Molecular Phylogenetics and Evolution 162 (2021) 107184

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Molecular Phylogenetics and Evolution

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Parallel diversification of the African tree toad genus (Bufonidae)

H. Christoph Liedtke a,*, Diego J. Soler-Navarro a, Ivan Gomez-Mestre a, Simon P. Loader b, Mark-Oliver Rodel¨ c a Ecology, Evolution and Development Group, Department of Wetland Ecology, Estacion´ Biologica´ de Donana˜ (CSIC), 41092 Sevilla, Spain b Life Sciences Department, Natural History Museum, London SW7 5BD, UK c Museum für Naturkunde – Leibniz Institute for Evolution and Science, Biodiversity Dynamics, Invalidenstr. 43, Berlin 10115, Germany

ARTICLE INFO ABSTRACT

Keywords: African diversity remains underestimated with many cryptic lineages awaiting formal description. An Central important hotspot of amphibian diversification is the Guineo-Congolian rainforest in Central Africa, its richness Amphibia attributable to present day and ancestral range fragmentation through geological barriers, habitat expansion and Diversity hotspot contraction, and the presence of steep ecological gradients. The charismatic Nectophryne tree toads present an Phylogeography interesting case study for diversificationin this region. The two formally described species comprising this genus Lower Guinea forests Guineo-Congolian forest show nearly identical geographic distributions extending across most of the Guineo-Congolian rainforest, but Sympatry show little morphological disparity. Both species harbour extensive genetic diversity warranting taxonomic re­ Character displacement visions, and interestingly, when comparing the subclades within each, the two species show remarkably parallel diversification histories, both in terms of timing of phylogenetic splits and their geographic distributions. This indicates that common processes may have shaped the evolutionary history of these lineages.

1. Introduction for physical barriers. Evidence for these have been found in numerous taxa. For example, the Sanaga River in southern in particular, The Guineo-Congolian rainforest constitutes the second largest but also the Dja, Ogoou´e and Mbini rivers (Fig. 1) have been implicated contiguous area of lowland tropical moist broadleaf forests after the as barriers to gene flowin primates (Anthony et al., 2007; Mitchell et al., Amazonian rainforest (Bele et al., 2015), and boasts an exceptional 2015), reptiles (Kindler et al., 2016), bats (Hassanin et al., 2015), birds species richness and endemism (Myers et al., 2000; Plana, 2004; White, (Huntley and Voelker, 2016) and (Jongsma et al., 2017; 1979). Amphibians are no exception and the region has some of the Leache´ et al., 2019). Centres of ancient forest refugia (Plana, 2004; highest diversity on all of continental Africa (Jenkins et al., 2013). The Fig. 1) and Pleistocene climate stability correlate with the diversification true diversity likely remains underestimated, with broad scale phylo­ patterns of forest dependent amphibian species (Bell et al., 2017; Charles genetic and biogeographic studies in recent years highlighting the need et al., 2018; Leach´e et al., 2019; Portik et al., 2017). A north–south for systematic and taxonomic revisions of major groups of African am­ seasonal inversion, often referred to as a ‘climatic hinge’ along the phibians (Jongsma et al., 2017; Leach´e et al., 2019; Liedtke et al., 2016; southern border of Cameroon as well as a coastal-inland climatic Portik et al., 2017). gradient, seems to have contributed to species diversification in rain The extraordinary species diversity in the Guineo-Congolian region forest trees (Hardy et al., 2013), primates (Mitchell et al., 2015), reptiles has most frequently been attributed to three biogeographic processes: (Freedman et al., 2010) and amphibians (Bell et al., 2017; Jongsma vicariance by rivers acting as either past or present barriers to gene flow, et al., 2017; Leach´e et al., 2019). vicariance by repeated habitat fragmentation and contraction, espe­ For amphibians, these processes have contributed to the diversity of cially of tropical forests, throughout the Oligocene – Miocene right up to representatives of (Portik et al., 2017), the Pleistocene, and dispersal across pronounced ecological gradients (Bell et al., 2017; Charles et al., 2018), Ranidae (Jongsma et al., 2017) followed by local adaptation, resulting in divergence without the need and Rhacophoridae (Leache´ et al., 2019), but not all species share the

* Corresponding author. E-mail address: [email protected] (H.C. Liedtke). https://doi.org/10.1016/j.ympev.2021.107184 Received 25 June 2020; Received in revised form 14 April 2021; Accepted 26 April 2021 Available online 29 April 2021 1055-7903/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184 same degree of sensitivity to the different processes. This idiosyncrasy 1981; Laurent, 1950; Tihen, 1960), form a monophyletic clade that appears to rest, at least in part, on the ecology of species. For example represents an evolutionarily independent lineage to all other African savanna specialized reed (Hyperolius spp.) experienced different bufonids (Liedtke et al., 2016). This represents a third amphibian diversification history to closely related forest specialists (Bell et al., example of a relatively recent Afro-Asian exchange, in addition to Chi­ 2017), and in turn, both types of reed frogs are less restricted by rivers as romantis and Amnirana (Leach´e et al., 2019). This clade of toads are all barriers than is the African foam-nest Chiromantis rufescens (Leach´e habitat specialists, many restricted to forests and Liedtke et al. (2017) et al., 2019) and the forest frog Scotobleps gabonicus (Portik et al., places their divergence from Asian lineages at the early Miocene which 2017). To gain a complete picture of the drivers of diversification of coincides with global expansion of forests and the physical connection of amphibians in this region therefore relies on investigating patterns in Africa and Eurasia through the Arabian Peninsula (Lonnberg,¨ 1929). phylogenetically and ecologically diverse taxa. Currently, there are two formally recognized species in the genus Nectophryne are small, arboreal forest toads (Amphibia: Bufonidae) Nectophryne: N. afra Buchholz and Peters, 1875 and N. batesii Boulenger, found across Central Africa and represent a phylogenetically and 1913 (Barbour, 1938; Frost, 2018). However, recent molecular work ecologically distinct taxon to the above listed amphibians. Species of this suggests cryptic genetic diversity in both taxa (Deichmann et al., 2017; genus are largely restricted to lowland forest and forest edges (Laurent, Liedtke et al., 2016). The two species show sympatric, essentially 1972) and have unique morphological adaptations to an arboreal life­ entirely overlapping distributions (IUCN, 2019; Laurent, 1972; Nobel, style. This includes digits heavily beset with lamellae comparable to 1924), an unexpected biogeographic pattern for sister species with those of geckos (Boulenger, 1900), and a specialized reproductive mode. apparently little morphological, behavioural or niche segregations. In Nectophryne lay relatively few, large eggs in water-filled tree cavities fact, species identification based on morphology alone is difficult and (Channing and Rodel,¨ 2019; Liedtke et al., 2014) and at least one species there are few definitive diagnostic features differentiating the two. The provides post-hatching parental care (Scheel, 1970), which is a rare original description of N. batesii diagnosed that “the snout of N. batesii, evolutionary occurrence in amphibians (Furness and Capellini, 2019; sp. n., [that] is shorter than that of N. afra, and, seen from below, pro­ Schulte et al., 2020; Vagi´ et al., 2019). Nectophryne, together with the jects far less considerably beyond the mouth; seen in profile, it is much genera , and in all likelihood the data deficient, less obliquely truncate” (Boulenger, 1913). Laurent (1987) found that in monotypic Laurentophryne parkeri (Blackburn et al., 2017; Grandison, addition to snout shape differences, N. batesii appears to have longer

