UNIVERSIDADE ESTADUAL DE CAMPINAS SISTEMA DE BIBLIOTECAS DA UNICAMP REPOSITÓRIO DA PRODUÇÃO CIENTIFICA E INTELECTUAL DA UNICAMP Versão do arquivo anexado / Version of attached file: Versão do Editor / Published Version Mais informações no site da editora / Further information on publisher's website: https://www.frontiersin.org/articles/10.3389/fgene.2019.00719/full DOI: 10.3389/fgene.2019.00719 Direitos autorais / Publisher's copyright statement: ©2019 by Frontiers Media. All rights reserved. DIRETORIA DE TRATAMENTO DA INFORMAÇÃO Cidade Universitária Zeferino Vaz Barão Geraldo CEP 13083-970 – Campinas SP Fone: (19) 3521-6493 http://www.repositorio.unicamp.br ORIGINAL RESEARCH published: 14 August 2019 doi: 10.3389/fgene.2019.00719 Extensive Cryptic Diversity Within the Physalaemus cuvieri–Physalaemus ephippifer Species Complex (Amphibia, Anura) Revealed by Edited by: Cytogenetic, Mitochondrial, and Annie Machordom, National Museum of Natural Sciences Genomic Markers (MNCN), Spain Reviewed by: Juliana Nascimento 1, Jucivaldo D. Lima 2, Pablo Suárez 3, Diego Baldo 4, Frank Glaw, Gilda V. Andrade 5, Todd W. Pierson 6, Benjamin M. Fitzpatrick 6, Célio F. B. Haddad 7, Bavarian State Collection of Zoology, Shirlei M. Recco-Pimentel 1 and Luciana Bolsoni Lourenço 1* Germany Stephane Boissinot, 1 Laboratório de Estudos Cromossômicos (LabEsC), Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, New York University Abu Dhabi, Universidade Estadual de Campinas, São Paulo, Brazil, 2 Núcleo de Biodiversidade (NUBIO), Instituto de Pesquisas United Arab Emirates Científicas e Tecnológicas do Estado do Amapá, Zoologia, Campus da Fazendinha, Macapá, Brazil, 3 Instituto de Biología Subtropical (CONICET-UNaM), Puerto Iguazú, Argentina, 4 Laboratorio de Genética Evolutiva, Instituto de Biología *Correspondence: Subtropical (CONICET-UNaM), Facultad de Ciencias Exactas, Químicas y Naturales, Universidad Nacional de Misiones, Luciana Bolsoni Lourenço Posadas, Argentina, 5 Departamento de Biologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal do [email protected] Maranhão-UFMA, São Luís, Brazil, 6 Department of Ecology & Evolutionary Biology, University of Tennessee, Knoxville, TN, United States, 7 Departamento de Zoologia and Centro de Aquicultura (CAUNESP), Instituto de Biociências, Universidade Specialty section: Estadual Paulista, São Paulo, Brazil This article was submitted to Evolutionary and Population Genetics, Previous cytogenetic and phylogenetic analyses showed a high variability in the frog a section of the journal taxa Physalaemus cuvieri and Physalaemus ephippifer and suggested the presence of Frontiers in Genetics undescribed diversity in this species complex. Here, by 1) adding specimens from the Received: 04 December 2018 Accepted: 09 July 2019 Brazilian Amazon region, 2) employing sequence-based species delimitation approaches, Published: 14 August 2019 and 3) including RADseq-style markers, we demonstrate that the diversity in the P. cuvieri–P. Citation: ephippifer species complex is even greater than previously suspected. Specimens from Nascimento J, Lima JD, Suárez P, Baldo D, Andrade GV, Pierson TW, Viruá and Western Pará, located at the Guiana Amazonian area of endemism, were Fitzpatrick BM, Haddad CFB, recovered as distinct from all previously identified lineages by the phylogenetic analyses Recco-Pimentel SM and based on mitochondrial DNA and RAD markers, a PCA from RAD data, and cytogenetic Lourenço LB (2019) Extensive Cryptic Diversity Within the Physalaemus analysis. The sequence-based species delimitation analyses supported the recognition of cuvieri–Physalaemus ephippifer one or two undescribed species among these Amazonian specimens and also supported Species Complex (Amphibia, Anura) Revealed by Cytogenetic, the recognition of at least three other species in the P. cuvieri–P. ephippifer species Mitochondrial, and Genomic Markers. complex. These new results reinforce the need for a comprehensive taxonomic revision. Front. Genet. 10:719. doi: 10.3389/fgene.2019.00719 Keywords: DNA-based species delimitation, RADseq, Amazonia, chromosome, Physalaemus Frontiers in Genetics | www.frontiersin.org 1 August 2019 | Volume 10 | Article 719 Nascimento et al. Species Delimitation in a Species Complex of Physalaemus INTRODUCTION recognized five groups in theP. cuvieri Clade: the P. biligonigerus species group, the P. cuvieri species group, the P. henselii species The neotropical region is known for its high species richness group, the P. gracilis species group, and the P. olfersii species (Myers et al., 2000), although the processes responsible for this group. Currently, the P. cuvieri species group encompasses nine richness remain under debate (see Haffer, 1997; Hoorn et al., species, among