# Historical biogeography highlights the role of Miocene landscape changes on the diversification of a clade of Amazonian tree frogs

Diego A. Ortiz, Conrad Hoskin, Fernanda Werneck, Alexandre Réjaud, Sophie Manzi, Santiago Ron, Antoine Fouquet

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## ► To cite this version:

Diego A. Ortiz, Conrad Hoskin, Fernanda Werneck, Alexandre Réjaud, Sophie Manzi, et al.. Historical biogeography highlights the role of Miocene landscape changes on the diversification of a clade of Amazonian tree frogs. Organisms Diversity and Evolution, 2022, 10.1007/s13127-022-00588-2. hal-03869364

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# Organisms Diversity & Evolution

## Historical biogeography of a diverse tree frog clade highlights a key role of Miocene landscape changes on Amazonian diversification

--Manuscript Draft--

<table><tr><td>Manuscript Number:</td><td>ODAE-D-21-00093</td></tr><tr><td>Full Title:</td><td>Historical biogeography of a diverse tree frog clade highlights a key role of Miocene landscape changes on Amazonian diversification</td></tr><tr><td>Article Type:</td><td>Original Article</td></tr><tr><td>Keywords:</td><td>Anura; Dryaderces; Hylidae; Neotropics; Osteocephalus; Pebas; Tepuihyla</td></tr><tr><td>Corresponding Author:</td><td>Diego Ortiz, MSc<br>James Cook University College of Science and Engineering<br>AUSTRALIA</td></tr><tr><td>Corresponding Author Secondary Information:</td><td></td></tr><tr><td>Corresponding Author's Institution:</td><td>James Cook University College of Science and Engineering</td></tr><tr><td>Corresponding Author's Secondary Institution:</td><td></td></tr><tr><td>First Author:</td><td>Diego Ortiz, MSc</td></tr><tr><td>First Author Secondary Information:</td><td></td></tr><tr><td>Order of Authors Secondary Information:</td><td></td></tr><tr><td>Order of Authors:</td><td>Diego Ortiz, MSc<br>Conrad Hoskin<br>Fernanda Werneck<br>Alexandre Réjaud<br>Sophie Manzi<br>Santiago Ron<br>Antoine Fouquet</td></tr><tr><td>Funding Information:</td><td>Center for the study of biodiversity in<br>Amazonia-Labex CEBA<br>(ref. ANR-10-LABX-25-01)<br>GUYAMAZON program (IRD, CNRS,<br>CTG, CIRAD and Brazilian FAPEAM)<br>(062.00962/2018)</td><td>Dr Antoine Fouquet<br>Dr Antoine Fouquet</td></tr><tr><td>Abstract:</td><td>The diversification processes that could explain why Amazonia currently hosts the most species-rich vertebrate fauna on earth remain poorly understood. We studied spatio-temporal speciation patterns in a diverse clade of tree frogs distributed throughout Amazonia (Anura: Hylidae: Osteocephalus, Tepuihyla, Dryaderces); and tested the hypothesis that mega wetlands that occupied western and central Amazonia during Miocene, prevented dispersals during extensive periods. We assessed the group's diversity through DNA-based (16S rRNA) species delimitation to identify Operational Taxonomic Units (OTUs) from 557 individuals encompassing all but three described taxa. We then selected one terminal for each OTU and sequenced their mitogenomes (~14,000 bp) to reconstruct a Bayesian, time-calibrated phylogeny and estimate their ancestral geographic distribution. The ancestral area of each genus was circumscribed to one of the major Amazonian biogeographic areas (western Amazonia, Guiana Shield, and Brazilian Shield, respectively) between ~10–20 Mya, suggesting that each of them diverged and evolved in isolation during this period around the Pebas mega wetland. Even after 10 Mya and the transition to the modern configuration of the Amazon River watershed, most speciation took place in situ (i.e., within each area). The spectacular diversification of Osteocephalus along Andean</td></tr></table>
foothills was probably driven by climatic stability, habitat diversity and the acquisition of new reproductive modes, but also included at least three recent trans-Amazonian dispersals (<6 Mya) of lineages breeding in ponds and riverbeds. Our findings clearly highlight the importance of the major hydrological changes that Amazonia underwent during Miocene on the diversification of organisms.

**Suggested Reviewers:**

Igor Kaefer
Instituto Nacional de Pesquisas da Amazônia: Instituto Nacional de Pesquisas da
Amazonia
kaeferigor@gmail.com
Experience on diversification of Amazonian amphibians

Philippe Kok
University of Lodz
pjrkok@gmail.com
Experience on systematics and evolution of Neotropical amphibians, particularly in the
Guiana Shield
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‘Original Article’

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2 **Historical biogeography of a diverse tree frog clade highlights a key role of Miocene**

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3 **landscape changes on Amazonian diversification**

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4 **Diego A. Ortiz¹,⁵, Conrad J. Hoskin¹, Fernanda P. Werneck², Alexandre Réjaud³, Sophie**

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5 **Manzi³, Santiago R. Ron⁴, Antoine Fouquet³**

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7 **¹College of Science and Engineering, James Cook University, Townsville, Australia**

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8 **²Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia, Manaus,**

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9 **Brazil**

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10 **³Laboratoire Evolution et Diversité Biologique, UMR 5174, CNRS, IRD, Université Paul**

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11 **Sabatier, Toulouse, France**

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12 **⁴Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del Ecuador,**

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13 **Quito, Ecuador**

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14 **⁵Corresponding author. Email: ortizdiego5@gmail.com**

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16 **Running title: Biogeography of *Osteocephalus*, *Tepuihyla* and *Dryaderces***

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18 **ORCID**

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19 **Diego A. Ortiz https://orcid.org/0000-0003-2917-4787**

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20 **Conrad J. Hoskin https://orcid.org/0000-0001-8116-6085**

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21 **Fernanda P. Werneck https://orcid.org/0000-0002-8779-2607**

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22 **Alexandre Réjaud https://orcid.org/0000-0001-8883-703X**

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23 **Sophie Manzi https://orcid.org/0000-0001-5907-6999**

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24 **Santiago R. Ron https://orcid.org/0000-0001-6300-9350**

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25 **Antoine Fouquet https://orcid.org/0000-0003-4060-0281**

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26 Abstract

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27 The diversification processes that could explain why Amazonia currently hosts the most species-rich vertebrate fauna on earth remain poorly understood. We studied spatio-temporal speciation patterns in a diverse clade of tree frogs distributed throughout Amazonia (Anura: Hylidae: *Osteocephalus*, *Tepuihyla*, *Dryaderces*); and tested the hypothesis that mega wetlands that occupied western and central Amazonia during Miocene, prevented dispersals during extensive periods. We assessed the group's diversity through DNA-based (16S rRNA) species delimitation to identify Operational Taxonomic Units (OTUs) from 557 individuals encompassing all but three described taxa. We then selected one terminal for each OTU and sequenced their mitogenomes (~14,000 bp) to reconstruct a Bayesian, time-calibrated phylogeny and estimate their ancestral geographic distribution. The ancestral area of each genus was circumscribed to one of the major Amazonian biogeographic areas (western Amazonia, Guiana Shield, and Brazilian Shield, respectively) between ~10–20 Mya, suggesting that each of them diverged and evolved in isolation during this period around the Pebas mega wetland. Even after 10 Mya and the transition to the modern configuration of the Amazon River watershed, most speciation took place *in situ* (i.e., within each area). The spectacular diversification of *Osteocephalus* along Andean foothills was probably driven by climatic stability, habitat diversity and the acquisition of new reproductive modes, but also included at least three recent trans-Amazonian dispersals (<6 Mya) of lineages breeding in ponds and riverbeds. Our findings clearly highlight the importance of the major hydrological changes that Amazonia underwent during Miocene on the diversification of organisms.

