Workshop Biodiversity and Biobank – FAPESP / ABC / SI

FAPESP, São Paulo, Brazil, 16 August 2017

Large-scale biodiversity genomics

initiatives in Brazil:

From DNA barcodes to whole genome

sequences

Eduardo Eizirik

Faculdade de Biociências, PUCRS, Brazil

Instituto Pró-Carnívoros, Brazil

Brazil

Area: 8,515,767 km2

Population: 190,732,694

Megadiverse country

- Estimated to harbor ~1.8 mi spp.

(10-17% of the world’s total)

[Lewinsohn & Prado 2005]

- Known fauna:

~9k vertebrates (711 mammals,

1,900 birds, 732 non-avian

reptiles, 973 amphibians, 3,133

continental fish, 1,376 marine

fish)

Map: wikipedia.org

Applications of DNA Barcoding in

Biodiversity Conservation

1. Gathering data on components of native biodiversity;

1.1. Baseline data (e.g. community composition and

dynamics, geographic distribution, trophic interactions).

1.2. Monitoring of biodiversity in impacted areas.

2. Gathering data on threats to native biotas.

- e.g. invasive species, pathogens, wildlife trafficking.

3. Helping to enforce actions aimed at curbing threats to

biodiversity.

- e.g. wildlife forensic analyses.

E. Eizirik, CBOL regional mtg. Brazil, 2007

The effort towards Large-scale DNA barcoding of

Brazilian biodiversity

Initial Proposal (2005):

Beginning of the Brazilian DNA barcoding network

- Large-scale inventory of Brazilian biodiversity

- Multi-center project designed in 2005 to boost taxonomic

research in Brazil using the DNA barcode concept as a catalyst to

integrate field collections, museum-based biodiversity research,

genome center networks and bioinformatics advances.

- 6 museums, 14 Centers of Molecular Biodiversity, ~300 people

Large-scale inventorying of Brazilian biodiversity (2005) Sampling strategy

15 sites:

-10,000 samples/site:

- fish

- amphibians

- reptiles (incl. birds)

- mammals

- spiders

- Leguminosae

Phase 1:

~US$ 3,000,000

2010-2014

2010 – BrBOL launched

>100 participating

groups

~500 people involved

- All major Brazilian museums

and natural history collections.

- Bioinformatics center

- Biomedical institution

- Agricutural research agency

The two largest museums did

not have molecular biology

laboratories.

- Implemented in both via

funding from the BrBOL

network.

Projeto ‘Tetrapoda’ - BrBOL

Barcoding Tetrapoda:

4 sub-groups

Amphibians (C. Haddad)

Reptiles (H. Zaher)

Birds (C. Miyaki)

Mammals (E. Eizirik)

Participating labs

Mammals

Birds

Non-avian reptiles

Amphibians

DNA barcoding of Brazilian tetrapods (2010-2014)

25 participating

institutions

> 100 people involved

4 Major Natural

history collections in

Brazil

French Guiana joined BrBOL

Group Barcoded Individuals Barcoded Species

Amphibians 5,100 450

Non-avian reptiles 2,608 816

Birds 3,508 1,253

Mammals 2,122 344

Total 13,338 2,863

DNA barcoding of Brazilian tetrapods

Results: DNA Barcode (COI) library construction (2010-2015)

DNA barcoding of Atlantic Forest amphibians

(M. Lyra, C. Haddad)

~3800 inds/vouchers

- 386 species

(71% of the known

species).

- 59/63 genera

Geographic distribution of mammal DNA barcodes

Large-scale DNA barcoding in Brazil

Good news:

1. We got started and scratched the surface

2. An unprecedented community of biodiversity scientists has

been assembled and integrated in Brazil.

3. There is capacity in the country to move forward.

Challenges ahead:

1. Securing continuous, large-scale funding.

2. Improving governance and organizational structure.

3. Scaling up and speeding up to tackle the magnitude of the task

and the pace of habitat loss in the country.

