Molecular Phylogenetics and Evolution 65 (2012) 547–561 Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev From Amazonia to the Atlantic forest: Molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges ⇑ Antoine Fouquet a,b, , Daniel Loebmann c, Santiago Castroviejo-Fisher d, José M. Padial d, Victor G.D. Orrico e, Mariana L. Lyra e, Igor Joventino Roberto f, Philippe J.R. Kok g,h, Célio F.B. Haddad e, Miguel T. Rodrigues b a CNRS-Guyane – USR 3456, Immeuble Le Relais – 2, Avenue Gustave Charlery, 97300 Cayenne, French Guiana b Departamento de Zoologia, Universidade de São Paulo, Instituto de Biociências, Caixa Postal 11.461, CEP 05508-090 São Paulo, SP, Brazil c Laboratório de Vertebrados Terrestres, Universidade Federal do Rio Grande, Instituto de Ciências Biológicas, Av. Itália Km 8, Carreiros, CEP 96.203-900 Rio Grande, RS, Brazil d Department of Herpetology, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024-5192, United States e Departamento de Zoologia, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho, Av. 24-A, 1515, Bela Vista, Caixa Postal 199, CEP 13506-900 Rio Claro, SP, Brazil f Departamento de Ciências Físicas e Biológicas, Laboratório de Zoologia, Universidade Regional do Cariri (URCA), Rua Cel. Antônio Luiz Pimenta, 1161, CEP 63105-000 Crato, Ceará, Brazil g Department of Vertebrates, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000 Brussels, Belgium h Biology Department, Unit of Ecology and Systematics, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium article info abstract Article history: Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of glo- Received 12 May 2012 bal climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have Revised 13 July 2012 revealed that the actual number of species in South American tropical forests is largely underestimated, Accepted 14 July 2012 but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 Available online 26 July 2012 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and min- ute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazo- Keywords: nia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominal Adelophryne species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Amazonia Atlantic forest Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, Cryptic species and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction Neotropical diversity confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzel- Phyzelaphryne aphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molec- ular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13 Ma minimum. This striking biogeographic pattern coincides with major events that have shaped the face of the South American continent, as we know it today. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction clock for many biologists before this invaluable heritage vanishes. This is particularly critical in the tropics, which host the bulk of the Life is facing its 6th mass extinction (Barnosky et al., 2011), and diversity on Earth (Gaston and Williams, 1996) but still remain the description of the world’s biodiversity is a race against the largely under-documented regarding the actual magnitude of their biological diversity and the mechanisms responsible for its origin (Balakrishnan, 2005). Tropical forests of South America are crucial ⇑ Corresponding author at: CNRS-Guyane – USR 3456, Immeuble Le Relais – 2, Avenue Gustave Charlery, 97300 Cayenne, French Guiana. because they are believed to host more species than anywhere else E-mail address: [email protected] (A. Fouquet). in the world (Gaston and Williams, 1996; Myers et al., 2000; 1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.07.012 548 A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561 Primack and Corlett, 2005; Wilson, 1992). Despite being flagged Almeida et al., 2011). Such puzzling distribution led Hoogmoed among the famous ‘‘biodiversity hotspots’’ as a priority area for and Lescure (1984) to question the homogeneity of Phyzelaphryni- conservation some 15 years ago (Mittermeier et al., 1998), the nae, which has been later hypothesized to consist of remnants of a Atlantic forest of Brazil is still being degraded at a fast/steady pace, once more diverse and broadly distributed clade (Gonzalez-Voyer particularly in its northern part (Ribeiro et al., 2009). Even the et al., 2011). These frogs have been poorly represented in recent ef- immense Amazonia, with a large part of its surface remaining rel- forts to document phylogenetic relationships among Terrarana, atively intact, deeply suffers from human activities and is reduced and anurans in general (Hedges et al., 2008; Heinicke et al., in size at an extremely fast pace (Malhi et al., 2008). 2009); only one Adelophryne and one Phyzelaphryne samples have The biodiversity hosted by both areas is still so poorly docu- been so far included (Hedges et al., 2008; Heinicke et al., 2009). mented that we do not know what is lost with each exploited hect- The obvious reason for this is that it is challenging to gather a are of forest (Tuomisto et al., 1995; da Silva et al., 2005; Carnaval meaningful sampling because these frogs are very small et al., 2009). Moreover, in the last decades many studies have re- (SVL = 11 mm in A. pachydactyla to a maximum of 23 mm in A. pat- vealed a large underestimation of species number actually occur- amona), secretive (some species are locally very common, like A. ring in these regions (Giam et al., 2012). This has been gutturosa, but hard to find because of their microhabitat [Kok particularly striking for amphibians, with many species now recog- and Kalamandeen, 2008]) and with very restricted distributions nized as localized endemics or even isolated micro-endemics occur- (i.e. a few localized patches over an entire continent). These di- ring in small patches of these forests (e.g. Fouquet et al., 2012a, b; rect-developing frogs (Cassiano-Lima et al., 2011; MacCulloch Funk et al., 2012). Given that more than one third of the amphibian et al., 2008) are found exclusively in or under the forest litter (A. species are currently threatened by extinction – thus more than in maranguapensis breeds in bromeliads [Cassiano-Lima et al., any other vertebrate group (Stuart et al., 2004, 2008) – assessing the 2011]) from lowlands to mountain forests up to 1400 m a.s.l. Their actual biodiversity represented by South American amphibians is natural history is extremely poorly documented. Cassiano-Lima becoming a major challenge (Wake and Vredenburg, 2008). et al. (2011) recently provided some information on the reproduc- Estimating the South American biodiversity is not only a matter tion and development of A. maranguapensis, and MacCulloch et al. of counting species, but also of accounting for the depth of the rela- (2008) as well as Kok and Kalamandeen (2008) reported the obser- tionships among species, the so-called ‘‘phylogenetic diversity’’ vation of A. gutturosa laying a single very large egg. (Faith, 1992; Crozier, 1997; Purvis and Hector, 2000). Recent stud- Adelophryne and Phyzelaphryne (Phyzelaphryninae) can be dis- ies have revealed that many South American lineages are in fact tinguished from other Terrarana by the shape of terminal digits. millions of years old. Even among closely related South American However, the morphological distinction between these two genera amphibian species (e.g. Grant et al., 2006; Heinicke et al., 2007; is somewhat ambiguous (Hoogmoed and Lescure, 1984) and the Fouquet et al., 2012a) or within species (e.g. Fouquet et al., 2007, monophyly of Adelophryne has never been formally tested. Molec- 2012b; Funk et al., 2012) the prevalence of deep divergence re- ular analyses included only one Adelophryne and one Phyzelaph- vealed by molecular phylogenetics and phylogeography has been ryne, and these samples formed unambiguously a natural group – astonishing. The extent of unrecognized species that are geograph- i.e. Phyzelaphryninae (Hedges et al., 2008). However, considering ically restricted and could represent millions of years of indepen- the reduced number of terminals included and the