingensis and B. xiangnanensis were both in Mt. Yangming (Hunan province, China), and B. cheni, B. jinggangensis, and B. lini were all found in Mt. Jinggang (Jiangxi Province, China). However, these sympatric species were all clustered in the same phylogenetic clade (Lyu et al., 2023).

There are more than 300 species in Megophryidae; however, the available mitogenome data are still very limited. As of now, only 25 complete and nine nearly complete mitogenomes belonging to 26 recognized species of Megophryidae are available from NCBI. The phylogenetic tree presented in this study, based on 28 mitogenomes in Megophryidae, demonstrated that intergeneric and intrageneric relationships can be reliably elucidated with high supporting values. It suggests that mitogenomes serve as valuable molecular markers for constructing a robust phylogenetic tree of Megophryidae. It is worth noting that the advent of NGS has revolutionized genomics research by enabling the sequencing of entire genomes with ever‐increasing throughput and decreasing costs (Van Dijk et al., 2014). With the application of NGS, we anticipate that the complete evolutionary history of Megophryidae will gradually be unveiled when more mitogenomes, such as the two Boulenophrys species in this study, become available in the future.

AUTHOR CONTRIBUTIONS

Hongmei Xiang: Conceptualization (equal); data curation (lead); formal analysis (equal); investigation (equal); methodology (equal); software (lead); visualization (lead); writing – original draft (lead); writing – review and editing (equal). Qiang Zhou: Investigation (equal); methodology (equal); visualization (supporting); writing – original draft (supporting); writing – review and editing (supporting). Wei Li: Formal analysis (supporting); investigation (equal); resources (equal); writing – review and editing (supporting). Juan Shu: Formal analysis (supporting); investigation (equal); resources (equal); writing – review and editing (supporting). Zhirong Gu: Formal analysis (supporting); investigation (equal); resources (equal); writing – review and editing (supporting). Wansheng Jiang: Conceptualization (lead); data curation (equal); formal analysis (equal); funding acquisition (lead); investigation (equal); methodology (supporting); project administration (lead); software (supporting); visualization (supporting); writing – review and editing (lead).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

We would like to thank Mr. Ying Liu, Mr. Biwu Qin, and Ms. Mingyao Zhang for their assistances in field works. This work was supported by the National Natural Science Foundation of China (32060128) and Zhilan Foundation (2020040371B/2022010011B).

Xiang, H. , Zhou, Q. , Li, W. , Shu, J. , Gu, Z. , & Jiang, W. (2024). Insights into phylogenetic positions and distribution patterns: Complete mitogenomes of two sympatric Asian horned toads in Boulenophrys (Anura: Megophryidae). Ecology and Evolution, 14, e11687. 10.1002/ece3.11687

DATA AVAILABILITY STATEMENT

The final complete mitogenomes, along with annotated information for both species, have been deposited under GenBank accession numbers OQ830572 (B. sangzhiensis) and OQ830573 (B. tuberogranulata).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The final complete mitogenomes, along with annotated information for both species, have been deposited under GenBank accession numbers OQ830572 (B. sangzhiensis) and OQ830573 (B. tuberogranulata).

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