Phylogenetic relationships of the Chinese torrent frogs (Ranidae: Amolops) revealed by phylogenomic analyses of AFLP-Capture data

Journal Pre-proofs Phylogenetic relationships of the Chinese torrent frogs (Ranidae: Amolops) revealed by phylogenomic analyses of AFLP-Capture data Zhaochi Zeng, Dan Liang, Jiaxuan Li, Zhitong Lyu, Yingyong Wang, Peng Zhang PII: DOI: Reference:

S1055-7903(20)30025-7 https://doi.org/10.1016/j.ympev.2020.106753 YMPEV 106753

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Molecular Phylogenetics and Evolution

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30 October 2019 13 January 2020 28 January 2020

Please cite this article as: Zeng, Z., Liang, D., Li, J., Lyu, Z., Wang, Y., Zhang, P., Phylogenetic relationships of the Chinese torrent frogs (Ranidae: Amolops) revealed by phylogenomic analyses of AFLP-Capture data, Molecular Phylogenetics and Evolution (2020), doi: https://doi.org/10.1016/j.ympev.2020.106753

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Phylogenetic relationships of the Chinese torrent frogs (Ranidae: Amolops) revealed by phylogenomic analyses of AFLP-Capture data Zhaochi Zeng1, 2, Dan Liang1, 2, Jiaxuan Li1, Zhitong Lyu1, Yingyong Wang1,*, Peng Zhang1,* 1. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China 2. Both authors contributed equally to this work *Corresponding author: Yingyong Wang. #205, The Museum of Biology, School of Life Sciences, Sun Yatsen University, No. 135, Xingang Xi Road, Guangzhou 510275, China. Email: [email protected] Peng Zhang. #434, School of Life Sciences, Sun Yat-sen University, Higher Education Mega Center, Guangzhou 510006, China. Tel: 86-20-39332782; Email: [email protected]

Highlights 1. Comprehensive phylogenetic analyses of the Chinese Amolops frogs were performed with both anonymous nuclear data and mitochondrial genome data obtained from AFLP-Capture. 2. Two major clades representing eastern and western Chinese Amolops species were revealed. 3. Three previously proposed species groups (the A. mantzorum, A. monticola and A.

marmoratus groups) were not monophyletic, suggesting a need for further investigation and revision. 4. The ancestor of the Chinese Amolops was estimated to have appeared in the late Eocene or early Oligocene, and the speciation of the Chinese Amolops was often related to geological events including island formation and mountain uplifts. 5. Combined analysis adding mitochondrial sequences from GenBank revealed the dispersal routes of the Amolops frogs into China from the Indochinese Peninsula.

Abstract The torrent frog genus Amolops contains nearly sixty species distributed in swift mountain streams throughout southeast Asia. The taxonomy of this genus has proven complicated due to unstable morphological diagnostic characters. The relationships of Amolops species and species groups were not readily resolved with a small number of molecular markers. Here, we applied the novel AFLP-Capture approach and acquired two large datasets (242 anonymous nuclear sequences and the mitochondrial genome) from 70 Chinese Amolops samples to study their relationships. The phylogenies inferred from the nuclear data and the mitochondrial data were both robust and revealed a primary phylogenetic split between eastern and western Chinese Amolops species. The relationships of the six species groups were clarified. While the three species groups in east China (the A. ricketti, A. daiyunensis and A. hainanensis groups) were monophyletic, the three species groups in the west (the A. mantzorum, A. monticola and A. marmoratus groups) were not monophyletic, suggesting a need

for further investigation and revision. The robust phylogenies also provided new insights into species relationships, especially for the A. mantzorum group, which has been difficult to resolve due to multiple speciation events occurring approximately 78 million years ago. The divergence times estimated with the nuclear data indicated that the ancestor of the Chinese Amolops appeared in the late Eocene or early Oligocene, and that speciation events in the Chinese Amolops were often related to geological events (e.g. the uprising of mountains and the formation of islands). By including the mitochondrial sequences from GenBank, a more comprehensive Amolops phylogeny was constructed that reflected the origin of the Chinese Amolops. Based on all these results, a dispersal scenario of the torrent frogs was hypothesized. Our research serves as the first example of using AFLP-Capture to obtain a large amount of data for shallow-scale phylogenetic and taxonomic studies, which should be useful for other nonmodel organism groups.