Fig. 1. Geographic distribution and sampling effort of Nectophryne afra (above) and N. batesii (below). Maps show IUCN Red List predicted species ranges, morphological and genetic sampling of current study and the known or suspected (“?”) type localities. Political borders, major rivers, current forest extent and past forest refugia (sensu Plana, 2004) are also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184 extremities, and he found that there may be sexual dimorphism in (accession numbers: MT721160-MT721174, MT724570-MT724593, forearm width as well as geographic variation in thigh width when MT733062-MT733084, SI1). comparing N. batesii from western and eastern populations. The gene dataset was supplemented with sequences from GenBank, This study aims to shed light on the evolutionary history of the including COI sequences where available. The complete dataset con­ charismatic genus of tree toad Nectophryne from Central Africa using sisted of 132 sequences (12S: 43 samples; 16S: 46 samples; COI: 15 multiple lines of evidence including mitochondrial and nuclear DNA samples; RAG1: 26 samples; 12 samples having sequences for all four sequence data, morphology and spatial distribution data. Specifically, loci). Wolterstorffinaparvipalmata was included as an outgroup, sourcing we assess whether 1) the morphological traits used to diagnose the two sequences from GenBank, given its phylogenetic affinityto Nectophryne species are reliable, and 2) whether there is any evidence for geographic (Liedtke et al., 2017). or climatic niche partitioning between the two species. Furthermore, we 3) assess the correlation of phylogeographic divergence patterns within 2.3. Phylogenetic inference and across species with known geographic and ecological barriers, and 4) compare the biogeographic history of these unique toads with other 12S and 16S sequences were aligned using the auto settings in SAT´e Central African amphibians that have vastly different ecologies. As these (Liu et al., 2012) with MAFFT as the aligner and RAXML with a GTRCAT toads are phylogenetically, biologically and ecologically distinct to model as the tree estimator. Gap positions in the 16S alignment were cut other studied amphibians in this region, they will add a valuable insight out using GBlocks (Castresana, 2000). To findthe open reading frame for to the understanding of biogeographic patterns of amphibians in the coding genes, COI and RAG1 sequences were aligned and translated Guineo-Congolian rainforest. using TranslatorX (Abascal et al., 2010), using MUSCLE as the aligner. All loci were concatenated and the optimal partitioning scheme was 2. Material and methods determined using PartitionFinder v2.1.1 (Lanfear et al., 2017) based on corrected Akaike Information Criterion (AICc) scores, implementing the 2.1. Sample and specimen acquisition greedy search algorithm with branch lengths unlinked and testing only MrBayes-compatible models. Non-coding genes and each codon position For this study we compiled 44 tissues and/or DNA samples for ge­ for coding genes were treated as individual partitions and the returned netic analyses and 67 adult Nectophryne preserved specimens for optimal scheme was: partition 1: 12S, 16S, CO1 codon 1 and 2, RAG1 morphological analyses. These adult specimens included the type series codon 1, 2 and 3; partition 2: CO1 codon 3. The final alignment had a for both N. afra and N. batesii, and were made available from the length of 2673 bases (12S: 378 bp, 16S: 522 bp, COI: 840 bp, RAG1: 933 following collections: California Academy of Science (CAS), Museum of bp). Comparative Zoology, Harvard (MCZ), Museo Nacional de Ciencias An additive tree on the concatenated alignment was inferred using Naturales, Madrid (MNCN), Museo delle Science, Trento (MUSE), MrBayes v3.2.1 (Ronquist et al., 2012) using the partitioning scheme Museum of Vertebrate Zoology, Berkeley (MVZ), North Carolina and substitution models suggested by PartitionFinder, with Museum of Natural Sciences (NCSM), the Natural History Museum, W. parvipalmata set as the outgroup and an unconstrained branch length London (NHM), Smithsonian Institution, Washington D.C. (USNM), and setting with an exponential prior mean = 10. Using a MCMC the pro­ Museum für Naturkunde, Berlin (ZMB), and research collection of N.L. gram sampled every 1000 iteration of a chain length of 3 000 000, Gonwouo (LG; Yaounde,´ Cameroon). The samples cover the full running 4 chains for 2 runs. The consensus tree was constructed with geographic range of Nectophryne (Fig. 1), though notably, no samples contype = allcompat. were included from the central parts of the Congolian Rainforest, only A time calibrated tree was inferred using BEAST v2.5.1 (Drummond for N. batesii were specimens obtained from the eastern most parts of and Bouckaert, 2015). Loci were partitioned as above and site models their range, the Albertine Rift (only two individuals), and for N. afra, no were inferred and marginalized using the bModelTest method in BEAST genetic material was available for specimens west of the Niger River. (Bouckaert, 2017). Molecular clock models were estimated separately All raw data related to specimens examined (locality, morphology, for mitochondrial (12S, 16S, CO1) and nuclear (RAG1) markers. A path genetics) are provided as supporting information (SI1). sampling analysis (Baele et al., 2012) was used to compare the marginal likelihood estimates of inferences with strict versus relaxed clocks, the 2.2. DNA amplification and sequencing former performing better (50 steps with 250,000 iterations each, Bayes Factor = 160.58). We used gamma priors for the strict clocks and a DNA was extracted from toe, muscle or liver samples stored in > 96% calibrated yule prior for the tree (Gernhard, 2008). The tree was cali­ ethanol or RNAlater using a TECAN Freedom EVO system (TECAN, brated to geological units of time based on the posterior distribution of Mannedorf,¨ Switzerland). Fragments of two mitochondrial genes (12S, node age estimates for the most recent common ancestor of Nectophryne 16S) and one nuclear gene (RAG1) were amplifiedvia Polymerase Chain and Wolterstorffina from a larger, fossil calibrated bufonid phylogeny Reaction (PCR) using puRETaq Ready-to-go PCR Beads (GE Healthcare) published by Liedtke et al. (2017). This phylogeny estimated the di­ in 25 µL reactions with primers and cycling conditions detailed in the vergences times of the Nectophryne splitting from Wolterstorffinato have supporting information (SI 2). RAG1 reactions were stabilized by adding occurred at a mean of 15.3 million years with the posterior ages for that 2 µL of Q-Solution (Qiagen, Venlo, The Netherlands). Unincorporated node being log normally distributed with a standard deviation of 1.15 primers and dNTPs were removed from PCR products using Antartic million years. This distribution was set as a prior constraint for that node (Exo-Sap purification) and the subsequent sequencing reactions were in the BEAST inference. We conducted a total of four MCMC searches performed using the BigDye kit by Thermo Fisher Scientific (Waltham, with 5 million generations each, sampling every 5000th iteration, MA, USA) on both forward and reverse sequences using the manufac­ including an additional MCMC search on priors only and convergence turer recommended PCR conditions. The PCR products were purified and effective sample sizes (ESS) of parameters in the log files were using Sephadex (GE Healthcare, Chicago, USA) and Sanger sequencing visually inspected using Tracer. Posterior trees were combined with a of these products in both directions was performed using an ABI PRISM burnin of 10% per run using LogCombiner v2.5.1, and summarized as a 3130xl Genetic Analyzer by Thermo Fisher Scientific (Waltham, MA, maximum clade credibility (MCC) tree based on median node heights USA). All lab work was conducted at the Biological Station of Donana.˜ using TreeAnnotator v2.5.1. Sequence quality was assessed by visual inspection of chromatograms using Geneious v.11.0.5 (https://www.geneious.com). Reverse- 2.4. Haplotype networks and isolation by distance complementary strands were aligned and contigs and primers were trimmed. All newly generated sequences were accessioned on GenBank Haplotype networks were constructed using the R package pegas