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48 **Keywords** Anura, *Dryaderces*, Hylidae, Neotropics, *Osteocephalus*, Pebas, *Tepuihyla*

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51 INTRODUCTION

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The Neotropics harbor the world's highest species diversity for many animal and plant
groups. The origin and distribution of this astonishing diversity has intrigued biologists for
almost two centuries, notably in Amazonia which encompass >6.5 million km² mostly
covered by rainforest (e.g., Wallace, 1854). Our understanding of the diversification
processes in the Neotropics has accumulated for particular groups over the last decades,
altogether suggesting that Amazonia acted as the major biodiversity source for the entire
continent (Antonelli et al., 2018). Nevertheless, the historical diversification of major
lineages in the Neotropics has been mostly tackled at the continental scale, whereas what
happened within Amazonia is still largely speculative (Cracraft et al., 2020) despite its central
role as cradle. This is at least partly due to the difficulty in including a sufficiently dense
sampling across this broad region and ultimately taxonomically complete and rigorous
temporal estimations of lineage diversification throughout Neogene (2.6-23 Mya).
Several diversification hypotheses have been proposed to explain the origin of
Amazonian diversity. The ones that involve major landscape changes that lead to allopatric
speciation have received most attention (for a review see Leite & Rogers, 2013). For
example, the riverine barrier hypothesis (Wallace, 1854) postulates that populations of
terrestrial organisms were isolated following the formation of the largest Amazonian rivers.
Paleogeographic data suggests that this hypothesis is limited to explain speciation since ~10
Mya to the present, following the establishment of the modern Amazon River watershed
(Albert et al., 2018; Hoorn et al., 2010). Another hypothesis involving speciation predating
10 Mya is the marine incursions hypothesis (Webb, 1995) which postulates that during the
middle Miocene, an extensive mega wetland with marine influence occupied western-central
Amazonia (Pebas system), and thus formed a barrier of unsuitable habitats for surrounding
rainforest organisms between 10-23 Mya (Albert et al., 2018; Hoorn et al., 2010).

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76 Paleogeographic reconstruction by Bicudo et al. (2019), based on models incorporating
1 77 Andean tectonics and mantle dynamics, also suggests an interior wetland with marine
2 3 influence along the eastern side of northern Andes for approximately the same timeframe.
4 78 However, it is important to keep in mind that large uncertainties surround Amazonia's hydro-
5 geological history, as illustrated by different models in Bicudo et al. (2019). The end of Pebas
6 system (draining north to the Caribbean Sea) was succeeded by the onset of the
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8 However, it is important to keep in mind that large uncertainties surround Amazonia's hydro-
9 geological history, as illustrated by different models in Bicudo et al. (2019). The end of Pebas
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101 lowlands and their diversification occurred *in situ* in Amazonia during Miocene (e.g.,
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2 *Adenomera*, Fouquet et al., 2014; *Allobates*, Réjaud et al., 2020; *Amazophrynella*, Rojas et
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4 al., 2018; Phyzelaphryninae, Fouquet et al., 2012; *Synapturanus*, Fouquet et al., 2021a). In
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general, the earliest diversification events of these ground- and understory-dwelling frogs are
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10 seemingly influenced by the presence of ancient mega wetlands (e.g., Pebas system),
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12 separating western vs. eastern Amazonian clades (>10 Mya); and subsequently to the
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14 establishment of the trans-continental Amazon River (~10 Mya), separating northern vs.
15 southern Amazonian clades. However, there have been few biogeographic studies of the
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17 exceptionally diverse arboreal frog fauna in Amazonia, for which biogeographic patterns are
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27 Amazonian dispersals and thus paralleling the pattern observed in birds and primates but in
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37 The diverse tree frog clade formed by the genera *Osteocephalus* (28 species), *Dryaderces*
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41 2018) is endemic to Amazonia and the Pantepui region (Duellman et al., 2016; Jungfer et al.,
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116 The diverse tree frog clade formed by the genera *Osteocephalus* (28 species), *Dryaderces*
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virtually unexplored. Duellman et al. (2016) estimated the divergences among the three genera between ~25–28 Mya and the crown age of *Osteocephalus* at 18.2 Mya (15.5–20.9), but these estimates are suspected to be largely overestimated based on recent genomic studies (Feng et al., 2017; Hime et al., 2021).

The spatial distribution of the three constituent genera produces a striking pattern. *Dryaderces* is endemic to the Brazilian Shield and *Tepuihyla* is restricted to the Pantepui region (Guiana Shield) and adjacent north-western Amazonia, but *Osteocephalus* has diversified extensively throughout Amazonia and is particularly diverse along the Andean foothills and in western Amazonia (Duellman, 2019). Within *Osteocephalus*, five species groups have been so far recognised based on their monophyly and reproductive modes (Jungfer et al., 2013): the *O. alboguttatus* and *O. planiceps* groups are restricted to western Amazonia and reproduce in phytotelmata. In contrast, the remaining three groups are widespread across Amazonia, with the reproduction of the *O. buckleyi* group being associated to streams whereas that of the *O. leprieurii* and *O. taurinus* groups associated to ponds, with the noticeable exception of *Osteocephalus oophagus* Jungfer & Schiesari, 1995 within the latter group which is a phytotelmata-breeder (Jungfer et al., 2013). The large variation in breeding ecology in *Osteocephalus* is of particular interest because it may have contributed to its extensive diversification, enabling co-occurrence of species (Jungfer et al., 2013).

Based on the fact that the crown age of the most recent common ancestor between *Osteocephalus* and *Trachycephalus* (another genus in Lophiohylinae) has been estimated to date back about 23 Mya using genomics (Feng et al., 2017), the diversification of our focal clade must have taken place through the Neogene. The origin of the three major clades (genera) distributed in the Amazonian areas described above (i.e., western Amazonia, Guiana Shield, and Brazilian Shield) either predates 10 Mya and implies a role of the Pebas system as barrier, or is more recent and implies that their divergences occurred across major
151 Amazonian hydrological features notably rivers. Given the higher diversity of *Osteocephalus* 1
2 in western Amazonia, we hypothesize that this region acted as a centre of diversification and 3
4 that depending on the breeding ecology of the different species groups, they dispersed 5
6 throughout the Amazon River watershed more or less efficiently after the demise of the Pebas 7
8 and Acre systems. 9
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To test these assumptions, we gathered a spatially dense sampling of mitochondrial
sequences (16S rRNA) from 557 individuals of the Amazonia-wide tree frog clade formed by
*Osteocephalus*, *Tepuihyla*, and *Dryaderces* that were used to delimit species. We
subsequently obtained entire mitogenomes (one individual per species) to reconstruct a time-
calibrated phylogeny and estimate their ancestral geographic distribution.

