Assembly and Evolution of the Amazonian Biota and Its Environment: an Integrative Approach

Assembly and evolution of the Amazonian Biota and its environment: An integrative approach

Lúcia G. Lohmann (Universidade de São Paulo) Joel Cracraft (American Museum of Natural History)

FAPESP 2012/50260-6 NSF 1241066 !"#$%&'($)*+,#))$-#"$.#/+

Brazil Canada >/2?*"'2%$%*+%*+DF#+G$4)#+ >/2?*"'279+#5+C#"#/7#+ >/2?*"'2%$%*+B*%*"$)+%*+=#2H'+ United States >/2?*"'2%$%*+B*%*"$)+%#+G$"H+ 01*"2($/+34'*41+#5++ >/2?*"'2%$%*+K'7$%4$)++ ++6$74"$)+82'7#"9+ ++%*+,$1L2/$'+ ,279+>/2?*"'279+6*:+;#"<+ 34'*4+G$"$*/'*+K1N)2#+=#*)%2+ B2*)%+34'*41+#5++ ++6$74"$)+82'7#"9+ J/'.747#+6$(2#/$)+%*++ ++G*'O42'$'+%$+01$PQ/2$++ 32%%)*+C*//*''**++ ++D7$7*+>/2?*"'279+ Argentina 6$74"$)+82'7#"9+34'*41++ ++E#'+0/A*)*'+,#4/79+ ,I6J,KC&J/'.747#+D4L*"2#"++ %*+K/7#1#)#A2$M+C4(41H/+ 6*:+;#"<+!#7$/2($)+=$"%*/+ Great Britain >/2?*"'279+#5+32(@2A$/+ >/2?*"'279+#5+K%2/-4"A@+ >/2?*"'279+#5+,#)#"$%#+ The Amazon Basin

• One of the most biodiverse areas on Earth but little is still known about the processes that led to such great diversity

• Many uncertainties remain about its geological history, age of formation, and extension of its aquatic systems

• Some models claim that the Amazon was established during the Miocene while others have established its origin in the Pleistocene

• Broad Objective: Achieve a new evolutionary and environmental synthesis of Amazonia

of 20 3 !""#$%&'(")"&)*+"$#,*&*(-.."$%")&*-..)&/01&&Meeting these scientific challenge calls for +$'"%1-#2"&*10))34+)*+5.+$-16&)'74+")&integrative cross-disciplinary studies PART I Characterization of Amazonian biodiversity

- How is biodiversity spatially distributed across Amazonia?

- How are species distributions organized into patterns of endemism?

- What are the biotic and abiotic environmental associations with those diversity patterns? Herbaria with significant Amazonian collections

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- Existing Amazonian Plant Specimens: ca. 1.5 million - Collection Density: 0.15-0.20 specimens/km2 (contrast with England: ca. 28 specimens/km2) Digitizing Specimens

Aggregating Data Data Limitations - Misidentified specimens - Specimens without coordinates - Specimens with wrong coordinates Vertebrate Data sets

• There are at least 400.000 records of vertebrates from Amazonia available at GBIF, of these:

Birds = 170.000 records Primates = 6.000 records (31.000 georeferrenced) (2.000 georeferrenced) Collaborating Institutions

• AMNH hold ca. 67.000 specimens • FMNH holds ca. 58.000 • INPA, Museu Goeldi, MZUSP & other Brazilian collaborators Amphilophium Arrabidaea Arrabidaea paniculatum affinis rego

Arrabidaea Anemopaegma Adenocalymma chica laeve bracteatum What are the patterns of diversity and endemism within groups? Are those patterns congruent across groups? Do these patterns relate to the environmental history of Amazonia? Data sets will be made available through Sinbiota and through “The Evolutionary Atlas of Amazonian Biodiversity” a WebPortal that is being constructed as part of this project Atlas of Amazonian Biodiversity

Main Goals:

• To communicate what is known and what is not known about Amazonian biodiversity and evolution.

• To inspire people to understand this incredible landscape and its plant and animal life from an evolutionary perspective. PART II Phylogenetic and phylogeographic history of selected Amazonian taxa

- What has been the evolutionary history of the Amazonian biota and how was it generated?

- Selected Organisms:

i. Butterflies (selected clades of Nymphalidae & Riodinidae)

ii. Primates (Callicebus, Cacajao, Chiropotes, Mico, Saimiri, Saguinus)

iii. Birds (selected clades of Amazonian birds)

iv. Plants (Bignoniaceae & Lecythidaceae) Mimetic butterflies

Advantages of this system:

- Well-sampled across their ranges

- Well-understood from a systematic perspective

- Geographically variable with congruent distributions

- Recently-enough diverged to allow for plausible molecular- clock estimates !"#$(<&=$><($ -?(""(,,((&;*+*(&.")@($,)*A4 !"#$%&'$()*+,&-./01!234 5+$)"+&678+,&-19/!:.;34 Phylogenetic relationships of the butterfly family Nymphalidae based on nDNA & mtDNA data

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H("7,&!"#$%&'$() !"#$"%&'()'% !"24'-"#$$"-.('%012-3% Phylogeography and speciation in Heliconius hermathena (Lepidoptera; Nymphalidae; Heliconiini) in Amazonian sand forests (“campinaranas”) Selected Neotropical Monkeys

- Estimate temporal and spatial diversification patterns of selected genera, especially:

(a) Callicebus (b) Cacajao (c) Chiropotes (d) Mico (e) Saimiri (f) Saguinus

- Correlate diversification patterns to physical barriers along the geographic distribution of taxa Jean Boubli (University of Salford, UK) Horácio Schneider & Iracilda Sampaio (UFPA) Phylogeny of the New World Titi Monkeys (Callicebus)

• Sampling • 15 species • 73 individuals

J. Boubli et al. (2014, MPE) Putting our results into perspectives

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Mico – Callithrix 5.96 3.83 8.59 4 Cebus – Sapajus 6 3.13 9.35 ‘New’ Cacajao -Chiropotes 6.91 4.56 9.34

