Species Species OTUs OTUs OPUs OPUs

Species Species

OTUsOTUs

OPUsOPUs

Rosselló-Mora & Amann 2001, FEMS Rev. 25:39-67

Taxa circumscription depends on the

observable charactersMost plants & animals

Rest of eukaryotes

Prokaryotes

Sexual isolation (biological SC)

Morphology (taxonomic SC)

Genealogy / Genetics / Phenotype

TheThe CONCEPTCONCEPT is theis the IDEAIDEA, what embraces a unit, what embraces a unit

(generally immutable)(generally immutable)

TheThe DEFINITIONDEFINITION is theis the WAYWAY to embrace a unit to embrace a unit

(changes with technical developments)(changes with technical developments)(depends on the observable characters)(depends on the observable characters)

SPECIESSPECIES CONCEPT versus DEFINITION CONCEPT versus DEFINITION

TheThe CONCEPTCONCEPT is theis the IDEAIDEA, what embraces a unit (generally immutable), what embraces a unit (generally immutable)

monophyletic group of isolates

genomically coherent

sharing high similarity in many independent phenotypic features

TheThe DEFINITIONDEFINITION is theis the WAYWAY to embrace a unit (changes with technical developments)to embrace a unit (changes with technical developments)

monophyly gene sequence analysis (i.e. 16S rRNA)

genomic coherence DDH

phenotype (biochemical tests, chemotaxonomy…)

CONCEPT vs DEFINITION CONCEPT vs DEFINITION

phylogenetic coherence

RNAr 16SFunctional genes (MLSA)

Genomic analyses

70-50%70%

genomic coherence

Reasociación DNA-DNAG+C, AFLP, MLSA

Genomic comparisons(ANI; AAI)

100%

60%

70%

80%

50%

phenotypic coherence

metabolismchemotaxonomy

spectrometry (Maldi-Tof; ICR-FT/MS)

16S rRNA gene sequence (gold sdt)

97% - 98.7% identity threshold

all organisms must be monophyletic

DDH (gold standard)

70% similarity threshold

96% ANI

identificative phenotypic property

chemotaxonomic markers

metabolic homogeneity

Tindall et al., 2010 IJSEM 60:249-266

How do we define / circumscribe species for prokaryotes

validation of a name requires DEPOSIT of a type strain in TWO

international collections

type material should be AVAILABLE to all the scientific community

type strains are the REFERENCE for any taxonomic work

The benefits of having to isolate organisms for taxonomic studies:The benefits of having to isolate organisms for taxonomic studies:

The problems of having to isolate organisms for taxonomic studies:The problems of having to isolate organisms for taxonomic studies:

it is impossible to “formally” classify uncultured organisms yet

just the Candidatus status is recognized

difficulties to homogenize names for the scientific community

Pedrós-Alió, 2006 TRENDS Microbiol 14:257-263Pedrós-Alió, 2006 TRENDS Microbiol 14:257-263

MOLECULAR TECHNIQUESMOLECULAR TECHNIQUES

generally identification of units (species) by means of 16S rRNA genesgenerally identification of units (species) by means of 16S rRNA genes

generally inform about the highly abundant organismsgenerally inform about the highly abundant organisms

it is not clear where to set a threshold of what is a speciesit is not clear where to set a threshold of what is a species

Red => knowable diversityRed => knowable diversity / / black => seed bank, unknown, difficult to knowblack => seed bank, unknown, difficult to know

Species Species microbial molecular ecology microbial molecular ecology

≠ disciplines use ≠ size of their basic units

≠ observational methods

Rosselló-Móra, 2011 Environ Microbiol 14:318-334

Taxonomists phylogenetic / genomic / phenotypic coherence

Ecologists 97% identity OTUs too wide for taxonomists

Evolutionary microbiologists much more strict too narrow for taxonomists

Compile microdiversity

into OTUs at 97% identityEcotype; early stage of

speciation A stable framework needs PRAGMATISM

OTUs OTUs OPERATIONAL TAXONOMIC UNITSOPERATIONAL TAXONOMIC UNITS

Clustering by sequence identity thresholdClustering by sequence identity threshold

Clustering at XX% Clustering at XX% identityidentity

Quiime …Quiime …

MetagenomeMetagenome OTU 1OTU 1 OTU 2OTU 2 OTU 3OTU 3 OTU 4OTU 4 SingletonsSingletonsdoubletonsdoubletons

