1. The Barcode Gap Speciation or Phylogeography? BANBURY 3 ? Graham Stone, Richard Challis, James Nicholls, Jenna Mann, Sonja Preuss Mark Blaxter Institute of Evolutionary Biology, Edinburgh University

2. BANBURY 3 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’.

3. BANBURY 3 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.

4. BANBURY 3 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.

5. BANBURY 3 Phylogeographic analyses of closely related species can, coincidentally, allow stringent testing of the of the DNA barcoding approach.

6. BANBURY 3 Oak gallwasps 1000 species worldwide, highly hostplant specific induce characteristic gall structures Nuclear gallwasp genes determine gall structure: distinctive gall morphologies reliably identify species.

7. BANBURY 3 They support ecologically closed communities of natural enemies

8. Individual species are found across the Western Palaearctic BANBURY 3

9. ..with populations in multiple glacial refugia BANBURY 3 Turkish refugia Iranian refugia

10. Comparative phylogeography Concordance: same origin, direction and timescale Concordance: same origin and direction, different timescales/demographies Discordance: different origin and different direction (± same timescale) BANBURY 3

11. Gallwasp phylogeography BANBURY 3 Andricus kollari Allozyme allele frequency data 13 loci, 2100 individuals 70 sites Intraspecific variation in widespread species corresponds to refugia

12. Gallwasp phylogeography BANBURY 3 Andricus kollari 433 bp Cytb, 160 individuals 2-3MY

13. Introgression and backcrossing creates barcode mismatches BANBURY 3 Andricus kollari 433 bp Cytb, 160 individuals 2-3MY

14. Parasitoid phylogeography European haplotypes Eastern haplotypes 1.8 MY BANBURY 3

15. The Andricus quercuscalicis clade Andricus quercuscalicis Andricus dentimitratus Andricus quercustozae Andricus caputmedusae BANBURY 3..is a group of closely-related oak gallwasp species with highly diagnostic gall phenotypes

16. Cytochrome b sequence resolves each species into glacial refuge clades Outgroup individuals from Turkey 433 bp Cytochrome b (all seqs ORF) K2P NJ BANBURY 3 Andricus quercustozae

17. But the geographic origin of outgroups influences relationships between these clades Outgroup individuals from TurkeyOutgroup individuals from C. Europe BANBURY 3

18. Analyse data for all 4 closely- related species (n=600 individuals, 221 haplotypes) K2P NJ BANBURY 3

19. 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 NJ BANBURY 3

20. Molecular Operational Taxonomic Units (MOTU)s identified using MOTU_define

21. Colour blocks show 8bp MOTU’s Identify MOTU’s using MOTU_define Little sign of a genuine barcoding gap in these data BANBURY 3

22. Colour blocks show 8bp MOTU’s MOTU’s rarely correspond to species Colour blocks show 8bp MOTU’s Andricus quercustozae BANBURY 3

23. Colour blocks show 8bp MOTU’s Andricus caputmedusae MOTU’s rarely correspond to species BANBURY 3

24. Colour blocks show 8bp MOTU’s Andricus dentimitratus MOTU’s rarely correspond to species BANBURY 3

25. Colour blocks show 8bp MOTU’s Andricus quercuscalicis MOTU’s rarely correspond to species BANBURY 3

26. Colour blocks show 8bp MOTU’s This is true irrespective of the threshold sequence difference for MOTU’s Colour blocks show 8bp MOTU’s BANBURY 3

27. Colour blocks show 8bp MOTU’s This is true irrespective of the threshold sequence difference for MOTU’s Colour blocks show 8bp MOTU’s BANBURY 3

28. .. 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 270 Andricus dentimitratus 332 Andricus quercustozae158 BANBURY 3

29. Instead, multispecies MOTU’s correspond to geographic regions BANBURY 3

30. Geographic grouping rules out sorting of ancestral polymorphism No evidence for role of symbionts Wolbachia, Cardinium, Spiroplasma, Flavobacteria. What generates the observed pattern? BANBURY 3

31. Nuclear sequence data do not support multispecies clades sorted by refuge 702 bp Nuclear Long wavelength opsin gene What generates the observed pattern? BANBURY 3

32. 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? BANBURY 3 0.99 0.95

33. 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? BANBURY 3 0.99 0.95

34. Conclusions 1 BANBURY 3 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.

35. BANBURY 3 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

36. BANBURY 3 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

37. BANBURY 3 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…