From Genes to Genomes:Progress and Pitfalls in Barcoding the

“Kingdom” Protista

Linda Amaral-Zettler, PhDNIOZ Royal Netherlands Institute for Sea Research & Utrecht University

7th International Barcode of Life Conference, Kruger National Park, South Africa

E.O. Wilson, “The Father of BioDiversity"

“If I could do it all over again, and relive my vision in the twenty-first century, I would be a microbial ecologist”

© Jim Harrison

1024 stars in the Universe 1029 microbes in the Ocean

Photomicrograph by Jed Fuhrman

Life in a drop* of water~ 10,000 Protists~ 1,000,000 Bacteria~ 1,000,000 Archaea~10,000,000 Viruses

*=1 ml

“Kingdom” Protista

Caron et al., 2017

Protists span a diversity of life styles, sizes and trophic levels

In the mid-1800’s, Haeckel is attributed to be the first to describe the evolutionary relationships among living organisms, a genealogy of life, as analogous to a tree.

PLANTAE PROTISTA ANIMALIA The Three Kingdoms

The Evolving Concept of Protistan Phylogeny

Caron et al., Nature Reviews Microbiology 2017

Archaea

Bacteria

Eukarya

Modified from Norm Pace and Mitch Sogin

The Molecular“Tree of Life”

According to Ribosomal RNA

Woese (1928-2012)

Pace

Sogin

Phylogenetic Diversity Study

5’CTTACAGACCCTCATCCT3’

C AT GT A GC TG C C TC C C A AC -T GT - GC CG T C TT T C G AG AT G- A GC TG C C TC C C A AG -T GT A CC TG C C TC C C A A

Organism AOrganism BOrganism COrganism D

rRNA GENE

PCR Amplify Genes

Clone Genes

Sequence Genes

Infer Phylogenetic Trees

Align Sequences

Eukaryotic Biofilm Phylogenetic Diversity

Animals

Fungi

Viridiplantae

Stramenopiles

Alveolata

Amoebae

Red Algae

?

ChlamydomonasChlorella

Diatoms

Ciliates

Euglena cf. mutabilisRotifers

Cercozoa

Euglenozoa Vahlkampfiid

LabyrinthulidBicosoecidCarteria

Amaral-Zettler et al., Nature (2002) 417: 137

A Comprehensive Tree of Life?

Hinchcliff, Smith et al. PNAS 2015

1,000,000,000,000,000,000,000,000,000,000Microbial Cells in the Oceans

Photomicrograph by Jed Fuhrman

A Global Census of Marine MicrobesLinda Amaral-Zettler et al., 2010

We have sampled less than 1/1018 of the world’s oceans!

Similar low coverage in other environments

Mitch Sogin Jan de Leeuw

ICoMM’s Tag Sequencing Strategy

• Sequence only short section of hypervariable regions

• Each “tag”is a proxy for a microbe • Query each tag against a reference

data base• Identify taxonomic source of each

Tag to infer community composition.

Provides massively parallel ability to count different kinds of microbes in a community but it is NOT A PHYLOGENETIC TOOL!

Photo credit: Tom Kleindinst

¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾Coastal¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾

¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾Freshwater¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾ARCNTLCWT HB

CCE

SBC PIE GCE FCE VCR

PAL

Trop

ical

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Ope

n O

cean¾¾¾¾¾¾¾

¾¾¾¾¾¾¾¾¾¾¾¾

Polar ¾¾¾¾¾¾¾¾¾¾¾¾¾

CCE DOCUMENT

DESCRIBE DISCOVER

DIVERSITY

MCR

MCR MCM

Microbial Inventory Research Across Diverse Aquatic MIRADA LTERS

V6 V6

V9V9

Eukaryal

V9

BACTERIA & ARCHAEA EUKARYA

http://www.rna.icmb.utexas.edu

V4

V6 V6

V9V9

Eukaryal

V9

BACTERIA & ARCHAEA EUKARYA

htp://www.rna.icmb.utexas.edu

V4

De Vargas et al., Science, 2015.

Estimated 127,500 Protist Plankton Species

(V9 survey)

Pawlowski et al., 2012

Recognized 26,010 to 74,373: Estimated: 140,000 to 1.6 million

V4 Protist pre-barcodes

Pawlowski et al., 2012

UniEukUniEuk isanopen,inclusive,community-basedandexpert-driveninternational

initiativetobuildaflexible,adaptive,universaltaxonomicframeworkforEukarya

PartnerInstitutions MainFunders

Scientific & Technical Advisory Board

Colomban de Vargas – France, SB RoscoffPelin Yilmaz – Germany, MPI BremenJuliet Brodie – UK, NHM LondonVirginia Edgcomb – USA, WHOIEunsoo Kim – USA, AMNH New YorkSina Adl – Canada, U SaskatchewanGuy Cochrane – UK, EMBL-EBIJavier del Campo – Canada, UBCStefan Geisen – Netherlands Inst. of EcologyFrank Oliver Glöckner – Germany, MPIAlastair Simpson – Canada, Dalhousie U