Keywords: sequence capture, molecular dating, taxonomy, biogeography, cascade frogs

1. Introduction The Asian ranid frog genus Amolops Cope, 1865 is a group of torrent frogs that inhabit rapid-flowing mountain streams or waterfalls in the region from Nepal and India to western and southern China and south to the Malay Peninsula (Frost, 2019). They are characterized by large abdominal suckers in tadpoles and enlarged digital

pads in adults, which are useful for maintaining position in torrents (Matsui, 1986). The delimitation and taxonomy of this genus have proven complicated (Yang, 1991). Due to adult morphological similarity, some Amolops species are easily confused with species of related genera that live in similar habitats. In fact, several species previously attributed to Amolops have been moved to the genus Odorrana, e.g., O. chapaensis and O. tormota (Chen et al., 2005; Ngo et al., 2006; Cai et al., 2007). Within the Amolops genus, similar external adult morphology can be observed among some species (Jiang et al., 2016), while highly variable morphology is found in other closely related species (e.g., A. granulosus and A. loloensis, Fu et al., 2014) or even in different populations of a species (e.g., A. mantzorum, Fei et al., 2009 and Figure S1). These variations pose problems in the delimitation and taxonomy of Amoplos species based solely on morphological characters, such as misidentifications and synonyms. On the other hand, due to their peculiar habitats and limited mobility, Amoplos are easily influenced by geographical barriers. Cryptic species are frequently reported, which exacerbates the difficulties in the delimitation and taxonomy of this genus (Lyu et al., 2019a; Onn et al., 2018). Currently, there are 59 recognized species in the genus Amolops, 34 of which are distributed in China and 26 are endemic to China (Frost, 2019; Lyu et al., 2019b; Pham et al., 2019; Yu et al., 2019). Fei et al. (2005; 2009) divided the Chinese Amolops species into six species groups (the A. ricketti, A. daiyunensis, A. hainanensis, A. mantzorum, A. monticola and A. marmoratus species groups) based on morphological characters. These divisions were later accepted by other researchers

and applied to the Amolops species distributed outside China (Stuart et al., 2010, Dever et al., 2012). While the monophyly of the A. ricketti, A. daiyunensis and A. hainanensis groups is supported by recent phylogenetic studies (Lyu et al., 2019), the monophyly of the A. marmoratus, A. monticola and A. mantzorum groups remains unverified. The species content of the A. monticola and A. mantzorum groups and the relationship of these two groups are particularly puzzling because samples from the A. monticola group are often nested within the A. mantzorum group in available phylogenetic analyses (Cai et al., 2007; Ngo et al., 2006). In addition, members of the A. mantzorum group show high variability in morphology; their relationships are difficult to clarify, and the status of several species of this group remains disputable (Fei et al., 2009; Lu et al., 2014). A robust and comprehensive Amolops phylogeny will help to resolve the taxonomic problems outlined above. However, most of the available phylogenetic studies on this genus have involved limited taxon sampling. The markers used were mostly a few mitochondrial markers that may not reflect true species relationships due to maternal inheritance. In two recent studies, a couple of nuclear protein-coding (NPC) gene markers have been used together with the mitochondrial markers (Lu et al., 2014; Staurt et al., 2010). However, the relatively conserved NPC marker was unable to provide sufficient phylogenetic signals to resolve the intrageneric relationships. Compared to coding sequences, the fast-evolving noncoding nuclear sequences are more effective in resolving shallow-scale relationships or species complexes, as has been demonstrated in the studies of neoaves, laurasiatherian

mammals and advanced snakes, etc (Chen et al., 2017; Jarvis et al., 2014; Li et al., 2017). Besides using predesigned intron markers or capture probes to obtain noncoding sequences for phylogenetic analyses, a novel approach known as AFLP (amplified fragment length polymorphism)-Capture, which uses homemade baits generated from the AFLP fragments of one species to capture a large number of homologous anonymous sequences in closely related species, has recently been reported (Li et al., 2019; Vos et al., 1995). This AFLP-Capture approach is cost effective and applicable to all organisms without the need for prior genome information (Li et al., 2019); thus, it is particularly suitable for the study of the interspecific relationships of the Amolops genus. In this study, we collected 70 Amolops samples from 49 different localities in China, representing 22 species and all 6 species groups. By applying the AFLPCapture approach, we obtained not only anonymous nuclear data from the Amolops samples but also mitochondrial genome data. Phylogenomic analyses with both datasets were conducted and compared. The divergence timescale of the Chinese Amolops species was estimated with the nuclear dataset. We also integrated the online Amolops sequences with our mitochondrial data and performed a combined analysis that encompassed ~66% of all known Amolops species. This is the currently most comprehensive phylogenetic study on the genus Amolops. Based on our results, the intrageneric relationships and the species-dispersal routes were discussed.

2. Materials and methods

2.1. Taxon sampling We conducted multiple field excursions to obtain samples of Amolops species in south and southwest China from 2013 to 2018 and collected 70 individuals from 49 different localities, representing 22 species (Figure 1; Table S1). This sampling included ~62% of the known Amolops species in China (Frost., 2019). Hylarana latouchii and Pelophylax nigromaculatus were collected as outgroup species. All specimens were photographed and euthanized. Muscle tissues were obtained from each specimen and preserved in 95% ethanol. Then the specimens were fixed in 10% buffered formalin, later transferred to 70% ethanol and deposited at the Museum of Biology of Sun Yat-sen University (SYS).

