Programa de Pós-Graduação em Genética, Conservação e ... · allowed the Great American Biotic...

Instituto Nacional de Pesquisas da Amazônia – INPA

Programa de Pós-Graduação em Genética, Conservação

e Biologia Evolutiva

Filogenia e biogeografia de três famílias de aves do

Neotrópico

Mateus Ferreira

Manaus, Amazonas

Março, 2018

Mateus Ferreira


Neotrópico

Orientador: Dra. Camila Cherem Ribas

Tese apresentada ao Instituto Nacional de

Pesquisas da Amazônia como requisito para

obtenção do grau de doutor em Genética,

Conservação e Biologia Evolutiva.

Manaus, Amazonas

Março, 2018

iii

F383 Ferreira, Mateus


Neotrópico / Mateus Ferreira. --- Manaus: [sem editor],

2018.

121 f. : il. color.

Tese (Doutorado) --- INPA, Manaus, 2018. Orientadora : Camila Cherem Ribas. Programa : Genética, Conservação e Biologia

Evolutiva.

1. Biogeografia. 2. Genômica. 3. Aves neotropicais. I.

Título.

CDD 598.7

Sinopse:

Neste trabalho foram realizados estudos sobre a relação

filogenética entre todas as espécies de três famílias de aves do

Neotrópico. Abordamos aspectos sobre a distribuição geográfica

das linhagens genéticas encontradas, conflitos entre os diferentes

marcadores genéticos e a subestimação da diversidade

taxonômica dos táxons estudados.

Palavras-chave: Bucconidae, Galbulidae, Trogonidae, Pantropical,

genômica, filogeografia, UCE

iv

Agradecimentos

Agradeço primeiramente a minha orientadora Camila Ribas, pela paciência e confiança

que depositou em mim durante esses anos de orientação. Sem sombra de dúvidas, esse trabalho

não seria possível sem essa amizade e parceria.

Ao meu co-orientador, Joel Cracraft, com quem tive a sorte de trabalhar durante o meu

doutorado sanduíche. Pelas excelentes conversas e orientações sobre biogeografia e sobre os

padrões e processos que moldaram a diversidade de aves no mundo.

À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e ao

programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, do Instituto

Nacional de Pesquisas da Amazônia, pela concessão da bolsa de doutorado no país e bolsa

sanduíche (# 88881.133440/2016-01), que tornaram este projeto possível.

Aos curadores e responsáveis pelas coleções científicas que gentilmente cederam

material para que este trabalho fosse desenvolvido: Alexandre Aleixo, Fátima Lima e Antonita

Santana (MPEG); Marlene Freitas (INPA); Nate Rice (ANSP), Cristina Miyaki (LGEMA),

Donna Dittman e Robb Brumfield (LSU), Paul Sweet e Tom Trombone (AMNH), Mark

Robbins (KU), John Bates e Ben Marks (FMNH), Brian K. Schmidt (USNM), Sharon Birks

(UWBM). E, a todas as pessoas envolvidas nas expedições de coleta dessas institutições.

Ao projeto “Dimensions US-Biota: Assembly and evolution of the Amazon biota and

its environment: an integrated approach”, um projeto financiado conjuntamente pela Fundação

de Amparo à Pesquisa de São Paulo (FAPESP #2012/50260-6) e pelo National Science

Fundation (NSF DEB 1241056). Cujo apoio e financiamento foram essenciais para a execução

das várias etapas desse doutorado.

A todos os colegas do EBBA, pela constante ajuda e pelas excelentes discussões e

incentivos, e pelo café, especialmente pelos cafés: Robs, Fernanda, Rafael, Claudinha, Érico,

Erik, Lídia, Renatinha, Jessica, Nelson, Carol, Waleskinha e todo mundo que passou por aqui.

Ao pessoal que me aguentou durante esse doutorado: Mariana Tolentino, Leandro, Marina

Maximiano, Ana, Marizita (Marina Carmona) Derek, Miquéias, Pedro, Cadu e Manu. Em

especial à Romina, pela caminhada lado a lado durante toda a execução desse projeto, pelos

puxões de orelha quando eu precisei e por ter me aguentado todo esse período.

Ao pessoal do LTBM, Giselle e Paula, pela excelente companhia, pelos cafés e ajudas

quando precisei.

v

To everyone who received me at the AMNH during my sandwich fellowship: Lydia,

Bill, Tom, Paul, Gabi, Brian, Luke, and Peter. A special thanks to Jessica and Laís for all the

support and friendship during my time in NY.

Também gostaria de agradecer ao Laboratório Nacional de Computação Científica

(LNCC/MCTI) por fornecer recursos de computação de alto desempenho através do

supercomputador SDumont, fundamentais para as análises realizadas neste estudo.

Por fim, um agradecimento especial para a minha família, que me apoiou

incondicionalmente em todo esse percurso, e cuja ajuda foi essencial para a finalização deste

doutorado.

vi

“Nothing in biology makes sense except in the light of evolution”

Theodosius Dobzhansky

“Life and Earth evolve together”

Leon Croizat

vii

Resumo

O Neotrópico é uma das regiões com os maiores índices de biodiversidade do planeta e muito

tem se questionado sobre a origem de tamanha diversidade. Acredita-se que os padrões de

diversidade atual dentro da região sejam um resultado da complexa história geomorfológica e

climática da região. Entre os eventos geomorfológicos mais discutidos estão o soerguimento

dos Andes e consequente reestruturação da drenagem continental, e o fechamento do Istmo do

Panamá, que permitiu a troca intercontinental de biotas. Neste trabalho foram selecionadas três

famílias de aves do Neotrópico. A família Trogonidae tem uma distribuição Pantropical,

ocorrendo também nas regiões subtropicais e tropicais da África e Ásia, no entanto, a maior

diversidade encontra-se justamente na região Neotropical. As famílias Bucconidae e Galbulidae

são duas famílias irmãs endêmicas do Neotrópico. Foram selecionadas amostras de todas as

espécies e quase todas as subespécies descritas para os três grupos. Para as espécies amplamente

distribuídas foram selecionadas amostras ao longo de toda a distribuição e uma análise prévia

para verificar a estrutura filogeográfica de cada grupo, com base nesses resultados, foram

selecionadas amostras para o sequenciamento de milhares de loci de regiões Ultra Conservadas

(Ultraconserved Elements, UCE). Dessa forma, compilamos três estudos nessa tese. No

primeiro capítulo, foi estudado um complexo de aves da família Galbulidae associada aos

ambientes de areia branca na região Amazônica. Através da comparação entre marcadores

moleculares com diferentes métodos de herança, DNA mitocondrial e nuclear (UCE), pudemos

observar um conflito entre esses dois marcadores. Através deste conflito foi possível propor um

modelo de diversificação para os ambientes de areia branca na região. No segundo capítulo

analisamos a diversificação global da família Trogonidae, com o auxílio dos UCEs

reconstruímos a relação filogenética entre todas as espécies da família e estimamos uma árvore

datada da diversificação de Trogonidae. No terceiro e último capítulo, analisamos os padrões

de diversificação das famílias Galbulidae e Bucconidae através de uma abordagem

filogeográfica e filogenética. Neste trabalho pudemos observar que a diversidade do grupo se

encontra claramente subestimada.

viii

Abstract

The Neotropical region has one of the highest biodiversity index in the planet and several

hypotheses have been proposed to explain the origin of such diversity. Currently, landscape

and climatic evolution are credited to be the two main processes responsible for shaping the

patterns. Landscape evolution includes, for example, the Andean uplift and consequent

continental drainage reconfiguration, and the closure of the Isthmus of Panama, which

allowed the Great American Biotic Interchange. In the present study we selected three

Neotropical families of birds. Trogonidae has a Pantropical distribution, members of this

family inhabit tropical and subtropical regions of Africa, Asia, however, the highest diversity

is currently found in the Americas. Galbulidae and Bucconidae are sister families and

endemics to the Neotropics. WE sampled all species and almost all subspecies currently

recognized for this three families, and for widespread species we thoroughly sampled

throughout their distributions to uncover hidden phylogeographic patterns. Based on these

results, we selected the samples to sequence thousands of Ultraconserved Elements (UCE).

Thus, we compiled three studies for this thesis. In the first chapter, we studied one Galbulidae

species complex associated with the Amazonian White-sand environments. We compared

between molecular markers that have different heritage systems, the mtDNA and nuDNA

(UCE), where we recovered contrasting histories between markers, and based on these results

we proposed a diversification model for the White-sand environments. In the second chapter,

we analyzed the global diversification of Trogonidae, employing thousands of UCE loci to

propose a phylogenetic hypothesis between all species currently recognized, and we also

estimated a fossil calibrated time tree for Trogonidae diversification. At last, in the third

chapter, we analyzed the diversification patterns for Galbulidae and Bucconidae using a

phylogeographic/phylogenetic approach. In this chapter it was clear how these groups

diversity in underestimated by currently taxonomic approach.

ix

Sumário

Agradecimentos ....................................................................................................................... iv

Resumo .................................................................................................................................... vii

Abstract .................................................................................................................................. viii

Introdução Geral ...................................................................................................................... 1

Objetivos .................................................................................................................................... 7

Capítulo 1 .................................................................................................................................. 8

Abstract ............................................................................................................................................................ 10

1. Introduction ............................................................................................................................................. 11

2. Methods ................................................................................................................................................... 13

2.1. Taxon sampling ................................................................................................................................ 13

2.2. DNA extraction, amplification and sequencing ............................................................................... 13

2.3. Phylogenetic analysis and haplotype networks ............................................................................... 14

3. Results ...................................................................................................................................................... 16

3.1. Sanger sequencing and haplotype networks ................................................................................... 16

3.2. mtDNA genome and time tree ......................................................................................................... 17

3.3. UCE sequencing, supermatrix analysis and Species trees ................................................................ 17

4. Discussion ................................................................................................................................................ 18

4.1. mtDNA and nuDNA incongruence ................................................................................................... 18

4.2. Biogeography of WSE avifauna ....................................................................................................... 21

4.3. Evolution in the White-sand ecosystems ......................................................................................... 22

5. Conclusion ................................................................................................................................................ 24

Acknowledgements .......................................................................................................................................... 24

Funding ............................................................................................................................................................. 25

References ........................................................................................................................................................ 25

Capítulo 2 ................................................................................................................................ 37

Abstract ............................................................................................................................................................ 39

Introduction ..................................................................................................................................................... 40

Results .............................................................................................................................................................. 42

UCE sequencing .......................................................................................................................................... 42

Phylogenetic inference ................................................................................................................................ 42

Time-calibrated tree ................................................................................................................................... 43

Discussion......................................................................................................................................................... 43

Phylogenomic contribution to the reconstruction of Trogonidae diversification .................................. 43

Diversification and biogeography of Trogons .......................................................................................... 44

Africa and Asia diversification .................................................................................................................. 46

Neotropical diversification ......................................................................................................................... 47

Conclusion ....................................................................................................................................................... 49

Materials and Methods ................................................................................................................................... 49

x

Taxon sampling and DNA extraction ........................................................................................................ 49

UCE and exons assembly ........................................................................................................................... 50

Phylogenetic relationships and species tree analysis................................................................................ 50

Dating analysis ............................................................................................................................................ 51

Acknowledgements.......................................................................................................................................... 51

References ........................................................................................................................................................ 52

Capítulo 3 ................................................................................................................................ 66

Abstract ............................................................................................................................................................ 67

Introduction ..................................................................................................................................................... 68

Material and Methods .................................................................................................................................... 70

Sampling and DNA isolation ........................................................................................................................ 70

Phylogeographic structure and UCE sampling ............................................................................................ 71

UCE assembly ............................................................................................................................................... 71

Phylogenetic relationship ............................................................................................................................. 72

Results .............................................................................................................................................................. 72

Phylogeographic results ............................................................................................................................... 72

UCE sequencing ........................................................................................................................................... 73

Phylogenetic results ...................................................................................................................................... 73

Discussion......................................................................................................................................................... 74

Phylogenetic results ...................................................................................................................................... 74

Galbulidae systematics ................................................................................................................................. 75

Conclusion ....................................................................................................................................................... 83

Acknowledgements.......................................................................................................................................... 83

References ........................................................................................................................................................ 84

Síntese Geral ......................................................................................................................... 102

Referências Bibliográficas ................................................................................................... 103

1

Introdução Geral

O Neotrópico é uma das regiões biogeográficas com uma das maiores biodiversidades

do mundo (Jetz et al., 2012; Holt et al., 2013), mesmo que uma grande parcela dessa diversidade

ainda seja desconhecida (Kier et al., 2005; Hopkins, 2007; Barrowclough et al., 2016). Na

região Neotropical, os biomas Mata Atlântica, Cerrado e Amazônia despontam como hotspots

de biodiversidade altamente ameaçados pela ação humana (Myers et al., 2000; Mittermeier et

al., 2003; Colombo e Joly, 2010). Em especial para a região Amazônica, que abrange mais de

40% da área total do Neotrópico, desde que Wallace (1852), fez suas primeiras observações

sobre a importância dos rios na delimitação da distribuição de diferentes espécies de primatas,

vários trabalhos foram realizados demonstrando a importância dos afluentes do rio Amazonas

na estruturação da diversidade alfa da região (Vanzolini e Willians, 1970; Cracraft, 1985;

Haffer, 1985; Ávila-Pires, 1995). A comparação e aparente congruência dos padrões de

distribuição geográfica permitiu a elaboração de algumas hipóteses sobre quais processos

poderiam ter dado origem a esses padrões (revisões em Haffer (1997) e Leite e Rogers (2013)),

incluindo as variações climáticas do Pleistoceno, em especial o Último Máximo Glacial (LGM

– Last Glacial Maximum, ca. 20.000 anos) (Haffer, 1969; Brown et al., 1974); a influência das

incursões marinhas (Nores, 1999; 2004); e a formação dos rios da bacia Amazônica (Bates et

al., 2004; Ribas et al., 2012). Com o advento da filogeografia (Avise et al., 1987; Avise, 2009)

e técnicas de datação molecular (Bromham e Penny, 2003; Bromham et al., 2017) novas teorias

foram propostas e além da congruência entra a distribuição geográfica o tempo de

diversificação também passou a fazer parte da comparações (Donoghue e Moore, 2003). Como

consequência, a teoria dos refúgios associados aos eventos climáticos do LGM foi parcialmente

rejeitada, já que as espécies se mostraram mais antigas que os ciclos glaciais mais recentes

(Colinvaux et al., 2000; Bush e Oliveira, 2006). As incursões marinhas, por outro lado, seriam

muito antigas para explicar a origem das espécies (Hoorn, 1993), favorecendo o modelo da

hipótese dos rios como barreira.

Atualmente, no entanto, o que sabemos sobre a complexidade da diversidade Amazônica

sugere que mais de um processo está por trás de sua origem (Bush, 1995; Bates et al., 2008;

Smith et al., 2014). Todos os eventos que moldaram a paisagem do Neotrópico ao longo do

tempo podem ter influenciado a diversificação da biota, por exemplo: A) o fim do “isolamento

2

esplêndido” (Simpson, 1980; Dacosta e Klicka, 2008) após o estabelecimento do Istmo do

Panamá (Haug e Tiedeman, 1998; Coates e Stallard, 2013; Lessios, 2015; Odea et al., 2016).

B) O soerguimento da cadeia de montanha dos Andes (Garzione et al., 2008; Hoorn et al., 2010;

Horton, 2018), que influenciou drasticamente não só a drenagem da bacia Amazônica (Hoorn

e Wesselingh, 2010; Latrubesse et al., 2010; Shephard et al., 2010; Nogueira et al., 2013; Hoorn

et al., 2017), como também o clima de todo o continente (Hartley, 2003; Ehlers e Poulsen,

2009; Insel et al., 2009). C) A influência das flutuações climáticas do Pleistoceno também

voltou a fazer parte das discussões, especialmente com relação ao estabelecimento de diferentes

regimes de precipitação dentro do continente (Cheng et al., 2013; Wang et al., 2017).

Dessa forma, faz-se necessário investigar não somente a evolução do modelo através das

variáveis biológicas, mas também quais processos físicos podem ter influenciado a sua

diversificação (Baker et al., 2014). Por exemplo, o estabelecimento do atual curso

transcontinental do rio Amazonas, ainda bastante discutido na literatura, varia entre o final do

Mioceno (10 – 7 Ma) (Hoorn e Wesselingh, 2010; Hoorn et al., 2017), início do Plioceno (~5

Ma) (Latrubesse et al., 2010), ou ainda, durante o Pleistoceno (2,5 Ma) (Nogueira et al., 2013;

Rossetti et al., 2015). Nesse sentido, estudando um gênero de aves (Psophiidae: Psophia) que

é restrita aos ambientes de terra-firme, e dessa forma suscetível às mudanças na configuração

drenagem, Ribas et al. (2012) propuseram um modelo de diversificação da fauna de terra firme

ao correlacionar os eventos de diversificação das espécies do gênero ao estabelecimento de

barreiras associadas aos principais afluentes da bacia, favorecendo o modelo do

estabelecimento do rio Amazonas durante o Pleistoceno. O modelo proposto por Ribas et al.

(2012) sugere que para compreender os fatores que influenciaram a evolução da paisagem,

como o efeito da formação de um determinado rio na diversificação de espécies de terra-firme,

deve-se buscar padrões congruentes, temporais e espaciais, de diversificação em grupos que

serão de fato afetados diretamente pela barreira (e.g. Polo, (2015)). Em contraponto, análises

que buscam explicar a diversificação na Amazônia através de um único processo, como por

exemplo, a importância dos rios como barreira utilizando uma ampla gama de táxons com

nichos variados (Oliveira et al., 2017; Santorelli et al., 2018; Smith et al., 2014) tendem a

refutar esta teoria, já que diferentes organismos respondem de diferentes maneiras aos

processos e eventos históricos. Dessa forma, aceitando que a diversificação na Amazônia é

inerentemente complexa, o teste de hipóteses deve ser feito de maneira dirigida, ou seja, deve-

se buscar grupos taxonômicos que tenham sido potencialmente influenciados pelas barreiras

em questão. Só assim será possível estabelecer a importância biológica de um determinado

3

evento e gerar dados importantes para o estabelecimento dos modelos de evolução

geomorfológica da região (Baker et al., 2014).

Essa iluminação recíproca entre os processos físicos e bióticos, no entanto, só é possível

levando em consideração o fato de que qualquer evento de diversificação só pode ser

correlacionado com um evento biogeográfico se duas condições forem respeitadas: 1) as

unidades biológicas utilizadas devem ser comparáveis entre si e devem representar linhagens

com uma história evolutiva única; 2) a relação filogenética entre essas linhagens deve

representar de fato a história de diversificação do grupo.

A primeira condição refere-se ao fato de que as unidades utilizadas no estudo devem

representar linhagens independentes. Geralmente, entende-se que espécies devem ser a unidade

básica para qualquer estudo de ecologia, evolução, ou biogeografia, no entanto, essa prática

pode ser particularmente problemática na Amazônia, uma vez que grande parte das espécies

amplamente distribuídas pela região na realidade representam um complexo de linhagens

evolutivas independentes (Ribas et al., 2012; D’horta et al., 2013; Fernandes et al., 2013;

Fernandes et al., 2014; Hrbek et al., 2014; Boubli et al., 2015; Thom e Aleixo, 2015; Byrne et

al., 2016; Carneiro et al., 2016; Boubli et al., 2017; Ferreira et al., 2017; Lima et al., 2017;

Ribas et al., 2018). Para aves, em particular, esse déficit entre a taxonomia atualmente

reconhecida e a real diversidade está diretamente relacionado ao fato de que a definição daquilo

que reconhecemos como espécie ainda é muito influenciado pelo tipo de conceito de espécie

utilizado, em especial o conceito biológico de espécie (Mayr, 1942), que implica no

reconhecimento de metapopulações isoladas reprodutivamente. No entanto, o reconhecimento

de isolamento reprodutivo em populações naturais é particularmente difícil, especialmente em

populações alopátricas, onde é impossível observar naturalmente esse contato. Mesmo em

populações parapátricas, o contato e estabelecimento de uma zona híbrida não necessariamente

ameaça o statu quo das espécies envolvidas (Weir et al., 2015). Especialmente, porque a

capacidade de hibridização entre espécies, mesmo distantes, parece ser uma característica

sinapomórfica para aves (Grant e Grant, 1992; Gill, 1998; Harrison e Larson, 2014).

O conceito de espécie, mesmo sendo um dos assunto centrais para os estudo de evolução

e ecologia, permanece ainda sem definição clara e é sem dúvida um dos pontos mais discutidos

dentro da biologia (Mayr, 1976; Donoghue, 1985; Isaac et al., 2004; De Queiroz, 2005; Aleixo,

2007; Joseph et al., 2008; Aleixo, 2009; De Queiroz, 2011; Cellinese et al., 2012; De Queiroz,

2012; Willis, 2017). Ressaltando o impacto dessa escolha entre um conceito ou outro e do nosso

atual conhecimento sobre a taxonomia de aves, um estudo recente demonstrou que a diversidade

4

das aves é, pelo menos, duas vezes maior do que a atualmente reconhecida (Barrowclough et

al., 2016). Por exemplo, dentro da Neotrópico, um dos padrões mais observados é a existência

de espécies amplamente distribuídas, compostas por diferentes subespécies morfologicamente

distintas e geograficamente estruturadas, as quais foram, no entanto, agrupadas dentro de uma

mesma espécie devido a existência da possibilidade dessas populações hibridizarem caso

entrem em contato.

