Amphibia-Reptilia 41 (2020): 349-359 brill.com/amre Hidden diversity within the broadly distributed Amazonian giant monkey frog (Phyllomedusa bicolor: Phyllomedusidae) Edvaldo Pereira Mota1, Igor Luis Kaefer2, Mario da Silva Nunes1, Albertina Pimentel Lima3, Izeni Pires Farias1,∗ Abstract. Phyllomedusa bicolor is a large-sized nocturnal tree frog found in tropical rainforests throughout much of the Amazonian region of Brazil, Colombia, Bolivia, Peru, Venezuela, and the Guianas. Very little is known about P. b i c o l o r genetic diversity and genealogical history of its natural populations. Here, using a sampling design that included populations covering most of its distributional range, we investigated the spatial distribution of genetic variability of this species, and we tested the hypothesis that P. b i c o l o r is composed of deeply structured genetic groups, constituting more than one lineage across the Brazilian Amazonia. The results suggested two main lineages in two geographic mega-regions: Western and Eastern Amazonia, the latter consisting of three population groups distributed in the Guiana and Brazilian Shields. The present findings have implications to taxonomy, to understanding the processes that lead to diversification, and to defining strategies of conservation and medicinal use of the species. Keywords: cryptic diversity, genetic diversity, Phyllomedusidae, phylogeography, tropical rainforest. Introduction in general, forest specialist species and species with low dispersal capacity tend to be geneti- Surveys of molecular biodiversity suggest high cally structured (Smith and Green, 2005; Ro- levels of intraspecific diversity within anurans dríguez et al., 2015). In summary, trait, distribu- from the Neotropics (Fouquet et al., 2007; Funk, tion, and life history can influence genetic diver- Caminer and Ron, 2012; Motta et al., 2018) gence between populations and may, ultimately, which often upon further examination trans- influence the phylogeographic patterns of the late to species diversity (e.g. Caminer et al., species. Besides, the taxonomic limits within 2017; Rojas et al., 2018). The nominal species Amazonian species complexes as well as the ge- to present such a pattern are often categorized ographic factors promoting and maintaining dif- as complexes of cryptic species, being gen- ferentiation among populations remain poorly erally those with wide geographical distribu- understood (Wesselingh et al., 2010; Kaefer et tion, small body size and low dispersal abilities al., 2013; Fouquet et al., 2014). (Gehara et al., 2014); however, species with re- In this context, several species of Amazo- stricted distribution cannot be discarded either nian anurans with a wide distribution turn out (Guayasamin et al., 2017). Also, such evolution- to be geographically structured in many evolu- ary differentiation is likely favored by habitat tionary lineages, some of which deserving spe- specificity (Vences and Wake, 2007; Kaefer et cific status (Kaefer et al., 2013; Rojas et al., al., 2013; Maia, Lima and Kaefer, 2017), i.e. 2018). Phyllomedusa bicolor (Boddaert, 1772) (Anura, Phyllomedusidae) is a relatively large- 1 - Laboratório de Evolução e Genética Animal, Instituto sized nocturnal tree frog found in tropical rain- de Ciências Biológicas, Universidade Federal do Ama- zonas, Manaus, Brazil forests throughout a broad geographic area, oc- 2 - Departamento de Biologia, Instituto de Ciências Bi- curring in the Amazonian region of Brazil, ológicas, Universidade Federal do Amazonas, Manaus, Colombia, Bolivia, Peru, Venezuela, and the Brazil 3 - Coordenação de Biodiversidade, Instituto Nacional de Guianas; it also occurs in the Cerrado habitat of Pesquisas da Amazônia, Manaus, Brazil Maranhão state, Brazil (Frost, 2019). The cur- ∗Corresponding author; e-mail: [email protected] rently accepted hypothesis that individuals of Downloaded from Brill.com10/16/2020 07:21:33PM via free access © Koninklijke Brill NV, Leiden, 2020. DOI:10.1163/15685381-bja10003 350 E.P. Mota et al. P. bicolor from geographically distant locations obtained from the primers MZV15-L (Moritz, Wright and represent the same taxon awaits support from Brown, 1992) and Control-IP-H (Goebel, Donnelly and Atz, 1999) to amplify a portion of the Cytb gene, and primers for genetic data. Skin secretions from this frog have the nuclear gene RAG1 (Rag1_phyFL and Rag1_phyR). been used by indigenous people from Brazil and The amplification of the 16S and Cytb was performed Peru for centuries as a ritualistic healing sub- in a final volume of 15 μl containing 5.5 μl of ddH2O, stance, and its use has spread via urban ex- 1.5 μl of dNTP (1.5 mM), 1.5 μl of 10X buffer (Tris-HCl 100 mM, KCl 500 mM), 1.5 ml of each primer (0.2 mM), pansion into alternative therapy clinics (Silva, 1.5 μlofMgCl2 (25 mM), 1 μl of DNA (approx. 