Molecular Phylogenetics and Evolution 65 (2012) 547–561

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Molecular Phylogenetics and Evolution

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From Amazonia to the Atlantic forest: Molecular phylogeny ofPhyzelaphryninae frogs reveals unexpected diversity and a strikingbiogeographic 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 Guianab Departamento de Zoologia, Universidade de São Paulo, Instituto de Biociências, Caixa Postal 11.461, CEP 05508-090 São Paulo, SP, Brazilc 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, Brazild Department of Herpetology, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024-5192, United Statese 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, Brazilf 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, Brazilg Department of Vertebrates, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000 Brussels, Belgiumh Biology Department, Unit of Ecology and Systematics, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

a r t i c l e i n f o

Article history:Received 12 May 2012Revised 13 July 2012Accepted 14 July 2012Available online 26 July 2012

Keywords:AdelophryneAmazoniaAtlantic forestCryptic speciesNeotropical diversityPhyzelaphryne

1055-7903/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ympev.2012.07.012

⇑ Corresponding author at: CNRS-Guyane – USR 34Avenue Gustave Charlery, 97300 Cayenne, French Gu

E-mail address: fouquet.antoine@gmail.com (A. Fo

a b s t r a c t

Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of glo-bal climate change and the pace of environmental alteration. Recent molecular phylogenetic studies haverevealed that the actual number of species in South American tropical forests is largely underestimated,but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6spp.), 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-nia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominalspecies of the subfamily as well as for newly discovered populations in order to (1) test the monophyly ofPhyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily,and (3) investigate their historical biogeography and diversification. Phylogenetic reconstructionconfirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzel-aphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forestand southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninaeis, at least, twice the currently recognized species diversity, with almost every geographically isolatedpopulation representing an anciently divergent candidate species. Such results highlight the challengesfor 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 Mioceneto the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Maminimum, 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 SouthAmerican continent, as we know it today.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Life is facing its 6th mass extinction (Barnosky et al., 2011), andthe description of the world’s biodiversity is a race against the

ll rights reserved.

56, Immeuble Le Relais – 2,iana.uquet).

clock for many biologists before this invaluable heritage vanishes.This is particularly critical in the tropics, which host the bulk of thediversity on Earth (Gaston and Williams, 1996) but still remainlargely under-documented regarding the actual magnitude of theirbiological diversity and the mechanisms responsible for its origin(Balakrishnan, 2005). Tropical forests of South America are crucialbecause they are believed to host more species than anywhere elsein the world (Gaston and Williams, 1996; Myers et al., 2000;

548 A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561

Primack and Corlett, 2005; Wilson, 1992). Despite being flaggedamong the famous ‘‘biodiversity hotspots’’ as a priority area forconservation some 15 years ago (Mittermeier et al., 1998), theAtlantic forest of Brazil is still being degraded at a fast/steady pace,particularly in its northern part (Ribeiro et al., 2009). Even theimmense Amazonia, with a large part of its surface remaining rel-atively intact, deeply suffers from human activities and is reducedin size at an extremely fast pace (Malhi et al., 2008).

The biodiversity hosted by both areas is still so poorly docu-mented that we do not know what is lost with each exploited hect-are of forest (Tuomisto et al., 1995; da Silva et al., 2005; Carnavalet al., 2009). Moreover, in the last decades many studies have re-vealed a large underestimation of species number actually occur-ring in these regions (Giam et al., 2012). This has beenparticularly striking for amphibians, with many species now recog-nized as localized endemics or even isolated micro-endemics occur-ring in small patches of these forests (e.g. Fouquet et al., 2012a, b;Funk et al., 2012). Given that more than one third of the amphibianspecies are currently threatened by extinction – thus more than inany other vertebrate group (Stuart et al., 2004, 2008) – assessing theactual biodiversity represented by South American amphibians isbecoming a major challenge (Wake and Vredenburg, 2008).

Estimating the South American biodiversity is not only a matterof counting species, but also of accounting for the depth of the rela-tionships among species, the so-called ‘‘phylogenetic diversity’’(Faith, 1992; Crozier, 1997; Purvis and Hector, 2000). Recent stud-ies have revealed that many South American lineages are in factmillions of years old. Even among closely related South Americanamphibian species (e.g. Grant et al., 2006; Heinicke et al., 2007;Fouquet et al., 2012a) or within species (e.g. Fouquet et al., 2007,2012b; Funk et al., 2012) the prevalence of deep divergence re-vealed by molecular phylogenetics and phylogeography has beenastonishing. The extent of unrecognized species that are geograph-ically restricted and could represent millions of years of indepen-dent evolution, and whether these undetected species arethreatened in the Atlantic forest and Amazonia are questions thatstill cannot be answered.

A large proportion of South American frogs are terraranans, i.e.the New World direct-developing frogs, with more than 900 spe-cies (Heinicke et al., 2007, 2009; Hedges et al., 2008). For example,the genus Pristimantis holds �400 nominal species and representsthe most species-rich genus among terrestrial vertebrates (Hei-nicke et al., 2007; Hedges et al., 2008), whereas other genera ofTerrarana have very few and are restricted to very small areasdespite being of similar age (Gonzalez-Voyer et al., 2011). WithinTerrarana, Eleutherodactylidae provides another striking exampleof such unbalance. This family holds more than 200 species, thegenus Eleutherodactylus (including subgenera Syrrophus, Euhyas,Peloruis, Schwartzius) holding more than 190 species and theremaining 16 species belonging to three other much smaller gen-era: Diasporus (n = 9), Adelophryne (n = 6) and Phyzelaphryne (n = 1).

The geographical distribution of these four genera provides astriking pattern given that they are all allopatric from southernUSA to the Atlantic forest in Brazil. Eleutherodactylus has diversifiedin the Caribbean and the southern part of North America, while itssister group, Diasporus, occurs in Central America and Chocó(Colombia). The two other genera are also found allopatricallyand display an intriguing distribution. Phyzelaphryne is a mono-typic genus previously known from only a few localities south ofthe Amazon River (Heyer, 1977; Heyer and Gascon, 1995; De laRiva et al., 2000), and Adelophryne contains six nominal speciesscattered in the northern periphery of Amazonia and in isolatedpatches of the remnants of the northern part of the Atlantic forestof Brazil. Most Adelophryne and Phyzelaphryne species are knownonly from their type locality and very few additional scatteredpopulations (Loebmann et al., 2011; Ortega-Andrade, 2009;

Almeida et al., 2011). Such puzzling distribution led Hoogmoedand Lescure (1984) to question the homogeneity of Phyzelaphryni-nae, which has been later hypothesized to consist of remnants of aonce more diverse and broadly distributed clade (Gonzalez-Voyeret al., 2011). These frogs have been poorly represented in recent ef-forts to document phylogenetic relationships among Terrarana,and anurans in general (Hedges et al., 2008; Heinicke et al.,2009); only one Adelophryne and one Phyzelaphryne samples havebeen so far included (Hedges et al., 2008; Heinicke et al., 2009).The obvious reason for this is that it is challenging to gather ameaningful sampling because these frogs are very small(SVL = 11 mm in A. pachydactyla to a maximum of 23 mm in A. pat-amona), secretive (some species are locally very common, like A.gutturosa, but hard to find because of their microhabitat [Kokand Kalamandeen, 2008]) and with very restricted distributions(i.e. a few localized patches over an entire continent). These di-rect-developing frogs (Cassiano-Lima et al., 2011; MacCullochet al., 2008) are found exclusively in or under the forest litter (A.maranguapensis breeds in bromeliads [Cassiano-Lima et al.,2011]) from lowlands to mountain forests up to 1400 m a.s.l. Theirnatural history is extremely poorly documented. Cassiano-Limaet al. (2011) recently provided some information on the reproduc-tion and development of A. maranguapensis, and MacCulloch et al.(2008) as well as Kok and Kalamandeen (2008) reported the obser-vation of A. gutturosa laying a single very large egg.

