The east Asian Asiatic toad (also known by the latin name Bufo gargarizans) lives in a wide range of habitats across East Asia including forests, meadows and cultivated land. However, it remains unclear how these toads evolved and became so widespread – partly because it has proved difficult for researchers to clearly define the species and what distinguishes it from other closely-related species of toads (collectively known as Bufo toads).

Othman et al. combined several bioinformatics techniques to study Asiatic toads and 38 other species of bufonid toads from across the globe. This approach found that Bufo toads first emerged in eastern Asia between 14 and 10 million years ago. This coincides with a point in time when large swathes of land in central Asia turned from adequate to sustain toad populations into desert, suggesting the change in climate prompted the toads to migrate eastwards from central Asia. The Bufo toads then divided into two groups of species: one in mainland East Asia and the other in Japan.

Furthermore, the study revealed there is more genetic diversity – that is, more variety in the DNA of individuals – in Asiatic toads than previously thought. The findings also help to define several other species of Bufo toads more clearly and describe a new toad species restricted to the Korean Peninsula, northeastern China and the Russian Primorye region: the Sakhalin toad (Bufo sachalinensis).

This work demonstrates that a large-scale study of many species across the globe can be used to understand how the species evolved and more clearly distinguish one species from another. The findings of Othman et al. will be of interest to both professional and citizen scientists interested in the natural history of Asia. Furthermore, as several species of Bufo toads are in decline in the wild, they provide evidence that may aid future efforts to conserve them.

Evolutionary history is critical in explaining the diversification within and between species, and the dynamics between species and environments in ecological zones (Ricklefs, 2006). Factors such as past geological events, natural dispersion, and anthropogenic changes (Dufresnes et al., 2020; Othman et al., 2020) can drive and induce different evolutionary scenarios such as physiological adaptation, genetic variability, and phenotypic divergence (Luquet et al., 2015). However, understanding phylogeography and the processes contributing to genetic divergence can be more challenging in taxonomic groups distributed across vast ranges. This difficulty results from the ecological responses to variability in environmental conditions and geographical features (Zhao and Yu, 2012). Thus, integrating macroevolution of species groups and microevolution within populations is necessary to understand the evolutionary mechanisms of complex study systems (Li et al., 2018).

Amphibians are an excellent model for studying the factors affecting distribution and evolution. Specifically, true toads in the family Bufonidae (bufonids) are well suited to this area of study due to their high species diversity (Rojas et al., 2018) and adaptive response to past climate change that led to an evolutionary-recent global radiation (Van Bocxlaer et al., 2010). Numerous phylogeographic and systematic studies have characterized the evolutionary diversifications of bufonids, notably in the Holarctic (Dufresnes et al., 2019; Garcia-Porta et al., 2012; Pauly et al., 2004; Recuero et al., 2012; Stöck et al., 2006), the Neotropics (Pramuk, 2006), and in the Western Ghats (Van Bocxlaer et al., 2009). However, Asian Bufo is generally used as an outgroup (Garcia-Porta et al., 2012; Recuero et al., 2012) resulting in only a few studies focusing on the spatiotemporal origin of Bufo in the Eastern Palearctic. Consequently, the resolution of evolutionary diversification patterns in Asian Bufo lineages is limited, and the existing phylogenetic studies are generally geared toward regional sampling and mitochondrial markers (Borzée et al., 2017; Chen et al., 2013; Liu et al., 2000; Macey et al., 1998; Yu et al., 2014). Although some recent taxonomic revisions have used multi-locus data (Fong et al., 2020; Zhan and Fu, 2011), the lack of primary fossils calibrations has resulted in molecular dating estimates that mostly depend on paleogeological events and secondary calibrations. Perhaps as a result, contradicting biogeographical hypotheses have been posited regarding the Asian lineage of Bufo (Borzée et al., 2017; Fu et al., 2005; Igawa et al., 2006; Macey et al., 1998) and inconsistent phylogenetic species boundaries have been proposed (Macey et al., 1998; Liu et al., 2000; Fu et al., 2005; Pyron and Wiens, 2011; Zhan and Fu, 2011).

To date, three biogeographical hypotheses relevant to the initial emergence of the Bufo genus in the Eastern Palearctic have been proposed: (hypothesis 1) the split between a Western and Eastern lineage due to the desertification of Central Asia during the Middle Miocene, c. 12.00 Mya (Garcia-Porta et al., 2012); (hypothesis 2) vicariant speciation at the time of the earliest emergence of a high-altitude Bufo group distributed in Eastern Tibet, followed by a subsequent dispersal to low elevations in the Late Miocene, c. 10.00–5.00 Mya (Macey et al., 1998); and (hypothesis 3) the isolation of an insular endemic clade on the Japanese Archipelago following the drift of the archipelago away from the Eurasian continent in the Late Miocene, c. 6.00 Mya (Igawa et al., 2006). Although differing in the details, the hypotheses proposed here agree on the Miocene paleogeological events as key factors in the segregation of the Eastern lineage of Bufo.

Comparable to the Western Bufo, two or more species of Eastern Palearctic Bufo form species complexes with similar morphologies and unclear taxonomic boundaries, such as the B. gargarizans and B. japonicus complexes (Matsui, 1986; Zhan and Fu, 2011; Arntzen et al., 2013). The divergence of B. gargarizans and B. japonicus complexes from the other Bufo in East Asia, and the radiations within each species complex coincide with the Plio-Pleistocene climatic oscillations (Fu et al., 2005), sea-level fluctuations, and ice age glaciations (Borzée et al., 2017), and selection pressure in the Anthropocene (Hase et al., 2013). Most Bufo species distributed on the East Asian mainland are restricted to the Qinghai-Tibetan Plateau (QTP) and adjacent high-altitude areas. The major exception to this is the B. gargarizans complex that is widely distributed in Northeast Asia. The species colonized its current range from a single source through long dispersal on the Asian mainland (Fu et al., 2005; Zhan and Fu, 2011). Concomitantly, the species expanded and formed a continental lineage of Bufo in septentrional East Asia, here defined as the northern-most range of current ‘B. gargarizans,’ including clades distributed in the Korean Peninsula and the Russian Far East (Matsui, 1986; Maslova, 2016). In contrast to the Asian mainland clades, Bufo septentrional East Asian clades show a marked impact of Pleistocene glaciations on range shifts (Borzée et al., 2017), and possible ice age refugia on the Korean Peninsula, and in the southernmost Amur River Basin (Borzée et al., 2017; Fong et al., 2020). One of the clades is also likely to have been isolated on the Korean Peninsula since the Last Glacial Maximum (LGM), with limited genetic exchange with other clades. This is plausible because of the Yellow Sea level rise during the LGM (Li et al., 2016), as reflected by the monophyly of the Korean B. gargarizans clade (Borzée et al., 2017).

