[From the U.S. Government Printing Office, www.gpo.gov]







                                        ENETIC


                                        ANALYSIS OF


                                       -AMERICAN SHAD

                                        ENTERING


                                        CHESAPEAKE


                                          AY








   Bonnie L. Brown, PhD                  A REPORT SUBMITTED TO THE

   Robert W. Chapman, PhD                CHESAPEAKE BAY COMMISSION





                      . . ......         .....
                                       .. . .... ....









                       ..........
                                      T






                       "N
             ... .............
              ............ ..... ....

















                                         BY CHESAPEAKE SCIENTIFIC


                                         INVESTIGATIONS FOUNDATION, INC.





              0                          SEPTEMBER, 1991

            0                    0


          0









                                         GENETIC ANALYSIS OF AMERICAN SHAD
                                                     ENTERING CHESAPEAKE BAY












                                  Principal Investigators:



                                  Bonnie L. Brown, Ph.D.                                                  Robert W. Chapman, Ph.D.
                                  Department of Biology                                                   Department of Biology
                                  Virginia Commonwealth University                                        East Carolina University
                                  Richmond, VA 23284-2012                                                 Greenville, NC 27858-4353







                                                                       A report submitted to:


                                                              The Chesapeake Bay Commission
                                                                          September, 1991





                                                                                   By:


                                                 Chesapeake Scientific Investigations Foundation, Inc.









                        This contracted research was funded by the Pennsylvania Delegation to the Chesapeake Bay Commission, the Virginia Council on the
                        Environment (COE) and the Virginia Marine Resources Commission. COE funding was provided through the Coastal Resources
                        Management Program through grant # NA90AA-H-CZ796 of the National Oceanic and Atmospheric Administration under the Coastal
                        Zone Management Act of 1972 as amended.









                         TABLE OF CONTENTS



                         Acknowledgements                  ........................................................................................              ii
                         Executive Summary                    .......................................................................................            1

                         Introduction          ................................................................................................                  4

                         Laboratory Analysis                  .......................................................................................            6

                         Data Analysis              ...............................................................................................              7

                         Results       ......................................................................................................                    9

                         Discussion          .................................................................................................                   11

                         Conclusion          ................................................................................................                    13

                         Significant Points                .........................................................................................             16

                         References          .................................................................................................                   17



                         Figure 1              Map of the Chesapeake Bay region showing location of American shad
                                               samples taken by commercial fishermen                              .............................................  20

                         Figure 2              Graphic depiction of restriction fragment patterns for American shad                                  ............ 22
                         Figure 3              Diagram of estimated contribution by baseline populations to Virginia@s
                                               1991 Atlantic Ocean harvest of American shad                                ...................................... 28

                         Table 1               Summary of American shad stocked into Susquehanna River during the
                                               period 1982-1991 by river of origin                           ..................................................  29
                         Table 2               Mitochondrial DNA haplotypes of American shad harvested outside
                                               Chesapeake Bay in 1991                        ...............................................................     30
                         Table 3               Chi-square comparisons for American shad in baseline samples                                     .................. 31
                         Table 4               Results of accuracy testing of GIRLSEM for synthetic mixed-sample
                                               shad mtDNA data                   .......................................................................         32

                         Table 5               Estimated stock composition of two mixed-fishery groups of American
                                               shad     .......................................................................................                  33

                         Appendix A            Genotypes of 1991 intercept fishery American shad                                  ................................ 34
                         Appendix B            Distribution of American shad mtI)NA haplotypes in intercept and
                                               baseline samples               .........................................................................          37





                                                                                       i









                  AC KNOW LEDGEM ENTS



                          This study was conducted for the Chesapeake Bay Commission. Funds were
                  provided by the Chesapeake Bay Commission, the Virginia Council on the Environment
                  (Coastal Zone Management Grant # NA90AA-H-CZ796), and the Virginia Marine
                  Resources Commission (in-kind services) to perform genetic analysis of American shad,
                  Alosa sapidissima, harvested by Virginia watermen from waters outside Chesapeake Bay in
                  Virginia's ocean gill net fishery. Genetic analysis was employed to ascertain, to the
                  maximum extent possible, the natal stream and spawning destination of shad which are
                  susceptible to harvest in the Atlantic Ocean intercept fishery. Genotypes were compared to
                  shad from ten baseline target rivers and from Susquehanna River previously analyzed
                  under MDNR contract number PR90-004-004. Inclusion of Albemarle Sound, Cape Fear
                  and Santee River baseline populations was made possible by a supplemental grant from
                  Atlantic States Marine Fisheries Commission's Interstate Fisheries Management Program
                  (ISFMP) grant number 91-1(PASRH). We thank J. Pella for editing an earlier version of
                  this report and for providing the GIRLSEM algorithm which we used to analyze the genetic
                  data produced by this study. We also thank Barney Jernigan for preparing other essential
                  computer programs and for his assistance with data analysis. A great many other people
                  also contributed to the study by providing biological specimens of American shad including
                  but not limited to:



                          Walt Ambrogetti, USFWS
                          Tom Backman, USFWS
                          Bob Brandt, NY State Dept. of Environmental Conservation
                          Rick Eager, USFWS
                          Chris Frese, RMC Environmental Services
                          Joe Loesch, Virginia Inst. of Marine Science
                          Art Lupine, NJ Freshwater Fisheries Lab
                          Ted Meyers, USFWS
                          Roy Miller, DE Div. of Fisheries & Wildlife
                          Robert O'Reilly, Virginia Marine Resources Commission
                          Jim Owens, Virginia Inst. of Marine Science
                          Fritz Rhode, NC Div. of Marine Fisheries
                          Dick St. Pierre, USFWS
                          Mark Tisa, MA Div. Fisheries & Wildlife
                          Glenn Ulrich, SC Wildlife & Marine Resources Div.
                          Dale Weinrich, MD Dept. of Natural Resources
                          Sara Winslow, NC Div. of Marine Fisheries









               EXECUTIVE SUMMARY


                       Genetic analysis was employed to examine the stock composition of American shad, Alosa
               sapidissima, harvested outside Chesapeake Bay in Virginia's Atlantic Ocean intercept fishery
               during the Spring of 1991. Genotypes of intercept fish were compared to fish from thirteen
               American shad populations in order to estimate the relative percentage of shad from each baseline
               population in the intercept sample. Techniques involved restriction endonuclease digestion of
               mitochondrial DNA (mtDNA) purified from shad egg tissue, a common methodology used for
               examining population dynamics in fishes. MtDNA genotypes were obtained for 158 individuals
               from three locations near the mouth of Chesapeake Bay: Rudee Inlet, Chincoteague, and Quinby,
               VA. The thirteen baseline populations included 549 American shad from spawning aggregations in
               ten target rivers (Connecticut, Delaware, Hudson, Nanticoke, Pamunkey, James, Chowan,
               Savannah, St. Johns and Santee Rivers), from the Susquehanna Flats in upper Chesapeake Bay,
               and from Susquehanna River shad lifted over Conowingo Dam in 1990 and 1991.

                       Yearly, since 1982, the Susquehanna has been stocked with shad from several of the
               following rivers: Columbia, Connecticut, Delaware, Hudson, James and Pamunkey. Genetic
               analysis of Susquehanna shad in 1990 and 1991 indicated that the Susquehanna shad population- is
               now a mixed-stock composed of native Susquehanna, native Chesapeake Bay, Connecticut,
               Hudson, Pamunkey and Santee lineages. Therefore, in the genetic analysis of the Atlantic Ocean
               fishery, occurrence of those lineages which have been stocked into the Susquehanna is taken to
               indicate the presence of Susquehanna fish.

                       Since many shad have mtDNA genotypes which are common to several if not all drainages,
               every fish cannot be uniquely identified to a river system. Therefore, when a mixed assemblage of
               fish is examined (such as an ocean fishery), genotypes of the entire group are statistically analyzed
               for comparison to baseline genetic data for potential source populations. The analysis provides an
               estimate of the most likely composition of the group in question. We have employed a standard
               statistical treatment, maximum likelihood analysis, which was modified by Dr. J. Pella and co-
               workers to interpret mixed-fishery genetic data. This analysis has been successfully employed to
               manage the west coast salmon fishery for many years.

                       Most baseline American shad populations share one common mtDNA type, but each
               contains unique types as well. These unique types of mtDNA made it possible to estimate the
               percentage of each baseline stock represented in the migrating coastal group of shad. Nineteen
               percent of American shad sampled outside Chesapeake Bay had unique mtDNAs themselves and
               could not be classified as originating from any of our current baseline populations. The remaining
               81 % of intercept shad were compared to the baseline populations in a maximum likelihood analysis
               of stock contribution. Composition of this portion of the intercept group was estimated to be due
               to contributions of the following seven stocks: St. Johns ( 2 + 2 %), Pamunkey ( 2 + 2 %),



                                                          1









               Chowan ( 5 ï¿½ 5 % ), Santee ( 19      12 %), Hudson ( 9 + 5 %), Susquehanna ( 31      + 10 17o) and
               C
                ,onnecticut (32 + 11 %). No contribution was detected from the other baseline shad populations.

                       Given the stocking history and composition of the Susquehanna River population as shown
               in Tables I and 5, it is possible that portions of the estimated contributions for some baseline
               stocks, particularly Santee River, are actually due to the presence of Susquehanna shad in the
               intercept sample. For example,      the genetic analysis does not distinguish individual Pamunkey
               sliad from Pamunkey River from those which were stocked into Susquehanna River. Statistical
               analysis of the genetic data employs frequencies of Pamunkey genotypes as they occur iri both the
               Pamunkey and Susquehanna to estimate the most likely relative contribution of each of the two
               stocks. Therefore, in the absence of compelling information such as tag returns or additional
               genetic data, the estimated contribution to the intercept fishery of river stocks previously introduced
               into the Susquehanna are taken as the actual preliminary estimates of those rivers' contributions to
               the intercept fishery. This conservative approach avoids underestimating contributions by those
               stocks. However, in the case of Santee River we do have compelling evidence to support the
               assumption that the contribution estimated for Santee River (originally included in the analysis as
               an outgroup) is due at least in part to the presence of Susquehanna shad in the intercept samples.
               First, Jesien and Hocutt (199 1) report that for shad tagged off Rudee Inlet, 28 % of tag retu ms
               were from north of Chesapeake Bay and only 17% of returns came from areas south of the
               tagging location (all from North Carolina). The remining 55% of shad tag returns were from fish
               which moved into Chesapeake Bay. This information, combined with knowledge of genetic
               similarity between the Susquehanna and Santee stocks (Chapman and Brown, 1991), knowledge
               of Santee River shad migration and spawning (G. Ulrich, pers. communication), and the
               geographic separation of Santee stocks from the intercept fishery, indicates that it is appropriate to
               group the Susquehanna and Santee contribution estimates (Wood et al. 1987). Thus, we estimate
               that Virginia's 1991 intercept fishery was comprised of at least 41 % Susquehanna shad (0.81 x
               (31suF, + 1%antee %

                      Despite small sample sizes, a trend is evident in the genetic composition of shad harvested
               north of the Bay mouth (Chincoteague and Quinby) and those harvested south of the Bay mouth
               (Rudee). Chi-square analysis indicates that the stock compositions of shad harvested from the
               two regions are significantly different. In fact, a large portion of Rudee Inlet shad were of Virginia
               origin while the Chincoteague and Quinby harvests were composed primarily of Susquehanna,
               Hudson and Santee lineages.

                      The estimates provided in this report should be considered preliminary for four important
               reasons. First, several of the baseline samples are very small and may not adequately represent
               those groups of American shad. Second, contributions attributed by genetic analysis to Santee and
               some other rivers may be due in part to the presence of Susquehanna shad which appear to be
               largely of Connecticut, Nanticoke and Santee River descent. Third, since the Susquehanna River
               population has not attained genetic stability, it is possible that contribution estimates for this river


                                                          2









               will vary from year to year as the resurgent Susquehanna population approaches a stable genetic
               equilibrium causing the estimated composition of the intercept fishery to vary. Finally, the fact that
               19% of intercept shad were distinguished by unique genotypes indicates that either the existing
               baseline populations have been inadequately characterized or that one or more potential source
               populations have not been included in the baseline data set. These significant points are discussed
               in the report and lead to recommendations for procedural refinements which must be incorporated
               into future American shad research.


                      To consider the potential effect of the coastal intercept fishery one would need several years
               of estimates of the stock composition and magnitude of both Maryland and Virginia's coastal
               harvests. However, a rough estimate can be made based on Virginia's preliminary estimate of their
               1991 ocean shad harvest and the present genetic data. The estimated amount of intercept shad
               harvested in 1991 was 405,612 pounds (at 4.5 pounds per fish, this is 90,136 shad). Multiplying
               by the factor of 0.41, approximately 36,955 intercepted shad were of Susquehanna origin. This
               value exceeds the number of shad lifted over Conowingo Dam in the Spring of 199 1. If these
               findings are verified by future monitoring, tagging and genetic evaluation then the most
               conservative action would be to restrict shad harvests along the Atlantic coast.









              INTRODUCTION


                     The American shad, Alosa sapidissima, is an anadromous member of the herring family
              (Clupeidae), which ranges from the Gulf of St. Lawrence to Florida (Walburg and Nichols, 1967).
              During its springtime spawning runs the species has been subjected to substantial commercial and
              recreational fishing pressure throughout its range, particularly in Chesapeake Bay tributaries and
              by Maryland and Virginia's ocean fisheries. In addition, shad populations of almost every
              Chesapeake Bay drainage have been further restricted by dams which block migration to their
              spawning habitat in fresh water transition zones. As a result of fishery exploitation, loss of
              spawning and nursery habitat, and possibly environmental degradation such as stream
              acidification, harvests of shad in Pennsylvania, Maryland, the District of Columbia and Virginia
              declined precipitously during the period 1965-1988 (Stagg, 1986; Gibson, Crecco and Stang,
              1988).

                     A great deal of effort has been expended to revive stocks of Chesapeake Bay American
              shad. Conservation and restoration measures were enacted in Pennsylvania, Maryland and the
              District of Columbia in the early 1980s. Some agencies required season, gear and by-catch
              restrictions along with creel limits to reduce fishing effort (Maryland's shad fishery was closed in
              1980). Concurrently, the issues of habitat loss and degradation were addressed by installing
              permanent fish passage facilities such as the one at Conowingo Dam, removing some obstructions
              to migrating fish, re-stocking fish into historical spawning habitats, establishing stock assessment
              and monitoring programs, and operating dam turbines in a manner which maintained minimum
              flow and standard dissolved oxygen levels (CEC, 1989).

                     Prior to these efforts, American shad migrating upstream in Susquehanna River had been
              few in number. Throughout the 1970s shad transported by the trap/lift assembly at Conowingo
              Dam averaged 127 fish per year (ASMFC, 1988). Yearly release of shad fry and of live pre-
              spawned adult shad from six other source rivers accompanied the lift operation beginning in 1982
              (Table 1). By 1989, more than 6000 migrating shad were reported to have been hauled upstream
              above all dams to the historical Susquehanna spawning areas.

                     In view of the apparent success of shad management efforts in Susquehanna River, a
              program of study was proposed to the Maryland DNR Chesapeake Bay Research and Monitoring
              Division's Power Plant Topical Research Program designed to examine population dynamics
              underlying the resurgent American shad population in Susquehanna River using molecular genetic
              techniques (Chapman and Brown, 1991). Mitochondrial DNA genotypes of American shad being
              moved over Conowingo Dam were compared to genotypes of shad from the source rivers, from
              other Chesapeake Bay rivers and from several southern east coast shad populations. Variation in
              n-dtochondrial DNA was analyzed and employed to estimate the percent contribution by any of
              these shad populations to the increasing Susquehanna stock.



                                                       4









                     Population genetic data collected for the Maryland DNR study were employed in the
              present study for Chesapeake Bay Commission to estimate the relative percentage of American
              shad from each baseline population being harvested in Virginds Atlantic Ocean intercept fishery
              during the Spring of 199 1. The baseline populations available for comparison with the Spring
              1991 coastal fishery were: Delaware River, DE (DEL), Hudson River, NY (HUD), Connecticut
              River, CT (CT), Nanticoke River, MD (NAN), Pamunkey River, VA (PAM), James River, VA
              (JAM), Chowan River, NC (CHO), Savannah River, SC (SAV), Santee River, SC (SAN), St.
              Johns River, FL (STJ), the Susquehanna Flats in upper Chesapeake Bay (SF), and Susquehanna
              River shad lifted over Conowingo Darn in 1990 (S90) and 1991 (S91). This report summarizes
              the seven-month research project, provides estimates of proportions of American shad from each
              of the groups outlined above which comprise the Spring 1991 coastal harvest, and outlines
              management and research implications of these data.