2.2. Data acquisition A sequence-capture approach with homemade baits from AFLP fragments (AFLP-Capture) was applied to obtain a large number of anonymous loci from the Amolops and outgroup samples. DNA extraction. Total genomic DNA was extracted from the muscle tissues preserved in 95% ethanol with a TIANamp Genomic DNA Kit (TIANGEN Inc., Beijing, China) following the manufacturer's protocol. All the genomic DNA was diluted to a concentration of 10-50 ng/μl with 1× TE and stored at -20°C for further use. AFLP bait preparation. We chose one sample (SYS a002328) of A. sinensis with high-quality DNA extract (fragments ~ 20 KB) to prepare the AFLP baits.

Briefly, the genomic DNA was digested with two restriction enzymes (MluI and SbfI) and ligated with restriction-associated double-stranded Y adapters. After selective amplification with different combinations of four primers (two biotinylated forward primers: MluI-F-SA and MluI-F-ST and two reverse primers: SbfI-R-SC and SbfI-RSG), the amplicons were pooled and purified and then immobilized on streptavidin beads. The bait-coated beads were resuspended in 50 μL TET buffer and stored at 4°C. These AFLP bait preparation steps are described in greater detail in Li et al. (2019). Library preparation. Genomic DNA (100 ng) was randomly fragmented using 1 μL NEBNext dsDNA fragmentase (NEB) at 37°C for 14 min in a 20 μL reaction volume for each sample. The fragmented products were then purified using AMPure XP beads. The purified DNA was used to prepare an Illumina sequencing library with the NEBNext Ultra DNA Library Prep Kit (NEB) according to the manufacturer's instructions. Each sample was labeled with a unique index sequence so that the samples could be pooled for hybridization capture. Hybridization capture and sequencing. For hybridization capture, 500 ng of the library pooled from 4 samples (125 ng per sample) and 20 μL of bait-coated beads were used in one reaction. The reaction conditions followed previously published protocols (Li et al., 2013; Maricic et al., 2010; Peñalba et al., 2014), but each sample was enriched only one time. The hybridization reaction started at 65°C and was decreased by 3°C every 6 h over 36 h, ending at 50°C, as described by Li et al. (2019).

After hybridization, the captured libraries were amplified for 12 cycles, then pooled in equal amount and sequenced on an Illumina HiSeq X-ten platform using paired-end 150-bp mode.

2.3. Sequence processing Sequence assembly. After demultiplexing and quality trimming using Trimmomatic (Bolger et al., 2014) and FastUniq (Xu et al., 2012), the clean reads of each sample were assembled into contigs using the SPAdes v.3.8.1 genome assembler (Bankevich et al., 2012) and then filtered with CD-Hit-EST (Fu et al., 2012) to reduce redundancy. The sequencing depths for the filtered contigs were calculated with SAMtools version 1.4.1. (Li et al., 2009). Only contigs with a length ≥ 200 bp and an average sequencing depth ≥ 10× were retained for further analysis. Orthologous sequence identification. There were likely both nuclear and mitochondrial sequences in the contigs of all samples. We first sorted out mitochondrial sequences by subjecting the assembled contigs to BLAST searches against the reference sequence, the mitochondrial genome of Amolops ricketti from GenBank (Accession No. KJ546429). For most samples, the mitochondrial contigs covered nearly the whole mitochondrial genome. The mitochondrial genes including 2 rRNA genes, 13 protein-coding genes and 22 tRNA genes (combined into one super gene) were identified from the sequences of each sample and aligned using MUSCLE v3.8 (Edgar, 2004) with default settings. For the remaining nuclear contigs, a mutual best-hit (MBH) strategy (program =

BLASTn, expectation value = 1E-10; NCBI BLAST+ version 2.6.0, Boratyn et al., 2013) was used to identify 1:1 orthology groups (OGs) from all samples. We defined orthology as being likely if bidirectional BLAST best hits were found between the query species and the reference sequence. To make full use of the data, we chose the contig set from the sample for AFLP-Capture bait preparing (SYS a002328) and the largest contig set among all from sample SYS a005363 as reference, and obtained two OG sets via the MBH strategy. The two nuclear OG sets were then combined to obtain the final nuclear OG set. Alignments were done with MUSCLE v 3.8. The ambiguously aligned regions at the terminals were trimmed, and anomalous sequences were removed from each OG with in-house Python scripts following Li et al. (2019). Only loci with data from over 30% of the Amolops samples were retained for further analysis.

2.4. Phylogenetic analysis The nuclear and mitochondrial datasets were concatenated, respectively, and analyzed with maximum likelihood (ML) method implemented in RAxML v.8.0 (Stamatakis, 2014). PartitionFinder (Lanfear et al. 2012) was used to select partitioning schemes and the best-fit models, which suggested one partition for the nuclear dataset and six partitions for the mitochondrial dataset (three partitions each for 12S rRNA gene, 16S rRNA gene and the tRNA super gene, and three partitions for each codon positions of all protein-coding genes). The nucleotide substitution models were GTR+GAMMA and GTR+GAMMA+I for the nuclear and the

mitochondrial datasets, respectively. Branch supports were evaluated with 500 rapid bootstrapping replicates. The species-tree analysis was performed for the nuclear dataset using ASTRAL v.4.7.8 (Mirarab et al., 2014b) under the coalescent model. For each locus, the best ML tree and 200 bootstrapping trees were estimated by using RAxML under the GTR + GAMMA model. These ML trees were used as input files for ASTRAL to calculate the final species tree and branch support.