5’5’ 3’3’V1 & V2V1 & V2 V5 & V6V5 & V6

Different groups use different variable zonesDifferent groups use different variable zones metagenomes of different zones are not comparablemetagenomes of different zones are not comparable perhaps identical sequences of different stretches may match different OTUs (green highlighted)perhaps identical sequences of different stretches may match different OTUs (green highlighted)

High identity does not mean common ancestryHigh identity does not mean common ancestry

100%100%

100% 100% reconditioningreconditioning

99%99%98%98%97%97%

Acinas et al., 2004 Nature 430:551-554Acinas et al., 2004 Nature 430:551-554

Clone librariesClone libraries

great phylotype diversitygreat phylotype diversity PCR errors (reconditioning)PCR errors (reconditioning) microdiversity (several operons?)microdiversity (several operons?)

grouping through % identitygrouping through % identity

OTU (Operational Taxonomic Unit)OTU (Operational Taxonomic Unit)

97% one species?97% one species?

OTUs OTUs OPERATIONAL TAXONOMIC UNITSOPERATIONAL TAXONOMIC UNITS

97% sequence identity threshold97% sequence identity threshold

RECOMMENDATIONS FOR THE USE OF OTUS

Use 99% or 98.7% identity (IS AT THE RESOLUTION OF A TAXONOMIC SPECIES)

Ecologists 97% identity OTUs too wide for taxonomists

Evolutionary microbiologists much more strict too narrow for taxonomists

Yarza et al., Nature Revs. 2014. 12: 635-645

SEQUENCING ERRORS

SILVA REF 112 DATABASE

OPUs OPUs OPERATIONAL PHYLOGENETIC UNITSOPERATIONAL PHYLOGENETIC UNITS

Grouping after phylogenetic inference (using parsimony tool of ARB)Grouping after phylogenetic inference (using parsimony tool of ARB)

Clustering at XX% Clustering at XX% identityidentity

Quiime …Quiime …

MetagenomeMetagenome OTU 1OTU 1 OTU 2OTU 2 OTU 3OTU 3 OTU 4OTU 4 SingletonsSingletonsdoubletonsdoubletons(representatives)(representatives)

Selected sequences are inserted in a tree Selected sequences are inserted in a tree preexisting using parsimony inferencepreexisting using parsimony inference different sizes, zones, may affiliate togetherdifferent sizes, zones, may affiliate together if no use of ARB, one can select the best if no use of ARB, one can select the best sequences from the databases matching sequences from the databases matching OTUs and reconstruct properlyOTUs and reconstruct properly

One OPU will contain different OTUs of One OPU will contain different OTUs of different length, zone, sample, etc…different length, zone, sample, etc…

OPUs OPUs subjective subjective best solution to measure diversity best solution to measure diversity

Rosselló-Móra & López-López, 2008. In: Accessing Uncultivated Microorganisms ASM PressRosselló-Móra & López-López, 2008. In: Accessing Uncultivated Microorganisms ASM Press

López-López et al., 2010 Environ Microbiol Reports 2:258-271López-López et al., 2010 Environ Microbiol Reports 2:258-271

Pernthaler & Amann. 2005. Microbiol Mol Biol Rev 69:440-461Pernthaler & Amann. 2005. Microbiol Mol Biol Rev 69:440-461

OPU OPU Operational Phylogenetic Unit Operational Phylogenetic Unit

similar to similar to ““Operational Phylogenetic-based Microbial PopulationsOperational Phylogenetic-based Microbial Populations”” (Pernthaler & Amann) (Pernthaler & Amann)

somehow subjective, but may reflect better ecologically relevant populationssomehow subjective, but may reflect better ecologically relevant populations

OPUs OPUs reduce diversity but may reflect metabolic groups reduce diversity but may reflect metabolic groups

OPUsOPUs

Reduce diversity measuresReduce diversity measures

somehow more tedious and subjective (not always negative)somehow more tedious and subjective (not always negative)

avoid the use of artificial thresholdsavoid the use of artificial thresholds

Species Species OTUs OTUs OPUs OPUs

Species are taxonomic units based on well characterized isolates Species are taxonomic units based on well characterized isolates

Molecular microbial ecology Molecular microbial ecology sequences sequences 97% identity = OTU (artificial threshold) 97% identity = OTU (artificial threshold)

I recommend you to use a cutoff value of 99% for OTU clusteringI recommend you to use a cutoff value of 99% for OTU clustering

OPUs avoid rigid thresholds & may reflect better metabolic and/or ecological typesOPUs avoid rigid thresholds & may reflect better metabolic and/or ecological types