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162 MATERIALS AND METHODS
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163 DNA-based species delimitation
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164 We analysed a 16S rRNA fragment as operational delimitation criterion to identify major
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DNA-based species delimitation

We analysed a 16S rRNA fragment as operational delimitation criterion to identify major mitochondrial DNA (mtDNA) lineages that may represent independent species (i.e., general lineage species concept; de Queiroz, 1999, 2007). This locus has been widely used in amphibian systematics (Vences et al., 2005). We generated new sequences from recent field collections (n = 52) using laboratory protocols described in Electronic supplementary material 1– ‘Sequencing of 16S rRNA’). The new sequences were combined with available GenBank sequences of our focal clade (n = 505). This resulted in 16S sequences from 557 individuals (*Osteocephalus*: n = 508; *Tepuihyla*: n = 34; and *Dryaderces*: n = 15); GenBank accessions and associated information to all analyzed sequences is available in Electronic supplementary material 2). The entire dataset was aligned using the E-INS-i strategy in MAFFT 7 (Katoh & Standley, 2013). The alignment was manually reviewed and its extremes trimmed in Aliview 1.26 (Larsson, 2014) to keep the majority of homologous nucleotides
176 (583 bp) among individuals. This dataset encompassed the entire geographic range for the
1 177 three genera (see Electronic supplementary material 3) and included 36 out of 39 currently
2 3 recognized species (92%), with the absence of *Osteocephalus duellmani* Jungfer, 2011
4 178 (which may be a junior synonym of *Osteocephalus festae* Peracca, 1904; Ortiz & Ron, 2020);
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8 179 *Osteocephalus germani* Ron et al., 2012; and *Tepuihyla luteolabris* Ayarzagüena et al., 1993.
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13 The species delimitation was undertaken using three single-locus methods (ABGD,
14 182 mPTP, and GMYC). The Automatic Barcode Gap Discovery (ABGD) is a distance-based
15 183 method which infers a 'gap' existing between intra- and interspecific genetic distances to
16 184 partition the dataset (Puillandre et al., 2012). The analysis was conducted using an
17 185 intraspecific divergence prior 'P' of 0.1-10% (P = 0.001-0.1), X (relative gap width) = 1, and
18 21 number of steps = 30 (Réjaud et al., 2020). We identified the 'gap' at 0.3% (i.e., distances
20 22 above P = 0.003 were interspecific), and then selected the 11th recursive partition (P = 0.005)
23 24 in the ranking of distances, which corresponds to the end of a plateau for number of groups
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26 'species' and thus it was considered stable (Puillandre et al., 2012). Partitions with 'P' values
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28 30 higher than this resulted into very few groups and merging several recognized species.
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37 The multi-rate Poisson Tree Processes (mPTP) is a tree-based method that incorporates
38 192 different levels of intraspecific divergence without input thresholds (Kapli et al., 2017; Zhang
39 40 et al., 2013). We first reconstructed a Maximum Likelihood tree in RAxML 8.2 (Stamatakis,
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42 43 2014) using one partition, 1000 iterations, and the GTRCAT approximation. The outgroup
44 45 was defined as in GMYC analysis (see below). After removing the outgroup, we performed
46 195 mPTP using 50 million iterations of Markov chain Monte Carlo (MCMC), sampling each
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54 The General Mixed Yule Coalescent (GMYC) is also a tree-based method that classifies
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201 2009; Pons et al., 2006). We first reconstructed an ultrametric phylogeny in BEAST 2.6
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7 (Drummond et al., 2006). The substitution model for 16S (one partition) was defined using
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12 al., 2016). We used *Trachycephalus coriaceus* Peters, 1867 as outgroup to constrain the root
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14 age of the tree with a normal prior distribution. This secondary calibration corresponds to the
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time to the crown age of the most recent common ancestor (TMRCA) between
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*Osteocephalus* and *Trachycephalus*, estimated at 23.52 Mya (15.1–32.5) by Feng et al.
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20 (2017) based on 95 nuclear genes (~88,000 bp) and relevant fossil calibration. We ran four
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> 200) and convergence of parameters among independent runs were verified in Tracer 1.7
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(Rambaut et al., 2018). We combined and re-sample the independent tree files (excluding
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9000 trees. From this file, we extracted the Maximum Clade Credibility (MCC) tree in
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TreeAnnotator 2.6 (Bouckaert et al., 2019) excluding an additional initial 10% as burn-in.
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After removing the outgroup, we performed GMYC on the ultrametric tree using the ‘splits’
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R-package (Ezard et al., 2009), an interval of 1–10 Mya, and the single-threshold method
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(Fujisawa & Barraclough, 2013).
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Operational Taxonomic Units (OTUs) were defined by applying a majority rule
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consensus to the results of the three delimitation methods (i.e., a cluster of sequences
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delimitation methods). Finally, we assigned the OTUs to known taxa based on *a priori*
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sequence identification, type locality, and known geographic range (Frost, 2021).
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Dated mitogenomic phylogeny

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across multiple mitochondrial genes (12S, 16S, ND1, ND2, COI, and CytB; Electronic supplementary material 2). Finally, we also generated a complete mitogenome for the outgroup *Trachycephalus coriaceus*.

From the complete mitogenomes and GenBank accessions, we extracted the non-coding (12S and 16S rRNAs) and all coding (CDS) regions considering the reading frame (therefore removing D-Loop and tRNAs) using Geneious v5.4 (https://www.geneious.com). We aligned each gene independently in MAFFT 7 using the strategies: E-INS-i for rRNA genes (multiple conserved domain and long gaps); and G-INS-i for protein-coding genes (global homology; Katoh et al., 2019). Finally, we concatenated the independent alignments in Geneious v5.4. Our final alignment consisted of 51 terminals and 14,098 characters (50% of missing data): 17 terminals including the outgroup had complete mitogenome (~14,000 bp), and 34 terminals had in average 3,555 bp (1,176–5,155). We predefined four partitions: one for combined 12S and 16S rRNAs, and one for each codon position of concatenated protein-coding genes. We estimated the best-fit substitution model for each partition using Bayesian Information Criteria (BIC) as implemented in PartitionFinder 2.1.