2.5. Divergence-time estimation Divergence-time estimation was conducted using the MCMCTREE program in the PAML v4.8 package (Yang, 2007) with the uncorrelated rate model (clock = 2). The ML topology from our nuclear dataset was used as the reference tree. Because no fossils of Amolops or neighboring genera have yet been discovered, we used the inference time from Feng et al. (2017) to calibrate the clock, and set the root age to 33.7 million years ago (Mya). A binning strategy was used to reduce sampling error that divided the nuclear data into 10 bins according to evolutionary rates (measured as their overall mean p-distances) (Bayzid & Warnow, 2013; Mirarab et al., 2014a; Zhang et al., 2018). The ML estimates of branch lengths for the 10 bins of alignments were obtained by using the BASEML program in PAML under the GTR + GAMMA model. On the basis of the mean tree depth of the 10 bins, the gamma-Dirichlet prior for the overall substitution rate (rgene gamma) was set at G (1, 3.334), and the gamma-Dirichlet prior for the rate-drift parameter (sigma2 gamma) was set at G (1,

4.5). The MCMC run was first executed for 100,000 generations as burn-in and then sampled every 150 generations until a total of 100,000 samples were collected. Two replicate MCMC runs using random seeds were performed to examine whether similar results were obtained.

2.6. Combined-data analysis The Amolops mitochondrial sequences produced in previous studies were retrieved from GenBank. To reduce redundancy, no more than three samples of the same species from the same sampling area were selected. GenBank accession numbers of the selected sequences are provided in Table S2. A total of 163 sequences of 5 mitochondrial genes (12S rRNA, 16S rRNA, COI, ND2 and CYTB) representing 106 samples were combined with our mitochondrial sequences obtained from AFLPCapture. The combined matrix was aligned using MUSCLE v 3.8. Phylogenetic analysis was performed with RAxML with the settings indicated above.

2.7. Species delimitation The Bayesian species-delimitation method was used to detect signals of species divergence for the putative cryptic species suggested by the phylogenies (Leaché and Fujita, 2010; Rannala and Yang, 2013; Yang and Rannala, 2010). This analysis was conducted using the program BP&P v.3.4 (Rannala and Yang, 2013; Yang, 2015) with both our nuclear and mitochondrial data. The sequence alignments were analyzed under the multispecies coalescent model (MSC) with a reversible-jump

Markov chain Monte Carlo (rjMCMC) algorithm. Gamma-distributed priors (G) were used to specify the population size (θ; θ = 4Nμ, where N is the effective population size and μ is the mutation rate per site per generation) and tree age (τ) (Leaché and Fujita, 2010). Three population-size /tree-age combinations were used in the analyses to allow a range of speciation histories: G (1, 10) for both θ and τ (representing large population size/deep divergence); G (1, 10) for θ and G (2, 2000) for τ (representing large population size/shallow divergence); and G (2, 2000) for both θ and τ (representing small population size/shallow divergence). Each analysis was run for 250,000 generations with a sampling frequency of 5, and the first 10,000 generations were discarded as burn-in. All analyses were run twice to check for consistency.

3. Results 3.1. Statistics of the AFLP-Capture data In this study, we adopted the newly developed AFLP-Capture technique to acquire data for the phylogenetic study of the Chinese Amolops. Home-made baits were prepared with the AFLP fragments from one Amolops sample to capture the homolgous sequences in the other samples through DNA hybridization, which readily enriched a large number of loci in multiple samples. After high-throughput sequencing, approximately 92 Gb of sequence data were obtained. Through a series of bioinformatic processing steps, we finally acquired two molecular datasets for studying the phylogeny of the Amolops species: an anonymous nuclear dataset and a mitochondrial genome dataset. The nuclear dataset contained 242 OGs, for which the

final alignment was 439,733 bp long with 49.7% missing data. The alignment of the mitochondrial sequences was ~15.4k bp long, containing 2 rRNA genes, 13 proteincoding genes and one tRNA supergene (combining 22 tRNAs). There was only 2% missing data for the mitochondrial dataset. Detailed statistics of the Amolops AFLPCapture data are shown in Table S1.

