THE BENEFITS AND CONSEQUENCES OF RESTORATION USING SELECTIVELY-BRED, DISEASE-TOLERANT OYSTERS Matthew P. Hare and Colin Rose Department of Biology University of Maryland Abstract: (250 words or less) Restoration efforts in Chesapeake Bay are now focused on targeted population supplementation using artificially selected, disease tolerant C. virginica to combat high mortalities from parasitic diseases. This tactic has potential benefits and considerable risks. I will argue that genetic testing of recruitment is necessary to evaluate and manage the genetic risks of supplementation with inbred oysters, and under some circumstances it also can provide a much needed direct measure of overall restoration efficacy. Results will be presented from a highly collaborative effort to genetically monitor DEBY-strain restoration plantings in two Chesapeake subestuaries. The results indicate that the DEBY broodstock currently used to produce restoration oysters is genetically bottlenecked and contributing to severe inbreeding in those tributaries where restoration has had the greatest numerical impact. The consequences of this inbreeding will be discussed and recommendations will be offered for the precautionary use of artificially selected strains in restoration.

Talk Outline Rationale behind supportive breeding using artificially-selected oyster strains pros and cons Risks are now predictable Modeling impacts of supportive breeding Inbred broodstock – a critical factor Recommendations Gone from ignorance to one of the best examples of genetic analysis of supportive breeding efficacy and risks

The Experts Weighed In, 2000

Supportive Breeding Goals and Impacts Increase population size limit early mortality in hatchery, then release juvenile “seed” oysters Hatchery broodstock and restoration seed represent a genetically bottlenecked subset of population Genetic diversity summarized by inbreeding effective population size, Ne Planting density important for sessile/sedentary spp with external fertilization

Consensus Points from Allen & Hilbish 2000 “stocking programs [supportive breeding] will be important…” for restoration success “Diseases…are a major limitation” for restoration “Continued use of selected disease resistant stocks is warrented…” Benefits Genetic measurement of restoration efficacy Disease tolerance  greater longevity of seed oysters “genetic rehabilitation”, infusing desirable alleles Risks Increase overall inbreeding Decrease genetic variation Lower mean fitness of population “Effective population size of wild populations is an essential parameter to predict genetic effects, but is unknown”

Broodstock Sources and Numbers of Seed Planted in Virginia Supportive Breeding 3.7x107 Oysters Planted 1999 workshop where it was decided that there’s not much to lose taking this risky approach 1. Live longer on reef, 2. inject alleles for disease-tolerance into wild pop, 3. be able to genetically identify progeny of restoration oysters T. Leggett, Chesapeake Bay Foundation

Risks of Supportive Breeding Can severely reduce total Ne total Ne with % hatchery contribution x: 1 x2 (1-x)2 Ne(tot) Ne(hatchery) Ne(wild) No estimates for these parameters in 1999 when ‘genetic rehabilitation’ recommended ― = ― + Ryman & Laikre 1991 Single generation Ne, assuming equal fitness of hatchery seed and wild oysters

Great Wicomico River, 2002 Tidal physics suggest ‘trap-like’ for retention of larvae http://www.vims.edu/mollusc/cbope/vabasin.htm

Predicted Consequences Wang & Ryman 2001 Closed hatchery line Ne(wild) = 1500 95% CI = 422 -  Rose et al. 2006 5-10% contribution from supportive breeding in 2002, Great Wicomico, VA Hare et al. 2006 5% contribution graphed N(census)  annually If overall population size does not increase then Ne will not recover – it will asymptote at a low level Census N = 20 million This applies Wang & Rymans MODEL 1, random selection of broodstock from Bay, so doesn’t incorporate added inbreeding in closed hatchery line. Hare & Rose in prep.

Effective Population Size – Hatchery Hatchery amplification spawn broodstock from primary line create lots of DEBY juveniles plant these “seed” oysters WILD oysters collected in DElaware BaY and exposed to disease = DEBY strain annual restoration broodstock hatchery amplification represents bottleneck that would occur if wild broodstock were used. Additional reduction in Ne expected from selection and drift in selection line. Disease tolerant oysters selected, = “PRIMARY” DEBY line 6 generations of selection

Effective Population Size – Hatchery Hatchery amplification spawn broodstock from primary line create lots of DEBY juveniles plant these “seed” oysters WILD oysters collected in DElaware BaY and exposed to disease = DEBY strain annual restoration broodstock hatchery amplification represents bottleneck that would occur if wild broodstock were used. Additional reduction in Ne expected from selection and drift in selection line. Disease tolerant oysters selected, = “PRIMARY” DEBY line 6 generations of selection

hatchery amplification of DEBY strain Loss of Alleles, locus 2j24 BEFORE Wild population n = 300 Allele AFTER hatchery amplification of DEBY strain n = 96 seed oysters Cannot distinguish between affects of selection versus hatchery amplification here