Adelophryne and Phyzelaphryne (Phyzelaphryninae) can be dis-tinguished from other Terrarana by the shape of terminal digits.However, the morphological distinction between these two generais somewhat ambiguous (Hoogmoed and Lescure, 1984) and themonophyly of Adelophryne has never been formally tested. Molec-ular analyses included only one Adelophryne and one Phyzelaph-ryne, and these samples formed unambiguously a natural group –i.e. Phyzelaphryninae (Hedges et al., 2008). However, consideringthe reduced number of terminals included and the frequent mor-phological conservatism or parallel evolution in general morphol-ogy observed in other groups of Terrarana distributed in similarenvironments (e.g. Psychrophrynella, Phrynopus) (Hedges et al.,2008; Gonzalez-Voyer et al., 2011), it would not be surprising tofind within Phyzelaphryninae relationships that contradict the cur-rent taxonomy.

For example, some specimens from Colombia were allocated tothe genus Phyzelaphryne in their original description (Heyer, 1977),and the advertisement call was described based on the Colombianmaterial, but Hoogmoed and Lescure (1984) demonstrated that thecall of P. miriamae described by Heyer (1977) in fact pertains toAdelophryne adiastola. Conversely, Lynch (2005) identified as A. adi-astola specimens from Leticia that might actually belong to thegenus Phyzelaphryne (see below). Moreover, the biogeographic pat-tern observed within Adelophryne – i.e. occurring in northernAmazonia and the Atlantic forest, is mystifying given that theAtlantic forest is separated from Amazonia by a northeast–south-west belt of open or dry formations (Prado and Gibbs, 1993; da Sil-va et al., 2004), which currently acts, and has acted in the past, as abarrier to biotic exchanges between these two forest blocks (Costa,2003; Mori et al., 1981). Many ancient clades are endemic to one orthe other of these regions, having very few species in common(Duellman, 1999). The patterns provided by new insights in theunderstanding of evolutionary relationships in many groups likeDendrophryniscus/Amazophrynella (Fouquet et al., 2012b), Allobates(Santos et al., 2009), Leposoma (Pellegrino et al., 2011), and Vitreor-ana (Guayasamin et al., 2008, 2009) suggest that small vertebratesdistributed in Amazonia and the Atlantic forest, i.e. having frag-mented range restricted to forest habitat, could display tens of mil-lions of years of divergence. Moreover, highly conservative orparallel morphological evolution in small, dull-colored, terrestrial,leaf litter-associated amphibian species has been repeatedly

A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561 549

highlighted as a source of cryptic diversity (Fouquet et al., 2007;Vieites et al., 2009).

Therefore, given the conservation importance of amphibians inthe region, and the numerous gaps in our understanding of thediversity and the origin of Phyzelaphryninae, we propose to (1) testthe monophyly of Phyzelaphryninae within Terrarana and themonophyly of Adelophryne and Phyzelaphryne, (2) estimate speciesdiversity within these genera and (3) investigate their biogeogra-phy and evolutionary history.

2. Materials and methods

2.1. Sampling

Tissue samples of all nominal species within Phyzelaphryninaewere taken from thigh muscle or liver (in one case from eggs) andpreserved in 95% ethanol (Table 1). Specimens were collected fromtheir type locality or from the closest localities possible, and aredeposited in different collections (Table 1). We also collected tissuesamples from specimens of newly discovered populations thatwere tentatively identified as belonging to one of the nominalspecies within Phyzelaphryninae. Sequences of some specimensof Phyzelaphryne miriamae and Adelophryne gutturosa were re-trieved from GenBank. Genomic DNA was extracted using PromegaDNA extraction kit. A total of 31 Adelophryne and 14 Phyzelaphryneindividuals were included. We follow the classification of Pyronand Wiens (2011) for Terrarana.

We targeted three mitochondrial (Cytb; COI; 12S–16S) andthree nuclear loci (RAG1; POMC; TYR) that were already partlyavailable for main Terrarana (n = 14), Hyloidea lineages (n = 14)and five outgroups (Table 1), which were collated together withPhyzelaphryninae for a total of 80 terminals. Data not presentedhere for A. baturitensis from Serra de Baturité (type locality) andSerra de Maranguape show that these populations are very similarto the ones included herein from Serra da Ibiapaba.

In order to reduce missing data for the other Terrarana and otherHyloidea terminals, we concatenated sequences from different spe-cies or even genera (Pelodryadinae, Centrolenidae, Sooglossus/Nasikabatrachus) when monophyly of the group involved wasunambiguous. The only early-diverging lineage within Terraranathat was not represented is Ceuthomantidae because we consid-ered the available data too limited for nuclear DNA (missing POMC,TYR and most RAG-1) to be included in our analyses; the position ofCeuthomantidae is also well supported (Heinicke et al., 2009). Wealso completed the matrix directly from biological material (Table1) and therefore produced sequence data for two loci or more formost Terrarana terminals (Holoaden, Brachycephalus, Oreobates,Haddadus, Barycholos and Eleutherodactylus) up to all the loci forEuparkerella, for which no sequences were previously available.

Fragments were amplified by standard PCR techniques; detailedinformation about the primers is available in Table 2. Sequencingwas performed using ABI Big Dye V3.1 (ABI, Foster City, USA) andresolved on an automated sequencer at IQUSP and GenomicEngenharia corp. (São Paulo, Brazil) and Macrogen Inc. (Korea). Se-quences were edited and aligned with CodonCode Aligner v.3.5.2.Novel sequences were deposited in GenBank (Table 1).

We generated 278 new sequences of terraranans (Table 1).Within Phyzelaphryninae some terminals harbor substantial miss-ing data. Nevertheless, preliminary analyses suggested that thesewere not impeding resolution given that these missing data wereevenly distributed among main lineages (Lemmon et al., 2009;Wiens and Morrill, 2011; Wiens, 1998, 2003; Simmons, 2012).For other terminals, missing data were limited to a maximum oftwo complete loci for Bryophryne (Cytb; RAG-1) and one completelocus for three terminals (Hypodactylus and Phrynopus for COI;

Nasikabatrachus/Sooglossus for POMC). A 345 bp portion of the Cytbfragment remained missing for six Terrarana terminals and Gas-trotheca; and a portion of RAG-1 for seven Terrarana lineages.

2.2. Data analyses

2.2.1. AlignmentMost data consisted of coding regions, and alignment was

unambiguous. We observed the insertion/deletion of one codonin RAG-1 fragment for Hyloidea/outgroup and several codon inser-tion/deletions in POMC, but none of them led to ambiguous align-ment after checking the reading frame. For the 12S–16S fragmentwe performed alignment with MAFFT v6 (Katoh et al., 2002) underdefault parameters except for the use of the L-INS-i strategy, whichis adapted to sequences with one conserved domain and long gaps.We obtained a final 5841 bp alignment. We used Bayesian analysisand Maximum Parsimony to investigate phylogenetic relationshipsamong terminals.

2.2.2. Bayesian analysisWe divided the dataset into seven partitions: one for each co-

don position of the mtDNA (1388/3 bp) and the nuDNA codinggenes (2515 bp/3) and one for the 12S/16S fragment (1938 bp).The choice of this partitioning was driven by the coding natureof mtDNA (Cytb, COI) and nuDNA (RAG1, POMC, TYR) loci and com-parable rates of evolution (Mueller, 2006; Hoegg et al., 2004; Fou-quet et al., 2012a; see results). Many studies, indeed, reported thatpartitioning by both gene and codon position gave the best fit tothe data (Caterino et al., 2001; Brandley et al., 2005). A more inclu-sive partitioning would have joined very different patterns ofmolecular evolution, and greater partitioning would likely causeoverparameterization (Marshall, 2010; Sullivan and Joyce, 2005).

We used the software jModeltest version 0.1.1 (Posada, 2008;Guindon and Gascuel, 2003) to select the substitution model thatbest fits each of these partitions under Akaike’s Information Crite-rion (Akaike, 1974). The seven resulting models (Suppl. Mat.) wereemployed in a Bayesian analysis with MrBayes 3.2 (Huelsenbeckand Ronquist, 2001; Ronquist and Huelsenbeck, 2003). The Bayes-ian analysis consisted of two independent runs of 2.0 � 107 gener-ations, starting with random trees and 10 Markov chains (onecold), sampled every 1000 generations. We also performed sepa-rate runs for mtDNA and nuDNA using the same partitions andmodels and 2.0 � 107 generations for each run (Suppl. Mat.). Ade-quate burn-in was determined by examining likelihood scores ofthe heated chains for convergence on stationarity, as well as theeffective sample size of values in Tracer 1.5 (Rambaut and Drum-mond, 2007). We discarded 10% of the generations/trees. We con-sidered relationships strongly supported when posteriorprobabilities were equal to or higher than 0.95.