              LABORATORY ANALYSIS


                     The laboratory procedures described in this report are intentionally brief Detaile4i
              instructions for the extraction and digestion of mitochondrial DNA can be found in Chapman and
              Brown (1990).

                     During the Spring of 1991, mitochondrial DNA (mtDNA) was extracted from American
              shad harvested in Virginia's Atlantic Ocean intercept fishery outside the mouth of Chesapeake Bay.
              On ten occasions during the period 5-27 March, shad were obtained from commercial fishermen
              landing at three locations: Rudee Inlet, Chincoteague and Quinby/Wachapreague (see Figure 1).
              Ovaries from each individual fish were removed and placed in a Ziplock Baggie along with an
              envelope containing a scale sample and pertinent data on the location of capture, size of fish, etc.
              Each baggie was sealed and placed on wet ice for transportation to the laboratory at East Carolina
              University in Greenville, NC.

                     All shad samples arrived at the laboratory on the day they were collected. Within, one day
              of sampling, approximately 10 g of egg tissue from each individual fish were processed -to isolate
              and purify mtDNA. The mtI)NA was rehydrated in 150 gl of sterile distilled water and aliquots of
              8.5 gl mtl)NA from each fish were combined with 0.5 Unit of the following restriction
              endonucleases (Aat 1, Apa 1, Bcl 1, Bgl I, Dra 1, EcoR 1, EcoR V, Hind 111, Kpn 1, Pst 1, Pvu II,
              Sal 1, Sma 1, Sst II, and Xba 1) along with 1 jil of the appropriate buffer supplied by the
              manufacturer. Each digest was incubated at 37 OC for 3-4 hours and contained a total volume of
              10 gl. Reactions were stopped with I gl of STOP solution (0.89 M Tris, 0.89 M boric acid, 0.02
              M EDTA, 0.25 % bromophenol blue, 50 % glycerol and 1 % SDS) and were electrophoresed
              overnight through 0.8 % agarose gels. The DNA in gels was stained with ethidium bromide and
              photographed under ultra-violet light as described by Chapman and Powers (1984).

                     Restriction digest patterns were recorded for each restriction endonuclease digest of each
              fish's mtDNA. Digestion patterns were assigned upper-case alphabetic symbols. Then, each
              individual was assigned a composite "haplotype" consisting of the letters designating the restriction
              fragment patterns produced by digestion with each of the fifteen enzymes.













                                                       6









                DATA ANALYSIS


                        Details of the mathematical properties of the algorithms used to perform statistical analyses
                can be found in Sokal and Rohlf (1981), Roff and Bentzen (1989), Fournier et al. (1984), Pella
                (1986), Pella and Milner (1987) and Wood et al. (1987).

                        Each fish's haplotype is a multiple characterization of that fish's mitochondrial genome and
                is transmitted in a manner analagous to human surnames. As in other animals, variation in shad
                mtDNA is typified by the occurrence of rare haplotypes in each population (Bentzen et al., 1988
                and 1989). If chi-square contingency tests were to be performed the rare haplotypes would be
                lumped and only the most frequent haplotypes would be employed in the analysis. This practice of
                pooling rare mtDNA haplotypes results in a severe loss of information relevant to geographic and
                temporal genetic variation. To resolve this problem, Roff and Bentzen (1989) presented a chi-
                square analysis which does not require pooling of rare variants. The analysis generates Monte
                Carlo distributions of expected chi-square from unpooled mtDNA data allowing high levels of
                significance even when sample sizes are small.

                        Haplotype frequencies were employed to determine whether baseline samples were
                distinguishable by the mtDNA genetic analysis and to examine basic genetic relationships between
                the baseline shad populations and the intercept population. First, chi-square statistics for
                heterogeneity of mtDNA haplotype frequencies were calculated (Roff and Bentzen, 1989). The
                chi-square analysis was conducted by initially treating all of the populations as one large
                assemblage. Based on the finding of significant heterogeneity, successively smaller sets of
                populations were analyzed until no further heterogeneity was detected. Finally, chi-square was
                determined between each pair of populations.

                        The actual contribution of each baseline population to the intercept sample was estimated
                from mtDNA information by conditional maximum likelihood estimation of stock composition.
                This approach has been extensively used in population genetics since the early 1930s (Fisher,
                1958; Crow and Kimura, 1970) and was reviewed by Pella and Millner (1987). The algorithm
                we used, called GIRLSEM, was proposed by Fournier et al. (1984) and modified by J. Pella of
                NMFS (Pella, 1986) for interpretation of mixed-fishery genetic data for salmon stocks from
                California to Alaska. The assumptions of maximum likelihood analysis are: 1) baseline stocks
                which potentially contribute to the mixture are genetically distinguishable, 2) sampling of baseline
                stocks is sufficiently precise to identify a significant portion of genetic diversity within each, 3) all
                source populations represented in the mixture are part of the baseline data set, and 4) a sufficiently
                large random sample is obtained from the mixed-stock fishery. The accuracy of this program for
                mtDNA data was tested by analyzing several artificial mixed populations of known composition
                created by randomly sampling the baseline haplotypes (with replacement) prior to the analysis of
                mixed shad populations. This accomplished two purposes. First, it tested the general robustness
                of the program's output with our baseline mtDNA data and, second, it identified populations


                                                             7









              whose contributions were consistently over- or under-estimated. Data were entered into the
              COULSEM analysis by treating haplotypes as "multiple alleles at a single locus." The contribution
              of all 13 baseline populations was then estimated based on the distribution of all 162 haplotypes.














































                                                        8










               RESULTS


                      A total of 158 individual shad collected during a one-month period from three locations
               near the mouth of Chesapeake Bay were processed. Fifteen restriction endonuclease digests were
               performed per individual for a total of 2,430 digests. Thirty-three restriction fragment profiles
               were observed in the intercept samples, encompassing seventy-eight separate restriction fragments
               of the Alosa sapidissima mitochondrial genome. Whenever possible the profiles for an enzyme
               were compared to those obtained by Bentzen et al. (1988 and 1989). Restriction fragment profiles
               for each enzyme are illustrated in Fig. 2.

                      Genotypes for each individual intercept shad are shown in Appendix A while those for
               individual baseline shad can be found in Chapman and Brown (1991). Some individuals in both
               studies were heteroplasmic for mtDNA size and/or site variation. That is, single shad sometimes
               had mtDNA molecules of different size and/or nucleotide sequence. This phenomenon was
               previously reported for shad by Bentzen et al. (1988 and 1989). In all tables of this report, a
               single-enzyme genotype denoted as "A/B" indicates the heteroplasmic combination of genotypes A
               and B in an individual. The same is true for genotypes A/C, A/E, etc. Haplotypes, the composites
               of all fifteen genotypes, for each intercept shad are shown in Table 2 and are also listed in
               Appendix B alongside haplotypes for all of the baseline populations. Thirty-two different
               haplotypes were detected in the intercept samples. Seventy-nine shad had either the common
               haplotype AAAAAAAAAAAAAAA or one of the many unique baseline haplotypes. Haplotypes of
               eighteen other individuals in the intercept sample were unique. In some instances the mtDNA
               preparation was of inadequate quantity or quality to perform all fifteen digests. Sixty-five
               additional shad were incompletely characterized and were not included in the final analysis
               (missing data are shown by "-" in Appendix A).

                      Results of a chi-square analysis based on the distribution of haplotypes are presented in
               Table 3. The first tier of the chi-square analysis was performed with the entire baseline sample as
               one assemblage (overall chi-square of 2445.18, P < 0.0001) indicating that significant differences
               existed within the aggregation. The assemblage was successively decomposed by population to
               the point where no further heterogeneity was detected. In the end, pairwise comparisons between
               baseline populations were necessary as each baseline sample (except SF) was found to be
               significantly different from virtually every other baseline sample. Significant differences between
               baseline samples were taken to indicate that they were distinct genetic stocks. An additional chi-
               square analysis found that shad harvested from Rudee Inlet were significantly different from those
               taken at Chincoteague and Quinby (chi-square of 94.78, P < 0.001) while no significant difference
               was detected between the Chincoteague and Quinby samples (chi-square of 19.45, P = .717).

                      Tests for general robustness of the GIRLSEM program were performed on multiple
               simulated mixed-stock populations created by random selection from the 549 shad genotypes in the
               baseline data set. Simulated mixture sizes ranged from 52 to 356. Table 4 lists the results of one


                                                          9









             such simulation. The simulations indicated that contributions of three baseline populations
             (Connecticut, Chowan and Delaware) were consistently misrepresented in the synthetic
             populations. This was expected since while most of the baseline populations surveyed had 18% or
             more unique individuals, our samples for Chowan and Connecticut Rivers had low proportions of
             urdque individuals. Statistical analysis indicated that Connecticut River was consistently under-
             represented while Delaware River was consistently over-represented.

                    Table 5 lists the estimated composition of the 1991 Virgiia intercept shad fishery.
             Incompletely characterized shad and the nineteen percent of intercept shad whose haplotypes were
             urdque (not traceable to in any baseline population) were withheld from the final analysis. Thus,
             the estimated contributions in Table 5 must be decreased by 19% to obtain the actual estimated
             contribution for each source population. By multiplying the maximum likelihood results in Table
             5 by a factor of 0.8 1, it is estimated that the following populations comprised the 1991 intercept
             harvest: Unknown (19%), Chowan (4%), St. Johns (M), Pamunkey (2%), Hudson (7%),
             Santee (16%), Connecticut (26%) and Susquehanna (25%). These estimates are depicted
             graphically in Figure 3.

                    Finally, although sample sizes precluded maximum likelihood analysis of the composition
             of Rudee Inlet, Chincoteague and Quinby samples individually, a qualitative analysis of their
             composition is in order. Examination of haplotypes other than the common
             AAAAAAAAAAAAAAA presented in Appendix B, indicates that most shad analyzed from Rudee
             Inlet shared haplotypes with Pamunkey River and a few shared haplotypes with Susquehanna
             River. Conversely, most of the Chincoteague and Quinby shad were typified by Susquehanna,
             Hudson and Santee haplotypes.























                                                     10









               DISCUSSION


                      American shad examined for this study and for the larger MDNR study were of diverse
               genetic composition. In order to evaluate composition of the intercept fishery it was first necessary
               to evaluate composition of Susquehanna and other baseline stocks. Chapman and Brown (199 1)
               reported chi-square evaluations between the baseline stocks employed here. As shown in Table 3,
               discrimination between populations was excellent-, virtually every pairwise comparison was
               significantly different at P < 0.001. They also reported that the existing Susquehanna River stock
               is comprised of shad descended from native Chesapeake Bay stocks, several east coast rivers
               stocked into the Susquehanna system, and lineages genetically resembling the Santee River stock.
               In addition, while samples collected within several baseline rivers in 1990 showed no temporal
               genetic variation, Susquehanna samples for 1990 and 1991 were found to be significantly different
               from one another (chi-square = 59.92, P<0.001) indicating that the Susquehanna population is not
               in a state of genetic equilibrium. Another important finding of that sudy was that shad from upper
               Chesapeake Bay were genetically similar to shad of several southern lineages, particularly from
               Santee River.


                       Chi-square analysis indicated American shad landed at different Atlantic Ocean locations
               were significantly different as would be expected for a mixed population. Of particular interest is
               the fact that shad landed at Rudee Inlet were different from those landed along the Virginia portion
               of the, Delmarva Penninsula while shad harvested along the Delmarva Penninsula by the intercept
               fishery (Chincoteague and Quinby) were not significantly different from one another. Since the
               intercept fishery is presently managed as a single unit, and since the intent of this study was to
               evaluate the composition of the entire intercept fishery, the three samples were not separated when
               conducting the maximum likelihood analysis of stock composition. Small sample sizes also
               precluded estimating the probable destination of shad landed at the Rudee Inlet, Chincoteague and
               Quinby locations separately with an acceptable degree of confidence. The final sample sizes for
               Rudee, Chincoteague and Quinby are small (n = 15, 40, 42, respectively) after excluding partial
               and unique haplotypes (17%, 7 %, and 9%, respectively) and the standard deviation values for
               these estimates were proportionally large. However, the qualitative trend indicated by the chi-
               square analysis is apparent. More shad analyzed from Rudee Inlet appeared to be of Virginia
               origin than of Susquehanna origin, while the Chincoteague and Quinby harvests were primarily
               composed of lineages typifying the Susquehanna.
                       Nineteen percent of the intercept fish could not be classified by our current baseline data
               set. This indicates that either these haplotypes actually exist in the present baseline populations but
               were missed in the baseline samples or that intercept fish with unique haplotypes were from
               populations not included in the present baseline data set. The former possibility indicates that
               despite the highly significant chi-square values between baseline populations, these reference
               populations may not have been sampled intensely enough to detect all mtDNA diversity. This



                                                       I I









              possibility was examined by performing the analysis described by Hebert et al. (1988) for
              detection of clonal diversity (data not shown). For populations other than those in upper
              Chesapeake Bay, this analysis indicated that a sample of approximately 40 individuals was
              sufficient to detect a significant portion of mtDNA diversity. Therefore, as suspected, some
              baseline populations were inadequately sampled. The latter possibility is equally likely; other
              source populations which could potentially be represented in the intercept fishery were not included
              in the baseline data set.


                     Conditional maximum likelihood estimates of the composition of the remaining 8 1 % of the
              intercept fishery sample indicate that those intercept shad were predominantly of Susquehanna,
              Connecticut, Santee and Hudson River origin (Table 5). Small contributions by other locations,
              from both Chesapeake Bay and from other southern rivers, were also detected in tile overall
              analysis. Given the mixed-stock nature of the Susquehanna, the estimates reported here are
              actually the maximum contribution by Susquehanna River and the minimum contribution by those
              stocks which have been introduced to the Susquehanna system. Calculated standard deviation
              values were considered acceptable for the management requirements of this species and were
              further improved by combining estimates for stocks which were genetically similar (i.e.
              Susquehanna and Santee).

                     On first impression, the contribution by Santee River stock makes little sense. Shad from
              this system are not likely to be found off the Virginia coast in March while they are spawning in
              Santee River. Inspection of the data in Table 5 and Appendix B, however, shows that the Santee
              and Susquehanna Rivers share haplotypes which are not found in other populations. In addition,
              there is other compelling evidence to support the assumption that the estimated contribution
              attributed to Santee may actually be due to misclassification of Susquehanna shad. First, in a study
              of shad tagged off Rudee Inlet, Jesien and Hocutt (1991) report that only 17% of tag returns came
              fi-om locations south of Chesapeake Bay and no returns were obtained from tributaries within the
              general geographic region of Santee River. Second, there is clear genetic similarity between the
              samples taken from Susquehanna River, Susquehanna Flats and Santee River (Chapman and
              Brown, 1991). Third, current knowledge of the timing of shad migration and spawning indicates
              that the possibility is extremely remote that the Santee stock would be found off the Virginia coast
              during the Spring of 1991. Other maximum likelihood analyses (data not shown) indicated that
              when Santee River was eliminated from the baseline data, the estimated contribution of
              Susquehanna River increased to 49 %. Therefore, for the present study estimated proportions of
              shad from Santee were combined with those for Susqehanna in order to improve estimates of
              contribution. Combining these groups based on patterns of similarity, suggested by Wood et al.
              (1987), changed the estimate of contribution for Susquehanna River to 41 + 10% and.significantly
              decreased standard deviation due to sampling variation.






                                                      12









               CONCLUSIONS


                      This project involved a genetic survey to identify which, if any, target populations were
               harvested by Virginia's Atlantic Coast intercept fishery. The study was conducted concurrently
               with one of the most comprehensive investigations to date of a mixed fishery employing mtDNA
               analysis. The results presented here are of immediate interest to the community of managers and
               scientists who regulate the American shad fishery in Chesapeake Bay. Furthermore, these data
               constitute a minimum framework which, if expanded, will allow development of a long-term
               monitoring program which could eventually match that for west coast salmon in its effectiveness.

