DNA Barcoding Case Study #5 Crustaceans

1. Kaitlin Zwicker Jessica Fredericks Karen Ssebazza DNA BarcodingCase Study #5Crustaceans

2. J. D. S Witt, D. L. Threloff, P. D. N Hebert • DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation (2006) • Used DNA barcoding to study the diversity of the crustacean subgenus Hyalella in the Great Basin of California and Nevada U.S.A.

3. Hyalella genus • Taxonomically difficult amphipod crustaceans • Freshwaters of North and South America • Short generation times • Habitat gradients create divergent selection pressures Taken from United States Department of Agriculture. Photo by Scott Bauer.

4. Subgenus Hyalella • Consists of 9 species • 8 narrowly distributed • H. azteca • Most common • All throughout North America • Many cryptic species • H. sandra and H. muerta • Each endemic to 2 locations in Death Valley National Park Witt and Hebert (2000)

5. Great Basin California and Nevada • Once a network of lakes and rivers • Now fragmented into a series of spring systems • Species of subgenus Hyalella were collected from 52 sites United States Fish and Wildlife Services

6. Finding Provisional Species • Provisional Species: • Require recognition, need to be investigated as a species • Without DNA Barcoding • Poor sp. boundaries • Difficult to recognize morphological differences • With DNA Barcoding • Flags overlooked lineages

7. Using DNA Barcoding • Species Screening Threshold (SST) • 10xSST • 10x average intrapopulation mitochondrial cytochromecoxidase I gene (COI) haplotype divergence • Previous studies with Hyalella show intraspecific divergence that fall below 10xSST • Conservative

8. Methods: Site Collections • DNA extracted from 4-10 individuals from each population • H. azteca • 49 sites • H. sandra, H. muerta • 2 springs in Death Valley National Park • Outgroup • Subgenus Austrohyalella • Found in South America Witt, Therloff and Hebert (2006)

9. Methods: COI gene analysis • Amplified (PCR) 680-bp • Sequenced • Aligned • Calc. sequence divergence for each population • Averaged all populations • X 10 average = 10xSST value • Neighbor-joining (NJ)

10. Methods: 28S rDNA • 33 individuals sequenced for 28s rDNA gene • 1400-bp amplified, sequenced and aligned • Paired with COI data to check for congruence between two populations located at the same site • Maximum likelihood phylograms 28S and COI

11. Results: COI Analysis • 282 COI sequences from 52 locations • 295bp parsimony informative out of 637bp aligned • 128 haplotypes • Sequence divergences were as high as 35.2 % • 2 sites contained 2 lineages

12. Results: COI Analysis • Mean intrapopulation sequence divergence = 0.375% • SST= 3.75% • H. muerta • No provisional species • H. sandra • 2 provisional species • H. azteca • 33 provisional species Witt, Threloff, Hebert (2006)

13. Results: Phylogenetic Analysis • Max nucleotide divergence (5%) is much lower than COI • Same site lineages are not sister taxa Witt, Threloff, Hebert (2006)

14. Conclusions on Barcoding • Why it was useful: • Does not over-split like phylogenetic criteria • SST is conservative • Closest provisional species pair is 4.4% • Finds provisional species • Fast to recognize species diversity to aid biodiversity loss strategies • Objections: • Does not have a defined species concept – cannot determine a species • It will take a long to catch up taxonomically

15. Conclusions on Conservation and Species Diversity • Many species of the Hyalella subgenus are endemic to the Southern Great Basin • Due to ground water exploitation: • 50% of all endemic aquatic organisms have population losses • 16 extinct • Aquatic habitats are small and uncommon • (Sada & Vinyard, 2002) • Conservation efforts for some fish may have impacts on invertebrates

16. References: • Witt J.D.S, Threloff D.L, Hebert P.D.N. “DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation” Molecular Ecology. 2006 15:3073-3082 • Witt J.D.S, Hebert P.D.N. “Cryptic specoes diversity and evolution in the amphipod genus Hyalella within central glaciated North America: a molecular phylogenetic approach” Can J. Fish Aquat. Sci. 2000 57:687-698 • United States Department of Argiculture: Agriculture Research Service 2009. Hyaellaaztecaimage. Downloaded from http://www.ars.usda.gov/is/graphics/photos on 17/3/2010 • United States Fish and Wildlife Services 2009. Great Basin Map. Downloaded from http://www.fws.gov/ on 17/3/2010

17. Questions??