Demographic Processes in the Montane Atlantic Rainforest

Molecular Phylogenetics and Evolution 62 (2012) 880–888

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Molecular Phylogenetics and Evolution

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Demographic processes in the montane Atlantic rainforest: Molecular and cytogenetic evidence from the endemic frog Proceratophrys boiei ⇑ Renata Cecília Amaro a,b, , Miguel Trefaut Rodrigues a, Yatiyo Yonenaga-Yassuda b, Ana Carolina Carnaval c,d a Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brazil b Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brazil c Department of Biology, The City College of New York, New York and CUNY Graduate Center, NY 10031, USA d Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720-3160, USA article info abstract

Article history: Historical climatic refugia predict genetic diversity in lowland endemics of the Brazilian Atlantic rainfor- Received 7 June 2011 est. Yet, available data reveal distinct biological responses to the Last Glacial Maximum (LGM) conditions Revised 4 November 2011 across species of different altitudinal ranges. We show that species occupying Brazil’s montane forests Accepted 7 November 2011 were signiﬁcantly less affected by LGM conditions relative to lowland specialists, but that pre-Pleistocene Available online 16 November 2011 tectonics greatly inﬂuenced their geographic variation. Our conclusions are based on palaeoclimatic distribution models, molecular sequences of the cytochrome b, 16S, and RAG-1 genes, and karyotype data for Keywords: the endemic frog Proceratophrys boiei. DNA and chromosomal data identify in P. boiei at least two broadly Proceratophrys boiei divergent phylogroups, which have not been distinguished morphologically. Cytogenetic results also Phylogeography Atlantic forest indicate an area of hybridization in southern São Paulo. The location of the phylogeographic break Brazil broadly matches the location of a NW–SE fault, which underwent reactivation in the Neogene and led Cytogenetics to remarkable landscape changes in southeastern Brazil. Our results point to different mechanisms Hybrid zone underpinning diversity patterns in lowland versus montane tropical taxa, and help us to understand Last Glacial Maximum the processes responsible for the large number of narrow endemics currently observed in montane areas of the southern Atlantic forest hotspot. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction In the Neotropics, new advances in palaeoclimatic reconstruction and statistical genetics are enabling multi-disciplinary ap- Many evolutionary biogeographic studies emphasize the role of proaches in evolutionary biogeography and expanding the field Pleistocene refugia, particularly during the Last Glacial Maximum of biodiversity prediction. Recent phylogeographic studies of low- (LGM), in species diversification and persistence through time (e.g. land and mid-altitude species of the Brazilian Atlantic rainforest Hewitt, 2000; Moritz et al., 2009). In the northern hemisphere, the revealed higher genetic diversity (Cabanne et al., 2007, 2008; climatic and biological effects of glacial–interglacial cycles have Carnaval et al., 2009; Fitzpatrick et al., 2009; Grazziotin et al., been extensively studied and are well described (Milot et al., 2000; 2006; Martins et al., 2007; Pellegrino et al., 2005) and higher Late Hickerson and Ross, 2001; Alexandrino et al., 2002; Fedorov and Quaternary demographic stability in the northern portion of the Stenseth, 2002; Griswold and Baker, 2002; Hoffman and Blouin, forest (Carnaval et al., 2009; Grazziotin et al., 2006). The southern 2004; Kotlik et al., 2004). Comparatively little is known about the half of the biome, well-known for its topographically complex environmental and demographic changes that occurred in tropical mountain ranges, appeared climatically unsuitable for the target zones. Data from the best-studied tropical system to date, the species under LGM conditions, and showed genetic signatures of Australian Wet Tropics, indicate extensive range contraction in recent population expansion – reflecting colonization from north- forest species during the LGM, and species persistence in montane ern refugia following the glacial period (Carnaval et al., 2009). forest refugia (Bell et al., 2010; Hugall et al., 2002; Graham et al., It remains to be seen whether currently montane Atlantic forest 2006; Moussalli et al., 2009). Similar processes have been detected taxa experienced similar demographic changes in the LGM. Con- in African systems (Bowie et al., 2006; Lawson, 2010). trary to what is observed in the Australian system, preliminary data suggest distinct responses to Pleistocene climatic change in lowland versus montane species in coastal Brazil. Carnaval et al. ⇑ Corresponding author at: Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brazil. (2009), for instance, observed differences in the spatial range of E-mail address: [email protected] (R.C. Amaro). climatically stable areas for species of distinct altitudinal ranges.