Lagothrix-Brachyteles 9.53 6.10 Genus 13.44

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1)**.2"342.0-45)6"6)7,*" Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Spatial and temporal patterns of diversiﬁcation on the Amazon: A test of the riverine hypothesis for all diurnal primates of Rio Negro and Rio Branco in Brazil

a,b, b c,d c,e Jean P. Boubli ⇑, Camila Ribas , Jessica W. Lynch Alfaro , Michael E. Alfaro , Maria Nazareth F. da Silva b, Gabriela M. Pinho f, Izeni P. Farias f a School of Environment and Life Sciences, 315 Peel Building, University of Salford, Salford M5 4WT, UK b Instituto Nacional de Pesquisas da Amazonia INPA, Manaus, Brazil c Institute for Society and Genetics, 1321 Rolfe Hall, University of California, Los Angeles, CA 90095, USA d Department of Anthropology, University of California, Los Angeles, CA 90095, USA e Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095, USA f Universidade Federal do Amazonas, Laboratório de Evolução e Genética Animal, Manaus, AM, Brazil article info abstract

Article history: The role of Amazonian rivers as drivers of speciation through vicariance remains controversial. Here we Received 1 October 2013 explore the riverine hypothesis by comparing spatial and temporal concordances in pattern of diversiﬁ- Revised 27 August 2014 cation for all diurnal primates of Rio Negro and its largest tributary, Rio Branco. We built a comprehensive Accepted 9 September 2014 comparative phylogenetic timetree to identify sister lineages of primates based on mitochondrial cyto- Available online xxxx chrome b DNA sequences from 94 samples, including 19 of the 20 species of diurnal primates from our study region and 17 related taxa from elsewhere. Of the ten primate genera found in this region, three Keywords: had populations on opposite banks of Rio Negro that formed reciprocally monophyletic clades, with Amazonia roughly similar divergence times (Cebus: 1.85 Ma, HPD 95% 1.19–2.62; Callicebus: 0.83 Ma HPD 95% Platyrrhini Phylogeography 0.36–1.32, Cacajao: 1.09 Ma, 95% HPD 0.58–1.77). This also coincided with time of divergence of several River barrier allopatric species of Amazonian birds separated by this river as reported by other authors. Our data offer Riverine hypothesis support for the riverine hypothesis and for a Plio-Pleistocene time of origin for Amazonian drainage sys- Vicariance tem. We showed that Rio Branco was an important geographical barrier, limiting the distribution of six primate genera: Cacajao, Callicebus, Cebus to the west and Pithecia, Saguinus, Sapajus to the east. The role of this river as a vicariant agent however, was less clear. For example, Chiropotes sagulata on the left bank of the Rio Branco formed a clade with C. chiropotes from the Amazonas Department of Venezuela, north of Rio Branco headwaters, with C. israelita on the right bank of the Rio Branco as the sister taxon to C. chiropotes + C. sagulata. Although we showed that the formation of the Rio Negro was important in driving diversiﬁcation in some of our studied taxa, future studies including more extensive sampling of markers across the genome would help determine what processes contributed to the evolutionary history of the remaining primate genera. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction New World, with the greatest concentration in the Amazon Basin. The origins of such high species diversity remain poorly under- Of the more than 685 taxa (species and subspecies) of recog- stood. One of the ﬁrst proponents of a mechanism to account for nized primates (Mittermeier et al., 2013) approximately one third the high primate species diversity in Amazonia was the British nat- (164 taxa, 20 genera, 5 families; Paglia et al., 2012) are found in the uralist Alfred R. Wallace. While on a collecting expedition to Brazil in the mid 19th century, Wallace noticed that primate species on opposite banks of large Amazonian rivers substituted one another Corresponding author at: School of Environment and Life Sciences, Room 315, ⇑ Peel Building, University of Salford, Salford M5 4WT, UK. (Wallace, 1852). Based on that observation he proposed that these E-mail addresses: [email protected], [email protected] (J.P. Boubli), rivers acted as barriers to the dispersal of animals. In particular, he [email protected] (C. Ribas), [email protected] (J.W. Lynch Alfaro), proposed that the three largest Amazonian rivers (the Amazon, [email protected] (M.E. Alfaro), [email protected] (M.N.F. da Silva), Madeira and Negro), divided the region into four districts charac- [email protected] (G.M. Pinho), [email protected] (I.P. Farias). http://dx.doi.org/10.1016/j.ympev.2014.09.005 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Boubli, J.P., et al. Spatial and temporal patterns of diversiﬁcation on the Amazon: A test of the riverine hypothesis for all diurnal primates of Rio Negro and Rio Branco in Brazil. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.09.005 ?7*3:,+-*[email protected],-B L)IG*&=+"K 6(J&=+"K

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C C ? C Selected lineages of Amazonian birds

• Eight focal lineages: (a) Ramphastidae (b) Capitonidae (c) Tyrannidiae (d) Furnariidae (e) Thamnophilidae (f) Formicariidae (g) Troglodytidae (h) Turdidae

• Main Collaborators: Joel Cracraft (AMNH) John Bates (FMNH) Camila Ribas (INPA) Alex Aleixo (MPEG) Using ultraconserved elements (UCEs) &-2>'%"?@AB%C

- Highly conserved DNA elements shared among evolutionary distant taxa ,.D.

- Useful for reconstructing the &-2>'%"EF>-)G)H'G evolutionary history and ,2"%E'.-'G"I.-!(2G'G

- 32 samples sequenced, over .+.KF%)%L 500 loci recovered and used in .K)=+3'+," ,2"&-2>'% pilot phylogenetic analyses

- 246 samples that are ready to be sequenced by Rapid Genomics (Florida, USA) ,.D.".K)=+3'+,% 12-"&-2>'% APhylogenyofBirdsBasedonOver1,500LociCollected by Target Enrichment and High-Throughput Sequencing

John E. McCormack1*, Michael G. Harvey1,2, Brant C. Faircloth3, Nicholas G. Crawford4, Travis C. Glenn5, Robb T. Brumfield1,2 1 Museum of Natural Science, Louisiana State University, Baton Rouge, Louisiana, United States of America, 2 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America, 3 Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America, 4 Department of Biology, Boston University, Boston, Massachusetts, United States of America, 5 Department of Environmental Health Science, University of Georgia, Athens, Georgia, United States of America A Phylogeny of Birds from 1,500 Loci