A segunda condição está relacionada aos problemas de conflitos entre a história de um

único gene e a história da espécie (Degnan e Rosenberg, 2009; Knowles, 2009). Esse conflito

tem se tornado cada vez mais evidente em face do desenvolvimento de técnicas de

sequenciamento massivo em paralelo (Metzker, 2010). Apesar de estarem se tornando mais

acessíveis, o sequenciamento e análise do genoma completo para organismos não modelo ainda

é impraticável para trabalhos que requerem amostragem de muitos indivíduos. Dessa forma,

algumas técnicas de se utilizar representações reduzidas foram desenvolvidas. Duas abordagens

dominam o cenário atualmente, uma delas é a utilização de enzimas de restrição para sítios

específicos ao longo de todo o genoma, denominada RAD-seq (restriction-site-associated DNA

sequencing) (Davey et al., 2011); e a outra é a utilização de sondas de RNA desenvolvidas para

capturar regiões específicas do genoma (Grover et al., 2012; Lemmon et al., 2012; Lemmon e

Lemmon, 2013). Uma das abordagens de sequenciamento de captura é a técnica que utiliza

sondas específicas para regiões do genoma ultra conservadas (do inglês, Ultra Conserved

Elements, UCE) (Faircloth et al., 2012). Essas regiões ultra conservadas foram selecionadas

pois permitem a utilização de um mesmo conjunto de sondas para realizar estudos em diversos

níveis taxonômicos, pois apesar das regiões centrais serem altamente conservadas, as regiões

flanqueadoras possuem variação suficiente tanto para recuperar relações mais antigas

(Mccormack et al., 2012; Crawford et al., 2015; Faircloth et al., 2015), quanto mais recentes

(Bryson et al., 2016; Manthey et al., 2016), inclusive utilizadas em radiações adaptativas

(Meiklejohn et al., 2016), onde altos níveis de separação incompleta de linhagens (do inglês,

Incomplete Lineage Sorting, ILS) sejam esperados (Degnan e Rosenberg, 2006; Oliver, 2013).

De modo a tentar então lançar alguma luz sobre os possíveis eventos que moldaram a

diversificação da biota Neotropical, foram selecionadas três famílias de aves: Trogonidae,

Galbulidae e Bucconidae. As três famílias possuem representantes por toda a região

Neotropical, incluindo várias espécies, ou grupo de espécies, amplamente distribuídas. A

família Trogonidae tem distribuição Pantropical, estando ausente apenas da região Australiana.

Representantes dessa família, popularmente conhecidos como surucuás, são aves de médio

5

porte e sua dieta varia entre insetívora e onívora, apresentam plumagem com coloração bastante

chamativa, e são reconhecidas por serem más dispersoras, não sendo capazes de realizar voos

de longa distância (Collar, 2017). Apesar de apresentarem plumagem bastante distinta, a

morfologia interna da família é bastante conservada e a sua monofilia nunca foi questionada

(Livezey e Zusi, 2007; Collar, 2017). No entanto, a relação entre trogonídeos e outras aves não

passeriformes já foi bastante controversa (Cracraft, 1981; Maurer e Raikow, 1981; Monteros,

2000; Mayr, 2003; Livezey e Zusi, 2006). Atualmente, aceita-se que a família seja uma das

primeiras linhagens a diversificar dentro da radiação de Coracimorphae sendo grupo irmão de

todas as outras famílias do grupo Core Landbirds (Jarvis et al., 2015; Prum et al., 2015).

Atualmente são reconhecidas 45 espécies (Collar, 2017; Gill e Donsker, 2018; Ramsen et al.,

2018) distribuídas em sete gêneros. A região Neotropical contém a maior diversidade da

família, com quatro gêneros e cerca de 30 espécies. A região Asiática contém dois gêneros e 12

espécies, e por último, a região Africana, possui um gênero com três espécies. Apesar da maior

diversidade da família ser encontrada no Neotrópico, a existência de fósseis na Europa (Mayr,

1999; Kristoffersen, 2002; Mayr, 2005) sugere uma origem no Paleártico e posterior dispersão

e colonização da distribuição atual. Diversos trabalhos já tentaram abordar a relação

filogenética entre os representantes da família (Monteros, 1998; Johansson e Ericson, 2005;

Moyle, 2005; Dacosta e Klicka, 2008; Ornelas et al., 2009; Hosner et al., 2010), entretanto,

nenhum foi capaz de resolver a relação entre os gêneros. O último trabalho publicado (Hosner

et al., 2010), e o único a incluir representantes de todos os gêneros, recuperou uma parafilia

entre regiões geográficas, sugerindo um cenário biogeográfico bem mais complexo, em que a

região Neotropical, por exemplo, tenha sido ocupada por pelo menos três linhagens distintas.

As famílias Galbulidae e Bucconidae formam um clado já bem estabelecido, tanto com

caracteres morfológicos (Livezey e Zusi, 2007), quanto dados moleculares (Hackett et al., 2008;

Prum et al., 2015). Dentro da ordem Piciformes, são as únicas famílias com representantes com

distribuição exclusivamente neotropical, formando o grupo irmão das outras famílias de

Piciformes (Prum et al., 2015). A família Galbulidae é composta por aves de pequeno a médio

porte, asas arredondadas e um bico longo e afilado utilizado para capturar insetos durante o

voo. Possui 19 espécies distribuídas em cinco gêneros diferentes (Tobias, 2017; Gill e Donsker,

2018; Ramsen et al., 2018). As espécies da família são geralmente agrupadas em oito grupos

zoogeográficos, seis desses grupos representam complexos de espécies com distribuições

alopátricas ou parapátricas, e dois são espécies amplamente distribuídas (Collar, 2017). A

família Bucconidae também inclui aves de pequeno a médio porte, asas curtas e arredondadas,

6

tendo como característica uma cabeça relativamente grande, atualmente são reconhecidas 35

espécies para a família distribuídas em nove gêneros (Gill e Donsker, 2018; Ramsen et al.,

2018). Os trabalhos de filogeografia desenvolvidos com representantes da família Bucconidae

– Malacoptila (Ferreira et al., 2017), Monasa e Nonnula (Soares, 2016) e Nystalus (Duarte,

2015) – demonstraram que a diversidade reconhecida pela taxonomia tradicional para esses

grupos é subestimada, já que existem muito mais linhagens genéticas geograficamente isoladas

do que táxons reconhecidos, demonstrando a importância da condução dos estudos de

filogeografia para elucidar a delimitação taxônomica dessas espécies amplamente distribuídas.

Dessa forma, o presente trabalho tem por objetivo reconstruir a relação filogenética entre

todas as linhagens dessas três famílias de modo a reconstruir a história de diversificação desses

três grupos. Para tanto, foram amostrados indivíduos ao longo da distribuição de todas as

espécies amplamente distribuídas para uma análise prévia da estrutura genética de cada uma

dessas linhagens. Com base nos resultados obtidos previamente foram selecionadas amostras

representativas de cada um desses agrupamento, tentando incluir, sempre que possível, um

representante para cada um dos táxons reconhecidos. Para essas amostras foram sequenciados

mais de 2000 loci de UCE, e com base nessa representação reduzida do genoma foram

realizadas análises para a reconstrução filogenética dos grupos.

7

Objetivos

O objetivo geral foi investigar a história biogeográfica da região Neotropical com base

nas relações filogenéticas entre todos os táxons atualmente reconhecidos para as famílias

Trogonidae, Bucconidae e Galbulidae baseado em dados de sequenciamento genômico. Sendo

que para isso foi necessário:

Capítulo 1: revisar a taxonomia e compreender os processos de isolamento e fluxo

gênico em um contexto espacial;

Capítulo 2: compreender a origem de táxons Neotropicais em uma família amplamente

distribuída;

Capítulo 3: compreender a estrutura filogeográfica de espécies amplamente distribuídas

em duas famílias Neotropicais para com isso obter uma reconstrução filogenética representativa

da diversificação do grupo.

8

Capítulo 1

Ferreira, M.; Fernandes, A.M.; Aleixo, A.; Antonelli,

A.; Olsson, U.; Bates, J.M.; Cracraft, J.; Ribas, C.C.

Evidence for mtDNA capture in the jacamar Galbula

leucogastra / chalcothorax species-complex and

insights on the evolution of white-sand environments

in the Amazon basin. Molecular Phylogenetics and

Evolution (doi: 10.1016/j.ympev.2018.07.007)

9

Manuscript submission to Molecular Phylogenetics and Evolution Contribution type: Original article

Evidence for mtDNA capture in the jacamar Galbula leucogastra / chalcothorax species-complex and insights on the evolution of white-sand ecosystems in the Amazon basin. Ferreira, Mateus a*; Fernandes, Alexandre M. b; Aleixo, Alexandre c; Antonelli, Alexandre d,e,f,g; Olsson, Urban d,f; Bates, John M. h; Cracraft, Joel i; Ribas, Camila C. j a Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, INPA, Manaus, AM, Brazil b Unidade Acadêmica de Serra Talhada, UFRPE, Serra Talhada, PE, Brazil c Coordenação de Zoologia, Museu Paraense Emílio Goeldi, Belém, PA, Brazil d Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden e Gothenburg Botanical Garden, Gothenburg, Sweden f Gothenburg Global Biodiversity Centre, Gothenburg, Sweden g Harvard University, Department of Organismic and Evolutionary Biology, Cambridge, MA, USA. h Integrative Research Center, Field Museum of Natural History, Chicago, IL, USA i Department of Ornithology, American Museum of Natural History, New York, NY, USA j Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil *Corresponding author Correspondence: Mateus Ferreira, Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia, CEP 69080-971, Manaus-AM, Brazil E-mail: [email protected]

mailto:[email protected]

10

Abstract

Jacamar species occur throughout Amazonia, with most species occupying forested habitats.

One species-complex, Galbula leucogastra / chalcothorax, is associated to white sand

ecosystems (WSE). Previous studies of WSE bird species recovered shallow genetic structure

in mtDNA coupled with signs of gene flow among WSE patches. Here, we characterize

diversification of the G. leucogastra/chalcothorax species-complex with dense sampling

across its distribution using mitochondrial and genomic (Ultraconserved Elements, UCEs)

DNA sequences. We performed concatenated likelihood and Bayesian analysis, as well as a

species-tree analysis using *BEAST, to establish the phylogenetic relationships among

populations. The mtDNA results recovered at least six geographically-structured lineages,

with G. chalcothorax embedded within lineages of G. leucogastra. In contrast, both

concatenated and species-tree analyses of UCE data recovered G. chalcothorax as sister to

all G. leucogastra lineages. We hypothesize that the mitochondrial genome of one of the G.

leucogastra lineage (Madeira) was captured into G. chalcothorax in the past. We discuss how

WSE evolution and the coevolution of mtDNA and nuclear genes might have played a role in

this apparently rare event.

Keywords: Amazonia, Galbulidae, jacamars, mtDNA capture, UCE, White-sand ecosystems

11

1. Introduction

White-sand ecosystems (WSE) represent a unique type of habitat within Amazonia,

covering an area of approximately 5% of the Amazon basin (Adeney et al., 2016). Contrasting

to the apparently continuous upland forest habitats found all over the basin, the WSE consist

of patches of differentiated habitats scattered across the landscape and isolated by the

forest matrix (Adeney et al., 2016). WSE comprise a continuum from open non-forested

habitats, such as campinas, with a predominance of grass and shrubland, to denser

vegetation, called campinaranas and varillales. In general, these communities grow on

nutrient poor and highly acidic soils, usually associated with quarzitic sand, even though

some clay and silt can also be found with varying amounts of organic matter (Adeney et al.,

2016). These complex environments, however, do not share a single history, since different

patches of WSE may have different geological origins (Prance and Schubart 1978; Frasier et

al., 2008). Podzolization, a natural process in which nutrients are leached away from the top

layers of soil, leaving only sand (Sauer et al., 2007), appears to be a principal cause of in loco

formation of the white-sand soils, especially in northeastern Amazonia (Nascimento et al.,

2004). In contrast, in central, northwestern, and southern Amazonia, white sand soils can be

found as fluvial deposits of ancient rivers (Roddaz et al., 2005), or abandoned ancient

paleochannels (Latrubesse, 2002; Cordeiro et al., 2016).

The insular characteristic of WSE intrigued researchers as to how the ecosystem and

its specialized biota evolved, how it responded to Pleistocene glacial cycles, and whether the

specialized biota is able to disperse through the surrounding forest matrix (Brown and

Benson, 1977; Anderson, 1981; Capurucho et al., 2013; Matos et al., 2016). Besides its

characteristic fragmentation, WSE are more physiologically stressful and challenging from an

ecological and evolutionary perspective, making them much more taxonomically selective,

with overall diversity being smaller when compared with adjacent forest areas (Borges,

2003; Fine et al., 2010; Laranjeiras et al., 2014; Adeney et al., 2016), although several new

species endemic to this habitat have recently been described (Whitney and Alonso, 1998,

2005; Alonso and Whitney, 2001; Cohn-Haft and Bravo, 2013; Cohn-Haft et al., 2013). Even

though the patches of WSE may have distinct geomorphological origins, the associated biota

presents varying degrees of association with WSE. While some plants have a loose

association with WSE (Fine and Baraloto, 2016), others are tighly associated with them, such

12

as species of Pagamea (Rubiaceae) (Vicentini, 2016). The same can be observed amongst

other organisms (Cohn-Haft, 2008; Vriesendorp et al., 2006), such as birds (Borges et al.,

2016a; Borges et al., 2016b). Therefore, considering the distinct geomorphological

characteristics of WSE and the association of the biota with these environments, recent

climatic and landscape changes must have had an important influence on the evolution and

distribution of WSE and their associated biota (Capurucho et al., 2013).

The few phylogeographic studies of WSE birds that have been undertaken, show

little genetic diversity with no geographic structure throughout Amazonia (Green-tailed

Goldthroat, Polytmus theresiae, Matos et al., 2016); or shallow but geographically structured

genetic diversity, with significant migration rates between some populations (Red-

shouldered Tanager, Tachyphonus phoenicius, Matos et al., 2016; Black Manakin, Xenopipo

atronitens, Capurucho et al., 2013). In general, results obtained so far for WSE birds suggest

that: (1) black-water flooded forest (igapó), due to similarities to WSE in vegetation

structure, may facilitate dispersal between isolated WSE patches; and, (2) Pleistocene glacial

periods, especially the Last Glacial Maximum, are temporally correlated with geographical

expansion of populations of species specialized in WSE.

These studies have been based on mtDNA markers (Capurucho et al., 2013), or on a

combination of mtDNA and a single nuclear marker (Matos et al., 2016). Until recently, most

phylogeographic studies have employed mtDNA. Its characteristic maternal inheritance,

comparatively small effective population size, rapid rate of mutation, and lack of

recombination, have long made mtDNA markers ideal for phylogeographic studies (Avise et

al., 1987; Avise, 2009), in contrast to single or few nuclear markers which usually provide

very little phylogenetic information. However, there are potential biases and limitations

associated with mtDNA (Zink and Barrowclough, 2008), including the potentital to overlook

hybridization and introgression (Carling and Brumfield, 2008). The use of large quantities of

nuclear markers has become the alternative to overcome these problems. One such strategy

has been the use of probes for Ultraconserved Elements (Faircloth et al., 2012; McCormack

et al., 2013) to sample homologous genomic regions across individuals (Faircloth et al.,

2012). These markers have so far been successfully used to study both very old radiations

(Moyle et al., 2016), and recent ones (Smith et al., 2014; Harvey et al., 2016; Manthey et al.,

2016).

13

Here, we investigate the diversification of a jacamar species-complex specialized in

WSE using genomic data. The jacamars (family Galbulidae) occur exclusively in the

Neotropics, with 19 species and 5 genera, mostly associated with wooded, lowland forest

habitats (Stotz et al., 1996; Tobias, 2017). In Amazonia, most species are restricted to upland

(terra firme) and flooded (varzea and igapó) forests, with only two species (Bronzy Jacamar,

Galbula leucogastra and Purplish Jacamar, G. chalcothorax) known to occur in WSE (Borges

et al., 2016a). Galbula leucogastra and G. chalcothorax were previously considered

subspecies of a single species (Peters, 1948; Haffer, 1974), but were split by Parker and

Remsen (1987), based on diagnostic plumage and size differences. A phylogeny of the family,

based on multiple gene regions, indicates that G. leucogastra and G. chalcothorax are sister-

species with high support (Witt, 2004). Here we first investigate the distribution of mtDNA

diversity within these two species by sampling individuals from throughout their

distributions. Then, based on these results, we gathered sequences of thousands of genomic

markers (UCE) for a subset of samples to reconstruct their history of diversification and

make inferences about the evolution of WSE.

2. Methods

2.1. Taxon sampling

We sampled 35 individuals covering almost the entire distribution of the Galbula

leucogastra / chalcothorax complex (Table S1). As outgroups, we used one sample of Yellow-

billed Jacamar G. albicollis (Witt, 2004). All tissues sequenced are represented by voucher

specimens deposited in ornithological collections (Table S1).

2.2. DNA extraction, amplification and sequencing

DNA was extracted using a modified phenol-chloroform protocol (Sambrook and

Russel, 2001). We used published DNA primers (Sorenson et al., 1999) to amplify and

sequence two mitochondrial genes (Cytochrome b [cytb], and NADH subunit 2 [ND2]) for all

individuals following standard PCR protocols. For a subset of individuals (see below) we

extracted DNA using the DNeasy kit (Qiagen Inc.) following the manufacturer’s protocol, and

sent the extracts to RapidGenomics® (Gainsville, FL) for sequencing, using a probe set

targeting 2321 loci of Ultra Conserved Elements (UCE) plus 98 conserved exons from genes

that were previously used in phylogenetic analysis (Harvey et al., 2017). Some of the exons

14

used were flanked by introns, which are more variable, and were the focus of this capture.

More information about the capture and sequencing of UCE loci can be found in Faircloth et

al. (2012).

2.3. Phylogenetic analysis and haplotype networks

Phylogenetic analysis of the mtDNA genes using the complete dataset (cytb and

ND2, N=35) was performed using Bayesian Inference (BI) implemented in MrBayes 3.2.6

(Ronquist et al., 2012). Both genes were concatenated and the best partition scheme and

substitution model were selected by PartitionFinder 2.1.1 using the Bayesian Information

Criteria (BIC) (Lanfear et al., 2016). We partitioned the genes by codon position, considering

possible saturation in the codon’s third position. Four parallel simultaneous runs were

performed, for a total of 4x107 generations, with trees sampled every 1000 generations. We

discarded the first 10% of trees as burn-in after checking the ESS values of each run in Tracer

1.6 (Rambaut et al., 2014). We used TCS v1.21 (Clement et al., 2000) to reconstruct

haplotype networks.

2.3.1. UCE and exons assembly and supermatrix approach

Based on the results of the mtDNA, we selected eight samples for UCE sequencing

(Table 1). The raw data received from Rapid Genomics were processed using the Phyluce

script pack (Faircloth 2016). Sequences with adapter contamination, and those of low-

quality, were trimmed using illumiprocessor (Faircloth, 2013) and Trimmomatic (Bolger et

al., 2014). After the sequences were ‘cleaned’ we employed Trinity RNASeq assembler

r201331110 (Grabherr et al., 2011) to assemble the contigs using a de novo method. The

contigs were then compared with the UCE database to identify which UCE loci were

sequenced. Since Trinity does not recover information on heterozygote loci we performed a

second round of assembly using the contigs that were identified as a reference to map the

clean reads back to it using the Bowtie2 (Langmead et al., 2009; Langmead and Salzberg

2012) plugin in Geneious R7.1 (Kearse et al., 2012). The consensus sequence of each

individual, derived from the reads, mapped back to each reference, was called using a

threshold of 75% with a depth of at least 5 reads. We then aligned each locus using MAFFT

(Katoh and Standley, 2013) with default options, and prepared the input matrix for the

subsequent analysis. To infer the phylogenetic relationship among all samples we

concatenated all the UCE loci and employed RAxML v8.2 (Stamatakis, 2014) under a

15

Maximum Likelihood analysis. Since we recovered almost all UCE loci for each sample, we

only used loci that were shared among all samples, with the final matrix having 2271 loci.

This matrix was analyzed by running RAxML to search for the optimal tree, under the fast hill

climbing algorithm, and bootstraping was performed with the autoMRE algorithm in the

program.

The 98 exons targeted were from 47 different genes. Because some of the

sequences included intronic regions, which are prone to indels, de novo assembly was not an

option. Therefore, we mapped all the probes to the Paradise Jacamar (Galbula dea) genome

(B10K Project), identified the genes that were targeted, and then used the whole gene-

sequences to map the reads back following the same approach that we used for UCE loci.

2.3.2. Mitochondrial genome assembly and time tree

As a byproduct of the UCE sequencing we also recovered the complete mtDNA

genome. We mapped all the contigs, assembled by Trinity, from each specimen to two

reference mtDNA genomes from representatives of close related families, the Downy

Woodpecker, Dryobates pubescens (Aves, Picidae; GenBank: NC_027936.1), and the Ivory-

billed Araçari, Pteroglossus azara (Aves, Ramphastidae; GenBank: DQ780882.1, Prum et al.,

2015). After we identified the contigs from each individual we used those contigs to map

back the reads of that same specimen, again using Bowtie2 to check for coverage depth.

Incongruences found between reads and contigs were checked manually. The complete

mtDNA genomes were then aligned using MAFFT (Katoh and Standley, 2013) under default

options. The mtDNA genomes downloaded from GenBank were used to import annotations.

Coding regions were manually checked for codon translations and translated protein

sequences were compared to check for frame shifts and stop codons. We employed the

concatenated coding regions in BEAST 1.8.2 (Drummond et al., 2012) to estimate a time tree

calibrated with the cytochrome b mutational rate of 0.0105 (normal distribution, SD=0.0034)

substitution.lineage-1.million years-1 (Weir and Schluter, 2008). The best partition scheme

and substitution model were selected by PartitionFinder 2.1.1 under the Bayesian

Information Criteria (BIC) (Lanfear et al., 2016). Two independent runs of 108 generations

were performed sampling trees every 1000 generations. Convergence, posterior

distributions, and ESS values were checked in Tracer 1.6 (Rambaut et al., 2014).