Data from the broadly distributed lowland Hypsiboas albomarginatus and Hypsiboas semilineatus indicate nearly complete absence of suitable habitat in the south during the LGM. In contrast, models and molecular data from Hypsiboas faber, a treefrog ranging up to 1000+ m above sea level, indicate population persistence in southern refugia during the LGM. Together, these data raise the yet un- tested hypothesis that currently montane (cold-tolerant) forest taxa were able to withstand the LGM climate and persist in the southern Atlantic forests ca. 21,000 years ago (21kya), whereas nowadays lowland-restricted taxa (species presumably unable to tolerate cold temperatures) were forced to retract to lower latitudes. This would explain the large number of narrow montane endemics and the contrasting low levels of intraspecific diversity in lowland taxa, currently seen in the southern range of the biome. To shed light on the demographic processes that occurred in montane areas of eastern South America during cold phases of Fig. 1. Cross-species comparison of climatic tolerances (locality data from Prado and Pombal, 2008; Carnaval et al., 2009, and current study; bioclimatic variables the Late Pleistocene, we test the hypothesis that populations cur- extracted from Worldclim (Hijmans et al., 2005)). Yellow diamonds = Hypsiboas rently able to occupy higher elevations in the southern Atlantic for- albomarginatus, black squares = H. faber, pink triangles = Proceratophrys boiei. ests were not as severely impacted by Pleistocene glacial periods relative to lowland specialists. To that end, we combine species distribution models, phylogeographic analyses, and cytogenetic data for the litter frog Proceratophrys boiei, an Atlantic rainforest endemic. This middle-sized cycloramphid species (average SVL McNeil, 1982) was used as a threshold-independent method for ca. 40 mm) is restricted to the southern half of the forest, reaching model evaluation. up to 1200+ m asl (Prado and Pombal, 2008). Adults of P. boiei lay Once the AUC values of distribution models under current cli- their eggs in renovated water next to small forest streams, where mate attested satisfactory model performance, climatic retrojec- the larvae develop (Pombal and Haddad, 2005; Prado and Pombal, tions were performed under 21kya scenarios using the 2008). Relative to the treefrogs used in Carnaval et al.’s (2009) Paleoclimatic Modelling Intercomparison Project (PMIP1) ECHAM3 study, P. boiei occupies regions of colder winters and drier condi- atmospheric general circulation model (Deutsches Klimarechen- tions during the dry season (Fig. 1), likely reflecting fundamental zentrum Modellbetreuungsgruppe, 1992), and under the newly differences in their physiological tolerances. We first develop dis- developed CCSM and MIROC 21kya climate models available tribution models for the species under current climatic conditions through PMIP2, at 2.5 arc-minutes resolution (Braconnot et al., and retroject them to climatic scenarios for the LGM period 2007; bioclimatic variables downloaded from http://www.worldc- (21kya). We use the models to infer past demographic trajectories, lim.org/past). Threshold values that maximized Kappa under cur- which we test with phylogeographic data from two mitochondrial rent climatic conditions were used to convert the continuous genes (16S and cytochrome b) and one nuclear marker (RAG-1). Be- palaeomodel outputs into presence/absence maps. cause the genetic data reveal two deeply