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 2012/50260-6NSF 1241066

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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

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Meeting these scientific challenge calls for 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:

Primates = 6.000 records(2.000 georeferrenced)

Birds = 170.000 records(31.000 georeferrenced)

Collaborating Institutions

• AMNH hold ca. 67.000 specimens

• FMNH holds ca. 58.000

• INPA, Museu Goeldi, MZUSP & other Brazilian collaborators

Arrabidaearego

Arrabidaeachica

Arrabidaeaaffinis

Anemopaegmalaeve

Amphilophiumpaniculatum

Adenocalymmabracteatum

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 IIPhylogenetic 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

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Phylogenetic relationships of the butterfly family Nymphalidae based on nDNA & mtDNA data

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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

Horácio Schneider &Iracilda Sampaio (UFPA)

Jean Boubli(University of Salford, UK)

Phylogeny of the New World Titi Monkeys (Callicebus)

J. Boubli et al. (2014, MPE)

• Sampling• 15 species• 73 individuals

Putting our results into perspective

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Cebus – Sapajus 6 3.13 9.35

Cacajao -Chiropotes 6.91 4.56 9.34

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

Jean P. Boubli a,b,!, Camila Ribas b, Jessica W. Lynch Alfaro c,d, Michael E. Alfaro c,e,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, UKb Instituto Nacional de Pesquisas da Amazonia INPA, Manaus, Brazilc Institute for Society and Genetics, 1321 Rolfe Hall, University of California, Los Angeles, CA 90095, USAdDepartment of Anthropology, University of California, Los Angeles, CA 90095, USAeDepartment of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095, USAfUniversidade Federal do Amazonas, Laboratório de Evolução e Genética Animal, Manaus, AM, Brazil

a r t i c l e i n f o

Article history:Received 1 October 2013Revised 27 August 2014Accepted 9 September 2014Available online xxxx

Keywords:AmazoniaPlatyrrhiniPhylogeographyRiver barrierRiverine hypothesisVicariance

a b s t r a c t

The role of Amazonian rivers as drivers of speciation through vicariance remains controversial. Here weexplore the riverine hypothesis by comparing spatial and temporal concordances in pattern of diversifi-cation for all diurnal primates of Rio Negro and its largest tributary, Rio Branco. We built a comprehensivecomparative phylogenetic timetree to identify sister lineages of primates based on mitochondrial cyto-chrome b DNA sequences from 94 samples, including 19 of the 20 species of diurnal primates fromour study region and 17 related taxa from elsewhere. Of the ten primate genera found in this region, threehad populations on opposite banks of Rio Negro that formed reciprocally monophyletic clades, withroughly similar divergence times (Cebus: 1.85 Ma, HPD 95% 1.19–2.62; Callicebus: 0.83 Ma HPD 95%0.36–1.32, Cacajao: 1.09 Ma, 95% HPD 0.58–1.77). This also coincided with time of divergence of severalallopatric species of Amazonian birds separated by this river as reported by other authors. Our data offersupport for the riverine hypothesis and for a Plio-Pleistocene time of origin for Amazonian drainage sys-tem. We showed that Rio Branco was an important geographical barrier, limiting the distribution of sixprimate genera: Cacajao, Callicebus, Cebus to the west and Pithecia, Saguinus, Sapajus to the east. The roleof this river as a vicariant agent however, was less clear. For example, Chiropotes sagulata on the left bankof the Rio Branco formed a clade with C. chiropotes from the Amazonas Department of Venezuela, north ofRio Branco headwaters, with C. israelita on the right bank of the Rio Branco as the sister taxon to C. chi-ropotes + C. sagulata. Although we showed that the formation of the Rio Negro was important in drivingdiversification in some of our studied taxa, future studies including more extensive sampling of markersacross the genome would help determine what processes contributed to the evolutionary history of theremaining primate genera.

! 2014 Elsevier Inc. All rights reserved.