3 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184 v0.11 (Paradis, 2010), using pairwise sequence differences for both the Discovery V8 stereo microscope (Carl Zeiss AG, Oberkochen, Germany). concatenated mitochondrial loci (12S, 16S and COI; 46 samples) and the Only adult specimens (24 N. afra and 43 N. batesii) were included and nuclear locus (RAG1; 26 samples). Networks were constructed for each ten external morphological measurements were taken: snout-urostyle species separately and samples were coded based on the five biogeo­ length (SUL), internarial distance (InD), inter-orbital distance (IoD; at graphic areas defined below (section 2.8). the narrowest part), eye diameter (EyD), canthus length (CaL), head Isolation-by-distance analyses were also performed for the two spe­ width (HeW; at the widest part), femur length (FeL), tibia length (TiL), cies separately, using the shortest geographic distances between GPS tarsus length (TaL) and foot length (FoL; distance between proximal end coordinates (calculated using the distm function in the R package geo­ of the inner metatarsal tubercle to the distal end of the longest phalange, sphere v1.5–10 (Hijmans, 2019), with a ’Vincenty’ (ellipsoid) great- including toe disc). All statistical analyses were performed in R. SUL, circle-distance function) and a genetic distance matrix reflecting the TiL, FoL and IoD were square root transformed to better conform to patristic distances estimated by MrBayes (i.e. branch lengths). Correla­ normality. The morphological space was explored with a principal tions between the two distance matrices for each species were tested component analysis and MANOVAs, followed by univariate comparisons using a permutational Mantel’s test (999 replicates) using the ade4 made using either a Student’s t-test or an ANOVA. To investigate dif­ package (Bougeard and Dray, 2018). The localities from the Albertine ferences in allometry (i.e. differences in morphological features inde­ Rift were excluded for this analysis as they presented clear geographic pendent of body size), linear regressions of clades or species on traits outliers that skewed the analysis. were performed with SUL (body size) as a covariate, testing for a sig­ nificant interaction term between them. If the interaction was not sig­ 2.5. Identification of genetic clades nificant, it was dropped from the model. Qualitative features including the number of tubercles and number Preliminary genetic barcoding of specimens suggested substantial of lamella and their colour on feet and hands (ordered from outer-most mitochondrial (16S rRNA) genetic divergence within the two formally to inner-most digit), extent of digital webbing and colour and patterns of recognized species. Based on the concatenated sequence tree estimated the ventrum were also recorded, but as these did not show consistent with BEAST, the species clades were sub-divided into genetic clades with variation between genetic clades, they were not included in further completely supported phylogenetic monophyly and geographic unity. analysis, though made available in SI1. No clear secondary sexual Uncorrected pairwise sequence distance was also checked for 16S characters could be determined and so sex was only assigned in cases (keeping only non-gap positions using GBLOCKS), to test if individuals where ova were visible either through previously made incisions or within subclades show less then 3% sequence divergence. This threshold distended abdomens (n = 12). Although sexual dimorphism was not has been shown to correlate well with species divergence in amphibians apparent, we cannot completely rule it out. Laurent (1987) found that (Vences et al., 2005), although it should not be treated as a hard males have thicker forearms and narrower eyelids, but these being threshold. These identified genetic clades were used for subsequent partly soft tissue traits, are strongly influenced by preservation condi­ biogeographic, morphological and phylogenetic species delimitation tion. In this study, the low number of indisputably sexed individuals did analyses. not permit robust analyses to test sexual dimorphism in morphological traits. In all plots we indicate which are females, but we did not address 2.6. Species delimitation sexual dimorphism statistically. To determine morphological features that are informative for species We used a multispecies coalescent approach implemented in BPP identification, we compared the morphology of specimens for which v4.3.0 (Flouri et al., 2020) to perform joint species tree inference and genetic barcodes were also available (12 N. afra and 17 N. batesii). species delimitation (analysis type A11). Specifically, BPP was utilized Qualitative inspection of specimens revealed few such informative to confirm the robustness of the five genetic clades identified by the morphometric characters and species assignation of non-barcoded BEAST and MrBayes inferences as phylogenetic operative taxonomic specimens was ultimately based on head shape (as detailed in the type units. This method calculates posterior probabilities for competing description of N. batesii) as well as the presence or absence of a lighter models of species delimitation accounting for incomplete lineage sorting coloured line connecting the upper lip to the tip of the snout (from here using a Bayesian framework. The alignments of all mitochondrial loci on referred to as the medial cleft). Compared to N. batesii, N. afra showed were concatenated and treated as a single locus and the RAG1 alignment a more protruding rostrum when viewed laterally and had a more as a second locus, and individuals were assigned to one of the five ge­ defined line along the medial cleft and upper lip (Fig. 2). As genetic netic clades of interest. The hypothesized relationship of these clades for subclades showed geographic allopatry (see results), subsequent the guide tree was based on the topologies obtained from these phylo­ genetic inferences. The different BPP species model priors were tested where priors 0 and 1 assigned equal probabilities for rooted trees, but with (prior 1) or without (prior 0) labelled histories, and priors 2 and 3 assigned equal probabilities for the number of species, dividing proba­ bilities uniformly (prior 3) or in proportion to the labelled histories (prior 2). The default, diffused inverse gamma priors for population size (theta; 3, 0.004) and the root (tau; 3, 0.004) were used as little infor­ mation was available for setting more informative priors. The MCMC chains were run for 100,000 iterations with a sampling frequency of 2 after a burn in of 8,000 iterations. Each of the four species models were run twice to check convergence of results.