                      The genetic analysis provides an intitial. "snapshot" of Virginia's intercept fishery indicating
               that approximately one-half of the shad harvested were destined for Susquehanna River. Due to
               the magnitude of effort and funds expended by all of the Bay states to rebuild shad stocks, policy
               makers and managers are sure to inquire: "Does the Virginia ocean shad harvest potentially affect
               the Susquehanna stock?" This question can be addressed by considering the estimate of shad
               harvested by Virginia's ocean fishery in 1991 (405,612 lb @ 4.5 lb per fish = 90,136 shad)
               multiplied by the estimated contribution of the Susquehanna to this harvest. Using the estimated
               41% contribution of Susquehanna shad, it can be calculated that 36,956 (+ 3,703) shad were
               intercepted off the Virginia coast in 1991.** Since the total number of shad lifted over Conowingo
               Dam was 22,083, restriction of the intercept fishery could potentially boost the Susquehanna
               population by 100%. Of course, this outcome is conditioned upon the assumption that these shad
               would not be captured in other areas of the Bay prior to reaching the Conowingo fish lift.

                      One possible test of this conclusion would be to eliminate the intercept fishery for just one
               year and assess the increase in the Susquehanna population over and above that increase which is
               expected based on the present trajectory of,population growth. The increase in shad lifted should
               be statistically analyzed to determine if the putative increase can be differentiated from the currently
               predicted increase without decreased fishing effort. Personnel involved with the ASMFC Shad
               and River Herring Scientific and Statistical Committee are familiar with shad population models
               and the statistics necessary to perform such an analysis.

                      Ile joint MD/VA shad tagging study at Rudee Inlet has produced some preliminary results
               which should be compared with the genetic data. The two studies are complementary in a very
               important way. Fifty-six percent of tag returns during the last six months have been from lower
               Chesapeake Bay; primarily from York River and its tributaries (Jesien and Hocutt, 1991). By
               comparison, genetic analysis estimates that more than half of the Rudee Inlet harvest is of
               American shad from Pamunkey River (a tributary of York River). Such close agreement indicates
               that the qualitative estimates of stock contribution for this location are correct.




                  The 95% confidence intervals for this estimate are 19,289 and 54,622.


                                                       13









                    Another aspect of the complementary relationship between the tagging and genetic analyses
             isassociated with the fact that no tags have been returned from upper Chesapeake Bay. There is
             no fishing effort for shad in upper Chesapeake Bay since both MD and PA have closed their shad
             fisheries. Yet the genetic analysis of the ocean catch shows an upper Bay component
             (Susquehanna River) in addition to a Pamunkey component. Thus, one analysis complements the
             other. In the future, tagging could address some critical issues raised by the genetic analysis. For
             example, both the Susquehanna Flats and the Susquehanna River shad populations have large
             Santee River components. It cannot be detern-iined fi-om the present data whether the Santee
             component of the intercept fishery is due to shad actually migrating from or to Santee River (not
             likely given the current state of knowledge) or whether it is due to Susquehanna shad with Santee
             haplotypes (very likely). If tagging endeavors are repeated once fishing resumes in upper
             Chesapeake Bay then both possibilities can be evaluated.

                    Like most other scientific investigations the present genetic analysis raises many new
             questions. Although the baseline data were adequate to address the majority of intercept fish
             harvested in 1991, they must be expanded before the entire fishery can be evaluated. We
             recommend increasing all baseline sample sizes to include at least 50 individuals and assessing
             existing Chesapeake Bay shad populations which were not examined in the present study
             (Rappahannock, Potomac, Patuxent, Choptank, Chester, etc.). In addition, it is clear that one
             sample is not adequate to formulate far-reaching management decisions. Shaklee et al. (1990) have
             examined mixed-stock fisheries of Pacific salmon. They found that stock composition varies
             substantially from year-to-year for mixed-stock assemblages. It would be prudent to assume that
             migratory mixtures of American shad stocks behave in a similar manner to Pacific coast salmonid
             stocks.


                    Depending on the Bay States' goals, we can suggest the necessary actions to undertake.
             First, if the sole purpose is to make an immediate management decision regarding coastal intercept
             shad fisheries then at least one more survey should be made of the Virginia fishery accompanied by
             at least two years of investigation of Maryland's ocean shad fishery. Analyses should proceed by
             sacrificing 150 fish per site per year, extracting mtDNA, and digesting mtDNA with the same 15
             enzymes employed in the present study. The resulting haplotypes should be compared to an
             expanded data base. This should provide adequate information to confidently make decisions
             pertaining to the intercept fishery.

                    If goals are broader and the States wish not only to manage and regulate the intercept
             fisheries but to monitor all Chesapeake Bay shad populations, then we would suggest a modified
             research/monitoring program. That program involves conducting the research described above to
             address the immediate issue of the impact of the intercept fisheries. However, we would also
             recommend converting previously collected samples to a new format (abbreviated as "PCR")
             which would allow all subsequent genetic analyses to be made from material obtained by
             amplifying mtDNA from non-lethal biopsy tissue samples (an obvious benefit when genetic


                                                     14









               analysis is associated with tagging). Tissue samples could be archived and analyzed at any time in
               the future.


                      Shad populations are dynamic entities influenced by both natural and anthropogenic
               factors. Genetic analysis offers a means not just to estimate the percent composition of mixed
               assemblages of shad but also to monitor the success of stocks as they respond to ecological
               changes and to various management practices. Like tagging, annual collection of catch-effort
               statistics, and other management programs, a program of genetic population analysis requires long-
               term commitment at the very least to collect and archive samples. Most biological monitoring
               programs require a minimum of four years of sequential monitoring before any type of trend can be
               determined. After that, analyses can be performed annually or bi-annually to re-evaluate important
               groups of Chesapeake Bay and intercept shad.




































                                                       15









             SIGNIFICANT POINTS


             *Virginia's 1991 intercept fishery was comprised of at least 41 % Susquehanna shad
                (0.81 x (31s,, + 19sm,,, % A



             *This estimate should be considered preliminary for two important reasons. First, the
             Susquehanna River population does not appear to have attained genetic stability and it is possible
             that contribution estimates for this River will vary from year to year. Second, statistical analysis
             indicates that several of the baseline samples are very small and may not completely represent those
             groups of American shad.



             *Preliminary genetic analysis indicates that the Rudee Inlet harvest differs from harvests along
             Virginia's portion of the Delmarva Penninsula.



             *These findings should be verified by a joint NlD/VA investigation which would expand and
             enhance the current genetic baseline data set, identify specific river stocks within both states'
             intercept fisheries, and establish a long-term shad monitoring program (perhaps associated with
             joint tagging efforts).
























                                                      16









               REFERENCES




               Altukhov, Y.P. and E.A. Salmenkova. 1987. Stock transfer relative to natural organization,
                      management, and conservation of fish populations. Chapter 14 In: Population genetics and
                      fishery management. N. Ryman and F. Utter, eds. Univ. of Washington Press, Seattle.
               ASMFC. 1988. 1988 Supplement to American shad and river herrings fishery management plan.
                      Fisheries Management Rept. No. 12 of the Atlantic States Marine Fisheries Commission.
                      October 1988, Washington, DC.
               ASMFC. 1990. 1990 Shad and river herring workshop, October 16-17, 1990, Nags Head, NC.
                      Atlantic States Marine Fisheries Commission, Washington, DC.
               Avise, J.C., J.E. Neigel and J. Arnold. 1984. Demographic influences on mitochondrial DNA
                      lineage survivorship in animal populations. J. Mol. Evol. 20: 99-105.
               Backman, T. National Fisheries Research and Development Laboratory, USFWS, Wellsboro, PA.
               Bentzen, P., W.C. Leggett and G.G. Brown. 1988. Length and restriction site heteroplasmy in the
                      mitochondrial DNA of American shad (Alosa sapidissima). Genetics 118: 509-518.
               Bentzen, P., G.G. Brown and W. C. Leggett. 1989. Mitochondrial DNA polymorphism,
                      population structure, and life history variation in American shad (Alosa sapidissima). Can.
                      J. Aquat. Sci. 46: 1446-1454.
               Birky, C.W., T. Maruyama and P. Fuerst. 1983. An approach to population and evolutionary
                      genetic theory for genes in mitochondria and chloroplasts, and some results. Genetics 103:
                      513-527.
               Brown, B.L. and K.T. Paynter. 1991. Mitochondrial DNA analysis of native and selectively
                      inbred Chesapeake Bay oysters, Crassostrea virginica. Marine Biology (in press).
               CEC. 1989. Chesapeake Bay Alosid Management Plan. An agreement corrunitment report from
                      the Chesapeake Executive Council. July 1989, Annapolis, MD.
               Chapman, R.W. and B.L. Brown. 1990. Mitochondrial DNA isolation methods. Chapter 4 In:
                      Electrophoretic and isoelectric focusing techniques in fisheries management. D.H.
                      Whitmore, ed. CRC Press, Boca Raton, FL.
               Chapman, R.W. and B.L. Brown. 199 1. Genetic investigation of the resurgence of American
                      shad, Alosa sapidissima, in Susquehanna River. Report submitted to Maryland Dep. of
                      Natural Resources, Chesapeake Bay Research and Monitoring Division, Annapolis, MD.
               Chapman, R.W. and D.A. Powers. 1984. A method for rapid isolation of mitochondrial DNA
                      from fishes. Maryland Sea Grant Tech. Rept. No. UM-SG-TS-84-05.
               Crow, J.F. and M. Kimura. 1970. An introduction to population genetics theory. Harper and
                      Row. NY.







                                                     17









             Dadswell, M.J., G.D. Melvin and P.J. Williams. 1983. Effect of turbidity on the temporal arid
                     spatial utilization of the inner Bay of Fundy by American shad (Alosa sapidissinia)
                     (Pisces:Clupeidae) and its relationship to local fisheries. Can. J. Fish. and Aquat. Sci. 40
                     (Suppl. 1): 322-330.
             Fisher, R.A. 1958. The genetical theory of natural selection. Dover Press, NY.
             Flagg, L. 1990. Review of Atlantic States Marine Fisheries Commission Fishery Management
                     Plan for American shad and river herring. pp 76-82 In: Atlantic States Marine Fisheries
                     Commission 1990 shad and river herring workshop. Oct. 16-17, 1990, Nags Head,NC.
             Fournier, D.A., T.D. Beacham, B.E. Riddell and C.A. Busack. 1984. Estimating stock
                     composition in mixed stock fisheries using morphometric, meristic, and electrophoretic
                     characteristics. Can. J. Figh. and Aquat. Sci. 41: 400-408.
             Gibson, M., V. Crecco an dD. Stang. 1988. Stock assessment of American shad from selected
                     Atlantic coast rivers. Special Report No. 15 of the Atlantic States Marine Fisheries
                     Commission, Washington, DC.
             Gibson, M. Rhode Island Div. of Fish and Wildlife, W. Kingston, RI.
             Hebert, P.D., R.D. Ward and L.J. Weider. 1988. Clonal-diversity patterns and breeding-systern
                     variation in Daphnia pulex, an asexual-sexual complex. Evolution 42: 147-159.
             Jesien, R. and C. Hocutt. 1991. Movement of American shad in the Chesapeake Bay. Progress
                     Report to MDNR, Annapolis, MD.
             Leggett, W.C. and R.R. Whitney. 1972. Water temperature and the migration of American shad.
                     Fish. Bull. (US) 70:659-670.
             Melvin, G.D., J.D. Michael and J.D. Martin. 1985. Fidelity of American shad, Alosa sapidissima,
                     (Osteichthyes:Clupeidae) to its river of previous spawning. Can. J. Fish. and Aquat Sci.
                     42:1-19.
             Ne'i, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York.
             Pella, J.J. 1986. The method of fitting expectations applied to computation of conditional
                     maximum likelihood estimates of stock composition from genetic marks. US
                     DOC/NOAA/NMFS/NWAFC Auke Bay Laboratory, Auke Bay, AK. Rept. No. 171.
             Pella, J.J. and G.B. Milner. 1987. Use of genetic marks in stock composition analysis. Chapter
                     10 In: Population genetics and fishery management. N. Ryman and F. Utter, eds. Univ. of
                     Washington Press, Seattle.
             Roff, D.A. and P. Bentzen. 1989. The statistical analysis of mitochondrial DNA polymorphisms:
                     Chi-square and the problem of small samples. Mol. Biol. Evol. 6: 539-545.
             Shaklee, J. C. Busack, A. Marshall, M. Miller and S. Phelps. 1990. The electrophoretic analysis
                     of mixed-stock fisheries of Pacific salmon. pp. 235-265 In: (Z-1. Ogita and C. Marke,
                     eds.) Isozymes: Structure, Function, and Use in Biology and Medicine. Prog. in C-lin. and
                     Biol. Res. Vol. 344. Wiley-Liss, Inc. New York.
             Sokal, R.R. and F. J. Rohlf. 1981. Biometry. W.H. Freeman and Co., San Francisco.




                                                       18









              Stagg, C. 1986. An evaluation of the information available for managing Chesapeake Bay
                     fisheries: preliminary stock assessments, Vol. I and IL Univ. Maryland, CBL, UMCEES
                     [CBLI Ref. No. 85-29.1.
              Takahata, N. and T. Maruyama. 198 1. A mathematical model of extranuclear genes and the genetic
                     Nariability maintained in a finite population. Genet. Res., Camb. 37: 291-302.
              Walburg, C.H. and R.P. Nichols. 1967. Biology and management of the American shad and
                     status of the fisheries. Atlantic coast of the United States 1960. US Fish and Wildl. Ser.
                     Special Sci. Rept. Fish. No. 550.
              Wood, C.C., S. McKinnell, T.J. Mulligan and D.A. Fournier. 1987. Stock identification with the
                     maximum-likelihood mixture model: sensitivity analysis and application to complex
                     problems. Can J. Fish. Aquat. Sci., 44: 866-8 8 1.







































                                                     19








             ]Figure 1.   Map of the Chesapeake Bay region showing locations of American shad
                          samples taken by commercial fishermen in 1991.






























        I




















                                                           20

























                                                                                               C  D C'



                                                             &ALTIMORE






                                                                                   m


                                              WASHINGTON












                                                                                A



                                                                                                            DL











                                                                                                         @A?
                                                                                                                  Chincoteague



                                                                                                     j
                                                                                                           0 inby
                                                                                                             u



                                   'NICKMOND






                                                                                          Af


                                                                                             Rudee Inlet


                                      Scalc Of WdM
                                                 30                       NCiiFOLK
                                                  I




                                                                               21









              ]Figure 2.   Graphic depiction of restriction fragment patterns for American shad.














































                                                              22













                         Aat I                          Apa I                            Bcl I

                    A B C        D          A B C D           E    F G            A B C        D E      F

                                                                                                               Mtn


         5-                                                                                                       5
         4-                                                                                                       4

         3                                                                                                        3


         2-                                                                                                       2

         1.6-                                                                                                   -1.6








         0.5-                                                                                                   -0.5














                                   Bgl I                  Dra I                Eco R I

                            A B     D    E   F           A   B          A B      C D      E   F



                 5-                                                                                       5
                 4-                                                                                       4

                 3                                                                                        3


                 2                                                                                        2
                                                                                                                  It
                                                                                                       -1.6








                0.5-                                                                                   -0.5













                                Eco R V              Hin d III               Kpn I

                           A B C        D          A B      C         A B C        D E



                5-                                                                                   5
                4-                                                                                   4

                3                                                                                    3


                2-                                                                                   2

                1.6-                                                                              -1.6







                0.5-                                                                              -0.5
















                                   Pst I                     Pvu Id                   Sal I

                          A B       C   D   E    F         A   B C              A    B C      D


               5-                                                                                           5
               4-                                                                                           4

               3                                                                                            3


                                                                                                            2
               2-
               1.6-                                                                                      -1.6






               0.5-                                                                                       -0.5
















                                 Sma I               Sst 11                 Xba I


                              A B C               A    B   C           A    B C      D E



                                                                                                        5
                  5-
                  4-                                                                                    4

                  3                                                                                     3

                                                                                                                     rl-
                  2-                                                                                    2            r1l
                  1.6-                                                                               -1.6



                  1 -                                                                               - 1



                  0.5-                                                                               -0.5









              Figure 3.    Diagram of estimated contribution by baseline populations to Virginia's
                           1991 Atlantic Ocean harvest of American shad.












