Bill Birky Department of Ecology and Evolutionary Biology and Genetics Program

1. Is There Meaningful Life Without Sex?Species, Diversity, and Natural Selection in Asexual Organisms Bill Birky Department of Ecology and Evolutionary Biology and Genetics Program The University of Arizona

2. The Species Problem Meets the Sex Problem Eukaryotes: most or all asexual organisms arose from sexual ancestors. Theory: Asexual mutants may have an advantage over sexual conspecifics, but sexual species probably have a higher speciation rate and/or lower extinction rate.

3. Why we need a good asexual species concept • The Biological Species Concept (BSC) is widely accepted and works well for many sexual organisms. • We must have a comparable species concept in asexuals before we can ask and answer fundamental questions: • Are sexual organisms more diverse than asexual? • Genetic and phenotypic diversity within species • Species diversity • Is natural selection less effective in asexual organisms? • Ka/Ks • HKA and MK tests of neutrality

4. More reasons why we need a good asexual species concept • Test “everything is everywhere” theory of the biogeography of microorganisms • Fenchel & Finlay (2004 BioScience 54:777) Most species <1mm are distributed globally. • But many organisms in this size range are probably asexual and have poorly-defined species. • Test the relationship between effective population size and genome structure • Lynch and Conley (2003 Science 302:1401) Microogranisms have extremely large effective population sizes (Ne) hence very effective selection and simple genomes. • But (Daubin and Moran 2004 Science 306:978) Lynch and Conery’s estimates of Ne are based on poorly-defined species.

5. More reasons why we need a good asexual species concept • Systematics! • Many eukaryotes are asexual • Epidemiology • Many parasites are asexual

6. Biological diversity is discontinuousThe earliest species definition: clusters of phenotypically similar individuals.

7. Biological diversity is discontinuous • These clusters are real: • Ernst Mayr et al.: New Guinea natives see mostly same clusters in birds as trained taxonomists. • Ed Wilson: they don’t recognize ant clusters because it’s not a survival trait. But it is a survival trait for Ed. • Difference also seen in urban cultures: In the teenage mallcrawler culture the ability to recognize urban plumage patterns and track their evolution is a survival trait.

8. Asexual organisms show phenotypic clusters, but speciation in asexuals is controversial Studies of speciation have focused on sexually-reproducing organisms and emphasized reproductive isolation as an important step. Asexual organisms have been neglected. This has led to misconceptions about species and speciation in asexuals. One common misconception is that an asexual lineage must form a continuum of variation due to mutation… No clusters. This is based on an over-simplified model of asexual reproduction ...

9. A common erroneous model of asexual reproduction But this model assumes that all cells reproduce in synchrony with no cell death. Ancestor: asexual mutant or allopolyploid in sexual species

10. In real life, some individuals reproduce more than others, just by chance (random genetic drift) Drift causes gaps and clusters in the tree, but it does not cause speciation. But that’s not all ...

11. In real life, some individuals reproduce more than others because of their genotype (natural selection) Truncating selection maintains a single species with genotypic variation determined by the mutation rate and effective population size. The latter is determined by the carrying capacity of the environment and the demographics of the organism. But how do we get speciation?

12. Diversifying selection or allopatry can cause speciation Diversifying selection for adaptation to different niches (together with random drift) produces interesting gaps and clusters that would usually be considered species. Each cluster is a population which is an independent arena for evolution (mutation, drift, and selection). Geographic isolation by itself can also do this, but the gaps won’t persist in sympatry unless the clades are adapted to different niches.

13. Distinguishing gaps separating species from gaps within species • Colored clusters were produced by divergent selection or long-term physical isolation. These are independently-evolving populations and are species. • Transient gaps within species are produced by random drift. These average 2Ne generations deep; the 95% CI is 4Ne generations. • Gaps between species that are > 4Ne generations deep are produced by divergent selection and/or long-term allopatry. These gaps separate populations that have been evolving independently long enough to become reciprocally monophyletic, i.e. to complete speciation.