divergent lineages within Molecular data included partial sequences of 16S, cytochrome b the species, we collect cytogenetic data in search of concordant (cyt b), and RAG-1 in 81 specimens of P. boiei from 30 localities chromosome-level variation and evidence of hybridization across (Table S1 in Supporting Information). Outgroups were Proceratoph- phylogroups. rys goyana, Proceratophrys laticeps, Proceratophrys renalis, Macroge- nioglottus alipioi, Odontophrynus americanus, Thoropa taophora and Cycloramphus acangatan. Approximately 650 bp of the cyt b gene 2. Material and methods were amplified with primers CB1-L and CB3-H (Palumbi, 1996), 620 bp of 16S were obtained with 16SAR and 16SBR (Palumbi, Species-level taxonomy follows Prado and Pombal (2008). Spe- 1996), and 430 bp of the nuclear gene RAG-1 were amplified with cies-wide distribution models under current and LGM (21kya) cli- R1GFF and R1GFR (Faivovich et al., 2005). Protocols for DNA extrac- matic periods were performed with MAXENT using default settings tion, amplification, sequencing, and sequence editing followed (Phillips et al., 2006), as per Carnaval and Moritz (2008). Point Amaro et al. (2009). locality data were obtained through field work (see Appendix), The matrix of 611 bp of aligned sequences of cyt b and 428 bp of and complemented from literature records (Prado and Pombal, RAG-1 showed no indels. The 16S alignment was based on second- 2008). Ninety-eight presence records, each from a unique locality, ary structure and yielded 553 alignment positions. All datasets were used in model construction (74 training points, 24 testing were combined in a matrix of 1592 bp and used in a Bayesian phy- points); 100 absence points were randomly selected as pseudo-ab- logenetic analysis in MrBayes 3.0b4 (Ronquist and Huelsenbeck, sence data for model testing. 2003) as described in Amaro et al. (2009). A best-fit model of nucle- Models were based on seven bioclimatic variables (annual otide substitution for each data partition was selected using the mean temperature, temperature seasonality, mean temperature Akaike information criterion (AIC) implemented in MrModeltest of the warmest and coldest quarters, annual precipitation and pre- v.2.2 (Nylander, 2004): HKY + I + G for cyt b and RAG-1, and GTR + G cipitation of the driest and wettest quarters) downloaded from the for 16S. Maximum likelihood (ML) bootstrapping (3000 replicates) WORLDCLIM 1.4 database (Hijmans et al., 2005). The original bio- was performed in Treefinder (Jobb, 2008) using default settings and climatic layers were cropped to span from latitude 12°470Nto models HKY + G for cyt b and RAG-1 and J2 for 16S that were also 34°460S and longitude 78°310Wto35°W to ensure that the training estimated in Treefinder. The majority 50% consensus trees saved points encompassed the full range of palaeoclimatic values. with posterior probabilities and bootstrap values on the nodes were Maximum Kappa (Cohen, 1960) was employed to establish a visualized using FigTree 1.3.1 (http://tree.bio.ed.ac.uk/). threshold and transform continuous outputs under current cli- Using the cyt b dataset, we estimated the time to the most re- matic conditions into a presence/absence map. The area under cent common ancestor (TMRCA) for each clade using relaxed the receiver operating characteristic curve (AUC; Hanley and Bayesian molecular clock with uncorrelated lognormal rates (Beast 882 R.C. Amaro et al. / Molecular Phylogenetics and Evolution 62 (2012) 880–888