2.2.3. Maximum parsimonyOf the 5841 total characters of the matrix, 2285 are constant,

538 variable characters are parsimony-uninformative and 3018are parsimony-informative using gaps as a fifth character state.The mtDNA partition totaled 3326 characters (1060 are constant,1974 parsimony informative) and the nuDNA partition totaled2515 characters (1225 are constant, 1044 parsimony informative).

We employed PAUP 4.0b10 (Swofford, 2002) to search for theshortest tree with the heuristic search option, tree bisection–reconnection (TBR) for branch swapping on 100 random-additionsequence replicates. We subsequently computed 500 nonparamet-ric bootstrap pseudoreplicates (Efron, 1979; Felsenstein, 1985). Wealso performed separate runs for mtDNA and nuDNA using thesame scheme (Suppl. Mat.). We considered relationships stronglysupported when MP bootstrap percentages equaled or exceeded70% (Hillis and Bull, 1993). We also ran a similar analysis treating

Table 1Sequence details including vouchers and accession numbers used for the Bayesian analysis.

Genus Species Cytb COI 12S 16S RAG1a RAG1b POMC TYR Locality Stat Lat. Long.

Adelophryne baturitensis CFBHT11100/JX298317

CFBHT11100/JX298245

CFBHT11100/JX298277

CFBHT11100/JX298145

CFBHT11100/JX298096 CFBHT11100/JX298197 Tiangua CE �3.709925 �40.934057

Adelophryne baturitensis CFBHT11101/JX298318

CFBHT11101/JX298246

CFBHT11101/JX298278

CFBHT11101/JX298146

CFBHT11101/JX298097 CFBHT11101/JX298198 Tiangua CE �3.709925 �40.934057

Adelophryne baturitensis CFBHT11110/JX298319

CFBHT11110/JX298247

CFBHT11110/JX298279

CFBHT11110/JX298147

CFBHT11110/JX298098 CFBHT11110/JX298199 Ibiapina CE �3.909126 �40.866494

Adelophryne baturitensis CFBHT11339/JX298377 CFBHT11339/JX298322

CFBHT11339/JX298250

CFBHT11339/JX298282

CFBHT11339/JX298150

CFBHT11339/JX298150

CFBHT11339/JX298101 CFBHT11339/JX298202 Ubajara CE �3.842332 �40.89323

Adelophryne baturitensis MTR14012/JX298375 MTR14012/JX298320

MTR14012/JX298248 MTR14012/JX298280 MTR14012/JX298148

MTR14012/JX298148 MTR14012/JX298099 MTR14012/JX298200 Ibiapaba CE �5.078753 �40.933371

Adelophryne baturitensis MTR14013/JX298376 MTR14013/JX298321

MTR14013/JX298249 MTR14013/JX298281 MTR14013/JX298149

MTR14013/JX298100 MTR14013/JX298201 Ibiapaba CE �5.078753 �40.933371

Adelophryne sp. 2 PEU80/JX298379 PEU80/JX298323 PEU80/JX298283 PEU80/JX298151 PEU80/JX298103 PEU80/JX298204 WenceslauGuimares

BA �13.68852 �39.483175

Adelophryne sp. 3 MTR20222/JX298378 MTR20222/JX298102 MTR20222/JX298203 Rio Patipe, APAGuaibim

BA �13.32164 �39.016328

Adelophryne sp. 1 CFBHT11716/JX298380 CFBHT11716/JX298324

CFBHT11716/JX298251

CFBHT11716/JX298284

CFBHT11716/JX298104 CFBHT11716/JX298205 Caruaru PE �8.25498 �35.904408

Adelophryne maranguapensis CFBHT14103/JX298325

CFBHT14103/JX298252

CFBHT14103/JX298285

CFBHT14103/JX298152

CFBHT14103/JX298105 CFBHT14103/JX298206 Maranguape CE �3.89029 �38.712502

Adelophryne maranguapensis CFBHT14119/JX298381 CFBHT14119/JX298326

CFBHT14119/JX298253

CFBHT14119/JX298286

CFBHT14119/JX298153

CFBHT14119/JX298153

CFBHT14119/JX298106 CFBHT14119/JX298207 Maranguape CE �3.89029 �38.712502

Adelophryne sp. 5 CFBHE234/JX298383 CFBHE234/JX298328

CFBHE234/JX298254 CFBHE234/JX298288 CFBHE234/JX298155

CFBHE234/JX298155 CFBHE234/JX298108 CFBHE234/JX298209 Mariana MG �20.3663 �43.444848

Adelophryne sp. 5 CFBHE235/JX298329

CFBHE235/JX298255 CFBHE235/JX298289 CFBHE235/JX298109 CFBHE235/JX298210 Mariana MG �20.3663 �43.444848

Adelophryne sp. 5 MTR17521/JX298382 MTR17521/JX298327

MTR17521/JX298287 MTR17521/JX298154

MTR17521/JX298107 MTR17521/JX298208 PE Rio Doce,Marliéria

MG �19.70991 �42.729192

Adelophryne sp. 5 MTR21918/JX298330

MTR21918/JX298156

MTR21918/JX298110 MTR21918/JX298211 Serra do Cipo MG �19.54879 �43.550606

Adelophryne sp. 4 MTR13570/JX298384 MTR13570/JX298331

MTR13570/JX298256 MTR13570/JX298290 MTR13570/JX298157

MTR13570/JX298111 MTR13570/JX298212 Faz. NovaAlegria,Trancoso

BA �16.531111 �39.118056

Adelophryne sp. 6 MTR15919/JX298385 MTR15919/JX298332

MTR15919/JX298291 MTR15919/JX298158

MTR15919/JX298158 MTR15919/JX298112 MTR15919/JX298213 Serra Bonita,Camacan

BA �15.3901 �39.5630

Adelophryne sp. 6 CFBH23672/JX298386 CFBH23672/JX298333

CFBH23672/JX298257

CFBH23672/JX298292

CFBH23672/JX298159

CFBH23672/JX298159

CFBH23672/JX298113 CFBH23672/JX298214 Una BA �15.2716 �39.069843

Adelophryne pachydactyla MTR16244/JX298388 MTR16244/JX298335

MTR16244/JX298259 MTR16244/JX298294 MTR16244/JX298161

MTR16244/JX298161 MTR16244/JX298115 MTR16244/JX298216 Serra dasLontras,Arataca

BA �15.1833 �39.3452

Adelophryne pachydactyla MTR5988/JX298387 MRT5988/JX298334

MTR5988/JX298258 MRT5988/JX298293 MRT5988/JX298160

MRT5988/JX298160 MRT5988/JX298114 MRT5988/JX298215 Serra doTeimoso,Jussari

BA �15.210915 �39.480972

Adelophryne sp. 7 MTR13808/JX298389 MTR13808/JX298336

MTR13808/JX298295 MTR13808/JX298162

MTR13808/JX298162 MTR13808/JX298116 MTR13808/JX298217 Serra do Navio AP 0.912857 �52.007933

Adelophryne patamona PK1875/JX298164 PK1875/JX298118 PK1875/JX298219 MountMaringma

Gu 5.219169 �60.575209

Adelophryne patamona PK1969/JX298390 PK1969/JX298337 PK1969/JX298260 PK1969/JX298296 PK1969/JX298163 PK1969/JX298163 PK1969/JX298117 PK1969/JX298218 MountMaringma

Gu 5.219169 �60.575209

Adelophryne patamona(Paratype)

ROM43035/JX298339

ROM43035/JX298262

ROM43035/JX298298

ROM43035/JX298166

ROM43035/JX298120 MountWokomung

Gu 5.089576 �59.827538

Adelophryne patamona(Holotype)