2.3.3. Species-tree analysis

16

Considering the possibility that concatenation might result in highly supported but

inaccurate results (Kubatko and Degnan 2007; Weisrock et al., 2012, but see Gatesy and

Springer 2014), we performed a species-tree analysis, which infers the most likely species-

tree based on individual gene trees, using the StarBEAST2 (Ogilvie et al., 2017) template in

the BEAST v.2.4.6 package (Bouckaert et al., 2014). Even though StarBEAST2 was developed

to deal with large amounts of data, we selected only the loci that had more than four

parsimony informative sites (PIS) among our samples. This latter step reduces the total time

of analysis and also avoids including loci lacking phylogenetic signal, which would create

noise in the analysis. We employed PartitionFinder2 (Lanfear et al., 2016) to check for the

best partition scheme and substitution model. Trees models were unlinked, except for exons

from the same gene, in which case we linked tree models across different partitions. Since

recombination is not expected to happen inside one gene, all exonic regions recovered

belonging to the same gene were considered to be connected in the species-tree (ST)

analysis. We used a Yule model of speciation, and ploidy was set to 2.0, unless genes were

from the Z chromosome (in which case, ploidy=1.5). We also included the complete mtDNA

as a single locus, with ploidy=1.0.

3. Results

3.1. Sanger sequencing and haplotype networks

We sequenced 996 bp and 1013 bp, respectively, of the cytb and ND2 dataset. The

best partitioning scheme consisted of four partitions (cytb_pos1 = K80+I;

ND2_pos2+cytb_pos2 = HKY; ND2_pos3+cytb_pos3 = GTR+G; ND2_pos1 = HKY+I). All

sequences were deposited in GenBank under the accession numbers MH484353-MH484422.

The BI analysis, and the haplotype network, recovered eight allopatric mtDNA lineages (six of

populations of G. leucogastra and two of G. chalcothorax), six represented by well-

supported clades, and two represented by single individuals (Fig. 1). Although all clades

corresponding to the allopatric lineages had strong support, basal relationships among them

were poorly supported, the only exceptions being the sister relationships between Guiana

and Negro clades of G. leucogastra and between G. chalcothorax and the Madeira lineage of

G. leucogastra.

17

Haplotypes networks were recovered for the different mtDNA clades using the

concatenated matrix of cytb and ND2 in which all missing data were discarded (final matrix =

1304 bp). The six haplotype networks recovered by TCS were separated from each other by

at least 19 connection steps. In almost all networks mutations were concentrated on

terminal branches (star-like networks) suggesting recent population expansion, with little to

no genetic diversity within lineages, except for the G. leucogastra Madeira lineage and for G.

chalcothorax, for which we recovered a different haplotype for each specimen. Samples

from different banks of the Tapajós River are separated by six mutational steps (Fig. 1: light

and dark green), and samples of G. chalcothorax (Fig.1: light and dark brown) exhibit almost

the same number of mutations among them as they do with respect to the haplotypes from

the Madeira lineage.

3.2. mtDNA genome and time tree

We recovered the complete mitochondrial genome from all samples sequenced for

UCEs. In contrast to our cytb+ND2 tree, the tree based on all the mtDNA coding genes was

highly supported (Fig. 2). Molecular dating indicates that diversification of the mtDNA

lineages started in the Middle Pleistocene, at about 1.5 million years ago (mya) (95%HPD =

2.4 - 0.75). Although all nodes were recovered with high support, the first three splits

occurred in a short period of time, with short internodes, suggesting a rapid radiation among

lineages from southern, northern and western Amazonia (Fig. 2). The earliest divergence is

suggested to have been between populations separated by the Amazon River (Fig. 2). In

both mtDNA analyses (cytb+ND2 and mtDNA genome), G. chalcothorax was recovered as the

sister-group to the G. leucogastra lineage from the west bank of Madeira River, with their

divergence dating to around 0.74 mya (95%HPD = 1.21 – 0.38), therefore rendering G.

leucogastra paraphyletic. The lineages from the north bank of the Amazon River were also

recovered as sister-groups, and diverged roughly around the same time, 0.61 mya (95%HPD

= 1 – 0.31). The most recent divergence occurred between lineages separated by the Tapajós

River at 0.28 mya (95%HPD = 0.47 – 0.13).

3.3. UCE sequencing, supermatrix analysis and Species trees

Raw reads resulted from the sequencing were deposited at the National Center for

Biotechnology Information (NCBI) Sequence Read Archive (PRJNA476145). The complete

UCE matrix, which included only those loci shared among all samples, contained 2271 UCE

18

loci, with mean locus length of 543.06 bp (see Table 1 for total number of reads, Trinity

contigs, UCE and exon loci recovered from each sample; for alignments, total number of loci,

and locus information, see Table 2). The concatenated RAxML tree recovered G.

chalcothorax as sister to all other samples of G. leucogastra with high bootstrap support

(p=100, Fig. 3). Thus, the earliest divergence is here suggested to have occurred between an

eastern and a western population, unlike the pattern suggested by the mitochondrial data.

The first split within G. leucogastra is between lineages north and south of the Amazon

River, followed by a split across the Madeira River (p=100), and then younger splits across

the Tapajós (p=96) and the Aripuanã (p=76).

For the StarBEAST species-tree we used 124 loci that had more than four parsimony

informative sites. The species-tree was identical in topology to the concatenated RAxML UCE

phylogeny, with some differences in statistical support, including two nodes without strong

support in the species-tree (p<0.95) (Fig. 3). In both the concatenated and the species-trees,

we found contrasting differences compared to the mtDNA genome tree. Besides the nature

of the earliest split in the complex, the most significant one is that the nuclear data recover

G. leucogastra as monophyletic and sister to G. chalcothorax with strong statistical support;

which contrasts with the mtDNA genomic tree where G. leucogastra was paraphyletic, and

G. chalcothorax was sister to the G. leucogastra Madeira lineage (Fig. 2). Furthermore, in the

UCE trees the G. leucogastra Aripuanã lineage (Fig. 3, dark pink) was strongly clustered with

samples distributed east of the Madeira River (Fig. 3).

4. Discussion

4.1. mtDNA and nuDNA incongruence

Historically, the Purplish Jacamar (G. chalcothorax) was considered a subspecies of

the Bronzy Jacamar (G. leucogastra) (Peters, 1948; Haffer, 1974). Parker and Remsen (1987)

proposed that the two taxa be recognized as separate species based on their distinct

phenotypes: G. leucogastra is bronzy-green, with some suffused metallic blue, and a white

belly, whereas G. chalcothorax is tinged reddish-purple, and has a black belly with only the

feathers tips being white. Although these color characters seem to fluctuate across

populations, G. chalcothorax is distinctly larger than G. leucogastra (Haffer, 1974). Parker

and Remsen (1987) also suggested that Haffer (1974) did not recognize G. chalcothorax as a

19

full species because of the supposition they would interbreed if the two taxa came together,

but they also noted (p. 98) that “… the absence of major river barriers between their ranges

suggests that no interbreeding occurs or would occur”.

The structure recovered by the mtDNA data within G. leucogastra, with five well

supported mtDNA clades, suggests that current taxonomic treatment under-represents the

diversity within this species, which currently includes only two subspecies: G. l. leucogastra

and G. l. viridissima, described based on few individuals from the Tapajós river (Griscom and

Greeway, 1941). Surprisingly, mtDNA data also revealed that G. leucogastra specimens

comprising the Madeira clade, the geographically closest to G. chalcothorax, are sister to G.

chalcothorax with high support, but with no shared haplotypes among species (Fig. 1, 2). In

contrast, the UCE concatenated RAxML tree as well as the UCE species-tree recovered G.

leucogastra and G. chalcothorax as reciprocally monophyletic sister species, with the

Madeira lineage of G. leucogastra sister to G. leucogastra lineages from SE Amazonia (i.e.

Aripuanã and Tapajós lineages, Fig. 3). Multiple explanations have been proposed to account

for conflicts in mitochondrial and nuclear genes histories (summarized in Table 3).

Incomplete lineage sorting (ILS), usually referred as one of the main cause for this

kind of discordance, is often evidenced by the paraphyly or polyphyly of single gene trees in

phylogeographic studies. The existence of ancient polymorphism is potentially observed in

recent events of diversification, where there has been insufficient time to sort all alleles for

the genes and populations studied (McKay and Zink, 2010). This is especially true in the

nuclear DNA (nuDNA), because of larger populations sizes (2Ne) and the tendency to take

twice as much time to coalesce (4Ne) when compared with mtDNA (Moore, 1995). Our

phylogeographic results seem consistent with the idea that ILS is not affecting the patterns

recovered, at least with respect to the mtDNA, since there were no shared haplotype among

the different lineages (Fig. 1). However, another effect of ILS is discordance among gene

trees histories in ancient diversification events (Oliver, 2013). At the base of our

mitochondrial genome time tree we observe three diversification events close in time, which

could have caused conflict among gene trees histories. However, when we compare the

results from the mtDNA (Fig. 2) with the UCE dataset (Fig. 3), the only difference found is the

position of Galbula chalcothorax (LSU2803). If ILS was the cause for the discordance

observed here, we would expect discordance in topology between the mtDNA and UCE data,

20

and greater discordance between the concatenated RAxML tree and the species tree

analysis. The differences found between the two UCE analyses were mainly in the support of

the relationships among G. leucogastra lineages, with the species tree analysis recovering

two nodes with low support (Fig. 3), these same nodes exhibit this pattern in the mtDNA

tree (Fig. 2). The position of G. chalcothorax, however, was recovered by both analyses with

high support, as sister to all G. leucogastra specimens. Therefore, ILS cannot be used to

explain the discordance between the mtDNA and nuDNA trees, especially regarding the

position of G. chalcothorax.

Another process that can cause similar results as ILS is gene flow. Our hypothesis for

this incongruence is that an ancient event of hybridization between G. chalcothorax and the

Madeira lineage of G. leucogastra caused the introgression of the Madeira lineage mtDNA

into the G. chalcothorax lineage, replacing its “original” mtDNA lineage. This mitochondrial

capture may have been influenced by the populational and ecological context of

differentiation within WSE. Even though the ranges of G. leucogastra and G. chalcothorax

appear to be currently allopatric (Tobias 2017), they approach each other between the Purus

and Juruá rivers (Fig. 1). Therefore, past gene flow may have been possible during drier

climatic periods in SW Amazonia (see below) (Mayle et al., 2004; Bush, 2017). MtDNA clades

found within G. leucogastra are more structured and differentiated than the clades found

within the other WSE birds, but all of them agree in recovering a well supported clade in

northern Amazonia, and with the Madeira River being an important barrier in the south

(Capurucho et al., 2013; Matos et al., 2016). The maintenance of such structured mtDNA

lineages may indicate that little or no gene flow is presently ongoing between the lineages,

suggesting that the forest matrix is a strong barrier for these birds.

Although mtDNA lineages may reflect species boundaries (Hill, 2017), recent studies

have shown a number of cases in which apparent mtDNA paraphyly is not just derived from

improper taxonomy (McKay and Zink, 2010) but also from mtDNA introgression among

adjacent populations (Drovetski et al., 2015; Shipham et al., 2015, 2017; Dias et al., 2018;

see also Toews and Brelsford, 2012). In most cases in which mitochondrial sweeps are

reported, they happened within known zones of hybridization (Dias et al., 2018; Drovetski et

al., 2015; Shipham et al., 2015, 2017). However, genetic and phenotypic data for the Galbula

leucogastra/chalcothorax suggest that there is no current hybrid zone corresponding to the

21

conflict between UCE and mtDNA signal reported here. Male-biased dispersal could explain

the mixing of nuDNA without disrupting mtDNA structure. Such sex-biased traits are

commonly used to explain discordances between mtDNA and nuDNA (Excoffier, 2009; Toews

and Brelsford, 2012). However, in a recent review of this process, Bonnet et al. (2017)

simulated several scenarios and observed that the only way to have massive discordance in

all simulations, such as the one observed in our results, without detectable nuclear

introgression, is when there is positive selection acting on mitochondrial lineages. In

addition, the mtDNA can accumulate deleterious mutations quickly, and in small

populations, drift could spread these deleterious mutations across the whole population in

short periods of time. Therefore, small populations may accumulate several deleterious

mutations and the “defective” mtDNA lineage can be supplanted by a foreign mtDNA lineage

(Hailer et al., 2012; Llopart et al., 2014; Hulsey et al., 2016; Sloan et al., 2017). This

hypothesis can be more plausible if effects of the mtDNA sweep are more beneficial than the

disadvantageous effects of mitonuclear incompatibilities (Sloan et al., 2017). Furthermore,

isolation could lead to coevolution of mitochondrial and the nuclear background genes

involved in cellular respiration, which could function as a post-zygotic barrier to gene flow,

due to Bateson-Dobzhansky-Muller Incompatibility (BDMI) (Orr, 1996). Given the

fragmented distribution of WSE in Amazonia, it is possible that the occupation of new

patches, or the fragmentation of previously continuous habitats into smaller patches of WSE

due to landscape evolution, followed by some time in allopatry, could lead to the mtDNA

structure we observe today and consequent coevolution of the nuclear background.

4.2. Biogeography of WSE avifauna

In phylogeographic studies of the Black Manakin (Xenopipo atronitens, Pipridae),

Capurucho et al. (2013) found the largest mtDNA divergences to correspond to populations

found across the Branco and Amazonas rivers. Similar results were observed for the Red-

shouldered Tanager (Tachyphonus phoenicius, Thraupidae, Matos et al., 2016), but with

greater isolation between opposite margins of the Amazon river. The divergence times

estimated between northern and southern lineages within X. atronitens and T. phoenicius

were 0.92 and 0.88 Ma, respectively, both slightly younger than the mean age estimate we

obtained for the first divergence on the mtDNA tree (~1.5 Ma, 95%HPD = 0.75 - 2.4) in G.

leucogastra, but with overlap of confidence intervals. Another WSE specialist studied, the

22

Green-tailed Goldenthroat (Polytmus theresiae, Trochilidae), showed no genetic structure,

but exhibited strong signs of recent population expansion (Matos et al., 2016). Evidence of

recent gene flow among otherwise isolated populations of the aforementioned species

contrast with the highly-structured lineages recovered here. The phylogeographic structure

found in the G. leucogastra/chalcothorax is, in fact, more similar to phylogeographic

patterns found in understory birds of terra-firme environments (Ribas et al., 2012;

Fernandes et al., 2013; Fernandes et al., 2014; Thom & Aleixo, 2015; Ferreira et al., 2017).

Although we found evidence for an ancient capture event of mtDNA lineages, there is no

evidence of current gene flow between G. leucogastra and G. chalcothorax. This may be

evidence that the current forest cover separating these two taxa differs from the forest

cover that existed when the mtDNA capture occurred (Cowling et al., 2001; Arruda et al.,

2018). Alternatively, it is possible that some intrinsic incompatibility has developed between

the two taxa. Xenopipo atronitens and G. leucogastra/chalcothorax are found in both WSE

and black-water flooded forest, T. phoenicius in WSE and savannas, and P. theresiae in WSE,

black-water flooded forest and savannas (Borges et al., 2016b). In both X. atronitens and T.

phoenicius, the authors suggest that the use of black-water flooded forests would facilitate

the connection between patches of WSE, consequently increasing the gene flow among

adjacent populations (Capurucho et al., 2013; Matos et al., 2016). Even though G.

leucogastra and G. chalcothorax are also found in black-water flooded forests (Borges et al.,

2016b), the presence of the congeneric species-complex specialized in flooded forests –

Galbula galbula, G. tombacea, G. cyanescens, and G. ruficauda – may be restraining the

dispersal of individuals of G. leucogastra/chalcothorax, due to ecological competitive

exclusion. In addition, when compared to the other WSE species, G. leucogastra and G.

chalcothorax are the only exclusive insectivores, meaning that they need not have as

extensive foraging areas as do frugivores or nectarivores (Levey and Stiles, 1992), and hence

they are potentially more prone to isolation and differentiation (Burney and Brumfield,

2009).

4.3. Evolution in the White-sand ecosystems

In western Amazonia, white sand formations predate Andean uplift, and are

probably a result of westward rivers flowing from the Guiana and Brazilian shields to the

Pacific Ocean, during the Early Miocene (Hoorn, 1993). These sandy sediments of western

23

Amazonia were reorganized and recycled multiple times within the basin during the Andean

uplift, and most of these sediments are now covered by more recent clay-rich sediments

derived from the Andes making them very scattered today. This mosaic of sediments is

reflected in soils with distinct edaphic conditions, which influence floristic composition that

ultimately influences local bird communities (Pomara et al., 2012). In the eastern Amazonia,

most WSE occurs on podzolic soils and abandoned paleochannels (Prance and Schubart

1978; Latrubesse, 2002; Nascimento et al., 2004; Sauer et al., 2007; Frasier et al., 2008;

Cordeiro et al., 2016).

Phylogeographic studies of WSE specialized birds suggest a history related to north

eastern Amazonia (Guiana region) and dispersal from there to other parts of the basin during

the Pleistocene (Whitney and Alonso, 1998; Capurucho et al., 2013; Matos et al., 2016). Also,

most of WSE birds have sister groups inhabiting other open vegetation habitats and not the

adjacent Amazonian humid forest formations, such as terra-firme or varzea (Rheindt et al.,

2008; Capurucho et al., 2013; McGuire et al., 2014; Matos et al., 2016). This suggests the

colonization of Amazonian WSE by lineages that had already evolved in open habitats,

instead of repeated adaptations in multiple lineages from neighboring humid forest. In this

sense, Galbula leucogastra and Galbula chalcothorax are unlike other WSE taxa since all

other Galbula species are found in forest habitats (Witt, 2004; Tobias, 2017).

The WSE were probably more widespread throughout the continent before Andean

uplift, thus extant WSE lineages of birds may be resilient species capable of enduring the

reconfiguration of the Amazon basin (Campbell et al., 2006; Hoorn et al., 2010; Nogueira et

al., 2013). The pattern of greater genetic diversity in the east we observe today should be

then related to the fact that during the Pleistocene climatic cycles, eastern Amazonia

experienced greater fluctuations in precipitation (Wang et al., 2017). Although these cyclical

oscillations were not enough to entirely replace forest with savannas (Bush, 2017; Wang et

al., 2017), they may have affected forest structure (Cowling et al., 2001; Barthe et al., 2017;

Arruda et al., 2018). This could have facilitated contact between different patches of WSE in

the east, especially for birds that can use black-water flooded forest, allowing them to

expand their distribution and colonize previously unoccupied patches of WSE.

In contrast, the paleoclimatic record suggests that western Amazonia remained as

humid as it is today throughout the Pleistocene (Cheng et al., 2013; Wang et al., 2017).

24

Nowadays, after the main phases of Andean uplift, the existence of WSE in western

Amazonia would occur only in scattered patches in recycled quartzite soils reminiscent of

ancient fluvial deposits (Hoorn, 1993; Latrubesse, 2002). This scenario, however, contrasts

with the postulated past gene flow between G. chalcothorax and G. leucogastra. An

explanation to this biological evidence of a different landscape in the past in southwestern

Amazonia, may be related to the subduction of the Nazca Ridge under the South American

plate. This event may have caused the uplift of the Fitzcarrald Arch (Espurt et al., 2010),

affecting the drainage system and causing the erosion of the clay-rich sediment layer and

exposure the nutrient poor sediment layer below. This change in the edaphic condition,

coupled with climate oscillations may have periodically expanded WSE distribution in

southwestern Amazonia, facilitating the contact between currently isolated lineages of G.

leucogastra and G. chalcothorax.

5. Conclusion

Here we showed an instance of clear discordance between phylogenetic relationships

recovered using mtDNA and nuclear data in our study taxa. Nuclear data agrees with current

taxonomy, which is based on phenotypic patterns, while the mtDNA relationships seem to

be related to an old event of mtDNA capture. The capture event relates to what is currently

known about the distinct biogeographical histories of WSE in Eastern and Western

Amazonia, especially regarding the past distribution of WSE in western Amazonia

throughout the Pleistocene. While these results raise important issues about apparent

discordances between mtDNA clades and current taxonomy, they also show that interesting

biogeographic histories can be uncovered when enough genetic data with different and

independent histories are available. Nonetheless, this study will be an important

contribution of NGS for studies for recent speciation and taxonomy.

Acknowledgements

We thank the curator and curatorial assistants of the Academy of Natural Science of Drexel

University, Philadelphia, USA (ANSP); Field Museum of Natural History, Chicago, USA

(FMNH); Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil (INPA); Lousiana State

University Museum of Natural Science, Baton Rouge, USA (LSUMZ); and Museu Paraense

25

Emílio Goeldi, Belém, Brazil (MPEG), for loaning tissue samples under their care. We thank S.

W. Cardiff and N. Rice for helping us with LSUMZ and ANSP specimens, respectively. We are

also grateful for all collectors involved in fieldwork throughout Amazonia who made this

paper possible. We also thank the Bird 10k Project Committee for allowing access to the

genome sequence of Galbula dea. We thank J. M. G. Capurucho and S. H. Borges for early

inputs on this paper and the two anonymous reviewers. The authors also acknowledge the

National Laboratory for Scientific Computing (LNCC/MCTI, Brazil) for providing HPC

resources of the SDumont supercomputer, which have contributed to the research results

reported within this paper.

Funding

Support to M.F.’s graduate research was provided by CAPES PhD and PDSE (#

88881.133440/2016-01) fellowships, and by AMNH Frank M. Chapman Memorial Fund. Post-

doctoral fellowship to A.M.F. was provided by CNPq (#500488/2012-6). A. Aleixo and C.C.R.

are supported by CNPq research productivity fellowships. Research was partly covered by

grants to C.C.R. (PEER/USAID program, cycle 5), A. Aleixo (CNPq # 471342/2011-4 and

FAPESPA # ICAAF 023/2011) and A.Antonelli from the European Research Council under the

European Union’s Seventh Framework Programme (FP/2007-2013, ERC Grant Agreement n.

331024), the Knut and Alice Wallenberg Foundation through a Wallenberg Academy

Fellowship, the Swedish Research Council (2015-04857), the Swedish Foundation for

Strategic research, the Faculty of Sciences at the University of Gothenburg, the Wenner-

Gren Foundations, and the David Rockefeller Center for Latin Amarican Studies at Harvard

University. A.Aleixo, C.C.R., J.M.B., J.C. and M.F. were supported by the grant Dimensions

US-Biota-São Paulo: Assembly and evolution of the Amazon biota and its environment: an

integrated approach, co-funded by the US National Science Fundation (NSF DEB 1241056) to

J.C. and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant

#2012/50260-6) to Lucia Lohmann.