We reconstructed a time-calibrated phylogeny using BEAST 2.6. Initially, we used a similar tree model setting as described for GMYC delimitation method (see above); however, some inconsistencies were detected on the parameters’ posterior, probably caused because of the large CI of our calibration prior. Therefore, we used the Calibrated Yule tree model instead as this allows a proper sampling when a single calibration point is used (Heled & Drummond, 2012), as in our case. In absence of relevant fossils for calibration in our focal clade, we constrained the tree root prior using the secondary calibration node estimated by Feng et al. (2017): TMRCA *Osteocephalus-Trachycephalus*, mean = 23.52 Mya, SD = 3.6 Mya, and normal distribution. Each partition was parametrized using the substitution model informed by PartitionFinder 2.1, but excluding the parameter of invariant sites proportion (Jia
276 et al., 2014; Stamatakis, 2016). We used a linked, uncorrelated relaxed clock lognormal of
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2 rate variation among branches for all partitions (Drummond et al., 2006; Duchêne et al.,
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4 2020). MCMC parametrization in BEAST, runs assessment, and obtention of the MCC tree
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7 was similar as described for GMYC method (see above), except that each chain ran 100
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10 million steps.
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15 In addition, using the same file with 9,000 trees (mitogenomic analysis) resulting from
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17 LogCombiner 2.6, we constructed a lineage through time (LTT) plot in ‘ape’ R-package
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19 (Paradis & Schliep, 2019) to explore changes in lineage accumulation over time. Finally, we
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21 conducted a Maximum Likelihood analysis on the mitogenomic matrix in RAxML 8.2
22 (Stamatakis, 2014), using 1000 iterations, and the GTRCAT approximation for the four
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288 Ancestral area reconstruction
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289 We estimated the ancestral geographic range for the genera based on the mitogenomic
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34 phylogeny using the ‘BioGeoBEARS’ R-package (Matzke, 2013). We coded the current
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36 distribution of our ingroup terminals using three areas: western Amazonia, Guiana Shield,
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38 and Brazilian Shield. These areas differ in geological history (Hoorn & Wesselingh, 2010),
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44 The areas are approximately delimited by the rivers: Negro, Madeira, and the lower course of
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46 the Amazon (see Electronic supplementary material 3). The analysis compares the likelihood
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48 of several models of ancestral state reconstruction and their fit to the data, weighted by the
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51 Akaike’s information criterion (AICc; Akaike, 1973). Six models were evaluated: Dispersal-
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54 Extinction Cladogenesis model (DEC), a likelihood version of the Dispersal-Vicariance
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56 Analyses (DIVALIKE), and a Bayesian range-evolution model (BAYAREALIKE). The
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281 In addition, using the same file with 9,000 trees (mitogenomic analysis) resulting from
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282 LogCombiner 2.6, we constructed a lineage through time (LTT) plot in ‘ape’ R-package
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283 (Paradis & Schliep, 2019) to explore changes in lineage accumulation over time. Finally, we
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284 conducted a Maximum Likelihood analysis on the mitogenomic matrix in RAxML 8.2
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289 We estimated the ancestral geographic range for the genera based on the mitogenomic
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34 phylogeny using the ‘BioGeoBEARS’ R-package (Matzke, 2013). We coded the current
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36 distribution of our ingroup terminals using three areas: western Amazonia, Guiana Shield,
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38 and Brazilian Shield. These areas differ in geological history (Hoorn & Wesselingh, 2010),
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44 The areas are approximately delimited by the rivers: Negro, Madeira, and the lower course of
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46 the Amazon (see Electronic supplementary material 3). The analysis compares the likelihood
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54 Extinction Cladogenesis model (DEC), a likelihood version of the Dispersal-Vicariance
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290 We estimated the ancestral geographic range for the genera based on the mitogenomic
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34 phylogeny using the ‘BioGeoBEARS’ R-package (Matzke, 2013). We coded the current
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36 distribution of our ingroup terminals using three areas: western Amazonia, Guiana Shield,
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38 and Brazilian Shield. These areas differ in geological history (Hoorn & Wesselingh, 2010),
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44 The areas are approximately delimited by the rivers: Negro, Madeira, and the lower course of
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46 the Amazon (see Electronic supplementary material 3). The analysis compares the likelihood
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54 Extinction Cladogenesis model (DEC), a likelihood version of the Dispersal-Vicariance
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56 Analyses (DIVALIKE), and a Bayesian range-evolution model (BAYAREALIKE). The
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founder-event speciation (+J). We included these latter three models in the model selection in

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face of recent debate about using this parameter (Klaus & Matzke, 2020; Ree & Sanmartín,

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# RESULTS

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## Species delimitation (16S)

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There were considerable differences in the estimated number of species found among the

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three methods: mPTP = 16, ABGD = 42, and GMYC = 159. The mPTP result (16 species)

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was the most conservative, but several recognized species (morphologically diagnosable)

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clustered together. The ABGD result (42 species) was more in agreement with current

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taxonomy. The GMYC method was deemed to over-split the dataset (159 species). The

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majority rule consensus over the three methods was consistent with the intermediate result

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provided by ABGD, delimiting 42 OTUs (Fig. 1a). Results of each method and consensus are

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in Electronic supplementary material 2 and 4.

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A ‘Taxonomic account’ describing the correspondence between the 42 OTUs and known

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taxa is in Appendix, including a detailed rationale for the addition of eight terminals to the

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mitogenomic matrix, thus leading to 50 putative species. Out of these, 14 OTUs could not be

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associated to any known taxon and were labelled as ‘aff.’ relative to their closest taxon in the

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mitogenomic phylogeny. The geographic distribution of all 50 putative species is illustrated

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in Electronic supplementary material 3. Additionally, interspecific, uncorrected p-distances

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are reported in Electronic supplementary material 1– ‘Table S1’, and Appendix– ‘Genetic

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distances (16S)’.

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## Dated mitogenomic phylogeny

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325 Thirty nine out of 50 nodes in the Bayesian phylogeny were highly supported (~80%), displaying posterior probabilities (PP) ≥ 0.95 (Fig. 1a). Nodes with PP < 0.9 were mostly bracketed by short branches and occurred between clades/terminals with and without complete mitogenome. The topology and bootstrap support (BS) of the Maximum Likelihood (ML) phylogeny was overall consistent with the Bayesian phylogeny, except for four low-supported nodes whose positions varied between analyses (Fig. 1a). One of these corresponds to the ‘*O. mimeticus* group’, which clustered as sister to the *O. buckleyi* group (in Bayesian), and as sister to the *O. leprieurii* group (in ML). We have considered the clade containing *Osteocephalus mimeticus* Melin, 1941 and *O. aff. mimeticus* as a new species group (i.e., ‘*O. mimeticus* group’), and not as part of the *O. buckleyi* group as suggested by Jungfer et al. (2013), because it displays an age of divergence similar to the other five traditional species groups (mean ≥ 10 Mya). In another low-supported node, the clade composed by *Osteocephalus buckleyi* Boulenger, 1882 + *Osteocephalus vilmae* Ron et al., 2012, completely swaps its position with the clade composed by *Osteocephalus camufatus* Jungfer et al., 2016 + *Osteocephalus helenae* Ruthven, 1919 + *O. aff. helenae* 1 + *O. aff. helenae* 2 in the Bayesian and ML trees (Fig. 1a).