2.7. Morphometry

Linear morphometric measurements were taken on preserved spec­ imens. This allows for the quantification of morphological differences between genetic clades, but also to assign non-barcoded specimens, Fig. 2. Illustration of extreme examples of the head shape in Nectophryne afra most importantly the type material, to genetic clades. Measurements (top, collection ID: NCSM 77617) and N. batesii (bottom, collection ID: NCSM were made using dial callipers to the nearest 0.01 mm, with the aid of a 77619), showing pointed snout with white line in N. afra (indicated).

4 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184 subclade assignation of non-barcoded individuals was based on collec­ encompassing territories of , Gabon and Republic of tion locality. Congo, and East LGF referring to any region east of the Ubangi river, We also explored the viability of using statistical approaches for skirting the northern bank of the Congo river across northern Demo­ morphological species determination. A discriminant model was trained cratic and parts of Central African Republic to the with the morphological data of barcoded individuals to then predicting Albertine Rift bordering East Africa (Fig. 3a). the species class of non-barcoded individuals. However, leave-one-out Species distribution models were constructed to evaluate geographic cross validation of the training model resulted in poor model accuracy and environmental niche overlap between the two species. The models and hence in unreliable species predictions. The results of this analysis were constructed using MaxEnt v3.4.1 (Phillips et al., 2006) executed were therefore instead used to quantify the degree of morphological from the dismo v1.1–4 package in R (Hijmans et al., 2017) and based on distinctiveness or similarity between species and between subclades. all individuals whose species assignment was genetically confirmedand Specifically, all transformed morphometric measurements (see above) had reliable locality information in this study (19 N. afra and 25 were subjected to a Principal Component Analysis (PCA) followed by a N. batesii). All nineteen BioClim variables and the digital elevation Regularized Discriminant Analysis (RDA) performed using the R pack­ model from the WorldClim v2 repository (https://worldclim. ages caret v6.0–85 and klaR v0.6–15, with either species or subclade as org/version2) were used as predictors at 30 s resolution and the the grouping variable. modelling area was restricted to the outlining political borders of all countries with recorded localities for both species. Models were then 2.8. Biogeographic areas and species distribution modelling used to predict the suitability of occurrence for every pixel in the modelling area. Niche overlap was estimated by comparing the two To investigate biogeographic congruencies with genetic structuring, species predictions using Schoener’s D and Warren’s I statistics (Warren specimens were assigned to five regions which are delimited by one or et al., 2008) in the dismo package, which range from 0 (no overlap) to 1 more known biogeographic barriers to amphibians as described in the (complete overlap). Geographic overlap was calculated by first con­ introduction. These regions were Bioko island and four regions within structing binary presence-absence maps using the 10 percentile training the Congolian and Lower Guinean Forest (LGF); North LGF referring to presence threshold, then projecting these to Albers equal area, and then any forest north of the Sanaga river, Central LGF spanning from the dividing the shared area where both species were predicted to occur, by Sanaga river to the southern border of Cameroon, which coincides with the total area where at least one of the species was predicted to occur. river barriers such as the Mbini and Dja rivers as well as a proposed climatic hinge, South LGF referring to regions south of this line

Fig. 3. Phylogeography of Nectophryne Tree Toads. A) Hypothesized biogeographic areas within the lower Guineo-Congolian forest ecosystem (LGF). B) Time calibrated BEAST phylogenetic inference based on concatenated sequences. Nodes with strong support (posterior probabilities ≥ 0.95) are indicated with a dot and node bars represent 95% highest posterior densities for node ages. Tips are annotated with the five identified genetic clades (Northern, Central and Southern for N. batesii and Northern and Southern for N. afra), biogeographic areas (colours referencing to areas on the map) and voucher numbers. C) Uncorrected pairwise 16S sequence differences for N. afra, N. batesii and the five genetic clades.