3.2. The Chinese Amolops phylogeny inferred from the AFLP-Capture data The large number of anonymous nuclear loci and the complete mitochondrial genome sequences recovered from the AFLP-Capture experiment enabled comprehensive phylogenetic analyses and parallel comparison between the nuclear and mitochondrial results. The torrent frog phylogenetic tree inferred from the concatenated ML analysis of the 242 nuclear OGs was generally well resolved, with 86% of the nodes exhibiting bootstrap support over 95% (Figure 2). The phylogeny inferred from the mitochondrial sequences also showed high bootstrap support at most of the nodes and yielded a topology very similar to the nuclear tree (Figure 3). Both the nuclear and mitochondrial phylogenies showed clearly two major clades of the Chinese Amolops species. These two major clades largely corresponded to the geographical distribution patterns of the torrent frogs: one clade comprises samples collected from southeast China, in the area east of the Tibetan Plateau and the Yunnan-Guizhou Plateau, while the other clade mainly contained samples from the Yunnan-Guizhou Plateau and the east margin of the Tibetan Plateau in southwest China. Thus, we temporarily named these two clades the East Clade and the West

Clade, respectively. Notably, although A. chunganensis was nested within the West Clade, the samples of this species were collected at several discrete sites across south China (see discussion below). Among the six species groups previously proposed based on morphological characters (Fei et al., 2009), three (the A. ricketti, A. daiyunensis and A. hainanensis species groups) belonged to the East Clade, while the remaining three (the A. mantzorum, A. monticola and A. marmoratus species groups) constituted the West Clade. All three species groups in the East Clade were recovered as monophyletic (BS = 100) in both our nuclear and mitochondrial phylogenies, in line with the morphological delimitation, and the A. hainanensis group was the sister taxon to the other two species groups (BS = 100). In the West Clade, the A. marmoratus species group was the sister taxon to a clade comprising the A. mantzorum and A. monticola groups. It is noteworthy that A. viridimaculatus, a species that used to be considered a member of the A. mantzorum group, was grouped with an unknown species sampled in Medog, Tibet, China, rather than with the other members of the A. mantzorum group, and A. viridimaculatus and the unknown species together formed the sister group to a clade comprising the A. mantzorum and A. monticola groups in both our nuclear and mitochondrial phylogenies. Such placement of A. viridimaculatus renders the A. mantzorum species group nonmonophyletic. The differences between the nuclear and mitochondrial phylogenies were the arrangement of some species within the A. ricketti group and the A. mantzorum group (indicated with red lines in Figure 3). The coalescent species tree estimated with ASTRAL using the nuclear data was

also well resolved, with 82% of the nodes presenting bootstrap support over 90 (Figure S2). The species relationships in the nuclear species tree were very similar to those in the nuclear ML tree. The incongruences between the nuclear species tree and the nuclear ML tree mainly occurred within the A. mantzorum group. However, the nodal supports for the incongruent clades were generally low (BS ≤ 50) in the species tree. By conducting the Bayesian species-delimitation analyses using both the nuclear and mitochondrial data, we verified the two unknown samples collected from Medog, China as a cryptic lineage diverged from A. viridimaculatus. Furthermore, we noticed that samples of A. hongkongensis from Hong Kong and from Guangdong province formed two distinct clades and exhibited apparent genetic differences in both the mitochondrial tree and the nuclear tree (data not shown). Bayesian speciesdelimitation analyses supported that the two molecular clades of A. hongkongensis represented two cryptic species (Figure S3).

3.3. Divergence times of the Chinese Amolops The divergence timescale of the Chinese Amolops was estimated using the nuclear dataset. Our results indicated that the ancestor of the Chinese Amolops appeared in the late Eocene or early Oligocene (31.9 Mya; 95% HPD: 34.1-26.8 Mya; Figure 4). The A. hainanensis species group, including two species (A. hainanensis and A. torrentis) endemic to Hainan Island, was estimated to have separated from its congeners in the Oligocene (24.6 Mya; 95% HPD: 20.1-28.6 Mya), consistent with

the putative formation time of Hainan Island, which was connected to northern Vietnam and southern Guangxi at least in the Eocene (Zhu, 2016), and separated from the continent in the late Oligocene (Mo and Shi, 1987). For the difficult-to-resolve A. mantzorum species group, our estimation showed that most species of this group diverged within 10 - 5 Mya. These species are distributed in the mountains at the eastern edge of the Tibetan Plateau, which have undergone intense uplift since 10 Mya (Favre et al., 2014). Thus, these results supported the notion that speciation of the A. mantzorum group was related to the isolation caused by the uplift of the mountains.

3.4. Combined mitochondrial analysis revealed the origin of the Chinese Amolops Our Amolops sampling was mostly from China, while many Amolops species are distributed on the Indochinese Peninsula as well as the Malay Peninsula (Frost 2019). To increase the taxon sampling outside China and to trace the origin of the Chinese Amolops, we retrieved the available Amolops mitochondiral data from GenBank and combined them with our mitochondrial data. In total, 163 sequences of 5 mitochondrial genes (12S rRNA, 16S rRNA, COI, ND2 and CYTB) representing 106 samples were added. The final matrix contained 39 species from 91 localities, 12 of which are distributed outside China. In the phylogeny inferred from the combined mitochondrial dataset, A. cremnobatus and A. larutensis from the Indochinese Peninsula and the Malay Peninsula formed the sister taxon to a clade comprising all other Amolops. Then, the

