Species boundaries, phylogeography and

conservation genetics of the red-legged frog (Rana

aurora/drytonii) complex

Presented by: Chris Burton & Matt Meyer

Presentation Overview

• Introduction – Matt

• Materials and Methods – Chris

• Results – Chris

• Discussion and Implications - Matt

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

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”

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

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

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)

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

Results

• 47of 107 sequnces were unique• All individuals showed low frequency of

guanine. – f(G)=0.15• Optimal Model selection HKY+G

Sequence

Variance

Phylogeny

• Several well supported clades

• Demonstrate a sister-group between aurora and cascadae

• aurora and cascadae are not a monophyletic group

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

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

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

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)

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

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

Questions???