Abstract Evolutionary relationships among birds in Neoaves, the clade comprising the vast majority of avian diversity, have vexed systematists due to the ancient, rapid radiation of numerous lineages. We applied a new phylogenomic approach to resolve relationships in Neoaves using target enrichment (sequence capture) and high-throughput sequencing of ultraconserved elements (UCEs) in avian genomes. We collected sequence data from UCE loci for 32 members of Neoaves and one outgroup (chicken) and analyzed data sets that differed in their amount of missing data. An alignment of 1,541 loci that allowed missing data was 87% complete and resulted in a highly resolved phylogeny with broad agreement between the Bayesian and maximum-likelihood (ML) trees. Although results from the 100% complete matrix of 416 UCE loci were similar, the Bayesian and ML trees differed to a greater extent in this analysis, suggesting that increasing from 416 to 1,541 loci led to increased stability and resolution of the tree. Novel results of our study include surprisingly close relationships between phenotypically divergent bird families, such as tropicbirds (Phaethontidae) and the sunbittern (Eurypygidae) as well as between bustards (Otididae) and turacos (Musophagidae). This phylogeny bolsters support for monophyletic waterbird and landbird clades and also strongly supports controversial results from previous studies, including the sister relationship between passerines and parrots and the non-monophyly of raptorial birds in the hawk and falcon families. Although significant challenges remain to fully resolving some of the deep relationships in Neoaves, especially among lineages outside the waterbirds and landbirds, this study suggests that increased data will yield an increasingly resolved avian phylogeny.

Citation: McCormack JE, Harvey MG, Faircloth BC, Crawford NG, Glenn TC, et al. (2013) A Phylogeny of Birds Based on Over 1,500 Loci Collected by Target Enrichment and High-Throughput Sequencing. PLoS ONE 8(1): e54848. doi:10.1371/journal.pone.0054848 Editor: Nadir Alvarez, University of Lausanne, Switzerland Received September 26, 2012; Accepted December 17, 2012; Published January 29, 2013 !! Copyright: ß 2013 McCormack et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestrictedFigure use, 1. distribution, Neoaves and species reproduction used in in this any medium, study. Species provided are the listed original in authorTable 1. and Numbers source are match credited. those in table and on the tips of phylogenies. Funding:IllustrationsThis research are wasbased supported on photos by NSF (see grant Acknowledgments). DEB-0841729 to RTB. DEB-1242260 to BCF and TCG and an Amazon Web Services grant to BCF, NGC, JEM, and TCGdoi:10.1371/journal.pone.0054848.g001 provided partial support for computational analyses. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competingusing Interests: the STARThe authors (Species have declaredTrees fromthat no Average competing Ranksinterests exist. of Generally, the Bayesian and ML phylogenies for the 1,541 locus * E-mail:Coalescences) [email protected] method [48]. We performed 1,000 multi-locus, alignment were similar in their topology and amount of resolution non-parametric bootstrap replicates for the STAR tree by (Fig. 2a; see Fig. S1 for fully resolved trees). Of the 31 nodes, 27 resampling nucleotides within loci as well as resampling loci (87%) were highly supported in the Bayesian tree (.0.95 PP), whereas a subset of 20 of those nodes (65%) were also highly within the data set [49]. We only performed the species treemitochondrial DNA [8]. However, rapidly evolving markers Introductionanalysis on the alignment with no missing data due to concerns supported in the ML tree (.75% bootstrap score). An additional 7 introduce a new set of problems to the inference of ancient about how missing loci might affect a coalescent analysis. nodes (23%) appeared in both the Bayesian and ML trees, but The diversification of modern birds occurred extremely rapidly, radiations: through time, substitutions across rapidly evolving with all majorTo orders assess and phylogenetically most families informative becoming distinct indels, within we scanned a support in the ML tree was low (bisected nodes in Fig. 2a). Four markersnodes overwrite (16%) had older either substitutions, low support resulting in both in trees signal (and saturation thus are short windowalignments of 0.5 by to eye 5 millionin Geneious years 5.4 around (Biomatters the Cretaceous- Ltd, Aukland, New Zealand), recording indels that were 2 bp or more in lengthandcollapsed homoplasy in Fig.[9]. 2a) To or address had high this support challenge, in the some Bayesian researchers tree, but Tertiary boundary [1–4]. As with other cases of ancient, rapid havedid inferred not appear ancient in phylogeny the ML using tree (white rare genomic nodes in changes, Fig. 2a). like A radiation,and resolving shared between deep evolutionary two or more relationships ingroup taxa. in We birds then has mapped informative indels onto the resolved 416-locus Bayesian phylog-retroposonphylogram insertions for the and 1,541 indels, locus because Bayesian rare tree changes (Fig. S2) are showed unlikely long posed a significant challenge. Some authors have hypothesized eny. to occurterminal in branchesthe same and way short multiple internodes times, near thereby the base minimizing of the tree, that the initial splits within Neoaves might be a hard polytomy that homoplasyconsistent [10,11]. with previous Though studies successful suggesting in some an ancient, cases [12], rapid will remain irresolvable even with expanded data sets (reviewed in Results retroposonsradiation are of Neoaves. often insufficiently numerous to fully resolve [5]). However, several recent studies have suggested that expanded relationshipsFor the between data set taxa requiring that no rapidly missing radiated data, we [13], recovered and genomic andWe provide taxonomic summary coverage statistics will for lead sequencing to an increasingly and alignment inalthough416 UCE often billedloci across as being 29 homoplasy-free, Neoaves species we and now the know chicken that resolvedTable avian 1. tree We of obtained life [2,6,7]. an average of 2.6 million reads per samplesharedoutgroup. retroposon Enrichments insertions for can three be due species to independent performed relatively events Using(range DNA = sequence 1.1–4.9 million). data to Thesereconstruct reads assembledrapid radiations into an like average[14].poorly (Table 1; Micrastur, Trogon, and Vidua), and we excluded the Neoavesof 1,830 phylogeny contigs per presents sample a (range practical = 742–2,418). challenge Anon averageseveral (per Athese second samples challenge to boost to reconstructing the number of ancient, loci recovered. rapid radiations The average is fronts. First,sample) short of 1,412 speciation of these intervals contigs matchedprovide little the UCE time loci for fromthelength randomness of these inherent 416 loci to was the 397 process bp, and of the gene total sorting concatenated (i.e., substitutionswhich to we accrue designed on target internal capture branches, probes (range reducing = 694–1,681). the coalescentalignment stochasticity), length was which 165,163 occurs characters even (includingwhen gene indels) histories with phylogeneticThe average signal for length rapid of speciationUCE-matching events. contigs Traditionally, was 429 base the pairsare estimated7,600 informative with 100% sites. accuracy Bayesian [15]. and ML The trees amount differed of conflict more in solution(bp) to this (range problem = 244–598), has been and to collectthe average additional coverage sequence of UCE-amongtheir gene-tree topology and topologies resolution due than to was coalescent observed stochasticityfor the 1,541 locus trees above (Fig. 2b; see Fig. S3 for fully resolved trees). Of data, preferablymatching from contigs a rapidly was 71 evolving times (range molecular = 44–138). marker The such percentage as increases as speciation intervals get shorter [16]. Hemiplasy refers of original sequencing reads that were ‘‘on target’’ (i.e., helped the 28 nodes, 24 (86%) were highly supported in the Bayesian tree build UCE-matching contigs) averaged 24% across samples (range (.0.95 PP), whereas only a subset of 14 (50%) was highly . PLOS ONE= 15% | www.plosone.org - 35%). 1supported in the ML January tree ( 201375% | bootstrap Volume 8 score). | Issue We 1 |recovered e54848 When we selected loci allowing 50% of species for a given locus an additional three nodes (11%) in both the Bayesian and ML to have missing data, the final data set contained 1,541 UCE loci trees, but support for these nodes in the ML tree was low (bisected and produced a concatenated alignment that was 87% complete nodes in Fig. 2b). Twelve nodes (43%) disagreed between the across 32 Neoaves species and the chicken outgroup. The average Bayesian and ML trees, a frequency much higher than the 16% length of these 1,541 loci was 350 bp (min = 90, max = 621), and disagreement we observed from the 1,541 locus analysis. the total concatenated alignment length was 539,526 characters The STAR species tree from the 416 locus data set (Fig. 3; Fig. (including indels) with 24,703 informative sites. S3c) was much less resolved and had lower support values than either the Bayesian or ML tree estimated for these data. There has