remaining Amolops species were divided into two clades, largely corresponding to the aforementioned East Clade and West Clade. Apparently, there were many more species belonging to the West Clade than to the East Clade. In the East Clade, A. spinapectoralis from Vietnam separated from all others at the basal split while in the West Clade, the A. marmoratus group, containing species from Myanmar, Thailand and Yunnan China, separated from all others at the basal split. Besides the A. marmoratus group, the A. monticola group in the West Clade also contains species from both China and the Indochinese Peninsula. These results suggest that the ancestors of the Chinese torrent frogs had come from the south, i.e., the Indochinese Peninsula, along different routes, and further diversified in different environments. Furthermore, the inclusion of more species in the tree indicated that not only the A. mantzorum species group but also the A. monticola and A. marmoratus groups in the West Clade were not monophyletic. A. medogensis, a species formerly considered a member of the A. marmoratus group, was clustered with A. viridimaculatus (an A. mantzorum group member) and an unknown species but not with the other A. marmoratus group members. A. chayuensis, a species formerly considered a member of the A. monticola group, separated at the second basal split of the West Clade, far away from the other members of the A. monticola group. Monophyly of all Amolops species except A. bellulus was supported in the combined mitochondrial tree; A. bellulus was paraphyletic with respect to A. nyingchiensis. Moreover, in our combined mitochondrial tree, several samples used in previous studies were positioned inappropriately (termini indicated with blue in

Figure S4): (i) A. wuyiensis KIZ F93009 was nested within A. ricketti; (ii) A. tuberodepressus CAS 234058 was nested within A. jinjiangensis; (iii) A. kangtingensis (A. xinduqiao) 0700319 and CIB-998, A. jinjiangensis 0700313 and IOZ4373 were nested within A. mantzorum; and (iv) A. granulosus KIZ C92161 was nested within A. lifanensis. These disparities probably represented misidentifications due to the conserved morphology of the Amolops species.

4. Discussion In this study, we conducted a comprehensive phylogenetic study of the genus Amolops with large sampling. The use of the AFLP-Capture technique simultaneously generated sequences of hundreds of anonymous nuclear loci and complete mitochondrial genomes for phylogenetic analysis. The amount of data analyzed herein surpassed all previous studies (Matsui et al., 2006; Ngo et al., 2007; Cai et al., 2007). Two major clades (the East and West Clades) were consistently recovered for the Amolops species in China. Among the six species groups previously proposed based on morphological characters, the three species groups in the East Clade were all recovered as monophyletic, but none of the three groups in the West Clade were monophyletic, suggesting a need for revision. According to our analysis, A. viridimaculatus, A. medogensis and A. chayuensis no longer belong to the species groups to which they were previously assigned, that is, the A. manzorum, A. marmoratus and A. monticola species groups, respectively. To accommodate these extra species, two new species groups shall be proposed: one for A. viridimaculatus,

A. medogensis and the unknown species, the other for A. chayuensis. In addition, according to our combined mitochondrial tree, the A. monticola and A. marmoratus species groups shall be expanded to accommodate species outside China; also, new species groups may need to be proposed for species that are phylogenetically outside the existing species groups, including A. spinapectoralis and a clade comprising A. cremnobatus and A. larutensis. Nevertheless, all of these suggestions for species group revisions require more evidence from future studies using nuclear data from more samples (e.g. A. monticola) plus detailed morphological analyses. The A. mantzorum species group contained species mostly distributed along the eastern margin of the Tibetan Plateau, where mountains and valleys shape a complex topography. The species relationships of this group inferred from our nuclear data and the mitochondrial data were quite different from each other, suggesting a complicated evolutionary history of the frogs. They had probably experienced multiple, nearly concurrent speciation events caused by the rapid uplift of the Tibetan Plateau (Favre et al., 2014). Further detailed studies on different Amolops populations in this area are needed. For the several recently identified species, such as A. tuberodepressus, A. jinjiangensis and A. shuichengensis (Lu et al., 2014; Lyu et al., 2019b), their validities are supported by both our nuclear and mitochondrial data. However, our combined mitochondrial analysis showed that samples of A. kangtingensis used by Lu et al. (2014) (collected from Luhuo, Yajiang, Jiagenba in Sichuan, China) were nested within the clade of A. mantzorum. Thus, the validity of A. xinduqiao [defined as the A. kangtingensis population in western Mt. Zheduo in the Yalong River Basin (including

the localities of Jiagenba, Pengbuxi, Xinduqiao of Kangding plus Luhuo and Yajiang)] requires further study. Cryptic species in the genus Amolops have been increasingly identified in recent years. For example, Chan et al. (2017) found two cryptic species (A. australis and A. gerutu) in the A. larutensis species complex on the Malay Peninsula. A. albispinus, A. sinensis and A. yatseni were once regarded as A. ricketti (Lyu et al., 2019a), and A. indoburmanensis and A. afhanus are cryptic species identified from the A. mamoratus complex (Dever et al., 2012). In this study, our analyses supported that samples of A. hongkongensis from Hong Kong and Guangdong province are two cryptic species, probably resulting from the geographical isolation between Hong Kong and Guangdong province (Figure S3). Samples of A. bellulus from two previous studies may also represent two cryptic species, the sampling localities of the two A. bellulus samples are separated by a river, the Nu Jiang, which likely serves as a geographical barrier for speciation. Thus, these A. bellulus samples are worth further investigation. Furthermore, our unknown Amolops samples collected from the area of Medog were supported by both the nuclear and mitochondrial data as a new species. All of these results indicate that the biodiversity of the genus Amolops remains underestimated. Further field work and detailed population studies are encouraged. From the comprehensive Amolops phylogeny that we constructed using the combined mitochondrial data, a dispersal scenario of the torrent frogs emerges (Figure S5). A clade containing species from Malaysia, Thailand and Vietnam is the sister taxon to all other Amolops, suggesting that the Amolops frogs probably originated on