5 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184

3. Results

3.1. Molecular phylogenetics, genetic subclades and biogeography

Both the MrBayes and BEAST phylogeny were largely resolved (nodes with posterior probability ≥ 0.99; Fig. 3b, SI 3) with the crown age of the Nectophryne genus (and thus the split between N. afra and N. batesii) dating at 10.57 million years (95%HPD: 11.42–19.73). At around 3–5 million years ago, the phylogeny splits into fiveclades; two for N. afra (northern and southern clade; 3.45 mya) and three for N. batesii (southern clade 4.61 mya, and central and northern clade; 3.40 mya). The BPP species delimitation analysis unequivocally sup­ ports these five clades. The results were consistent across the different priors used, producing individual clade supports with posterior proba­ bilities ≥ 0.99 and an overall model with fivespecies as performing best, also with posterior probabilities ≥ 0.99 (SI 3). In N. afra, the two sub­ clades reflect a geographic split with a northern clade constituting in­ dividuals from Bioko and regions north of the Sanaga River in Cameroon (North LFG; Fig. 3a & b), and a southern clade constituting individuals south of the Mbini River (South LGF; Fig. 3a & b) from Gabon and the Republic of Congo. This southern clade also contains the only barcoded individual available from the geographically central region of southern Cameroon (0766LG; Central LGF; Fig. 3a & b), but its phylogenetic Fig. 4. Haplotype network for Nectophryne afra and N. batesii for concatenated placement is not well resolved (BEAST posterior probability 0.61; SI 3). mitochondrial loci and the nuclear RAG1 locus. Node size reflects haplotype Further genetic sampling would be needed to determine whether this frequency and colours reflectgeographic regions. Edge length and labels reflect region constitutes a well-supported clade and how it relates to the number of mutations. Alternative links are shown as grey dashed lines. remaining lineages. In N. batesii, the subclades reflect the same geographic divisions of 3.2. Morphometry well-supported North and South LGF clades, but in this species the geographically intermediate region forms its own, well supported third 3.2.1. Species designation of non-genetic specimens clade consisting of three barcoded individuals from southern Cameroon Qualitative inspection of genetically barcoded specimens yielded few (Central LGF) as well as the only barcoded individual from the Albertine characteristics that were deemed useful for species assignation. The Rift in eastern Democratic Republic of the Congo (East LGF). Based on pointed, overhanging snout of N. afra compared to N. batesii used as the this phylogenetic inference, this central clade is most closely related to diagnostic feature in the latter’s type description, although clear in the northern clade. extreme cases proved to be a continuous trait with overlap across spe­ Nectophryne batesii from Bioko island are monophyletic with a clade cies. More consistent was a lighter coloured line from the top lip to the age of 0.61 Mya (0.77–2.04) whereas in N. afra, Bioko individuals are tip of the snout in N. afra although this was less visible in older speci­ polyphyletic, representing two independent colonisations or one back mens (Fig. 2). colonisation (key nodes are not fully resolved), but again representing When using the quantitative morphometric measurements of geno­ relatively recent migrations with a maximum node age of 0.57 Mya typed individuals to train a Regularized Discriminant Analysis model (0.27–0.95). (RDA; with PCA as pre-processing collinear variables), the optimized The uncorrected 16S genetic distances showed up to 12.9% pair-wise model had a predictive accuracy of 62.84% for the leave-one-out cross distances between N. afra and N. batesii, with a maximum of 5.03% validation. This highlights the fact that the two species are not unam­ within N. afra and 5.81% within N. batesii (Fig. 3c). The northern N. afra biguously differentiable based on the morphometric data collected. clade showed the lowest maximum intraclade distance (1.68%) followed Under the optimized model, all N. afra were correctly assigned, but four by the northern and central N. batesii clades, with the southern clades of N. batesii were misclassifiedas N. afra (Fig. 5). When predicting species both species showing maximum intraclade distances above 3%. assignation of non-barcoded specimens, including the type specimens For both species, the mitochondrial haplotype networks (Fig. 4) for both species, and comparing these to our prior assignations, there suggested that there is generally little admixing between biogeographic were two (out of twelve) discordances for N. afra, including the type regions with the exception of Bioko and North LGF in N. afra. In both specimen ZMB 8472, and five (out of 26) discordances for N. batesii, species, the Central LGF populations from Cameroon are most closely including one syntype (BMNH 1947.2.19.39) with an overall prediction related to the South LGF Gabon populations, and the Bioko populations accuracy of 83.58% (SI5). are most closely related to the North LGF populations. For RAG1 too, the Bioko and North LGF populations for both species are admixed, but here, 3.2.2. Species-level comparison in both N. afra and N. batesii, the Central LGF haplotypes are most Subjecting the transformed measurements to a rigid rotation via a similar to the North LGF haplotypes. Similarly, there was also no Principal Component Analysis (PCA) showed extensive overlap in consensus on the placement of the East LGF haplotype of N. batesii, morphological space of the two species (Fig. 6). No single variable which for mitochondrial loci is more closely related to a South LGF contributes substantially more than others to PC1 which explained haplotype whereas for RAG1 shares the same haplotype as the Central 76.1% of the variance, suggesting that this first axis largely represents LGF Cameroon populations. overall size, with N. batesii being the slightly larger species. The most When comparing genetic distances (pairwise patristic distance of prominent loading of PC2 (6.9% of the variance) is canthus length fol­ additive MrBayes tree) with geographic distance (km), a significant lowed by internarial and interorbital space (PCA loadings in SI6), sug­ isolation-by-distance effect is observed in both N. afra (observed cor­ gesting these traits may differ between the two species. relation = 0.845, simulated p = 0.001) and N. batesii (observed corre­ Despite the PCA showing only marginal morphological disparity lation = 0.853, simulated p = 0.001; SI 4). between species, statistical morphological differences could be dis­ cerned. Nectophryne afra tends to be smaller than N. batesii (all

6 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184

Fig. 5. Posterior probabilities of predicted Nectophryne species (bottom panel) and clades (top panel) based on morphometric Regularized Discriminant Analysis models. Bars represent all individuals with morphometric data and are annotated with hollow circles if genetically barcoded and hollow squares if specimens are part of the type series.

width and eye diameter were also significantly different, with no sig­ nificantinteraction with body size (different regression intercepts; SI6). In addition to being smaller, N. afra therefore also has a narrower head and smaller eyes, and particularly when body sizes are small, this species also has longer and pointier snout and potentially also longer feet (metatarsus plus longest phalange).

3.2.3. Clade-level comparison Due to low sample sizes of genetically barcoded specimens when dividing the dataset into subclades, these analyses were performed using all measured specimens (see methods on how specimens without sequence data were classified). The two N. afra clades showed measur­ able body size differences, with from the northern clade being significantlysmaller than their southern counterparts (northern N. afra clade mean SUL = 17.4 ± 2.5; southern N. afra clade mean SUL = 20.2 ± 1.9; Table 1; t = -3.129, df = 19.328, p-value = 0.005). A MANOVA on the morphology principal components did not find significant shape differences in multivariate space between these two clades however (Pillai = 0.619, approx. F = 2.116, df = 1,22, p = 0.103). That said, relative to body size, the southern clade does appear to show a trend = = = Fig. 6. PCA biplot comparing Nectophryne afa (green) and N. batesii (blue) towards longer tibia (t 2.349, p 0.029), shorter canthus lengths (t = = = morphology. Filled points are individuals with only morphological data, Filled -1.280, p 0.214) and wider inter-orbital spaces (t 1.931, p 0.067; points with halos are also genetically barcoded. Polygons with shading show SI7). These differences, although slight, give the impression that the the convex hull of only barcoded individuals of each species, outlined polygons southern clade of N. afra approaches the larger, more robust phenotype show the convex hull of all individuals per species. Type specimens are high­ of N. batesii, especially in head shape, an observation that was also made lighted with hollow squares around the point, and confirmed females with an when comparing the specimens by eye while taking measurements. overlaid white cross. (For interpretation of the references to colour in this figure For N. batesii, size differences between clades were less clear. The legend, the reader is referred to the web version of this article.) smallest individuals again came from the northern clade and the largest from the southern clade, however an ANOVA supported no significant specimens: t = -2.697, df = 49.269, p-value = 0.01; genetically barcoded differences in body size (F = 2.208, df = 2,40, p = 0.123). Clades did only: t = -1.814, df = 26.773, p-value = 0.081) with mean body sizes of significantly differ in overall shape however (MANOVA on principal 18.1 ± 2.4 mm versus 20.1 ± 3.4 mm respectively (barcoded samples components: Pillai = 1.129, approx. F = 4.144, df = 2,40, p < 0.001), only; Table 1). Moreover, a Multivariate Analysis of Variance (MAN­ and linearly regressing individual variables on clade and body size found OVA) on the principal component scores of the PCA showed a significant significantinteraction terms for tibia length (t = -2.158, p = 0.016) and species effect (Pillai = 0.470; approx. F = 4.958; df = 1,65; p < 0.001), eye diameter (t = -2.534, p = 0.016) when comparing the southern to but this was not significant when looking only at barcoded individuals the northern clade and significantclade effects for femur (t = 4.658, p < (Pillai = 0.488; approx. F = 1.713; df = 1,27; p = 0.154). Linear re­ 0.001) and foot lengths (t = 2.357, p = 0.024). The central clade has a gressions for species on individual morphological measurements with more prominent head compared to the northern clade, with head width, body size (SUL) as a covariable (SI6) returned a significantspecies-body eye diameter, inter-orbital and inter-narial distances all being signifi­ size interaction term for canthus length and interorbital distance (p < cantly larger (t = 2.576, p = 0.014; t = 2.207, p = 0.34; t = 2.23, p = 0.05) for both the barcoded-only and the complete datasets as well as 0.032; t = 3.028, p = 0.004). As in N. afra, the northern clade of foot length for only the complete dataset (different regression slopes; SI N. batesii therefore again appears to be smaller and more slender than 6), indicating differences in allometry in these traits. In addition, head the two more southern clades.