Dave Caron – USA, U Southern CaliforniaSandra Baldauf – Sweden, U UppsalaSonya Dyhrman – USA, Columbia U, NYLaura Katz – USA, Smith College, MAConnie Lovejoy – Canada, U Laval, Québec Alexandra Worden – USA, MBARIJohn Archibald – Canada, Dalhousie UDavid Bass – UK, NHM, LondonPatrick Keeling – Canada, UBCJan Pawlowski – Switzerland, U Geneva

Steering Committee Advisory Council

Micah Dunthorn – Germany, U KaiserslauternLinda Amaral-Zettler – The Netherlands, NIOZClaire Gachon – UK, SAMSLaure Guillou – France, SB RoscoffLine Le Gall – France, MNHN ParisLaura Wegener Parfrey – Canada, UBCMatthew Brown – USA, Mississippi SUEnrique Lara – Switzerland, U NeuchatelFrédéric Mahé – France, CIRADRamon Massana – Spain, ICMConrad Schoch – USA, NIH/NLM/NCBIAlexandros Stamatakis – Germany, HITS

Cédric Berney, Taxonomy Coordinator UPMC & CNRS, SB Roscoff, FranceAndreea Ciuprina, System Implementer Jacobs University, Bremen, GermanyChristian Quast Max Plank Institute, Bremen, GermanyJeena Rajan, Clara Amid EMBL-EBI, UKMartin Kolisko UBC, CanadaLucas Czech, Pierre Barbera HITS, GermanySara Bender, Jon Kaye Gordon & Betty Moore Foundation

www.eukref.org

www.unieuk.org

OpencalltocontributeV418SrRNA genedatasetstoparticipateinEukBank v1.0Deadline:Dec.31,2017

Caron et al., 2017

PICO

M A C R O

Haptophytes

Sargassum

2 µm

2,000 µm

Phylum Haptophyta(Order: Isochrysidales)

Emiliania huxleyi Gephyrocapsa oceanica Pseudoisochrysis paradoxa Ruttnera lamellosa Isochrysis galbana

Cornwall, UK BrayaSø, Greenland

MARINE FRESH

Alkenone paleothermometry

Emiliania huxleyi Gephyrocapsa oceanica Pseudoisochrysis paradoxa Chrysotila lamellosa Isochrysis galbana

Prahl and Wakeham 1987; Prahl et al. 1988; Castenada et al. 2011

= 0.04T – 0.11

Questions:

• What is the global diversity of lacustrine alkenone-producing haptophyte algae?

• How is haptophyte diversity recorded in lake sediment records?

• Why are Global Group I haptophytes hyperdiversecompared to their brackish and marine cousins?

Susie TherouxCollaborator: Yongsong Huang

Yinsui Zheng Nora RichterBilly D’Andrea

Rela

tive

inte

nsity

Lipid BiomarkersMicrocosms

DNA Biomarkers

In Situ Studies

METHODS

Global Alkenone-producing Haptophyte Diversity

Theroux et al., 2010 EPSL

= Group I

Group I

Group III

Group II

IsochrysidalesFRESH

MARINE

Nora Richter

“Global Group I” Diversity

Group III

Group II

Isochrysidales Diversity

Group I

Lipidomics Genomics

Barnes, Invertebrate Zoology

Sargassum: “A Golden Floating Rainforest”“On September 17, 1492 Columbus recorded "much more weed appearing…in which they found a live crab, which the Admiral kept. He says that these crabs are certain signs of land“

Sargassum

Plants

MI

CROB

E

S

Not all Microbes Are Microscopic!

AnimalsFungi

EUKARYA

Barcoding Brown Algae (stramenopiles)

NocturnalSea

The Sargassum Ecosystem

NocturnalSea

Sargassum Endemic Species

Sea Slug

Sea SlugSea Slug

Sea Slug

Shrimp

Crab

Pipefish

Triggersfish

Filefish

AnglerfishShrimp

Sea spider

Hydroids

worms

Laffoley et al. 2009

Combining Education and Research: Marine Biodiversity and Conservation

Schell et al. 2015

Laffoley et al. 2009

Johnson et al. 2012

Climate-Change relatedBloom?

Natural Range Expansion?