ROM43034/JX298338

ROM43034/JX298261

ROM43034/JX298297

ROM43034/JX298165

ROM43034/JX298119 ROM43034/JX298220 MountWokomung

Gu 5.089576 �59.827538

Adelophryne patamona ROM39578/GQ345201 ROM39578/EU186679

ROM39578/EU186679

ROM39578/GQ345296

ROM39578/GQ345262 ROM39578/EU186772 MountAyanganna

Gu 5.395223 �59.962406

Adelophryne adiastola AJC2463JX298391 AJC2463/JX298340

AJC2463/JX298263 AJC2463/JX298299 AJC2463/JX298167

AJC2463/JX298167 AJC2463/JX298121 AJC2463/JX298221 Com. PuertoVaupes

Col. 1.198056 �70.281389

Adelophryne gutturosa PK1168/JX298393 PK1168/JX298342 PK1168/JX298266 PK1168/JX298302 PK1168/JX298169 PK1168/JX298123 PK1168/JX298223 Muri Muricreek,KaieteurNP

Gu 5.27729 �59.432316

Adelophryne gutturosa PK2231/JX298392 PK2231/JX298341 PK2231/JX298264 PK2231/JX298300 PK2231/JX298168 PK2231/JX298122 PK2231/JX298222 La Escalera,Bolivar state

Ven 6.014069 �61.449308

Adelophryne gutturosa PK1362/JX298170 PK1362/JX298124 PK1362/JX298224 Elinkwa creek,KaieteurNP

Gu 5.27729 �59.432316

Adelophryne gutturosa ROM44051/JX298265

ROM44051/JX298301

Meamu River Gu 6.232029 �60.619926

Phyzelaphryne miriamae SMS629/JX298394 SMS629/JX298343 SMS629/JX298267 SMS629/JX298303 SMS629/JX298171 SMS629/JX298125 SMS629/JX298225 Com. SãoSebastião dosBargas

AM �3.78943 �59.034048

Phyzelaphryne miriamae MTR19141/JX298397 MTR19141/JX298347

MTR19141/JX298307 MTR19141/JX298175

MTR19141/JX298175 MTR19141/JX298129 MTR19141/JX298229 Moio Bamba,Margem DPurus

AM �4.720095 �62.133036

Phyzelaphryne miriamae MTR19437/JX298396 MTR19437/JX298346

MTR19437/JX298306 MTR19437/JX298174

MTR19437/JX298174 MTR19437/JX298128 MTR19437/JX298228 Moio Bamba,Margem D

AM �4.720095 �62.133036

550A

.Fouquetet

al./Molecular

Phylogeneticsand

Evolution65

(2012)547–

561

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A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561 551

Table 1 (continued)

Genus Species Cytb COI 12S 16S RAG1a RAG1b POMC TYR Locality Stat Lat. Long.

ornata DQ679269 DQ679269Odontophrynus americanus AF665/FJ685666 AF665/JX298366 JF1891/AY843704 JF1891/AY843704 AF665/JX298191 JF1891/AY844480 AF665/JX298141 AF665/JX298239Thoropa miliaris/

taophoraAF1434/FJ685662 CFBH3239/

DQ502874CFBH3239/DQ283331

CFBH3239/DQ283331

AF1434/JX298192 USNM209318/GQ345301

USNM209318/GQ345271

AF1434/JX298241

Rhinella margaritifera/arenarum

ROM40103/JX298409 ROM40103/JX298367

USNM268828/DQ158490

USNM268828/DQ158490

USNM268828/DQ158407

MACN38639/AY844370

KU215143/AY819080 MRT6313/JN692075

Allobates femoralis/granti/trilineatus

AfemSapoiv10a/DQ523152

OMNH36070/DQ502811

LSUMZ17436/EU342537

LSUMZ17436/EU342537

AF519/JX298193 UTAA56478/DQ503385

KU220660/AY819088 OMNH36070/DQ503156

Centrolenidae uranoscopa/colymbiphyllum/eurygnatha/valerioi/bejaranoi

MTR15819/JX298412 KRL0852/FJ766714

CFBH5729/AY843595 CFBH5729/AY843595 MTR15819/JX298194

UCR17418/EU663519 MTR15819/JX298142 MNK5242/AY844029

Paratelmatobius mantiqueira/cardosoi

ITH0938/JX298413 ITH0938/JX298372

CFBH240/EU224408 CFBH240/EU224408 ITH0938/JX298195

ITH0938/JX298195 ITH0938/JX298143 ITH0938/JX298242

Leptodactylus knudseni/pentadactylus/myersi

396MC/JX298414 396MC/JX298373 FC13095/AY326017 FC13095/AY326017 1890T/JX298196 1890T/JX298196 396MC/JX298144 109MC/JX298243

Amazoprynella bokermanni/minutus/sp.

MTR10040/JX298410 MTR10176/JX298368

QCAZ883/DQ158420 QCAZ883/DQ158420 QCAZ883/DQ158346

MJH7095/DQ503337 KU221827/AY819081 3035T/JX298240

Hyla japonica/arenicolor

IABHU6123/AB303949 IABHU6123/AB303949

LSUMZH-230/AY843633

LSUMZH-230/AY843633

??/FJ227068 LSUMZH-230/AY844420

PB42-7/HM152465 LSUMZH-230/AY844078

Phyllomedusa/Agalychnis

oreades/callidryas/tomopterna

CHUNB56875/GQ365966

KRL0917/FJ766570

WED55380/AY326045

WED55380/AY326045

KU221949/EF174319

MJH7076/AY844497 KU221949/AY819153 MJH7076/AY844157

Litoria/Cyclorana aurea/caerulea/meiriana

AM52744/AY843937+manyaDLSN-72386/EF125030

??/AY835904 DMH/AY326038 DMH/AY326038 TWR1007/EF174310

SAMA17215/AY844475

AM52744/GQ366037 AM52744/AY844130

Rana nigromaculata NC_002805 NC_002805 NC_002805 NC_002805 KUHE32995/AB526661

temporaria??/AY323776

KUHE32995/AB526647 FMNH232879/DQ282932

Kaloula pulchra/taprobanica

cplGEN/AY458595 cplGEN/AY458595 cplGEN/AY458595 cplGEN/AY458595 SIH-09/AY323772 SIH-09/AY323772 ZCMV11017/HM998968 AB611924

Microhyla heymonsi/sp. cplGEN/AY458596 cplGEN/AY458596 cplGEN/AY458596 cplGEN/AY458596 MVZ236751/EF396095

MVZ236751/EF396095

MNCN-DNA28462/HM998967

MVZ236751/EF395979

Calyptocephallela geayi JN3/JX298415 JN3/JX298374 AMNHA168414/DQ283439

AMNHA168414/DQ283439

MNCN8002/AY583337

MNCN8002/AY583337

??/AY819090 JN3/JX298244

Nasikabatrachus/Sooglossus

thomasseti/sahyadrensis/sechellensis

?/AY341742 ??/GU136124 UMMZ(#15)/DQ28344

RAN25162/DQ283452

MNHN2003.3412/DQ872921

MNHN2003.3412/DQ872921

UMMZ(#15)/DQ283028

Acronyms for newly added material: CFBHT = Celio F. B. Haddad Tissue collection; MTR, PEU, AF, FS, MCL, ITH = Miguel Trefaut Rodrigues field number; PK = Philippe Kok field numbers; SMS = Sergio Marques de Souza fieldnumbers; ROM = Royal Ontario Museum; AJC = Andrew J. Crawford field numbers; T = François Catzeflis field numbers; JN = José J. Nuñes field numbers; LSUMZ = Lousiana State University Museum of Zoology; MNCN = MuseoNacional de Ciencias Naturales; AndesA = Universidad de los Andes; MC = Christian Marty field numbers; JMP = Jose M. Padial field numbers.

552A

.Fouquetet

al./Molecular

Phylogeneticsand

Evolution65

(2012)547–

561

Table 2Primer details including primer name, sequences and authors.