1. Introduction

Of the more than 685 taxa (species and subspecies) of recog-nized primates (Mittermeier et al., 2013) approximately one third(164 taxa, 20 genera, 5 families; Paglia et al., 2012) are found in the

New World, with the greatest concentration in the Amazon Basin.The origins of such high species diversity remain poorly under-stood. One of the first proponents of a mechanism to account forthe high primate species diversity in Amazonia was the British nat-uralist Alfred R. Wallace. While on a collecting expedition to Brazilin the mid 19th century, Wallace noticed that primate species onopposite banks of large Amazonian rivers substituted one another(Wallace, 1852). Based on that observation he proposed that theserivers acted as barriers to the dispersal of animals. In particular, heproposed that the three largest Amazonian rivers (the Amazon,Madeira and Negro), divided the region into four districts charac-

http://dx.doi.org/10.1016/j.ympev.2014.09.0051055-7903/! 2014 Elsevier Inc. All rights reserved.

! Corresponding author at: School of Environment and Life Sciences, Room 315,Peel Building, University of Salford, Salford M5 4WT, UK.

E-mail addresses: j.p.boubli@salford.ac.uk, jeanpboubli@gmail.com (J.P. Boubli),camilaribas@gmail.com (C. Ribas), jlynchalfaro@ucla.edu (J.W. Lynch Alfaro),michaelalfaro@ucla.edu (M.E. Alfaro), mndasilva@gmail.com (M.N.F. da Silva),gabriela.m.pinho@gmail.com (G.M. Pinho), izeni@evoamazon.net (I.P. Farias).

Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

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

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 alldiurnal primates of Rio Negro and Rio Branco in Brazil. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.09.005

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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)

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- Highly conserved DNA elements shared among evolutionary distant taxa

- Useful for reconstructing the evolutionary history and population-level relationships of many organisms

- 32 samples sequenced, over 500 loci recovered and used in pilot phylogenetic analyses

- 246 samples that are ready to be sequenced by Rapid Genomics (Florida, USA)

!!

A Phylogeny of Birds Based on Over 1,500 Loci Collectedby Target Enrichment and High-Throughput SequencingJohn E. McCormack1*, Michael G. Harvey1,2, Brant C. Faircloth3, Nicholas G. Crawford4, Travis C. Glenn5,

Robb T. Brumfield1,2

1Museum of Natural Science, Louisiana State University, Baton Rouge, Louisiana, United States of America, 2Department of Biological Sciences, Louisiana State

University, Baton Rouge, Louisiana, United States of America, 3Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles,

California, United States of America, 4Department of Biology, Boston University, Boston, Massachusetts, United States of America, 5Department of Environmental Health

Science, University of Georgia, Athens, Georgia, United States of America

Abstract

Evolutionary relationships among birds in Neoaves, the clade comprising the vast majority of avian diversity, have vexedsystematists due to the ancient, rapid radiation of numerous lineages. We applied a new phylogenomic approach to resolverelationships in Neoaves using target enrichment (sequence capture) and high-throughput sequencing of ultraconservedelements (UCEs) in avian genomes. We collected sequence data from UCE loci for 32 members of Neoaves and oneoutgroup (chicken) and analyzed data sets that differed in their amount of missing data. An alignment of 1,541 loci thatallowed missing data was 87% complete and resulted in a highly resolved phylogeny with broad agreement between theBayesian 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 ledto increased stability and resolution of the tree. Novel results of our study include surprisingly close relationships betweenphenotypically divergent bird families, such as tropicbirds (Phaethontidae) and the sunbittern (Eurypygidae) as well asbetween bustards (Otididae) and turacos (Musophagidae). This phylogeny bolsters support for monophyletic waterbird andlandbird clades and also strongly supports controversial results from previous studies, including the sister relationshipbetween passerines and parrots and the non-monophyly of raptorial birds in the hawk and falcon families. Althoughsignificant challenges remain to fully resolving some of the deep relationships in Neoaves, especially among lineagesoutside the waterbirds and landbirds, this study suggests that increased data will yield an increasingly resolved avianphylogeny.

