From Genes to Genomes: Progress and Pitfalls in Barcoding the … · 2018-02-02 · From Genes to...
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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
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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
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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
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“Kingdom” Protista
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Caron et al., 2017
Protists span a diversity of life styles, sizes and trophic levels
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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
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The Evolving Concept of Protistan Phylogeny
Caron et al., Nature Reviews Microbiology 2017
Archaea
Bacteria
Eukarya
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Modified from Norm Pace and Mitch Sogin
The Molecular“Tree of Life”
According to Ribosomal RNA
Woese (1928-2012)
Pace
Sogin
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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
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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
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A Comprehensive Tree of Life?
Hinchcliff, Smith et al. PNAS 2015
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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
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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
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¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾Coastal¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾
¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾Freshwater¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾ARCNTLCWT HB
CCE
SBC PIE GCE FCE VCR
PAL
Trop
ical
¾¾¾¾¾¾¾¾
Ope
n O
cean¾¾¾¾¾¾¾
¾¾¾¾¾¾¾¾¾¾¾¾
Polar ¾¾¾¾¾¾¾¾¾¾¾¾¾
CCE DOCUMENT
DESCRIBE DISCOVER
DIVERSITY
MCR
MCR MCM
Microbial Inventory Research Across Diverse Aquatic MIRADA LTERS
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V6 V6
V9V9
Eukaryal
V9
BACTERIA & ARCHAEA EUKARYA
http://www.rna.icmb.utexas.edu
V4
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V6 V6
V9V9
Eukaryal
V9
BACTERIA & ARCHAEA EUKARYA
htp://www.rna.icmb.utexas.edu
V4
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De Vargas et al., Science, 2015.
Estimated 127,500 Protist Plankton Species
(V9 survey)
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Pawlowski et al., 2012
Recognized 26,010 to 74,373: Estimated: 140,000 to 1.6 million
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V4 Protist pre-barcodes
Pawlowski et al., 2012
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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
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www.eukref.org
www.unieuk.org
OpencalltocontributeV418SrRNA genedatasetstoparticipateinEukBank v1.0Deadline:Dec.31,2017
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Caron et al., 2017
PICO
M A C R O
Haptophytes
Sargassum
2 µm
2,000 µm
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Phylum Haptophyta(Order: Isochrysidales)
Emiliania huxleyi Gephyrocapsa oceanica Pseudoisochrysis paradoxa Ruttnera lamellosa Isochrysis galbana
Cornwall, UK BrayaSø, Greenland
MARINE FRESH
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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
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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
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Rela
tive
inte
nsity
Lipid BiomarkersMicrocosms
DNA Biomarkers
In Situ Studies
METHODS
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Global Alkenone-producing Haptophyte Diversity
Theroux et al., 2010 EPSL
= Group I
Group I
Group III
Group II
IsochrysidalesFRESH
MARINE
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Nora Richter
“Global Group I” Diversity
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Group III
Group II
Isochrysidales Diversity
Group I
Lipidomics Genomics
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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“
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Sargassum
Plants
MI
CROB
E
S
Not all Microbes Are Microscopic!
AnimalsFungi
EUKARYA
Barcoding Brown Algae (stramenopiles)
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NocturnalSea
The Sargassum Ecosystem
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NocturnalSea
Sargassum Endemic Species
Sea Slug
Sea SlugSea Slug
Sea Slug
Shrimp
Crab
Pipefish
Triggersfish
Filefish
AnglerfishShrimp
Sea spider
Hydroids
worms
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Laffoley et al. 2009
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Combining Education and Research: Marine Biodiversity and Conservation
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Schell et al. 2015
Laffoley et al. 2009
Johnson et al. 2012
Climate-Change relatedBloom?
Natural Range Expansion?
First Reports in Brazil 2011
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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
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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
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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:[email protected]
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
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Modified from Don Johnson
Population Genomics of Floating Sargassum
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Sargassum CruiseSummer 2018
NERRR/V Pelagia
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“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!