First Reports in Brazil 2011

Sargassum natans I

Sargassum natans VIII

Sargassum fluitans III

Sargassum vachellianum (NC027508)

Sargassum horneri (NC024613)

Sargassum fusiforme (NC024655)

Sargassum thunbergii (NC026700)

Sargassum muticum (NC024614)

Sargassum hemiphyllum (NC024861)

0.05

100

100

100

100

100

100

(a)

(b)

(c) (d)

(e)

(f)

Sargassum natans I

Sargassum natans VIII

Sargassum fluitans III

Sargassum natans VIII

YU-087994

YU-087997

YU-087893

S. natans IS. natans VIIIS. fluitans III

Amaral-Zettler et al., 2016 Ecology and Evolution

S. fluitans III

S. natans VIII S. natans I

99.7%identity 99.7%identity

99.98%identity

Amaral-Zettler et al., 2016, Ecology and Evolution

Mitochondrial Genomes34,727 bp

S. fluitans III

S. natans VIII S. natans I

99.7%identity 99.7%identity

99.98%identity

Amaral-Zettler et al., 2016, Ecology and Evolution

Mitochondrial Genomes34,727 bp

Ecology and Evolution 2016; 1–10  | 1www.ecolevol.org

Received:5August2016  |  Revised:24October2016  |  Accepted:25October2016DOI:10.1002/ece3.2630

O R I G I N A L R E S E A R C H

Comparative mitochondrial and chloroplast genomics of a genetically distinct form of Sargassum contributing to recent “Golden Tides” in the Western Atlantic

Linda A. Amaral-Zettler1,2 | Nicholas B. Dragone3 | Jeffrey Schell3 | Beth Slikas1 |  Leslie G. Murphy1 | Clare E. Morrall4 | Erik R. Zettler3,5

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.© 2016 The Authors. Ecology and EvolutionpublishedbyJohnWiley&SonsLtd.

1MarineBiologicalLaboratory,JosephineBayPaulCenterforComparativeMolecularBiologyandEvolution,WoodsHole,MA,USA2DepartmentofEarth,Environmental,andPlanetarySciences,BrownUniversity,Providence,RI,USA3SeaEducationAssociation,WoodsHole,MA,USA4St.George’sUniversity,Grenada,WestIndies5OekologiaEnvironmentalResearch&Education,Falmouth,MA,USA

CorrespondenceLindaA.Amaral-Zettler,MarineBiologicalLaboratory,JosephineBayPaulCenterforComparativeMolecularBiologyandEvolution,WoodsHole,MA,USA.Email:amaral@mbl.edu

Funding InformationThisworkwassupportedbyaUSNationalScienceFoundation(NSF)collaborativegranttoLAA-Z(OCE-1155571)andERZ(OCE-1155379),andanNSFTUESgrant(DUE-1043468)toLAA-ZandERZ.

AbstractOver the past 5years,massive accumulations of holopelagic species of the brownmacroalgaSargassum incoastal areasof theCaribbeanhavecreated “goldentides”thatthreatenlocalbiodiversityandtriggereconomiclossesassociatedwithbeachde-teriorationandimpactonfisheriesandtourism.In2015,thefirstreportidentifyingthecause of these extreme events implicated a rare form of the holopelagic speciesSargassum natans(formVIII).However,sincethefirstmentionofS. natans VIII in the 1930s,basedsolelyonmorphologicalcharacters,nomoleculardatahaveconfirmedthisidentification.Wegeneratedfull-lengthmitogenomesandpartialchloroplastge-nomesofallrepresentativeholopelagicSargassumspecies,S. fluitans III and S. natans I alongsidetheputativelyrareS. natans VIII,todemonstratesmallbutconsistentdiffer-encesbetweenS. natans I and VIII(7bpdifferencesoutofthe34,727).Ourcompara-tiveanalysesalsorevealedthatbothS. natans I and S. natans VIIIshareaveryclosephylogeneticrelationshipwithS. fluitans III(94-and96-bpdifferencesof34,727).Wedesignednovelprimersthatamplifiedregionsofthecox2 and cox3markergeneswithconsistentpolymorphicsitesthatenableddifferentiationbetweenthetwoS. natans forms(I and VIII)fromeachotherandbothfromS. fluitans III in over 150 Sargassum samplesincludingthosefromthe2014goldentideevent.Despiteremarkablegenesyntenyandsequenceconservation,thethreeSargassumformsdifferinmorphology,ecology,anddistributionpatterns,warrantingmoreextensiveinterrogationofholope-lagic Sargassumgenomesasawhole.

K E Y W O R D S

accumulations,chloroplastgenome,macroalgae,mitogenome,SargassoSea,strandings

1  | INTRODUCTION

ThebrownalgaSargassumisoneofthemostdiversemarinemacroal-gal generawith 351 recognized speciesworldwide (Guiry & Guiry,2016).Members of the genusSargassum arewidespread, andmost

speciesarebenthicwithholdfasts,withonlytworecognizedholope-lagic speciesSargassum natans (Linnaeus)GaillonandSargassum flu-itans (Boergesen) Boergesen that do not attach to substrates (ibid).Thesespecieshavegasvesiclesanddriftandreproducevegetativelyat the surface of the ocean (Dawes &Mathieson, 2008). They are

Modified from Don Johnson

Population Genomics of Floating Sargassum

Sargassum CruiseSummer 2018

NERRR/V Pelagia

“Discovery consists of seeing what everybody has seen and thinking what nobody has thought…”

Albert Szent-Gyorgyi, in Irving Good, The Scientist Speculates (1962)

Thanks for your attention!