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 TargetEnrichment 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: ! 2013 McCormack et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by NSF grant DEB-0841729 to RTB. DEB-1242260 to BCF and TCG and an Amazon Web Services grant to BCF, NGC, JEM,and TCG provided partial support for computational analyses. The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: mccormack@oxy.edu

Introduction

The diversification of modern birds occurred extremely rapidly,with all major orders and most families becoming distinct within ashort window of 0.5 to 5 million years around the Cretaceous-Tertiary boundary [1–4]. As with other cases of ancient, rapidradiation, resolving deep evolutionary relationships in birds hasposed a significant challenge. Some authors have hypothesizedthat the initial splits within Neoaves might be a hard polytomy thatwill remain irresolvable even with expanded data sets (reviewed in[5]). However, several recent studies have suggested that expandedgenomic and taxonomic coverage will lead to an increasinglyresolved avian tree of life [2,6,7].Using DNA sequence data to reconstruct rapid radiations like

the Neoaves phylogeny presents a practical challenge on severalfronts. First, short speciation intervals provide little time forsubstitutions to accrue on internal branches, reducing thephylogenetic signal for rapid speciation events. Traditionally, thesolution to this problem has been to collect additional sequencedata, preferably from a rapidly evolving molecular marker such as

mitochondrial DNA [8]. However, rapidly evolving markersintroduce a new set of problems to the inference of ancientradiations: through time, substitutions across rapidly evolvingmarkers overwrite older substitutions, resulting in signal saturationand homoplasy [9]. To address this challenge, some researchershave inferred ancient phylogeny using rare genomic changes, likeretroposon insertions and indels, because rare changes are unlikelyto occur in the same way multiple times, thereby minimizinghomoplasy [10,11]. Though successful in some cases [12],retroposons are often insufficiently numerous to fully resolverelationships between taxa that rapidly radiated [13], andalthough often billed as being homoplasy-free, we now know thatshared retroposon insertions can be due to independent events[14].A second challenge to reconstructing ancient, rapid radiations is

the randomness inherent to the process of gene sorting (i.e.,coalescent stochasticity), which occurs even when gene historiesare estimated with 100% accuracy [15]. The amount of conflictamong gene-tree topologies due to coalescent stochasticityincreases as speciation intervals get shorter [16]. Hemiplasy refers

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

using the STAR (Species Trees from Average Ranks ofCoalescences) method [48]. We performed 1,000 multi-locus,non-parametric bootstrap replicates for the STAR tree byresampling nucleotides within loci as well as resampling lociwithin the data set [49]. We only performed the species treeanalysis on the alignment with no missing data due to concernsabout how missing loci might affect a coalescent analysis.To assess phylogenetically informative indels, we scanned

alignments by eye in Geneious 5.4 (Biomatters Ltd, Aukland,New Zealand), recording indels that were 2 bp or more in lengthand shared between two or more ingroup taxa. We then mappedinformative indels onto the resolved 416-locus Bayesian phylog-eny.

Results

We provide summary statistics for sequencing and alignment inTable 1. We obtained an average of 2.6 million reads per sample(range = 1.1–4.9 million). These reads assembled into an averageof 1,830 contigs per sample (range = 742–2,418). An average (persample) of 1,412 of these contigs matched the UCE loci fromwhich we designed target capture probes (range = 694–1,681).The average length of UCE-matching contigs was 429 base pairs(bp) (range = 244–598), and the average coverage of UCE-matching contigs was 71 times (range = 44–138). The percentageof original sequencing reads that were ‘‘on target’’ (i.e., helpedbuild UCE-matching contigs) averaged 24% across samples (range= 15% - 35%).When we selected loci allowing 50% of species for a given locus

to have missing data, the final data set contained 1,541 UCE lociand produced a concatenated alignment that was 87% completeacross 32 Neoaves species and the chicken outgroup. The averagelength of these 1,541 loci was 350 bp (min= 90, max= 621), andthe total concatenated alignment length was 539,526 characters(including indels) with 24,703 informative sites.

Generally, the Bayesian and ML phylogenies for the 1,541 locusalignment were similar in their topology and amount of resolution(Fig. 2a; see Fig. S1 for fully resolved trees). Of the 31 nodes, 27(87%) were highly supported in the Bayesian tree (.0.95 PP),whereas a subset of 20 of those nodes (65%) were also highlysupported in the ML tree (.75% bootstrap score). An additional 7nodes (23%) appeared in both the Bayesian and ML trees, butsupport in the ML tree was low (bisected nodes in Fig. 2a). Fournodes (16%) had either low support in both trees (and thus arecollapsed in Fig. 2a) or had high support in the Bayesian tree, butdid not appear in the ML tree (white nodes in Fig. 2a). Aphylogram for the 1,541 locus Bayesian tree (Fig. S2) showed longterminal branches and short internodes near the base of the tree,consistent with previous studies suggesting an ancient, rapidradiation of Neoaves.For the data set requiring no missing data, we recovered