Leopardus geoffroyiScinax granulatus

Dietary analyses of wild cats using DNA barcodes of prey items

CPCN Pró-Mata, PUCRS, Brazil

Vriesea platynemaAechmea gamosepala

Meta-barcoding of Atlantic Forest bromeliad

tank waters

Meta-barcoding of Atlantic Forest bromeliad

tank waters

Biodiversity of the mineral province of Carajás (G. Oliveira)

DNA barcodes

Genomes

Metagenomes

Canga plantsCave invertebrates

Plant barcodes

16

144

0

2748

46

706

0

3179

0 0

179

2077

26 0 3

2285

0

500

1000

1500

2000

2500

3000

3500

BOLD ITV

Carajás

Circonuscus sp.

Cave invertebrates:1,074 specimensCirconiscus: 1 BOLD 70 ITV

Guilherme Oliveira – Environmental Genomics @ITV

Whole-genome sequencing of Brazilian biodiversity

The Jaguar Genome

Project

www.jaguargenome.org

Origin: 2011

Brazilian Congress of Genetics

ConGen course in the Pantanal

- Unique features among the Panthera

- Phenotypic diversity (size, coloration)

The jaguar (Panthera onca)

Galetti et al. 2013. Science

Conservation Issues

The Jaguar Genome

Project

www.jaguargenome.org

• Consortium of Brazilian Institutions

PUCRS, ESALQ/USP, FIOCRUZ,

IPC, CENAP, IDSM, IOP, UFSJD,

ZMQB

• Collaborators in 6 different countries

- USA

- Russia

- Ireland

- Portugal

- Spain

- Argentina

SEQUENCING ASSEMBLY ANNOTATION

Vagalume(Sorocaba Zoo)

100 bp

180 bp

3 kb

8 kb

3 lanes

1 lane

1 lane

2,660,456,270 reads

94x coverage

Sequencing the jaguar genome

• De novo assembly

• ALLPATHS

• 156,436 Contigs

• 7,521 Scaffolds

• 2.4 Gb

• Contig N50: 28.6 kb

• Scaffold N50: 1.52 Mb

SEQUENCING ASSEMBLY ANNOTATION

Sequencing the jaguar genome

• Maker2

• De novo prediction of 25,451 coding genes

• Validation of 96% with RNA-seq data (6

tissues) and other empirical evidence.

• Annotation of repetitive elements (e.g. 344,251

microsatellites), ncRNA and numts (nuclear

insertions of mtDNA)

SEQUENCING ASSEMBLY ANNOTATION

Sequencing the jaguar genome

Comparative genomics of genus Panthera:

Henrique Figueiró, PUCRS

Gang Li, William Murphy

Texas A&M University

Species tree

• Estimated based on 100-kb genomic windows, as well as

gene-by-gene data sets (13,183 shared loci).

lion (Panthera leo)

leopard (Panthera pardus)

jaguar (Panthera onca)

Snow leopard (Panthera uncia)

tiger (Panthera tigris)

• Extensive genealogical discordance in the Panthera, with remarkable spatial

variation across their genomes.

Tree 1 (species tree)

6

5

0

4

3

1

2

Tree 2 Tree 3

lion

jaguar

leopard

tiger

snow leopard

cat

jaguar

leopard

lion

tiger

snow leopard

cat

XAutosomes

Years

(M

ya)

lion

leopard

jaguar

tiger

snow leopard

cat

X X

P1 P2 P3 D s.e. Z

PLE PON PUN -0.2235 0.0043 -52.5

PPA PON PUN -0.1804 0.0042 -43.3

PPA PLE PUN 0.0567 0.0034 16.7

PLE PON PTI -0.1739 0.0055 -31.6

PPA PON PTI -0.1379 0.0051 -27.2

PPA PLE PTI 0.0482 0.0037 13.1

PTI PUN PON -0.1200 0.0040 -30.0

PTI PUN PPA -0.0742 0.0028 -26.8

PTI PUN PLE -0.0685 0.0026 -26.0

PPA PLE PON 0.0569 0.0048 11.8

P. leoP. tigris P. oncaP. pardusP. uncia

ABBA-BABA test

lion

jaguar

leopard

tiger

snow leopard

cat

jaguar

leopard

lion

tiger

snow leopard

cat

lion

leopard

jaguar

tiger

snow leopard

cat

Outlier window testScreen for divergence time outliers indicating introgressed segments

