DNA barcoding of fungi: a feasibility analysis Donal Hickey, Concordia University, Montreal, Canada

2of 20 General questions: Is DNA barcoding a taxonomic tool? - a phylogenetic tool? - a tool for simply assigning unidentified specimens to known species? - all of the above? - none of the above? Specific question: Will DNA barcoding work for fungi? The big question: Should we be using mitochondrial sequences DNA barcoding?

3of 20 First, let’s see how barcoding works in a case where we know the answer. Then we can move on, with more confidence, to cases such as fungi – where we definitely don’t know the answer.

4of 20 The CBOL brochure: What is wrong with this picture?

5of 20 Benchmarking DNA barcodes: an assessment using available primate sequences

6of 20 By using high bootstrap values, most of the branching pattern collapses, but the species resolution remains.

7of 20 The two closely related species of chimpanzee can be resolved by reducing the bootstrap cut-off to 95%

8of 20 Or, we can retain the 100% bootstrap and increase the sequence length – it’s a simple trade-off. In this case the “barcode” was extended to 1,500 bp

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10of 20 Let’s begin with long sequences (5 concatenated genes)

11of 20 Then, let’s use a single genes (CO1)

12of 20 How about Cytochrome b?

13of 20 CO1 Barcode (600 bp)

14of 20 Multiple strains within a species

15of 20 The relationship between barcode length and diagnostic value (Lepidopteran dataset)

16of 20 Barcodes for genome composition :GC content of animal mitochondria

17of 20 Are mitochondrial barcodes a bad idea?

18of 20 Count the green dots

19of 20 Numbers of mitochondrial genomes in the mammalian female germ line fluctuate between 1,000,000 per cell and 100 per cell. But the “bottleneck” in a single female is approximately 5,000 mit genomes. (Tim Wai, McGill)

20of 20 The bad news: Mitochondrial sequence variation does not reflect the population size and/or the breeding pattern of the population. “Panmictic population” has no meaning when applied to mitochondrial data.

21of 20 The good news: Since mitochondrial genes do not reflect the effects of sexual outbreeding, mitochondrial barcodes should work equally well in populations with different breeding structures. In other words, we shouldn’t worry too much about the fact that fungi, unlike birds, do not come in breeding pairs.

22of 20 The cautionary note. Since different lineages have: (i) different cell sizes, correlated with different numbers of mitochondria per cell; and (ii) different numbers of cell generations per organismal generation, we should expect to see large variations in the relative rates of mitochondrial and nuclear sequence evolution, even if the base mutation rates were the same.

23of 20 A modest proposal for a barcoder’s credo 1.Leave phylogenetics to the phylogeneticists (i.e., use their trees) 2.Leave taxonomy to the taxonomists (i.e. confine ourselves to their Latin binomial names). 3.Assign barcode sequences to known species names and hang them on independently derived trees (otherwise, we are crossing a line into DNA taxonomy and molecular phylogenetics).

24of 20 Visualization: the color scheme. Perfect match to verified voucher specimen: GREEN Perfect match to known species, but no verified voucher with exactly that barcode sequence: BLACK <0.25% mismatch to known sequence/specimen: YELLOW <0.50% mismatch to known sequence/specimen: ORANGE <0.75% mismatch to known sequence/specimen: RED <1.00% mismatch to known sequence/specimen: RED >1.00% mismatch to known sequence: PROBLEM! (seek help from a qualified professional)

25of 20 A field test

26of 20 * The field test Duhamel

27of 20 Acknowledgements Xiang Jia Min Mehrdad Hajibabaei Greg Singer The Canadian Taxpayer