416 UCE loci across 29 Neoaves species and the chickenoutgroup. Enrichments for three species performed relativelypoorly (Table 1; Micrastur, Trogon, and Vidua), and we excludedthese samples to boost the number of loci recovered. The averagelength of these 416 loci was 397 bp, and the total concatenatedalignment length was 165,163 characters (including indels) with7,600 informative sites. Bayesian and ML trees differed more intheir topology and resolution than was observed for the 1,541locus trees above (Fig. 2b; see Fig. S3 for fully resolved trees). Ofthe 28 nodes, 24 (86%) were highly supported in the Bayesian tree(.0.95 PP), whereas only a subset of 14 (50%) was highlysupported in the ML tree (.75% bootstrap score). We recoveredan additional three nodes (11%) in both the Bayesian and MLtrees, but support for these nodes in the ML tree was low (bisectednodes in Fig. 2b). Twelve nodes (43%) disagreed between theBayesian and ML trees, a frequency much higher than the 16%disagreement we observed from the 1,541 locus analysis.The STAR species tree from the 416 locus data set (Fig. 3; Fig.

S3c) was much less resolved and had lower support values thaneither the Bayesian or ML tree estimated for these data. There has

Figure 1. Neoaves species used in this study. Species are listed in Table 1. Numbers match those in table and on the tips of phylogenies.Illustrations are based on photos (see Acknowledgments).doi:10.1371/journal.pone.0054848.g001

A Phylogeny of Birds from 1,500 Loci

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

Bertholletia excelsa

Lecythidaceae

Scott Mori (NYBG) & Chris Dick

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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

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(Lohmann, 2006)

A NEW GENERIC Lucia G. Lohmann2,3,4 and Charlotte M. Taylor2

CLASSIFICATION OF TRIBEBIGNONIEAE (BIGNONIACEAE)1

ABSTRACT

The history of classification of the tribe Bignonieae and its genera are reviewed as context for a comprehensive new genus-levelclassification of the tribe Bignonieae (Bignoniaceae, Lamiales). This new classification is based on a well-supported phylogenybased on multiple molecular markers from both chloroplast and nuclear DNA, a morphological survey, and a broad sampling oftaxa. Genera are circumscribed here as clades that are well supported as monophyletic by molecular data and also recognizable byone or more morphological synapomorphies. Perianthomega Bureau ex Baill. is here transferred from Bignoniaceae tribe Tecomeaeinto Bignonieae, and 21 genera and a total of 393 species are recognized in Bignonieae: Adenocalymma Mart. ex Meisn. (82species), 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), MartinellaBaill. (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 herecircumscribed differently from previous classifications, in particular Memora Miers and Sampaiella J. C. Gomes are synonymizedwith Adenocalymma; Distictella Kuntze, Distictis Mart. ex Meisn., Glaziova Bureau, Pithecoctenium Mart. ex DC., andUrbanolophium Melch. are synonymized with Amphilophium; Cydista Miers, Clytostoma Miers ex Bureau, Macranthisiphon Bureauex K. Schum., Mussatia Bureau ex Baill., Phryganocydia Mart. ex Bureau, Potamoganos Sandwith, Roentgenia Urb., and SaritaeaDugand are synonymized with Bignonia; Macfadyena A. DC., Melloa Bureau, and Parabignonia Bureau ex K. Schum. aresynonymized with Dolichandra; Arrabidaea DC. is synonymized with Fridericia; Gardnerodoxa Sandwith is synonymized withNeojobertia; Leucocalantha Barb. Rodr. is synonymized with Pachyptera; and Ceratophytum Pittier, Periarrabidaea A. Samp.,Paragonia Bureau, Pseudocatalpa A. H. Gentry, and Spathicalyx J. C. Gomes are synonymized with Tanaecium. The generaAdenocalymma, Amphilophium, Fridericia, Dolichandra, and Tanaecium are formally emended here as to diagnosis andcircumscription. A natural key, complete morphological descriptions, and illustrations characterize the accepted genera, and fullgeneric synonymy and a catalogue of their component species summarize their basic nomenclature and geographic range. Threenew 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 brasiliensisBaill. Thirty-two generic names are newly synonymized, and 144 new nomenclatural combinations are made. A lectotype isdesignated for one genus, Periarrabidaea A. Samp., and 78 species names. One species name is neotypified, Memora campicolaPilg. ([ 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 andsupport during this study. We thank Peter Raven, Mick Richardson, Bette Loiselle, Richard Olmstead, John Pruski, AmyPool, Fred Barrie, Ivan Jimenez, 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 andnomenclature, Carmen Ulloa Ulloa for her contributions to Bignonieae taxonomy for the Checklist of the World, and VictoriaC. Hollowell for valient editing. Thanks also to Barbara Alongi for preparing line drawings, Alexandre Zuntini for greatassistance with the preparation of figures, and Sara Fuentes for her help with SEM. We are indebted to the curators ofvarious herbaria who made their collections available for this study, especially B, BM, BR, G, K, NY, P, M, MO, and W, andto 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); researchgrants by the American Society of Plant Taxonomists, the Botanical Society of America, the Federated Garden Clubs ofMissouri, Idea Wild, and the Whitney R. Harris World Ecology Center; postdoctoral support from the Center forConservation and Sustainable Development of the Missouri Botanical Garden; a pq-2 grant from Conselho Nacional deApoio a Pesquisa, Brazil (CNPq), a regular research grant by Fundacao˜ 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 aredeeply and sincerely indebted to the late A. H. Gentry, whose work forms the basis of most of this study, and to whom wededicate the present study.