1.6.1; Drummond and Rambaut, 2007). Due to lack of fossil calibra- 3. Results tions for this group, we used the protein-coding mtDNA mutation rate proposed by Crawford (2003) (0.957% per lineage per million Climate-based distribution models of P. boiei were built under years). Partitioned phylogenetic estimates were constructed using current environmental conditions (AUC = 0.990, Fig. 2 top left). the best-fitting model of nucleotide substitution, which was se- Irrespective of the 21kya climatic scenario used (ECHAM3, MIROC, lected using the AIC with an uncorrelated lognormal tree prior or CCSM), paleomodels indicated persistence of suitable climate for and the Birth and Death speciation process. We performed two the species through most of its range during the LGM (Fig. 2 top independent runs of 50 million generations sampling every right and bottom). Results obtained under the ECHAM3 climatic 1000th generation following a pre-burnin of 5000 generations. scenario predicted a slight contraction in the southern range of Scale operators were adjusted as suggested by the program. the species (Fig. 2 top right). Climatic models MIROC and CCSM Because the Bayesian analysis revealed well-supported clades (Fig. 2 bottom left and right, respectively) predicted climatic suit- corresponding to distinct geographical regions, haplotype diversity ability along the coast and hence no major demographic changes (Hd) and nucleotide diversity (p) were estimated for each group during the LGM. using DNAsp v5 (Librado and Rozas, 2009). For descriptive pur- Bayesian and ML reconstructions based on mitochondrial par- poses, overall genetic variation was partitioned into hierarchical tial sequences of cyt b and 16S, and also the combined matrix components among clades, among localities within clades, and including cyt b, 16S and RAG-1, recovered two highly supported, within localities, using analysis of molecular variance (AMOVA; geographically structured clades in P. boiei that meet in the vicin- Excoffier et al., 1992). Population substructure was quantified with ities of the Tietê river (Fig. 3). Clade South includes individuals the fixation index FST in the program Arlequin (Excoffier et al., from southern São Paulo (SP) state and Santa Catarina (SC), 2005) after 10,000 iterations. Coalescent-based tests of historical whereas clade North has specimens from northern SP, Rio de population expansion (Ramos-Onsins and Rozas, 2002) were esti- Janeiro (RJ), Minas Gerais (MG), and Espírito Santo (ES, Fig. 3; mated in DNAsp as per Carnaval and Bates (2007) to search for sig- Table 1). The northern clade is further divided into two well- natures of demographic changes, or lack thereof. Analyses used the supported groups: North 1 with representatives in ES and MG, pooled mtDNA data excluding gaps (1116 bp) for the whole spe- North 2 in northern SP, RJ and southern MG (Fig. 3, Table 1). cies, as well as per individual clade. We also explored relationships Mitochondrial-DNA based estimates of TMRCA were highly con- among haplotypes of each dataset using median joining networks cordant among runs and suggest a Late Miocene/Early Pliocene (Bandelt et al., 1999) obtained in NETWORK 4.5.1.6 (http:// divergence among northern and southern lineages: TMRCA of P. www.fluxus-engineering.com). For population-level analyses, boiei 7.86 Mya (95% CI 4.58–11.70), South phylogroup 2.86 Mya RAG-1 sequences were phased into haplotypes with PHASE (95% CI 1.18–4.99), North phylogroup 6.62 Mya (95% CI 3.27– (Stephens and Donnelly, 2003), using default conditions. 10.27), TMRCA of the North 1 and North 2 phylogroups are esti- Karyotypes were obtained for 39 individuals from 12 localities mated at 3.74 (1.70–5.95) Mya and 1.56 (0.74–2.62) Mya, (Table S1). Chromosome studies were performed after conven- respectively. tional Giemsa staining, C-banding, and Ag-NOR staining, following Median joining networks of the combined mtDNA dataset show Amaro-Ghilardi et al. (2008). 31 distinct haplotypes, striking geographic structure, and high