The taxonomic assignment of East Asian toads using their morphometric and geographic distribution included up to 11 distinctive species in the past (see the chronology of taxonomic updates in Figure 1—figure supplement 1). Seven recognized taxa within Bufo were assigned to East Asia, ranging from western People’s Republic of China (hereafter China) to continental East Asia and associated islands on the continental shelf, and the Amur River Basin. Additionally, four taxa were specific to the Japanese archipelago (Matsui, 1986). A systematics revision based on mitochondrial markers then simplified the East Asian mainland Bufo to five members, with synonimization of some monophyletic groups and invalidation of paraphyletic taxa (Liu et al., 2000). In a more recent multi-locus study (Pyron and Wiens, 2011), 10 species were considered valid, although some under sampled taxa remain unresolved (see the type locality distributions and chronology of systematic revision in Supplementary file 1A, Figure 1—figure supplement 1).

Clade sorting based on morphological and life-history characteristics also influences the tree topology of Asian Bufo. Stream-breeders such as B. andrewsi, B. torrenticola, B. stejnegeri, and the former ‘Torrentophryne’ all demonstrate adaptation to the lotic ecosystem and high-altitude environment (Liao et al., 2016). For instance, adults lack tympanums (Tsuji and Kawamichi, 1996). Ancestral state reconstruction indicates that the semi-aquatic natural histories of East Asian Bufo likely arose independently (Fong et al., 2020). Arguably, life-history characteristics may not reflect a phylogenetic relationship, however, such adaptive traits may still help offer explanations in the role of morphological and ecological characters on the systematics of Asian Bufo, and help revisit previous studies in cases of convergence.

Other factors to consider are anthropogenic activities as they have also impacted the current distribution and phylogeny of bufonids, especially in insular regions (Othman et al., 2020). An example is an intergradation between the two B. j. japonicus subspecies in Japan that were originally geographically isolated from each other. Historically, B. j. formosus occurred in the east and B. j. japonicus occurred in the west (Miura, 1995). This was followed by the invasion of northern Japan (i.e., Hokkaido and Sado) by the former after anthropogenic introductions (Hase et al., 2013; Suzuki et al., 2020). The distribution of B. gargarizans has similarly been strongly influenced by human activities, particularly through the traditional medicine trade (Zhan et al., 2020). As a result, contemporary genetic patterns in the species complex show muddled genetic signals (Lee et al., 2021). From this, it can be inferred that the past and ongoing trade may lead to the reduction in local diversity within the B. gargarizans complex.

The issues related to the taxonomic groupings in the B. gargarizans complex are majorly rooted in the inconsistency of the hierarchical systematic ranks used. For instance, the validity of the endemic Taiwan toad, also called Central Formosa toad, B. bankorensis as a species was inconsistent across taxonomic revisions despite its position within the B. gargarizans clade (Matsui, 1986; Liu et al., 2000; Yu et al., 2014). These inconsistencies confuse not only the interpretations of evolutionary history, but also the ecology. For instance, ecologists have regarded B. andrewsi as a species of its own with life-history traits related to adaptation to high altitude (Liao et al., 2016), but systematists considered B. andrewsi as a junior synonym of B. gargarizans (Fu et al., 2005). Clearly, an integrative re-evaluation of East Asian Bufo species is required. Species delimitation via coalescent methods is a promising tool for addressing these inconsistencies, and a notable improvement to conventional molecular phylogenetic analysis. This methodology has been used to untangle taxonomic issues linked to phenotypic variations and cryptic lineages in other Asian toads such as Ingerophrynus celebensis (Evans et al., 2008). Additionally, the inclusion in our species tree topology of some previously undersampled bufonids from the eastern tip of Tibet to the northern South East Asia such as B. pageoti, B. tuberculatus, a high elevation-restricted B. gargarizans subspecies, and B. gargarizans minshanicus (hereafter B. minshanicus), may increase the robustness of our analyses and resolve the inconsistent placement of these clades within the nomenclature.

The spatiotemporal origin of the East Asian Bufo lineage is uncertain and characterized by a limited understanding of the evolutionary processes involved. In addition, species delimitations present a serious taxonomic discrepancy within the B. gargarizans complex, resulting in repeated calls for taxonomic clarification at a fine scale. This manuscript is structured into two sections, with different taxonomic and geographic scales (Figure 1). The first section focuses on resolving the biogeography of Holarctic bufonid species, following the Bufonidae lineage since the breakdown of the West Gondwana. We use a combination of fossilized birth-death calibrations and multi-locus coalescent-based species tree methods to estimate the most probable time and routes of colonization of bufonids into the Palearctic (Figure 1). In the second section, we elucidate the biogeographic pattern of the East Palearctic Bufo and resolve the taxonomy of the B. gargarizans complex. Here, we evaluate the best species tree topology by testing five alternative hypotheses to recover the taxonomic relationship between Asian species of Bufo. We rely on an intensive and widespread sampling, integrated biogeographical analyses, niche differentiation between divergent clades, and model-based species delimitation approach to resolve the taxonomic boundaries of the B. gargarizans species complex, including the recently expanded septentrional East Asian clade (Figure 1).