2 Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, U.S.A.; charlotte.taylor@mobot.org.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; llohmann@usp.br.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)

cpDNA and mtDNA Genome Assembly

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TGCATATTATACGTCATTCTAAAGATAAGGAAGGGCTAAAGGTAAGGCGGGTTAAGTTAGGGCATCAGTTGCTTC

08 de maio de 2014 Page 6 of 62

Genome Annotation

Chloroplast genome

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Struthio camelusRhea americana

Tinamus guttatusNothoprocta

Dromaius novaehollandiaeCasuarius casuarius

Apteryx australis

Megapodius freycinetLeipoa ocellataCrax blumembachiiGuttera pucheraniCallipepla californicaRollulus rouloulGallus gallusBonasa umbellus

Chauna torquataAnseranas semipalmataDendrocygna arboreaAnser albifronsAnas platyrhynchos

Phoenicopterus ruberTachybaptus ruficollisPodiceps cristatusColumba liviaTreron calvaPterocles personatusMesitornis unicolor

Rhynochetus jubatusEurypyga heliasPhaethon lepturus

Otis tardaTauraco erythrolophus

Coccyzus americanusCentropus phasianinusCoua cristata

Guira guira

Aegotheles insignisHemiprocne mystaceaStreptoprocne zonarisChaetura pelagicaPhaethornis griseogularisFlorisuga mellivoraColibri coruscans

Nyctibius grandisSteatornis caripensis

Podargus strigoidesBatrachostomus septimusEurostopodus macrotisCaprimulgus longirostris

Opisthocomus hoazin

Psophia crepitansGrus canadensisHeliornis fulicaSarothrura insularisRallus limicola

Burhinus capensisChionis minorPluvianus aegyptiusHaematopus aterRecurvirostra americanaCharadrius vociferus

Thinocorus orbignyianusRostratula benghalensisJacana jacana

Scolopax rusticola

TurnixStiltia isabellaCatharacta skuaAlca tordaLarus marinus

Gavia immerAptenodytes forsteriSpheniscus humboldtiThalassarche bulleri

Pelecanoides magellaniOceanites oceanicus

Puffinus creatopus

Ciconia abdimiiTheristicus melanopisEgretta tricolorPelecanus erythrorynchosBalaeniceps rex

Anhinga anhingaPhalacrocorax

Fregata minorSula sula

Cathartes auraSagittarius serpentariusPandion haliaetusElanus caeruleusButeo jamaicensisNinox novaeseelandiaeStrix occidentalisTyto albaPhodilus badius

Leptosomus discolor

Colius colius

Trogon personatusTockus camurusBuceros bicornis

Phoeniculus purpureusUpupa epops

Galbula albirostrisBucco capensis

Megalaima ortiLybius hirsutusTrachyphonus erythrocephalusCapito nigerSemnornis frantzii