Fig. 2. P. boiei climate-based distribution models under current conditions (top left) and paleoscenarios ECHAM3 (top right), MIROC (bottom left) and CCSM (bottom right). Areas in black are suitable for species occurrence. R.C. Amaro et al. / Molecular Phylogenetics and Evolution 62 (2012) 880–888 883

Fig. 3. Bayesian tree topology of combined molecular data set (cyt b, 16S and RAG-1 genes; 50% majority-rule consensus phylogram). Posterior probability and ML bootstrap values marked above branches. Inset map portrays sampled localities, which were color-coded to depict representatives of phylogroups South (red), North 1 (green), and North 2 (blue). Arrow points to Iperó (SP), where karyotypic data show hybridization between the southern and northern phylogroups. Thick lines denote state boundaries; state acronyms are provided in text. Thin lines depict major rivers.

levels of diversity (Hd 0.9271, Fig. 4a). Forty-eight distinct haplo- reciprocal monophyly. Additional genetic diversity indices are pro- types of RAG-1 were present in the sample of 152 (phased) se- vided as Supplementary Information (Tables S2 and S3). AMOVA quences (Hd 0.8962, Fig. 4b), and only one was shared between analyses showed that about 74% of the cyt b genetic variation, phylogroups South, North 1 and North 2. Two RAG-1 haplotypes and 64% of 16S sequence variability, occurred among phylogroups were shared between phylogroups North 1 and South. All other nu- – yielding signiﬁcantly high values of FST (0.971 and 0.946, respec- clear haplotypes were exclusive to individual phylogroups, sug- tively, p < 0.001, Table S4). While among-phylogroup structure ex- gesting nuclear geographic genetic structure despite absence of plains just 8% of the total genetic variation in the RAG-1 gene, FST 884 R.C. Amaro et al. / Molecular Phylogenetics and Evolution 62 (2012) 880–888

Table 1 Summary of molecular and cytogenetic data with phylogroup assignment, number of individuals sampled per site, major chromosomal features, and number of individuals karyotyped. NOR = nucleolar organizer region; PCH = pericentromeric constitutive heterochromatin; Y = yes; N = no; juv. = juveniles; M = males; F = females.

Clade Locality, State # Sequences cytb, 16S, RAG-1 NOR location PCH in large blocks ZW:ZZ system # Karyotypes South Apiaí, SP 1, 1, 1 Pair 4 N N 1 juv. Buri, SP 1, 1, 1 Pair 4 N N 2 F Cananéia, SP 1, 1, 1 – – – – Caucaia do Alto, SP 7, 6, 7 Pair 4 N N 5 F Eldorado, SP 1, 2, 2 – – – – Embu, SP 1, 1, 1 – – – – Iperó, SP 4, 4, 4 Pair 4 N N 1 M, 2 F Iperó, SP 1, 1, 1 Pairs 4 and 8 N N 1 M Juquitiba, SP 2, 2, 2 Pair 4 N N 4 M, 1 juv. Piedade, SP 5, 6, 6 Pair 4 N N 1 M, 4 F Pilar do Sul, SP 1, 1, 1 – – – – Ribeirão Branco, SP 1, 1, 1 – – – – Ribeirão Grande, SP 4, 4, 3 – – – Tapiraí, SP 1, 1, 1 – – – 1 M Treviso, SC 1, 1, 1 – – – – North 1 Cristina, MG 0, 3, 3 – – – – Eugenópolis, MG 1, 1, 1 – – – – Mariana, MG 1, 1, 1 – – – – PN Caparaó, MG and ES 2, 8, 7 Pair 8 Y N 4 F, 1 juv. PE Rio Doce, Marliéria, MG 0, 1, 1 – – – – Muniz Freire, ES 0, 1, 1 – – – – North 2 Bananal, SP 3, 3, 3 – – – – Jambeiro, SP 3, 3, 3 Pair 8 Y Y 1 M, 1 F Piracaia, SP 0, 1, 1 – – – – Santa Branca, SP 1, 1, 1 Pair 8 Y Y 1 M Santana do Parnaíba, SP 0, 1, 1 Pair 8 Y Y 1 F Santo Antonio do Pinhal, SP 2, 3, 3 – – – – São Luís do Paraitinga, SP 4, 5, 5 – – – – Serra da Cantareira, SP 8, 10, 8 Pair 8 Y Y 6 M, 3 F Secretário, RJ 0, 1, 1 – – – – Camanducaia, MG 2, 3, 3 – – – – Cristina, MG 1, 1, 1 – – – –