                                                                                           Unknown
                                                                                           Susquehanna
                                                                                           Santee

                                                                                           Connecticut
                                                                                           Hudson

                                                IX
                                                                                           Chowan
                                                                                           Parnunkey
                                                                                           St. Johns





























                                                             28











              Table 1. Summary, by river of origin, of American shad stocked into the Susquehanna River
                         during the period 1982-199L Shad from Columbia River, WA are an introduced
                         population derived from the Hudson River stock. Information provided by the
                         Susquehanna River Coordinator.


                  A.                              Hatchery cultured fry (millions)

                             Year       Pamunkey             James      Columbia         Delaware      Hudson

                             1991                -                                          3.212        8.845
                             1990           0.178                -              -           3.565        6.000
                             1989           0.754            0.220          12.422          1.645        5.660
                             1988           0.655            0.029           8.467          0.949            -
                             1987           1.403            0.040           6.919          1.227            -
                             1986           2.433            0.210          11.184          1.243            -
                             1985           2.222            0.458           1.906          1.642            -
                             1984           4.289            0.509           7.162          0.380            -
                             1983           1.000            1.100           1.950              -            -
                             1982           1.151            0.740           6.949




                   B.                             Live pre -spawned adults

                             Year                    Susquehanna          Hudson      Connecticut

                             1991                            22083              -               -
                             1990                            14792              -               -
                             1989                            6590               -               -
                             1988                            4730               -               -
                             1987                            6900            6032               -
                             1986                            4080            4965               -
                             1985                              950           3158               64
                             1984                                0           3592              185
                             1983                                0           3123             1187
                             1982                              875             992            1573










                                                                29








             Table 2.     Mitochondrial DNA haplotypes of American shad harvested outside Chesapeake
                          Bay in 1991. Haplotypes are created by listing single-enzyme genotypes.
                          The order of enzymes in a haplotype is as listed on p. 6. Genotype assignments
                          in the form "A/B" or "A/0 are heteroplasmic combinations of genotypes "A and
                          B" or "A and C", respectively.


                          Haplotype                                                        Site
                                                                       Rudee         Chincoteague         Quinby
                          AAAAAAAAAAAAAAA                                  2                16                14
                          AAAAAAAAAAAAAAA/B                                0                1                 0
                          AAAAAAAAAAAAAAB                                  0                1                 0
                          AAAAAAAAAAABAAA                                  0                1                 1
                          AAAAAAAAAABAAAA                                  0                1                 1
                          AAAAAAAAA/BAAA/BAAA                              0                0                 1
                          AAAAAAAABAAAAAA                                  1                4                 5
                          AAAAAAAABAAAAAB                                  1                0                 0
                          AAAAAAAABAAAABA                                  1                0                 0
                          AAAAAAAABAABAAA                                  1                7                 3
                          AAAAAAAABEAAAAA                                  1                0                 0
                          AAAAAAAACAAAAAA                                  0                0                 1
                          AAAAAABABAAAAAA                                  0                0                 1
                          AAAAAABABAABAAA                                  0                0                 1
                          AAAAAACAAAAA/CAAB                                1                0                 0
                          AAAAAACAAAACAAA                                  1                0                 0
                          AAAAAACABAAAAAA                                  1                0                 0
                          AAAAAACABAAAAAB                                  1                0                 0
                          AAAAAACABAAA/CAAB                                1                0                 0
                          AAAAABAAAAAAAAA                                  0                1                 0
                          AAAABAAAAAAAAAA                                  0                1                 0
                          AAAFAAAAAAAAAAA                                  1                0                 0
                          ABAAAAAAAAAAAAA                                  0                1                 0
                          AGAAAAAAAAABAAA                                  0                1                 0
                          BABAAAAAEAAAAAA                                  0                0                 1
                          DAAAAAAAAAAAAAA                                  0                5                 8
                          DAAAAAAAAABAAAA                                  0                0                 2
                          DAAAAAAABAAAAAA                                  0                0                 1
                          DAAAAAAABAABAAA                                  0                0                 1
                          DAAAAAA/CAAA/CAAAAA                              1                0                 0
                          DAAAAABAAAAAAAA                                  0                0                 1
                          DAAAAABAAAAA/CAAB                                1                0                 0


                                                Totals                    15                40               42







                                                           30








                   Table 3.    Chi-square comparisons for American shad in baseline populations calculated per Roff and Bentzen
                            (1989). The first entry in each cell is the observed chi-square value, the second entry is the largest chi-
                            square obtained by simulation, and the third entry is the probability that the observed value was due to
                            chance. Rivers are abbreviated as follows: S90-Susquehanna at conowingo Dam in 1990,
                            S91 -Susquehanna at Conowingo Dam in 1991, JAM-Jamcs, NAN-Nanticoke, SF-Susquehanna
                            Flats, PAM-Pamunkey, CHO-Chowan, DEL-Delaware, CT-Connecticut, SAV-Savannah,
                            STJ-St. Johns, HUD-Hudson, SAN-Santee, COL-Columbia. Data are from Chapman and
                            Brown (1991).



                            S91      JAM    NAN         SF   PAM      CHO      DEL       CT SAV            STJ    HUD      SAN COL

                   S90     59.92   87.29    58.34    34.29   69.98    31.90    60.33   40.76    103.02   70.33    69.98    62.64   116.44
                           39.17   32.17    29.20    38.83   35.20    34.54    23.38   30.79    31.32    39.48    27.67    31.08   27.76
                           0.000   0.000    0.000    0.001   0.000    0.001    0.000   0.000    0.000    0.000    0.000    0.000   0.000

                   S91      --     39.06    35.55    11.07   30.70    16.08    31.89   18.52    51.62    34.33    34.43    30.60   67.05
                                   29.40    30.46    25.43   23.18    23.38    29.76   21.31    29.40    25.75    33.82    26.26   28.58
                                   0.000    0.000    0.362   0.000    0.052    0.000   0.003    0.000    0.000    0.000    0.000   0.000

                   JAM                      49.88    27.59   49.13    29.72    57.50   33.55    56.85    40.92    54.69    50.24   83.01
                                            30.81    25.93   26.14    25.72    28.56   20.20    31.65    24.43    31.07    30.16   35.61
                                            0.000    0.000   0.000    0.000    0.000   0.000    0.000    0.000    0.000    0.000   0.000

                   NAN                         --    19.53   37.36    23.62    52.13   38.00    60.18    38.98    47.92    48.21   69.06
                                                     21.85   26.68    21.88    30.90   20.78    39.00    29.06    29.78    29.59   29.68
                                                     0.004   0.000    0.000    0.000   0.000    0.000    0.000    0.000    0.000   0.000

                   SF                                  --    16.51      8.57   17.38   10.47    39.11    22.64    18.78    15.94   56.12
                                                             22.70    11.24    27.64   17.32    32.74    20.39    25.19    23.50   23.40
                                                             0.013    0.038    0.089   0.024    0.000    0.000    0.000    0.047   0.000

                   PAM                                                22.05    51.72   32.20    62.72    43.32    45.98    45.13   76.20
                                                                      19.75    26.79   21.83    33.09    22.31    31.23    24.64   30.21
                                                                      0.000    0.000   0.000    0.000    0.000    0.000    0.000   0.000

                   CHO                                                   --    16.36   10.69    43.46    21.34    22.30    15.58   56.98
                                                                               34.73   15.01    28.42    19.34    31.36    22.68   27.59
                                                                               0.117   0.020    0.000    0.000    0.009    0.028   0.000

                   DEL                                                           --    12.52    68.90    42.12    41.17    31.94   94.22
                                                                                       25.02    30.10    27.41    35.39    28.86   32.98
                                                                                       0.297    0.000    0.000    0.000    0.000   0.000

                   CT                                                                     --    48.83    26.82    22.05    18.60   69.00
                                                                                                24.77    17.84    21.35    20.85   22.72
                                                                                                0.000    0.000    0.000    0.004   0.000

                   SAV                                                                             --    51.61    69.79    63.48   90.50
                                                                                                         28.11    36.63    30.34   28.43
                                                                                                         0.000    0.000    0.000   0.000

                   STJ                                                                                      --    45.98    42.44   71.00
                                                                                                                  31.28    24.71   24.02
                                                                                                                  0.000    0.000   0.000

                   HUD                                                                                              --     36.22   79.56
                                                                                                                           27.82   26.06
                                                                                                                           0.000   0.000

                   SAN                                                                                                       --    83.00
                                                                                                                                   28.06
                                                                                                                                   0.000


                                                                              31









             Table 4.  Results of a typical maximum likelihood analysis of simulated mixture populations.
                       Tests were conducted to determine ability of the GIRLSEM algorithm toestiniate
                       stock composition of a mixed sample from shad mtDNA data. Mixture populations
                       of known composition were created by randomly sampling twelve baseline samples
                       with replacement. Final sample size for the analysis shown was 96. Data are taken
                       from Chapman and Brown (1991) which included Columbia River, WA (denoted
                       COL) as one of the baseline samples.



                       Source                     Contribution                         Ho:A=E         alpha
                                                 Actual        Estim.        SE            z           0.05


                       1 .COL                    0.066         0.067        0.036         0.016       accept
                       2. CT                     0.066         0.000        0.025       -2.604         reject
                       3. DEL                    0.104         0.359        0.058         4.405        reject
                       4. HUD                    0.142         0.149        0.051         0.156       accept
                       5. SF                     0.028         0.000        0.017       -1.672        accept
                       6. NAN                    0.066         0.074        0.037         0.222       accept
                       7. PAM                    0.076         0.066        0.037       -0.243        accept
                       8. JAM                    0.113         0.059        0.040       -1.353        accept
                       9. CHO                    0.047         0.000        0.022       -2.181         reject
                       10. SAN                   0.113         0.082        0.043       -0.718        accept
                       11. SAV                   0.094         0.074        0.040       -0.500        accept
                       12. STJ                   0.085         0.068        0.038       -0.428        accept























                                                                   32







            Table 5. Estimated stock composition of three groups of American shad. A) Virginia's intercept
                     fishery and B) Susquehanna River in 1990 and in 1991. Abbreviations are as listed
                     in Table 3.



                  A. Composition estimates for 81% of Virginia's 1991 intercept fishery. Nineteen percent of
                     intercept shad could not be characterized by the current baseline data. Columbia River
                     was not included in the ma3dmum likelihood analysis of Virginia's fishery because that
                     stock would not be represented. Fish of Columbia River descent are allocated by the
                     analysis to contributions by Susquehanna and Hudson Rivers.

                                    Source          Contribution       SD


                                    CT                  0.32           0.11
                                    DEL                 0.00           0.00
                                    HUD                 0.09           0.05
                                    SF                  0.00           0.00
                                    NAN                 0.00           0.00
                                    PAM                 0.02           0.02
                                    JAM                 0.00           0.00
                                    CHO                 0.05           0.05
                                    SAN                 0.19           0.12
                                    SAV                 0.00           0.00
                                    STJ                 0.02           0.02
                                    SUS                 0.31           0.10


                  B. Estimated composition of Susquehanna River shad samples collected in 1990 and
                     1991. Forty-eight percent of shad taken in 1990 and 31% of shad in 1991 were
                     unique to the Susquehanna system. Composition estimates are given for the
                     remaining 52% and 69%, respectively. The Columbia River shad stock (denoted COL)
                     was included in the analysis of Susquehanna shad because that stock has been
                     introduced into the Susquehanna system.

                                                          1 1990                             1991
                                    Source        Contribution         SD         Contribution      SD


                                    COL                 0.01           0.01             0.04        0.04
                                    CT                  0.03           0.06             0.50        0.13
                                    DEL                 0.00           0.00             0.00        0.00
                                    HUD                 0.00           0.00             0.00        0.00
                                    SF                  0.03           0.08             0.02        0.10
                                    NAN                 0.16           0.06             0.00        0.00
                                    PAM                 0.03           0.03             0.06        0.06
                                    JAM                 0.00           0.00             0.06        0.06
                                    CHO                 0.12           0.10             0.12        0.10
                                    SAN                 0.14           0.07             0.00        0.00
                                    SAV                 0.00           0.00             0.00        0.00
                                    STJ                 0.00           0.00             0.00        0.00


                                                               33








        Appendix A. Genotypes of American shad taken in the coastal intercept fishery outside
                Chesapeake Bay during the Spring of 1991. Enzyme titles are abbreviated
                versions of those listed on p. 6. Genotypes given with a "P represent heteroplasmic
                combinations of both genotypes listed. A dash "-" indicates incomplete data for
                determining that fish's genotype.

                                    Enzyme Genotype
          Location   ID  Aat Apa Bcl l3gl Dra Ed EcV Hin Kpn Pst Pvu Sal Sma Sst Xba

          Rudee Inlet 1  D A A A  A A B A  A A A  A/CAA B
                      2  D A A A  A A AICA A AICA A A A A
                      3    B A A  A A A A  A B A  A - - A
                      4    A A -  A A A A  - A A  - - - -
                      5  A A A F  A A A A  A A A  A A A A
                      6    B   A    A   -  A B -  B A A B
                      7    A        A   A  A - A  - - - A
                                  A A A    A A A  - - - A
                           A   A    A      A - - - - -  A
                     10      A      A      A - A  A - - -
                     11  A A A A  A A A A  A A A  A A A A
                     12  - A A A  - A - A  A A A  A
                     13  - - - - -  A - A  A A A  A - - -
                     14  A - A -  A A - A  A A A  A   - A
                     15  A - A A  A A A A  - A A  -   A A
                     16  A A A A  A A A A  B A A  B A A A
                     17  - A A A  - A - A  A A A  A - A A
                     18  A A A A  A A A A  B A A  A A A A
                     19  - A A -  - A - -  A A A  A   A A
                     20  - - - - -  A - -  A A A
                     21  - - - - -  A - - - - - - - - -
                     22  - A A A  - A - A  A A A  A - A A
                     23  - - - A  - A - A  A - - - - -  A
                     24  - A - A  - B - A  A - - - - -  A
                     25  A A A A  A A C A  A A A  C A A A
                     26  A A - A  A - - -  A - - - - -  A
                     27  A - - A  A A - -  A A A  - - - -
                     28  A - - A  A A - A  A - - - - -  A
                     29  A A A A  A A C A  A A A  A/CAA B
                     30  A - - A  A A - -  A - - - - -  A
                     31  D - A A  A A - -  A A -  A - - A
                     32  A A A A  A A A A  B A A  A A B A
                     33  - - - - -  A - - - - - - - - -
                     34  A A A A  A A C A  B A A  A A A A
                     35  A - - A  A A - -  A - - - - -  A
                     36  A A A A  A - C A  B A A  AJC-A B
                     37  A A A A  A A - -  A A A  A - - A
                     38  A - - A  A - - A  A A A  - - - A
                     39  - A - A  A A - A  B A A  A - A -
                     40  A A A A  A A C A  B A A  A A A B
                     41  A A A A  A A A A  B E A  A A A A
                     42  A A A A  A - A A  B A A  A - A B
                     43  - A A A  A - A A  B A A  B - A -
                     44  - A A A  A A/BAA  B A A  A - A -
                     45  A A A A  A A A A  B A A  A A A B
                     46  - - - - - - -  A  - - - - - - -
                     47  - A A A  - - A A  B - A  A
                     48  - - - A  - - - A  - - - - - - -
                     49  A - A A  A - A -  A A - - -  A -
                     50  - - - - - - - - - - - - - - -
                     51  - - - - - - - - - - - - - - -
                     52  A A A A  A A A A  A A A  A A A A




                                  34








        Appendix A. Continued

                                    Enzyme Genotype
        Location     ID Aat Apa Bcl Bgl Dra Ed EcV Hin Kpn Pst Pvu Sal Sma Sst Xba