the southern Indochinese Peninsula or the Malay Peninsula. Then, the frogs spread northward. One group moved across what is now Vietnam and Hainan Island into the east of China and formed the species of the East Clade; Vietnam and Hainan Island span, respectively, the first and second deepest splits within the East Clade. Another group of frogs migrated along the western Indochinese Peninsula to the southern edge of the Tibetan Plateau. Some frogs then spread southward back into the Indochinese Peninsula and diversified there, among which A. chunganensis and A. wenshanensis reentered China at different times, probably from southern Yunnan and Guangxi. A. chunganensis made its way into mountain streams in southeast China (Fei et al., 2009; Pope, 1929; Yuan et al., 2018); thus, A. chunganensis, though belonging to the West Clade, is not restricted to the mountains at the eastern margins of the plateaus and can be sampled at different sites across south China. This group of frogs formed the A. monticola species group. Other frogs of the West Clade spread further north along the eastern edge of the Tibetan Plateau and diversified with the rapid uplift of the mountains (Favre et al., 2014), which formed the A. mantzorum species group. This dispersal scenario, however, is based on the maternally inherited mitochondrial genes. Further study using nuclear data from more samples of Amolops species outside of China will reveal more of the Amolops dispersal history.

5. Conclusion We conducted a comprehensive phylogenetic study of the Amolops frogs in China by analyzing the anonymous nuclear data and the mitochondrial genome data

obtained from 70 samples. Two major clades were revealed for the Chinese Amolops species: one distributed in the east and southeast of China and the other mainly distributed in mountains along the eastern edges of the Tibetan Plateau and the Yunnan-Guizhou Plateau. The three species groups in the east (the A. ricketti, A. daiyunensis and A. hainanensis groups) were recovered as monophyletic, but not for the three groups in the west (the A. mantzorum, A. monticola and A. marmoratus groups), for which reevaluations and revisions are required. The phylogenies constructed in this study not only resolved many intrageneric relationships but also indicated that the biodiversity of the Amolops frogs deserves further investigation. The estimated divergence timescale of the Chinese Amolops showed that the ancestor of the Chinese Amolops appeared in the late Eocene or early Oligocene and that geological events had played a role in the Amolops speciation. By combining the online mitochondrial data in our analyses, we generated the most comprehensive Amolops phylogeny to date, encompassing ~66% of the known species. We found that the torrent frogs dispersed into China from the Indochinese Peninsula largely along two routes. Our study can serve as the basis for future taxonomy and conservation of this genus.

Acknowledgments We thank Jian Wang, Yao Li and Zuyao Liu for their help in the field work. We thank Jianhuan Yang for his provision of some samples. This work was supported by National Natural Science Foundation of China (No. 31872205 and 31672266 to P.

Zhang and No. 31601847 to D. Liang), the Natural Science Foundation of Guangdong Province (No. 2017A030313160 to D. Liang), and Comprehensive Scientific Survey of Luoxiao Mountains Region of Ministry of Science and Technology, P.R.China (No. 2013FY111500 to Y. Wang).

Data Accessibility The raw Illumina sequencing data generated in this study can be downloaded from the NCBI Sequence Read Archive under the BioProject Accession Number PRJNA566076.

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Figure legends Figure 1. Sampling map. Circles represent sampling localities. Numbers in circles correspond to the locality numbers in Table S1.

Figure 2. Phylogeny of the Chinese Amolops species inferred by maximum likelihood through concatenation analysis with 242 anonymous loci obtained from AFLPCapture. Squares at nodes indicate bootstrap support over 95.

Figure 3. Phylogenetic tree of the Chinese Amolops species based on maximum

likelihood analysis of mitochondrial genomes. Black dots represent bootstrap support above 90; red lines indicate clade relationships different from those inferred from the nuclear data.

Figure 4. Time-calibrated topology for the Chinese Amolops species and distribution ranges of the six species groups in China. Divergence times were estimated with MCMCTREE using the nuclear data. Numbers at nodes represent the mean node ages. Blue bars represent the 95% credibility intervals of the node ages with the exact values in brackets.

Figure S1. Amolops mantzorum individuals collected from different populations or even from the same population exhibit various color patterns.

Figure S2. ASTRAL species tree inferred from the 242 anonymous nuclear loci using ML gene trees as input. Numbers at nodes represent bootstrap branch support.