14. A new species concept for asexual organisms The preceding theory can be considered a new species concept for asexuals, tentatively called the Evolutionary Genetic Species Concept (EGSC). Suggestions for a better name are welcome. (Barraclough, Birky, Burt 2003 Evolution 57:2166-2172) This theory suggests that if we find populations separated from each other by ≥ 4Ne generations, we can be very sure that they have been evolving independently long enough to become reciprocally monophyletic (complete lineage sorting). They are independent arenas for mutation, selection, and drift.

15. The Evolutionary Genetics Species ConceptSpecial and General Forms • General form: Asexual species are populations that have been evolving independently long enough to become reciprocally monophyletic, because they are adapted to different niches or are allopatric. • Special form: Asexual species are reciprocally monophyletic populations that are adapted to different niches.

16. The species concept requires a species criterion • Species concepts are applied using species criteria, which are operational definitions of species. Recent theory tells us how to identify reciprocally monophyletic populations from DNA sequences of small samples. This is a species criterion for our species concept. Rosenberg 2003 Evolution 57:1465: If samples of ≥ 3 individuals from two populations are reciprocally monophyletic and are separated by ≥ 4Ne generations, then the entire populations are reciprocally monophyletic with probability ≥ 95%.

17. Species criterion The 4-times rule detects reciprocally monophyletic populations using DNA sequences from samples of ≥3 individuals.

18. Proposing a new species concept is hazardous traditional systematist strict cladist Bill

19. Application to bdelloid rotifers, an ancient asexual lineage • Rotifers are microscopic aquatic invertebrates, mainly freshwater. • Four classes differing in mode of reproduction:

20. Some remarkable features of bdelloids • Bdelloids are found anyplace that is wet even occasionally (Antarctica!). • Most bdelloids withstand dessication. • Bdelloids readily colonize new habitats (Surtsey, bird baths). • Bdelloids withstand huge doses of ionizing radiation. • Dessication or radiation fragments bdelloid chromosomes, which are re-assembled during recovery. • Bdelloids survive freezing at -80° without cryoprotectants.

21. Species have been recognized in bdelloids but not confirmed Bdelloids have been divided into 4 families, 19 genera, and over 350 species on the basis of morphology and behavior. BUT The demonstration of clades or species requires molecular data in invertebrates and protists, where morphology is often inadequate or misleading. Adineta Philodina

22. Applying species concept and criterion to bdelloid rotifers • 344 female bdelloids were isolated from nature and reared to produce clones in the lab (205 from Birky lab, 139 from Tim Barraclough’s group at Silwood Park). • Collection sites: • United States, Great Britain,The Netherlands, Austria, France, Germany, Italy, Switzerland, Quebec, Mexico, Tanzania, and New Zealand. • Sea level to 12,000 ft./3660 meters • All life zones from Sonoran to Alpine. • Lakes, permanent and temporary ponds and streams, moss, dust

23. Collecting Sites in U.S.

24. Collecting Sites Worldwide

25. Collecting Sites: Birky & Barraclough Labs

26. Some collecting sites, from moss at sea level in the Netherlands to 12,000 feet in the Snowy Range of Wyoming

27. Phylogenetic Analysis We sequenced 591 bp of the mitochondrial cox1 (COI) gene from each clone and made phylogenetic trees. Phylogenetic trees show a pattern of shallow clades separated mostly by deep branches ending in a basal soft polytomy. These clades are strongly supported by a variety of evolutionary models and methods. 59 clades are samples from simple reciprocally monophyletic populations as defined by the 4X rule, i.e. from different species.

28. Species detected in a smaller database • 188 isolates from the U.S., The Netherlands, and Italy • 33 clades (bold lines) are reciprocally monophyletic, have D/p ≥ 4, and contain no such clades within them: they are samples from simple reciprocally monophyletic populations. • These are species. • The singlets probably represent additional species. Enlarged fragment of tree

29. Tim Barraclough: detecting species byanalyzing branching rates Fit maximum likelihood model with 2 parameters p << 0.001 234 clusters, 95% CI 225-244

30. Some features of our species • They are not artefacts of incomplete sampling: adding more individuals, collecting sites or individuals per site has never split or joined species. • They are not artefacts of the cox1 gene fragment: 353 bp of the cob gene identifies the same species in a subset of 88 isolates. • They are not artefacts of the modest sequence length; adding the cob gene sequences to the cox1 sequences increases total sites from 591 to 944 bp and informative sites from 299 to 500 without changing species. • At least some of the species identified in this way are adapted to different niches by morphology, behavior, food preference, or temperature tolerance. They fit the special EGSC. (Birky et al. 2005 Hydrobiologia 546:29-45) • Correspondence to described morphospecies is imperfect, with a number of cryptic species and possibly some polyphyletic genera.