7 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184

Table 1 Means and standard deviations of morphological measurements (in mm) for Nectophryne afra, N. batesii and their sub clades. Summaries provided for all specimens measured as well as for only genetically barcoded samples. For description of abbreviated measurements, see material and methods. SUL FeL TiL TaL FoL InD IoD HeW EyD CaL N

N. afra 18.3 ± 2.7 8.6 ± 1.3 9.1 ± 1.3 5.4 ± 0.7 6.6 ± 1 1.3 ± 0.2 2.5 ± 0.3 5.7 ± 0.7 2.2 ± 0.3 1.8 ± 0.1 24 N. batesii 20.2 ± 2.9 9.9 ± 1.6 10.1 ± 1.5 5.8 ± 0.8 7.6 ± 1.5 1.4 ± 0.2 3 ± 0.4 6.4 ± 0.7 2.5 ± 0.3 1.9 ± 0.3 43 N. afra–north 17.4 ± 2.5 8.3 ± 1.4 8.5 ± 1.2 5.1 ± 0.6 6.4 ± 1.1 1.2 ± 0.2 2.4 ± 0.2 5.5 ± 0.7 2.2 ± 0.3 1.9 ± 0.1 16 N. afra–south 20.2 ± 1.9 9.3 ± 0.7 10.2 ± 0.8 6 ± 0.7 7 ± 0.7 1.4 ± 0.1 2.7 ± 0.3 6 ± 0.3 2.4 ± 0.2 1.8 ± 0.2 8 N. batesii –north 19.5 ± 3.6 9.1 ± 1.9 9.3 ± 1.9 5.5 ± 0.9 6.8 ± 1.9 1.3 ± 0.2 2.8 ± 0.6 6.1 ± 0.9 2.3 ± 0.5 1.8 ± 0.3 15 N. batesii –central 19.9 ± 2.3 9.6 ± 1 10.2 ± 1 5.8 ± 0.6 7.8 ± 1 1.4 ± 0.1 3 ± 0.3 6.4 ± 0.5 2.7 ± 0.2 1.8 ± 0.2 19 N. batesii –south 21.9 ± 2.5 11.5 ± 0.9 11.2 ± 0.8 6.2 ± 0.8 8.6 ± 1.4 1.4 ± 0.2 3.1 ± 0.3 6.7 ± 0.6 2.6 ± 0.1 2 ± 0.2 9 Barcoded only N. afra 18.1 ± 2.4 8.6 ± 1.3 9.1 ± 1.5 5.4 ± 0.8 6.4 ± 1 1.3 ± 0.2 2.5 ± 0.2 5.5 ± 0.7 2.1 ± 0.3 1.8 ± 0.1 12 N. batesii 20.1 ± 3.4 10 ± 1.8 10 ± 1.7 5.8 ± 1 7.3 ± 1.8 1.3 ± 0.2 2.8 ± 0.5 6.2 ± 0.8 2.4 ± 0.4 1.9 ± 0.3 17 N. afra–north 16.6 ± 2 7.9 ± 1.2 8.1 ± 1.1 5 ± 0.6 6 ± 1.1 1.2 ± 0.3 2.4 ± 0.2 5.1 ± 0.7 2 ± 0.3 1.8 ± 0.1 7 N. afra–south 20.1 ± 0.9 9.6 ± 0.7 10.4 ± 0.6 6 ± 0.6 7 ± 0.6 1.4 ± 0.1 2.7 ± 0.1 6 ± 0.1 2.3 ± 0.1 1.8 ± 0.1 5 N. batesii –north 19.1 ± 3.6 9.1 ± 1.8 9.2 ± 1.9 5.5 ± 1 6.6 ± 1.9 1.2 ± 0.2 2.6 ± 0.5 5.9 ± 0.8 2.2 ± 0.4 1.8 ± 0.3 9 N. batesii –central 20.1 ± 2.4 9.9 ± 0.5 10.2 ± 0.7 6.4 ± 0.3 7.4 ± 0.3 1.6 ± 0.2 3 ± 0.5 6.4 ± 0.3 2.6 ± 0.2 1.8 ± 0.2 3 N. batesii –south 21.9 ± 3.2 11.5 ± 1.1 11.2 ± 1 6.1 ± 1 8.4 ± 1.6 1.4 ± 0.2 3.1 ± 0.3 6.7 ± 0.7 2.6 ± 0.1 2 ± 0.3 5