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Author contibutions

M.F. and A.M.F. developed the sampling plan, extracted DNA and sequenced all samples. M.F.

performed all analysis. A.A.P., A.A., U.O., J.M.B., J.C. and C.C.R. were involved in intellectual merit,

funding, and writing. All authors participated in writing the manuscript.

Supporting information

Additional supporting information may be found in the online version of this article.

Table S1 Supplementary details of individuals.

33

Table 1 - Samples used for UCE sequencing, their voucher numbers, general locality, number of clean reads after Illumiprocessor, number of contigs assembled by Trinity, and total UCE loci recovered from Trinity.

Species Museum voucher Locality Clean reads Trinity contigs UCE loci

G. chalcothorax LSUMZ B2803 N of Napo River, Iquitos, Peru 1,524,126 6,537 2,230

G. leucogastra INPA A4182 145 Km WWS of Apuí, AM, Brazil 2,540,148 12,163 2,269

G. leucogastra INPA A4672 Right bank of Jatapú River, AM, Brazil 2,209,895 9,491 2,263

G. leucogastra LSUMZ B35619 Arapiuns River, PA, Brazil 4,394,658 10,853 2,246

G. leucogastra LSUMZ B9608 Nicolás Suarez, Pando, Bolívia 1,677,988 5,074 1,928

G. leucogastra MPEG 59360 Novo Airão, AM, Brazil 2,372,950 6,026 1,957

G. leucogastra MPEG 75618 Right bank of Tapajós River, PA, Brazil 1,346,149 6,117 2,263

G. leucogastra MPEG 73685 Novo Aripuanã, AM, Brazil 1,466,240 6,896 2,227

G. albirostris INPA A064 Amazonas, Brazil 2,809,416 16,718 2,256

Table 2 – Summary of each method, including number of loci, total length, mean length size of each loci, minimum and maximum length, number of Parsimony Informative sites.

Method Complete Exons Species Tree†

Number of loci 2271 47 124

Total length (bp) 1,233,287 47,580 80,085

Mean length size (bp) 543.06 849.64 645.85

Min - Max length (bp) 118 – 1,305 182 - 3093 347 – 3093

Number of PI sites (mean) 2003 (0.88) 190 (3.39) 744 (6)

†without the mtDNA

Table 3 – Possible causes of conflict in mitochondrial and nuclear DNA histories.

Inferred process Reference

Incomplete lineage sorting Funk and Omland, 2003; McKay and Zink, 2010;

Zink and Barrowclough, 2008

Incomplete sampling Shipham et al., 2015, 2017

Improper taxonomy McKay and Zink, 2010

Adaptive introgression Bock et al., 2014; Dobler et al., 2014

Demography or Sex-biased traits Bonnet et al., 2017; Daly-Engel et al., 2012;

Rheindt and Edwards, 2011; Sloan et al., 2017

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Figure 1 - Map of sequenced individuals, phylogenetic Bayesian tree recovered, and haplotype networks. The colors in the tree, map and networks are correspondent, and the tree and networks are based on two mtDNA genes (2009 bp, cytb and ND2). Posterior probabilities obtained at each node are indicated on the tree, red circles represent pp=1. The brown labeled points are G. chalcothorax, all other lineages are G. leucogastra. Terminal names in red are samples used in the UCE analysis. Circles sizes in the haplotypes network correspond to number of individuals sharing the haplotype. Maximum number of connection steps for the haplotypes networks is 19.

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Figure 2 – Chronogram recovered by BEAST using all mtDNA coding genes with a calibration derived from the mutational rate of the cytb gene (Weir and Schluter 2008). Posterior probabilities obtained at each node are indicated in the tree, red circles represent pp>98, associated confidence interval (95% HPD) for diversification time (blue bar), and the median time of divergence. Colors are correspondent with Figure 1.

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Figure 3 - Comparison between the concatenated UCE RAxML tree (left) and the StarBEAST2 species tree (right). Bootstrap support for the RAxML tree, and the posterior probability for the StarBEAST species tree, is show near the nodes. Colors are correspondent with Figure 1.

37

Capítulo 2

Ferreira, M.; Aleixo, A.; Bates, J. M.; Cracraft, J.;

Ribas, C. C. Phylogenomics of trogons (Aves:

Trogonidae) shed light on the Quaternary

biogeography of tropical forests and the connections

between Asia, North and South America. Manuscrito

formatado para Molecular Biology and Evolution

38

Manuscript submission to Molecular Biology and Evolution

Contribution type: Article

Phylogenomics of trogons (Aves: Trogonidae) shed light on the Quaternary

biogeography of tropical forests and the connections between Asia, North

and South America

Ferreira, Mateus1*; Aleixo, Alexandre2; Bates, John M.3; Cracraft, Joel4; Ribas, Camila C.5

1 Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, INPA,

Manaus, AM, Brazil 2 Coordenação de Zoologia, MPEG, Belém, PA, Brazil 3 Department of Ornithology, FMNH, Chicago, IL, USA 4 Department of Ornithology, AMNH, New York, NY, USA 5 Coordenação de Biodiversidade, INPA, Manaus, AM, Brazil

*Corresponding author

Correspondence: Mateus Ferreira, Coordenação de Biodiversidade, Instituto Nacional de

Pesquisas da Amazônia, CEP 69080-971, Manaus-AM, Brazil

E-mail: [email protected]


39

Abstract

The pantropical distribution of trogons always drew attention of biogeographers, with

species distributed all over the forests regions of subtropical and tropical Africa, Asia and

America, several studies tried to reconstruct the phylogenetic relationships without, however,

being able to achieve conclusive results. For the first time, all genera and almost all currently

recognized species, 43 out of 45, were sampled and sequenced for thousands of ultraconserved

elements (UCE) to reconstruct the family phylogenetic hypothesis. We analysed the

concatenated dataset using different treatments for missing data with RAxML and ExaBayes,

we also estimated a species tree using SVDquartets. We also estimated a fossil calibrated time

tree for trogons diversification sampling 177 individuals of the Core Landbirds for RAG1 and

RAG2 genes. Our results were congruent among all methods with high nodal support,

disagreement between treatments (Species Tree x concatenated) were observed only at the basal

nodes. In general, our results support the monophyly of the different biogeographical regions,

with Apaloderma species being sister to the Asian (Harpactes and Apalharpactes) and the

Neotropical trogons (Euptilotis, Pharomachrus, Priotelus, and Trogon). Trogonidae initial

diversifications occurred around 20 Ma, and continued till the Pleistocene, where most of the

Neotropical species appeared. Based on these results, we proposed how the climate changes

since the Late Oligocene influenced forest distributions and how the establishment of land

bridges between continents helped shape the family diversification.

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Introduction

The Trogonidae have some of the most colourful and exquisite plumages among birds.

Representatives of this family, usually known as trogons or quetzals, can be found in forested

tropical and subtropical regions of Africa, Asia and America (Collar 2017). The monophyly of

the family was never questioned due to the morphological homogeneity among species

(Livezey and Zusi 2007; Collar 2017), the most iconic feature that differentiate trogons and

quetzals from other birds is the heterodactyl foot, in which digits 1 and 2 are directed backwards

and the basal half of digits 3 and 4 are fused and directed forward (Maurer and Raikow 1981;

Mayr 2009). However, it is precisely this unique feature that makes trogons so difficult to relate

with extant birds. Despite several attempts to reconstruct the relationship between trogons and

other birds, most of the morphological (Cracraft 1981; Maurer and Raikow 1981; Mayr 2003;

Livezey and Zusi 2007) and the first molecular analyses (Monteros 2000; Hackett, et al. 2008;

McCormack, et al. 2013) were unable to recover conclusive results about their phylogenetic

relationships. Only recently, employing genomic representations, trogons were shown to be a

sister group to a clade containing mousebirds (Coliiformes), cuckoo rollers (Leptosomiformes)

and other Core Landbirds (Jarvis, et al. 2014; Prum, et al. 2015).

Although the relationship with other birds is partially resolved, the relationships within

the family are still pending conclusive results. Historically, the genera and species within each

biogeographic region were considered monophyletic. The highest diversity is found in the

Neotropical region, with four genera, Euptilotis, Pharomachrus, Priotelus and Trogon, and ~30

species ranging from southwestern USA to northern Argentina. The Indo-Malaysian region

comprises 2 genera, Apalharpactes and Harpactes, and 12 species, ranging from southern India,

Southeast Asia, Philippines, the Malay Peninsula, Borneo, Philippines, Sumatra and Java, while

the African region includes only one genus, Apaloderma and tree species. Although trogons are

currently found only in tropical and subtropical regions, fossil records indicate that they had a

wider distribution in the past. Two fossils from Europe, Primotrogon wintersteini (Mayr 1999)

from the Middle Oligocene, and ?P. pumilio (Mayr 2005), from the Middle Eocene, are credited

to be sister group to all other extant species (Mayr 2009). Whereas Septentrogon madseni

(Kristoffersen 2002), from the transitional Paleocene-Eocene Fur Formation in north-western

Denmark shares morphological characteristics that put him inside the Trogonidae. The presence

of these fossils in Europe suggests a widespread lineage occurring in regions that are currently

unsuitable for them. The similarity between fossils and extant trogons also indicates that this

lineage suffered little morphological changes through time. This apparent conservatism of

41

morphological characteristics also makes the inferences of phylogenetic relationships among

extant species difficult.

The first molecular phylogenetic hypothesis for trogons was based on two mitochondrial

genes and included 20 out of the ca. 45 species (Monteros 1998). This study supported the

hypothesis of monophyly of the biogeographic regions, recovering the Neotropical genera sister

to the Asian, with the African clade sister to these two (Monteros 1998). Following studies that

increased the number of genes and/or samples, however, couldn’t recover the monophyly of the

Neotropical genera, nor the relationship among the different regions (Johansson and Ericson

2005; Moyle 2005; DaCosta and Klicka 2008; Hosner, et al. 2010). The most recent paper

(Hosner, et al. 2010), and the first one to sample the genus Apalharpactes, recognized six clades

(Apaloderma, Apalharpactes, Harpactes, Pharomachrus/Euptilotis, Priotelus, and Trogon)

with uncertain relationships among them, but showing evidences of Apalharpactes being more

closely related with the African Apaloderma, than to the other Asian genus, Harpactes,

implying a very complex biogeographical pattern, with two independent colonizations of Asia.

A similar pattern suggested for the Neotropical genera, which group three distinct clades

(Hosner, et al. 2010).

This uncertainty regarding phylogenetic relationships so far was probably related to the

scarcity of signal due to a low number of loci employed in previous studies. Genomic analyses

using a reduced representation of the genome can increase phylogenetic information and avoid

confounding the histories of single genes with the species relationships (Degnan and Rosenberg

2009; Knowles 2009). Also, since the correct interpretation of biotic evolution can shed light

on the landscape evolution (Baker, et al. 2014), a robust and well supported phylogenetic

hypothesis is of extreme importance for defining hypothesis in biogeography (Donoghue and

Moore 2003; Lexer, et al. 2013). In this sense, a prominent approach to study systematics using

genomic markers is the use of probes for Ultraconserved Elements (UCE)(Faircloth, et al. 2012;

McCormack, et al. 2012; McCormack and Faircloth 2013; McCormack, et al. 2013; Faircloth,

et al. 2015). These probes, have been employed to reconstruct deep (Faircloth, et al. 2015;

Moyle, et al. 2016; Branstetter, et al. 2017; Esselstyn, et al. 2017) and shallow (Bryson, et al.

2016; Manthey, et al. 2016) phylogenetic relationships, even where high incomplete lineage

sorting is expected, such as in cases of rapid evolutionary radiation (Meiklejohn, et al. 2016).

Therefore, trogons represent a great study model on how genomic representation may

elucidate uncertain phylogenetic relationships, and to understand how the landscape evolution

shaped the family diversification, due to its pantropical geographic distribution and preference

42

for forested habitats. Here, we aim (1) to generate and unprecedent and robust analyses of

phylogenetic relationships within the Trogonidae family, using nearly complete sampling of all

recognized speces and a genomic representation of more than 2,000 UCE loci, (2) to investigate

the monophyly of main biogeographical regions, and (3) to reconstruct a calibrated tree to infer

the timing of diversification, and how it was influenced by the global events on geography and

climate.

Results

UCE sequencing

The reference sequences we extracted from the Apaloderma vittatum genome (Gilbert,

Jarvis, Li, Consortium, et al. 2014) included 2,228 loci. The mean number of sequences for

each individual was 2,080,592, and a mean number of UCE loci was 2,222, with only one toe

pad sample (AMNH 322898) recovering less than 2000 loci (Table 1). The complete matrix

contained 1421 loci, with mean locus length of 510.27 base pairs, and a total of 37,880

parsimony informative (PI) sites, mean of 26.6 per locus (Table 2). The incomplete matrices

with 95% and 75% completeness have 2,210 and 2,217 loci, with mean locus length of 499.77

and 495.95 base pairs, and 55,060 and 57,259 PI sites, with mean of 24.91 and 25.83 sites per

locus (Table 2).

Phylogenetic inference

The tree topologies were congruent among all methods and with high node support, apart

from the SVDq analyses, in which the basal nodes presented low support. The concatenated

RAxML and ExaBayes phylogenies recovered the Asian trogons sister to the Neotropical, and

these two sisters to the African clade with high support (Fig. 1). All the ExaBayes analyses,

including the complete and the two incomplete datasets, recovered the same topology with all

nodes with the maximum posterior probability (Fig. 1). Although the topologies recovered by

RAxML trees were congruent with ExaBayes, some of the basal nodes received low support.

The same was observed with SVDq.

Within the Asian group, Apalharpactes was sister to Harpactes, but with low support in

the RAxML (Table 3) analyses. Within Harpactes we recovered three groups: (1) the distinct

H. oreskios; (2) the two small-bodied species H. duvaucelli and H. orrhophaeus; and (3) the

large-bodied species, containing the other species, with clearly defined and high support

supported relationships (Fig. 1). The Neotropical clade was recovered with high nodal support

(Table 3), showing the quetzals, Euptilotis and Pharomachrus, as sister to Priotelus and Trogon

43

(Fig. 1). Pharomachrus moccino, the only Central America species, is sister to all other

Pharomachrus species. The two Andean species, P. antisianus and P. auriceps, are not closely

related (Fig. 1). Within Trogon, the most diverse genus in the family, we recovered 5 clades,

all of which include species at both sides of the Andes (Fig. 1).

Time-calibrated tree

The concatenated matrix of RAG1 and RAG2 sequences includes 4757 base pairs for 177

representatives of the Core Land birds (Claramunt and Cracraft 2015; Prum, et al. 2015)

(Supplementary Table 1). Phylogenetic analysis of this matrix recovered a well-supported tree.

Trogonidae diversification started in the Early Miocene, the first of four divergence events are

close to each other, around 20 Ma (Fig. 2). While the Asian species originated during the Late

Miocene/ Early Pliocene, most Neotropical species originated during the Late

Pliocene/Pleistocene (Fig. 2).

Discussion

Phylogenomic contribution to the reconstruction of Trogonidae diversification

Recovering basal relationships in the Trogonidae phylogeny has proven to be challenging,

and previous studies have failed to resolve the relationships among genera (Monteros 1998;

Mayr 2003; Johansson and Ericson 2005; Moyle 2005), either because of incomplete taxon

sampling or inadequate number of markers. Monteros (1998) using only two mtDNA genes

recovered a tree topology similar to the one we recovered, in which taxa from different

biogeographical regions were monophyletic. However, the relationships among genera were

not well supported, and Apalharpactes was not sampled. Johansson and Ericson (2005), and

then Moyle (2005), increased the sampling and added a few nuclear introns, yet there were few

improvements in phylogenetic resolution. Moyle (2005) recovered a paraphyletic Neotropical

group, with the quetzals being sister to all other genera, and the Asian and African group sister

to each other embedded within Trogon and Priotelus. Johansson and Ericson (2005) based on

a combined analysis of mtDNA and three nuclear introns recovered a topology similar to ours,

however, node support for the Neotropical group, and the node grouping Asia and the

Neotropics, received low to moderate support. Hosner, et al. (2010) were the first to include an

Apalharpactes sample, but their results were also inconclusive, as relationships among genera

were poorly supported and biogeographical groups, except for Africa, were not monophyletic.

Our phylogenetic results were the first to recover with moderate to high support the

relationship of almost all currently recognized species, as our analyses recovered most of the

44

nodes with high statistical support (Fig. 1). The nodes that did not receive full support at the

base of the tree (Table 3) are connected by short internodes, probably as a result of an ancient

rapid radiation (Whitfield and Lockhart 2007). Recurrent issues arising from rapid radiations

usually include incomplete lineage sorting (ILS), represented by conflict among gene trees due

to successive events of speciation in short periods of time, which can be accentuated by large

population sizes (Oliver 2013; Suh, et al. 2015). ILS probably was also the main cause of low

support in previous studies that employed few genetic markers, as they could have conflicting

histories (Knowles 2009; Oliver 2013) and probably lacked strong phylogenetic signal to

recover the deep phylogenetic relationships (Salichos and Rokas 2013). Evidence of gene tree

incongruence was strongly observed in the whole-genome analysis of bird diversification,

where there was no single gene tree that fully corroborated the combined topology (Jarvis, et

al. 2014). However, counterintuitive, increasing the number of markers does not necessarily

means an improvement in poorly supported nodes. Instead, expanding the number of markers

increases the probability of discordance among them (Oliver 2013), and thus, notably in events

of rapid radiation, some divergences are expected not to behave as a fully bifurcating tree, but

more like a network (Bapteste, et al. 2013; Suh, et al. 2015) because most genes will have

discordant histories due to ILS (Degnan and Rosenberg 2006). Therefore, concatenation may

be the best approach when the number of possible sites supporting a relationship is concentrated

in a few loci diluted in a high number of loci affected by ISL (Gatesy and Springer, 2014;

Springer and Gatesy, 2016). Nonetheless, based on our results, after the first events of

diversification, most of nodes were recovered with high statistical support for all analysis,

including the Neotropical node, which means that, even though we probably do not have enough

confidence to allege the correct order of events that trogons went through their initial

diversification, we may still infer some hypothesis based on current distribution and ecology.

Diversification and biogeography of Trogons

Trogons are still-hunting predators feeding on insects or small vertebrates, but most of

Asian and Neotropical species also feed on fruits, with quetzals being mostly frugivores. They

inhabit the midstory and canopy of tropical and subtropical forest, with some species occurring

in forested patches of open habitats (e.g. Trogon curucui). Most species are territorialists, with

small territories, and lack the capacity to fly over long distances, usually flying from perch to

perch in short sallies (Collar 2017). The morphological conservatism of fossils compared to

extant species suggests that trogons have not underwent large ecological shifts (Mayr 1999,

2003; Mayr 2005), hence their historical distribution probably was affected by the distribution

45

of suitable habitats through time. Although nowadays there is no continuous patch of suitable

habitats, i.e. forested habitat, between Africa, Asia and America, during the Early Miocene, due

to a warmer climate, most of the dry land was covered by forest habitats, such as the broad-leaf

deciduous (Mixed Mesophytic) forest that covered most of the Northern Hemisphere (Baskin

and Baskin 2016), and forests dominated by deciduous conifers that extended even over the

Article Circle (Jahren 2007; Jahren and Sternberg 2008).

The abundance of forests during the Tertiary is due to both warmer temperatures and

twice the current amount of CO2 concentrations (Zachos, et al. 2001). However, after the

Eocene Climatic Optimum (52 to 50 Ma), in which global mean temperatures were 8-10°C

higher, the world temperature started to cool down with two climatic aberrations, where the

amount of ice in polar regions increased drastically. The first one, known as Oi-1, happened

just above the limits between Eocene and Oligocene (34 Ma) (Zachos, et al. 2001), this

glaciation event caused rapid expansions of Antarctic continental ice-sheets and global

temperatures remained low until a warming trend at the end of Oligocene (Zachos, et al. 2001).

This warm phase that followed extended from the Late Oligocene until middle Miocene (~15

Ma) with the Mid-Miocene Climatic Optimum (17 to 15 Ma) and it was followed by a gradual

cooling, with the culmination in the Glacial cycles throughout the Plio/Pleistocene (Zachos, et

al. 2001). The second aberration, Mi-1, happened during this warm period at the end of the

Oligocene (~23 Ma), and was followed by a series of glaciation events (Zachos, et al. 2001),

period well within the confidence interval for the initial diversification events we recovered in

our time calibrated phylogeny. Both aberrations probably influenced the distribution and rates

of diversification in some groups that have similar distributions as trogons, such as ferns

(Bauret, et al. 2017; Hennequin, et al. 2017), and flowering plants (Li, et al. 2017). Interestingly,

other groups of birds that have similar distributions present different patterns of diversification

than trogons; woodpeckers (Aves: Picidae) and kingfishers (Aves: Alcedinidae) apparently

have dispersed to the New World from the Old World more than once, however these events

seem to be younger than those we recovered for trogons, around 15 to 5 Ma for woodpeckers

(Shakya, et al. 2017), and 10 to 5 Ma for kingfishers (Andersen, et al. 2017). This pattern

suggests that dispersal between Asia and America was possible during a long period of time,

probably experiencing cycles of connection and disconnection due to climatic variations

(Zachos, et al. 2001). Therefore, our temporal framework supports an ancestral lineage

distributed over the Palearctic region (Claramunt and Cracraft 2015), with dispersal to Asia,

46

Africa and America during a short period of time, causing the poorly supported nodes we

observed in our analysis.

Africa and Asia diversification

Even though African and Asian linages are as old as the Neotropical, only 6% and 31%

of species diversity are found in these areas, respectively. Although contentious, there are

probably many reason for the uneven diversity among areas. Monteros (1998) suggests that

competitive exclusion might play a role in this pattern, as African and Asian trogons need to

compete with other groups of frugivores birds, such as mousebirds (Colliformes), hornbills

(Bucerotidae), barbets (Megalaimidae and Lybiidae), turacos (Musophagidae), and several

families of passerines (Irenidae, Pycnonotidae, etc). While the Neotropical trogons are, along

cotingas (Cotingidae) and toucans (Ramphastidae), one of the most important family for seed

dispersal in this region (Collar, et al. 2017).

