The Barcode GapSpeciation or Phylogeography?

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Graham Stone, Richard Challis, James Nicholls, Jenna Mann, Sonja PreussMark Blaxter

Institute of Evolutionary Biology, Edinburgh University

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Phylogeography and DNA barcoding often use the same tools, but have different aims.

Barcoding• aims to identify species-specific sequences at a single locus.

• would like to capture the full diversity of sequence variation inherent in a taxon, but usually does not. • works best when ancestral polymorphism between sister lineages has been completely sorted, creating monophyletic sister clades and a ‘barcoding gap’.

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Mitochondrial barcoding traumas

Incomplete sorting of ancestral polymorphism.

Barcodes jump between species.

Introgression and sorting may only become obvious if you sample closely related taxa in depth. You cannot know the scale of this problem by sampling one taxon.

Empiricially, barcoding generally works.

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Phylogeography

•Reconstructs the spatial relationships between lineages over time

•Requires extensive within-species sampling

•Commonly combines mitochondrial and nuclear markers (allozymes, microsatellites, sequence)

•Struggles to find nuclear loci polymorphic enough to allow direct comparison with mitochondrial

sequence data.

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Phylogeographic analyses of closely related species can, coincidentally, allow stringent testing of the of the DNA barcoding approach.

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Oak gallwasps

• 1000 species worldwide, highly hostplant specific• induce characteristic gall structures• Nuclear gallwasp genes determine gall structure: distinctive gall morphologies reliably identify species.

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They support ecologically closed communities of natural enemies

Individual species are found across the Western Palaearctic

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..with populations in multiple glacial refugia

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Turkish refugiaIranian refugia

Comparative phylogeography

Concordance: same origin, direction and timescale

Concordance: same origin and direction, different timescales/demographies

Discordance: different origin and different direction (± same timescale)

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Gallwasp phylogeography

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Andricus kollari

Allozyme allele frequency data13 loci, 2100 individuals 70 sites

Intraspecific variation in widespread species corresponds to refugia

Gallwasp phylogeography

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Andricus kollari

433 bp Cytb, 160 individuals

2-3MY

Introgression and backcrossing creates barcode mismatches

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Andricus kollari

433 bp Cytb, 160 individuals

2-3MY

Parasitoid phylogeography

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Number of mismatches

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ObservedSimulated

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

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European haplotypes

Eastern haplotypes

1.8 MY

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The Andricus quercuscalicis clade

Andricusquercuscalicis

Andricusdentimitratus

Andricusquercustozae

Andricuscaputmedusae

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..is a group of closely-related oak gallwasp species with highly diagnostic gall phenotypes

Cytochrome b sequence resolves each species into glacial refuge clades

Outgroup individuals from Turkey

433 bp Cytochrome b(all seqs ORF)

K2P NJBANBURY 3

Andricusquercustozae

But the geographic origin of outgroups influences relationships between these clades

Outgroup individuals from TurkeyOutgroup individuals from C. Europe

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Analyse data for all 4 closely-related species (n=600 individuals, 221 haplotypes)

K2P NJBANBURY 3

Analyse data for all 4 closely-related species (n=600 individuals, 221 haplotypes)

Sequence divergence within this whole group matches that within single gallwasp species (e.g. A. kollari)

K2P NJBANBURY 3

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Molecular Operational Taxonomic Units (MOTU)s identified using MOTU_define

Colour blocks show 8bp MOTU’s

Identify MOTU’s using MOTU_define

Little sign of a genuine barcoding gap in these data

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Colour blocks show 8bp MOTU’s

MOTU’s rarely correspond to species

Colour blocks show 8bp MOTU’s

Andricus quercustozae

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Colour blocks show 8bp MOTU’s

Colour blocks show 8bp MOTU’s

Andricus caputmedusae

MOTU’s rarely correspond to species

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Colour blocks show 8bp MOTU’s

Colour blocks show 8bp MOTU’s

Andricus dentimitratus

MOTU’s rarely correspond to species

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Colour blocks show 8bp MOTU’s

Colour blocks show 8bp MOTU’s

Andricus quercuscalicis

MOTU’s rarely correspond to species

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Colour blocks show 8bp MOTU’s

This is true irrespective of the threshold sequence difference for MOTU’s

Colour blocks show 8bp MOTU’s

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Colour blocks show 8bp MOTU’s

This is true irrespective of the threshold sequence difference for MOTU’s

Colour blocks show 8bp MOTU’s

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.. And is true when phylogeny reconstruction uses more complex models

MrBAYES

HKY+G, partitioned by codon position, parameter estimates unlinked across partitions

Ln Bayes Factors against species monophyly:

Andricus caputmedusae 270Andricus dentimitratus 332Andricus quercustozae 158BANBURY 3

Instead, multispecies MOTU’s correspond to geographic regions

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Geographic grouping rules out sorting of ancestral polymorphism

No evidence for role of symbionts Wolbachia, Cardinium, Spiroplasma, Flavobacteria.

What generates the observed pattern?

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Nuclear sequence data do not support multispecies clades sorted by refuge

702 bp Nuclear Long wavelength opsin gene

What generates the observed pattern?

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Nuclear sequence data do not support multispecies clades sorted by refuge

MtDNA data

702 bp Nuclear Long wavelength opsin gene

What generates the observed pattern?

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0.99

0.95

Nuclear sequence data do not support multispecies clades sorted by refuge

MtDNA data

Observed mtDNA patterns are compatible with hybridisation and back-crossing to parental types within refugia

702 bp Nuclear Long wavelength opsin gene

What generates the observed pattern?

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0.99

0.95

Conclusions 1

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How common is this pattern?

Don’t know: we need studies of appropriate taxa. But gallwasps are unlikely to be an isolated case.

•Many radiations of species have occurred over the same timescale, especially insects.•Most widespread taxa show differentiation between

regional refugia. •Little is known for most taxa about current or past

hybridisation within refugia.•Expect to find more examples as phylogenetic density of barcode sampling increases.

Not safe to assume it is rare.

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A worst case scenario:

IF related taxa often hybridise in refugia, mtDNA barcoding will generate a molecular taxonomy of refugia, not species.

Barcoding gaps would then indicate phylogeographic breaks, not speciation.

Cool for reconstructing community history, bad for species barcodes.

Conclusions 2

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1. We should check how often this occurs, even in groups where we think barcoding works, by sampling over the full range of species.

2. We need nuclear sequence markers to corroborate mitochondrial barcodes.

Conclusions 3

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What could CBOL do with broader mt+nuclear sampling that it can’t do now?

•Reduce uncertainty in identification

•Facilitate array-based barcoding

•Identify sources and demographies of key target species.

•Assess multispecies concordance: biodiversity hotspots.

•Enhance phylogenetic utility

Look on the bright side…