Gene Primers Sequences Authors

Cytb CYB-05L GCCAACGGCGCATCCTTCTTCTT Meyer (1993)Cytb LGL765 GAAAAACCAYCGTTGTWATTCAACT Bickham et al. (1995)Cytb CbR2 GTGAAGTTRTCYGGGTCYCC Fouquet et al. (2012)COI dgLCO1490 GGTCAACAAATCATAAAGAYATYGG Meyer (1993)COI dgHCO2198 TAAACTTCAGGGTGACCAAARAAYCA Meyer (1993)12S t-Phe-frog ATAGCRCTGAARAYGCTRAGATG Wiens et al. (2005)12S t-Val-frog TGTAAGCGARAGGCTTTKGTTAAGCT Wiens et al. (2005)12S MVZ59 ATAGCACTGAAAAYGCTDAGATG Graybeal (1997)12S tRNAval GGTGTAAGCGAGAGGCTT Goebel et al. (1999)16S 16Sbr-H CCGGTCTGAACTCAGATCACGT Palumbi et al. (1991)16S 16SC-16L GTRGGCCTAAAAGCAGCCAC Darst and Cannatella (2004)RAG1a MartFL1 AGCTGGAGYCARTAYCAYAARATG Hoegg et al. (2004)RAG1a Ad2R ATTGGCTCTCCATGTTTCATAG This paperRAG1a AMPF2 ACNGGNMGICARATCTTYCARCC Hoegg et al. (2004)RAG1a RAG1C GGAGATGTTAGTGAGAARCAYGG Biju and Bossuyt (2003)RAG1a Ad1R CTTCACGCACCAACTTTTCATC This paperRAG1b Amp F1 ACAGGATATGATGARAAGCTTGT Hoegg et al. (2004)RAG1b Mart R6 GTGTAGAGCCARTGRTGYTT Hoegg et al. (2004)POMC POMC1 GAATGTATYAAAGMMTGCAAGATGGWCCT Wiens et al. (2005)POMC POMC2 TAYTGRCCCTTYTTGTGGGCRTT Wiens et al. (2005)TYR TYR1E GAGAAGAAAGAWGCTGGGCTGAG Bossuyt and Milinkovitch (2000)TYR TYR1C GGCAGAGGAWCRTGCCAAGATGT Bossuyt and Milinkovitch (2000)TYR TYR1H ACACTTCTGGGCATCTCTCC Bossuyt and Milinkovitch (2000)

A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561 553

gaps as missing data to check whether it could have a significantinfluence.

2.2.4. Single nuDNA locus networks and mtDNA genetic distancesIn order to support our proposed species delineation we also

computed a statistical parsimony network for each nuDNA locususing TCS 1.21 (Clement et al., 2000) with a 95% connection limit.The original alignments used previously were reduced to Phyzel-aphryninae and were also trimmed in order to reduce missing data.

Table 3p distances calculated among Phyzelaphryninae species using (a) Cytb; (b) 16S (the 463 b

(a) Cytb

A. baturitensisA. sp. 3 0.222A. sp. 2 0.231 0.202A. sp. 1 0.242 0.236 0.250A. maranguapensis 0.226 0.192 0.196 0.240A. sp. 5 0.273 0.307 0.255 0.305 0.297A. sp. 4 0.295 0.284 0.291 0.289 0.274 0.238A. sp. 6 0.289 0.298 0.289 0.287 0.270 0.250A. pachydactyla 0.273 0.286 0.281 0.296 0.283 0.244A. sp. 7 0.256 0.275 0.272 0.283 0.261 0.263A. patamona 0.278 0.253 0.267 0.269 0.253 0.285A. adiastola 0.276 0.284 0.300 0.276 0.274 0.277A. gutturosa 0.261 0.273 0.276 0.281 0.271 0.245P. miriamae 0.295 0.282 0.307 0.283 0.285 0.308P. sp. 1a 0.304 0.304 0.281 0.324 0.286 0.289

(b) 16S

A. baturitensisA. sp. 2 0.137A. sp. 1 0.118 0.144A. maranguapensis 0.128 0.170 0.138A. sp. 5 0.209 0.236 0.202 0.220A. sp. 4 0.184 0.195 0.180 0.209 0.138A. sp. 6 0.208 0.232 0.187 0.210 0.143 0.117A. pachydactyla 0.206 0.227 0.197 0.203 0.162 0.115A. sp. 7 0.195 0.219 0.192 0.212 0.171 0.132A. patamona 0.173 0.204 0.181 0.195 0.172 0.155A. adiastola 0.200 0.218 0.201 0.240 0.204 0.192A. gutturosa 0.207 0.194 0.184 0.233 0.204 0.172P. miriamae 0.197 0.234 0.210 0.239 0.231 0.205P. sp. 1a 0.213 0.235 0.246 0.262 0.245 0.231

We eventually kept 38 terminals and 889 bp for RAG1, 41terminals and 544 bp for POMC and 39 terminals and 532 bp forTYR. Because some haplotype groups were not connected to eachother within the 95% limit of probability of parsimony, we at-tempted to connect them by increasing the connection thresholdup to a maximum of 30 steps.

Genetic distances (p distance) were also computed for mito-chondrial loci using MEGA 5.1 (Tamura et al., 2011) and are shownin Table 3.

p ending).

0.2660.229 0.2270.260 0.263 0.2700.258 0.277 0.265 0.2260.253 0.264 0.279 0.236 0.2150.267 0.259 0.255 0.233 0.203 0.1840.281 0.265 0.285 0.245 0.281 0.243 0.2580.291 0.303 0.294 0.267 0.259 0.266 0.271 0.204

0.1230.156 0.1610.159 0.158 0.1410.196 0.202 0.182 0.1340.191 0.172 0.183 0.125 0.1170.217 0.204 0.204 0.184 0.199 0.1840.237 0.206 0.231 0.197 0.202 0.211 0.125

554 A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561

2.2.5. Molecular datingTo estimate timing of diversification within Phyzelaphryninae,

we undertook molecular dating with Beast 1.6.2 (Drummond andRambaut, 2007). We used two approaches: (1) concatenateddataset method and (2) multilocus species tree method (�BEAST;Heled and Drummond, 2010), both with a matrix focusing onEleutherodactylidae.

Preliminary analyses revealed close affinity among some termi-nals for which sequence data were incomplete, and were thus dis-carded. In other cases some closely related terminals werecomplementary and were combined to represent only one termi-nal. With this strategy we were able to obtain an almost completematrix representing all the main lineages in Phyzelaphryninaeexcept one from Valença (Bahia, Brazil; MTR20222). This lineagerepresents a candidate species (see results), but we preferred todiscard this terminal because of missing data and because it isnot needed to evaluate the broad temporal aspects of the diversifi-cation of the group. Nevertheless, the overall diversity withinPhyzelaphryninae is well represented.

Both analyses were calibrated on the crown age of Eleuthero-dactylinae (31.3 Ma; 44.7–21.1) estimated by Heinicke et al.(2007, 2009) based on a large sequence dataset and fossil/biogeo-graphic calibrations. This prior was set as a normal distributionwith mean and sd equal to the estimation from Heinicke et al.(2009). Monophyly of Eleutherodacylinae and Phyzelaphryninaewas enforced considering previous results. All partitions wereconsidered underestimated uncorrelated lognormal rates. The treeprior used the Birth and Death Process, with a UPGMA generatedstarting tree and the auto optimize option for operators. Wecomputed 108 generations, sampled every 1000 generations. Theconcatenated analysis used the same partitioning as previously;i.e. seven partitions (coding mtDNA by codons, 12S–16S, codingnuDNA by codons) each under GTR + G with linked tree prior forall trees. The multilocus dataset, however, was based on the fourloci with unlinked tree prior for all trees (mtDNA, RAG1, POMCand TYR) with each nuDNA coding gene sub-partitioned by codonposition. Each partition was considered under GTR + G, estimatedbase frequencies and four gamma categories.

We examined convergence on stationarity using Tracer 1.5(Rambaut and Drummond, 2007). For both analyses effectivesample sizes were >200 for all parameters except where priorand posterior jumped between alternative values. A few relativesubstitution rates were also with low ESS, jumping from high val-ues to zero (probably because no substitutions of these types areobserved) rendering the prior on the rate invalid (Drummondet al., 2002). Therefore, we computed additional 108 generationsrun with the prior distribution of these relative rates changed froma gamma to a uniform distribution bounded between 10�5 and 1.This made ESS > 150 for all parameters except alpha for CP1 and2 for TYR and RAG1 and their respective tree likelihoods for themultilocus analysis. Nevertheless, the time estimates were similaramong runs. The maximum clade credibility trees were computedwith Tree Annotator 1.6.2.