Pteroglossus aracari

Indicator variegatusPicumnus cirratusMelanerpes carolinus

Merops pusillus

Todus angustirostrisMomotus momotaAlcedo leucogasterHalcyon malimbicaChloroceryle americana

Brachypteracias leptoCoracias caudata

Cariama cristata

Micrastur gilvicollisDaptrius aterFalco peregrinusNestor notabilisCalyptorhynchus funereusPsittacus erithacusMyiopsitta monachusAlisterus scapularisAcanthisitta chlorisPitta sordidaSmithornis rufolateralisPsarisomus dalhousiaePhilepitta castanea

Sapayoa aenigma

Piprites chlorisPlatyrinchus coronatus

Rhynchocyclus brevirostrisOnychorhynchus coronatus

Oxyruncus cristatus

Tityra semifasciataCotinga cayana

Pipra coronataTyrannus tyrannus

Thamnophilus nigrocinereusTerenura sharpeiMelanopareia torquataConopophaga ardesiacaScytalopus magellanicusGrallaria ruficapillaSclerurus mexicanusFormicarius colmaFurnarius rufusMenura novaehollandiaeAtrichornis clamosusClimacteris erythropsPtilonorhynchus violaceusMalurus melanocephalusMeliphaga analogaPardalotus punctatus

Petroica cucullata

Irena cyanogaster

Orthonyx teminckii

Nicator chloris

Mohoua albicilla

Pomatostomus isidorei

Lanius excubitor

Vireo philadelphia

Ptilorrhoa caerulescens

Struthidea cinerea

Daphoenositta chrysoptera

Pachycephala hyperthra

Cyanocitta cristata

Melampitta lugubris

Manucodia atraParadisaea raggiana

Cracticus quoyiArtamus leucorhynchus

Oriolus xanthonotus

Coracina novaehollandiae

Rhipidura hyperthraDicrurus adsimilis

Elminia nigromitratus

Monarcha axillaris

Aegithina tiphiaTelophorus dohertyiPrionops plumatus

Batis mixta

Vanga curvirostris

Philesturnus carunculatusChaetops frenatusPicathartes gymnocephalus

Bombycilla garrulus

Cinclus cinclusCatharus ustulatusMuscicapa ferruginea

Sturnus vulgarisMimus patagonicus

Sitta carolinensisCerthia familiarisTroglodytes aedon

Remiz pendulinusParus major

Aegithalos iouschensisHirundo rustica

Regulus calendula

Acrocephalus newtoni

Megalurus palustrisSphenoeacus afer

Pycnonotus barbatus

Cisticola anonymus

Zosterops senegalensisSylvia nana

Alauda arvensis

Promerops cafer

Dicaeum aeneum

Nectarinia olivacea

Melanocharis nigra

Paramythia montium

Prunella collarisPloceus cucullatusPasser montanusMotacilla cinereaTaeniopygia guttata

Fringilla montifringillaCalcarius lapponicusEmberiza schoeniclusThraupis cyanocephala

Struthio sp.

DinornithidaeLithornithidae

Aepyornithidae

Gallinuloididae

Presbyornithidae

Fluvioviridavidae

PreficaParaprefica

Eurotrochilus

NamibiavisProtoazin

Rhynchaeitinae

PlesiocathartesSandcoleidaeChascacocolius

Primotrogon

PrimobuccoEocoraciasParacoracias

Palaeotodus

Messelirrisor

PhororhacoideaIdiornithidae

Halcyornithidae

80 60 40 20 0

South America

How old is the South American avifauna?

• Birds have been in South America’s warm & wet forest environments for a very long time

• Higher taxa tend to show deep origins of stem-lineages

• However, ages of genera are not good metrics for diversification dynamics

• The question is: “How old is the modern diversity?”