        Chincoteague  I A  A A A B  A A A A  A A A A  A A
                      2 D  A A A A  A A A A  A A A A  A A
                      3 A  A A A A  A A A A  A A A A  A A
                      4 D  A A A A  A A A A  A A A A  A A
                      5 A  A A A A  A A A A  A A B A  A A
                      6 A  A A A A  A A A B  A A A A  A A
                      7 D  A A A A  A A A A  A A A A  A A
                      8      A A A  A A A    A A A A  A
                      9      A A A  A A A    A A A A  A
                     10    A A A A  A A A    A - B - -A
                     11 A  A A A A  A A A A  A A A A  A A
                     12 A  A A A A  A A A A  A A A A  A A/B
                     13 A  A A A A  A A A A  A A A A  A A
                     14 A  A A A A  A A A A  A A A A  A A
                     15 A  A A A A  A A A A  A A A A  A B
                     16 -  - A A A  A A A -  A A A -  A -
                     17 D  A A A A  A A A A  A A A A  A A
                     18 -  A A A A  A A A A  A - - -  A -
                     19 -  A A A A  A A A A  A - - A  A -
                     20 D  A A A A  A A A A  A A A A  A A
                     21 A  A A A A  A A A A  A A A A  A A
                     22 A  A A A A  A A A A  A A A A  A A
                     23 A  A A A A  A A A A  A A A A  A A
                     24 A  A A A A  A A A A  A B A A  A A
                     25 A  A A A A  A A A A  A A A A  A A
                     26 A  A A A A  A A A A  A A A A  A A
                     27 A  A A A A  A A A A  A A A A  A A
                     28 A  A A A A  B A A A  A A A A  A A
                     29 A  A A A A  A A A A  A A A A  A A
                     30 A  A A A A  - - - A  - A A A  A -
                     31 -  - A - - - - - - - - - - - -
                     32 -  A A - - - - - - -   A A -  A -
                     33 -  A A - - - - - - -   A B -  A -
                     34 A  G A A A  A A A A  A A B A  A A
                     35 A  B A A A  A A A A  A A A A  A A
                     36 A  A A A A  A A A A  A A A A  A A
                     37 A  A A A A  A A A B  A A A A  A A
                     38 A  A A A A  A A A B  A A A A  A A
                     39 - - - - - - - -   A  - A A - - -
                     40 -  - A - - - - - - -   A A -  A -
                     41 A  A A A A  - - - B  A A B A  A A
                     42 A  A A A A  A A A A  A A A A  A A
                     43 A  A A A A  A A A B  A A B A  A A
                     44 A  A A A A  A A A B  A A B A  A A
                     45 A  A A A A  A A A B  A A B A  A A
                     46 A  A A A A  A A A A  A A A A  A A
                     47 A  A A A A  A A A B  A A A A  A A
                     48 A  A A A A  A A A B  A A B A  A A
                     49 A  A A A A  A A A B  A A B A  A A
                     50 A  A A A A  A A A A  A A A A  A A
                     51 A  A A A A  A A A B  A A B A  A A
                     52 A  A A A A  A A A B  A A B A  A A
                     53 A  A A A A  A A A A  A A A A  A A
                     54 -  A A A A  - A A -  A - A A  A -









                                  35








        Appendix A. Continued


                                    Enzyme Genotype
        Location      ID Aat Apa BcI Bgl Dra Ed EcV Hin Kpn Pst Pvu Sal Sma Sst Xba

        Quinby        1  A A A  A A A A  A A A A  A A A  A
                      2    A               A - A  A
                      3    A A  A - - -  A A A A  A A A  A
                      4    A - - - - - - - - - - - - -
                      5  A A A  A A A A  A B A A  B A A  A
                      6  - - - - - - -   A B A A  A A A  -
                      7  A A A  A A A B  A B A A  A A A  A
                      8  A A - - - - -   A A A -  A A A  A
                      9  - - - - - - - - - - - - - - -
                     10  A A A  A A A A  A A/BAA  A/13AA A
                     11  A A A  A A A A  A B A A  B A A  A
                     12  A A A  A A A B  A B A A  B A A  A
                     13  - - - - - - - - - -- - - - - -
                     14  - - - - - - - - - -- -   A - - -
                     15  - A - - -  A - - - - -   A -    A
                     16  - - - - - - - - -   A
                     17  A A A  A A A A  A A A A  A A A  A
                     18  A A A  A A A A  A B A A  A A A  A
                     19  D A A  A A A A  A A A B  A A A  A
                     20  - A - - - - -   A A A A  A A A  A
                     21  - A A  A A - - -  A A A  A - -  A
                     22  A A A  A A - A  A C A A  A A A  A
                     23  A A A  A A A A  A B A A  A A A  A
                     24  D A A  A A A A  A A A A  A A A  A
                     25  D A A  A A A A  A A A A  A A A  A
                     26  D A A  A A A A  A B A A  B A A  A
                     27  D A A  A A A B  A A A A  A A A  A
                     28  D A A  A A A A  A A A B  A A A  A
                     29  A A A  A A A A  A A A A  A A A  A
                     30  D A A  A A A A  A B A A  A A A  A
                     31  A A A  A A A A  A B A A  A A A  A
                     32  D A A  A A A A  A A A A  A A A  A
                     33  A A A  A A - A  A A A A  A - A  -
                     34  D A A  A A A A  A A A A  A A A  A
                     35  D A A  A A A A  A A A A  A A A  A
                     36  - - -  A A - -  A - A A  A A A  A
                     37  D A A  A A A A  A A A A  A A A  A
                     38  D A A  A A A A  A A A A  A A A  A
                     39  D A A  A A A A  A A A A  A A A  A
                     40  A - A  A - A A  - - A A  A - A  -
                     41  A A A  A A A A  A A A A  A A A  A
                     42  A A A  A A A A  A A A A  A A A  A
                     43  A A A  A A A A  A B A A  B A A  A
                     44  A A A  A A A A  A B A A  A A A  A
                     45  B A B  A A A A  A E A A  A A A  A
                     46  A A A  A A A A  A A A A  A A A  A
                     47  A A A  A A A A  A A A A  B A A  A
                     48  A A A  A A A A  A A A A  A A A  A
                     49  A A A  A A A A  A A A A  A A A  A
                     50  A A A  A A A A  A A A B  A A A  A
                     51  A A A  A A A A  A A A A  A A A  A
                     52  A A A  A A A A  A B A A  A A A  A
                     53  A A A  A A A A  A A A A  A A A  A
                     54  A A A  A A A A  A A A A  A A A  A
                     55  A A A  A A A A  A A A A  A A A  A
                     56  A A A  A A A A  A A A A  A A A  A




                                  36









             Appendix B. Distribution of mtDNA haplotypes in intercept and baseline American shad
                         population samples. Abbreviations for the intercept samples are Rudee Inlet- R,
                         Chincoteague-C, andQuinby-Q. Other abbreviations areas listed in Table 3.


                                                                  Population
             Haplotypes               R   C 0 S90 S91 SF COL CT DEL HUD NAN PAM JAM CHO SAN SAV STJ

             I AAAAAAAAAAAAAAA        2   16  14  17    6      15 22    14  3   5   5   9  14   3   6
             2 AAAAAAAAAAAAAAB            1      -   1
             3 AAAAAAAAAAAAAAA/B          1
             4 AAAAAAAAAAAAAAD                                                              3
             5 AAAAAAAAAAAAABA                   3                  1       1
             6 AAAAAAAAAAAAABA/B                 1
             7 AAAAAAAAAAAAABD                                              1
             8 AAAAAAAAAAAABAA                                              I       1           1   3
             9 AAAAAAAAAAAABBA                                                      3           9
             10 AAAAAAAAAAAACAA                                                                 1
             11 AAAAAAAAAAABAAA           1  1   2                  2       1           1   4
             12 AAAAAAAAAAABABA
             13 AAAAAAAAAAACBBA                                                                 1
             14 AAAAAAAAAAADAAA                                                                 1
             15 AAAAAAAAAABAAAA           1  1                      1   4
             16 AAAAAAAAAABAABA                                                     1
             17 AAAAAAAAAABABBA                                                                 1
             18 AAAAAAAAAA/BAAAAA                1
             19 AAAAAAAAAA/CAAAAA                1
             20 AAAAAAAAAA/CABAAA/B              1
             21 AAAAAAAAAC.AAAAA                                    I
             22 AAAAAAAAACAAAAA                                                     8               1
             23 AAAAAAAAADAAAAA                                  1
             24 AAAAAAAAAEAAAAA                                                                     1
             25 AAAAAAAAAFAAAAA                                                             1
             26 AAAAAAAAA/BAAA/BAAA          1
             27 AAAAAAAABAAAAAA       1   4  5   5               6  8   2       1   3       2   2   2
             28 AAAAAAAA13AAAAAB      1                                         2   1   2
             29 AAAAAAAABAAAABA       1              3
             30 AAAAAAAABAAABBA                                     1                           6
             31 AAAAAAAABAAACBA                                                                 I
             32 AAAAAAAABAABAAA       1   7  3       2  2        2  2   5       2   2       7   1
             33 AAAAAAAABAABABA                      1                      1
             34 AAAAAAAABAABACA                                                             1
             35 AAAAAAAABAABBAA                                                                     1
             36 AAAAAAAABAABBBA                                     1                           1
             37 AAAAAAAABCAAAAA                                                     1
             38 AAAAAAAABCAABBA                                                     1
             39 AAAAAAAA13CBBAAA                                                    1
             40 AAAAAAAABCIAABAAA                                                   1
             41 AAAAAAAABFAABBA                                                                 1
             42 AAAAAAAACAABAAA                                                     1
             43 AAAAAAAACAABBBA                                                                 1
             44 AAAAAAAADAAAAAA                                     I
             4.5 AAAAAAAADAAAABA                                            1
             46 AAAAAAAAECAAAAA                  1
             47 AAAAAAABAAAAABA                                         1
             48 AAAAAAABAAAABBA                                                                 1
             49 AAAAAAABEAAAAAA       1          1
             50 AAAAAAACAAAAAAA              1                          1
             51 AAAAAAA/CAAAAABAA/B              1
             52 AAAAAAA/CABAAAAAA                1
             53 AAAAAAA/CABEAAAAA                1



                                                           37








           Appendix B. Continued
                                                                    Population
              Haplotypes               R  C   Q S90 S91 SF COL CT DEL HUD NAN PAM JAM CHO SAN SAV STJ


             5.4 AAAAAABAAAAAAAA                  3                      4    3   1
             5.5 AAAAAABAAAABAAB                                              1
             515 AAAAAABAAACAAAA                                         2
             57 AAAAAABAAA/CAAABA                 1
             5.3 AAAAAABABAAAAAA              1   1
             59 AAAAAABABAABAAA               1
             60 AAAAAACAAAAAAAA                   4      2           1        4   3
             61 AAAAAACAAAAAAAA/B                 I
             6Z AAAAAACAAAAAAAD                   1
             63 AAAAAACAAAAABBA                                               1
             64 AAAAAACAAAABAAA                                                   1
             65 AAAAAACAAAACAAA        I
             66 AAAAAACAAAAA/CAAB      1
             67 AAAAAACABAAAAAA        1                                          2
             68 AAAAAACA.BAAAAAB       1
             69 AAAAAACABAAAABA                                                   1
             70 AAAAAACABAABAAA                                                   2
             71 AAAAAACABAABAAA                                                   1
             72 AAAAAACACAABAAA                                                   1
             73 AAAAAACABAAA/CAAB      1
             74 AAAAAACBAAAAAAA
             75 AAAAAACBAAAAABD                   1
             76 AAAAAACBAAAABBA                                               1
             77 AAAAAACBAAACBBA                                               1
             78 AAAAAACBACAAA33A
             79 AAAAAACBBAAAABA                                                                  1
             80 AAAAAACBBAAC/BBBA                                             1
             81 AAAAAACBDAAAAAA                                               1
             82 AAAAAACCAAAAAAA                                                      1
             83 AAAAAACCAAAAA13A                  1
             84 AAAAAACCAAAABBA                                               1
             85 AAAAAACCCAACBBA                                               1
             86 AAAAAADBBAAAABA
             8 7AAAAABAAAAAAAAA           1                                                         2
             88 AAAAABAAAA/CAAAAA                 1
             89 AAAAABAABAAAAAA
             90 AAAABAAAAAAAAAA           1
             91 AAAABBAAAAAAAAA
             92 AAAAIEAAAAAAAAAAA                            26          2
             93 AAAA/EAAAABAAAAAA                                        3
             94 AAAA/EAA13AAAAAAAA                            3
             95 AAAA/EAACAAAAAAAA                             5
             96 AAAAAIEACAAAAAAAA                 1
             97 AAABAAAAAAAAAAA
             98 AAABAAAAAAAABAA
             99 AAABAACAAAAAAAA
            100 AAACAAAAAAABBBA
            101 AAACAAAABAAABBA
            102 AAAEAAAAAAAAAAA
            103 AAAEAACCABAAAAA
            104 AAAFAAAAAAAAAAA        1
            105 AABAAAAABAAAAAA
            106 AACAAAAAAAAAAAA
            107 AACAAAAAAAJBAAAA
            108 AADAAAA-AA.@@                                                               2
            109 AADAAAAABAAAABA
            110 AADAAAAAAAAABBA
            111 AADAAAAACCABAAC




                                                             38









             Appendix B. Continued
                                                                                     Population
                 Haplotypes                     R     C     Q S90 S91 SF COL CT DEL HUD NAN PAM JAM CHO SAN SAV STJ


               112  AAEAAAAAAAAAAAA                                                                                     1    1
               113  AAFAAAAAACABBAA                                                                                          1
               114  ABAAAAAAAAAAAAA                   1
               115  ABAAAAAAAAAABAA                                                       2
               116  ABAAAAAAAAAABAA                                                                                               3
               117  ABAAAAAAAAACAAA                                                       1
               118  ABAAAAAAACAAAAA                                                                          2
               119  ABAAAAAABBAAABA                                  1
               120  ABAAAACAAABAAAA                                                                      1
               121  ABAAAACABBAAAAA                                                                      1
               122  ABAAABAAAAAABAA                                                                                               1
               123  ABACAAAAACAAAAA                                                       1
               124  ACAAAAAAAAAAAAA                                                                                          1    3
               125  ACAAAAAAAAAABAA                                                                                               1
               126  ACAAAAAAACAAAAA                                                                                          2
               127  ACABAAAAAAAACBA                                                                                          2
               128  ADDAAACBAAAAAAA                                                                      1
               129  AEAAAAAAAAAAAAA                                                                      1
               130  AEAAAACAAAAAAAA                                                                      1
               131  AEAAAACBBBAAAAA                                                                      1
               132  AA/EAAAACBAAAABBA                                1
               133  AFAAAAAAAA/BABAAA                           1
               134  AGAAAAAAAAABAAA                   1
               135  BAAAAABAAAAAAAA/D                           1
               136  BAAAAACCAAAAAAA                             1
               137  BABAAAAAEAAAAAA                         1
               138  BBAAAAAAAEAABAA                                                                                               1
               139  BBAABACABAAAABA                             1
               140  CAAAAAAABBABAAA                             1
               141  CAAAAAAACAAABBA                                                                                          1
               142  CAAAAAABBA/CAAABA                           1
               143  CAAAAACAAAAAAAA                             1                                        1
               144  CAAAAAA/CAAAAAAAA                           2
               145  CAAAAACAAAAAAAA/B                           1
               146  CAAAAACBAAAABBA                                                                                          1
               147  CAAAAACBAAABBBA                                                                                          1
               148  CAAAAACBAA/CAAAAA                           1
               149  CAAAABCBAAAAAAA                                                                                               1
               150  DAAAAAAAAAAAAAA                   5     8   4                                                       1
               151  DAAAAAAAAAAABAA                                                                                          1
               152  DAAAAAAABAABAAA                                       1
               153  DAAAAAAAAABAAAA                         2   1
               154  DAAAAAAAAA/CAAAAA                           1
               155  DAAAAAAAABAAAAA                             1
               156  DAAAAAAABAAAAAA                         1   1
               157  DAAAAAAABAABAAA                         1   1
               158  DAAAAAA/CBAAAAABA                           1
               159  DAAAAAA/CAAAICAAAAA 1
               160  DAAAAABAAAAAAAA                         1
               161  DAAAAABAAAAA/CAAB            1
               162  DAAAAACAAAAAAAA                             I



                    Totals                      15    40    42  75  10   11   36   24    48  39   24    31  38    12    38   45   26
                    Proportion Unique Individuals-    -     - 0.48 0.31 0.09 0.27 0.04 0.16 0.18 0.41 0.13 0.36 0.00 0.21 0.44 0.35






                                                                             39





                                                                                                          APPendix 17





                                                 ENETIC


                                                 ANALYSIS OF


                                                   --M
                              ........                ERICAN SHAD

                                                 tf4TERING


                                                      E S A P E A K E
                                ..               .......
                               ...               .......