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Our study addresses biogeographic scenarios explained in two different sections: (1) the biogeography of Holarctic bufonids and (2) the biogeography of Eastern Palearctic Bufo and the taxonomic revision of the species complexes.

Biogeography of Holarctic bufonids

The goal was to refine the time estimates of the split between clades of Holarctic bufonids, in coherence with fossil data (Figure 2, Figure 2—figure supplement 1 and Supplementary file 1B).

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Fossilized birth death dating

The dated species tree using fossilized birth-death showed that following the split of West Gondwana, bufonids first diverged into American clades and African-Eurasian clades. The subsequent splits of genera of both ancestors followed a contemporary timeline, with the first divergence dating from the Early Oligocene to Middle Miocene (Figure 2).

In Eurasia, radiations between the Oligocene and Early Miocene resulted in the segregation of the clades of North African origin c. 26.08 Mya (95% highest posterior density [HPD 95%]/Mya for all dating estimates in Table 1; Figure 2). The emergence of the main extant clade was dated from the Early to Middle Miocene. The monophyletic Bufotes emerged c. 21.72 Mya (Table 1; Figure 2) with the North African-Eurasian group diverging from the Western Himalayan Bufotes c. 19.81 Mya (Table 1; Figure 2). Later, the Iberian Epidalea and its sister genus the Eurasian Strauchbufo emerged c. 10.88 Mya (Table 1; Figure 2). The Palearctic Bufo split into two clades, the Western and the Eastern Palearctic Bufo between the Early and the Middle Miocene c. 14.49 Mya (Table 1; Figure 2). The credible interval showed a considerable overlap in the timing of radiations within Bufo during the Middle Miocene (10.00–15.00 Mya), indicated by the isolation of the European Bufo clade c. 11.03 Mya (Table 1; Figure 2), and a subsequent emergence of the Asian Bufo clade c. 9.99 Mya (Table 1; Figure 2).

Table 1

Clade	Key nodes	Dating analysis methods
		Fossilized birth-death (median [HPD 95%]/Mya)	Yule (median [HPD 95%]/Mya)
i	North Africa-Eurasia origin	26.08 [22.54‒32.51]	25.88 [22.55‒31.81]
ii	Neotropical origin	24.25 [13.21–39.07]	23.40 [13.10–35.61]
a (1)	Emergence of Bufotes (West Himalaya)	21.72 [18.90–25.25]	21.67 [19.63–27.68]
a (2)	Emergence of Bufotes (North Africa-Eurasia)	20.42 [18.10‒23.28]	20.37 [18.79–25.02]
c	Emergence of Palearctic Bufo	14.49 [9.76‒22.70]	14.52 [9.83‒22.58]
d	Radiation of western Palearctic Bufo	11.03 [9.14‒15.42]	11.04 [9.12–15.37]
e	Emergence and early radiation of eastern Palearctic Bufo	9.99 [4.66‒16.57]	10.11 [4.58–6.34]
b	Emergence of Epidalea and Strauchbufo (Eurasia)	10.88 [7.11‒17.78]	10.87 [7.03‒17.51]
h	Emergence of Rhinella (South America)	14.16 [11.16‒22.24]	14.00 [11.15–21.54]
g	Emergence of Incilius (Central America)	11.90 [7.15‒22.40]	11.80 [7.16–21.52]
f	Emergence of Anaxyrus (Nearctic)	9.56 [6.16‒18.14]	9.53 [6.13–17.58]

The earliest split among the American bufonids segregated the Neotropical clades predominantly during the Early Miocene c. 24.25 Mya (Table 1; Figure 2). The most basal divergence was Rhinella in the southern continent c. 14.16 Mya (Table 1; Figure 2), from which the younger Central American Incilius c. 11.90 Mya (Table 1; Figure 2), and Nearctic Anaxyrus may have diverged c. 9.56 Mya (Table 1; Figure 2). The alternative calibrated time tree analysis under a Yule speciation model resulted in a topology and time estimates that generally corresponded to that of the fossilized birth-death analysis (Table 1).

Evolutionary diversification of Eastern Palearctic Bufo

Here, we focused on reconstructing the historical biogeography of Eastern Palearctic Bufo and resolving the taxonomic boundaries of the species complex within the genus. The vast distribution and taxonomic inconsistencies in Eastern Palearctic Bufo warrant a careful examination of the hypotheses proposed by previous studies.

Optimum species tree topology for Palearctic Bufo

The vast distribution range of the Bufo genus in the Eastern Palearctic resulted in cryptic diversity. Hence, we aimed to resolve the topology of Palearctic Bufo lineages (N species =26) following five hypotheses (Figure 3 and Figure 3—figure supplement 1). We derived these hypotheses from ranges, life histories, geological events, and the likelihood of single or multiple origins. Here, our focal taxa included the Palearctic Bufo species and three group members of the ‘Torrentophryne’ genus, a clade paraphyletic to Bufo, to elucidate the validity of the genus and determine its relationship with Asian Bufo (Figure 3 and Figure 3—figure supplement 1). The nested sampling analyses supported an optimum tree topology for the Eastern Palearctic Bufo linked to Miocene geological events with the highest Marginal Likelihood estimation (MLE; Model C: Supplementary file 1C, Figure 3), followed in likelihood by the topology of a single origin for the East Asian Bufo clade (Model E: Supplementary file 1C, Figure 3). Whereas, the topologies structured by life history and morphological trait recovered the lowest likelihoods, and did not favor the monophyly of ‘Torrentophryne’ (Model B: Supplementary file 1C, Figure 3). The best-fit topology of Model C supported three well-resolved monophyletic clades, a Bufotes (PP: 1.0; Figure 3), a Western Palearctic Bufo clade (PP: 0.90; Figure 3), and an Eastern Palearctic Bufo clade (PP: 1.0; Figure 3). Despite weakly recovering the East Asian mainland Bufo clade, the species tree strongly supported the distinction of the Japanese Bufo subclades (PP: 1.0; Figure 3). The alternative topologies from the four suboptimum models (Models A, B, D, and E) were qualified by lower MLE values than that of Model C (details in Supplementary file 1C and Figure 3—figure supplement 1).