PLOS ONE | www.plosone.org 3 January 2013 | Volume 8 | Issue 1 | e54848 Lecythidaceae

Bertholletia excelsa Scott Mori (NYBG) & Chris Dick

! Bignoniaceae “Trumpet-creeper” family

\ Tribe Bignonieae (Bignoniaceae)

• Large clade of lianas (ca. 400 spp. & 21 genera) • Conspicuous component of the Amazonian flora • The most diverse and abundant clade of lianas in most Amazonian ecosystems • Occur in all the major ecological zones (from savannahs to wet forests) • Very diverse morphologically & ecologically 79 Adenocalymma

2 Neojobertia 40 Amphilophium

45 Anemopaegma

3 Pyrostegia

11 Mansoa Tribe Bignonieae Tribe 28 Bignonia

10 Dolichandra Callichlamys 16 Tanaecium

69 Fridericia

6 Xylophragma

16 Cuspidaria

15 Tynanthus (Lohmann, 2006) 13 Lohmann Lundia

4 Pachyptera Manaosella (2006) (2006) 15 Pleonotoma

2 Martinella

3 Stizophyllum Perianthomega A NEW GENERIC Lu´cia G. Lohmann2,3,4 and Charlotte M. Taylor2 CLASSIFICATION OF TRIBE BIGNONIEAE (BIGNONIACEAE)1

ABSTRACT The history of classification of the tribe Bignonieae and its genera are reviewed as context for a comprehensive new genus-level classification of the tribe Bignonieae (Bignoniaceae, Lamiales). This new classification is based on a well-supported phylogeny based on multiple molecular markers from both chloroplast and nuclear DNA, a morphological survey, and a broad sampling of taxa. Genera are circumscribed here as clades that are well supported as monophyletic by molecular data and also recognizable by one or more morphological synapomorphies. Perianthomega Bureau ex Baill. is here transferred from Bignoniaceae tribe Tecomeae into Bignonieae, and 21 genera and a total of 393 species are recognized in Bignonieae: Adenocalymma Mart. ex Meisn. (82 species), Amphilophium Kunth (47), Anemopaegma Mart. ex Meisn. (45), Bignonia L. (28), Callichlamys Miq. (1), Cuspidaria DC. (19), Dolichandra Cham. (8), Fridericia Mart. (67), Lundia DC. (13), Manaosella J. C. Gomes (1), Mansoa DC. (12), Martinella Baill. (2), Neojobertia Baill. (2), Pachyptera DC. ex Meisn. (4), Perianthomega (1), Pleonotoma Miers (17), Pyrostegia C. Presl (2), Stizophyllum Miers (3), Tanaecium Sw. (17), Tynanthus Miers (15), and Xylophragma Sprague (7). Several genera are here circumscribed differently from previous classifications, in particular Memora Miers and Sampaiella J. C. Gomes are synonymized with Adenocalymma; Distictella Kuntze, Distictis Mart. ex Meisn., Glaziova Bureau, Pithecoctenium Mart. ex DC., and Urbanolophium Melch. are synonymized with Amphilophium; Cydista Miers, Clytostoma Miers ex Bureau, Macranthisiphon Bureau ex K. Schum., Mussatia Bureau ex Baill., Phryganocydia Mart. ex Bureau, Potamoganos Sandwith, Roentgenia Urb., and Saritaea Dugand are synonymized with Bignonia; Macfadyena A. DC., Melloa Bureau, and Parabignonia Bureau ex K. Schum. are synonymized with Dolichandra; Arrabidaea DC. is synonymized with Fridericia; Gardnerodoxa Sandwith is synonymized with Neojobertia; Leucocalantha Barb. Rodr. is synonymized with Pachyptera;andCeratophytum Pittier, Periarrabidaea A. Samp., Paragonia Bureau, Pseudocatalpa A. H. Gentry, and Spathicalyx J. C. Gomes are synonymized with Tanaecium. The genera Adenocalymma, Amphilophium, Fridericia, Dolichandra,andTanaecium are formally emended here as to diagnosis and circumscription. A natural key, complete morphological descriptions, and illustrations characterize the accepted genera, and full generic synonymy and a catalogue of their component species summarize their basic nomenclature and geographic range. Three new names are published: B. neouliginosa L. G. Lohmann replaces Phryganocydia uliginosa Dugand; B. neoheterophylla L. G. Lohmann replaces Cydista heterophylla Seibert; and Tanaecium neobrasiliense L. G. Lohmann replaces Sanhilaria brasiliensis Baill. Thirty-two generic names are newly synonymized, and 144 new nomenclatural combinations are made. A lectotype is designated for one genus, Periarrabidaea A. Samp., and 78 species names. One species name is neotypified, Memora campicola Pilg. ([ Adenocalymma campicola (Pilg.) L. G. Lohmann). Key words: Neotropical flora, Lamiales, Bignoniaceae, Bignonieae, Adenocalymma, Amphilophium, Anemopaegma, Arrabidaea, Bignonia, Dolichandra, Fridericia, Tanaecium.