Inside Africa, except for Apaloderma narina which has six recognized subspecies, the

other two, A. vittatum and A. aequatoriale are monotypic (Collar 2017). However, no

phylogeographic study was conducted to evaluate genetic structure within these species, with

recent studies using other organism as models showing shallow genetic structure probably

originated by aridification of the continent as a response of Plio/Pleistocene climatic

fluctuations (Bowie, et al. 2004; Bowie, et al. 2006; Voelker, et al. 2010). The diversification

event we recovered between A. vittatum and A. narina happened around 7.4 Ma (Fig. 2) and

precedes the beginning of the most drastic climatic fluctuations of the Pliocene, making any

assumption of what may have caused this very hard, in particular considering that Africa has

been geomorphologically stable for the last 40 Ma (Potts and Behrensmeyer 1992). Also, A.

vittatum inhabits the montane forests, while A. narina and A. aequatoriale, inhabits the

lowlands, and although we could not sample A. aequatoriale, previous work recovered it as

sister species to A. narina (Hosner, et al. 2010). Suggesting that other mechanisms may be

responsible for Apaloderma species diversification (Moritz, et al. 2000).

In contrast with previous studies (Hosner, et al. 2010), our analyses recovered the

monophyly of Asian trogons. Although the bootstrap support was moderate for this node in the

likelihood analysis, it was recovered with high statistical support in the Bayesian analysis

(Table 3). This suggest that after the initial diversification of the family, at least two Paleartic

lineages (Claramunt and Cracraft 2015) colonized the Sundaland, the continental shelf that

extended from SE Asia and comprises the Malay Peninsula, and the islands of Borneo, Java,

and Sumatra. The time of diversification we found for Apalharpactes and Harpactes is

47

consistent with the Hymalayan uplift acceleration, derived from India-Asia continental collision

(Hall 2012; Hu, et al. 2017), and with the intermittent glaciations that followed the Mi-1

glaciation at the Oligocene-Miocene boundary (Zachos, et al. 2001). These two events

combined may have shaped Asian trogons diversification, however, making assumptions about

Haparctes diversification involves a very complex history, and it is difficult based on extant

species distribution to make any assumption about possible biogeographic barriers. Current

geography of SE Asia and the Sunda islands can be misleading, the Sunda shelf was once

exposed and covered by forest (Hall 2012; Bruyn, et al. 2014), and sea-level fluctuations were

responsible for islands “formation” and connectivity, especially during the climatic fluctuations

of the Pleistocene (Woodruff 2010). This mechanism is suggested as a possible explanation for

Southeast Asia bird diversification (Lim, Rahman, et al. 2010; Lim, Zou, et al. 2010; Lim, et

al. 2017). However, most of the Harpactes diversification events precede the Pleistocene, and

occurred between the Mid-Miocene Climatic Optimum (17-15 Ma) (Zachos, et al. 2001) and

the Early Pliocene, much older than the diversification events of the Neotropical clade, for

example. The only phylogeographic study conducted so far, with the Philippine Trogon

(Harpactes ardens), demonstrated geographical structure among different island matching

subspecies distribution (Hosner, et al. 2014), whereas H. kasumba, H. diardii and H.

erythrocephalus showed little to no genetic variation in the mtDNA for the few samples used

(Hosner, et al. 2010). Therefore, further studies, with broad sampling are necessary to

understand how the Pleistocene climate, and sea level fluctuation, influenced population

structure, which in turn may shed some light on the initial diversification of this genus.

Neotropical diversification

For the first time, Neotropical trogons were recovered as a monophyletic group with high

statistical support (Monteros 1998; Johansson and Ericson 2005; Moyle 2005; Hosner, et al.

2010). Although most of extant diversity is currently found in Central and South America,

trogons arrived first in the Americas through the Beringia Bridge, northwest North America,

and colonized the whole west coast, during a period when there were vast forests covering

North America (Baskin and Baskin 2016). Therefore, tracing back the events related with the

initial divergences would require extensive palaeontological investigation. The overall trend we

observe in this clade diversification is that Central American lineages occupied South America

through the Panamanian Isthmus, and most of divergence events postdate the Mid-Miocene

Climatic Optimum (17-15 Ma), which marks the beginning of the cooling trend that escalated

to the Plio-Pleistocene glaciations. Also during this period, there was extensive orogenic

48

activity in Mexico, including the uplift of Sierra Madre Occidental (34 – 15 Ma) (Ferrari, et al.

2007) and the formation of the Trans-Mexican Volcanic Belt (35 – 2.5 Ma) (Ferrari, et al. 2000).

Both events triggered climatic changes, which in turn influenced the establishment of major

biomes in Mexico (Ferrari, et al. 1999), that have been shown to have influenced diversification

in Amazillia hummingbirds (Ornelas, et al. 2014), and some plants (Lavin, et al. 2004; Becerra

2005; Arakaki, et al. 2011).

Another major event that shaped Neotropical trogons diversification was the

establishment of the connection between North and South America, through the uplift of the

Isthmus of Panama. The Great American Biotic Interchange allowed inter-continental exchange

of biotas that were previously isolated in both continents and is of great importance for shaping

bird assemblages and diversification (Weir, et al. 2009; Smith and Klicka 2010). Early studies

suggested that the connection was only fully established at 3 Ma (Haug and Tiedeman 1998;

Coates and Stallard 2013; Odea, et al. 2016), however, even though contentious in the literature

(Farris, et al. 2011; Montes, et al. 2012; Bacon, et al. 2013; Bacon, et al. 2015a, b; Hoorn and

Flantua 2015; Lessios 2015; Montes, et al. 2015; Odea, et al. 2016), this date was broadly used

as a calibration point in phylogenetic studies attempting to integrate and synthesize patterns of

dispersion across the Isthmus (review in Bacon, et al. (2015a)). Our results suggest that trogon

dispersion across the Isthmus started as early as 6.5 Ma, with the split of Pharomachrus

moccino from the other Pharomachrus species, and happened at least six additional times

within Trogon diversification, all of them after 4 Ma. These results are also supported by a

former study using only one mitochondrial marker for Trogon (DaCosta and Klicka 2008).

Finally, the most notorious accomplishment of Neotropical trogons was to colonize the

Greater Antilles. The genus Priotelus, which includes species endemic to the islands of Cuba,

P. temnurus, and Hispaniola, P. roseigaster, split from Trogon around 17 Ma (Fig. 2). Trogons

are well known for being weak fliers, so the chances of the ancestor of Priotelus to have

dispersed through the ocean to colonize not just one, but two Caribbean islands are low. One

possible explanation is the land bridge that once connected Central America to South America,

known as GAARlandia (Greater Antilles + Aves Ridge) land bridge (Iturralde-Vinent 1994,

2006). Although this land connection is credited to be much older (35 – 33 Ma) (Alonso, et al.

2011; Rícan, et al. 2013; Nieto-Blázquez, et al. 2017) than the split of Priotelus and Trogon,

during the Middle-Late Miocene, the emerged islands that were part of the land bridge were

still connected by shallow seas (Iturralde-Vinent 2006), and sea levels fluctuations may have

facilitated the dispersal to these islands. Fabre, et al. (2014) studying Caribbean rodents found

49

a similar age (16.5 Ma) for the subfamily of rodents that occupy the Greater Antilles. However,

the sister group is from South America, and the authors suggested that the ancestor of this group

colonized the Caribbean Islands via rafting. Our results imply in a more complex scenario for

the Greater Antilles colonization, and further studies are required to evaluate this late

connection.

Conclusion

In this study we recovered the phylogenetic relationships among almost Trogonidae taxa

using a genomic approach. Coupled with our fossil calibrated time tree, we were able to propose

a model of diversification that related not only how the climate change since the Late Oligocene,

but also the connections between continents, shaped the family diversification. The monophyly

of the different biogeographical regions was recovered, and even though some nodes at the base

of the tree received low support, the pattern of rapid radiation is clear at the initial stages of

trogons diversification. Also, even though trogons are currently restricted to subtropical and

tropical regions, they were widespread lineages in the past, and their diversification was

influenced by forest distribution through time. Our results also identified some interestingly

new questions to be pursued: Are Neotropical trogons species really younger than African and

Asian, or is it just a sampling artifact? What was the influence of past sea level fluctuations in

the diversification of Harpactes? Is competition preveting diversification in Apaloderma?

Materials and Methods

Taxon sampling and DNA extraction

We sampled 48 individuals comprising all genera and currently recognized species of the

Trogonidae family, except for the African Bare-cheeked Trogon (Apaloderma aequatoriale),

and the narrow endemic Javan Trogon (Apalharpactes reinwardtii) (Collar 2017; Gill, et al.

2018; Remsen, et al. 2018). All samples are represented by voucher specimens deposited in

ornithological collections at the American Museum of Natural History (AMNH), Academy of

Natural Sciences of Drexel University (ANSP), Field Museum of Natural History (FMNH),

Instituto Nacional de Pesquisas da Amazônia (INPA), Kansas University (KU), Laboratório de

Genética e Evolução Molecular de Aves - USP (LGEMA), Louisiana Museum of Natural

History (LSUMZ), Museu Paraense Emílio Goeldi (MPEG), Smithsonian Institution National

Museum of Natural History (USNM) and Burke Museum (UWBM) (Appendix S1).

50

DNA from fresh tissue was extracted with the DNeasy kit (Qiagen Inc.), following the

manufacture’s protocol. For taxa lacking fresh tissues we cut toepad clips from museum

specimens with a sterile surgical blade and processed in a dedicated room for ancient DNA

(aDNA Lab, AMNH). Toepads were rinsed with 100% ethanol, and ultra-pure water prior to

digestion to remove any inhibitor that could cause problems in downstream procedures. We

then extracted DNA with the DNeasy kit (Qiagen Inc.), replacing the regular silica columns

with the QIAquick columns, to ensure maximum DNA yield. All extracts were sent to Rapid

Genomics (Gainsville, FL) for library prep and target-capture sequence 2321 loci of

Ultraconserved Elements (UCE) plus 98 conserved exons from 46 genes that were previously

employed in phylogenetic analyses (Hackett, et al. 2008; Kimball, et al. 2009; Harvey, et al.

2017).

UCE and exons assembly

The raw sequence data were processed with the Phyluce script pack (Faircloth 2016). We

employed illumiprocessor (Faircloth 2013) and Trimmomatic (Bolger, et al. 2014) to remove

adapter contamination and low-quality reads. To assemble a reference genome, we mapped the

UCE and exons probes back to the Apaloderma vittatum genome (Gilbert, Jarvis, Li,

Consortium, et al. 2014) using the script phyluce_probe_run_multiple_lastzs_sqlite, and then,

phyluce_probe_slice_sequence_from_genomes to extract the probe region plus 500 base pairs

from each flanking region. Apaloderma exonic regions were identified based on the Gallus

gallus genes, and annotations of CDS and exons were copied to the reference sequences inside

Geneious version R10.2.3 (Kearse, et al. 2012). With these sequences as a reference we mapped

back the clean reads of each individual employing Bowtie2 (Langmead and Salzberg 2012)

plugin 7.2.1 inside Geneious. The consensus sequences were called with the highest quality

threshold and a depth of at least 4 reads. Each locus was aligned with MAFFT (Katoh and

Standley 2013) under default parameters.

Phylogenetic relationships and species tree analysis

Since the intergeneric relationship among trogons are still mostly unresolved (Monteros

1998; Johansson and Ericson 2005; Moyle 2005; Hosner, et al. 2010), we first performed a

maximum likelihood analyses in RAxML v8.2 (Stamatakis 2014), and a Bayesian Inference

analyses in ExaBayes v.1.4 (Aberer, et al. 2014), using the concatenated matrix with three

treatments for missing data: a complete matrix, where no missing data was allowed, and two

where the missing data was allowed, a 95% and 75% completeness matrix, in which each locus

should have at least 95% or 75%, respectively, of all individuals in the matrix. As outgroups

51

we selected one mousebird (Colius striatus, (Gilbert, Jarvis, et al. 2014b)), and a roller

(Leptosomus discolor, (Gilbert, Jarvis, et al. 2014a)), suggested by recent studies as the closest

relatives to the Trogonidae family (Jarvis, et al. 2014; Prum, et al. 2015). We also estimated a

species tree using the SVDquartets (Chifman and Kubatko 2014) implemented in PAUP*

v4a(build157) (Swofford 2002), that samples quartets of individuals for each gene tree and infer

an unrooted phylogeny, performing a species tree using a coalescent approach. We

exhaustively sampled all quartets and performed a 100 bootstrap to quantify the support for

each node.

Dating analysis

To date the Trogonidae phylogeny we employed the slow evolving recombination-

activating genes (RAG-1 and RAG-2) and a dense sampling for the Core Landbirds group

(Telluraves), with the same calibration points used by Claramunt and Cracraft (2015). The

concatenated matrix was partitioned by codon and the best partition and substitution model

schemes were selected by PartitionFinder2 (Lanfear, et al. 2017).

Acknowledgements

The authors thankfully acknowledge all the curators and curatorial assistants of the

American Museum of Natural History, New York, USA (AMNH), Academy Academy of

Natural Science of Drexel University, Philadelphia, USA (ANSP); Field Museum of Natural

History, Chicago, USA (FMNH); Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil

(INPA); Kansas University (KU), Laboratório de Genética e Evolução Molecular de Aves –

USP (LGEMA), Lousiana State University Museum of Natural Science, Baton Rouge, USA

(LSUMZ); and Museu Paraense Emílio Goeldi, Belém, Brazil (MPEG), Smithsonian Institution

National Museum of Natural History (USNM), for borrowing tissue samples under their care.

We are also grateful for all collectors involved in the fieldwork that make this paper possible.

We thank L. Moraes for early input on this paper. MF acknowledge CAPES for his PhD

fellowship, and CAPES PDSE fellowship (# 88881.133440/2016-01) and the support from the

AMNH Frank M. Chapman Memorial Fund. The authors also thanks the grant Dimensions US-

Biota-São Paulo: Assembly and evolution of the Amazon biota and its environment: an

integrated approach, co-funded by the US National Science Fundation (NSF DEB 1241056) to

J.C. and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant

#2012/50260-6) to Lucia Lohmann. AA and CCR are supported by CNPq research productivity

fellowships. The authors acknowledge the National Laboratory for Scientific Computing

52

(LNCC/MCTI, Brazil) for providing HPC resources of the SDumont supercomputer, which

have contributed to the research results reported within this paper.

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Table 1 – Samples used in this study, the museum voucher numbers, locality and geographical coordinates

(when available), number of UCE reads, and loci recovered for each sample.

Species Museum voucher Locality Clean

reads

UCE

loci

Apaloderma vittatum SRP028834 Tanzania: Udzungwa Mts. - 2,228*

Apaloderma narina AMNH DOT-12430 Liberia: Lofa, Ziggida (08°02'15.5"N 9°31'49.5"W) 3,607,056 2,228

Apalharpactes mackloti LSUMZ B-49104 Indonesia: Sumatra 1,664,511 2,220

Apalharpactes mackloti AMNH 633881 Indonesia: Sumatra, Bandar-Baroe (03°15'57.6''N 98°30'49.9''E) 2,758,684 2,080

Harpactes ardens USNM 607340 Philippines: Barrio Via, Sitio Hot Springs, Baggao Mun. (17°50'N,

122°01'E) 1,193,041 2,208

Harpactes diardii AMNH DOT-563 Malaysia: Sabah, Klias Forest Reserve (05°19’34’’N

115°40’25’’E) 3,601,173 2,226

Harpactes oreskios ANSP 16308 Malaysia: Sabah, Mendolong (04°54'27.6"N 115°47'04.5"E) 5,208,017 2,228

Harpactes orrhophaeus AMNH DOT-15159 Malaysia: Sabah, Mt. Lucia (04°27’37.8’’N 117°55’20.4’’E) 4,250,801 2,228

Harpactes duvaucelli LSUMZ B-38592 Malaysia: Sabah, Imbak Valley, ca 60 km S Telupid (5°06’N

117°01’51’’E) 887,312 2,222

Harpactes fasciatus AMNH 778649 India: Dangs, Bhawandagad 5,386,424 2,218

Harpactes erythrocephalus AMNH DOT-12240 Vietnam: Quang Nam, Ngoc Linh Range (15°11’00’’N

108°02’00’’E) 2,126,329 2,224

Harpactes wardii AMNH 307761 Myanmar: Laukkaing 5,151,969 2,198

Harpactes whiteheadi LSUMZ B-52627 Malaysia: Sabah, Tambuman, Mt. Trus Madi (05°35’09’’N

116°29’26’’E) 11,299,280 2,228

Harpactes kasumba AMNH DOT-15326 Malaysia: Sabah, Ulu Tungud Forest Reserve, Melian Range

(05°50’48’’N 117°10’57’’E) 4,264,359 2,228

Euptilotis neoxenus AMNH DOT-11080 USA: Arizona, Ramsey Canyon Preserve (31°26'50.2"N

110°18'25.8"W) 1,955,116 2,186

Pharomachrus pavoninus INPA A-1993 Brazil: Amazonas, Parque Nacional do Jaú (01°49’50’’S

61°35’45’’W) 2,080,592 2,215

Pharomachrus auriceps

hargitti AMNH 175988 Ecuador: Baeza, Arriba (0°27’54’’S 77°53’44.9’’W) 6,034,956 2,210

Pharomachrus auriceps

auriceps FMNH 473723 Peru: Rodriguez de Mendoza (06°S 77°W) 2,620,376 2,221

Pharomachrus fulgidus AMNH 322895 Venezuela: Near village of Junquito on Colonia Tovar Rd

(10°27’23’’N 67°04’31’’W) 4,665,318 1,864

Pharomachrus moccino AMNH 326512 Honduras: Mt Pucca, Gracias (14°34’43’’N 88°38’30’’W) 5,630,314 2,215

Pharomachrus antisianus ANSP 19429 Ecuador: Napo, 12 km NNE El Chaco; Mirador 5,651,764 2,228

Priotelus temnurus ANSP 20257 Cuba 1,644,934 2,220

Priotelus roseigaster KU 8098 Dominican Republic: Parque Nacional Sierra Baoruco, Pueblo

Viejo (18°12’N 71°32’W) 1,431,709 2,221

Trogon clathratus USNM 613996 Panama: Bocas del Toro, Los Planes (08°35’43’’N 82°14’16’’W) 3,200,785 2,158

Trogon mesurus ANSP 19305 Ecuador: Esmeraldas, 20 km ENE Muisne (0°38’51’’N

79°59’59’’W) 7,341,190 2,142

Trogon massena KU 2073 Mexico: Campeche, Silvituc (18°13’48’’N 90°12’W) 1,689,867 2,224

Trogon comptus LSUMZ B-11829 Ecuador: Esmeraldas, El Placer (0°52’N 78°33’W) 2,072,859 2,228

Trogon melanurus INPA A-1955 Brazil: Amazonas, Parque Nacional do Jaú (01°49’50’’S

61°35’45’’W) 2,451,461 2,225

Trogon viridis INPA A-5240 Brazil: Pará, Aveiro, left bank Tapajós River (03°42.3’S

55°35.5’W) 1,893,902 2,226

Trogon chionurus LSUMZ B-28571 Panama: Colón, Achiote Road (09°13’32’’N 80°0’56’’W) 1,879,103 2,225

Trogon melanocephalus USNM 646857 El Salvador: La Paz, Aeropuerto Internacional El Salvador

(13°25’57’’N 89°03’50’’W) 1,521,530 2,224

Trogon citreolus UWBM 101087 Mexico: Michoacán, Lazaro Cardenas, La Mira (18°05.71’N

102°23.71’W) 1,311,613 2,224

Trogon bardii LSUMZ B-71992 Costa Rica: Osa, Los Charces (08°40’19’’N 83°30’19’’W) 2,036,944 2,226

Trogon violaceus MPEG CN437 Brazil: Pará, Alenquer, ESEC Grão-Pará (0°09’S 55°11’W) 1,251,316 2,222

Trogon caligatus LSUMZ B-66270 Peru: Tumbes, El caucho Biological Station (3°49’25’’S

80°15’37’’W) 4,878,667 2,150

Trogon ramonianus INPA A-5449 Brazil: Pará, Santarém, Rio Arapiuns (3°19’S 55°20’W) 2,665,900 2,228

Trogon curucui INPA A-5286 Brazil: Pará, Aveiro, left bank Tapajós River (3°42.3’S

55°35.5’W) 1,157,694 2,221

Trogon aurantius LGEMA 15782 Brazil: Minas Gerais, RPPN Serra do Caraça (20°07’01’’S

43°29’16’’W) 1,162,924 2,213

Trogon surrucura MPEG SC015 Brazil: Santa Catarina, Blumenau, Vila Itoupava (26°39’59’’S

49°05’41’’W) 2,005,634 2,224

Trogon rufus tenellus LSUMZ B-26564 Panama: Colón, Gamboa (9°09’25’’N 79°45’36’’W) 4,118,529 2,228

Trogon rufus amazonicus INPA A-5284 Brazil: Pará, Aveiro, left bank Tapajós River (3°42.3’S

55°35.5’W) 3,892,857 2,228

Trogon rufus chrysochlorus LGEMA 9557 Brazil: São Paulo, Ubatuba (23°23’24’’S 45°05’24’’W) 1,161,086 2,225

59

Trogon elegans FMNH 434014 El Salvador: Sonsonate: Izalco, Canton Las Laja (13°45’35’’N

89°40’21’’W) 475,853 2,201

Trogon mexicanus FMNH 343220 Mexico: Jalisco, Puerto los Mazos, Sierra de Manantlan

(19°28’09’’N 103°56’51’’W) 1,322,925 2,222

Trogon aurantiiventris LSUMZ B-41625 Panama: Bocas del Toro, Chiriqui (8°47’29’’N 82°12’35’’W) 6,441,454 2,228

Trogon collaris puella FMNH 394272 Mexico: Oaxaca, San Gabriel Mixtepec, Sierra de Miahuatlan

(16°09’56’’N 97°01’29’’W) 292,340 2,114

Trogon collaris collaris MPEG CN450 Brazil: Pará, Alenquer, ESEC Grão-Pará (0°09’S 55°11’W) 1,361,638 2,221

Trogon personatus LSUMZ B-48503 Guyana: Potaro-Siparuni, Kopinang Mountain (4°57’54’’N

59°54’49’’W) 1,826,664 2,228

Table 2 – Summary information of each method, including number of loci, total length of the concatenated

alignment, mean length size per locus, minimum and maximum length, and the total number of the Parsimony

Informative (PI) sites. Complete 75% 95%

Number of loci 1421 2110 2217

Total lenght 725090 1054512 1099526

Mean length size 510.27 499.77 495.95

Min-max length 259-1145 162-1145 162-1145

Number of PI sites 37,880 55,060 57,259

Table 3 – Node support for recalcitrant nodes in the Trogonidae phylogeny. RAxML ExaBayes SVDq

75% 95% complete 75% 95% complete 95%

Asian + Neotropical 70 62 84 1.0 1.0 1.0 -

Apalharpactes + Harpactes 60 46 52 1.0 1.0 1.0 -

Neotropical 100 100 100 1.0 1.0 1.0 100

60

Figure 1 – Phylogeny of Trogonidae inferred with ExaBayes summarizing the results from other analyses. The

circle at each node represent the statistical support for the RAxML analyses and the species tree reconstruction

inferred by SVDq. Green lines represent distribution shifts from Central America to South America. Trogon

species were group in five species groups highlighted with grey boxes: “rufus”, “collaris”, “melanurus”, “viridis”,

and “violaceus”.