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Phylogeny and population structure of East Asian Bufo

We first inferred the haplotype network of both mtDNA and nuclear data using the median-joining method. The haplotype of the concatenated mtDNA resulted in 98 haplotypes (see genetic diversity and neutrality tests in Supplementary file 1D; Figure 4A). The AMOVA based on the six monophyletic clades (N populations =8) recovered from the mtDNA phylogenetic analyses supported the population structure and showed that 55.48% of the molecular variance was attributed to differences among clades (df =5). We found 22.08% of the molecular variance among populations within clades (df =7) and 22.44% of the variance was found within individuals (df =213; Supplementary file 1E). The average fixation index over all the loci tested showed that F_SC =0.50, F_ST =0.78, and F_CT =0.55, and that there was a negative correlation between geographical distance and genetic differentiations (N populations =13; Pearson’ r=–0.059, Figure 4—figure supplement 1). The analysis of the nuclear data (POMC-RAG-1-Rho) from eight populations resulted in a single group of haplotypes (N=54) with a haplotype diversity (Hd) of 0.972 (Figure 4B), an average pairwise difference of 7.667 (±4.533), and a nucleotide diversity of 0.007 (±0.005). Additionally, the Mantel test for nuclear data revealed a low correlation between the geographic distance and genetic variation for the diploid populations of the East Asian Bufo (N population=8; Pearson’s r=0.072, Figure 4—figure supplement 1).

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To increase the phylogenetic resolution of Bufo across East Asia, we enlarged the sampling range to septentrional Eastern Asia where it covered the distributions of B. sachalinensis sachalinensis in the Amur River Basin and the B. sachalinensis cf. sachalinensis subclade restricted to the Korean Peninsula (currently ‘B. gargarizans’). We also conducted two independent molecular phylogenetic analyses using concatenated mtDNA fragments of the control region (CR) and NADH dehydrogenase 2 (ND2); and concatenated the nuDNA of the gene fragments proopiomelanocortin (POMC), recombination activating gene 1 (RAG-1) and rhodopsin (Rho). The phylogenetic trees derived from the mtDNA (N taxa =221) and nuDNA data (N=44) inferred six monophyletic clades, respectively. The mtDNA and nuDNA trees recovered discordant topologies, and they resulted in different composition of clades (Figure 4C and D). We found B. gargarizans from the Asian mainland to be segregated into multiple clades scattered across the nuDNA tree (see details in Table 2, Figure 4). We highlighted other discordance found between the monophyletic B. j. formosus and B. j. japonicus in the mtDNA tree (Clades 1 and 2; Figure 4C), in which both Japanese B. japonicus clustered in a clade based on the nuDNA tree (Clade 4; Figure 4D). In addition, nuDNA resolved B. bankorensis and the septentrional East Asia clades of B. sachalinensis as monophyletic (Figure 4D). Despite these inconsistencies, we found both mtDNA and nuDNA trees to similarly manifest a segregation between the septentrional East Asian clades of B. sachalinensis and B. gargarizans (Figure 4).

Table 2

Distribution range	Clade	Description of cladistics
		Concatenated mtDNA (CR-ND2)	Concatenated nuDNA (POMC-RAG1-Rho)
Japanese Archipelago	Bufo japonicus formosus	Monophyletic (Clade 1)	Grouped together in a clade of Japanese Bufo
Japanese Archipelago	Bufo japonicus japonicus	Monophyletic (Clade 2)
Korean Peninsula	Bufo stejnegeri	Monophyletic (Clade 3)	B. stejnegeri were grouped with Korean B. gargarizans in a nested clade of southeastern mainland and septentrional East Asian B. gargarizans (Clade 3)
Northeastern Mainland Asia	Bufo gargarizans	Monophyletic (Clade)	Contained multiple clades of B. gargarizans restricted to the southeastern and northeastern Asian mainland
Eastern Mainland	Bufo gargarizans	Polyphyletic with a clade of B. bankorensis distributed in Taiwan Island(Clade 5)	Formed multiple clades across Eastern Asian Bufo lineages. A clade grouped with northeastern B. gargarizans. Another clade is polyphyletic with of B. bankorensis of Taiwan Island and B. gargarizans distributed in septentrional East Asia (Clade 3).
Taiwan Island	Bufo bankorensis
Central Mainland	Bufo gargarizans		Formed two distinctive clades: A clade that restricted to western Mainland (Clade 2). Belonged to monophyletic groups of southeastern, western, and northeastern Mainland B. gargarizans
Septentrional East Asia (Korean Peninsula)	Bufo sachalinensis cf. sachalinensis	Monophyletic (Clade 6)	Monophyletic (Clade 3)
Septentrional East Asia (Amur River Basin)	Bufo sachalinensis sachalinensis	Monophyletic (Clade 6)	Monophyletic (Clade 3)

Additionally, we reconstructed a 16S rRNA only tree to include an individual B. gargarizans from Vietnam. The individual did not cluster with East Asian lowlands B. gargarizans, rather it was nested inside a clade of high elevation-restricted species including B. andrewsi and B. tibetanus (Clade C: Figure 4—figure supplement 2).

Diploid genotype clusters

The structure analysis based on the 1030 bp of multilocus POMC-RAG-1-Rho supported two clusters (K=2) within East Asian Bufo, with Mean (LnProb) equal to –827.838 and mean (similarity score) among 10 runs equal to 0.974 (Figure 4D). The first cluster included the populations of Bufo in the western, central, southeastern, and northeastern Asian mainland along with the Japanese Bufo (Figure 4D). We recorded a negligible amount of admixture between population clusters of B. gargarizans in the western mainland, eastern mainland, and the Japanese Archipelago (see admixture portions in Supplementary file 1F and Figure 4D). However, we found significant amounts of admixture in the central and southeastern mainland Asia (Supplementary file 1F, Figure 4D). The second cluster was restricted to B. sachalinensis of septentrional East Asia with significant admixture, ranging from the Korean Peninsula to the Amur River Basin and Sakhalin Island (Supplementary file 1F, Figure 4D). This cluster also included the population of B. bankorensis distributed in Taiwan Island (Figure 4D).