341 Our phylogeny (Fig. 1a) is generally consistent with previous studies (Blotto et al., 2020; Duellman et al., 2016; Jungfer et al., 2013; Ron et al., 2016), but improves their resolution on three major aspects: 1) *Osteocephalus* is sister to *Tepuihyla*, with high support (PP = 0.95; BS = 76), instead of *Dryaderces*; 2) most internal nodes within *Tepuihyla* are highly supported, and show a different position for *Tepuihyla exophthalma* Smith & Noonan, 2001 and *Tepuihyla warreni* Duellman & Hoogmoed, 1992 which diverged earlier than *Tepuihyla tuberculosa* Boulenger, 1882 + *T. shushupe*; and 3) internal nodes among *Osteocephalus* species groups are highly supported (except for the ‘*O. mimeticus* group’ position).

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349 *Dryaderces* is estimated to have diverged from *Tepuihyla* + *Osteocephalus* at 19.5 Mya (11.8–26.9); and *Tepuihyla* from *Osteocephalus* at 17.4 Mya (10.4–24.2). Crown ages of the three genera are: *Osteocephalus* [13.1 Mya (7.6–18.4)], *Dryaderces* [11.3 Mya (5.6–17.6)], and *Tepuihyla* [9.8 Mya (5.3–14.7)]. Age information for all nodes depicted in Figure 1a is in Electronic supplementary material 1– ‘Table S2’. Lineage through time plot shows a constant speciation rate (Fig. 1b).

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supplementary material 5), indicating that this node corresponds to the minimum age of colonization of *Tepuihyla* from the Guiana Shield to western Amazonia (mean 8 Mya).

From ~10 Mya to the present, the diversification of each genus continued mostly within their respective ancestral areas. In *Osteocephalus*, the inferred areas for all six species groups during ~10–5 Mya indicate that their ancestors co-occurred in western Amazonia (Fig. 2a,d,e). Posteriorly, the diversification for three out of the six species groups continued essentially within western Amazonia (*O. alboguttatus*, *O. planiceps*, and ‘*O. mimeticus*’ groups). In contrast, one lineage in each of the *O. buckleyi*, *O. leprieurii*, and *O. taurinus* groups dispersed out of western Amazonia relatively recently (last ~6 Mya), and diversified in the rest of Amazonia too (Fig. 2a). These latter three species groups currently contain species restricted to one of the three Amazonian areas, as well as widespread species in all areas (Fig. 2a; Electronic supplementary material 3). The initial dispersals out of western Amazonia are seemingly not be synchronous among the three latter groups; however, some temporal overlap is observed for more recent dispersals at nodes with mean age between ~2–3 Mya (Fig. 2a). The nodes associated to all dispersals display some ambiguity on the inferred ancestral areas in DEC model (Fig. 2a) and across models (see Electronic supplementary material 5, and Appendix– ‘Comments on dispersals within *Osteocephalus*’).

Combined, the models suggest that the dispersion sequence of *Osteocephalus* was: *O. taurinus* group first, from western Amazonia to Guiana Shield and subsequently to Brazilian Shield; *O. buckleyi* group second, from western Amazonia to Brazilian Shield and subsequently to Guiana Shield; and the terminal of *Osteocephalus leprieurii* Duméril and Bibron, 1841 third, from western Amazonia to Guiana Shield and Brazilian Shield equally likely.

## DISCUSSION
398 **Species diversity**

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399 Our final delimitation (n = 50 putative species) altogether with three missing taxa (O.
400 duellmani, O. germani, and T. luteolabris) indicates that the three genera may harbour 53
species. The 14 unidentified OTUs (candidate species) represent a 36% increase of the
current known diversity for the three genera combined (39 species; Frost, 2021; Ortiz & Ron,
402 2018). Most of these 39 species are morphologically diagnosable which makes unlikely the
possibility of taxonomic inflation within the group. The ratio of potential undocumented
diversity in our focal clade (36%) is relatively low compared to what has been found in other
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406 widespread Neotropical frog groups using 16S (e.g., Fouquet et al., 2014, 2021a; Gehara et
407 al., 2014; Vacher et al., 2020). We believe that the main reason for this lower ratio comes
408 from a better taxonomic knowledge because our focal group has attracted more attention in
409 recent years (Blotto et al., 2020; Chasiluisa et al., 2020; Jungfer et al., 2013; Moura et al.,
410 2021; Ron et al., 2012, 2016).

411 Although our DNA-based delimitation is mostly based on a single evidence line
412 (monophyly of lineages using a single-locus) and is thus prone to false positives and
413 negatives (e.g., Hickerson et al., 2006), many OTUs are already confirmed by the overall
correspondence with current taxonomy (Appendix– ‘Taxonomic account’). However, we
414 could identify four OTUs representing cases of false negative since they included more than
415 one nominal species that were readily differentiated by their morphology and advertisement
calls. Therefore, even if this study does not represent an integrative taxonomic review per se,
416 it provides a robust framework for future studies. These further investigations using nuclear
417 DNA (nDNA) and phenotypic data are needed not only to clarify the status of the 14
418 unidentified OTUs but also of widespread ‘species’ that may represent species complexes
419 (i.e., O. helenae, O. leprieurii, and O. taurinus; see Appendix– ‘Taxonomic account’).
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422 Finally, vast under sampled regions remain in Amazonia and Pantepui region, notably in

423 Colombia and Venezuela which may harbour undocumented species.

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## Diversification around the Pebas mega wetland

425

426 Our results suggest that the three genera diverged and were isolated from each other for at

427 least ~10–20 Mya (early–middle Miocene). This period coincides with the existence of the

428 Pebas mega wetland system and the ancestral area of each genus (and the bulk of their subsequent diversification) match the putative suitable rainforest areas during this period

430 (Fig. 2a,b). Therefore, this major hydrological barrier seems to largely explain the deep

431 spatio-temporal pattern of divergence observed in our focal clade. Additional support for the

432 hypothesis that Miocene mega wetlands represented a major barrier preventing dispersals and

433 diversification in western Amazonia comes from the fact that the three genera started to

434 diversify during the last stages of Pebas system (the period that preceded the onset of the

435 transcontinental Amazon River, triggered by intensified Andean uplift). These early

436 diversifications within genera (~10–13 Mya; Fig. 1a) remained mostly *in situ*, except for

437 *Tepuihyla* that may have dispersed westward around this period following the disappearance

438 of the barrier once formed by the northward-drained Pebas (Fig. 2b,c). This specific

439 timeframe was also relevant for the highest diversification period of rocket frogs (*Allobates*)

440 in western Amazonia (10–14 Mya; Réjaud et al., 2020). Since lacustrine habitats are

441 unsuitable for both arboreal and terrestrial frog groups, the retreat of the Pebas system around

442 this period may have favoured both groups.