3. Results

3.1. Monophyly of Phyzelaphryninae and genera

Relationships are well resolved within Eleutherodactylidae withonly two poorly sustained nodes as well as among other Terraranawith only three poorly sustained nodes (Fig. 1a). However, deeperrelationships among Hyloidea remain ambiguous. Both methods(BA and MP) reveal very deep divergence among well-definedgroups within Phyzelaphryninae using the three matrix configura-tions (Fig. 1a). This subfamily is itself strongly supported as a clade,

sister group of Eleutherodacylinae. Adelophryne is confirmed asbeing monophyletic and as the sister group of Phyzelaphryne.Moreover, Adelophryne is represented by three deeply divergentand well-sustained clades that are geographically circumscribedto Northern Amazonia Clade (NAMC), Northern Atlantic ForestClade (NAFC; from Ceará to Bahia) and Southern Atlantic forestClade (SAFC; from Bahia to Minas Gerais) (Figs. 1a and 2). Eachof these four major clades harbors deep subdivisions.

Terrarana is well supported as monophyletic in all methods.Relationships among main Terrarana clades are mostly similar toHedges et al. (2008) and slightly different from Pyron and Wiens(2011). Relationships among Brachycephalidae, Eleutherodactyli-dae and Craugastoridae remain poorly resolved using the totaldataset. However, within Craugastoridae the interrelationshipsamong subfamilies are relatively different from those shown inPyron and Wiens (2011) with (1) Craugastorinae strongly sus-tained as the sister group to the other Craugastoridae, (2) Hypo-dactylus forming a strongly sustained clade with Pristimantinaeand Strabomantinae, (3) this last clade being the sister group ofHoloadeninae with Euparkerella as the sister group of Holoadenand (4) Strabomantinae weakly sustained as nested withinPristimantinae.

3.2. Species diversity/candidate species

By combining evidence from tentative identification of thespecimens, references, phylogenetic position, time of divergenceand geographical locations, we identify ‘‘cryptic species’’ and flagthese lineages as candidate species. We use the term ‘‘cryptic’’ ina relaxed definition given that we did not examine thoroughlythe morphological differences that may exist between the segre-gated entities; that is why we use the term candidate species. Nev-ertheless, we argue that these differences are very subtle, which isemphasized by the misidentifications already documented (Heyer,1977; Hoogmoed and Lescure, 1984; Hoogmoed et al., 1994; Lynch,2005), and that the term ‘‘cryptic’’ can be used in a previousdefinition: ‘‘two or more distinct species previously classified asa single one due to overall morphological similarity that preventsimmediate obvious distinction’’ (Bickford et al., 2007; Pfenningerand Schwenk, 2007). We provide details below and in discussionjustifying our species delineation.

Phyzelaphryne (Southern Amazonian Clade, SAMC) is subdividedin two well-supported clades. One is distributed on the east sidefrom the right margin of Purus River to Abacaxis River (Fig. 2).The other one is situated on the west from the left margin of thePurus River to Leticia at the border between Brazil, Colombia andPeru. This genus is for the first time reported from these two lattercountries. Actually, Lynch (2005) previously found the species inColombia, but erroneously referred it to Adelophryne adiastolainstead of Phyzelaphryne. Levels of divergence between the twoPhyzelaphryne clades and the absence of allelic sharing in the threenuDNA loci (Fig. 3) strongly suggest additional specific subdivi-sions. We tentatively associate the eastern clade to the nominalspecies given that the type locality lies in the Madeira river catch-ment. This clade appears strongly structured across catchments ofthe Madeira, Purus (right margin) and Abacaxis rivers with well-differentiated pairs of lineages.

The western clade associated to a candidate species is evenmore deeply subdivided between the Colombian populations andthe two Brazilian ones (Juruá, Purus left margin) with no allelicsharing in any of the three nuDNA loci (Fig. 3). Nevertheless, con-sidering the absence of other lines of evidence that could corrobo-rate the hypothesis of additional species in this clade, weconservatively assign them to a single species.

The Northern Amazonian Clade (NAMC) is recovered as thesister group of all the Adelophryne representatives of the Atlantic

(a)

(b)

Fig. 1. (a) Phylogenetic reconstruction based on Bayesian analysis of concatenated loci. Bootstrap supports from MP analyses are also indicated after posterior probabilities. �

stand for pp values higher than 0.99 and bootstraps % higher than 99. Hyphens (-) indicate nodes not recovered with MP and values in red indicate poorly supported nodes i.e.pp < 0.95 and node not recovered with MP. (b) Topologies obtained from Bayesian analysis and MP for main Phyzelaphryninae lineages.

A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561 555

forest from Bayesian analysis, but under MP this clade is sustainedas the sister group of the SAFC instead (Fig. 1b and Suppl. Mat.).Surprisingly, the population identified as A. gutturosa from Serrado Navio (Amapá, Brazil) is in fact recovered as the sister groupof all the other species of this clade and thus renders A. gutturosaparaphyletic. We therefore refer the Serra do Navio population toan undescribed species of Adelophryne. Another noteworthy resultis that the previously published sequences of A. gutturosa (Heinickeet al., 2009) obtained from an individual from Mount Ayanganna(Guyana) in fact correspond to A. patamona. DNA sequencesobtained from the holotype (ROM 43034) and the paratypes of A.patamona are included herein (Table 1), allowing us to be certainthat the previous identification was erroneous. Interestingly, theColombian species A. adiastola is recovered nested within thisNAMC as the sister species of A. gutturosa, both forming a cladegrouped with A. patamona.

The Northern Atlantic Forest Clade (NAFC) gathers A. baturiten-sis, A. maranguapensis (two species described from elevationallyisolated moist forests in Ceará state, northeastern Brazil), onepopulation from the state of Pernambuco that has been identifiedas A. baturitensis (Loebmann et al., 2011), and two populations fromBahia state. Relationships among species within that group remainlargely unresolved. The two neighbor populations from Bahia stateare highly divergent and form a strongly supported clade. More-over, they do not share any alleles for the nuDNA loci (Fig. 3).

The southern Atlantic Forest Clade (SAFC) includes the lastnominal species A. pachydactyla and no less than three additionalhighly divergent lineages corresponding to newly discovered pop-ulations, extending the range of the genus ca. 650 km straight line

southwards to the Brazilian state of Minas Gerais. Given the incon-sistency in morphological identification, level of divergence,absence of shared nuDNA alleles (Fig. 3) and geographical loca-tions, we call these populations a candidate species. We tentativelyassign the population from Serra das Lontras and Serra do Teimosoto the nominal species (A. pachydactyla) given that their morpho-logical characteristics agree with Hoogmoed et al. (1994), andthe geographical proximity to the type locality.

3.3. Molecular dating

Both ‘‘concatenated’’ and ‘‘multilocus’’ approaches led to similartopologies, notably supporting the NAMC as the sister group ofNAFC + SAFC with high posterior probabilities. Time estimatesare, however, younger from the multilocus analysis than fromthe concatenated one. Phyzelaphryninae crown age is recoveredbetween 40.5 My old (concatenated) and 27.4 My old (multilocus)thus originating during late Eocene/early Oligocene. Major cladesof Adelophryne are recovered to have diverged between 25.8 Ma(concatenated) and 16.5 Ma (multilocus) thus during earlyMiocene. The NAMC diversified earlier than the other major cladesgiven that the four species originated between 20 (concatenated)and 13.4 Ma (multilocus) while NAFC, SAFC and SAMC diversifiedlater between 14.6–12.8 (concatenated) and 7.2–8 Ma(multilocus).

The fact that the concatenated analysis yielded older divergencetimes than did the multilocus analysis is consistent with an expec-tation that the average coalescence time for the various gene lin-eages should exceed somewhat the divergence times of the

Fig. 2. Map of the distribution of sampling localities (circles), and type localities (stars). Additional records from the literature are illustrated in Ecuador, Brasil and Colombiafor A. adiastola (Ortega-Andrade, 2009), Brazil ES for A. cf. pachydactyla (Almeida et al., 2011) and in Bolivia ad Brazil PA for P. miriamae – (from Heyer, 1977; precise localitynot mentioned, De la Riva et al., 2000).