Effective Population Size - Hatchery Population bottlenecks generate ephemeral correlations among alleles at unlinked genes gametic phase disequilibrium Magnitude of correlations depends on bottleneck Ne Waples 1991 method; Nb(LD) = 8 microsatellite loci, binned into biallelic data n 300 48 100 82 100 Wild Primary LCR02 DEBY GWR02 DEBY LCR04 DEBY LD(28) 0 5 9 11 23 Nb(LD) 5.76 4.69 3.80 4.86 95% CI 3.05 2.66 2.17 2.77  10.27 7.50 6.04 7.73 1 3 x (r2 – 1/S) R2 is the arithmetic mean of squared correlations between alleles at all pairwise combinations of loci, and S is the harmonic mean of sample sizes Nb is effective number per year, Ne is effective number per generation (accounting for repro output in >1 year). No simple relationship between the two, but if multiple estimates of Nb are consistent, it suggests that harmonic mean Nb = Ne would be similar to Nb

Effective Population Size - Hatchery Waples 1991 method 8 microsatellite loci, binned into biallelic data n 300 48 100 82 100 Wild Primary LCR02 DEBY GWR02 DEBY LCR04 DEBY LD(28) 0 5 9 11 23 Nb(LD) 5.76 4.69 3.80 4.86 95% CI 3.05 2.66 2.17 2.77  10.27 7.50 6.04 7.73

Effective Population Size - Hatchery Waples 1991 method 8 microsatellite loci, binned into biallelic data n 300 48 100 82 100 Wild Primary LCR02 DEBY GWR02 DEBY LCR04 DEBY LD(28) 0 5 9 11 23 Nb(LD) 5.76 4.69 3.80 4.86 95% CI 3.05 2.66 2.17 2.77  10.27 7.50 6.04 7.73

Consequences of Supportive Breeding Closed hatchery line Ne(hatchery) = 5 Initial reduction in Ne of 75 – 95% Recovery is slower than implied here, assumes constantly growing census N If overall population size does not increase then Ne will not recover – it will asymptote at a low level Census N = 20 million This applies Wang & Rymans MODEL 1, random selection of broodstock from Bay, so doesn’t incorporate added inbreeding in closed hatchery line. Hare & Rose in prep.

What’s wrong with small Ne ?? No problem with longstanding small Ne Problems caused by Ne Severity of problems depends on genetic load 8 to 14 highly deleterious recessives per genome in Pacific oyster, C. gigas Ne Reduced survivorship, fecundity

Inbreeding Depression in Oysters Common view: high fecundity buffers oysters from inbreeding depression High early mortality allows ‘purging’ of load Correct view: even slight inbreeding causes measurable reduction in fitness-related traits Unrelated parents, F = 0 by definition, First cousin mating is F = 0.0625, sibling cross F = 0.203 Evans et al. 2004 Aquaculture 230:89-98 40 families, n = 402

Recommendations I Two of three motivations for using selected strains were speculative, still no data on DEBY fitness in restoration setting Spawn wild oysters and use procedures that  Ne(hatchery) Continue use of DEBY oysters only in rivers where genetic monitoring can be used to test efficacy of changing planting strategies Except for genetic tracking, the benefits are speculative possibilities – much more speculative than the risk of reduced Ne in the event that restoration is highly successful

Recommendations II There is no empirical justification for using DEBY x wild broodstock crossings Speculative benefits Disables genetic monitoring of restoration Known inbreeding risks still apply

Collaborators C. Rose Department of Biology & K. Paynter D. Meritt S.K. Allen, Jr. M.D. Camara R. Carnegie M. Luckenbach K.S. Reece Department of Biology & Horn Point Laboratory, University of Maryland Virginia Institute of Marine Science College of William and Mary, Virginia

Oyster Disease Research Program, NOAA, Sea Grant with help from… Maryland Oyster Recovery Partnership Charlie Frentz Virginia Chesapeake Bay Foundation Rob Brumbaugh Tommy Leggett VA Marine Resources Division Jim Wesson VIMS: Cheryl Morrison Wendy Ribeiro Missy Southworth Hare Lab, UMD: Jenna Murfree Paulette Bloomer Natasha Sherman Gang Chen Maria Murray Peter Thompson Safra Altman Kristina Cammen Andrew Ascione Ninh Vu Katie Shulzitski Oyster Disease Research Program, NOAA, Sea Grant