10 ng) and Monteiro and Bernarde, 2019). Even though 0.3 μl of Taq DNA polymerase. The amplification cycles Phyllomedusa bicolor is a pharmacologically were performed using the following conditions: (1) initial denaturation at 92°C for 1 minute, (2) 35 cycles consisting important taxon, no study has assessed the ge- of denaturation at 92°C for 1 minute, annealing of primers at netic diversity and genealogical history of natu- 50°C for 40 seconds, and extension at 72°C for 1.5 minutes, ral populations of this species, and many of its as well as (3) final extension at 72°C for 5 minutes. The PCR products were purified with the exonuclease few published DNA sequences are not assigned and alkaline phosphatase enzymes, following the manu- to any specific geographic location, given that facturer’s protocol (Fermentas). Sequencing reactions were they have been obtained from pet trade speci- performed using the Big Dye Terminator Cycle Sequencing Kit, following the manufacturer’s protocol (Life Technolo- mens. Therefore, we assess the genetic diver- gies). Products were resolved on the ABI3130xl automatic sity of Phyllomedusa bicolor populations by sequencer (Life Technologies) and edited and aligned using sequencing mitochondrial and nuclear genetic the software BioEdit (Hall, 1999). markers. Assuming its large geographical dis- tribution, we aim to test whether populations of Data analysis P. bicolor constitute the same evolutionary unit A network of haplotypes was generated using a con- + throughout its range. catenated data of all molecular markers (16S rRNA Cytb+RAG1) in the program HAPLOVIEWER for the re- construction of the genealogical relations between individ- uals (Salzburger, Ewing and von Haeseler, 2011). Materials and methods Single locus discovery and phylogeography Sampling and data collection To test whether populations of Phyllomedusa bicolor com- Sampling occurred in the years 2008 to 2009 and was per- prise just one evolutionary unit, we used the ultramet- formed in sampling sites across five states in the Brazilian ric consensus tree topology generated in BEAST as in- Amazonia. A total of 117 individuals were obtained from put for single-locus species discovery analyses: bGMYC, a 11 different sampling sites (supplementary table S1), which Bayesian implementation of the GMYC (Reid and Carstens, include populations that cover a large part of the species dis- 2012), and the Poisson tree process (PTP) model for species tribution in the Amazonian biome. delimitation (Zhang et al., 2013) as implemented in mPTP We collected samples at night around permanent and (Kapli et al., 2017). For these analyses, we used only unique semi-permanent puddles as well as around ponds near haplotypes from the concatenated data set. The phylogeo- streams. At least seven individuals were collected from graphic analysis in BEAST was conducted using concate- each area. The sampling was conducted through auditory nated data from all sampled individuals. Clade support was and visual searches with a headlamp. Tissue samples were assessed with 1,000 non-parametric bootstraps. For these collected from one of the toes, preserved in 95% ethanol analyses, we used the TIM+G+I model of molecular evo- and deposited in the Animal Genetics Tissue Collection – lution selected by the program jMODELTEST (Posada, CTGA-ICB/UFAM Campus (CGEN, Resolution No. 75 of 2008). August 26, 2004) Laboratory of Animal Genetics and Evo- Using the concatenated data we calculated the time of lution of the Federal University of Amazonas, Manaus, most recent common ancestor (TMRCA) for each group Brazil. of haplotypes using a coalescence history approach inves- Genomic DNA extraction was performed according to tigated by Markov Chain Monte Carlo (MCMC) using the the protocol described by Sambrook, Fritsch and Maniatis BEAST v1.8 program (Drummond and Rambaut, 2007). (1989). We used sets of primers to amplify two regions of The analyses were performed assuming a relaxed molecu- the mitochondrial DNA (mtDNA) and one nuclear gene. lar clock lognormal type, which assumes independent rates Amplification was performed via polymerase chain reaction on different branches (Drummond et al., 2006). Prior trees (PCR) using primers for 16S rRNA mitochondrial gene were modeled according to the Yule process of speciation. (Palumbi, 1996), Cytochrome b (Cytb) specific primers The chain (MCMC) used in the BEAST analysis of di- developed for Phyllomedusa bicolor using amplifications vergence had a size of 10 million steps, with a consensus Downloaded from Brill.com10/16/2020 07:21:33PM via free access Genetic diversity of giant monkey frog Phyllomedusa bicolor 351 tree recorded every 10,000 steps and a burn-in of 5%. The constant.