61

Figure 2 – Time-calibrated phylogeny of Trogonidae inferred from the concatenated dataset of RAG1 and RAG2

genes using BEAST. This tree represents part of the tree calibrated using (Claramunt and Cracraft 2015)

calibrations, complete taxon data in Supplementary Table 1. The basal nodes were constrained to match the UCE

topology, all other nodes have a red circle, if the posterior probability is 1.0, or the posterior is written next to the

node. Timings of major splits are shown next to each node. Blue bars represent the 95% HPD estimates of node

height. Green lines represent distribution shifts from Central America to South America. The top-right figure

represents the whole tree with calibration points as red circles.

62

Supplementary Table S1 – Table containing taxonomic information on all specimens employed in the RAG time

tree. The RAG1 and RAG2 column refers to GenBank accession numbers for these two genes. Taxonomy follows

del Hoyo, et al. (2017).

Order Family Species RAG1 RAG2

Passeriformes Thraupidae Thraupis cyanocephala AY057035 AY443236

Passeriformes Emberizidae Emberiza schoeniclus AY056992 AY443143

Passeriformes Passeridae Passer montanus AF143738 AY443198

Passeriformes Prunellidae Prunella collaris AY057024 AY443213

Passeriformes Dicaeidae Dicaeum aeneum AY443282 AY443139

Passeriformes Regulidae Regulus calendula AY057028 AY443220

Passeriformes Irenidae Irena cyanogaster AY056999 AY443158

Passeriformes Nectariniidae Nectarinia olivacea AY057009 AY443180

Passeriformes Turdidae Catharus ustulatus AY443265 AY443114

Passeriformes Cinclidae Cinclus cinclus AY056985 AY443119

Passeriformes Mimidae Mimus patagonicus AY057005 AY443173

Passeriformes Sturnidae Sturnus vulgaris AY057032 AY443232

Passeriformes Troglodytidae Troglodytes aedon AY057038 AY443241

Passeriformes Certhiidae Certhia familiaris AY056983 AY443115

Passeriformes Sittidae Sitta carolinensis AY443332 AY443227

Passeriformes Sylviidae Sylvia nanna AY057033 AY443233

Passeriformes Pycnonotidae Pycnonotus barbatus AY057027 AY443219

Passeriformes Hirundinidae Hirundo rustica AY443290 AY443154

Passeriformes Aegithalidae Aegithalos iouschensis AY056976 AY443103

Passeriformes Locustellidae Megalurus palustris AY319988 AY799840

Passeriformes Remizidae Remiz pendulinus AY443328 AY443222

Passeriformes Promeropidae Promerops cafer AY443323 AY443212

Passeriformes Monarchidae Monarcha axillaris AY057006 AY443176

Passeriformes Laniidae Lanius excubitor AY443293 AY443160

Passeriformes Artamidae Artamus leucorhynchus AY056980 AY443109

Passeriformes Artamidae Artamus cyanopterus AY443262 AY443108

Passeriformes Artamidae Cracticus quoyi AY443278 AY443135

Passeriformes Vangidae Vanga curvirostris AY057040 AY443244

Passeriformes Platysteiridae Batis mixta AY443263 AY443110

Passeriformes Vireonidae Vireo philadelphia AY057041 AY443245

Passeriformes Melanocharitidae Melanocharis nigra AY057002 AY443167

Passeriformes Melanocharitidae Melanocharis vesteri AY443299 AY443168

Passeriformes Orthonychidae Orthonyx teminckii AY057012 AY443309

Passeriformes Climacteridae Climacteris erythrops AY443268 AY443121

Passeriformes Menuridae Menura novaehollandiae AY057004 AY443171

Passeriformes Furnariidae Furnarius rufus AY056995 AY443149

Passeriformes Rhinocryptidae Scytalopus magellanicus AY443331 AY443226

Passeriformes Thamonophilidae Terenura sharpei JX213518 JX213481

Passeriformes Pipridae Piprites chloris FJ501717 FJ501897

Passeriformes Pipridae Piprites pileata JF970177 KC157559

Passeriformes Pipridae Lepidothrix coronata FJ501655 FJ501835

Passeriformes Pipridae Antilophia galeata FJ501600 FJ501780

Passeriformes Oxyrunchidae Oxyruncus cristatus FJ501689 FJ501878

Passeriformes Cotingidae Cotinga cayana FJ501623 FJ501803

63

Passeriformes Cotingidae Laniisoma elegans FJ501651 FJ501831

Passeriformes Cotingidae Phoenicircus nigricollis FJ501705 FJ501885

Passeriformes Tyrannidae Tyrannus tyrannus AF143739 AY443243

Passeriformes Sapayoidae Sapaoya aenigma DQ320606 DQ320573

Passeriformes Dendrocolaptidae Dendrocolaptes certhia FJ461166 FJ460982

Passeriformes Pittidae Pitta sordida AY443219 AY443206

Passeriformes Acanthisittidae Acanthisitta chloris AY056975 AY443102

Psittaciformes Psittacidae Psittacus erithacus EF517674 EF517687

Psittaciformes Psittacidae Alisterus scapularis KT954426 EF517677

Psittaciformes Psittacidae Melopsittacus undulatus XM_005150647.1 XM_005150646.1

Psittaciformes Psittacidae Micropsitta brujinii EF517673 EF517681

Psittaciformes Psittacidae Amazona aestiva LMAW01003202 LMAW01003202

Psittaciformes Psittacidae Myopsitta monachus DQ143328 -

Psittaciformes Psittacidae Agapornis personata EF517672 EF517679

Psittaciformes Cacatuidae Calyptorhynchus funereus KT954425 EF517680

Psittaciformes Strigopidae Nestor notabilis XM_010020228.1 XM_010020229.1

Falconiformes Falconidae Falco peregrinus AY461399 KT954538

Falconiformes Falconidae Falco cherrug XM_005441067.1 XM_005441068.2

Falconiformes Falconidae Daptrius ater AY461397 KT954537

Falconiformes Falconidae Micrastur gilvicollis AY461403 KT954536

Cariamiformes Cariamidae Cariama cristata XM_009699718.1 XM_009699720.1

Piciformes Ramphastidae Pteroglossus aracari KT954416 KT954525

Piciformes Capitonidae Capito niger KT954414 KT954523

Piciformes Semnornidae Semnornis frantzii KT954415 KT954524

Piciformes Lybiidae Trachyphonus erythrocephalus KT954413 KT954522

Piciformes Lybiidae Lybius hirsutus KT954412 KT954521

Piciformes Megalaimidae Megalaima oorti KT954411 KT954520

Piciformes Picidae Melanerpes carolinus KT954418 KT954527

Piciformes Picidae Picoides pubescens XM_009905561.1 XM_009905562.1

Piciformes Picidae Picumnus cirratus AF295195 -

Piciformes Indicatoridae Indicator variegatus KT954417 KT954526

Piciformes Bucconidae Bucco capensis MPEG_ARA018

Piciformes Bucconidae Nystalus maculatus MPEG_MARJ045

Piciformes Bucconidae Nonnula rubecula INPA_A4705

Piciformes Bucconidae Monasa atra INPA_A8299

Piciformes Bucconidae Chelidoptera tenebrosa MPEG_JTW1160

Piciformes Bucconidae Hapaloptila castanea LSU_12059

Piciformes Bucconidae Micromonacha lanceolata LSU_4489

Piciformes Bucconidae Cyphos macrodactylus MPEG_AMA354

Piciformes Bucconidae Notharchus tectus LSU_28765

Piciformes Bucconidae Hypnellus bicinctus FMNH_339641

Piciformes Bucconidae Nystactes tamatia MPEG_JRT134

Piciformes Bucconidae Notharchus ordii LSU_25460

Piciformes Bucconidae Notharchus hyperrhynchus MPEG_GAPTO296

Piciformes Bucconidae Malacoptila fulvogularis FMNH_321031

Piciformes Bucconidae Malacoptila rufa LSU_103572

Piciformes Galbulidae Jacamalcyon tridactyla MPEG_800

Piciformes Galbulidae Brachygalba lugubris MPEG_293

Piciformes Galbulidae Jacamerops aureus MPEG_JAP375

64

Piciformes Galbulidae Galbacyrhynchus purusianus INPA_A1429

Piciformes Galbulidae Galbula dea INPA_A2288

Piciformes Galbulidae Galbula leucogastra MPEG_AMZ190

Piciformes Galbulidae Galbula ruficauda MPEG_MARJ109

Piciformes Galbulidae Galbula cyanescens MPEG_PUC159

Piciformes Galbulidae Galbula albirostris MPEG_JAP616

Piciformes Galbulidae Galbula cyanicollis MPEG_FLJA056

Coraciformes Alcedinidae Chloroceryle americana KT954422 KT954533

Coraciformes Alcedinidae Halcyon malimbica DQ111819 KT954532

Coraciformes Alcedinidae Alcedo leucogaster DQ111794 KT954531

Coraciformes Momotidae Momotus momota KT954421 KT954530

Coraciformes Todidae Todus angustirostris KT954420 KT954529

Coraciformes Coraciidae Coracias caudata AF143737 AY443126

Coraciformes Brachypteracidae Brachypteracias leptosomus KT954423 KT954534

Coraciformes Meropidae Merops pusillus KT954419 KT954528

Coraciformes Meropidae Merops nubicus XM_008938323.1 XM_008938322.1

Bucerotiformes Upupidae Upupa epops KT954409 KT954517

Bucerotiformes Phoeniculidae Phoeniculus purpureus KT954408 KT954516

Bucerotiformes Bucerotidae Buceros rhinoceros XM_010145185.1 XM_010145184.1

Bucerotiformes Bucerotidae Buceros bicornis KT954407 KT954515

Bucerotiformes Bucerotidae Tockus camurus KT954406 KT954514

Leptosomatiformes Leptosomidae Leptosomus discolor XM_009958543.1 XM_009958545.1

Colliformes Coliidae Colius colius KT954404 KT954512

Colliformes Coliidae Colius striatus XM_010201405.1 XM_010209029.1

Strigiformes Strigidae Strix occidentalis DQ482641 KT954508

Strigiformes Strigidae Ninox novaeseelandiae KT954400 KT954507

Strigiformes Tytonidae Tyto alba XM_009975325.1 XM_009975324.1

Strigiformes Tytonidae Phodilus badius KT954402 KT954510

Accipitrifromes Accipitridae Buteo jamaicensis EF078718 KT954506

Accipitrifromes Accipitridae Elanus caeruleus EF078724 KT954505

Accipitrifromes Pandionidae Pandion haliaetus EF078706 KT954504

Accipitrifromes Sagittaridae Sagittarius serpentarius KT954399 KT954503

Accipitrifromes Cathartidae Cathartes aura EF078766 KT954502

Accipitrifromes Accipitridae Aquila chrysateos XM_011594630.1 XM_011594629.1

Accipitrifromes Accipitridae Haliaeetus albicilla XM_009928640.1 XM_009928639.1

Accipitrifromes Accipitridae Haliaeetus leucocephalus XM_010586008.1 XM_010586006.1

Trogoniformes Trogonidae Apaloderma vittatum XM_009874816.1 XM_009869619.1

Trogoniformes Trogonidae Apaloderma narina AMNH_DOT12430

Trogoniformes Trogonidae Apalharpactes mackloti LSU_49104

Trogoniformes Trogonidae Apalharpactes mackloti AMNH_633881

Trogoniformes Trogonidae Harpactes ardens AY625239 -

Trogoniformes Trogonidae Harpactes ardens USNM_607340

Trogoniformes Trogonidae Harpactes diardii AMNH_DOT563

Trogoniformes Trogonidae Harpactes oreskios AY625238 -

Trogoniformes Trogonidae Harpactes oreskios ANSP_16308

Trogoniformes Trogonidae Harpactes orrhopheus AY625241 -

Trogoniformes Trogonidae Harpactes orrhopheus AMNH_DOT15159

Trogoniformes Trogonidae Harpactes duvaucelli LSU_38592

Trogoniformes Trogonidae Harpactes fasciatus AMNH_778649

65

Trogoniformes Trogonidae Harpactes erythrocephalus AMNH_DOT12240

Trogoniformes Trogonidae Harpactes wardii AMNH_307761

Trogoniformes Trogonidae Harpactes whiteheadii LSU_52627

Trogoniformes Trogonidae Harpactes kasumba AMNH_DOT15326

Trogoniformes Trogonidae Euptilotis neoxenus AMNH_DOT11080

Trogoniformes Trogonidae Pharomachrus pavoninus LSU_4986

Trogoniformes Trogonidae Pharomachrus auriceps hargitti AMNH_175988

Trogoniformes Trogonidae Pharomachrus auriceps auriceps FMNH_473723

Trogoniformes Trogonidae Pharomachrus fulgidus AMNH_322895

Trogoniformes Trogonidae Pharomachrus moccino AMNH_326512

Trogoniformes Trogonidae Pharomachrus antisianus ANSP_19429

Trogoniformes Trogonidae Priotelus temnurus ANSP_20257

Trogoniformes Trogonidae Priotelus roseigaster KU_8098

Trogoniformes Trogonidae Trogon clathratus USNM_613996

Trogoniformes Trogonidae Trogon mesurus ANSP_19305

Trogoniformes Trogonidae Trogon massena KU_2073

Trogoniformes Trogonidae Trogon comptus LSU_11829

Trogoniformes Trogonidae Trogon melanurus INPA_A1995

Trogoniformes Trogonidae Trogon viridis INPA_A5240

Trogoniformes Trogonidae Trogon chionurus LSU_28571

Trogoniformes Trogonidae Trogon melanocephalus USNM_646857

Trogoniformes Trogonidae Trogon citreolus UWBM_101087

Trogoniformes Trogonidae Trogon bardii LSU_71992

Trogoniformes Trogonidae Trogon violaceus MPEG_CN437

Trogoniformes Trogonidae Trogon caligatus LSU_66270

Trogoniformes Trogonidae Trogon ramonianus INPA_A5449

Trogoniformes Trogonidae Trogon curucui INPA_A5286

Trogoniformes Trogonidae Trogon aurantius LGEMA_15782

Trogoniformes Trogonidae Trogon surrucura MPEG_SC015

Trogoniformes Trogonidae Trogon elegans FMNH_434014

Trogoniformes Trogonidae Trogon rufus amazonicus INPA_A5284

Trogoniformes Trogonidae Trogon rufus tenellus LSU_26564

Trogoniformes Trogonidae Trogon rufus chrysochlorus LGEMA_9557

Trogoniformes Trogonidae Trogon mexicanus FMNH_343220

Trogoniformes Trogonidae Trogon aurantiiventris LSU_41625

Trogoniformes Trogonidae Trogon collaris puella FMNH_394272

Trogoniformes Trogonidae Trogon collaris collaris MPEG_CN450

Trogoniformes Trogonidae Trogon personatus LSU_48503

66

Capítulo 3

Ferreira, M.; Aleixo, A.; Bates, J. M.; Cracraft, J.;

Ribas, C. C. Phylogeography and phylogenomics of

jacamars (Aves: Galbulidae) and puffbirds (Aves:

Bucconidae) reveal underestimation of species

diversity and recurrent biogeographic patterns in the

Neotropics. Manuscrito formatado para Zoological

Journal of Linnean Society

67

Manuscript submission to Zoological Journal of Linnean Society

Contribution type: Article

Phylogeography and phylogenomics of jacamars (Aves: Galbulidae) and

puffbirds (Aves: Bucconidae) reveal underestimation of species diversity

and recurrent biogeographic patterns in the Neotropics

Ferreira, Mateus1*; Aleixo, Alexandre2; Bates, John M.3; Cracraft, Joel4; Ribas, Camila C.5

1 Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, INPA,

Manaus, AM, Brazil 2 Coordenação de Zoologia, MPEG, Belém, PA, Brazil 3 Department of Ornithology, FMNH, Chicago, IL, USA 4 Department of Ornithology, AMNH, New York, NY, USA 5 Coordenação de Biodiversidade, INPA, Manaus, AM, Brazil

*Corresponding author

Correspondence: Mateus Ferreira, Coordenação de Biodiversidade, Instituto Nacional de

Pesquisas da Amazônia, CEP 69080-971, Manaus-AM, Brazil

E-mail: [email protected]

Short running title: Galbuliformes phylogenomic

Abstract

Galbulidae (jacamars) and Bucconidae (puffbirds) are sister families endemic to the

Neotropical region. Together they comprise 57 species and more than a 100 described

subspecies. Both families have their highest diversity in Amazonia. Within Galbulidae, most

species have restricted and parapatric / allopatric distributions in relation to other closely related

species, while within Buccondiae, species are widespread and polytypic. In this study, we

obtained DNA sequence data for over 400 samples, and used previous published results, of all

widespread species to uncover phylogeographic patterns. Then, based on these results, we

selected and sequenced thousands of Ultraconserved Elements to reconstruct the phylogenetic

relationships among these phylogeographic groups and propose the first phylogenetic

hypothesis for these two families with dense taxon sampling. Our phylogeographic results

recovered phylogeographic breaks in almost all studied groups, most of them associated with

the main tributaries of the Amazon River, and many corresponding to already described

subspecies. We then reconstructed phylogenetic relationships based on over 2,000 UCE loci

using a concatenated approach in a Bayesian Inference framework. Overall, most nodes had


68

high support, and the relationships among genera, species and instraspecific diversity were

discussed. We propose the recognition of all subspecies that received support from the

phylogeographic and phylogenomic approaches as distinct species. We found evidence of

paraphyly of several species and proposed taxonomic changes to deal with that.

Introduction

Species usually are the basic unit of any study in evolutionary biology. Considering they

should represent the lowest and only non-arbitrary rank above individuals, species are the basic

operational unit for comparing any intrinsic evolutionary aspect, such as physiology, behaviour,

morphology, etc. However, we still lack a broad and comprehensive concept for species

recognition (Cellinese, Baum & Mishler, 2012; de Queiroz, 2007; de Queiroz, 2012). In birds,

taxonomy has been historically influenced by the Biological Species Concept (Mayr, 1942;

Mayr, 1976), based on reproductive isolation as the main criterion for species delimitation.

Therefore, since this concept was adopted several distinct allopatric populations were lumped

as subspecies due to morphological similarities pending further investigation to prove the

absence of gene flow (Peters, 1945; Peters, 1948). This implies that allopatric and parapatric

populations, even if diagnosably distinct, should only be recognized as full species if there is

evidence of reproductive isolation (Gill, 2014).

In the Neotropical region, and especially in Amazonia, one of the main issues that

obscures the recognition of diversity patterns is the fact that most widespread species are in fact

complexes of taxa, usually diagnosable and geographically structured, that are lumped under

the same species name due to their morphological similarities and physical isolation. Many of

these polytypic species, when thoroughly sampled, prove to include distinct lineages,

sometimes not even closely related to each other (Bravo, Chesser & Brumfield, 2012; Bravo,

Remsen, Whitney & Brumfield, 2012; Fernandes, Wink, Sardelli & Aleixo, 2014; Isler, Bravo

& Brumfield, 2013; Lopes, Chaves, Aquino, Silveira & Santos, 2017; Lutz, Weckstein, Patane,

Bates & Aleixo, 2013; Ribas, Aleixo, Nogueira, Miyaki & Cracraft, 2012; Ribas, Aleixo,

Gubili, d'Horta, Brumfield & Cracraft, 2018; Tobias, Bates, Hackett & Seddon, 2008). The

recognition of these hidden lineages is critical for appropriate hypothesis formulation in

macroevolution and biogeography (Donoghue & Moore, 2003; Lexer, Mangili, Bossolini,

Forest, Stölting, Pearman, Zimmermann, Salamin & Carine, 2013). For example, Amazonian

areas of endemism were recognized based on congruent distribution patterns of bird species

69

(Borges & Da Silva, 2012; Cracraft, 1985), and have been used as a basis to formulate

hypothesis of biotic diversification in Amazonia (Haffer, 1969; Haffer, 1974; Haffer, 1997).