Genealogy

Intriguing overlap of pathways enriched in both introgressedsegments and genes with signatures of positive selection

Design of a customized Panthera exome capture array

• 19,000 genes = 36Mb

• Nimblegen Capture Kit

• Annotation

> > >= =

• 113 jaguar individuals sequenced in 2 HiSeq lanes

• Average coverage of 8x

• 160,000 SNPs – 67,000 SNPs after filtering

• 30 individuals used to assess intra-specific

variation in genes with signatures of introgression

Assessment of intra-specific variation

Collaboration with

Rasmus Nielsen

(UC Berkeley)

Coalescent simulations to test for positive selection in genes bearing signatures of inter-species introgression

Ongoing fronts – Jaguar Genome Project

- Continued analyses of the exome data set.

- Analysis of a GBS data set covering multiple

Brazilian regions.

- Analysis of whole genomes from additional

individuals:

- Guatemala (Chromium – 10xG)

- Arizona (Discovar)

- Amazonia, Atlantic Forest, Caatinga

(Illumina HiSeqX).

Other felid genome sequencing projects

Puma

(Puma concolor)

Pampas cat

(Leopardus colocolo)

Ocelot

(Leopardus pardalis)Margay

(Leopardus wiedii)

Collaborations with:

Beth Shapiro (UCSC)

Priscilla Villela (EcoMol)

Luiz Coutinho (ESALQ/USP)

Greg Barsh (HudsonAlpha)

Bill Murphy (Texas A&M)

Chris Kaelin (Stanford)

Pedro Galetti (UFSCar)

Warren Johnson (Smithsonian)

Klaus Koepfli (Smithsonian)

Other ongoing genome sequencing projects

Maned wolf

(Chrysocyon brachyurus)

Bush dog

(Speothos venaticus)

Neotropical otter

(Lontra longicaudis)

Humpback whale

(Megaptera novaeangliae)

South American fur seal

(Arctocephalus australis)

Luis Claudio Marigo

South American foxes

(Lycalopex spp.)

Fernanda Valdez

Cristine Trinca

Henrique Figueiró

Marina Favarini

Tiago Ferraz

Maísa Bertoldo

Paulo Chaves

Flavia Tirelli

Fernanda Michalski

Anne Schmidt-Küntzel

Maria Eduarda Appel

Bromeliad meta-barcoding

Taiz Simão

Laura Utz

Adriana Giongo

Eric Tripplett

Renata Medina

Acknowledgments - Barcodes

BrBOL

Claudio Oliveira

Guilherme Oliveira

Cristina Miyaki

Fabricio Santos

Aristóteles Góes

Mariana Oliveira

Paulo Buckup

Ana Maria Espin

Fernando Monteiro

Jorge Porto

Hussam Zaher

Célio Haddad

Mariana Lyra

Felipe Grazziotin

Alexandre Aleixo

Marcelo Weksler

Benoit de Thoisy

Camila Ribas

Pedro C. Estrela

Yuri Leite

Larissa Oliveira

Alexandre Percequillo

Aleksey Komissarov

Rodrigo Teixeira

Adauto Nunes

Leandro Silveira

Fernando Azevedo

Emiliano Ramalho

Graham Hughes

Oliver Ryder

Greg Barsh

Chris Kaelin

Marta Svartman

Acknowledgements – Genome Sequencing

Henrique Figueiró

Fernanda Trindade

Maíra Rodrigues

Lucas G. Silva

Vera de Ferran

Sarah Santos

Cristine S. Trinca

Daniel Kantek

Laura Heidtmann

Gustavo Lorenzana

Support: CNPq, CAPES, FAPERGS,

FAPESP, Tetrapak

William Murphy

Gang Li

Rasmus Nielsen

Steve O’Brien

Guilherme Oliveira

Luiz Coutinho

Agostinho Antunes

Emma Teeling

Toni Gabaldón

Patricia Saragueta

Ronaldo G. Morato

Robert Wayne

Klaus Koepfli

Bridgett VonHoldt

Warren Johnson

Sandro Bonatto

C. Scott Baker

Larissa Oliveira