1. Species boundaries, phylogeography and conservation genetics of the red- legged frog (Rana aurora/drytonii) complex Presented by: Chris Burton & Matt Meyer

2. Presentation Overview Introduction – Matt Materials and Methods – Chris Results – Chris Discussion and Implications - Matt

3. R. Aurora Originally classified as two distinct species: R. aurora (Northern red- legged frog) and R. draytonii (California red- legged frog) Reclassified R. aurora as a single polytypic species with two subspecies, R. a. aurora & R. a. draytonii Currently R. a. aurora and R. a. draytonii are conspecific subspecies R. a. aurora R. a. draytonii

4. R. aurora Once widespread in Sierra Nevada and San Joaquin Valley Currently only 6 known, recently discovered, populations still in existence in these areas R. a. draytonnii – threatened under US Endangered Species Act. – Enormous economic and ecological consequences Mark Twain’s – “The Celebrated Jumping Frog of Calaveras County”

5. Range Restricted to Pacific Coast of North America from southern British Columbia to northern Mexico. Broad Zone of Intergradation – several hundred kilometers in northern California http://www.californiaherps.com/anurans/maps/rauroramap.jpg

6. Materials and Methods 108 Specimen from six taxa Sample 50 sites that span the range of aurora and draytonii – (1 to 4 from each site) Included three outgroup taxa –R. boylii –R. muscosa –R. catesbeiana Specimen

7. Molecular Methods DNA was extracted Primers were developed to amplify a fragment of cytochrome b mtDNA from all taxa Species specific Primers cytb1-ra cytb1-rm cytb1-rb cytb2-ra Individual were sequenced in both directions Sequences ranged from 297 to 397bp (most ~ 350bp)

8. Analysis Parsimony analysis was ran on both short and full fragments Likelihood analysis used MODELTEST for a common 287bp fragment Bootstrap proportions (BP) were used to asses the strength of the trees Parametric bootstrapping used to test a prior hypthesis of relationships

9. Results 47of 107 sequnces were unique All individuals showed low frequency of guanine. – f(G)=0.15 Optimal Model selection HKY+G Sequence Variance

10. Phylogeny Several well supported clades Demonstrate a sister- group between aurora and cascadae aurora and cascadae are not a monophyletic group

11. Bootstrap Likelihood of Subset of Unique Sequences 15 sequences – 3 of each major group Show monophyly of individual taxon and the monophyly of aurora and cascadea clade

12. Parametric Bootstrap Analysis Test Hypothesis that Rana a. aurora and R. a. draytonii are sister taxa Search for a model tree, with aurora + draytonii forming an exclusive clade

13. The aurora/draytonii contact zone Sample effort was supplemented –To approximate the width of the contact zone –To identify biogeographical barriers Found that the two over lap over a several-km region Pure aurora found from Big River north Pure draytonii from Mills Creek south In between both were found Breeding dynamics or restriction of overlap zone can not be determined due to only one or a few indiviuals being sequenced per site However, mtDNA contact zone can be determined to be narrow with no obvious barriers to gene flow

14. Discussion and Conclusions mtDNA data supports the separate species hypothesis based on: Relatively deep differentiation and reciprocal monophyly of aurora and draytonii The sister group relationship of aurora and cascadae and the exclusion of draytonii Assuming the data reflects the correct order of speciation: Split 1 – between northern (aurora & cascadae) and southern frogs (draytonii) Split 2 – between coast range (aurora) and interior cascade mountains (cascadae)

15. Biogeography Past studies have shown two distinct phylogeographical splits along the Pacific Coast in California –North/South break –Northern California break These phylogeographic boundaries relatively coincide with the north and south ends of the aurora and draytonii contact zone Data reflects history of species, not just mitochondrion

16. Species Conservation / Implications As a result of the data, many of the population that were thought to be intergrades are not. –draytonii (protected species) extends farther north –Confirmation with nuclear markers could result in a conservation status adjustment Single draytonii population in southern California –Only 3 adult males –Captive breeding –Data suggests more closely related to distant draytonii populations rather than closest ones

17. Questions???