Considering that any biogeographic study should be based on a taxonomy that correctly

recognizes the evolutionary units included in the studied groups, for the present study we

densely sampled all recognized taxa within two sister families of birds restricted to the

Neotropical region. Galbulidae and Bucconidae form a clade, sometimes recognized in its own

order Galbuliformes, that diverged from all the other Piciformes during the early Eocene and

diverged from each other in the Late Eocene (Prum, Berv, Dornburg, Field, Townsend,

Lemmon & Lemmon, 2015). Although the ancestor was from the Afrotropical region the two

families’ entire diversification happened inside the Neotropical region (Claramunt & Cracraft,

2015). Hence, making these two families excellent models to understand how landscape

evolution of the Neotropical region influenced diversification. However, there are no

phylogenetic hypotheses about relationships within these two families, and the few

phylogeographic studies conducted so far with Bucconidae species showed that the diversity is

highly underestimated by current species limits (Almeida, 2013; Duarte, 2015; Ferreira, Aleixo,

Ribas & Santos, 2017; Soares, 2016). Although Galbulidae species were never subjected to

phylogeographic studies, with 19 species distributed in 5 genera, jacamar distributions were

used as models by Haffer (1974), together with other families, when he proposed his theory for

Amazonian diversification (Haffer, 1974). Haffer recognized eight zoogeographic groups, five

were composed of species complexes, and two were widespread polytypic species. Bucconidae,

in turn, are composed of 38 species distributed in 12 genera. However, half of those species

consist of polytypic groups lumped as subspecies due to morphological similarities. Groups

such as the White-fronted Nunbird, Monasa morphoeus, and the Rusty-breasted Nunlet,

Nonnnula rubecula, are composed of several subspecies, which in fact still underestimate the

phylogeographic structure recovered for them (Soares, 2016). On the other hand, Malacoptila

species are widespread species for which only a few subspecies were described, however,

phylogeographic patterns indicated a great underestimation of taxonomic diversity. For

example, for a single species, the Rufous-necked Puffbird (M. rufa), that only includes two

subspecies described, ten distinct genetic lineages were recovered (Ferreira et al., 2017). Due

to these first results, the present study focused on sampling all named taxa described for these

two families, and sampling all widespread species throughout their distribution to uncover

phylogeographic patterns. Based on these results, we selected samples representing all

phylogeographic groups and sequenced thousands of Ultraconserved Elements (UCE)

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(Faircloth, McCormack, Crawford, Harvey, Brumfield & Glenn, 2012; McCormack &

Faircloth, 2013; McCormack, Harvey, Faircloth, Crawford, Glenn & Brumfield, 2013) to

recover their phylogenetic relationships. Our aims are (1) to characterize the phylogeographic

patterns and population structure within widespread species, recognizing the cryptic diversity

within them, when present; (2) propose a densely sampled phylogenetic hypothesis for these

two families; and (3) discuss patterns of diversification in the entire clade.

Material and Methods

Sampling and DNA isolation

We sampled 436 individuals from almost all named taxa currently recognized within

Galbuliformes (Gill & Donsker, 2018; Peters, 1948; Piacentini, Aleixo, Agne, Mauricio,

Pacheco, Bravo, Brito, Naka, Olmos, Posso, Silveira, Betini, Carrano, Franz, Lees, Lima, Pioli,

Schunck, do Amaral, Bencke, Cohn-Haft, Figueiredo, Straube & Cesari, 2015; Rassmussen &

Collar, 2002; Remsen, Areta, Cadena, Claramunt, Jaramillo, Pacheco, Pérez-Emen, Robbins,

Stiles, Stotz & Zimmer, 2018 Tobias, 2017), and when available, we used published sequences

to select samples for UCE sequencing. All samples are represented by voucher specimens

deposited at the ornithological collections of the American Museum of Natural History

(AMNH), Academy of Natural Sciences of Drexel University (ANSP), Field Museum of

Natural History (FMNH), Instituto Nacional de Pesquisas da Amazônia (INPA), Kansas

University (KU), Laboratório de Genética e Evolução Molecular de Aves - USP (LGEMA),

Louisiana Museum of Natural History (LSUMZ), Museu Paraense Emílio Goeldi (MPEG),

Smithsonian Institution National Museum of Natural History (USNM) and Burke Museum

(UWBM) (Table S1).

DNA from fresh tissue was extracted with the DNeasy kit (Qiagen Inc.), following the

manufacturer’s protocol. For taxa lacking fresh tissues we sampled toe pad clips from museum

specimens at the American Museum of Natural History (AMNH). Toe pads were cut from

specimens with a sterile surgical blade and processed in a dedicated room for ancient DNA

(aDNA Lab, AMNH). They were rinsed with 100% ethanol, and twice with ultra-pure water

prior to digestion to remove any inhibitor that could cause problems in downstream procedures.

We then extracted DNA with the DNeasy kit (Qiagen Inc.), replacing the regular silica columns

with the QIAquick (Qiagen Inc.) columns, to ensure maximum DNA yield.

71

Phylogeographic structure and UCE sampling

Widespread species that lacked previous studies were sampled throughout their

distributions to uncover phylogeographic structure. We amplified one mitochondrial gene

(NADH subunit 2 – ND2) following conventional PCR protocols and sequenced both strands

with BigDye® Terminator v3.1 in an ABI 3130/3130XL automated capillary sequencer

(Applied Biosystems®) following manufacturer’s protocols. The sequences were edited on

Geneious version 10.2.3 (Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton,

Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes & Drummond, 2012) and aligned with

MAFFT (Katoh & Standley, 2013) under default parameters. We analysed each species

complex independently. Within Galbulidae we analysed five species complexes: 1)

Brachygalba and Jacamaralcyon; 2) Jacamerops; 3) Galbula dea; 4) Galbula cyanicollis, G.

chalcocephala, and G. albirostris; and 5) G. ruficauda, G. pastazae, G. cyanescens, G.

tombacea, and G. galbula. We used a previous study to select samples for G. leucogastra and

G. chalcothorax (Ferreira et al., submitted). For Bucconidae, we gathered data in this study for

five polytypic species or species complexes: 1) Bucco capensis; 2) Cyphos macrodatylus; 3)

Notharchus tectus; 4) Notharchus ordii, N. hyperrhynchus, N. macrorhynchus, N. swainsoni,

and N. pectorales; and 5) Chelidoptera tenebrosa. Sample selection for the genera Monasa,

Nonnula, Malacoptila, and Nystalus was based on previous studies (Almeida, 2013; Duarte,

2015; Ferreira et al., 2017; Soares, 2016). The best evolutionary model for each matrix was

selected by jModelTest 2.1.10 (Darriba, Taboada, Doallo & Posada, 2012). We performed a

Bayesian inference analysis (BI) implemented in MrBayes 3.2.6 (Ronquist, Teslenko, van der

Mark, Ayres, Darling, Hohna, Larget, Liu, Suchard & Huelsenbeck, 2012) with four parallel

simultaneous runs consisting of a total of 4x107 generations, sampling trees every 1000

generations. ESS values, stationarity, and convergence among runs were checked in Tracer 1.6

(Rambaut, Suchard, Xie & Drummond, 2014). Based on these results we selected our samples

for UCE sequencing. All extracts were sent to Rapid Genomics (Gainsville, FL) for library prep

and target-capture sequencing of 2321 loci of Ultraconserved Elements (UCE) (Faircloth et al.,

2012; McCormack et al., 2013).

UCE assembly

The raw sequence data were processed with the Phyluce script pack (Faircloth, 2016).

We employed illumiprocessor (Faircloth, 2013) and Trimmomatic (Bolger, Lohse & Usadel,

2014) to remove adapter contamination and low-quality reads. We assembled our targeted

regions using a reference genome for each family. For Bucconidae, we used the Collared

72

puffbird (Bucco capensis), and for Galbulidae, the Paradise jacamar (Galbula dea) genomes.

We mapped the UCE probes back to each genome using the script

phyluce_probe_run_multiple_lastzs_sqlite, and then, phyluce_probe_slice_sequence_from_g-

enomes to extract the probe region plus 500 base pairs from each flanking region (Faircloth,

2016). With these sequences as a reference we mapped back the clean reads of each individual

employing Bowtie2 (Langmead & Salzberg, 2012) plugin 7.2.1 inside Geneious version 10.2.3

(Kearse et al., 2012). The consensus sequences were called with the highest quality threshold

and a depth of at least 4 reads. Each locus was aligned with MAFFT (Katoh & Standley, 2013)

under default parameters.

Phylogenetic relationship

Even though the sister relationship between Galbulidae and Bucconidae is well

established (Hackett et al., 2008; Livezey & Zusi, 2007; Prum et al., 2015), we used the

Rhinoceros hornbill (Buceros rhinoceros, Bucerotidae)(Gilbert, Jarvis, Li, Li, Avian Genome

Consortium, Wang & Zhang, 2014b), the Northern Carmine bee-eater (Merops nubicus,

Meropidae)(Gilbert, Jarvis, Li, Li, Avian Genome Consortium, Wang & Zhang, 2014c), and

the Downy woodpecker (Picoides pubescens, Picidae)(Gilbert, Jarvis, Li, Li, Avian Genome

Consortium, Wang & Zhang, 2014a) as outgroups. To recover the phylogenetic relationships,

we performed a Bayesian Inference analysis in ExaBayes v1.4 (Aberer, Kobert & Stamatakis,

2014) employing the concatenated matrix of all UCE loci with 75% completeness, where only

loci that had at least 75% of all individuals were selected. Four parallel chains consisting of

4x107 generations were performed.

Results

Phylogeographic results

With a few exceptions, we obtained the whole ND2 sequence for all samples.

Phylogenetic trees and maps of samples and lineages’ distributions can be found in the

Supplementary Material (Figures S1-S10). Overall, most species complexes contained

phylogeographic structure in the mtDNA that matches known areas of endemism for birds. The

only two widespread species that apparently lacked phylogeographic structure were Cyphos

macrodactylus and Chelidoptera tenebrosa. The phylogeographic breaks were more

conspicuous in birds with stronger association with terra-firme forests [Fig. S2-S4, S6,

Malacoptila spp. (Ferreira et al., 2017), Monasa morphoeus and Nonnula rubecula (Soares,

2016)]. However, species associated with other habitats, such as várzeas, open habitats (i.e.

73

non-forested) or white-sand environments also showed structure [Fig. S1, S5, S8-S9, also

Galbula leucogastra/chalcothorax, Nystactes (Almeida, 2013), Nystalus spp. (Duarte, 2015),

and Nonnula ruficapilla (Soares, 2016)]. Nonetheless, some lineages are represented by a single

individual and additional samples should be collected and analysed to make further

assumptions. It is also worth to note that some species were paraphyletic in the mtDNA. The

most remarkable are the complex Brachygalba lugubris, B. albogularis (Fig. S1), G. albirostris,

G. cyanicolis, and G. chalcocephala (Fig. S4), G. ruficauda, G. cyanescens (Fig. S5);

Notharchus tectus, N. subtectus (Fig. S8); N. hypperhynchus, N. swainsoni, N. macrorhynchus

(Fig. S9).

UCE sequencing

The reference sequences we assembled from the Collared puffbird (Bucco capensis) and

the Paradise jacamar (Galbula dea) genomes included 2226 and 2279 sequences, respectively.

The mean number of sequences was 2,240,885 reads; and a mean number of 2191 UCE loci per

sample (Table S1). The matrix for Galbulidae contained 2165 loci, while for Bucconidae, the

matrix had 2158 loci.

Phylogenetic results

In general, the ExaBayes tree is well supported, with most of the nodes with lower support

found near the tips (Fig. 2, 3). Galbulidae consisted of two clades, the first comprises

Jacamaralcyon and Brachygalba, and the other, Jacamerops, Galbacyrhynchus, and Galbula

(Fig. 1). Within Bucconidae, some genera were paraphyletic. Bucco, that previously included

four species (Gill & Donsker, 2018; Peters, 1948; Piacentini et al., 2015; Remsen et al., 2018),

comprises three distinct genera as previously suggested by morphological characters

(Rassmussen & Collar, 2018): B. capensis Linneus, 1766 is the family and genus type and more

closely related to Nystalus; Cyphos macrodactylus von Spix, 1824, is sister to the clade that

comprises Notharchus, Hypnelus, and Nystactes; and finally, Nystactes tamatia (J. F. Gemelin,

1788), and N. noanamae (Hellmayer, 1909), more closely related with Hypnelus species (Fig.

3). Notharchus was also paraphyletic, with Hypnelus and Nystactes embedded within it. N.

tectus and N. subtectus were sister to Hypnelus, Nystactes, and the remaining Notharchus

species (Fig. 1, 3).

The relationships within genera in the UCE trees (Fig. 2, 3) mostly agreed with the

mtDNA phylogeographic structure. Most notably is the paraphyly of Brachygalba lugubris in

relation to B. albogularis (Fig. 2), and the polyphyletic status of Galbula ruficauda, in which

the lineages from Central America (G. melanogenia), and northern South America (G. pallens,

74

G. ruficauda), including G. pastazae, are sister group to the clade comprising the species group

of G. albicollis (albicollis, chalcocephala, cyanicollis) and G. galbula (galbula, pastazae,

cyanescens, rufoviridis). Also, in contrast with the mtDNA, the two samples of G. cyanescens

are sister to G. heterogyna and G. rufoviridis from the Brazilian Shield, instead of being

embedded between them (Fig. S5). For puffbirds, the UCE tree also recovered the paraphyly of

N. tectus subspecies (Fig. 3), and for the hyperrhynchus group (Fig. S9), we recovered N.

macrorhynchus sister to N. swainsoni and N. hyperrhynchus, rendering the Amazonian group

paraphyletic.

Discussion

Phylogenetic results

Our dense sampling coupled with the use of UCE loci provided good insights about

genera and species relationships. We sampled all species, and almost all subspecies, for the two

families, and characterized the spatial distribution of mtDNA lineages for all widespread

species. Predominantly, our results indicate a severe disparity between currently recognized

species and the potential number of independent evolutionary units within these clades.

Avian taxonomy has historically been greatly influenced by the Biological Species

Concept (BSC), which assumes that reproductive isolation is required for recognition of species

status (de Queiroz, 2005). This condition, can be easily detected in sympatric taxa, however,

for parapatric and allopatric populations, natural observations are very hard to detect.

Consequently, many morphologically distinct taxa have been lumped into species complexes,

pending further analysis to prove them different. Thus, the null hypothesis for species

recognition has been of peer-reviewed publications proving that essential reproductive isolation

is true among allopatric populations. It implies that we should be looking for reasons that

differentiate allopatric populations, either through genetic evidence or some other characteristic

that would lead to reproductive isolation, rather than assuming that they already are

reproductive isolated, because they are not in contact, and looking for evidence proving the

contrary (Gill, 2014). Albeit avian taxonomy and systematics is probably the best known among

vertebrates, there are still many taxa to be described (Barrowclough, Cracraft, Klicka & Zink,

2016), and although species concept, or criteria, are amid one of the most controversial topics

in biology (Aleixo, 2007; Dayrat, Cantino, Clarke & de Queiroz, 2008; de Queiroz, 2012), the

appropriate understanding of a lineage’s evolutionary history is essential to several fields,

including conservation and biogeography (Avendaño, Arbeláez-Cortés & Cadena, 2017; Ribas,

75

Gaban-Lima, Miyaki & Cracraft, 2005; Tobias, Bates, Hackett & Seddon, 2008), especially in

the emergent field of geogenomics (Baker, Fritz, Dick, Eckert, Horton, Manzoni, Ribas,

Garzione & Battisti, 2014). Therefore, we are confident our results provide great insight about

Galbulidae and Bucconidae systematics and will enable future biogeography studies to uncover

how the landscape evolution of South America shaped this group’s diversity.

Galbulidae systematics

Galbulidae currently recognized diversity includes 19 species distributed in 5 genera

(Tobias, 2017). Our results, however, show that this diversity is severely underestimated. In

addition to the fact that most widespread species have genetic lineages structured

geographically, we also found evidence that, at least four species are para- (Brachygalba spp.)

or polyphyletic (Galbula ruficauda complex). Conceding that we recognize all subspecies that

were monophyletic in our analyses and elevate them to species status, the species diversity of

Galbulidae practically doubles, from 19 to 37 species, including at least six new taxa that need

to be formally described. Biogeographically, there is also some noteworthy patterns that arouse

from the mtDNA data. All widespread species presented some degree of genetic structure in

the known areas of endemism in Amazonia (Borges & Da Silva, 2012; Cracraft, 1985). Most

of the larger Amazonian tributaries, including rivers such as the Negro, Madeira, Solimões, and

Amazonas delimit lineages in opposite margins, however, if they were responsible for causing

these divergences still need to be investigated.

According to our phylogenetic hypothesis for Galbulidae, there are now eight main

groups of species:

1. Brachygalba and Jacamaralcyon

Brachygalba and Jacamaralcyon species were recovered as sisters to all other jacamars.

The monotypic Jacamaralcyon species, Jacaramaralcyon trydactyla (Viellot, 1817), is

endemic to the Atlantic Forest, inhabiting semi-deciduous or gallery forest. This species was

recovered as sister to all other Brachygalba species (Fig. 1), which prefer forest edges and open

habitats throughout the Amazon basin and north South America. B. goeringii Sclatter, PL &

Salvin, 1869 and B. salmoni Sclatter, PL & Salvin, 1879 represent two distinct lineages within

Brachygalba radiation (Fig. 2), with very distinct plumages and restricted distributions in

northern South America. B. goeringii was recovered as sister to all other Brachygalba species,

and B. salmoni, as sister group to the species group of B. lugubris (naumburgae, obscuriceps,

lugubris, and melanosterna) and B. albogularis (von Spix, 1824), from the Amazon basin (Fig.

2). Because B. albogularis was embedded within B. lugubris lineages, we recommend that the

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current subspecies of B. lugubris should now be elevated to species status. This way, we resolve

the paraphyly of B. lugubris, and fully recognize all its diversity. Further studies are necessary

to completely understand B. obscuriceps Zimmer, JT & Phelps, 1947 and B. naumburgae

Chapman, 1931 distributions, especially regarding the relationship between B. lugubris, and B.

l. fulviventris Sclater, PL, 1891 and B. l. caquetae Chapman, 1917.

2. Jacamerops

Jacamerops individuals are so distinct from the other jacamars that were once considered

to belong to a separate subfamily Jacameropinae. Although this treatment is no longer followed,

Jacamerops are by far the bulkiest jacamars, inhabiting the midstory and canopy of continuous

forest in the Amazon basin. Among the four subspecies recognized, J. a. ridgway Todd, 1943

formed a well supported clade in both analyses. (Fig. 2, S2), while J. a. aureus (Statius Müller,

PL, 1776) was monophyletic in the mtDNA analysis (Fig. S2) but paraphyletic in the UCE

analysis, with the two individuals from the Guiana Shield as sister to all other J. aureus

individuals (Fig. 2). Since the type from J. a. aureus is British Guiana (Peters, 1948), we

consider that only this group should be recognized as J. aureus, while the second lineage should

receive a new name (Fig. 2). An interesting biogeographic pattern that arouse from Jacamerops

data was the sister relationship between J. penardi Bangs & Barbour, 1922, from Central

America, and J. isidori Deville 1849, from the Madeira-Solimões interfluve. A similar pattern

was found in the Hylophylax species complex (Fernandes et al., 2014). Finally, J. ridgwayi

Todd, 1943 requires further study to fully evaluate all diversity present in this group, our results

suggest the presence of at least 4 mtDNA lineages, each separated by the main rivers of the

Brazilia Shield.

3. Galbalcyrhynchus

Galbacyrhynchus species are endemic to floodplain forests from Western Amazon.

Galbalcyrhynchus purusianus Goeldi, 1904 was considered conspecific with G. leucotis Des

Murs, 1845, and they were actually considered male and female forms of the same species.

Nonetheless, the parapatric distribution and the apparently lack of intermediate forms (Haffer,

1974) render these two taxa the status of distinct species (Fig. 2).

4. Galbula dea complex

Previously allocated in the genus Urogalba, Galbula dea individuals are the most

morphologically distinct among Galbula species. Our results recovered six distinct mtDNA

lineages (Fig. S3) that matches with the UCE results (Fig. 2), in which four already have

associated names. G. dea (Linnaeus, 1758) from the Guiana Shield; G. brunneiceps (Todd,

77

1943) from the Negro-Solimões interfluve; G. phainopepla (Todd, 1943) from the Solimões-

Madeira interfluve; and G. amazonum (Sclater, PL, 1855). The last lineage, from the Madeira-

Tapajós interfluve was considered to be part of G. d. brunneiceps (Peters, 1948), however, since

the type locality from G. brunneiceps is Manacapurú, Rio Solimões, Brazil, we suggest that a

new name should be given to this lineage.

5. G. leucogastra/chalcothorax

This complex includes the only jacamars that inhabit white-sand environments (Adeney,

Christensen, Vicentini & Cohn-Haft, 2016) in the Amazon basin. Although highly structured

throughout its distribution (Ferreira et al., submitted) this group lacks morphological

distinctiveness among genetic lineages, thus further systematic and taxonomic work is required

before the proposition of any change in nomenclature.

6. Galbula albirostris, G. chalcocephala, and G. cyanicollis

These tree species were formerly considered conspecifics in G. albirostris Latham, 1790

(Peters, 1948), later Haffer (1974) recognized G. cyanicollis Cassin, 1851, based on the lack of

interbreeding between these two forms. Our results support the recognition of all three species,

with G. albirostris restricted to the Guiana Shield, east of Negro River; G. chalcocephala

Deville, 1849 in between the west bank of lower Negro river, west of Branco River, and north

of Solimões all the way down to the west bank of the upper Ucayali River (Harvey, Seeholzer,

Cáceres A, Winger, Tello, Camacho, Aponte Justiniano, Judy, Ramírez, Terrill, Brown, León,

Bravo, Combe, Custodio, Zumaeta, Tello, Bravo, Savit, Ruiz, Mauck & Barden, 2014); and at

last, G. cyanicollis, along the south bank of Amazon River. This group of species, in contrast

with other jacamars, only inhabits the interior of forests, mainly in terra-firme habitats. Not

surprisingly, the mtDNA showed lineages separated by the main Amazonian tributaries (Fig.

S4). However, some lineages presented some interesting biogeographic patterns, such as the

distinct lineage at the lower portion of Madeira-Tapajós interfluve, that is also found in other

groups of birds, such as Rhegmatorhina berlespchi (Ribas et al., 2018), Malacoptila rufa

(Ferreira et al., 2017), and Glyphorhynchus spirurus (Fernandes, Gonzalez, Wink & Aleixo,

2013). Another pattern, that has not been reported before for birds, is the distinct lineage

between the Purus and Tapajós Rivers (Fig. S4). This is the first evidence of a lineage of and

understory terra-firme bird that has n structure related to the Madeira River.