556 A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561

population lineages (Liu et al., 2009). An independent test of ourdivergence-time estimates is to ask whether they predict reason-able rates of evolution for the mitochondrial Cytb gene, whose evo-lutionary rate has been calibrated in many prior studies ofvertebrates. An expected evolutionary of 2.1% sequence divergenceper million years has been obtained by comparing multiple pairs ofsister species whose separation was caused by formation of theIsthmus of Panama (reviewed by Reece et al., 2010). Using the datain Table 3, we compare our estimated divergence times with thoseobtained using the Cytb calibration for the eight interspecificbranching events in Fig. 4 that we estimate to be less than 20 mil-lion years; these are the cases for which substitutional saturationof Cytb should be minimal. For the five nodes within the NAFCand SAFC, divergence times estimated from the Cytb calibrationare very close to our estimates from the concatenated analysis, dif-fering by no more than 8%. For the three nodes within the NAMCand SAMC, divergences estimated by the Cytb calibration are closerto the multilocus estimated dates, being identical in one case, 21%higher in a second case, and 17% lower in the remaining case. Theseresults support the fidelity of our estimated divergence times asbeing consistent with the expected evolutionary rate of Cytb.

4. Discussion

4.1. Cryptic diversity and conservation

With up to eight candidate species detected, our results indicatea >100% increase in the species diversity of the group, which likelystill remains underestimated. This high number of candidate

species is even more surprising considering the few and scatteredlocalities that have so far been sampled; they represent only a tinyportion of the potential distribution of these two genera. This esti-mate matches previous DNA-based attempts to evaluate the actualspecies richness in tropical amphibians (Fouquet et al., 2007;Vieites et al., 2009; Jansen et al., 2011; Funk et al., 2012) and alsomatches sudden increases in species richness of several Terraranagenera (e.g. Brachycephalus Pombal, 2010). Our species delineationis based on the convergence of evidence from identification of thespecimens, references, phylogenetic position, time of divergenceand geographical location. Fine-tuned species delineation wouldgreatly benefit from an ‘‘integrative taxonomy’’ approach (Dayrat,2005; Will et al., 2005; Padial et al., 2010), but this approach liesbeyond the scope of our paper given that thorough examinationof the morphological variation as well as vocalization comparisonswould require material not yet at hand. Nonetheless, in all casesthe levels of divergence and concordance among several unlinkedloci leave little doubt that these populations correspond to previ-ously undetected species.

The divergence time between nominal species (e.g. between A.adiastola and A. gutturosa or between A. maraguapensis and A. batu-ritensis) is similar to or lower than that between our candidate spe-cies (Fig. 4). The case of NAMC is particularly compelling given thatAdelophryne sp. 7 (Serra do Navio, Amapá) is the sister lineage to allthe other species of the clade with a TMRCA estimated between 20(concatenated) and 13.4 Ma (multilocus). Hoogmoed et al. (1994)already noticed that the animals from Serra do Navio that theyassigned to A. gutturosa are slightly different from the type materialfrom Guyana. Within NAFC, relationships among the species re-main unclear, but divergences are similar among them (Fig. 4) with

Fig. 3. Statistical parsimony networks for each nuDNA locus and each major clade. Haplotypes are shown as circles proportional in size to haplotype frequency. Each nominaland candidate species are delimited by a color filled rectangle.

A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561 557

a minimum estimate around 6.5 Ma (multilocus), which is muchhigher than the divergence generally observed among sister spe-cies of frogs (e.g. Fouquet et al., 2007; Vences et al., 2005; Vieiteset al., 2009).

Moreover, in addition to being highly divergent from both A.maranguapensis and A. baturitensis, the isolated population fromPernambuco is more than 500 km away from any nominal Adeloph-ryne population (Loebmann et al., 2011), and the two populationsfrom Bahia are more than 1000 km away from any samplednominal Adelophryne population of the same clade. Therefore, weargue that all three lineages represent candidate species, hereincalled Adelophryne sp. 1–3. The distinction between Adelophrynesp. 2 and 3 is, however, more arbitrary given that we miss datafor Adelophryne sp. 3, but based on available mt and nuDNA se-quences, divergence is also very deep (20% with Cb) (Fig. 1a; Table4). The populations clustering into the SAFC comprise only one

nominal species: Adelophryne pachydactyla. The latest divergenceis estimated around 10.5 My old (concatenated) and 5.4 My old(multilocus); all the highly divergent lineages are recovered onboth mt and nuDNA, and at least the populations from MinasGerais are morphologically different (Felipe Leite pers. com.).Therefore, the three additional lineages undoubtedly representcandidate species. The 13.2–8 My separating the two Phyzelaph-ryne clades are also compelling evidence for the existence ofdistinct species. Subdivision of the western candidate species ofPhyzelaphryne into several species-level entities is also very likelygiven the estimated 3 My of divergence and the lack of nuDNAallele sharing.

In addition to the newly detected lineages/species, the old diver-gence times between Phyzelaphryne and Adelophryne (40–30 Ma),among the three Adelophryne major clades (25–16 Ma), and amongthe species within the different clades (all >6 Ma) – particularly in

(a)

(c) (d)

(b)

Fig. 4. Bayesian time-calibrated, maximum clade-credibility tree using relaxed clock with (a) concatenated partitioned dataset (b) multilocus species tree (�Beast).Calibration point is indicated with yellow circle. Posterior probabilities are indicated above the nodes, while the median of the posterior distributions of the ages of the nodesare indicated below. Ninety-five percent credibility intervals are indicated with blue bars. (c) Posterior distribution of the mean rate of substitution of each locus from themultilocus species tree analysis. (d) Simplified tree of the topology obtained from each locus from the multilocus species tree analysis.

558 A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561

NAMC – are striking. Such results highlight the inherent difficulty instudying amphibian diversity and evolutionary trends based onmorphology alone, because it can be extremely conserved (Cherryet al., 1977, 1978; Emerson, 1986) and is often homoplastic (Boss-uyt and Milinkovitch, 2000; Parra-Olea and Wake, 2001; Guayas-amin et al., 2008).

Species from the Atlantic forest are also characterized by extre-mely old divergences among populations previously consideredconspecific, and it is likely that more species remain to bediscovered in that biome as well as in Amazonia. Actually, the doc-umented record of A. cf. pachydactyla from Espírito Santo, Brazil byAlmeida et al. (2011), as well as the single population identified asA. adiastola from Ecuador by Ortega-Andrade (2009) deserve spe-cial attention, as they could correspond to additional candidatespecies.

Revealing such remarkable diversity in a clade morphologicallyhighly homogeneous stresses the challenge for conservation thatwe are facing, given that all of these species have highly restricteddistributions, sometimes in isolated highlands like Ceará andPernambuco, and that human impact or climate change is a realthreat for such species/populations (Corlett, 2012). The situationin the northern Atlantic forest being particularly worrying (Ribeiroet al., 2009), the northern fragments deserve prime conservationpriority (Carnaval et al., 2009; Ribeiro et al., 2009).

4.2. Biogeography

The biogeographic pattern in Eleutherodactylidae is particularlystriking with a first split between Eleutherodactylinae, occurring inMiddle America and the Caribbean, and Phyzelaphryninae, found

in Amazonia and Atlantic forest, that dates back to the Eocene,some 46 Ma according to Heinicke et al. (2009), and estimated be-tween 44.2 (concatenated) and 32.8 (multilocus) Ma in this work.This divergence has already been discussed by Heinicke et al.(2007) and was attributed to an ancient overseas dispersal fromSouth America towards Middle America and the proto Caribbean.

Two subsequent events are most noteworthy: the basal split of(1) Phyzelaphryninae and of (2) Adelophryne. (1) The divergencebetween the genera Phyzelaphryne and Adelophryne dates back to40–30 Ma, which corresponds to the Eocene/Oligocene boundaryi.e. one of the major extinction events related to an abrupt coolingof the global climate (Prothero, 1994). Given a likely northernAmazonian origin of Adelophryne (see below) and southern Amazo-nian origin for Phyzelaphryne, this split likely originates from anorth/south fragmentation of the range due to climate change. Thisis a period of southern uplift of the Andes (Hoorn et al., 2010a,b),isolation of Antarctica and the creation of a circumpolar current,dramatic drop of the sea level, and major climatic changes (Or-tiz-Jaureguizar and Cladera, 2006). This period also correspondsto the prevalence of large grazing herbivores and ‘modernization’of other faunal aspects during the mid-Cenozoic, reflecting adapta-tion to major environmental changes, including increased aridityand cooling (Flynn and Wyss, 1998). Late Eocene–early Oligocenealso witnessed the spread of open vegetation at the expense ofthe rainforest that previously dominated the southern South Amer-ican continent (Roig-Juñent et al., 2006; Romero, 1986). Such con-ditions were unlikely favorable to forest-restricted frogs withdirect development in the forest litter and may be responsiblefor the initial disjunction within the group that today occurs onopposite sides of the Amazon River. Interestingly, the origin of

Tabl

e4

Sequ

ence

deta

ilsin

clud

ing

vouc

hers

and

acce

ssio

nnu

mbe

rsus

edfo

rth

em

olec

ular

dati

ng.