1 L. G. L. is extremely grateful to her Ph.D. advisers Drs. Elizabeth A. Kellogg and Peter F. Stevens for all their help and support during this study. We thank Peter Raven, Mick Richardson, Bette Loiselle, Richard Olmstead, John Pruski, Amy Pool, Fred Barrie, Ivań Jimeńez, Olga Martha Montiel, Bob Magill, Alexandre Zuntini, Miriam Kaehler, Fabiana Firetti, Joao˜ Semir, and Maria Mercedes Arbo for their significant input and help, Roy Gereau for assistance with morphology and nomenclature, Carmen Ulloa Ulloa for her contributions to Bignonieae taxonomy for the Checklist of the World, and Victoria C. Hollowell for valient editing. Thanks also to Barbara Alongi for preparing line drawings, Alexandre Zuntini for great assistance with the preparation of figures, and Sara Fuentes for her help with SEM. We are indebted to the curators of various herbaria who made their collections available for this study, especially B, BM, BR, G, K, NY, P, M, MO, and W, and to the late W. G. D’Arcy for essential reference materials. Support for this study came from Ph.D. fellowships to L. G. L. from Conselho de Auxılio´ a` Pesquisa, Brazil (CAPES), the University of Missouri–St. Louis, the Missouri Botanical Garden, and the Compton Foundation; a Dissertation improvement grant from NSF (the National Science Foundation); research grants by the American Society of Plant Taxonomists, the Botanical Society of America, the Federated Garden Clubs of Missouri, Idea Wild, and the Whitney R. Harris World Ecology Center; postdoctoral support from the Center for Conservation and Sustainable Development of the Missouri Botanical Garden; a pq-2 grant from Conselho Nacional de Apoio a` Pesquisa, Brazil (CNPq), a regular research grant by Fundaçao˜ de Amparo a Pesquisa do Estado de Sao˜ Paulo, Brazil (FAPESP, 2011/50859-2), and a collaborative Dimensions of Biodiversity-BIOTA grant supported by FAPESP (2012/50260-6), National Science Foundation (NSF), and National Aeronautics and Space Administration (NASA). We are deeply and sincerely indebted to the late A. H. Gentry, whose work forms the basis of most of this study, and to whom we dedicate the present study. 2 Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, U.S.A.; [email protected]. 3 University of Missouri–St. Louis, Department of Biology, 8001 Natural Bridge Road, St. Louis, Missouri 63121, U.S.A. 4 Current address: Universidade de Sao˜ Paulo, Instituto de Biociencias,ˆ Departamento de Botanica,ˆ Rua do Matao,˜ 277, CEP 05508-090, Sao˜ Paulo, SP, Brazil; [email protected]. doi: 10.3417/2003187

ANN.MISSOURI BOT.GARD. 99: 348–489. PUBLISHED ON 15 MAY 2014. Next-generation sequencing using an Illumina HiSeq Platform

- Extracted total genomic DNA from herbarium specimens

- DNA was fragmented to construct short-insert libraries (~300 pb) with NEBNext DNA Library Prep Master Mix and NEBNext Multiplex oligos

- Libraries were quantified using qPCR with a Kappa Library Quantification Kit

- 21 species were pooled together in one lane of an Illumina HiSeq 2000 system for sequencing

- 4 libraries were sequenced (= 84 species)

mit_500-1600-psbA-mit Sequencher™ "Ade_cymbalum.SPF"

cpDNA and mtDNA Genome Assembly

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For cp genome we used Olea europeae as reference +",-"./012/ 89:;9/0120// 34567!"/ <)<",)$"/ 1" For mt genome We used Mimulus guttatus as reference

T G C A T A T T Contigs were joined into larger T G C A T A T T A T A C G T C AT T C T A A A G A T A A G G T G C A T A T T A T A C G T C A T T C T A A AcontigsG A T A A G G A inA G Sequencher 5.3.2 T G C A T A T T A T A C G T C A T T C T A T G C A T AT T A T A C G T C A T T C T A A A G A T A A G G A A G G G C T A A A G G T A A G G C G G G T G C A T AT T A T A C G T C A T T C T A A A G A T A A G G A A G G G C T A A A G G T A A G G C G G G T G C A T A T T A T A C G T C A T T C T A A A G A T A A G G A A G G G C T A A A G G T A A G G C G G G T G C A T A T T A T A C G T C A T T C T A A A G A T A A G G A A G G G C T A A A G G T A A G G C G G G T T A A G T T A G G G C A T C A G T T G C T T C 2 8 0 | 2 9 0 | 3 0 0 | 3 1 0 | 3 2 0 | 3 3 0 | 3 4 0 | 3 5 0 | 3 6 0 | 3 7 0 | 3 8 0 | 3 9 0

T G C A T A T T A T A C G T C A T T C T A A A G A T A A G G A A G G G C T A A A G G T A A G G C G G G T T A A G T T A G G G C A T C A G T T G C T T C

08 de maio de 2014 Page 6 of 62 Genome Annotation

Chloroplast genome Initial annotation was performed using Dual Organellar GenoMe Annotator (DOGMA; Wyman et al., 2004);

Mitochondrial genome We used MITOFY program to search/blast in databases for protein- coding genes, tRNAs, and rRNAs genes known from seed plant mitochondrial genomes. !"#$%&&&% #'()*+,-.(,%,/'%01)2')2-34/1(%/1+,)-5%)6%"738)*13*%,393

D3E'.83'.F3=,,B38',38,6&*-.)3'7F8*-(3*G3#6.H*B7.B3=7*I7J,-F78(K

D3E'.83'.F3=,,B38',3=7*+,*+-.&'7<3'7F8*-(3*G3#6.H*B7.L3.F37BG,--,I3G-*63 &'()*+,B,M<3M6,D8-,,3.BI3=7*+,*+-.&'7<3.B.)(F,FK Broad scale patterns