                                                   :.Ay





                                          .... ...... ..



           Bonnie L. Brown, PhD                  A REPORT SUBMITTED TO THE

                                                                                                                q
           Robert W. Chopmon, PhD                CHESAPEAKE BAY COMMISSION





                               . . . . . .. . . . . . . . . .
                              . . . . . . . . . . . . . . . . . . . . . . . . . .



                                                                               fA
                       .:: . . . . . . . . . . . . .
                       . . . . . . . . . . .




                               .........


                                                                            TT




                                                                 4, j4
                    .. ............. ...........
                       . .. ........
                                                                              fka
                                                                        al@A









                                         0       BY CHESAPEAKE SCIENTIFIC

                                                 INVESTIGATIONS FOUNDATION, INC.
                                                                                                411









































                                                 SEPTEMBER, 1991








                                             GENETIC ANALYSIS OF AMERICAN SHAD
                                                        ENTERING CHESAPEAKE BAY







                      Principal Investigators:



                                         Bonnie L. Brown, Ph.D.                                               Robert W. Chapman, Ph.D.
                                         Department of Biology                                                Department of Biology
                                         Virginia Commonwealth University                                     East Carolina University
                                         Richmond, VA 23284-2012                                              Greenville, NC 27858-4353








                                                                      A report submitted to:

                                                              The Chesapeake Bay Commission
                                                                          September, 1991




                                                                                   By:

                                                 Chesapeake Scientific Investigations Foundation, Inc.













                      This contracted research was funded by the Pennsylvania Delegation to the Chesapeake Bay Commission, the Virginia Council on the
                      Environment (COE) and the Virginia Marine Resources Commission. COE funding was provided through the Coastal Resources
                      Management Program through grant # NA90AA-H-=96 of the National Oceanic and Atmospheric Administration under the Coastal Zone
                      Management Act of 1972 as amended.









               ACKNOWLEDGEMENTS



                      This study was conducted for the Chesapeake Bay Commission. Funds were
               provided by the Chesapeake Bay Commission, the Virginia Council on the Environment
               (Coastal Zone Management Grant # NA90AA-H-CZ796), and the Virginia Marine
               Resources Commission (in-kind services) to perform genetic analysis of American shad,
               Alosa sapidissima, harvested by Virginia watermen from waters outside Chesapeake Bay in
               Virginia@s ocean gill net fishery. Genetic analysis was employed to ascertain, to the maximum
               extent possible, the natal stream and spawning destination of shad which are susceptible to
               harvest in the Atlantic Ocean intercept fishery. Genotypes were compared to shad from ten
               baseline target rivers and from Susquehanna River previously analyzed under MDNR
               contract number PR90-004-004. Inclusion of Albemarle Sound, Cape Fear and Santee River
               baseline populations was made possible by a supplemental grant from Atlantic States Marine
               Fisheries Commission's Interstate Fisheries Management Program (ISFMP) grant number
               91-1(PASRH). We thank J. Pella for editing an earlier version of this report and for providing
               the GIRLSEM algorithm which we used to analyze the genetic data produced by this study.
               We also thank Barney Jernigan for preparing other essential computer programs and for his
               assistance with data analysis. A great many other people also contributed to the study by
               providing biological specimens of American shad including but not limited to:

                      Walt Ambrogetti, USFWS
                      Tom Backman, USFWS
                      Bob Brandt, NY State Dept. of Environmental Conservation
                      Rick Eager, USFWS
                      Chris Frese, RMC Environmental Services
                      Joe Loesch, Virginia Inst. of Marine Science
                      Art Lupine, NJ Freshwater Fisheries Lab
                      Ted Meyers, USFWS
                      Roy Miller, DE Div. of Fisheries & Wildlife
                      Jim Owens, Virginia Inst. of Marine Science
                      Fritz Rhode, NC Div. of Marine Fisheries
                      Dick St. Pierre, USFWS
                      Mark Tisa, MA Div. Fisheries & Wildlife
                      Glenn Ulrich, SC Wildlife & Marine Resources Div.
                      Dale Weinrich, MD Dept. of Natural Resources
                      Sara Winslow, NC Div. of Marine Fisheries









                                                           2








                EXECUrfVE SUMMARY


                       Genetic analysis was employed to determine the stock composition of American shad,
                Alosa sapidissima, harvested outside Chesapeake Bay in Virginia!s Atlantic Ocean intercept
                fishery during the Spring of 1991. Genotypes of intercept fish were compared to fish from
                fourteen American shad populations in order to estimate the relative percentage ofshad from
                each baseline population in the intercept sample. Techniques involved restriction
                endonuclease digestion of mitochondrial DNA (mtDNA) purified from shad egg fissue, a
                common methodology used for examining population dynamics in fishes. MtDNA genotypes
                were obtained for 158 individuals from three locations near the mouth of Chesapeake Bay:
                Rudee Inlet, Chincoteague, and Quinby, VA. The fourteen baseline populations included 585
                American shad from spawning aggregations in eleven target rivers (Columbia, Connecticut,
                Delaware, Hudson, Nanticoke, Pamunkey, James, Chowan, Savannah, St. Johns and Santee
                Rivers), from the Susquehanna Flats in upper Chesapeake Bay, and from Susquehanna River
                shad lifted over Conowingo Dam in 1990 and 1991.

                       Since many shad have mtDNA genotypes which are common to several if not all
                drainages, every fish cannot be uniquely identified to a river system. Therefore, when a
                mixed assemblage of fish is examined (such as an ocean fishery), genotypes of the entire
                group are statistically analyzed for comparison to baseline genetic data for potential source
                populations. The analysis provides an estimate of the most likely composition of the group in
                question. The standard statistical treatment which has been developed to interpret mixed-
                fishery genetic data was created by Dr. J. Pella and co-workers for west coast salmon and
                has been successfully employed to manage that fishery for many years.

                       Most baseline American shad populations share one common mtDNA type, but each
                contains unique types as well. These unique types of mtDNA made it possible to estimate
                the percentage of each baseline stock represented in the migrating coastal group of shad.
                Nineteen percent of American shad sampled outside Chesapeake Bay had unique mtDNAs
                themselves and could not be classified as originating from any of our current baseline
                populations. The remaining 81% of intercept shad were compared to the baseline populations
                in a maximum likelihood analysis of stock contribution. Composition of this portion of the
                intercept group was estimated to be due to contributions of the following six stocks: James
                (1+13 %), St. Johns (4+4 %), Pamunkey (7+6 %), Santee (7+14 %), Hudson (15+9 %), and
                Susquehanna (65+12 %). No contribution was detected from the other baseline shad
                populations. Thus, we estimate that Virginia's 1991 intercept fishery was comprised of
                approximately 53% Susquehanna shad (0.81 X 65%); perhaps a great deal more if the Hudson
                and Santee contributions are due to presence of Susquehanna Flats fish in the intercept
                sample.

                       These estimates should be considered preliminary for two important reasons. First,
                the Susquehanna Flats baseline sample is very small and may not adequately represent that
                group of American shad. Contributions attributed by genetic analysis to Hudson and Santee


                                                            3









               Rivers may be due in part to the presence of Susquehanna Flats shad which appear to be of
               Hudson and Santee River descent. However, the composition of Susquehanna Flats shad is
               not yet known with certainty due to small sample size from that area. Second, the
               Susquehanna River population does not appear to have attained genetic stability and it is
               possible that contribution estimates for this River will vary from year to year as the resurgent
               Susquehanna population approaches a stable genetic equilibrium.

                     Despite small sample sizes, a trend is evident in the genetic composition of shad
               harvested north of the Bay mouth (Chincoteague and Quinby) and those harvested south of
               the Bay mouth (Rudee). Seventy percent of Rudee Inlet shad were of Virginia origin while
               the Chincoteague and Quinby harvests were composed primarily of Susquehanna, Hudson
               and Santee shad.


                      To consider the potential effect of the coastal intercept fishery we would need several
               years of estimates of the stock composition and magnitude of both Maryland and Virginia@s
               coastal harvests. However, a rough estimate can be made based on Virginia!s preliminary
               estimate of their 1991 ocean shad harvest and the present genetic data. The estimated
               amount of shad harvested in 1991 was 405,612 pounds. Multiplying by a factor of 0.53,
               approximately 215,000 pounds were of Susquehanna origin. This is roughly twice the
               poundage of shad lifted over Conowingo Dam in the Spring of 1991. If these findings are
               verified by future monitoring, tagging and genetic evaluation then the most conservative
               action would be to restrict shad harvests along the Delmarva Peninsula.

























                                                         4











                        The American shad, Alosa sapidissima, is an anadromous member of the herring
                 family (Clupeidae), which ranges from the Gulf of St. Lawrence to Florida (Walburg and
                 Nichols, 1967). During its springtime spawning runs the species has been subjected to
                 substantial commercial and recreational fishing pressure throughout its range, particularly in
                 Chesapeake Bay tributaries and by Maryland and Virginia's ocean fisheries. In addition,
                 shad populations of almost every Chesapeake Bay drainage have been further restricted by
                 dams which block migration to their spawning habitat in fresh water transition zones. As a
                 result of fishery exploitation, loss of spawning and nursery habitat, and possibly
                 environmental degradation such as stream acidification, harvests of shad in Pennsylvania,
                 Maryland, the District of Columbia and Virginia declined precipitously during the period 1965-
                 1988 (Stagg, 1986; Gibson, Crecco and Stang, 1988).

                        A great deal of effort has been expended to revive stocks of Chesapeake Bay
                 American shad. Conservation and restoration measures were enacted in Pennsylvania,
                 Maryland and the District of Columbia in the early 1980s. Some agencies required season,
                 gear and by-catch restrictions along with creel limits to reduce fishing effort (Maryland's shad
                 fishery was closed in 1980). Concurrently, the issues of habitat loss and degradation were
                 addressed by installing permanent fish passage facilities such as the one at Conowingo, Dam,
                 removing some obstructions to migrating fish, re-stocking fish into historical spawning
                 habitats, establishing stock assessment and monitoring programs, and operating. dam
                 turbines in a manner which maintained minimum flow and standard dissolved oxygen levels
                 (CEC, 1989).


                        Prior to these efforts, American shad migrating upstream in Susquehanna River had
                 been few in number. Throughout the 1970s shad transported by the trap/lift assembly at
                 Conowingo Dam averaged 127 fish per year (ASMFC, 1988). Yearly release of shad fry and
                 of live pre-spawned adult shad from six other source rivers accompanied the lift -operation
                 beginning in 1982 (Table 1). By 1989, more than 6000 migrating shad were reported to have
                 been hauled upstream above all dams to the historical Susquehanna spawning areas.

                        In view of the apparent success of shad management efforts in Susquehanna River, a
                 program of study was proposed to the Maryland DNR Chesapeake Bay Research and
                 Monitoring Division's Power Plant Topical Research Program designed to examine population
                 dynamics underlying the resurgent American shad population in Susquehanna River using
                 molecular genetic techniques (Chapman and Brown, 1991). Mitochondrial DNA genotypes of
                 American shad being moved over Conowingo Dam were compared to genotypes of shad from
                 the source rivers, from other Chesapeake Bay rivers and from several southern east coast
                 shad populations. Variation in mitochondrial DNA was analyzed and employed, to estimate
                 the percent contribution by any of these shad populations to the increasing Susquehanna
                 stock.




                                                             5









                     Population genetic data collected for the Maryland DNR study were employed in the
              present study for Chesapeake Bay Commission to estimate the relative percentage of
              American shad from each baseline population being harvested in Virginia's Atlantic Ocean
              intercept fishery during the Spring of 1991. The baseline populations available for comparison
              with the Spring 1991 coastal fishery were: Columbia River-WA, Delaware River-DE, Hudson
              River-NY, Connecticut River-CT, Nanticoke River-MD, Parnunkey River-VA, James River-
              VA, Chowan River-NC, Savannah River-SC, Santee River-SC and St. Johns River-FL, the
              Susquehanna Flats in upper Chesapeake Bay, and Susquehanna River shad lifted over
              Conowingo Dam in 1990 and 1991. This report summarizes the seven-month research
              project, provides estimates of proportions of American shad from each of the groups outlined
              above which comprise the Spring 1991 coastal harvest, and outlines management and
              research implications of these data.








                LABORATORY ANALYSIS


                       The laboratory procedures described in this report are intentionally brief. Detailed
                instructions for the extraction and digestion of mitochondrial DNA can be found in Chapman
                and Brown (1990).

                       During the Spring of 1991, mitochondrial DNA (mtDNA) was extracted from
                American shad harvested in Virginia's Atlantic Ocean intercept fishery outside the mouth of
                Chesapeake Bay. Shad were obtained from commercial fishermen landing at three locations:
                Rudee Inlet, Chincoteague and Quinby/Wachapreague (see Figure 1). Ovaries from each
                individual fish were removed and placed in a Ziplock Baggie along with an envelope
                containing a scale sample and pertinent data on the location of capture, size of fish, etc. Each
                baggie was sealed and placed on wet ice for transportation to the laboratory at East Carolina
                University in Greenville, NC.

                       All shad samples arrived at the laboratory on the day they were collected. Within one
                day of sampling, approximately 10 g of egg tissue from each individual fish were processed to
                isolate purify mtDNA. The mtDNA was rehydrated in 150 gl of sterile distilled water and
                aliquots of 8.5 W mtDNA from each fish were combined with 0.5 Unit of the following
                restriction endonucleases (Aat I, Apa I, Bcl I, Bgl I, Dra I, EcoR 1, EcoR V, Hind III, Kpn I,
                Pst I, Pvu 111, Sal I, Sma 1, Sst H, and Xba 1) along with 1 gl of the appropriate buffer supplied
                by the manufacturer. Each digest was incubated at 37 OC for 3-4 hours and contained a total
                volume of 10 gl. Reactions were stopped with .1 ul of STOP solution (0.89 M Tris, 0.89 M
                boric acid, 0.02 M EDTA, 0.25 % bromophenol blue, 50 % glycerol and 1 % SDS'
                                                                                                .) and were
                electrophoresed overnight through 0.8 % agarose gels. The DNA in gels was stained with
                ethidium bromide and photographed under ultra-violet light as described by Chapman and
                Powers (1984).


                       Restriction digest patterns were recorded for each restriction endonuclease digest of
                each fish's mtDNA. Digestion patterns were assigned upper-case alphabetic symbols. Then,
                each individual was assigned a composite "haplotype" consisting of the letters designating
                the restriction fragment patterns produced by digestion with each of the fifteen enzymes.













                                                           7









               DATA ANALYSIS


                      Details of the mathematical properties of the algorithms used to perform statistical
               analyses can be found in Sokal and Rohlf (198 1), Roff and Bentzen (1989), Fournier et al.
               (1984), Pella (1986), and Pella and Milner (1987).

                      Each fish's haplotype is a multiple characterization of that fish's mitochondrial genome
               and is transmitted in a manner analagous to human surnames. As in other animals, variation
               in shad mtDNA is typified by the occurrence of rare haplotypes in each population (Bentzen
               et al., 1988 and 1989). If chi-square contingency tests were to be performed the rare
               haplotypes would be lumped and only the most frequent haplotypes would be employed in the
               analysis. This practice of pooling rare mtDNA haplotypes results in a severe loss of
               information relevant to geographic and temporal genetic variation. To resolve this problem,
               Roff and Bentzen (1989) presented a chi-square analysis which does not require pooling of
               rare variants. The analysis generates Monte Carlo distributions of expected chi-square from
               unpooled mtDNA data and allows high levels of significance even when sample sizes are
               small.


                      Haplotype frequencies were employed to determine basic genetic relationships
               between the baseline shad populations and the intercept population. First, a series of chi-
               square statistics for heterogeneity of mtDNA haplotype frequencies were calculated (Roff
               and Bentzen, 1989). The chi-square analysis was conducted by treating all of the populations
               as one large assemblage. Then, based on the finding of significant heterogeneity,
               successively smaller sets of populations were analyzed until no further heterogeneity was
               detected.