levels were also significantly high for this marker (FST = 0.50823, 4. Discussion p < 0.001). Signatures of population expansion were not detected either at 4.1. Population genetic structure in P. boiei the species level (pooled data from northern and southern phylogroups, R2 = 0.971, p = 0.480), nor in the North or South phylogroup Mitochondrial and nuclear sequence analyses suggest indepen- alone (North group R2 = 0.080, p = 0.076; South group R2 = 0.075, dent evolutionary trajectories across the Tietê river (Figs. 3 and 4, p = 0.059; Table S5). Table 1), a result also reflected in P. boiei’s chromosomal arrange- The karyotype of P. boiei is composed of 22 meta and submeta- ments (Fig. 5, Table 1). While previous studies had described dis- centric chromosomes (Fig. 5a). Meiotic spreads show cells in meta- tinct arrangements within the species (Amaro-Ghilardi and phase I with 11 bivalents, in general ring-like bivalents, and in Yonenaga-Yassuda, 2002; Silva et al., 2003; Ananias et al., 2007), metaphase II with 11 chromosomes (Fig. S1). Heteromorphic sex ours is the first to describe the geographical extent of their occur- chromosomes are not detected on mitotic or meiotic analyses rences. Despite cytogenetic and molecular evidence of deep geo- using conventional staining. FISH signals of (TTAGGG)n are exclu- graphical structure, a recent study of Proceratophrys species did sively detected in the telomeric regions of all chromosomes of P. not find morphometric distinctions across the phylogroups here boiei (Fig. 5b). uncovered (Prado and Pombal, 2008). It remains to be seen whether Samples from phylogroups South and North had nucleolar orga- the examination of additional morphological characters, or call var- nizer regions (NORs) in different chromosome pairs: individuals iation across populations, will reveal phenotypic differences within belonging to the southern phylogroup showed NORs in the short P. boiei that are congruent with the structure here described. arm of pair 4; specimens from the northern phylogroup presented Molecular data recovered two subgroups within the northern NORs in the short arm of pair 8. One individual from Iperó (SP, phylogroup (North 1 and North 2; Fig. 3), a result which agrees South phylogroup, ID numbe AF2129) showed NORs in the short with the cytogenetic data (Fig. 5). The heteromorphism found only arms of both pairs 4 and 8 (Table 1; Fig. 5c and d). in females of North 2 phylogroup suggests an early process of dif- Constitutive heterochromatin is present in the pericentromeric ferentiation into a ZZ:ZW system of sexual chromosomes, where region of chromosomes in all phylogroups. However, individuals sexual chromosomes appear to have the same size and morphol- from the northern clade exhibit large blocks of constitutive hetero- ogy under conventional staining, but can be distinguished after chromatin in pericentromeric and proximal regions (Fig. 5e and f), C-banding (chromosome Z with C-bands in the pericentromeric re- whereas individuals from the southern phylogroup do not (Fig. 5g; gion, and chromosome W nearly completely heterochromatic). Table 1). Moreover, in females of the North 2 phylogroup, one chro- Although the small North 2 karyotype dataset precludes us from mosome of pair 1 is almost entirely heterochromatic, whereas its affirming that the ZZ:ZW system is widespread within the homologue shows a pericentromeric C-band block (Fig. 5f; Table 1). phylogroup, this heteromorphism has been detected in two other This heteromorphism is not detected either in males of the same localities within the geographical range of the subgroup (Amaro- localities, nor in individuals from the remaining phylogroups. Ghilardi and Yonenaga-Yassuda, 2002; Ananias et al., 2007). R.C. Amaro et al. / Molecular Phylogenetics and Evolution 62 (2012) 880–888 885

Fig. 4. Proceratophrys boiei mtDNA (a) and nuclear (b) haplotype networks. Localities and haplotype descriptions follow Table S1. Mutational steps between alleles are indicated on lines.

