1. Genetic Considerations Regarding the Planned Introduction of C. ariakensis into the Chesapeake Bay Jan Cordes, Jie Xiao, and Kimberly Reece Virginia Institute of Marine Science College of William and Mary Dafeng River, China

2. Emerging Interest in C. ariakensis Aquaculture  1970s- Accidentally imported to U.S. west coast along with shipment of C. gigas and C. sikamea from Japan.  Current limited propagation of C. ariakensis on west and east coasts of U.S.  Interest in expanding C. ariakensis aquaculture to replace ailing C. virginica industry on the U.S. east coast. Exotic Introduction  Current interest in introducing C. ariakensis into the Chesapeake Bay.  2003- Congress mandates preparation of an Environmental Impact Statement (EIS) to examine risks and benefits of introduction. Being developed by Army Corps of Engineers, the states of Maryland and Virginia, the EPA, NOAA, and the USFWS.

3. Emerging Interest in C. ariakensis Genetic Concerns Regarding Proposed Introduction  How accurate are Crassostrea oyster species identifications based on morphology, and how reliable are published studies on C. ariakensis life history? If approved, can we insure that ONLY C. ariakensis will be imported?  Is there genetic structure among wild populations of C. ariakensis across its native range and does this effect the choice of source for broodstock importation?  Do current U.S. hatchery stocks exhibit reduced genetic variability and what are the genetic relationships of these stocks to wild populations? ie. Are genetic bottlenecks in hatchery stocks a cause for concern? Projects Addressing these Concerns  Developed genetic identification key for Indo-Pacific Crassostrea species.  Developed microsatellite markers for genetic population structure analysis of wild C. ariakensis populations.  Used microsatellites to investigate genetic variation in U.S. hatchery stocks and assess their relationships to wild populations.

4. S. com S. cuc L. cri C. vir C. bel C. sp C. ire L C. mad C. gry C. gig C. sik C. ari C. hon L Restriction Enzyme Digests of COI Region with Dde I Species I.D. Key Species Identification Key  Based on a phylogenetic analysis of 12 oyster species using ITS-1 and COI gene region sequences  PCR/RFLP based molecular key Uses d ITS-1 and COI sequences to develop a two marker that unambiguously distinguished among 9 Crassostrea species

5. People’s Republic of China India Japan Reported range of C. ariakensis/ C. rivularis * * C. ariakensis and C. rivularis synonymized (Carriker and Gaffney, 1996) C. rivularis (Thailand) C. rivularis (Malaysia) C. rivularis (India) C. ariakensis (southern China) C. ariakensis (Japan, South Korea, northern China) Geographic Range and Species I.D.

6. People’s Republic of China India Japan Reported range of C. ariakensis/ C. rivularis * * C. ariakensis and C. rivularis synonymized (Carriker and Gaffney, 1996) C. madrasensis (Thailand) C. belcheri (Malaysia) C. gryphoides (India) C. ariakensis and C. hongkongensis (southern China) C. ariakensis (Japan, South Korea, northern China) Geographic Range and Species I.D.

7. Archived Samples Identified as C. ariakensis in the Field Species content according to molecular ID Sample location C. ariakensis C. hongkongensis C. gigas C. sikamea C. belcheri C. gryphoides C. madrasensis Sac.sp?Unknown S. Korea XXX JapanXXX N. China XX S. China XX ThailandX MalaysiaX IndiaX Prevalence of C. ariakensis misidentifications Geographic Range and Species I.D.

8. Genetic Variation among Wild Populations * Performed by Genetic Identification Services, Inc., Chatsworth, Ca, USA. Marker Development  Developed m icrosatellite-enriched libraries. *  Sequenced 68 microsatellite-containing clones. *  Designed primers for 38 tri- and tetranucleotide microsatellite loci. *  Tested primers for ability to amplify and exhibit polymorphism using samples of C. ariakensis and C. hongkongensis.  Tested promising loci for null alleles issues using 8 family crosses of C. ariakensis.  To date have optimized 8 loci with an end goal of 10-12 markers.  Used 4 of these loci for preliminary population structure analysis.

9. Population pairwise F st (above) and P-values (below). * indicates Non-significant values. IRKRYRDR IR-0.0220.0140.026 KR<0.001-0.01*0.014 YR<0.0010.007*-0.025 DR<0.001 - Genetic Variation among Wild PopulationsSampleLD HW E IR 0 of 6 none KR 1 of 6 none YR 3 of 6 none DR 0 of 6 none Linkage disequilibrium and significant deviations from Hardy- Weinberg Equilibrium (HWE)

10. Factorial Correspondence Analysis (FCA) by Individuals IR DR YR KR Genetic Variation among Wild Populations

11. Genetic Variation in Hatchery Stocks vs. Wild Populations Project Goals  Used same 4 microsatellite markers from population structure analysis.  Conducted preliminary analysis to: 1.Determine levels of genetic variation in hatchery stocks vs. wild populations. 2.Determine genetic relationships among hatchery stocks and wild populations, particularly comparing variation in hatchery stocks to that observed in their source populations.