The effect of introgression

The cytonuclear discrepancy (Figure 4) may be the result of introgression or/and incomplete lineage sorting. Thus, we further evaluated the pattern of introgression in our nuclear data (POMC-RAG1-Rho, N=44, nucleotide length =1030 bp) using ABBA or BABA test. To do so, we employed Patterson’s D-statistic to compare the number of allelic ABBA and BABA sites. Here, the D-statistic value we obtained was equal to 1, with the ABBA-BABA pattern calculated among the sites failing to reach equal frequencies (50:50). The probability of specific sites carring the allelic patterns of ABBA or BABA was equivalent to 1, and the number of segregating sites that fit the pattern of ABBA or BABA in at least one population was equivalent to 0. The unsymmetrical frequencies violate the assumption that only incomplete lineage sorting affects the Bufo nuclear tree, and showed the possibility of introgression as a significant factor in shaping the nuclear genetic structure.

Molecular dating and ancestral range

Despite topological discordance, all nuDNA and mtDNA trees consistently revealed a distinction between the clades of Eastern Central Asia and septentrional East Asia in the B. gargarizans complex. Thus, we also provided dating estimates using the nuDNA data set as an alternative to the mtDNA estimates (Table 3). In the context of dating estimates, we considered mtDNA estimates more informative than nuclear estimates based on two factors: (1) the higher number of taxa in the mtDNA tree than in the nuDNA tree resulting to a clearer phylogeographic structure with higher support values for the clades recovered and (2) the clades recovered from the mtDNA tree highly matched the best-supported topology of species tree of Eastern Palearctic Bufo (Figure 2). Overall dating for the major B. gargarizans clades from the nuDNA data set (especially the 95% HPD ranges) were in agreement with the mitochondrial estimates, with nuDNA ingroup nodes slightly younger than mtDNA (Table 3).

Table 3

Key events (node number)	mtDNA (CR-ND2)			nuDNA (POMC-RAG-1-Rho)
	Relaxed molecular clock			Strict molecular clock
	Yule prior (median [HPD 95%]/Mya)	Birth-death prior (median [HPD 95%]/Mya)	Mean(median [HPD 95%]/Mya)	Birth-death prior(median [HPD 95%]/Mya)
Root age of East Asian Bufo	10.47 [7.88‒13.40]	17.93 [11.81‒26.63]	14.20 [9.46‒20.02]	10.12 [6.60‒12.77]
Emergence of Japanese Bufo (A1)	7.75 [5.98‒9.52]	8.70 [6.97‒10.44]	8.23 [6.48‒9.98]	7.64 [4.99‒8.92]
Crown clade of B. j. formosus	3.71 [1.71‒5.92]	4.15 [2.38‒6.13]	3.93 [2.05‒6.03]	‒
Crown clade of B. j. japonicus	2.24 [0.72‒4.33]	1.58 [0.57‒2.84]	1.91 [0.65‒3.59]	‒
Crown clade of B. stejnegeri	3.32 [1.54‒5.24]	2.22 [0.72‒4.12]	2.77 [1.13‒4.68]	4.09 [1.46‒5.43]
MRCA of East Asian mainland Bufo (A2)	8.38 [6.13‒10.88]	14.25 [9.67‒19.44]	11.32 [7.90‒15.16]	‒
Crown clade of B. tibetanus ‒ B. andrewsi	5.21 [3.25‒7.45]	5.26 [3.01–7.66]	5.24 [3.13‒7.55]	‒
Stem node of B. gargarizans complex (A3)	6.85 [4.70‒9.32]	11.25 [7.28‒16.08]	9.04 [5.99‒12.70]	‒
Stem clade of Chinese mainland of B. gargarizans	5.19 [3.16‒6.72]	5.34 [3.45‒7.83]	5.27 [3.31‒7.28]	‒
Segregation between B. gargarizans inhabiting high (B. minshanicus) and low elevation (B. g. gargarizans)	4.70 [3.23‒6.53]	2.68 [1.82‒3.26]	3.69 [2.53‒4.90]	3.77 [2.39‒3.83]
Crown clade of B. andrewsi and B. gargarizans inhabiting high elevated range (B. minshanicus)	2.89 [2.18‒3.54]	2.55 [1.86‒3.28]	2.72 [2.02‒3.41]	2.79 [1.18‒3.10]
Crown clade of B. g. gargarizans in the southeastern Mainland	3.14 [1.92‒4.49]	2.56 [1.48‒3.86]	2.85 [1.70‒4.18]	3.05 [1.04‒3.45]
Nested clades of B. gargarizans popei and B. g. gargarizans in the Central, southeastern and northeastern Mainland	2.21 [1.37‒3.20]	1.28 [0.71‒1.95]	1.85 [1.04‒2.58]	3.65 [1.18‒5.28]
Crown clade of B. bankorensis	1.34 [0.74‒1.94]	1.28 [0.72‒1.84]	1.31 [0.73‒1.89]	1.80 [0.50‒1.80]
Stem clade of septentrional East Asian B. sachalinensis	1.95 [1.55‒2.34]	1.81 [1.38‒2.28]	1.88 [1.47‒2.31]	2.21 [0.93‒2.16]
Crown clade of Korean B. sachalinensis cf. sachalinensis	1.58 [1.18‒1.93]	1.22 [0.82‒1.61]	1.40 [1.00‒1.77]	‒
Crown clade of Russian B. sachalinensis sachalinensis	1.06 [0.62‒1.53]	0.46 [0.19‒0.79]	0.76 [0.41‒1.16]	‒