31. Are asexual organisms less diverse than sexual organisms? The average nucleotide diversity in 59 bdelloid species is significantly lower than the average in 10 monogonont species.

32. Effects of incomplete sampling on estimates of nucleotide diversity Would our values of nucleotide diversity change if we had sampled more different sites or larger numbers of individuals per site? Preliminary analysis of a subset of the data shows a correlation between nucleotide diversity and number of clones per site, and also number of sites. However, the R2 values are small. Note that most published values of nucleotide diversity are based on rather small samples and the effects of sample size have not been examined.

33. Looking for the Hill-Robertson Effect A major evolutionary advantage of sex is that it helps avoid the Hill-Robertson effect: the chance fixation of detrimental mutations, and loss of beneficial mutations. This can be viewed as a reduction in Ne in asexuals compared to sexuals, for the same census size N. One consequence is Muller’s ratchet, the accumulation of detrimental mutations. Preliminary results estimates of Ka/Ks in the hsp80 nuclear gene (Mark Welch & Meselson 2001) and the cox1 mitochondrial gene (Birky et al. 2005) are similar in bdelloids and monogononts, so there is no evidence for the ratchet. We will re-do the cox1 analyses using the larger dataset on interspecific distances only. Have the bdelloids avoided the H-R effect? If so, how?

34. Do bdelloids escape the consequences of asexual reproduction by having a very large effective population size? • It has been suggested that bdelloids avoid the consequences of the Hill-Robertson effect by having a huge census size N and therefore a large Ne. • This argument is suspect a priori, because there is evidence that organisms with large N do not have correspondingly large Ne. • For mitochondrial genes in bdelloids and monogononts, p ≈ Neu or Ne = p /u. From our cox1 data, Ne = 7 X 10-3/u for bdelloids and • 18 X 10-3/u for monogononts. Preliminary results suggest u is about twice as high in monogononts, so Ne is similar in monogononts and bdelloids, and is similar to other invertebrates.

35. Some Extensions • The 4X rule can be used to find bacterial species in at least two sets of sequence data. • Finding species by using DNA sequences is not the end of taxonomy! Species found in this way should be studied to find morphological traits that distinguish them reliably. Just as in traditional systematics, the behavior, ecology, and distribution of the species should be studied. • The cox1 sequence we use is the same one proposed for DNA barcode projects. The sequences can be used to place newly collected individuals in species already found with the 4X rule. They could also be used to determine the number and abundance of species using environmental PCR. Barcodes are a tool for systematics, not a substitute for studying the biology of the organisms.

36. I GRATEFULLY ACKNOWLEDGE • Tim Barraclough and Elizabeth Herniou for collaborating and sharing data. • The University of Arizona for generous startup funds that financed most of these studies. • The Society of Sigma Xi for a Grant-in-Aid. • Undergraduate Biology Research Program and the MacNair Scholarship program for support undergraduate students. • Heather Maughan for doing most of the original Ka/Ks tests and for continuing advice and assistance in every aspect of this work. • The undergrad students who did most of the work in my lab: Josh Adams, Brad Askam, Chris Brownlee, Ryan Couch, Lea Gemmel, Elena Henry, Linnea Herbertson, Katy Marlor, Julia Perry, Cindy Wolf. • Many people for helpful suggestions, constructive criticism, and data, including: Tim Barraclough, Doug Futuyma, Africa Gomez, David Hillis, Davis and Wayne Maddison, Brian McGill, Claudia Ricci, Mike Rosenzweig, Claus-Peter Stelzer, Bob Wallace, Mike Worobey, and Liz Walsh. • The Paerthenogenesis Network for the PARTNER conferences. • Matt Meselson and David Mark Welch for starting it all.