When performing a regularized discriminant analysis on principal by chance (AUC N. afra: 0.986, N. batesii: 0.966). For both species, components of the full morphological dataset with the fiveclades as the precipitation of the wettest month and wettest quarter (BIO13 and grouping variable, the optimal model had a leave-one-out cross valida­ BIO16) were the variable with the highest percent contribution, and tion accuracy of 55.68%. This reiterates the extensive morphological when mapping areas of high environmental suitability (Fig. 7), both overlap between clades. When comparing assigned to predicted clades, species show similar distribution hotspots around coastal Central Africa all clades except the northern N. batesii clade had a balanced accuracy of especially Cameroon and Equatorial Guinea. The predicted distributions > 90% with the southern N. afra clade showing the highest sensitivity for N. afra showed higher suitability further south along the coast line (100%) and specificity (96.66%) suggesting that this was the most than N. batesii. Nectophryne batesii showed higher suitability areas more morphologically distinct clade. The northern N. batesii clade showed the inland in Cameroon and Republic of Congo. Despite these differences in lowest balanced accuracy (63.33%) and was the only clade that showed predicted distribution, the two species show high degrees of niche noticeable low cross-species sensitivity, with 20% being misclassifiedas similarity, with Schoener’s D = 0.71 and Warren’s I = 0.93. When belonging to the northern N. afra clade (Fig. 5). This corroborates the restricting predicted occurrences by the 10th percentile training pres­ trend observed that there is a convergence in morphology across species ence, the species’ geographic ranges overlap by 59.4%. in this northern region. The type specimens for both species were correctly assigned to clades 4. Discussion by the RDA predictions, indicative that they are morphologically similar to specimens in their corresponding genetic clades although it is This study uses an integrative approach to investigate the evolu­ important to note that the N. batesii type series of eleven specimens tionary and phylogeographic history of the genus Nectophryne. Our makes up 57.9% of the central clade and the results should therefore be analysis of genetics, morphology, and spatial distribution reveals un­ treated with caution. It is also worth noting that the holotype for N. afra, expected evolutionary patterns for the two described species, given which had a higher posterior probability of being N. batesii for the their>10 million years of independent evolution. The two species are species-level analysis (0.73), here too had only a 0.68 posterior proba­ sympatric throughout much of their geographic range, show extensive bility of belonging to the southern N. afra clade and a 0.30 posterior ecological niche overlap, and show only marginal morphological dif­ probability of belonging to the geographically equivalent, central ferences, despite substantial genetic variation. This case of sympatry and N. batesii clade. limited morphological divergence is noteworthy, and contrary to ex­ pectations of character displacement processes predicted to occur (Schluter, 2000). 3.3. Geographic range and species distribution modelling Our primary aims were to quantify discernible morphological dif­ ferences between species, assess the relevance for known geographic Two Nigerian specimens of Nectophryne (ZMB 84,664 and ZMB and ecological barriers for phylogeographic divergences, and to 84666) were genetically barcoded as N. batesii, thereby extending this compare phylogenetic divergence patterns of subclades across species. ’ species geographic range west of the Cross River, into , thought The species description of N. batesii (Boulenger, 1913) defined snout ¨ previously only to be inhabited by N. afra (Onadeko and Rodel, 2009). length and shape as the main distinguishing feature from N. afra. Here Similarly, five Nectophryne specimens from Bioko (BMNH 2005.1861, we confirm that N. afra have proportionally longer and more pointed NCSM 87721, NCSM 87737, NCSM 87765, NCSM 87769) were geneti­ snouts than N. batesii, but caution that this is not an infallible morpho­ cally barcoded as N. batesii and together with barcoded specimens from logical diagnostic feature. In addition, we find that N. afra is generally the collection of the Natural History Museum of Madrid, we here report smaller (although this is driven mostly by one geographic clade) and ’ this species presence on Bioko for the first time based on genetic relative to body size, these toads have narrower inter-orbital spaces and ´ sequence data (see also recent comment in Sanchez-Vialas et al., 2020). longer feet. However, both taxa overlap in all measured traits and the Both species have been recorded in the Democratic Republic of the discriminant analysis could not accurately classify individuals to species Congo south of the Congo river (de Witte, 1934; Laurent, 1952), which based on morphology alone. Among the misclassified specimens in the together with the sequenced material from the Democratic Republic of discriminant analysis was the holotype for N. afra. This could indicate – the Congo (MTSN 7987 88) and southern Gabon (MCZ:Herp:A-149203, that a taxonomic revision of this species is required, but more likely, that ZMB 86039) requires that the IUCN ranges for both species be extended the little morphological variation resulted in insufficient predictive southwards (Fig. 1). Despite both generally being described as lowland power of the model. species, one individual (ZMB 82855) was collected on Mt. Manengouba Despite the few tangible morphological differences between species, in Cameroon at approximately 2020 m.a.s.L. (SI1). historical records document instances of sympatric occurrences of The species distribution modelling performed better than expected

8 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184

Fig. 7. Predicted suitable ranges of Nectophryne afra (left) and N. batesii (right) based on MaxEnt environmental niche models. Yellow backgrounds show the modelling area and the colour gradients from yellow to black show environmental suitability for each species. Points show localities of individuals used for modelling and polygon outlines show IUCN range maps. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

N. afra and N. batesii (Laurent, 1972; Nobel, 1924). Moreover, the genus consists of at least five phylogenetically determined operative expert-predicted distribution maps by the IUCN Red List effectively taxonomic units. Any such action however, is complicated by the fact suggest the two species are sympatric throughout most of their range. that relatively few specimens exist in collections from key geographic Here, for the first time using genetic barcoding, we confirm that both regions, especially with genetically viable material. Moreover, we found species do indeed occur together or in close proximity. In fact, this few tangible morphological diagnostic features in the specimens exam­ sympatry occurs more widely across mainland localities than previously ined (though sample sizes were small) and genetic signals consistent known, and even on Bioko Island, thought until recently to only be with isolation-by-distance and incomplete lineage sorting. In general, inhabited by N. afra (Sanchez-Vialas´ et al., 2020). The environmental the conserved morphology of African bufonids has made their classifi­ niche models corroborate that N. afra and N. batesii show extensive cation difficult (Clarke, 2001). Nonetheless, recent efforts to improve environmental niche overlap, though the extent of the MaxEnt predicted our understanding of the diversity of habitat-specialist bufonids like suitable areas for both species is much more restricted than the IUCN Nectophryne have led to the revision of numerous groups (e.g. Ceríaco ranges. Although this is not unexpected given the qualitative nature of et al., 2018; Menegon et al., 2004; Rodel¨ et al., 2004). Molecular bar­ the IUCN ranges, the modelled distributions should be interpreted with coding especially has contributed significantlyto this effort by exposing caution due to the few locality records used relative to the large cryptic lineages, such as in the Capensibufo Cape Toads, where formerly modelling area and a notable scarcity of genetically barcoded records two species were shown to harbour at least fivedistinct units (Channing from eastern parts of their ranges. For example, both species have been et al., 2017; Tolley et al., 2010). Cryptic lineages of Nectophryne iden­ recorded in the Albertine Rift and in other parts of eastern Democratic tified in this study should therefore be the focus of future taxonomic Republic of the Congo (de Witte, 1934; Laurent, 1952), but the models work. failed to predict areas of high suitability in these regions. Intriguingly, the identified subclades in each species show remark­ The geographic and ecological co-occurrence of N. afra and N. batesii, ably parallel phylogenetic histories, both in terms of timing of phylo­ as well as their lack in morphological divergence poses the interesting genetic splits and their geographic distributions, pointing towards a question of what other barriers are in place to prevent hybridization of common process or processes that may have shaped the evolutionary these species. For many anuran amphibians, male vocalization serves as history of these lineages. Three such processes have been proposed in a prezygotic isolation mechanism. For example, in relatively young the literature to explain the diversity of the Guineo-Congolian region for species of sympatric Leptopelis tree frogs, morphological disparity is a variety of taxa, namely: speciation through allopatry driven by past or equally scarce, but male advertisement calls diverge (Portillo and present physical barriers, past contraction of forest habitat or dispersal Greenbaum, 2014). For Nectophryne too, mating calls are anecdotally across ecological gradients. Recent demographic modelling of amphib­ distinct, though documented audio recordings are scarce. The relevance ians in this region have suggested that all three processes have shaped of calls for mate attraction in this species is unknown, as the calls are their evolution and that differences in species’ ecology may explain described as not very complex and males do not have developed vocal idiosyncrasies in these patterns (Bell et al., 2017; Leache´ et al., 2019). sacs (Amiet, 1976; Amiet and Goutte, 2017; Channing and Rodel,¨ 2019). We findthat divergence of subclades in both Nectophryne species support Both species also lack all major components of the tympanic middle ear, the above hypothesized scenarios, but, as has been the case in previous which does not rule out acoustic communication, but has consequences studies (e.g. Leache´ et al., 2019), it is difficult to distinguish between for their perception of airborne sounds (Pereyra et al., 2016). Relatively these processes, because the separations between forest refugia, the lo­ little is known about other aspects of the life history of Nectophryne, but cations of steep ecological gradients and major rivers often coincide interestingly, in contrast to the conformity in adult phenotypes, juve­ geographically. niles colour patterns differ starkly (Blackburn and Droissart, 2008; Based on our dating estimates, the divergence of N. afra and N. batesii Boulenger, 1906; Channing and Rodel,¨ 2019). has occurred between 11.41 and 19.73 Mya and the identified sub- Within both species of Nectophryne we found extensive genetic di­ clades originating within a window of 2.33 to 6.25 Mya. These phylo­ versity and we could identify at least two allopatric and genetically genetic divergences hence predate the last glacial maxima and are delimited clades within N. afra and three within N. batesii. The presence therefore not a good fitfor the Pleistocene refugia hypothesis. However, of cryptic lineages that likely require taxonomic action has previously the Guinea-Congolian forest was likely fragmented repeatedly as far been suggested (Deichmann et al., 2017) and here we confirmthat this back as the Miocene (Edwards et al., 2010; Morley, 2000; Portillo et al.,