Here, we compared the three hypotheses related to the phylogeography of the B. gargarizans complex, explained by: QTP vicariance and dispersal (Macey et al., 1998), dominance of long dispersals (Fu et al., 2005), and ice-age refugia (Borzée et al., 2017). Our dating and ancestral range estimates supported the contribution of vicariance and dispersal to the earliest diversification of the East Asian Bufo. We dated the events concerning the basal clade of Eastern Asian Bufo from the Early to Late Miocene c. 14.20 Mya (see HPD 95% in Table 3; Figure 5). The events were subsequently followed by the emergence and isolation of the Japanese Bufo from the Eastern Asian lineage in Late Miocene (Figure 5A1), the Japanese Bufo group then splitting into two distinct species, B. j. formosus and B. j. japonicus, c. 8.23 Mya (Table 3, Figure 5). These events were followed by the radiations within the East Asian Bufo lineage resulting in the independent divergence of B. stejnegeri in the Korean Peninsula, c. 8.90 Mya (Table 3, Figure 5) and a split between the high altitudes East Asian clades: B. tibetanus, B. andrewsi, and B. gargarizans c. 11.32 Mya (Table 3, Figure 5). These East Asian clades are present in the areas of the QTP and likely to have dispersed from high elevation areas (Figure 5A2), resulting in the divergence between B. tibetanus and B. andrewsi, dated between the Pliocene and the Late Miocene c. 5.24 Mya (Table 3, Figure 5).

Figure 5

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We found the combined effects of long dispersal and ice-age refugia to contribute equally to the radiation of the B. gargarizans complex in the Plio-Pleistocene, although the trade of the species over the last millennium has muddled the genetic signature (Figure 5A3). The deepest split within the B. gargarizans complex is estimated to have occurred around the same timeframe and at high elevation, delineating the B. minshanicus clade (B. gargarizans subspecies) in the southwestern to central mainland Asia (i.e., Sichuan and Shaanxi) c. 5.27 Mya (Table 3; Figure 5). Later, B. gargarizans may have dispersed to lower latitudes with the splitting from the monophyletic clade of B. gargarizans gargarizans ranging from southeastern to northeastern mainland Asia (i.e., Shanghai, Zhejiang, Hubei, Jinan, and Shenyang) c. 2.85 Mya (Table 3; Figure 5). A widely dispersed B. gargarizans clade further diverged in the central, southeastern, and northeastern mainland Asia (i.e., Sichuan, Shaanxi, Hubei, and Jiangsu, Dalian) c. 1.85 Mya (Table 3; Figure 5), nested within the B. g. popei clade (Figure 5). The B. bankorensis clade is a more recent divergence c. 1.31 Mya (Table 3; Figure 5), sharing a common ancestor with the southeastern B. g. gargarizans (Table 3, Supplementary file 1G, Figure 5).

We estimate the emergence of B. j. formosus in Japan to have occurred between the Early Pleistocene and the Late Miocene c.3.93 Mya (Table 3, Figure 5), followed by a recent divergence of B. j. japonicus between the Pleistocene and the Pliocene c. 1.91 Mya (Table 3, Figure 5). In Northeast Asia, the B. stejnegeri clade may have independently emerged at the Plio-Pleistocene boundary c. 2.77 Mya (Table 3, Figure 5). Finally, the B. gargarizans clade dispersed and diverged eastward of the Yellow Sea during the Pleistocene c. 1.88 Mya (Table 3, Figure 5), established a population on the Korean Peninsula c. 1.40 Mya (Table 3, Figure 5) and expanded further to the Amur River Basin c. 0.76 Mya (Table 3, Figure 5).

LGM population expansion

We tested the ice-age refugia hypothesis to infer the impact of past Yellow Sea fluctuations on the East septentrional Bufo clades. We demonstrated that the rise in the past Yellow Sea level resulted in population expansions for the Korean and Russian Bufo clades, as showed by significantly negative values of Tajima’s D and Fu’s Fs (refer to Clade 6; Supplementary file 1D). The results were consistent with the Bayesian Skyline Plot, which indicated a recent population expansion in the Amur River Basin clade c. 0.48 Mya (effective population size trajectory (mean [HPD]/Ne) 1.18 [0.30–2.94]) until present (Ne =3.73 [0.23–20.64]) with a mean likelihood: –1930.53 [HPD: −1942.89, –1918.43], Figure 6. In comparison, the populations of the Bufo distributed on the southwestern margin of Yellow Sea consistently declined since the Late Pleistocene c. 0.55 Mya (Ne =5.34 [13.46–1.124]) until present (Ne =3.43 [0.48–11.84]) with a mean of likelihood: –2954.63 [HPD: −2969.60, –2941.08], Figure 6.

Figure 6

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Ecological niche modeling

To clarify the divergence in ecological requirements between B. gargarizans and B. sachalinensis, we examined the niche overlap between the two clades (Figure 7). The habitat suitability model for B. gargarizans (Figure 7A) was characterized by an AUC of 0.9239±0.0185 and a TSS of 0.6741±0.0408, while the model for the B. sachalinensis (Figure 7B) was characterized by an AUC of 0.9632±0.0043 and a TSS of 0.8723±0.0115. The ‘I’ niche overlap statistic between the two models was 0.4198, while the ‘D’ statistic was 0.1566. These values were significantly lower than the average values of the null distribution, with the mean of ‘I’ in the null distribution being 0.9788 (p<0.0001) and the mean of ‘D’ in the null distribution being 0.8546 (p<0.0001). Here, our result showed the overlap to be significantly less than expected, and therefore supporting the segregation in environmental requirements between the two East Asian Bufo clades.

Figure 7 with 1 supplement see all

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Population migration in septentrional East Asia

We then examined the impact of gene flows and migration on local adaptation of the focal B. sachalinensis clades. Along the latitudinal gradient, our results suggested a weak gene flow outwards from the Amur River Basin and B. s. sachalinensis, in comparison with the symmetrical gene flow between the subspecies of B. sachalinensis restricted to the Korean Peninsula and B. gargarizans (Figure 8). This limited gene flow did not hinder the local adaptation of B. sachalinensis clade distributed at northern latitudes.