4.2. The São Paulo contact zone from Grazziotin et al. (2006) are also consistent with hybridization in snakes from a nearby site. Multi-species samples from the re- Similar to P. boiei, several species of forest vertebrates show gion and from along the Tietê river will be pivotal to verify the ex- well-defined phylogeographic breaks in southern São Paulo, all dat- tent of the region of secondary contact, and to confirm whether a ing back to the Early Pliocene or Late Pleistocene. Yet, no apparent suture zone occurs in this portion of the Atlantic rainforest (Moritz marked climatic, edaphic, vegetational, or topographic differences et al., 2009). are observed across the breaks. In a pattern strikingly similar to Congruent patterns of phylogeographical breaks in the southern our results, Grazziotin et al. (2006) recovered two phylogroups of Atlantic forest fauna suggest common biological responses to land- cyt b in the snake Bothrops jararaca that closely match P. boiei’s scape changes during the Neogene (Early Pliocene–Late Miocene). northern and southern split. Based on available samples, the con- While paleontological data point to a subtropical to temperate cli- tact zone in B. jararaca appears to be located slightly south of that mate in the region (Garcia et al., 2008), the period was marked by here reported for P. boiei, and the divergence between phylogroups intense tectonic activity. Particularly, the location of P. boiei’s was estimated around 3.8 Mya. In the Phyllomedusa burmeisteri frog phylogeographical break zone coincides with that of a NW–SE fault group, divergence between the northern group (P. burmeisteri and of the Southern Brazil Continental Rift, which was reactivated Phyllomedusa bahiana) and the southern group (Phyllomedusa dis- starting in the Miocene (Riccomini et al., 2010). Tectonic move- tincta, Phyllomedusa tetraploidea and Phyllomedusa iheringii) was ment in the fault zone lifted the southwestern block and lowered estimated around 5.0 Mya, and the location of the break coincides the northeastern side, altering local hydrographic basins through with the Guapiara lineament and the Cubatão shear zone (Brunes headwater captures. Former headwaters of the Tietê were then et al., 2010), located slightly south of the phylogeographical break captured by the Paraíba do Sul River; geological analyses based of P. boiei. Phylogeographic breaks located in areas close to that of on current topographic features point to a sharp vertical displace- P. boiei were also detected for the woodcreeper Xiphorhynchus ment of at least 160 m of altitude along the Tietê River (Riccomini and frogs of the Thoropa miliaris complex (Cabanne et al., 2007, et al., 2010). While exact dates and details of these processes are 2008; Fitzpatrick et al., 2009). Yet, estimates of divergence times largely unknown, available data indicate that geomorphological provided by those studies were smaller (0.5–0.3 Mya) than those and hydrological consequences of the NW–SE fault activity oc- reported here. Fine-scale sampling of these taxa, along with im- curred throughout the Pliocene and Pleistocene (Riccomini et al., proved multi-locus estimates of divergence times, will allow us to 2010), which could explain the observed differences in the location investigate the amount of spatial overlap of these contact areas of phylogeographic breaks, as well as associated divergence times, and to test whether observed levels of divergence could have been across local species of vertebrates. generated by a single, common vicariant event (Moritz et al., 2009). It remains to be demonstrated whether these changes in relief – Our karyotypic data provide evidence of hybridization between a vertical displacement, possibly tied to vegetational and landscape P. boiei’s two major phylogroups in Iperó (central São Paulo). Data changes in surrounding areas – sufficed as vicariant forces to 886 R.C. Amaro et al. / Molecular Phylogenetics and Evolution 62 (2012) 880–888