443 In *Osteocephalus*, the divergence of the ancestors of all six species groups (gr.) was

444 probably driven by the availability of new and diverse rainforest environments along Andean

445 slopes and nearby lowlands following the retraction of mega wetlands in western Amazonia

446 (Hoorn et al., 2010). Also, the concomitant acquisition of new reproductive modes
447 (phytotelmata- and stream-breeding) between 10.4–11.7 Mya from a probable pond-breeding ancestor (Jungfer et al., 2013), may have been important in their diversification and co-occurrence (Fig. 2d,e). According to our phylogeny, the evolution of phytotelmata-breeding from pond-breeding happened in at least two instances in *Osteocephalus* (in the ancestor of *O. alboguttatus* gr. + *O. planiceps* gr., and in *O. oophagus* within the *O. taurinus* gr.), and once in *Tepuihyla* (in the ancestor of *T. tuberculosa* + *T. shushupe*) (Fig. 2a). On the other hand, the evolution of stream-breeding from pond-breeding appeared once in *Osteocephalus* in the ancestor of *O. buckleyi* gr. + ‘*O. mimeticus* gr.’ (Fig. 2a). Therefore, we hypothesize that intensified Andean uplift around 10 Mya (and subsequently) might have provided new reproductive habitats that led to ecological divergence in *Osteocephalus*, such as higher densities of streams and water-filled cavities of bromeliads (which radiated extensively in the northern Andes; Givnish et al., 2014; Zizka et al., 2020).

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## Diversification and dispersion throughout the modern Amazon drainage

The speciation rate for the three genera has been constant (Fig. 1b) and most speciation events have occurred *in situ* with only a few trans-Amazonian dispersals. The onset of the modern Amazon River resulted in the Pebas mega wetland being first reduced in north-western Amazonia vs. the south-western region that remained vastly flooded into the Acre system (7–10 Mya; Hoorn et al., 2010). This large area probably remained unsuitable since it seems to have continued to prevent dispersals even for pond-breeders (see below). This suggests that the first connection for non-flying, rainforest organisms between the three major areas would have been established between western Amazonia and the Guiana Shield, while the Brazilian Shield remained more isolated from the rest, possibly explaining why *Dryaderces* did not disperse out of it. Interestingly, despite this being the first genus into diverge, most of its speciation occurred relatively recently in the lowlands (last ~3 Mya),
with the exception of the deep divergence of *D. pearsoni* (~11.3 Mya) which is the only *Dryaderces* species known to occur close to the Andes (Electronic supplementary material 3).

The relatively low and recent diversification of *Dryaderces* in the Brazilian Shield may be related to the significant climate instability of the eastern Amazonia during the Pleistocene (e.g., Cheng et al., 2013; Silva et al., 2019; Wang et al., 2017).

Assessing diversification within *Osteocephalus* is particularly insightful, given the diversity, distributional patterns, and variation in breeding mode. Our results suggest that the six *Osteocephalus* species groups formed <10 Mya in western Amazonia; however, only three of them dispersed and diversified eastwards relatively recently (last ~6 Mya; Fig. 2a).

These dispersions occurred in one lineage in each of these groups and do not seem to have been synchronous (Fig. 2a). The *O. taurinus* gr. probably dispersed first towards the Guiana Shield, supporting the result discussed above for *Tepuihyla* for an early connection between western Amazonia and the Guiana Shield. On the other hand, the *terra firme* expansion after the cease of Acre system (Albert et al., 2018) may explain the more recent dispersal of the *O. buckleyi* gr. from western Amazonia towards the Brazilian Shield (compared to that of the *O. taurinus* gr.). Nevertheless, the dispersion and diversification of the *O. buckleyi* gr. was not extensive in the latter area (Electronic supplementary material 3).

The *Osteocephalus* groups that diversified in western Amazonia and the Andean foothills (*O. alboguttatus* gr., *O. planiceps* gr., ‘*O. mimeticus* gr.’, and most of *O. buckleyi* gr.) have a high species diversity, and their species have relatively small distributions (Electronic supplementary material 3) and acquired ‘derived’ reproductive modes (i.e., phytotelmata- or stream-breeding vs. the likely ancestral state of pond-breeding; Fig. 2a). In contrast, the clades that contain few and widespread species in the three major Amazonian areas display the ‘ancestral’ pond-breeding mode (*O. leprieurii* within *O. leprieurii* gr., *O. taurinus* within *O. taurinus* gr., and *O. helenae* within *O. buckleyi* gr.; Fig. 2a). The latter species reproduces
497 in ponds associated to black-water flooded forest (*igapó*) on the margin of rivers. This pattern of distribution and reproductive mode suggests that life-traits within our focal clade may be correlated to dispersal potential across rivers (Fouquet et al., 2015) that in turn may modulate diversification.

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Resolving the details of dispersal and speciation events during the Pliocene and Pleistocene (last 5.3 Mya) is complicated because of spatio-temporal uncertainties over how the large tributaries of the Amazon River formed (Albert et al., 2018), whether or not these acted as dispersal barriers for these frogs, and the possibility of river captures (Pupim et al., 2019; Ruokolainen et al., 2019). Nevertheless, the high diversification within *Osteocephalus* suggests that climatic stability in western Amazonia and step environmental gradients along the Andean foothills probably played a significant role on its diversification (Cheng et al., 2013; Ron et al., 2012).

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## Conclusions

We assessed the biogeographic history of a diverse, Amazonia-wide clade of tree frogs. Our dense sampling and mitogenomic reconstruction represent the most robust and complete phylogenetic assessment of *Osteocephalus*, *Tepuihyla*, and *Dryaderces* to date. The biogeographic reconstruction suggest that the genera evolved in isolation around Miocene mega wetlands during a significant period of time. The diversification within genera <10 Mya need be further studied incorporating approaches of population genetics (including nDNA) and niche modelling. These approaches may be particularly relevant for the widespread species to unveil cryptic diversity and population structure, thus offering insights into this period of significant speciation in Amazonia. Our study provides a framework for future research on the systematics and evolution of morpho-ecological traits in these tree frogs.
521 frogs, and adds evidence to biogeographic assessment of arguably the world's most diverse

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522 region.

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523

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524 Acknowledgements

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525 This study is part of DAO's PhD dissertation at James Cook University (JCU), Australia.

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526 DAO thanks JCU for his PhD scholarship. FPW thanks CNPq (Productivity Fellowship) and

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527 the L'Oréal-UNESCO For Women In Science Program. We thank researchers, curatorial

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528 staff and herpetological collections for providing tissue samples: M. Rodrigues (Universidade

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529 de São Paulo, Brazil), A. Lima, C. Ribas, and M. Freitas (Instituto Nacional de Pesquisas da

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530 Amazônia, Brazil), R. Rojas (Universidad Nacional de la Amazonía Peruana, Peru), P. Kok

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531 (University of Lodz, Poland), J. C. Chaparro (Museo de Biodiversidad del Perú, Peru), B.

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532 Noonan (University of Mississippi, USA), I. Valente (Instituto de Desenvolvimento

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533 Sustentável Mamirauá, Brazil), D. Pavan (Ecosfera Consultoria e Pesquisa em Meio

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534 Ambiente Ltda., Brazil), and E. Courtois (Laboratoire Ecologie, Evolution, Interactions des

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535 Systèmes amazoniens, French Guiana). L. Moraes assisted with sample preparation for

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536 shipment; and D. Pazmiño with elaboration of Figures 3–5. DAO thanks to S. Ho for advice

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537 on BEAST analyses, and to organisers of the Taming the BEAST Down Under workshop

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538 2019.