                      The actual contribution of each baseline population to th e intercept sample was
               estimated from the recorded mtDNA information by the method of conditional maximum
               likelihood estimation of stock composition as proposed by Fournier et al. (1984) and by
               Ferris and Berg (1987). The basic concepts underlying this genetic analysis are similar to a
               traditional mark-recapture study. The algorithm we used was created by J. Pella of NMFS
               (Pella, 1986) for interpretation of mixed-fishery genetic data for west coast salmon. Called
               GIRLSEM, the computer program written in FORTRAN is an iteratively reweighted least
               squares algorithm to compute the conditional maximum likelihood estimate of composition of
               a group of fish of mixed ancestry. An additional benefit of this program is that sampling error,
               which leads to variability of the composition estimates, can be assessed by bootstrap
               resampling and by the infinitesimal jackknife procedure. The accuracy of this method was
               explored by analyzing several artificial populations of known composition created by sampling
               the baseline haplotypes (with replacement) prior to the analysis of mixed shad populations.






                                                           8











                        A total of 162 individual shad collected during a one-month period from three locations
                near the mouth of Chesapeake Bay were processed. Fifteen restriction endonuclease digests
                were performed per individual for a total of 2,430 digests. Thirty-three restriction fragment
                profiles were observed in the intercept samples, encompassing seventy-eight separate
                restriction fragments of the Alosa sapidissima mitochondrial genome. Wheneverpossible, the
                .profiles for an enzyme were compared to those obtained by Bentzen et al. (1988 and 1989).
                Restriction fragment profiles for each enzyme are illustrated in Fig. 2.

                        Genotypes for each individual intercept shad are shown in Appendix A. Haplotypes,
                the composites of all fifteen genotypes, for each intercept shad are shown in Table 2 and are
                also listed in Appendix B alongside haplotypes for all of the baseline populations. The
                frequencies of each haplotype for each of the three locations examined are shown in Table 2
                along with the frequencies of haplotypes in each of the baseline populations. Thirty-four
                different haplotypes were detected in the intercept samples. Seventy-nine shad had either
                the common haplotype AAAAAAAAAAAAAAA or one of the many unique baseline
                haplotypes. Haplotypes of eighteen individuals in the intercept sample were unique. In some
                instances the mtDNA preparation was of inadequate quantity or quality to perform an fifteen
                digests. Sixty-five shad were incompletely characterized and were not included in the final
                analysis (missing data are shown by "-" in Appendix A).

                        Results of a chi-square analysis based on the distribution of haplotypes are presented
                in Table 3. Ile first tier of the chi-square analysis was performed with the entire sample
                (baseline and intercept) as one assemblage (overall chi-square of 4765.41, P < 0.0001) and
                indicated that significant differences existed within the aggregation. The assemblage was
                successively decomposed by population to the point where no further heterogeneity was
                detected among the groups shown in Table 3. An important finding of this analysis was that
                shad harvested from Rudee Inlet were significantly different from those taken at
                Chincoteague and Quiriby (chi-square of 94.78, P < 0.001). Also, no significant difference
                was detected between the Chincoteague and Quinby samples (chi-square of 33.78, P = .717).

                        Prior to the final analyses, the GIRLSEM program was tested by compiling several
                subsets of known composition from the baseline haplotypes in order to evaluate accuracy and
                precision when estimating the composition of mixed stock samples from shad mtDNA
                haplotypes (Chapman and Brown, 1991). Results of these analyses are shown in Table 4.
                GIRLSEM analysis performed well under the artificial scenarios and accurately estimated the
                actual contribution of baseline populations in most instances. Estimates were statistically
                acceptable and required fewer than 100 iterations to converge. Instances where estimates
                were incorrect could be attributed to contributions by baseline populations which had a very
                low portion of unique individuals. One river in the present baseline data set, Connecticut
                River, falls into this category, where only one of 24 fish sampled was unique. In the trials,
                actual contribution by Connecticut was consistently attributed to Delaware River.


                                                             9








                     Incompletely characterized shad and the nineteen percent of intercept shad whose
              haplotypes were unique (not observed in any baseline population) were withheld from the
              final analysis. Haplotypes for the remaining intercept samples were compared to those of all
              baseline populations. Every haplotype which was shared between the intercept sample and
              any baseline population was used in final runs to estimate percent composition of the
              intercept sample. The number of intercept shad employed in this analysis was seventy-nine
              (81% of the total intercept sample). Separate conditional maximum likelihood analyses were
              also conducted for shad from each of the three sample locations. Table 5 lists the estimated
              composition of the 1991 Virginia intercept shad fishery. By multiplying the maximum
              likelihood results in Table 5 by a factor of 0.81, it is estimated that the following populations
              comprised the 1991 intercept harvest: Unknown (19.37%), James (0.81%), St. Johns (3.26%),
              Parnunkey (5.70%), Santee (5.70%), Hudson (12.22%), and Susquehanna (52.94%). These
              estimates are depicted graphically in Figure 3.





































                                                        10








                .DISCUSSION

                       American shad examined for this study and for the larger MDNR study are of diverse
                genetic composition. In order to evaluate composition of the intercept fishery it was first
                necessary to evaluate composition of Susquehanna and other Chesapeake Bay stocks.
                Chapman and Brown (1991) reported that the existing Susquehanna River stock is comprised
                of shad descended from both native Chesapeake Bay and southern Atlantic coast rivers and
                that the Susquehanna stock does not appear to be in a state of genetic equilibrium..

                       Chi-square analysis indicated American shad landed at different Atlantic Ocean
                locations were significantly different. Those landed at Rudee Inlet were different from shad
                landed along the Virginia portion of the Delmarva Peninsula. Shad harvested along the
                Delmarva Peninsula by the intercept fishery (Chincoteague and Quinby) were not
                significantly different from one another.

                       Nineteen percent of the intercept fish could not be classified by our current baseline
                data set. This indicates that the baseline data set must be expanded before this portion of
                the intercept fishery can be accurately evaluated. Conditional maximum likelihood estimates
                of the composition of the remaining 81% of the intercept fishery sample indicate that those
                intercept shad were predominantly of Susquehanna and Hudson River origin (Table 5). Small
                contributions by other locations, from both Chesapeake Bay and southern rivers, were also
                detected in the overall analysis. Although the standard deviation for the largest contributing
                population, Susquehanna, is marginally acceptable, other standard deviation values are large
                (Table 5). This situation occurs due to small sample sizes.

                       Small sample sizes made it difficult to estimate the probable destination of shad
                landed at the Rudee, Chincoteague and Quinby locations separately with an acceptable
                degree of confidence. The final sample sizes for Rudee, Chincoteague and Quinby are small
                (n = 15, 40, 42, respectively) after excluding partial and unique haplotypes (17%, 7%, and 9%,
                respectively) and the standard deviation values for these estimates are proportionally large
                (see Table 5). However, a general trend is apparent. Most shad analyzed from *'Fludee Inlet
                were estimated to be of Virginia origin (70% Pamunkey and James, 30% Susquehanna) while
                the Chincoteague and Quinby harvests were composed of Susquehanna, Hudson and Santee
                shad.









                 CONCLUSIONS


                         This project involved a genetic survey to identify which, if any, target populations were
                 harvested by Virginia's Atlantic Coast intercept fishery. 'Me study was conducted
                 concurrently with one of the most comprehensive investigations of a mixed fishery attempted
                 to date employing mtDNA analysis. The results presented here are of immediate short-term
                 use to the community of managers and scientists who regulate the American shad fishery in
                 Chesapeake Bay. Furthermore, these data constitute a minimum framework which when
                 expanded will allow development of a long-term monitoring program which could eventually
                 rival that for west coast salmon in its effectiveness.


                         The genetic analysis provides an intitial "snapshot" of Virginia@s intercept fishery
                 indicating that at least one-half of the shad harvested were destined for Susquehanna River.
                 Due to the magnitude of effort and funds expended by all of the Bay states to rebuild shad
                 stocks, policy makers and managers are sure to inquire: "Does the Virginia ocean shad
                 harvest potentially affect the Susquehanna stock?" This question can be addressed by
                 considering the preliminary estimate of shad harvested by Virginia's ocean fishery in 1991
                 (405,612 1b) multiplied by the proportion of those harvested which were estimated to be of
                 Susquehanna origin ( 53 % ) and comparing the figure to Susquehanna shad abundance in
                 1991 ( 22,083 shad were lifted over Conowingo Dam @ 4.5 lb = 99,374 lb ). Thus the
                 Virginia 1991 harvest may be twice as great as the quantity of Susquehanna shad moved
                 over Conowingo Dam. Seventy-eight percent of Virginia's ocean shad harvest occurs north of
                 the Bay mouth. Based on these values and the genetic results from Rudee Inlet vs the
                 Peninsula locations, the impact on the Susquehanna population could be greatly diminished by
                 restricting harvests along the Peninsula.

                         This raises a very important issue. We know that Maryland's ocean shad catch
                 increased from 40,000 lb in 1980 to 143,300 lb in 1988 (Flagg, 1990). It would be useful from
                 both management and policy standpoints to estimate the composition of that fishery as well.

                         The joint MD/VA shad tagging study at Rudee Inlet has produced some preliminary
                 results which should be compared with the genetic data. The two studies are complementary
                 in a very important way. Fifty-six percent of tag returns during the last six months have been
                 from lower Chesapeake Bay; primarily from York River and its tributaries (Jesien and Hocutt,
                 1991). By comparison the genetic analysis estimates that 66 % of the Rudee Inlet harvest is
                 of American shad from Pamunkey River (a tributary of York River). Such close agreement
                 would tend to indicate that the genetic estimates of stock contribution for this location are
                 correct.


                         Another aspect of how the tagging and genetic analyses complement one another is
                 associated with the fact that no tags have been returned from upper Chesapeake Bay. There
                 is no fishing effort for shad in upper Chesapeake Bay since both MD and PA have closed their



                                                              12








                shad fisheries. Yet the genetic analysis of the ocean catch provides the upper Bay
                component (Susquehanna River) in addition to the Pamunkey and James components.

                       In the future, tagging could address some critical issues raised by the genetic
                analysis. For example, both the Susquehanna Flats and the Susquehanna River shad
                populations have large Santee River components. It cannot be determined from the present
                data whether the Santee component of the intercept fishery is due to shad actually returning
                to Santee River or whether it is due to Susquehanna shad with Santee haplotypes. If tagging
                endeavors are repeated once fishing resumes in upper Chesapeake Bay then we can evaluate
                both possibilities.

                       Like most other scientific investigations the present ge netic analysis raises as many
                questions as it answers. Although the baseline data were adequate to address the majority
                of intercept fish harvested in 1991, they must be expanded before the entire fishery can be
                evaluated. We recommend increasing all baseline sample sizes to at least 50 and assessing
                existing Chesapeake Bay shad populations which were not examined in the present study
                (Rappahannock, Potomac, Patuxent, Choptank, Chester, etc.). In addition, it is clear that one
                sample is not adequate to formulate far-reaching management decisions. Shaklee et al.
                (1990) have examined mixed-stock fisheries of Pacific salmon. They found that stock
                composition varies substantially from year-to-year for mixed-stock assemblages. It would be
                prudent to assume that migratory mixtures of American shad stocks behave in a similar
                manner to Pacific coast salmonid stocks.


                       Depending on the Bay States' goals, we can. suggest the necessary actions to
                undertake. First, if the sole purpose is to make an immediate management decision regarding
                coastal intercept shad fisheries then at least one more survey should be made of the Virginia
                fishery accompanied by at least two years of investigation of Maryland's ocean shad fishery.
                Analyses should proceed by sacrificing 150 fish per site per year, extracting mtDNA, and
                digesting mtDNA with the same 15 enzymes employed in the present study. The resulting
                haplotypes should be compared to an expanded data base. This should provide adequate
                information to confidently make decisions pertaining to the intercept fishery.

                       If goals are broader and the States wish not only to manage and regulate the intercept
                fisheries but to monitor all Chesapeake Bay shad populations, then we would suggest a
                modified research/monitoring program. This program involves conducting the research
                described above to address the immediate issue of the impact of the intercept fisheries.
                However, we would also recommend converting previously collected samples to a new format
                (PCR) which would allow all subsequent genetic analyses to be made from material obtained
                by amplifying mtDNA from non-lethal biopsy tissue samples (an obvious benefit when
                genetic analysis is associated with tagging). Tissue samples could be archived and analyzed
                at any time in the future.




                                                          13








                      Shad populations are dynamic entities influenced by both natural and anthropomorphic
               factors. Genetic analysis offers a means not just to estimate the percent composition of
               mixed assemblages of shad but also to monitor the success of stocks as they respond to
               ecological changes and to various management practices. Like tagging, annual collection of
               catch-effort statistics, and other management programs, a program of genetic population
               analysis requires long-term commitment at the very least to collect and archive samples.
               Most biological monitoring programs require a minimum of four years of sequential monitoring
               before any type of trend can be determined. After that, analyses can be performed annually or
               bi-annually to re-evaluate important groups of Chesapeake Bay and intercept shad.







































                                                         14









                SIGNIFTCANT POINTS


                *Virginia's 1991 intercept fishery was comprised of approximately 53% Susquehanna shad
                   (0.81 X 65%), perhaps more.

                *This estimate should be considered preliminary for two important reasons. First, the
                   Susquehanna River population does not appear to have attained genetic stability and it is
                   possible that contribution estimates for this River will vary from year to year. Second, the
                   Susquehanna Flats baseline sample is very small and may not completely represent that
                   group of American shad.

                *Genetic analysis indicates that the Rudee Inlet harvest differs from harvests along
                   Virginia's portion of the Delmarva Peninsula.

                *These findings should be verified by a joint MD/VA investigation which would expand the
                   current genetic baseline data set, identify specific river stocks within both suites'
                   intercept fisheries, and establish a long-term shad monitoring program (associated with
                   joint tagging efforts).





























                                                          15











                Altukhov, Y.P. and E.A. Salmenkova. 1987. Stock transfer relative to natural organization,
                       management, and conservation of fish populations. Chapter 14 In: Population genetics
                       and fishery management. N. Ryman and F. Utter, eds. Univ. of Washington Press,
                       Seattle.
                ASMFC. 1988. 1988 Supplement to American shad and river herrings fishery management
                       plan. Fisheries Management Rept. No. 12 of the Atlantic States Marine Fisheries
                       Commission. October 1988, Washington, DC.
                ASMFC. 1990. 1990 Shad and river herring workshop, October 16-17, 1990, Nags Head, NC.
                       Atlantic States Marine Fisheries Commission, Washington, DC.
                Avise, J.C., J.E. Neigel and J. Arnold. 1984. Demographic influences on mitochondrial DNA
                       lineage survivorship in animal populations. J. Mol. Evol. 20: 99-105.
                Backman, T. National Fisheries Research and Development Laboratory, USFWS, Wellsboro,
                       PA.
                Bentzen, P., W.C. Leggett and G.G. Brown. 1988. Length and restriction site heteroplasmy in
                       the mitochondrial DNA of American shad (Alosa sapidissima). Genetics 118: 509-518.
                Bentzen, P., G.G. Brown and W. C. Leggett. 1989. Mitochondrial DNA polymorphism,
                       population structure, and life history variation in American shad (Alosa sapidissima).
                       Can. J. Aquat. Sci. 46: 1446-1454.
                Birky, C.W., T. Maruyama and P. Fuerst. 1983. An approach to population and evolutionary
                       genetic theory for genes in mitochondria and chloroplasts, and some results. Genetics
                       103: 513-527.
                Brown, B.L. and K.T. Paynter. .199 1. Mitochondrial DNA analysis of native and selectively
                       inbred Chesapeake Bay oysters, Crassostrea virginica. Marine Biology (in press).
                CEC. 1989. Chesapeake Bay Alosid Management Plan. An agreement commitment report
                       from the Chesapeake Executive Council. July 1989, Annapolis, MD.
                Chapman, R.W. and B.L. Brown. 1990. Mitochondrial DNA isolation methods. Chapter 4 In:
                       Electrophoretic and isoelectric focusing techniques in fisheries management. D.H.
                       Whitmore, ed. CRC Press, Boca Raton, FL.
                Chapman, R.W. and B.L. Brown. 1991. Genetic investigation of the resurgence of American
                       shad, Alosa sapidissima, in Susquehanna River. Report submitted to Maryland Dep. of
                       Natural Resources, Chesapeake Bay Research and Monitoring Division, Annapolis,
                       MD.
                Chapman, R.W. and D.A. Powers. 1984. A method for rapid isolation of mitochondrial DNA
                       from fishes. Maryland Sea Grant Tech. Rept. No. UM-SG-TS-84-05.
                Dadswell, M.J., G.D. Melvin and P.J. Williams. 1983. Effect of turbidity on the temporal and
                       spatial utilization of the inner Bay of Fundy by American shad (Alosa
                       sapidissima)(Pisces:Clupeidae) and its relationship to local fisheries. Can. J. Fish.
                       and Aquat. Sci. 40 (Suppl. 1): 322-330.
                Ferris, S.D. and W.J. Berg. 1987. The utility of mitochondrial DNA in fish genetics and fishery
                       management. Chapter I I In: Population genetics and fishery management. N. Ryman
                       and F. Utter, eds. Univ. of Washington Press, Seattle.