them P. cuvieri and P. ephippifer (see list in 2010; Álvarez-Presas et al., 2014; Fouquet et al., 2015; Garzón- Frost, 2018). Orduña et al., 2015), and a large part of this diversity is still Using DNA sequence data, Lourenço et al. (2015) recovered undescribed (Myers et al., 2000; Fouquet et al., 2007; Giam four distinct lineages among specimens first identified as either et al., 2012). Giam et al. (2012), based on the analysis of the Physalaemus cuvieri or Physalaemus ephippifer. These four taxonomic effort dedicated to the description of species over lineages correspond to karyological groups recognized previously time and the geographic distribution of the described species, by Quinderé et al. (2009), which were distinguishable particularly estimated that approximately 33% of the species of amphibians by the location of nucleolus organizer regions (NORs). Thus, were not described at that time, including those in the it appears that there is undescribed, cryptic diversity within neotropical forests, a biome supposedly hosting a great part of these two species. Miranda et al. (2019) also described deep these unknown species. Delimiting valid species, however, can phylogenetic structure among populations identified as P. cuvieri, be a complicated task, and DNA sequence data sets are useful corroborating the presence of undescribed species in this group. in this matter, as they enable the identification of historical Here, we follow the naming conventions of Lourenço et al. (2015) lineages in phylogenetic (or tree-based) analyses and inferences and refer to these four lineages individually as P. ephippifer and of genetic distances and gene flow statistics in non–tree-based lineages 1 to 3 of “P. cuvieri,” and collectively as the P. cuvieri–P. methods (see Wiens and Penkrot, 2002; Camargo et al., 2013; ephippifer species complex. examples in Elmer et al., 2007; Funk et al., 2012; Fouquet The lineages 1 to 3 of Physalaemus“ cuvieri” (L1–L3) have et al., 2012; Ortega-Andrade et al., 2015). These methods are primarily allopatric distributions; L1 occurs in northern and especially useful for cryptic species for which morphological northeastern Brazil, L3 was recognized based on specimens from characters provide insufficient or misleading evidence for just one locality (i.e., Porto Nacional, in central Brazil), and L2 species delimitation. occupies a broader area, which extends from the central state One such example includes the South American frogs assigned of Bahia to southern Brazil and northern Argentina (Lourenço to Physalaemus cuvieri or Physalaemus ephippifer (Anura, et al., 2015) (Figure 1, inset). Leptodactylidae). A previous phylogenetic study recognized two Physalaemus ephippifer, which is easily distinguished major clades in the genus Physalaemus, which were informally cytogenetically from L1 to L3 of “P. cuvieri” by the presence of referred to as the Physalaemus signifer Clade and Physalaemus heteromorphic sex chromosomes Z and W (Nascimento et al., cuvieri Clade (Lourenço et al., 2015). That study also tested the 2010), rendered P. cuvieri paraphyletic in the phylogenetic monophyly of the species groups previously proposed based on analyses, as it was recovered as sister to L1 of “P. cuvieri” phenetic analyses (Lynch, 1970; Nascimento et al., 2005) and (Lourenço et al., 2015). Physalaemus ephippifer occurs at FIGURE 1 | Geographic distribution of the specimens collected for this work (1–10). 1. Viruá National Park, State of Roraima. 2–4. Óbidos, State of Pará. 5–6. Alenquer, State of Pará. 7. Monte Alegre, State of Pará. 8–9. Prainha, State of Pará. 10. Santa Bárbara, State of Pará. The blue dot indicates Belém, the type locality of P. ephippifer. In the inset, a map at a smaller scale shows a broader area and includes all the sites previously sampled for the study of the species complex Physalaemus cuvieri–Physalaemus ephippifer (Lourenço et al., 2015). Frontiers in Genetics | www.frontiersin.org 2 August 2019 | Volume 10 | Article 719 Nascimento et al. Species Delimitation in a Species Complex of Physalaemus the mouth of the Amazon River, with the type locality in TABLE 1 | Voucher numbers and sampled sites of the specimens analyzed the Brazilian municipality of Belém (Figure 1). Although P. cytogenetically. ephippifer has also been reported in the Guianas, the Bolívar Taxon Sample site Voucher numbers region of Venezuela, and Suriname (Frost, 2018), the true extent county of the geographic distribution of this species is still unclear Physalaemus Santa Bárbara, ♀ SMRP 252.105 (ZUEC 21355) mt, (see comment in Frost, 2018). The specimens of P. ephippifer ephippifer State of Pará, Brazil SMRP 252.106 (ZUEC 21356), SMRP previously included in the cytogenetic (Nascimento et al., 2010) 252.113