Figure S3. Bayesian species delimitation based on both the nuclear and mitochondrial datasets. Analyses were conducted on A. the Guangdong population and the Hong Kong population of A. hongkongensis; and B. the unknown Amolops species and A. viridimaculatus. The values under each node are speciation probabilities for three combinations of the priors (θ and τ).

Figure S4. Amolops phylogeny inferred from the combined mitochondrial data using maximum likelihood. Black dots represent bootstrap support above 90. Termini marked with blue represent possible misidentifications in previous studies.

Figure S5. Putative dispersal scenario of the Amolops frogs. The currently West Clade and East Clade in China were formed as a result of the different migration route of the Amolops ancestors. Different color of arrows represented the possibly migration route of different Amolops species groups.

Table legends Table S1. Frog samples used in this study and the statistics for sequence processing.

Table S2. Specimens with mitochondrial marker sequences from GenBank.

Highlights 1. Comprehensive phylogenetic analyses of the Chinese Amolops frogs were performed with both anonymous nuclear data and mitochondrial genome data obtained from AFLP-Capture. 2. Two major clades representing eastern and western Chinese Amolops species were revealed. 3. Three previously proposed species groups (the A. mantzorum, A. monticola and A. marmoratus groups) were not monophyletic, suggesting a need for further investigation and revision. 4. The ancestor of the Chinese Amolops was estimated to have appeared in the late Eocene or early Oligocene, and the speciation of the Chinese Amolops was often related to geological events including island formation and mountain uplifts. 5. Combined analysis adding mitochondrial sequences from GenBank revealed the dispersal routes of the Amolops frogs into China from the Indochinese Peninsula.

East Clade West Clade

A. ricketti A. wuyiensis

A. ricketti group A. daiyunensis group A. hainanensis group A. mantzorum group A. moticola group A. marmoratus group

A. albispinus A. sinensis A. yatseni

0.02

A. yunkaiensis A. hongkongensis A. daiyunensis A. hainanensis A. torrentis A. jinjiangensis A. tuberodepressus A. granulosus A. loloensis A. shuichengicus A. mantzorum A. lifanensis A. nyingchiensis A. chunganensis Amolops sp. A. viridimaculatus A. afghanus Pelophylax nigromaculatus Hylarana latouchii

East Clade West Clade

A. ricketti

A. wuyiensis A. albispinus

A. ricketti group

A. yatseni

A. sinensis A. yunkaiensis A. hongkongensis A. daiyunensis A. torrentis A. hainanensis

A. daiyunensis group A. hainanensis group

A. mantzorum A. tuberodepressus A. jinjiangensis A. loloensis

A. mantzorum group

A. granulosus 74

A. shuichengicus A. lifanensis A. nyingchiensis A. chunganensis A. viridimaculatus Amolops. sp. H. latouchii

0.05

P. nigromaculatus

A. monticola group A. mantzorum group A. afghanus

A. marmoratus group

A. ricketti

8.9 (12.4 - 5.7)

A. wuyiensis

12.7 (16.4 - 9.2)

A. yatseni 17.6 (21.8 - 13.5)

A. sinensis

9.3 (12.8 - 6.3) 6.9 (10.0 - 4.4)

A. albispinus

19.1 (23.2 - 14.9)

A. yunkaiensis A. hongkongensis

10.3 (15.0 - 6.1)

24.6 (28.6 - 20.2)

A. daiyunensis A. hainanensis

9.4 (16.2 - 4.1)

A. torrentis A. shuichengicus 8.0 (11.3 - 5.2)

A. jinjiangensis 4.9 (7.4 - 3.0)

A. tuberodepressus

7.3 (10.5 - 4.7)

31.0 (34.1 - 26.8)

9.6 (13.3 - 6.4)

A. granulosus

7.7 (11.0 - 5.0)

A. loloensis

12.2 (16.4 - 8.3)

A. mantzorum 15.8 (20.3 - 11.3)

31.9 (34.5 - 27.6)

17.8 (22.4 - 12.9)

A. lifanensis A. nyingchiensis

10.9 (15.5 - 6.8)

A. chunganensis A. viridimaculatus

12.0 (16.9 - 7.4)

26.1 (30.1 - 21.2)

Amolops sp. A. afghanus Hylarana latouchii

21.2 (27.3 - 13.0)

Pelophylax nigromaculatus 30 Eocene

Oligocene

25

20

15 Miocene

10

5

0 Mya

Pliocene Quater nary

A. ricketti group A. daiyunensis group A. hainanensis group A. mantzorum group A. moticola group A. marmoratus group

Zeng Zhao-Chi: Investigation, Formal analysis, Writing - Original Draft. Liang Dan: Writing - Review & Editing, Project administration, Funding acquisition. Li Jia-Xuan: Methodology. Lyu Zhi-Tong: Resources. Wang Ying-Yong: Conceptualization, Funding acquisition, Resources. Zhang Peng: Conceptualization, Methodology, Supervision, Funding acquisition, Project administration, Writing - Review & Editing.