9 H.C. Liedtke et al. Molecular Phylogenetics and Evolution 162 (2021) 107184

2018), and indeed, the centre of genetic diversity for both species is seen them. Morphological variation is minimal among species and genetic in present day Gabon and eastern Cameroon, which coincide with large clades despite the genetic divergence observed. The distinct genetic regions of historic rainforest stability in the area (Fig. 1; Plana, 2004). clades in both N. afra and N. batesii are geographically isolated and, Our inference also suggests that the colonisation of Bioko by both interestingly, show near parallel phylogeographic histories. In both N. batesii and N. afra occurred more recently than 2.04 Mya, but before cases, the geographic divisions of clades correspond to the three pro­ 0.22 Mya, hence after the volcanic formation of the island and before the posed biogeographic scenarios described for this region, but based on last glacial maximum. Divergence date estimates derived from second­ the available data, it is difficult to distinguish between these. ary calibrations, as is the case here, should be treated with caution. How N. afra and N. batesii lineages themselves have diverged remains However, we have confidencein the estimated timing of events, because uncertain. Both show centres of genetic diversity in the same geographic the estimated dates for the colonization of Bioko do not predate the regions, highly sympatric distributions and relatively few external formation of the island and coincide with temporary formation of land morphological differences. To understand how these toads evolved, we bridges as well as divergence dates in other amphibians found on the suggest further investigations into their life history, more fine scaled mainland and Bioko (Charles et al., 2018). The colonization most likely characterization of their microhabitat use and studies on their trophic took place from the northern Gulf of Guinea region, where the land ecology. This would shed light on prezygotic isolation mechanisms that bridge connected Bioko to Cameroon. Potential ‘reverse colonization’ of may have driven the speciation of these curious amphibians and has the mainland from Bioko in other amphibians has been suggested (Bell allowed them to largely coexist with no evidence of hybridization. et al., 2017). This may be a further explanation for the pattern seen in N. afra, where multiple episodes of colonisations back and forth cannot CRediT authorship contribution statement be ruled out. Allopatric speciation through forest fragmentation pre- dating the Pleistocene and changes in sea level during the last glacial H. Christoph Liedtke: Conceptualization, Formal analysis. Diego J. maxima are therefore possible drivers of lineage diversification in Soler-Navarro: Formal analysis. Ivan Gomez-Mestre: Conceptualiza­ Nectophryne. tion, Supervision. Simon P. Loader: Conceptualization. Mark-Oliver The role of rivers in isolating Nectophryne clades is also relevant. The Rodel:¨ Conceptualization. main divisions of subclades in both species coincide with major rivers dividing the landscape. The Sanaga River in central Cameroon coincides Declaration of Competing Interest with an undisputed genetic and morphological divergence in both spe­ cies, as do the Mbini and Dja rivers along the Cameroon-Guinea border The authors declare that they have no known competing financial in N. batesii. Geological evidence suggests that major rivers in the region, interests or personal relationships that could have appeared to influence including the Sanaga have been largely stable since the Cenozoic the work reported in this paper. (Goudie, 2005; Ngueutchoua and Giresse, 2010), which coincides well with the relatively old phylogenetic origins of the five identified Nec­ Acknowledgements tophryne clades. Populations from the Central LGF region in southern Cameroon show We would like to thank all people working in collections who have evidence of incomplete lineage sorting or ongoing gene flow with granted and facilitated the loan of specimens and tissues. Our thanks respect to the northern and southern clades and both species show also to individual collaborators who have provided materials and isolation-by-distance patterns. Moreover, the largest morphological photos, Marius Burger, Matthias Dahmen, Nono LeGrand Gonwouo, distinction can be seen between the northern and southern clades. Such David Gower (who thanks the Bioko Biodiversity Protection Program for patterns are in line with the ecotone model that postulates that diver­ hosting and facilitating fieldwork; Equatorial Guinea research permit gence of lineages has occurred with ongoing gene flow (i.e. parapatry) 0177/014; export permit 036/014), Vaclav´ Gvoˇzdík, Michele Menegon, across an ecological gradient, in this case the climatic hinge along the Fabian Mühlberger, and Mareike Petersen. Thank you also to Eli southern border of Cameroon resulting in a north–south seasonal Greenbaum for constructive comments on the manuscript, Ana Piriz and inversion. Typically, genetic variation across ecotones is accompanied Jose Maria Gasent for help with lab work and to Paula Martin Art for by morphological variation, matching selective differences across these specimen illustrations (https://www.instagram.com/paulamartinart). ecological boundaries, but based on our data this is not the case. It is This work was financially supported by Ministerio de Economía, therefore less clear to what extent this third biogeographic process has Industria y Competitividad (MINECO) [grant number CGL2014-59206- contributed to the evolutionary history of Nectophryne. Rigorous genetic P]. demographic modelling would be required to test scenarios of speciation with gene flow (e.g. Barratt et al., 2018; Leache´ et al., 2019) and Appendix A. Supplementary data ecological studies would be needed to identify the causes of concomitant morphological change in both N. afra and N. batesii, such as differences Supplementary data to this article can be found online at https://doi. in diet, breeding sites, mate recognition and choice, as well as ontoge­ org/10.1016/j.ympev.2021.107184. netic allometry between clades. 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