Figure 8

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In particular, the migration trajectory obtained from the unlinked multi-locus data showed a lack of gene flow with a symmetrical pattern of gene exchange between the populations of Bufo distributed on the northeastern mainland and populations distributed on the Korean Peninsula (refer to migration rates and theta (Θ) estimates: Supplementary file 1H, Figure 8). We demonstrated a comparable and moderate gene flow when focused on the average population migration rate (M) of haploids (2 N m) and diploids (4 N m) of the two populations. The gene flow rate from the northeastern mainland toward the Korean Peninsula (M₁–M₃; Figure 8) was 0.106, and in the opposite direction (M₃–M₁; Figure 8) was 0.214 (Supplementary file 1H). Contrary to the symmetrical migration patterns between the northeastern mainland and Korean Peninsula, we estimated an asymmetrical migration pattern toward septentrional Eastern Asian B. s. sachalinensis in the Amur River Basin (Supplementary file 1H, Figure 8). We detected a higher rate of gene flow into the Amur River Basin, transferred from the eastern mainland and from the Korean Peninsula (Supplementary file 1H, Figure 8). However, we found a lower rate of gene flow from the Amur River Basin toward the northeastern mainland and the Korean Peninsula (Supplementary file 1H, Figure 8).

Additionally, the migration pattern between the two subpopulations of B. stejnegeri in Korea was also symmetrical, with a negligibly low rate of gene flow from the northern toward the southern population, and vice-versa (Supplementary file 1H, Figure 8).

Species boundaries and taxonomy updates

Here, we provided support to delimit the cryptic B. s. sachalinensis distributed in septentrional Eastern Asia (Amur River Basin). The path sampling and Bayes factor analyses generally supported lineage-splitting as the best speciation pattern as opposed to the lumping of the clades of East Asian Bufo (Supplementary file 1I, Figure 9). Out of the eight species delimitation models tested (models A–H; Supplementary file 1I, Figure 9), Bayes factors determined Model A to be the most favorable alternative, receiving the highest support out of all scenarios determined by the path sampling analyses (Supplementary file 1I, Figure 9). Our model supported the split of the East Asian Bufo genus into seven independent taxonomic units (Model A; MLE: –502.801, Bayes factor: 3.198; Supplementary file 1I, Figure 9). These correspond to B. j. japonicus, B. j. formosus, B. andrewsi, B. gargarizans, B. stejnegeri, B. bankorensis, and the clade we refer to as B. sachalinensis (see justification below). Model A also supported a species-level boundary between the two subspecies of B. japonicus: B. j. japonicus and B. j. formosus. The species delimitation model suggested the taxonomic merging of B. sachalinensis cf. sachalinensis clade in the Korean Peninsula with the B. s. sachalinensis clade in the Amur River Basin (Supplementary file 1I, Figure 9). Moreover, Model A validated the split between the clades distributed in septentrional East Asia from B. g. gargarizans clades in East Asia (Model A; Supplementary file 1I, Figure 9).

Figure 9

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Following multiple calls for a taxonomic revision and the various species descriptions and synonimization (Matsui, 1986; Igawa et al., 2006; Borzée et al., 2017; Lee et al., 2021), we suggest the following updates to the taxonomy. It is to be noted that we do not describe any new species as a long list of valid former names is available (Supplementary file 1I, Figure 1—figure supplement 1). We provided the taxonomy updates on the basis of species delimitation analysis (Figure 9), and the following recommendations were corroborated by the allopatric distribution of the delineated clades (Figure 9) and the differentiation in niche requirements (Figure 8). Thus, we presented the most robust taxonomic framework applied to this group since the description of B. gargarizans.

Here, we refer to Bufo gargarizans Cantor, 1842 (see Cantor, 1842) type locality in Supplementary file 1A and Figure 1—figure supplement 1 as B. gargarizans gargarizans (the Zhoushan toad based on the type locality) following the literature (Dubois and Bour, 2010). The subspecies is currently geographically widespread, a consequence of its synonimization with several clades of taxonomically unstable Bufo in the East Asian mainland, including the intergraded subspecies B. gargarizans popei Matsui, 1986, two high elevation-restricted Bufo species described under the epithets of B. andrewsi Schmidt, 1925, and B. minshanicus Stejneger, 1926 (see type localities and ranges in Supplementary file 1A, Figure 1—figure supplement 1). Here, we provide evidence for the independent evolution of B. andrewsi (Figure 9) and therefore the validity of B. andrewsi at the species level, against the previous synonimization (Fu et al., 2005). The distinction between B. bankorensis and B. gargarizans (Figure 8) is supported by the polyphyletic structure in their mtDNA lineage (Figures 4B and 5), further reinforced by the independent evolutionary lineage as shown by the multilocus BFD tree (Figure 9). Hence, we support the species status of B. bankorensis and reject their synonimization (Liu et al., 2000). The species was described under the epithet B. bankorensis Barbour, 1908 (Supplementary file 1A, Figure 1—figure supplement 1).

Most notably, we propose to redefine the Bufo clades in septentrional East Asia (Figure 9), which includes the clades referred to as B. sachalinensis cf. sachalinensis and B. s. sachalinensis in the analysis section. We therefore recommended the elevation of the septentrional East Asia clade currently named B. gargarizans to the species level, under the taxonomic epithet Bufo sachalinensis (Life Science identifier LSID urn:lsid:zoobank.org:pub:14938673-9CEB-4325-BDBC-83A0AC85B8A7, accessible at http://zoobank.org/References/14938673-9CEB-4325-BDBC-83A0AC85B8A7) and under the common name Sakhalin toad. We suggest the Chinese common name Dōng Běi Chán Chú for this species. The species was described under the name B. vulgaris sachalinensis Nikolsky, 1905 (Type locality: Sakhalin Island, Russia, syntypes ZISP 1934–1936 and MNKNU 26290; Supplementary file 1A, Figure 1—figure supplement 1). We dated the split within B. gargarizans from the Middle Pleistocene (Figure 5), and the two species segregated along a diversity of ecological requirements (Figure 9). We then propose the assignment of clades in the Korean Peninsula and the Amur River Basin to different subspecies under B. sachalinensis to reflect evident geographic and genetic distinction. This framework aligns with the lineage-splitting pattern suggested by Model B in the nuDNA species delimitation analysis which provides a model support value only marginally lower than that of Model A (Figure 9), while our mtDNA phylogeny provides robust support for subspecies-level differentiation of these groups. The population on the Korean Peninsula does not have precedence in the taxonomy and it is not the type locality to any Bufo that is not synonymous with B. stejnegeri (Supplementary file 1A, Figure 1—figure supplement 1). Hence, we refer to this evolutionarily significant unit as B. s. cf. sachalinensis.