(Lohmann et al. 2013) 1.0 Fridericia trailii 1.0 Fridericia speciosa Fridericia nigrescens 0.96 Fridericia pubescens Fridericia subincana 1.0 Fridericia cinerea Fridericia rego Fridericia conjugata 0.98 Fridericia spicata 1.0 Fridericia cinnamomea 1.0 Fridericia chica Fridericia dispar 0.99 Fridericia erubescens 1.0 Fridericia patellifera Fridericia leucopogon 1.0 1.0 Fridericia triplinervia Fridericia oligantha 0.95 1.0 Fridericia prancei Fridericia platyphylla 1.0 Xylophragma myrianthum Xylophragma harleyi 1.0 Lundia corymbifera 0.98 Lundia longa 1.0 Lundia nitidula 1.0 Lundia densiflora Lundia spruceana 1.0 1.0 Cuspidaria cinerea 1.0 Cuspidaria sceptrum Fridericia Cuspidaria inaequalis 0.95 and allies 1.0 Cuspidaria subincana 1.0 Cuspidaria convoluta clade 1.0 Cuspidaria lateriflora 0.99 Cuspidaria floribunda 1.0 Tynanthus polyanthus 1.0 1.0 Tynanthus villosus 1.0 Tynanthus elegans B Tynanthus panurensis 1.0 Tanaecium affine 0.95 Tanaecium tetragonolobum Tanaecium truncatum 0.99 Tanaecium pyramidatum 0.95 Tanaecium caudiculatum 1.0 Tanaecium bilabiatum 0.96 Tanaecium revillae Tanaecium selloi 1.0 Tanaecium crucigerum Pachyptera aromatica 0.95 Pleonotoma stichadenia 1.0 Pleonotoma jasminifolia Pleonotoma longiflora Manaosella cordifolia 1.0 Anemopaegma floridum 1.0 Anemopaegma foetidum 1.0 Anemopaegma laeve 1.0 Anemopaegma robustum 1.0 Pyrostegia dichotoma Pyrostegia venusta 1.0 1.0 Mansoa difficilis 1.0 Mansoa lanceolata 1.0 Mansoa hirsuta 1.0 Mansoa verrucifera Mansoa standleyi 1.0 Bignonia hyacinthina 0.98 0.99 Bignonia prieurei 1.0 Bignonia corymbosa Multiples Bignonia microcalyx 0.91 Bignonia aequinoctialis of four clade 1.0 1.0 Bignonia callistegioides Core Bignonia uleana Bignonieae 1.0 Bignonia bracteomana Bignonia capreolata clade 1.0 Bignonia nocturna 1.0 Amphilophium aschersonii 1.0 Amphilophium paniculatum Amphilophium rodriguesii 1.0 Amphilophium nunezii 0.99 1.0 Amphilophium bauhinioides 0.98 Amphilophium frutescens Amphilophium granulosum 1.0 Amphilophium crucigerum Amphilophium lohmanniae 1.0 Amphilophium elongatum Amphilophium magnoliifolium 1.0 Dolichandra steyermarkii 0.98 Dolichandra unguis-cati 1.0 Dolichandra quadrivalvis Dolichandra cynanchoides 1.0 1.0 Martinella obovata Martinella iquitoensis 1.0 Stizophyllum perforatum Stizophyllum inaequilaterum 1.0 Adenocalymma campicola Bignonieae 1.0 Adenocalymma adenophorum 1.0 Adenocalymma moringifolium Adenocalymma-Neojobertia 1.0 Adenocalymma magnificum 1.0 Adenocalymma bracteosum clade 1.0 Adenocalymma trichocladum 1.0 Adenocalymma salmoneum 1.0 1.0 Adenocalymma bracteatum Adenocalymma subincanum 1.0 1.0 1.0 Adenocalymma cymbalum Adenocalymma impressum 1.0 Neojobertia candolleana 1.0 Neojobertia mirabilis Perianthomega vellozoi Tabebuia sauvallei Tecoma capensis A Jacaranda arborea

60 50 40 30 20 10 Fridericia trailii A: eastern South America Fridericia speciosa Fridericia nigrescens Text B: South American dry areas Fridericia pubescens Fridericia subincana Fridericia cinerea C: lowland Amazonia Fridericia rego Fridericia conjugata D: western South America and Central America Fridericia spicata Text Fridericia cinnamomea E: North America Fridericia chica Fridericia dispar Fridericia erubescens AB Fridericia patellifera Fridericia leucopogon ABC Fridericia triplinervia Fridericia oligantha ABCD Fridericia prancei Fridericia platyphylla Xylophragma myrianthum AC Xylophragma harleyi Lundia corymbifera ACD Lundia longa Lundia nitidula BC Lundia densiflora Lundia spruceana Cuspidaria cinerea BCD Fridericia Cuspidaria sceptrum Cuspidaria inaequalis CD and allies Cuspidaria subincana clade Cuspidaria convoluta CE Cuspidaria lateriflora Cuspidaria floribunda Tynanthus polyanthus Tynanthus villosus Tynanthus elegans Tynanthus panurensis Tanaecium affine Tanaecium tetragonolobum Tanaecium truncatum Tanaecium pyramidatum Tanaecium caudiculatum Tanaecium bilabiatum Tanaecium revillae Tanaecium selloi Tanaecium crucigerum Pachyptera aromatica Pleonotoma stichadenia Pleonotoma jasminifolia Pleonotoma longiflora Manaosella cordifolia Anemopaegma floridum Anemopaegma foetidum Anemopaegma laeve Anemopaegma robustum Pyrostegia dichotoma Pyrostegia venusta Mansoa difficilis Mansoa lanceolata Mansoa hirsuta 5 Mansoa verrucifera Mansoa standleyi Bignonia hyacinthina Bignonia prieurei Bignonia corymbosa Multiples Bignonia microcalyx Bignonia aequinoctialis of four clade Bignonia callistegioides Core Bignonia uleana Bignonieae Bignonia bracteomana Bignonia capreolata clade Bignonia nocturna Amphilophium aschersonii Amphilophium paniculatum Amphilophium rodriguesii Amphilophium nunezii Amphilophium bauhinioides 4 Amphilophium frutescens Amphilophium granulosum Amphilophium crucigerum Amphilophium lohmanniae Amphilophium elongatum Amphilophium magnoliifolium Dolichandra steyermarkii Dolichandra unguis-cati Dolichandra quadrivalvis 2 Dolichandra cynanchoides Martinella obovata 6 Martinella iquitoensis Stizophyllum perforatum Stizophyllum inaequilaterum Bignonieae Adenocalymma campicola Adenocalymma adenophorum Adenocalymma moringifolium Adenocalymma magnificum Adenocalymma-Neojobertia Adenocalymma bracteosum clade Adenocalymma trichocladum Adenocalymma salmoneum 1 Adenocalymma bracteatum Adenocalymma subincanum Adenocalymma cymbalum Adenocalymma impressum Neojobertia candolleana 3 Neojobertia mirabilis Perianthomega vellozoi