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546 Declarations

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547 Funding

548 This research was funded by the Center for the study of biodiversity in Amazonia-Labex

549 CEBA (ref. ANR-10-LABX-25-01) under ‘SBOTA’ project granted to AF and DAO; and

550 GUYAMAZON program, an international French/Brazilian cooperation program (IRD,

551 CNRS, CTG, CIRAD and Brazilian Fundação de Amparo à Pesquisa do Estado do

552 Amazonas-FAPEAM 062.00962/2018) under ‘COMBIA’ project co-coordinated by AF and

553 FPW.

554

555 Data availability

556 Data associated to all analysed DNA sequences (vouchers, GenBank accessions, taxonomic

557 and geographic information) is available in Electronic supplementary material 2. Newly

558 generated DNA sequences were deposited in GenBank under accessions XXXX–XXXX (to

559 be add upon acceptance) for 16S, and XXXX–XXXX (to be add upon acceptance) for

560 mitogenomes; and final alignments are available in TreeBASE (link to matrices to be add

561 upon acceptance).

562

563 Conflict of interest

564 The authors declare that there is no conflict of interest.

565

566 Authors' contributions

567 DAO and AF conceived the study. DAO, FPW and AF acquired samples and funding. DAO,

568 SM and AF generated DNA sequences. DAO organized data and conducted analyses with

569 contributions of AR and AF. DAO led the writing and preparation of figures with

570 contributions of CJH, FPW, SRR and AF. All authors commented on the final draft.
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Code availability

Not applicable

Ethics approval

Not applicable

Consent to participate

Not applicable

Consent for publication

Not applicable
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910 FIGURES (MAIN TEXT)

<figure><img src="image_3.png" /></figure>
<table>
  <tr>
    <td>934</td>
    <td>Fig. 2 Panel a, ancestral area reconstruction for the diversification of <i>Osteocephalus</i>,</td>
  </tr>
  <tr>
    <td>1</td>
    <td><i>Tepuihyla</i>, and <i>Dryaderces</i> in Amazonia, resulting from the best-fit model (DEC) in</td>
  </tr>
  <tr>
    <td>2</td>
    <td>BioGeoBEARS and based on the mitogenomic phylogeny (Fig. 1a) and three areas (western</td>
  </tr>
  <tr>
    <td>3</td>
    <td>Amazonia 'W', Guiana Shield 'G', and Brazilian Shield 'B'). Species are grouped according</td>
  </tr>
  <tr>
    <td>4</td>
    <td>to the three genera and six <i>Osteocephalus</i> species groups (gr.), with their generalized</td>
  </tr>
  <tr>
    <td>5</td>
    <td>reproductive mode. Species displaying a reproductive mode being the exception within their</td>
  </tr>
  <tr>
    <td>6</td>
    <td>groups are depicted with asterisks (<i>O. vilarsi</i> apparently displays both modes within its</td>
  </tr>
  <tr>
    <td>7</td>
    <td>group; Ferrão et al. 2019). Two time slices show the hypothetic, ancestral distribution of the</td>
  </tr>
  <tr>
    <td>8</td>
    <td>lineages (coloured polygons) at 16 Mya (panel <b>b</b>) and 9 Mya (panels <b>c-e</b>) according to DEC</td>
  </tr>
  <tr>
    <td>9</td>
    <td>model and constrained by the extent of the Pebas and Acre mega wetlands. In panels <b>b-e</b>, the</td>
  </tr>
  <tr>
    <td>10</td>
    <td>current distribution of the lineages is also plotted from 16S records; the species are combined</td>
  </tr>
  <tr>
    <td>11</td>
    <td>in genera or species groups (see distribution of each species in Electronic supplementary</td>
  </tr>
  <tr>
    <td>12</td>
    <td>material 3). An arrow indicates the flow direction of the Amazon River, and at the right-</td>
  </tr>
  <tr>
    <td>13</td>
    <td>bottom of the continent is the time span for that particular landscape configuration following</td>
  </tr>
  <tr>
    <td>14</td>
    <td>Hoorn et al. (2010)</td>
  </tr>
  <tr>
    <td>15</td>
    <td></td>
  </tr>
  <tr>
    <td>16</td>
    <td></td>
  </tr>
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    <td>17</td>
    <td></td>
  </tr>
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    <td>18</td>
    <td></td>
  </tr>
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    <td>19</td>
    <td></td>
  </tr>
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    <td>20</td>
    <td></td>
  </tr>
  <tr>
    <td>21</td>
    <td></td>
  </tr>
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    <td>22</td>
    <td></td>
  </tr>
  <tr>
    <td>23</td>
    <td></td>
  </tr>
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    <td>24</td>
    <td></td>
  </tr>
  <tr>
    <td>25</td>
    <td></td>
  </tr>
  <tr>
    <td>26</td>
    <td></td>
  </tr>
  <tr>
    <td>27</td>
    <td></td>
  </tr>
  <tr>
    <td>28</td>
    <td></td>
  </tr>
  <tr>
    <td>29</td>
    <td></td>
  </tr>
  <tr>
    <td>30</td>
    <td></td>
  </tr>
  <tr>
    <td>31</td>
    <td></td>
  </tr>
  <tr>
    <td>32</td>
    <td></td>
  </tr>
  <tr>
    <td>33</td>
    <td></td>
  </tr>
  <tr>
    <td>34</td>
    <td></td>
  </tr>
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    <td>35</td>
    <td></td>
  </tr>
  <tr>
    <td>36</td>
    <td></td>
  </tr>
  <tr>
    <td>37</td>
    <td></td>
  </tr>
  <tr>
    <td>38</td>
    <td></td>
  </tr>
  <tr>
    <td>39</td>
    <td></td>
  </tr>
  <tr>
    <td>40</td>
    <td></td>
  </tr>
  <tr>
    <td>41</td>
    <td></td>
  </tr>
  <tr>
    <td>42</td>
    <td></td>
  </tr>
  <tr>
    <td>43</td>
    <td></td>
  </tr>
  <tr>
    <td>44</td>
    <td></td>
  </tr>
  <tr>
    <td>45</td>
    <td></td>
  </tr>
  <tr>
    <td>46</td>
    <td></td>
  </tr>
  <tr>
    <td>47</td>
    <td></td>
  </tr>
  <tr>
    <td>48</td>
    <td></td>
  </tr>
  <tr>
    <td>49</td>
    <td></td>
  </tr>
  <tr>
    <td>50</td>
    <td></td>
  </tr>
  <tr>
    <td>51</td>
    <td></td>
  </tr>
  <tr>
    <td>52</td>
    <td></td>
  </tr>
  <tr>
    <td>53</td>
    <td></td>
  </tr>
  <tr>
    <td>54</td>
    <td></td>
  </tr>
  <tr>
    <td>55</td>
    <td></td>
  </tr>
  <tr>
    <td>56</td>
    <td></td>
  </tr>
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    <td>57</td>
    <td></td>
  </tr>
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    <td></td>
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    <td></td>
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    <td>60</td>
    <td></td>
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    <td>61</td>
    <td></td>
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    <td></td>
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    <td></td>
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    <td>64</td>
    <td></td>
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    <td>65</td>
    <td></td>
  </tr>
</table>
959 FIGURES (APPENDIX)