7. G. melanogenia, G. pastazae, G. pallens, and G. ruficauda.

Although G. melanogenia Sclater, PL, 1852, was first described as a full species, it was

later lumped together with G. rufoviridis Cabanis, 1851 in G. ruficauda Cuvier, 1816 due to

78

morphological similarity (Peters, 1948). Our results, however, recovered this group as sister to

the clade containing G. cyanicolis and G. galbula complex (Fig. 2). Also, G. pastazae

Taczanowski and Berlepsch, 1885, probably the only jacamar to live in high altitudes, is

embedded between G. melanogenia and G. ruficauda (Fig. S5, 2). Therefore, our

recommendation is that G. melanogenia, from Central America and the Pacific coast of

Colombia and Ecuador, along with G. pallens Bangs, 1898 and G. ruficauda Cuvier, 1816

should be recognized as species. Further studies are required to check the validity of G. r.

brevirostris Cory, 1913.

8. Galbula galbula, G. tombacea, G. cyanescens and G. rufoviridis

This group is often regarded as G. galbula (Linnaeus, 1766) species group due to

morphological and ecology similarity. Usually associated with forest edges and floodplains

forest, while G. albirostris species group, its sister clade (Fig. 2), is usually associated with the

interior of terra-firme forests. Despite been associated with floodplain forests, and therefore,

not “bounded” by rivers, there are no previous reports of hybridization among these taxa. We

found, however, that the individual INPA A019 is phenotypically G. tombacea (checked by

M.F.), however, the mtDNA clustered with G. cyanescens Deville, 1849 (Fig. S5). This is the

only reported case of hybrids among this group, the other individual that could be a hybrid - G.

cyanescens, voucher MPEG MAD305 - is phenotypically G. cyanescens (checked by Fátima

Lima), even though the individual was collected in the right bank of Madeira River, supposedly

the limit between distributions of G. cyanescens and G. heterogyna Todd, 1932. Another

important pattern that we can observe in this group is the apparently discordance between the

mtDNA and UCE trees (Fig. S5, 3). Our mtDNA tree recovered G. cyanescens as one lineage

embedded within lineages of G. rufoviridis and G. heterogyna. It also recovered G. rufoviridis

as paraphyletic (Fig. S5). The UCE tree instead, recovered G. cyanescens as sister to G.

heterogyna and G. rufoviridis (Fig. 2). In addition, all samples we sequenced for G. rufoviridis

were recovered as monophyletic and sister to G. heterogyna. Thus, this might be an evidence

of mtDNA capture (Sloan, Havird & Sharbrough, 2017), in which probably G. cyanescens

captured the mtDNA lineage of G. heterogyna. However, further studies are required to

understand the direction and timing of this event.

Bucconidae systematics

Our results showed that, similar to the situation with Galbulidae, Bucconidae diversity is

underestimated. In addition, we found evidence of genera paraphyly. Phylogeographic patterns

recovered for widespread puffbird species varied from little to no genetic structure, as in

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Chelidoptera and Cyphos, to highly structured, as in Malacoptila (Ferreira et al., 2017),

Nonnula rubecula and N. ruficapilla, and Monasa morphoeus (Soares, 2016). Historically,

apart from the morphologically explicit genera Hapaloptila, Chelidoptera, Malacoptila,

Micromonacha, Monasa, and Nonnula, all other species were lumped in Bucco Brisson, and

later split in Notharchus and Hypnelus. Currently, although some authors recognize different

genera for former Bucco species (i.e. Cyphos, and Nystactes) (Rassmussen & Collar, 2018),

many others still keep several species within the genus Bucco (Gill & Donsker, 2018; Piacentini

et al., 2015; Remsen et al., 2018). Our results however recovered Bucco as polyphyletic, and

thus, we favor the recognition of Cyphos Spix, 1824 (which has priority over Argicus Cabanis

& Heine, 1863) and Nystactes Gloger 1827. Also, we recovered Notharchus specie as

paraphyletic, with the species group of N. tectus (Boddaert, 1783) as sister to the clade

containing Hypnelus, Nystactes and the other species of Notharchus. One way to resolve this

paraphyly would be to include Hypnelus and Nystactes in the genus Notharchus, however, both

Nystactes and Hypnelus species are morphologically distinct from any of Notharchus species.

Therefore, we propose the revalidation of Nothriscus Cabanis & Heine, 1863 to accommodate

Nothriscus tectus, N. subtectus and N. picatus, resolving the paraphyletic relationships found

within Notharchus species.

1. Bucco capensis and Nystalus

Bucco capensis Linnaeus, 1766 and Nystalus species were recovered as sister to all other

puffbirds. The sister relationship we recovered between B. capensis and Nystalus is validated

by the bill-tip morphology that was previously used to separate former Bucco species in the

genera Cyphos and Nystactes (Rassmussen & Collar, 2018). Our results for B. capensis samples

recovered three clades in the UCE tree (Fig. 3) in contrast to the four clades found in the mtDNA

analysis (Fig. S7). Our UCE analysis also favor the recognition of B. dugandi Gilliard, 1949

and suggest the presence of a new taxon yet undescribed. Nystalus relationships found here

were similar to a previous study that used only one mtDNA marker (Duarte, 2015), which

recovered N. maculatus (Gmelin, JF, 1788) and N. striatipectus (Sclater, PL, 1854) as sister to

all remaining species, and N. chacuru (Vieillot, 1816) as sister to N. radiatus (Sclater, PL, 1854)

and the N. striolatus species complex: N. obamai Whitney et al., 2013; N. striolatus (Pelzeln,

1856), and N. torridus Bond & Meyer de Schauensee, 1940. Further studies are required to fully

understand the distribution and relationship of Nystalus striolatus species complex.

2. Chelidoptera

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The Swallow-winged puffbird, Chelidoptera tenebrosa Pallas, 1782, is by far the most

distinct puffbird, aberrant both in morphology and in ecology. With swallow-like morphology,

they are highly specialized in aerial activity, and its flying proficiency is probably the cause for

the lack of genetic structure we found in the mtDNA (Fig. S10). However, we were unable to

sample UCE from the two toe pad samples, from the subspecies C. t. pallida Cory, 1913, from

Northwest Venezuela; and C. t. brasiliensis Sclater, PL, 1862, from the east coast in Brazil.

3. Monasa and Nonnula

Monasa and Nonnula were the focus of a recent phylogeographic study (Soares, 2016).

Species from both genera presented high levels of genetic structure in the mtDNA, and we

sampled one individual per mtDNA lineage that were uncovered previously. We recovered

Monasa as sister to Chelidoptera, and these two sisters to Nonnula (Fig. 1). Relationships inside

each genus (Fig. 3) were also congruent to Soares (2016). In addition to this previous study, we

were able to sample three toe pads representing three subspecies of M. morphoeus (Hahn &

Küster, 1823): M. m. morphoeus (Hahn & Küster, 1823) from the east coast of Brazil; M. m.

pallescens Cassin, 1860; and M. m. grandior Sclater, PL & Salvin, 1868, both from Central

America. However, their phylogenetic relationship with other subspecies of M. morphoeus was

uncertain (Fig. S11) and further studies are required to fully understand if the phylogeographic

structure found in the mtDNA matches the UCE. For Nonnula, our results support the paraphyly

of N. ruficapilla (Tschudi, 1844), with N. amaurocephala Chapman, 1921 embedded within it.

Both genera are being studied using broader sampling of individuals and molecular markers.

4. Malacoptila

Malacoptila UCE topology was congruent with the concatenated dataset topology from

Ferreira et al. (2017), placing M. fulvogularis Sclater, PL, 1854 as sister to all other species.

This result changes the previous biogeographic interpretations, and a more detailed study

focusing on this genus is necessary, to fully understand the relationship of Malacoptila species,

including the position of M. mystacalis (Lafresnaye, 1850), that in the concatenated UCE tree

was recovered as sister to all other species (Fig. 3). Since, M. mystacalis UCE contigs were

shorter due to DNA degradation common in toe pad samples (McCormack, Tsai & Faircloth,

2016), we assembled a small subset of Malacoptila samples to minimize the effects of missing

data, and yet, M. mystacalis was again, recovered as sister to all other species of Malacoptila

(Fig. S12). Further sampling of this narrow endemic species is required to confirm if this pattern

is true, or an artefact of toe pad sequencing error.

5. Hapaloptila

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The monotypic Hapaloptila castanea (Verreaux, J, 1866) was recovered as sister group

to Micromonacha, Cyphos, Nothriscus, Hypnelus, Nystactes, and Notharchus (Fig. 1, 3). Very

distinct in morphology, this species is specialized in cloud forests, usually above 1,500 m, and

even though it can be found in both sides of the Andes, no subspecies was ever described. The

two specimens we samples are from opposite sides of Andes, however a more focused work on

this species is required to understand the relationships among these apparently disjunct

populations.

6. Micromonacha

Micromonacha lanceolata (Deville, 1849) occurs in the middle and upper stories of

forests in both sides of the Andes, usually below 1,500 m. With populations also found in

Panama and Costa Rica. Although no subspecies is currently recognized (Rassmussen & Collar,

2018), populations from Central America were historically recognized in a distinct subspecies

M. l. austinsmithi Dwight and Griscom, 1942. Our results recovered the sample from Panama

as sister to the other two samples from Peru and Brazil, however, we refrain from making any

nomenclatural change pending better sampling of this group to fully understand its diversity.

7.Cyphos

Cyphos macrodactylus Spix, 1824 can only be found east of the Andes, mostly near water

inside terra-firme and varzea forests in Western Amazon. Our phylogeographic sampling

showed almost no genetic structure, only the westernmost sample showed some difference. If

this is, in fact, a phylogeographic structure, or just an artifact in sampling, still needs to be

investigated. The described subspecies C. m. caurensis (Cherrie, 1916) from the Caura River

region, Venezuela, is currently considered undifferentiated from the nominal form (Rassmussen

& Collar, 2018), and probably does not correspond to this phylogeographic break, additional

sampling is required for further assumptions.

8. Nothriscus

The three species included in the genus Nothriscus Cabanne & Heine 1863, N. tectus, N.

subtectus and N. picatus, were first described as full species, and later lumped and considered

conspecific as Nothriscus tectus (Boddaert, 1783) (Peters, 1948). Recently, N. subtectus

regained its status as full species (Rassmussen & Collar, 2018), but N. tectus and N. picatus are

still considered subspecies (Gill & Donsker, 2018; Remsen et al., 2018). Our results recovered

N. picatus as sister to a clade containing N. tectus and N. subtectus, both in the mtDNA and the

UCE tree. Biogeographically, implying that the two forms found in the Amazon, N. picatus and

N. tectus, are not sister. Therefore, we propose the recognition of these taxa as full species and

82

a more extensive work should be carried out to understand the limits of distribution of both

Amazonian species, and if there is any contact, what are the implications of it.

9. Hypnelus and Nystactes

The sister relationship of Hypnelus and Nystactes is supported by the autapomorphic bifid

bill tip in both genera, that is also present in Notharchus, although less pronounced in the later

(Rassmussen & Collar, 2018). Hypnelus species are restricted to northern South America, with

H. ruficollis (Wagler, 1829) having three subspecies, and H. bicinctus (Gould, 1837), two. Their

specific status has been questioned based on hybridization in part of their distribution (Donegan,

Quevedo, Verhelst, Cortés-Herrera, Ellery & Salaman, 2015), however without a thorough

genetic and geographic sampling, this decision remains questionable. Nystactes noanamae

(Hellmayr, 1909) and the species group of N. tamatia (Gmelin, JF, 1788), form the sister group

of Hypnelus (Fig. 1, 3). Nystactes noanamae, is a restricted-range species, present only in a

small portion of northwest Colombia, and currently considered Near-threatened by IUCN

(Rassmussen & Collar, 2018). Its sister species, N. tamatia, is associated with the flooded

forests in Amazonia, rarely found far from the water, even when in terra firme. Previous

phylogeographic study found six genetic lineages for N. tamatia, one lineage was composed by

only one sample though (Almeida, 2013). Nevertheless, our results corroborate the

relationships previously found, and further studies are being conducted to understand the

relationships and distribution of each lineage (Almeida, 2013).

10. Notharchus

Notharchus species can be grouped into three distinct groups based on distribution and

morphology. Notharchus ordii (Cassin, 1851), as sister to all other species, is restricted to

Amazonia, and unusually uncommon in collections. Its habitat preference and current

distribution is virtually unknown. The sampling we gathered for the mtDNA sequencing

actually represents all tissue samples available, and the apparent phylogeographic structure we

found (Fig. S9) may only represent an artifact of sampling. Notharchus pectorales (Gray, GR,

1846) is restricted to Northwest Colombia and East Panama. The last groups of species, is the

group centered in N. macrorhynchus (Gmelin, JF, 1788). The ND2 analyses recovered a

polytomy between the N. swainsoni (Gray, GR, 1846) N. macrorhynchus, and several lineages

of N. hyperrhynchus, including one lineage from Central America (Fig. S9). Our UCE tree, in

contrast, recovered N. macrorhynchus sister to N. swainsoni and N. hyperrhynchus. This result

corroborates the recognition of N. hyperrhynchus and N. swainsoni as full species and renders

the two Amazonian groups as non-sister lineages. Although the two subspecies of N.

83

hyperrhynchus seem to be paraphyletic in the UCE topology, the geographical relationship

seem to be reasonable, and a reappraisal of this subspecies distribution is desirable in further

studies.

Conclusion

The results presented here corroborate most of the diversity historically described in these

two families, but also hidden patterns that need further investigation. With our thorough

sampling of practically all widespread species and species complexes we were able to recover

the phylogeographic patterns for the entire diversification of jacamars and puffbirds. This study

is the first one to present a phylogenetic hypothesis for this two families employing a genomic

dataset. Based on this tree we resolved some relationships that were obscured by morphological

similarities among taxa, such as the recognition of the different species previously lumped into

Galbula ruficauda, and even revalidating four genera of puffbirds to accommodate paraphyletic

relationships found. Overall, the results presented here are another instance reinforcing the fact

that Neotropical bird diversity still is underestimated, and that we still need exploratory research

to fully comprehend diversity patterns, especially in the super complex Amazonian Basin,

which will be of extreme importance for future biogeographical interpretations and better

conservation planning.

Acknowledgements

The authors thankfully acknowledge all the curators and curatorial assistants of the

American Museum of Natural History, New York, USA (AMNH), Academy Academy of

Natural Science of Drexel University, Philadelphia, USA (ANSP); Field Museum of Natural

History, Chicago, USA (FMNH); Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil

(INPA); Kansas University (KU), Laboratório de Genética e Evolução Molecular de Aves –

USP (LGEMA), Lousiana State University Museum of Natural Science, Baton Rouge, USA

(LSUMZ); and Museu Paraense Emílio Goeldi, Belém, Brazil (MPEG), Smithsonian Institution

National Museum of Natural History (USNM), for borrowing tissue samples under their care.

We are also grateful for all collectors involved in the fieldwork that make this paper possible.

To J. McKay for helping with some laboratory procedures at the AMNH. MF acknowledge

CAPES for his PhD fellowship, and CAPES PDSE fellowship (# 88881.133440/2016-01) and

the support from the AMNH Frank M. Chapman Memorial Fund. The authors also thank the

grant Dimensions US-Biota-São Paulo: Assembly and evolution of the Amazon biota and its

84

environment: an integrated approach, co-funded by the US National Science Fundation (NSF

DEB 1241056) to J.C. and the Fundação de Amparo à Pesquisa do Estado de São Paulo

(FAPESP grant #2012/50260-6) to Lucia Lohmann; PEER-USAID Cycle 5 to CCR. AA and

CCR are supported by CNPq research productivity fellowships (#310880/2012-2 and

#308927/2016-8, respectively). The authors acknowledge the National Laboratory for

Scientific Computing (LNCC/MCTI, Brazil) for providing HPC resources of the SDumont

supercomputer, which have contributed to the research results reported within this paper.

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Figure 1 – Phylogeny of the Galbulidae and Bucconidae families inferred with ExaBayes. All nodes in this tree

receive the maximum posterior probability. The two genomes used as reference sequence were included in this

analysis.

89

Figure 2 – Phylogeny of the Galbulidae inferred by ExaBayes with the 75% completeness matrix. Node support

is indicated near it, if no support is indicated posterior probability is 1.0.

90

Figure 3 – Phylogeny of the Bucconidae inferred by ExaBayes with the 75% completeness matrix. Node support

is indicated near it, if no support is indicated posterior probability is 1.0.

91

Figure S1 – Phylogenetic relationship and map with sample distribution of Brachygalba and Jacamaralcyon

species. Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean

posterior probabilities of 1.0, values that differs are indicated near the node. Samples highlighted in red were the

samples selected for UCE analysis. The maps contain sample localities and approximate lineage distribution.

Colours are correspondent between the tree and the map.

92

Figure S2 – Phylogenetic relationship and map with sample distribution of Jacamerops aureus complex.

Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior

probabilities of 1.0, values that differs are indicated near the node. Samples highlighted in red were the samples

selected for UCE analysis. The maps contain sample localities and approximate lineage distribution. Colours are

correspondent between the tree and the map.

93

Figure S3 – Phylogenetic relationship and map with sample distribution of Galbula dea complex. Phylogenetic

tree was recovered by MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior probabilities

of 1.0, values that differs are indicated near the node. Samples highlighted in red were the samples selected for

UCE analysis. The maps contain sample localities and approximate lineage distribution. Colours are


94

Figure S4 – Phylogenetic relationship and map with sample distribution of the species complex of G. albirostris,

G. chalcocephala and G. albirostris. Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2

(1041bp). Red circles mean posterior probabilities of 1.0, values that differs are indicated near the node. Samples

highlighted in red were the samples selected for UCE analysis. The maps contain sample localities and

approximate lineage distribution. Colours are correspondent between the tree and the map.

95

Figure S5 – Phylogenetic relationship and map with sample distribution of the species complex of G. galbula, G.

tombacea, G. cyanescens, G. pastazae, and G. ruficauda. Phylogenetic tree was recovered by MrBayes using the

mtDNA gene ND2 (1041bp). Red circles mean posterior probabilities of 1.0, values that differs are indicated

near the node. Samples highlighted in red were the samples selected for UCE analysis. The maps contain sample

localities and approximate lineage distribution. Colours are correspondent between the tree and the map.

96

Figure S6 – Phylogenetic relationship and map with sample distribution of the species Bucco capensis.





97

Figure S7 – Phylogenetic relationship and map with sample distribution of the species Cyphos macrodactylus.





98

Figure S8 – Phylogenetic relationship and map with sample distribution of the species complex of Notharchus

tectus, N. subtectus, and N. picatus. Phylogenetic tree was recovered by MrBayes using the mtDNA gene ND2

(1041bp). Red circles mean posterior probabilities of 1.0, values that differs are indicated near the node. Samples

highlighted in red were the samples selected for UCE analysis. The maps contain sample localities and

approximate lineage distribution. Colours are correspondent between the tree and the map.

99

Figure S9 – Phylogenetic relationship and map with sample distribution of the species complex of Notharchus

ordii, N. pectorales, N. swainsoni, N. macrorhynchus, and N. hyperrhynchus. Phylogenetic tree was recovered by

MrBayes using the mtDNA gene ND2 (1041bp). Red circles mean posterior probabilities of 1.0, values that

differs are indicated near the node. Samples highlighted in red were the samples selected for UCE analysis. The

maps contain sample localities and approximate lineage distribution. Colours are correspondent between the tree

and the map.

100

Figure S10 – Phylogenetic relationship and map with sample distribution of the species Chelidoptera tenebrosa.





101

Figure S11 – Phylogenetic tree recovered for Monasa using a subset of samples to check for M. mystacalis

phylogenetic affinity. The same tree topology was recovered by RAxML and ExaBayes, the RAxML bootstrap

support were low overall.

Figure S12 – Phylogenetic tree recovered for Malacoptila using a subset of samples to check for M. mystacalis

phylogenetic affinity. The same tree topology was recovered by RAxML and ExaBayes with high support, with

only node receiving bootstrap support different from 100.

102

Síntese Geral

Neste trabalho coletamos dados que nos ajudaram a compreender a relação filogenética

de três famílias de aves do Neotrópico. A utilização de dados de sequenciamento genômico e a

inclusão de amostras representando quase todas as linhagens basais em cada família permitiu

realizar inferências sobre a importância de uma amostragem ampla, tanto num sentido de

amostras, quanto marcadores. No primeiro capítulo pudemos observar o impacto do conflito

entre marcadores moleculares com diferentes padrões de herança, e quais as implicações

biológicas deste conflito. Além disso, através da análise combinada da história dos dois

marcadores foi possível propor um modelo de evolução das áreas de vegetação aberta

relacionadas aos solos de areia branca dentro da bacia Amazônia. No segundo capítulo,

recuperamos a relação filogenética da família Trogonidae utilizando quase todas as espécies

descritas com base em uma matriz com mais de 2.000 marcadores moleculares. Com base

nesses resultados traçamos um modelo de como a evolução do clima desde o final do Oligoceno

e as conexões entre os continentes influenciaram a história de diversificação do grupo. Por fim,

no terceiro capítulo, analisamos a diversidade intraespecífica de duas famílias endêmicas do

Neotrópico e reconstruímos a primeira hipótese de relação filogenética para Galbulidae e

Bucconidae utilizando dados genômicos. Neste capítulo pudemos observar como a percepção

da diversidade nesses grupos é subestimada e influenciada pela taxonomia vigente, e que a

amostragem densa ao longo da distribuição de espécies amplamente distribuídas pode revelar

táxons e padrões ainda desconhecidos.

De modo geral, este trabalho reforça a complexidade dos padrões de diversidade da biota

Neotropical, e que ainda se faz necessário estudos para desvendar esses padrões, em especial

na Amazônia. Além disso, fica claro que a diversidade real da região ainda está mascarada pela

taxonomia vigente e revisões sistemáticas e taxonômicas são necessárias. Só através do

reconhecimento dessa diversidade escondida é que será possível, não só traçar hipóteses sobre

os processos que deram origem a tamanha diversidade, mas também traçar planos de

conservação que reconheçam a história evolutiva de cada um desses grupos.

103

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