50 40 30 20 10 0 Thraupis cyanocephala Emberiza schoeniclus Calcarius lapponicus Fringilla montifringilla How old is the South American avifauna? Motacilla cinerea Passer montanus Taeniopygia guttata Ploceus cucullatus Prunella collaris Dicaeum aeneum Regulus calendula Bombycilla garrulus Irena cyanogaster Nectarinia olivacea Muscicapa ferruginea Catharus ustulatus Cinclus cinclus Mimus patagonicus Sturnus vulgaris Troglodytes aedon Certhia familiaris Sitta carolinensis Sylvia nana Pycnonotus barbatus Zosterops senegalensis Acrocephalus newtoni Hirundo rustica Aegithalos iouschensis Cisticola anonymus Megalurus palustris Sphenoeacus afer Alauda arvensis Nicator chloris Parus major Remiz pendulinus Elminia nigromitratus Promerops cafer Petroica cucullata Monarcha axillaris Melampitta lugubris Struthidea cinerea • Birds have been in South Paradisaea raggiana Manucodia atra Cyanocitta cristata Lanius excubitor Dicrurus adsimilis Rhipidura hyperthra Artamus leucorhynchus Cracticus quoyi Vanga curvirostris Prionops plumatus Telophorus dohertyi Aegithina tiphia Batis mixta Oriolus xanthonotus Pachycephala hyperthra Ptilorrhoa caerulescens Paramythia montium Coracina novaehollandiae America’s warm & wet forest Daphoenositta chrysoptera Vireo philadelphia Melanocharis nigra Mohoua albicilla Philesturnus carunculatus Picathartes gymnocephalus Chaetops frenatus Pomatostomus isidorei Orthonyx teminckii Pardalotus punctatus South America Malurus melanocephalus Meliphaga analoga Ptilonorhynchus violaceus Climacteris erythrops Atrichornis clamosus Menura novaehollandiae Furnarius rufus Sclerurus mexicanus environments for a very long Formicarius colma Grallaria ruficapilla Scytalopus magellanicus Conopophaga ardesiaca Melanopareia torquata Terenura sharpei Thamnophilus nigrocinereus Platyrinchus coronatus Piprites chloris Oxyruncus cristatus Rhynchocyclus brevirostris Cotinga cayana Onychorhynchus coronatus Tityra semifasciata Pipra coronata Tyrannus tyrannus Philepitta castanea Psarisomus dalhousiae time Smithornis rufolateralis Sapayoa aenigma Pitta sordida Acanthisitta chloris Alisterus scapularis Psittacus erithacus Myiopsitta monachus Calyptorhynchus funereus Nestor notabilis Halcyornithidae Falco peregrinus Daptrius ater Micrastur gilvicollis Phororhacoidea Idiornithidae Cariama cristata Pteroglossus aracari Capito niger Semnornis frantzii Trachyphonus erythrocephalus Lybius hirsutus Megalaima orti Melanerpes carolinus Picumnus cirratus Indicator variegatus Bucco capensis Galbula albirostris • Higher taxa tend to show Chloroceryle americana Halcyon malimbica Alcedo leucogaster Momotus momota Palaeotodus Todus angustirostris Coracias caudata Brachypteracias lepto Primobucco Eocoracias Paracoracias Merops pusillus Upupa epops Phoeniculus purpureus Messelirrisor Buceros bicornis Tockus camurus Primotrogon deep origins of stem-lineages Trogon personatus Plesiocathartes Leptosomus discolor Sandcoleidae Chascacocolius Colius colius Phodilus badius Tyto alba Strix occidentalis Ninox novaeseelandiae Buteo jamaicensis Elanus caeruleus Pandion haliaetus Sagittarius serpentarius Cathartes aura Balaeniceps rex Pelecanus erythrorynchos Egretta tricolor Rhynchaeitinae Theristicus melanopis Ciconia abdimii Phalacrocorax Anhinga anhinga Sula sula Fregata minor Puffinus creatopus Pelecanoides magellani • However, ages of genera are Oceanites oceanicus Thalassarche bulleri Spheniscus humboldti Aptenodytes forsteri Gavia immer Eurypyga helias Rhynochetus jubatus Phaethon lepturus Alca torda Catharacta skua Larus marinus Stiltia isabella Turnix Jacana jacana Rostratula benghalensis Thinocorus orbignyianus Scolopax rusticola Recurvirostra americana not good metrics for Haematopus ater Charadrius vociferus Pluvianus aegyptius Chionis minor Burhinus capensis Sarothrura insularis Heliornis fulica Rallus limicola Grus canadensis Psophia crepitans Namibiavis Protoazin Opisthocomus hoazin Colibri coruscans Florisuga mellivora Phaethornis griseogularis Eurotrochilus diversification dynamics Chaetura pelagica Streptoprocne zonaris Hemiprocne mystacea Aegotheles insignis Prefica Steatornis caripensis Paraprefica Nyctibius grandis Caprimulgus longirostris Eurostopodus macrotis Batrachostomus septimus Podargus strigoides Fluvioviridavidae Coua cristata Centropus phasianinus Coccyzus americanus Guira guira Otis tarda Tauraco erythrolophus Mesitornis unicolor Pterocles personatus Treron calva Columba livia Podiceps cristatus Tachybaptus ruficollis Phoenicopterus ruber Bonasa umbellus • The question is: “How old is Gallus gallus Rollulus rouloul Callipepla californica Guttera pucherani Crax blumembachii Leipoa ocellata Megapodius freycinet Gallinuloididae Anas platyrhynchos Anser albifrons Dendrocygna arborea Presbyornithidae Anseranas semipalmata Chauna torquata Casuarius casuarius Dromaius novaehollandiae Aepyornithidae Apteryx australis the modern diversity?” Nothoprocta Tinamus guttatus Dinornithidae Lithornithidae Rhea americana Struthio sp. Struthio camelus

80 60 40 20 0 Finer scale patterns Biogeographic patterns of Psophia

<2-,E0%2M,E '.%,0N'%,"&.O'-+%

(Ribas, Aleixo, Nogueira, Miyaki & Cracraft, 2012) Species-level 3.0-2.7 Myr 2.7-2.0 Myr taxa are young