Finer scale patterns

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Biogeographic patterns of Psophia

(Ribas, Aleixo, Nogueira, Miyaki & Cracraft, 2012)

2.7-2.0 Myr3.0-2.7 Myr

2.0-2.1 Myr 1.3-0.8 Myr

1.0-0.7 Myr 0.8-0.3 Myr

Species-level taxa are young

(Ribas et al. 2012)

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GUESTEDITORIAL

Timing the diversification of theAmazonian biota: butterfly divergencesare consistent with Pleistocene refugiaIvonne J. Garz!on-Ordu~na*, Jennifer E. Benetti-Longhini andAndrew V. Z. Brower

Evolution and Ecology Group, Department of

Biology, Middle Tennessee State University,

Murfreesboro, TN 37132, USA

*Correspondence: Ivonne J. Garz!on-Ordu~na,Department of Biology, Middle Tennessee StateUniversity, Murfreesboro, TN 37132, USA.E-mail: ivonne.garzon@gmail.com

ABSTRACT

Rejection of the Pleistocene refugium hypothesis (PRH) as an explanation forthe 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 overestimatesof the time of species formation. We present divergence times between pairs of

sister species (131 pairs), and among pairs of sister species and their closest rel-

ative (56 triplets), from 35 genera of Neotropical butterflies. Our aim is torefocus 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 eventsleading to the formation of butterfly sister species occurred within the last

2.6 Myr, a result consistent with the temporal predictions of the PRH, suggest-

ing that the PRH cannot be completely discarded as a driver of Neotropicaldiversification.

KeywordsAmazonia, biodiversity, crown ages, diversification, Lepidoptera, mitochon-drial DNA, Neogene, Neotropics, Pleistocene refugium hypothesis.

INTRODUCTION

The reasons for the enormous numbers of species hosted by

Neotropical forests intrigued 19th-century naturalists, and

still puzzle systematists, ecologists, geologists and palaeontol-

ogists today. There are two contrasting positions regarding

the patterns and timing of biotic diversification in the Neo-

tropics in evolutionary time: one that emphasizes Neogene

(23–2.6 Ma) vicariance events as a result of major rearrange-

ments of the Amazonian landscape, and a second that points

to Pleistocene (< 2.6 Ma) climatic cycles as an engine of

diversification. While controversy over timing may not seem

to be a biogeographical issue per se, the abiotic processes

that could explain biotic distributions differ between these

two time periods in fundamental ways. Therefore, inferring

when diversification took place points to which geological

and/or climatic mechanisms may have been involved.

At the end of the Tertiary, the Neogene (23–2.6 Ma) was

a period of dramatic geological events in the Neotropics,

such as the uplift of the Andes, the formation of a large

lacustrine system in what is today western Amazonia (Lake

Pebas), shifts in the courses and watersheds of major rivers,

and the subsequent establishment of terrestrial conditions.

Authors such as Hoorn et al. (2010) have argued that this

tectonic activity caused changes in the landscape that pro-

vided biogeographical opportunities for new species interac-

tions, and generated new adaptive pressures that triggered

speciation. According to this scenario, most physical barriers,

such as mountains and rivers, were in their current positions

by the end of the Pliocene (2.6 Ma), and therefore vicariant

speciation events caused by those barriers must have

occurred earlier, implying that most current sister species

diverged prior to 2.6 Ma (but see Ribas et al., 2012).

In contrast, Haffer’s (1969) Pleistocene refugium hypothe-

sis (PRH) suggests that many extant Neotropical species

originated after the Neogene (< 2.6 Ma; Cohen et al., 2013)

as a result of environmental fluctuations driven by repeated

cycles of global cooling and warming. The PRH proposes

that cold spells during the Pleistocene caused the fragmenta-

tion and replacement of moist Amazonian forest by drier

grass savannas, isolating populations of forest obligate taxa,

allowing allopatric differentiation and ultimately driving an

ª 2014 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1doi:10.1111/jbi.12330

Journal of Biogeography (J. Biogeogr.) (2014)

comparison), the results of divergence timesfor selected species representing 35 genera

Garzón–Orduña & Brower 2014

Neogene

(Hoorn et al. 2010.)

Crown nodes with ages within the Neogene

New molecular data favor a more recent origin for the formation of the Amazonian

Biota

- 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?

PART IV: Characterization of the geological and environmental history of

Amazonia

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

Prance et al. 1982 Haffer et al. 1997

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5 to 9 My

Last 5 My andLGM

Present

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

Speleothem record and Amazonian climate suggests different forest corridors at different times

- West has suffered less drought in the last 4MY

DRY

WET

WET

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- E'.83I*37B8,+-.8,I3I.8.3G-*63&.),*=7*+,*+-.&'(L3+,B,M<DI,6*+-.&'7<3

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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