<table>
  <tbody>
    <tr>
      <td>1</td>
      <td>960</td>
      <td><strong>Fig. 3</strong> Dorsolateral views of <em>Osteocephalus. a O. alboguttatus sensu stricto</em>, QCAZ15972,</td>
    </tr>
    <tr>
      <td>2</td>
      <td>961</td>
      <td>Puyo-Canelos road, Pastaza, Ecuador. <strong>b O. mimeticus sensu stricto</strong>, AF4442 (sequenced),</td>
    </tr>
    <tr>
      <td>3</td>
      <td>962</td>
      <td>San José, San Martín, Peru. <strong>c O. buckleyi sensu stricto</strong>, Canelos, Pastaza, Ecuador. <strong>d O.</strong></td>
    </tr>
    <tr>
      <td>4</td>
      <td>963</td>
      <td><em>vilmae</em>, QCAZ51205, Pompeya-Iro road Km 80, Orellana, Ecuador. <strong>e O. mutabor</strong>,</td>
    </tr>
    <tr>
      <td>5</td>
      <td>964</td>
      <td>QCAZ56066, Lorocachi, Pastaza, Ecuador. <strong>f O. mutabor</strong>, QCAZ39588, Río Pucayacu,</td>
    </tr>
    <tr>
      <td>6</td>
      <td>965</td>
      <td>Pastaza, Ecuador. <strong>g O. taurinus sensu stricto</strong>, Reserva Ducke, Amazonas, Brazil. <strong>h O.</strong></td>
    </tr>
    <tr>
      <td>7</td>
      <td>966</td>
      <td><em>oophagus sensu stricto</em>, Reserva Ducke, Amazonas, Brazil. Photographs: <strong>a</strong>, <strong>d-f</strong> by Santiago</td>
    </tr>
    <tr>
      <td>8</td>
      <td>967</td>
      <td>Ron (BIOWEB, Licence: CC BY-NC-ND 4.0); <strong>b</strong> by Antoine Fouquet; <strong>c</strong>, <strong>g-h</strong> by Diego Ortiz</td>
    </tr>
    <tr>
      <td>9</td>
      <td>968</td>
      <td></td>
    </tr>
    <tr>
      <td>10</td>
      <td>969</td>
      <td><strong>Fig. 4</strong> Dorsolateral and ventral views of <em>Osteocephalus. a O. cabrerai</em>, CORBIDI120,</td>
    </tr>
    <tr>
      <td>11</td>
      <td>970</td>
      <td>Tarapoa, Sucumbíos, Ecuador. <strong>b O. helenae</strong>, AF2427 (sequenced), Nassau, Suriname. <strong>c O.</strong></td>
    </tr>
    <tr>
      <td>12</td>
      <td>971</td>
      <td><em>helenae</em>, Amapá, Brazil. <strong>d O. helenae</strong>, Voltaire, French Guiana. <strong>e-f O. aff.</strong> <em>helenae</em> 2 'morph</td>
    </tr>
    <tr>
      <td>13</td>
      <td>972</td>
      <td><em>cabrerai</em>', AF2357 (sequenced), Mapaou, French Guiana. <strong>g O. aff.</strong> <em>helenae</em> 2 'morph</td>
    </tr>
    <tr>
      <td>14</td>
      <td>973</td>
      <td><em>cabrerai</em>', AF3358 (sequenced), Bakhuis, Surinam. <strong>h O. aff.</strong> <em>helenae</em> 2 'morph <em>cabrerai</em>', St.</td>
    </tr>
    <tr>
      <td>15</td>
      <td>974</td>
      <td>Georges, French Guiana. Photographs: <strong>a</strong> by Pablo Venegas (BIOWEB, Licence: CC BY-NC-ND 4.0); <strong>b-h</strong> by Antoine Fouquet</td>
    </tr>
    <tr>
      <td>16</td>
      <td>975</td>
      <td></td>
    </tr>
    <tr>
      <td>17</td>
      <td>976</td>
      <td></td>
    </tr>
    <tr>
      <td>18</td>
      <td>977</td>
      <td><strong>Fig. 5</strong> Dorsolateral views of <em>Osteocephalus. a O. leprieurii sensu stricto</em>, Kaw mountain,</td>
    </tr>
    <tr>
      <td>19</td>
      <td>978</td>
      <td>French Guiana. <strong>b O. leprieurii</strong>, AF1824, Mana, French Guiana. <strong>c O. yasuni sensu stricto</strong>,</td>
    </tr>
    <tr>
      <td>20</td>
      <td>979</td>
      <td>QCAZ69010, Estación Científica Yasuní, Orellana, Ecuador. <strong>d O. yasuni</strong>, QCAZ55994,</td>
    </tr>
    <tr>
      <td>21</td>
      <td>980</td>
      <td>Lorocachi, Pastaza, Ecuador. <strong>e O. deridens</strong>, QCAZ56031, Lorocachi, Pastaza, Ecuador. <strong>f O.</strong></td>
    </tr>
    <tr>
      <td>22</td>
      <td>981</td>
      <td><em>fuscifacies</em>, QCAZ59887, Parque Nacional Llanganates, Pastaza, Ecuador. <strong>g O. aff.</strong> <em>leoniae</em> 2,</td>
    </tr>
    <tr>
      <td>23</td>
      <td>982</td>
      <td>AF4513 (sequenced), San Martín, Peru. <strong>h O. planiceps</strong>, QCAZ55881, Lorocachi, Pastaza,</td>
    </tr>
  </tbody>
</table>
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983 Ecuador. Photographs: **a–b, g** by Antoine Fouquet; **c–f, h** by Santiago Ron (BIOWEB,

984 Licence: CC BY-NC-ND 4.0)
<figure><img src="image_4.png" /><figcaption>Figure1</figcaption></figure>

a

Group

OTU
(16S)

Dryaderces

Tepuihyla

O. taurinus gr.

O. alboguttatus gr.

O. planiceps gr.

O. leprieurii gr.

'O. mimeticus gr.'

O. buckleyi gr.
<figure><img src="image_5.png" /><figcaption>Figure2</figcaption></figure>
Click here to access/download
**Supplementary Material**
Appendix.docx
a

<figure><img src="image_6.png" /></figure>

b

<figure><img src="image_7.png" /></figure>

c

<figure><img src="image_8.png" /></figure>

d

<figure><img src="image_9.png" /></figure>

e

<figure><img src="image_10.png" /></figure>

f

<figure><img src="image_11.png" /></figure>

g

<figure><img src="image_12.png" /></figure>

h

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>h</figcaption></figure>

<figure><img src="image_22.png" /></figure>
Click here to access/download
**Supplementary Material**
ESM1.pdf
Click here to access/download
**Supplementary Material**
ESM2.xlsx
Click here to access/download
**Supplementary Material**
ESM3.pdf
Click here to access/download
**Supplementary Material**
ESM4.pdf
Click here to access/download
**Supplementary Material**
ESM5.pdf
Click here to access/download
**Supplementary Material**
Cover letter_Ortiz.docx