the genetic data failed to detect statistically significant signatures of population expansion even when the southern phylogroup was analyzed separately (Table S5). Thomé et al. (2010) found similarly deep mitochondrial structure across the Atlantic forest range while examining the genetic differentiation and historical distribution of endemic toads belonging to the Rhinella crucifer group. In R. crucifer, an old divergence event between a southernmost lineage and other phylogroups was dated to the Pliocene, while remaining groups have apparently diverged throughout the Pleistocene (Thomé et al., 2010). Paleoecological distribution models supported a scenario of habitat fragmentation associated with glacial cycling, but some phylogeographic breaks coincided with putative barriers associated with neotectonic activity. As in P. boiei, data from R. crucifer do not show the southern Holocene colonization of the Atlantic forest from northern refugia previously detected in warm-adapted, Hypsiboas species by Carnaval et al. (2009). Rather, the Rhinella results suggested species persistence in southern Brazil during the LGM – an observation potentially related to its ability to tolerate and breed under cooler conditions. While Pleistocene climatic change strongly affected the ranges of currently widespread lowland forest endemics – to the point of explaining current patterns of genetic diversity – data from both distribution models and population genetics indicate that glacial conditions were much less stringent on taxa currently able to occupy montane habitats in coastal Brazil. Lowland species currently living in milder climates and higher temperatures were seemingly restricted to refugial areas and lower latitudes during the LGM, expanding their ranges following Holocene warming (Carnaval et al., 2009). Conversely, it appears that the physiological profile of montane taxa has shielded these species from the Pleistocene climatic changes that so strongly impacted Brazil’s coastal lowland groups. Taxa currently restricted to high elevations, and possibly taxa now limited to high latitudes, will likely show a pattern oppo- site to that observed in lowland forms: they were probably more widespread under cooler climates, and are currently restricted in their distribution (Rodrigues et al., 2009). Fig. 5. (a) Conventional Giemsa-staining karyotypes and metaphases of P. boiei; (b) Preliminary models of the Atlantic forest biome (Carnaval and distribution of telomeric sequence (TTAGGG)n after FISH, showing exclusively Moritz, 2008) failed to represent persistence of montane forests telomeric pattern; (c) pairs 4 and 8 showing secondary constrictions in conven- in southern Brazil during the LGM. This may be an artifact of the tional staining, and Ag-NOR location; (d) metaphase of hybrid individual from Iperó (SP), showing Ag-NORs in pairs 4 and 8; (e) karyotype of male (M) from North 2 modeling procedure. In the future, more refined models of the phylogroup (Santa Branca, SP): note constitutive heterocromatin in large blocks. Atlantic forest biome must take into consideration the many differ- Karyotypes of males (M) and females (F) of phylogroup North 1 show similar ent climatic profiles of the forest, hence capturing the different configuration; (f) karyotype of female from North 2 phylogroup (Serra da behaviors of lowland and montane areas. Comparative phylogeo- Cantareira, SP): note large blocks of constitutive heterochromatin and the heteromorphic ZZ:ZW pair (pair 1); (g) karyotype of female from the southern phylogroup graphic molecular studies and paleoclimate distribution models (Piedade, SP): note absence of large blocks of constitutive heterochromatin. Males of species of strikingly different thermal adaptations, especially of phylogroup South show similar configuration. mountaintop endemics, should confirm the generality of the diversity patterns and underpinning demographic processes that we promote the differentiation of parapatric mtDNA lineages of P. boiei here discuss. Physiological data on thermal preferences and toler- and other vertebrates. More precise dating is needed both in the ances of target species also will promote great insight into the pro- geological and molecular areas. Perhaps more intriguing is the cesses underlying diversity distribution in the face of past, present, mechanism behind the current parapatry of the southern and and future climate change in Brazil and beyond. northern phylogroups, now that erosion and post-Pliocene tectonics reduced the steep altitudinal differences generated through the Acknowledgments reactivation of the fault. In the absence of environmental differences across the contact area, this question must be resolved José Cassimiro, Felipe F. Curcio, Dante Pavan, Mauro Teixeira Jr., through further geological and molecular studies. Agustin Camacho, Pedro Nunes, Vanessa Verdade, Vinícius X. Silva, José Mario Ghellere, Marianna Dixo, Sandra Favorito, and Ricardo 4.3. LGM conditions and montane taxa Fuentes helped tremendously with fieldwork. Sabrina Baroni and Maíra Concistré assisted with technical support. Celio F. B. Haddad, Contrary to preliminary models of the forest and of lowland for- Hussam Zaher, Diego Santana, and João Luiz Gasparini shared est frogs (Carnaval and Moritz, 2008; Carnaval et al., 2009), the ge- tissues for the phylogeographic study. Claudio Riccomini and Maria netic data and MIROC and CCSM-based models of P. boiei suggest Judite Garcia provided invaluable insight on the geology of SE that it was able to maintain most of its current range during the Brazil. Roberto Vilela Roberto helped enormously in use of the LGM. Although ECHAM3-based paleomodels suggested a slight computer program Beast. Craig Moritz commented on early ver- contraction in the species’ southernmost range at 21kya (Fig. 2), sions of the manuscript. Funding provided by Fundação de Amparo R.C. 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