Spec

ies

Cb

CO

I12

S16

SR

AG

1aR

AG

1bPO

MC

TYR

A.b

atur

iten

sis

MTR

1401

3/JX

2983

76M

TR14

013/

JX29

8321

MTR

1401

3/JX

2982

49M

TR14

013/

JX29

8281

CFB

HT1

1339

/JX

2981

50C

FBH

T113

39/

JX29

8150

MTR

1401

3/JX

2981

00M

TR14

013/

JX29

8201

A.s

p.2

PEU

80/J

X29

8379

PEU

80/J

X29

8323

PEU

80/J

X29

8283

PEU

80/J

X29

8151

PEU

80/J

X29

8103

PEU

80/J

X29

8204

A.s

p.1

CFB

HT1

1716

/JX

2983

80C

FBH

T117

16/

JX29

8324

CFB

HT1

1716

/JX

2982

51C

FBH

T117

16//

JX29

8284

CFB

HT1

1716

/JX

2981

04C

FBH

T117

16/

JX29

8205

A.m

aran

guap

ensi

sC

FBH

T141

19/

JX29

8381

CFB

HT1

4119

/JX

2983

26C

FBH

T141

19/J

X29

8253

CFB

HT1

4119

//JX

2982

86C

FBH

T141

19/

JX29

8153

CFB

HT1

4119

/JX

2981

53C

FBH

T141

19/

JX29

8106

CFB

HT1

4119

/JX

2982

07A

.sp.

5C

FBH

E234

/JX

2983

83M

R17

521/

JX29

8327

CFB

HE2

34/J

X29

8254

CFB

HE2

34/J

X29

8288

CFB

HE2

34/J

X29

8155

CFB

HE2

34/J

X29

8155

CFB

HE2

34/J

X29

8108

CFB

HE2

34/J

X29

8209

A.s

p.4

MTR

1357

0/JX

2983

84M

TR13

570/

JX29

8331

MTR

1357

0/JX

2982

56M

TR13

570/

JX29

8290

MTR

1357

0/JX

2981

57M

TR13

570/

JX29

8111

MTR

1357

0/JX

2982

12A

.sp.

6M

R15

919/

JX29

8385

MR

1591

9/JX

2983

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A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561 559

the bufonid ‘‘range expansion phenotype’’, as coined by Van Bocxl-aer et al. (2010), corresponds to this period of habitat modification.Moreover, the late Eocene–early Oligocene period matches diver-gence times in the higher taxon Terrarana major clades that are al-most exclusively associated with forest habitat (Heinicke et al.,2009), with some of these clades being endemic to either theAtlantic forest or Amazonia.

(2) Despite somewhat conflicting signal among loci (Figs. 1a;4d), both concatenated and multilocus approaches favored unam-biguously NAMC as the sister group of the other Adelophryne. Suchpattern shown by the Atlantic forest Adelophryne – being actuallynested within otherwise Amazonian Phyzelaphryninae i.e. thatPhyzelaphryne descends from an Amazonian lineage and is the sis-ter taxon to Adelophryne occurring both in Amazonia and in theAtlantic forest – is a pattern never recovered previously. Given thatEleutherodactylinae likely originated by dispersal from northwest-ern South America (Heinicke et al., 2007) and that the Atlantic for-est Adelophryne are nested within Phyzelaphryninae, it seemslikely that Adelophryne originally was situated in northern Amazo-nia and subsequently dispersed to the Atlantic forest some 23–16 Ma. Nonetheless, we acknowledge that such short internodesat the base of Adelophryne allied with the conflicting results foundby MP, call for a deeper investigation based on a larger number ofunlinked nuclear loci and other sources of evidence. Nonetheless,the split between the NAMC and the Atlantic forest Adelophrynematches a period when the Purus Arch connected the GuianaShield and the Brazilian Shield (Hoorn et al., 2010b). Later (midMiocene), the Pebas system and the flowing paleo Amazon riverhave most likely prevented any possible route to the southeastfor such small-bodied terrestrial and direct-developing frogs.Moreover, the 20–15 My window corresponds to a period of highertemperature (Zachos et al., 2001). Such conditions may have al-lowed Adelophryne to disperse rapidly given the short internodebetween two Atlantic forest clades today in contact on each bankof the Rio de Contas (Bahia). Nonetheless, it is striking that Adel-ophryne could have dispersed over great distances between theGuiana Shield and southern Atlantic forest in such a short timeframe (<2 My).

Similarly, the north vs. south Atlantic forest pattern observedwithin Adelophryne is concordant with several studies of vicariantforms whose limits are more or less coincident with the Rio Docevalley (northern Espírito Santo state; Carnaval et al., 2009; Costa,2003; Pellegrino et al., 2005; Pinto-da-Rocha et al., 2005; da Silvaet al., 2004). Several plant taxa are restricted to either one of theseareas, producing a strong floristic differentiation between thenorthern and southern Atlantic forests (Oliveira-Filho and Fontes,2000). This pattern strikingly matches what is observed in othertaxa like Dendrophryniscus (Fouquet et al., 2012b) and Leposoma(Pellegrino et al., 2011). In these examples, divergence times alsoindicate that areas of environmental stability lasted for 20 My inthe Guiana Shield and in several parts of the Atlantic forest fromMiocene to Quaternary, a much longer time period than that mod-eled by Carnaval and Moritz (2008).

Acknowledgments

We are grateful to the many people and institutions that madethis study possible and Allan Larson (MPE AE) as well as the twoanonymous reviewers for their sound comments on the manu-script. Thanks to Renato Recoder, Marco A. Sena, Mauro TeixeiraJr., José Cassimiro da Silva, Agustin Camacho, Dante Pavan, GabrielSkuk, Vanessa Verdade, Roberta Damasceno, Renata Amaro, SergioMarques de Souza, Francisco dal Vechio, José Mario Guellere, TamiMott, Pedro M. S. Nunes, H. Bonfim, Sonia Machado, Felipe Leiteand Luciana Fusinatto for help in the field and/or for collectedinvaluable material. We also thank Erney Plessman de Camargo,

560 A. Fouquet et al. / Molecular Phylogenetics and Evolution 65 (2012) 547–561

José Maria da Silva, and Admilson Torres for their invaluable helpduring fieldwork in Amapá. We are also grateful to Manuel An-tunes Jr., Sabrina Baroni and Maira Concistré (Instituto de Biociên-cias da Universidade de São Paulo) who assisted with lab work.Instituto Chico Mendes de Conservação da Biodiversidade (ICM-Bio), Conselho Nacional de Desenvolvimento Científico and Tec-nológico (CNPq DL. Doctoral fellowship 140226/2006-0) andParque Nacional da Serra do Cipó granted collection permits andassisted in the field. Funding was provided by CNPq, Fundação deAmparo à Pesquisa do Estado de São Paulo (FAPESP, 2003/10335-8, 2011/50146-6 and 2010/51071-7; A. Fouquet PD scolarship2007/57067-9, V.D. Orrico PhD scholarship FAPESP 2007/57067-9, M.L. Lyra PD scholarship 2010/50124-0), Fundação Cearense deApoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) pro-vided I.J. Roberto’s scholarship. The work of S. Castroviejo-Fisherwas financed by a Universidad de los Andes (2009–2010) and aFulbright/Spanish Ministry of Education (2010–2012) post-doc-toral research contracts, and J. M. Padial’s research is funded by aGerstner Post-doctoral Fellowship at the American Museum ofNatural History. PJRK’s fieldwork in Guyana was made possiblethanks to the financial support of the Belgian Directorate-Generalof Development Cooperation with additional support from the KingLéopold III Fund for Nature Exploration and Conservation; speci-mens from Guyana were collected under Permit Numbers030605BR006 and 160107BR068, and exported under Permit Num-bers 191205SP01, 040406SP014, 040706SP0171 and 191207SP018issued by the Guyana Environmental Protection Agency.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2012.07.012.

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