2.0-2.1 Myr 1.3-0.8 Myr

1.0-0.7 Myr 0.8-0.3 Myr

(Ribas et al. 2012) ?7*3:,+-*[email protected],-B L)IG*&=+"K Primates 6(J&=+"K

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/(I$> =$+"8> Journal of Biogeography (J. Biogeogr.) (2014)

GUEST Timing the diversiﬁcation of the EDITORIAL Amazonian biota: butterﬂy divergences are consistent with Pleistocene refugia Ivonne J. Garzon-Ordu na*,~ Jennifer E. Benetti-Longhini and Andrew V. Z. Brower

Evolution and Ecology Group, Department of ABSTRACT Biology, Middle Tennessee State University, Rejection of the Pleistocene refugium hypothesis (PRH) as an explanation for Murfreesboro, TN 37132, USA the high biodiversity of Neotropical forest is based in part on the assertion that biotic elements of these forests evolved during the Neogene. That argument is justified, in turn, by the ages of crown groups (the age of the most recent com- mon ancestor of extant species of a clade). We consider the use of crown ages as a metric to reject the PRH to be an unfair test, because the circumscription of crown groups of interest is arbitrary, and their ages represent overestimates of the time of species formation. We present divergence times between pairs of comparison), the results of divergence times sister species (131 pairs), and among pairs of sister species and their closest rel- for selected species representing 35 genera ative (56 triplets), from 35 genera of Neotropical butterflies. Our aim is to refocus the discussion about the timing of diversification of the Neotropical biota on the time of the formation of extant species, a metric that is consistent and comparable across taxa. Our results show that 72% of speciation events leading to the formation of butterfly sister species occurred within the last 2.6 Myr, a result consistent with the temporal predictions of the PRH, suggesting that the PRH cannot be completely Garzón discarded–Orduña as a & driver Brower of Neotropical 2014 diversification.

*Correspondence: Ivonne J. Garzon-Ordu na,~ Keywords Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132, USA. Amazonia, biodiversity, crown ages, diversiﬁcation, Lepidoptera, mitochon- E-mail: [email protected] drial DNA, Neogene, Neotropics, Pleistocene refugium hypothesis.

Pebas), shifts in the courses and watersheds of major rivers, INTRODUCTION and the subsequent establishment of terrestrial conditions. The reasons for the enormous numbers of species hosted by Authors such as Hoorn et al. (2010) have argued that this Neotropical forests intrigued 19th-century naturalists, and tectonic activity caused changes in the landscape that pro- still puzzle systematists, ecologists, geologists and palaeontol- vided biogeographical opportunities for new species interac- ogists today. There are two contrasting positions regarding tions, and generated new adaptive pressures that triggered the patterns and timing of biotic diversification in the Neo- speciation. According to this scenario, most physical barriers, tropics in evolutionary time: one that emphasizes Neogene such as mountains and rivers, were in their current positions (23–2.6 Ma) vicariance events as a result of major rearrange- by the end of the Pliocene (2.6 Ma), and therefore vicariant ments of the Amazonian landscape, and a second that points speciation events caused by those barriers must have to Pleistocene (< 2.6 Ma) climatic cycles as an engine of occurred earlier, implying that most current sister species diversification. While controversy over timing may not seem diverged prior to 2.6 Ma (but see Ribas et al., 2012). to be a biogeographical issue per se, the abiotic processes In contrast, Haffer’s (1969) Pleistocene refugium hypothe- that could explain biotic distributions differ between these sis (PRH) suggests that many extant Neotropical species two time periods in fundamental ways. Therefore, inferring originated after the Neogene (< 2.6 Ma; Cohen et al., 2013) when diversification took place points to which geological as a result of environmental fluctuations driven by repeated and/or climatic mechanisms may have been involved. cycles of global cooling and warming. The PRH proposes At the end of the Tertiary, the Neogene (23–2.6 Ma) was that cold spells during the Pleistocene caused the fragmenta- a period of dramatic geological events in the Neotropics, tion and replacement of moist Amazonian forest by drier such as the uplift of the Andes, the formation of a large grass savannas, isolating populations of forest obligate taxa, lacustrine system in what is today western Amazonia (Lake allowing allopatric differentiation and ultimately driving an

ª 2014 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1 doi:10.1111/jbi.12330 Crown nodes with ages within the Neogene Neogene

(Hoorn et al. 2010.) New molecular data favor a more recent origin for the formation of the Amazonian Biota PART IV: Characterization of the geological and environmental history of Amazonia

- What has been the history of environmental change across Amazonia from the late Neogene to present?

- When did the Amazonian river drainage form?

- What was the Amazonian landscape like before the Amazon river formed and how did it change after the river formation?

The Refugia hypothesis suggests that South America was dry during the Pleistocene

Haffer et al. 1997 Prance et al. 1982

!"#$%&'()*+,-.&/*),+0+$-1".&2+0"3) (Cheng et al. 2013) Speleothem record and Amazonian climate suggests different forest corridors at different times

- Climate has varied differently in time in eastern and western Amazonia

5 to 9 My DRY WET Present

WET - West has suffered less Last 5 My and LGM drought in the last 4MY DRY History of the Amazonian drainage When did the current drainage system develop?

Upper Miocene: 10 to 8 mya (Hoorn et al. 1995, Lundberg et al. 1995)

Upper Pliocene/Pleistocene: less than 2.5 mya (Rossetti et al. 2005, Campbell et al. 2006, Nogueira, 2008, Latrubesse et al. 2010)

New geological data favor a more recent origin for the formation of the Amazonian drainage PART IV: Integrative studies

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D3P*Q3'.F38',3#6.H*B7.B3,BJ7-*B6,B83.BI378F3=7*8.3,J*)J,I38*+,8',-K http://amazoniabiodiversity.com/ Summary

• PART I. Compiled large data-bases with ca. 100.000 geo- referenced records of plants and vertebrates;

• PART II. Have reconstructed the phylogeny of ca. 20 different clades using Sanger sequencing. Have extracted DNA and generated substantial amounts of NGS sequence data that are now being used in new phylogenetic and phylogeographic studies;

• PART III. Molecular dating favors a recent origin for Amazonian forest species;

• PART IV. New geological data favor a more recent origin for the formation of the Amazonian drainage, as well as the formation of different forest corridors at different times;

• PART V. The integration of new geological and biological data are telling us similar stories about the past history of Amazonia