12. Genetic Variation in Hatchery Stocks vs. Wild Populations History of Crassostrea ariakensis in the USA 1970’s - Accidentally imported to the Pacific Northwest (Oregon) with C. sikamea and C. gigas seed from Japan in the 1970s. Called the ‘Oregon Strain’ (Breese and Malouf. 1977. Aquaculture 12:123-126). 1970’s present - Oregon Strain is hatchery propagated in the US. Reportedly very few individuals were used as broodstock for spawns in some years (Robinson and Langdon, personal communication). 1990 - Rutgers’ imports C. ariakensis Oregon Strain stock from the U.S. west coast 1999 - VIMS hatchery establishes WCA stock from Oregon Strain broodstock obtained from west coast U.S. 1999 - VIMS hatchery establishes NCA stock from northern Chinese C. ariakensis (Yellow River population, Shandong, P.R.C.) 1999 and 2000. VIMS hatchery establishes SCA stock from southern Chinese C. ariakensis. (Dafeng River population, Beihai, Guangxi, P.R.C.)

13. VIMS Hatchery Stocks SCA 1999 SCA 2000 NCA 1999 + F1F1 F1F1 WCA 1999 F1F1 TUI 2002 Pacific Northwest, USA ‘Oregon Strain’ Genetic Variation in Hatchery Stocks vs. Wild Populations Yellow River, northern China Dafeng River, Southern China

14. Allelic richness for four hatchery strains and four wild populations of C. ariakensis. Genetic Variation in Hatchery Stocks vs. Wild Populations Linkage disequilibrium and significant deviations from Hardy- Weinberg Equilibrium (HWE) Wild Populations Hatchery StocksSampleLD HW E IR 0 of 6 none KR 1 of 6 none YR 3 of 6 none DR 0 of 6 none SampleLD HW E TUI 1 of 6 4 WCA 3 of 6 2 NCA 5 of 6 1 SCA 0 of 6 1

15. NJ Tree Based on Cavelli-Sforza Genetic Distances. Bootstrap percentages based on 1000 iterations. Population pairwise F st (above) and P-values (below). *indicates non-significant values. TUINCAWCASCAIRKRYRDR TUI-0.2640.0910.2110.1980.2080.2050.221 NCA<0.001-0.166.00720.0100.0860.087 WCA<0.001 -0.1230.0850.1090.1000.120 SCA<0.001 -0.0710.0600.0660.045 IR<0.001 -0.0220.0140.026 KR<0.001 -0.01*0.014 YR<0.001 NS*-0.025 DR<0.001 - Genetic Variation in Hatchery Stocks vs. Wild Populations 100 77 73 5263 72 Hatchery Stocks Wild Populations NCA SCA WCA TUIKR YR IR DR

16. Factorial Correspondence Analysis (FCA) by Individuals c IR, KR, YR, DR TUI SCA WCA NCA Genetic Variation in Hatchery Stocks vs. Wild Populations

17. Conclusions  At least eight different species of Crassostrea are frequently misidentified as C. ariakensis throughout its reported range, at least two in China alone.  All published information on C. ariakensis range, distribution, life history, etc., should be viewed with caution given likelihood of misidentification.  Great care is necessary for proper ID of ANY live exotic oysters brought into U.S., whether for resale, aquaculture, or introduction.  Genetic structure does exist among wild C. ariakensis populations across its native range, suggesting that locally adapted populations may differ in their usefulness for aquaculture and/or introduction into the Chesapeake Bay.  Existing hatchery stocks show evidence of reduced genetic variation even after a single generation in the hatchery setting.  Existing hatchery stocks show evidence of accelerated genetic drift from their source wild populations.  Microsatellite markers show promise in their ability to assign hatchery individuals back to their source stock, and could be used to monitor the success of, and genetically track, any introductions.

18. Things to Think About  Is there precedence for introducing an exotic species for ecosystem rehabilitation?  Is C. ariakensis the best species for introduction and not just the most palatable?  Are there safeguards IN PLACE to insure that ONLY C. ariakensis is imported?  Do we know which native C. ariakensis population is best suited as a source for introduction?  If the restoration goal of an exotic introduction is to create an aquaculture industry or resuscitate a traditional-style fishery, what will be the impacts on the native oyster and the Chesapeake Bay ecosystem as a whole?  If the goal is to use an exotic species to restore oysters to a functional part of the Chesapeake Bay, do our hatcheries have the infrastructure to produce enough C. ariakensis oysters to jump start the populating of the entire Chesapeake Bay without producing highly inbred, genetically compromised seed?

19. Sample Collections Stan Allen Kelly Johnson Hyesuk An S. Klingbunga Ryan Carnegie Irv Kornfield M. He Mark Luckenbach Francis Obeirn X. Wu Ximing Guo Photo by R. Carnegie Supported by grants from the NOAA Non-Native Oyster Research Program Beijing outside the Forbidden City Acknowledgements