Our reconstruction of the Holarctic bufonids biogeography using species tree with fossilized birth-death calibration and phylogenetic hypotheses testing verified that the earliest split between Western and Eastern Palearctic Bufo occurred during the Middle Miocene (c. 14.46–10.00 Mya; Table 1), temporally matching with the maximum dust outflows in the deserts of Central Asia (Guo et al., 2002). Subsequently, we retraced the single-origin Asian Bufo in eastern Asia before the split between the continental and Japanese lineages, corroborated by the two Miocene paleogeological events: the rapid uplifts of regions adjacent to the QTP (Hengduan mountains; Xing and Ree, 2017) and the drift of the Japanese Archipelago (Barnes, 2003).

Our revisit of the three phylogeography hypotheses did not favor any specific hypothesis explaining the present geographic distribution of eastern Palearctic Bufo, in disagreement with the long colonization hypothesis (Zhan and Fu, 2011). Instead, our results provided support to the combination of the three elements: vicariance of the western mainland clade, followed by long dispersal, and possible refugia in northeastern Asia during the last ice age. We detected a loss of genetic structure within B. gargarizans clades, possibly due to introgression resulting from the trade of the species over a millennium (Lee et al., 2021). The recent segregation around the Yellow Sea, as shown by the species distribution models and migration pattern, also provides support to the delineation of a septentrional Asian Bufo clade associated with range shift toward northern latitudes. We therefore resurrect the previously described B. sachalinensis and elevate it to the species level. Our findings resolved the taxonomic boundaries in the B. gargarizans complex, and redefine the taxonomic and conservation units: B. g. gargarizans, B. s. sachalinensis, and B. sachalinensis cf. sachalinensis, with the latter waiting for a subspecies description.

This supplementary text includes the supportive information of materials and methods and results used to build our conclusion, such as the extended population genetic analyses, and the details of calibration points used for molecular dating estimation. We include the assessment of model selections for the species tree topology and species delimitation modeling. We also include the detailed information of all data sets used for the reconstruction of the paleogeographic maps for the molecular dating analyses.

All data generated or analysed during this study are either included in the manuscript and supporting files, or submitted online depositories. All Sequences generated in the present study were deposited to the Genbank database [https://www.ncbi.nlm.nih.gov/ 927 genbank/] under the accession number MW081664- MW081847 (CR), MW467646-MW467777 (ND2), MW489915-MW489964 (POMC), MW489986-MW490035 (RAG-1), MW507752-MW507780 (Rho). Input files in the form of BEAST XML generated for all molecular dating analyses and species delimitation modelling are available from the Mendeley Data repository http://dx.doi.org/10.17632/wdtw6kn2t4.1 (Othman et al., 2021).

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Author details

1. Siti N Othman

   1. Laboratory of Animal Behaviour and Conservation, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China
   2. Department of Life Sciences and Division of EcoScience, Ewha Womans University, Seoul, Republic of Korea

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3894-7553

2. Spartak N Litvinchuk

   Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation

   Contribution

   Conceptualization, Data curation, Resources, Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

3. Irina Maslova

   Federal Scientific Center of the East Asia Terrestrial Biodiversity Far Eastern Branch of Russian Academy of Sciences, Vladivostok, Russian Federation

   Contribution

   Project administration, Resources, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Hollis Dahn

   Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Canada

   Contribution

   Data curation, Resources, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Kevin R Messenger

   Herpetology and Applied Conservation Laboratory, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China

   Contribution

   Data curation, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

6. Desiree Andersen

   Department of Life Sciences and Division of EcoScience, Ewha Womans University, Seoul, Republic of Korea

   Contribution

   Formal analysis, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Michael J Jowers

   CIBIO/InBIO (Centro de Investigação em Biodiversidade e Recursos Genéticos), Universidade do Porto, Vairão, Portugal

   Contribution

   Data curation, Resources, Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8935-5913

8. Yosuke Kojima

   Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Japan

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

9. Dmitry V Skorinov

   Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

10. Kiyomi Yasumiba

    Tokyo University of Agriculture and Technology, Tokyo, Japan

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

11. Ming-Feng Chuang

    Department of Life Sciences and Research Center for Global Change Biology, National Chung Hsing University, Taichung, Taiwan

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7328-2577

12. Yi-Huey Chen

    Department of Life Science, Chinese Culture University, Taipei, Taiwan

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0987-2385

13. Yoonhyuk Bae

    Department of Life Sciences and Division of EcoScience, Ewha Womans University, Seoul, Republic of Korea

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

14. Jennifer Hoti

    1. Department of Life Sciences and Division of EcoScience, Ewha Womans University, Seoul, Republic of Korea
    2. Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

15. Yikweon Jang

    Department of Life Sciences and Division of EcoScience, Ewha Womans University, Seoul, Republic of Korea

    Contribution

    Funding acquisition, Project administration, Resources, Writing – review and editing

    For correspondence

    jangy@ewha.ac.kr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7213-7319

16. Amael Borzee

    Laboratory of Animal Behaviour and Conservation, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China

    Contribution

    Conceptualization, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review and editing

    For correspondence

    amaelborzee@gmail.com

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1093-677X

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