                                                           16








               Flagg, L. 1990. Review of Atlantic States Marine Fisheries Commission Fishery
                      Management Plan for American shad and river herring. pp 76-82 In: Atlandc States
                      Marine Fisheries Commission 1990 shad and river herring workshop. Oct. 16-17,
                      1990, Nags Head, NC.
               Fournier, D.A., T.D. Beacham, B.E. Riddell and C.A. Busack. 1984. Estimating stock
                      composition in mixed stock fisheries using morphometric, meristic, and electrophoretic
                      characteristics. Can. J. Fish. and Aquat. Sci. 41: 400-408.
               Gibson, M., V. Crecco, an dD. Stang. 1988. Stock assessment of American shad from selected
                      Atlantic coast rivers. Special Report No. 15 of the Atlantic States Marine Fisheries
                      Commission, Washington, DC.
               Gibson, M. Rhode Island Div. of Fish and Wildlife, W. Kingston, RI.
               Jesien, R. and C. Hocutt. 1991. Movement of American shad in the Chesapeake Bay.
                      Progress Report to MDNR, Annapolis, MD.
               Leggett, W.C. and R.R. Whitney. 1972. Water temperature and the migration of American
                      shad. Fish. Bull. (US) 70:659-670.
               Melvin, G.D., J.D. Michael and J.D. Martin. 1985. Fidelity of American shad, Alosa
                      sapidissima, (Osteichthyes:Clupeidae) to its river of previous spawning. C an. J. Fish.
                      and Aquat Sci. 42:1-19.
               Nei, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York.
               Pella, J.J. 1986. The method of fitting expectations applied to computation of conditional
                      maximum likelihood estimates of stock composition from genetic marks. LIS
                      DOC/NOAA/NMFS/NWAFC Auke Bay Laboratory, Auke Bay, AK. Rept. No. 171.
               Pella, J.J. and G.B. Milner. 1987. Use of genetic marks in stock composition anal, sis. Chapter
                                                                                              y
                      10 In: Population genetics and. fishery management. N. Ryman and F. Utter, eds. Univ.
                      of Washington Press, Seattle.
               Roff, D.A. and P. Bentzen. 1989. The statistical analysis of mitochondrial DNA
                      polymorphisms: Chi-square and the problem of small samples. Mol. Biol. Evol. 6: 539-
                      545.
               Shaklee, J. C. Busack, A. Marshall, M. Miller and S. Phelps. 1990. The electrophoretic
                      analysis of mixed-stock fisheries of Pacific salmon. pp. 235-265 In: (Z-1. Ogita and C.
                      Marke, eds.) Isozymes: Structure, Function, and Use in Biology and Medicine. Prog.
                      in Clin. and Biol. Res. Vol. 344. Wiley-Liss, Inc. New York.
               Sokal, R.R. and F. J. Rohlf. 1981. Biometry. W.H. Freeman and Co., San Francisco.
               Stagg, C. 1986. An evaluation of the information available for managing Chesapeake Bay
                      fisheries: preliminary stock assessments, Vol. I and II. Univ. Maryland, CBL,
                      UMCEES (CBLj Ref. No. 85-29.1.
               Takahata, N. and T. Maruyama. 1981. A mathematical model of extranuclear geries and the
                      genetic variability maintained in a finite population. Genet. Res., Camb. 37: 291-302.
               Walburg, C.H. and R.P. Nichols. 1967. Biology and management of the American shad and
                      status of the fisheries. Atlantic coast of the United States 1960. US Fish and Wildl.
                      Ser. Special Sci. Rept. Fish. No. 550.




                                                        17









              SUGGESTED PEER-REVIEWERS


              Richard St. Pierre
              Susquehanna River Coordinator
              USFWS
              1721 N. Front Street, Suite 105
              Harrisburg, PA 17102


              James Shaklee, Ph.D.
              Washington Dept of Fisheries
              115 General Administration Bldg.
              Olympia, WA 98504


              John Graves, Ph.D.
              Virginia Institute of Marine Science
              College of william and Mary
              Gloucester Point, VA 23062


              Paul Bentzen, Ph.D.
              Dept. of Biology
              Dalousie University
              Halifax, Nova Scotia
              CANADA B3H 4J1































                                                     18






















                   Figure 1. Map of Virginia@s Atlantic coastline showing locations of the
                              three landing areas sampled for American shad: Rudee Inlet,
                              south of the Bay mouth, Quinby and Chincoteague, north of
                              the Bay mouth.












                                                                                     Wei


                                                                    C&I



                                                                                   Vill
                                                                        iNW,-


                                                                       Qp






                                                                                                  OU
                                                                                                  MIDI










                                                                                                         Chincoteague



                                                                                       N."                   6
                                                                                                            0

                                                                                                Quirby








                                                                               A








                                                                                     udee Inlet


                                    Scak of Kan
                                                                    P401FOLK






















                   Figure 2.   Graphic representation of American shad restriction fragment
                               patterns. Names of restriction enzymes occur along the top margin
                               and are those listed in the text. Genotype names assigned to each
                               pattern are the capitol letters directly underneath each enzyme.
                               Bands of a molecular weight standard are shown along the left
                               and right margins of each page and the sizes of these bands are
                               indicated in kilobases (thousands of nucleotide base pairs).













                      Aat I                        Apa I                           Bcl I

                 A B C D                A B C D          E F G              A B C        D E     F
      9

                                                                                                           5
        4-                                                                                                 4

       3-                                                                                                  3


       2-                                                                                                  2








      0.5-                                                                                               -0.5














                            Bgl I                     Dra I               Eco RI

                     A   ME B    D    E   F          A B            A   B   C   D   E F



           5=                                                                                       5
           4-                                                                                       4

           3-                                                                                       3



           2-                                                                                       2

           1.6-









           0.5-                                                                                   -0.5












                               Eco R V              Hin d IH                 Kpn
                           A B C        D          A B      C         A B C D           E





                 3-                                                                                   3


                 2-                                                                                  2
                1.6-                                                                              -1.6






                0.5-                                                                              -0.5














                                Pst I                     Pvu H                   Sal I

                        A B      C   D    E   F         A B     C            A B C         D


                                                                                                         5
             5-
             4-                                                                                          4
             3                                                                                           3

             2-                                                                                          2
             1.6-                                                                                     -1.6





             o.5-                                                                                     -0.5


















                              Sma I              Sst 11                Xba I


                            A B C             A   B    c          A B      C   D E



                5                                                                                5
                4-                                                                               4


                3-                                                                               3



                2-                                                                               2

                1.6-                                                                          -1.6








                0.5-                                                                          -0.5





















                  Figure 3. Graphic illustrations of the percent composition of the 1991
                             ocean shad harvest. A. Total catch, B. Rudee Inlet,
                             C. Chincoteague and D. Quinby.








                         A                                B                                C                                D

    James                                                        Savannah







            Total Ocean Harvest                     Rudee Inlet                    Chincoteague                Quinby/wachapreague



                                                                     (3   txpknvwn
                                                                     )w   sus 1990
                                                                          So 1991

                                                                          Pamunkey
                                                                          James
                                                                          Santee
                                                                          Savannah
                                                                          St. John's


















                                      TabLe 1. Summary of American shad stocked into the Susquehanna River
                                                 during the period 1982-1991 by river of origin



                                       A.                 Hatchery cuLtured fry (mittions)


                                         Year    Pamunkey       James     CoLumbia    DeLaware      Hudson

                                         1991         -                                 3.212       8.845
                                         1990       0.178          -           -        3.565       6.000
                                         1989       0.754       0.220       12.422      1.645       5.660
                                         1988       0..655      0.029       8.467       0.949          -
                                         1987       1.403       0.040       6.919       1.227          -
                                         1986       2.433       0.210       11.184      1.243          -
                                         1985       2.222       0.458       1.906       1.642          -
                                         1984       4.289       0.509       7.162       0.380          -
                                         1983       1.000       1.100       1.950           -          -
                                         1982       1.151       0.740       6.949




                                       B.                 Live pre-spawned aduLts


                                         Year             Susquehanna Hudson       Connecticut


                                         1991                   22083          -            -
                                         1990                   14792          -            -
                                         1989                    6590          -            -
                                         1988                    4730          -            -
                                         1987                    6900        6032
                                         1986                    4080        4965           -
                                         1985                      950       3158           64
                                         1984                        0       3592           185
                                         1983                        0       3123          1187
                                         1982                      875         992         1573

















                                        Table 2. Mitochondriai DNA haplotypes of American shad
                                                     harvested outside Chesapeake Bay in 1991.


                                        HapLotype
                                                                    Rudee   Chincoteague     Quinby
                                        AAAAAAAAAAAAAAA                2           16           14
                                        AAAAAAAAAAAAAAA/B              0            1            0
                                        AAAAAAAAAAAAAAB                0            1            0
                                        AAAAAAAAAAABAAA                0            1
                                        AAAAAAAAAABAAAA                0            1            1
                                        AAAAAAAAA/BAAA/BAAA            0            0            1
                                        AAAAAAAABAAAAAA                1            4            5
                                        AAAAAAAABAAAAAB                1            0            0
                                        AAAAAAAABAAAABA                1            0            0
                                        AAAAAAAABAABAAA                1            7            3
                                        AAAAAAAABEAAAAA                1            0            0
                                        AAAAAAAACAAAAAA                0            0            1
                                        AAAAAABASAAAAAA                0            0            1
                                        AAAAAABABAABAAA                0            0            1
                                        AAAAAACAAAAA/CAAB                           0            0
                                        AAAAAACAAAACAAA                             0            0
                                        AAAAAACASAAAAAA,               1            0            0
                                        AAAAAACASAAAAAB                1            0            0
                                        AAAAAACASAAA/CAAR              1            0            0
                                        AAAAABAAAAAAAAA                0            1            0
                                        AAAASAAAAAAAAAA                0            1            0
                                        AAAFAAAAAAAAAAA                1            0            0
                                        ASAAAAAAAAAAAAA                0            1            0
                                        AGAAAAAAAAABAAA                0            1            0
                                        BABAAAAAEAAAAAA                0            0            1
                                        DAAAAAAAAAAAAAA                0            5            8
                                        DAAAAAAAAASAAAA                0            0            2
                                        DAAAAAAASAAAAAA                0            0            1
                                        DAAAAAAABAABAAA                0            0            1
                                        DAAAAAA/CAAA/CAAAAA            1            0            0
                                        DAAAAABAAAAAAAA                0            0            1
                                        DAAAAABAAAAA/CAAB              1            0            0
                                                                      15           40           42
















                                     Table 3. Selected chi-square comparisons for American shad in
                                        baseline and intercept populations. Abbreviations are as follows:
                                        R-Rudee, C-Chincoteague, Q-Quinby, SF-Susquehanna Flats, CHO-Chowan,
                                        SAV-Savannah, SAN-Santee, STJ-St. Johns, 90-Susquehanna at Conowingo
                                        Dam in 1990, 91-Susquehanna at Conowingo Dam in 1991.






                                                               Chi-square                     Number of
                                     Comparison               data     simuL.     P           hap     indiv



                                     ALL populations         4765.41             0.000        164      494


                                     R-C-0                    94.78    92.39     0.000        34        97
                                     C_Q                      19.45    33.78     0.717        22        82


                                     East coast pop.         2445.18  1349.23    0.000        124      361
                                     Ches. Bay-North          814.74   411.67    0.000        73       219
                                     Chesapeake Bay           169.13   200.76    0.095        52       361
                                     North of Ches. Bay       101.39   135.60    0.088        29       125
                                     South of Ches.  Bay      416.02   310.50    0.000        62       142


                                     90-91-SF                 213.00   243.04    0.007        97       166
                                     90-91                    77.38    112.81    0.467        99       155


                                     90-SF                    59.10    103.00    0.858        83       110
                                     90-SAN                   80.44    82.42     0.003        89       140


                                     91-SF-SAN                77.52    99.86     0.063        31       108
                                     91-SF                    22.10    32.77     0.036        24        67


                                     SF-SAN                   17.49    36.71     0.376        17        52
                                     SF-SAV                   39.11    49.66     0.039        29        56
                                     SF-STJ                   24.19    [email protected]     0.055        36        39
                                     SF-CHO                   15.27    21.83     0.063        11        31

































                                  Table 4. Trials and accuracy testing of GIRLSEM to determine ability of the
                                           algorithm to estimate stock composition of a mixed sample from
                                           mtDNA data. Mixture populations of known composition were
                                           created by sampling twelve baseline populations with replacement.








                                       Mixture 1.
                                        25% of all baseline populations sampled with replacement.
                                        36 hapiotypes compared.


                                                     Source N     Contribution                 Ho:A=E    alpha=
                                                                  Actual    Estim.    SE        z        0.001
                                                           96
                                                     1. COL       0.066     0.067     0.036    0.016     accept
                                                     2. CT        0.066     0.000     0.025    -2.604    accept
                                                     3. DEL       0.104     0.359     0.058    4.405     reject
                                                     4. HUD       0.142     0.149     0.051    0.156     accept
                                                     5. SF        0.028     0.000     0.017    -1.672    accept
                                                     6. WAR       0.066     0.074     0.037    0.222     accept
                                                     7. PAM       0.076     O.W       0.037    -0.243    accept
                                                     8. JAN       0.113     0.059     0.040    -1.353    accept
                                                     9. CHO       0.047     0.000     0.022    -2.181    accept
                                                     10. SAN      0.113     0.082     0.043    -0.718    accept
                                                     11. SAV      0.094     0.074     0.040    -0.500    accept
                                                     12. STJ      0.085     0.068     0.038    -0.428    accept


                                       Mixture 2.
                                        50% of all baseline populations sampled with replacement.
                                        40 haptotypes compared


                                                     SourceR      Contribution                 No:A=E    alpha=
                                                                  Actual    Estim.    SE        z        0.0001
                                                          164
                                                     1. COL       0.102     0.097     0.033    -0.152    accept
                                                     2. CT        0.059     0.000     0.018    -3.211    reject
                                                     3. DEL       0.122     0.304     0.0"     4.124     reject
                                                     4. HUD       0.108     0.142     0.036    0.931     accept
                                                     5. SF        0.031     0.093     0.026    2.381     accept
                                                     6. MAN       0.064     0.032     0.024    -1.355    accept
                                                     7. PAM       0.085     0.043     0.027    -1.586    accept
                                                     S. JAM       0.122     0.047     0.030    -2.468    accept
                                                     9. CHO       0.048     0.000     0.017    -2.879    accept
                                                     10. SAN      0.096     0.075     0.031    -0.687    accept
                                                     11. SAV      0.108     0.111     0.034    0.102     accept
                                                     12. STJ      0.055     0.057     0.025    0.086     accept


                                      Mixture 3.
                                       75% of all baseline populations sampled with replacement.
                                       72 haplotypes compared.


                                                     SourceR      Contribution                 Ho:A=E    alpha=
                                                                  Actual    Estim.    SE       z         0.001
                                                          258
                                                     1. COL       0.095     0.091     0.042    -0.095    accept
                                                     2. CT        0.060     0.000     0.024    -2.478    accept
                                                     3. DEL       0.146     0.295     0.059    2.537     accept
                                                     4. HUD       0.122     0.107     0.046    -0.330    accept
                                                     5. SF        0.027     0.033     0.025    0.224     accept
                                                     6. MAN       0.061     0.061     0.035    -0.002    accept
                                                     7. PAM       0.058     0.075     0.036    0.464     accept
                                                     8. JAM       0.109     0.081     0.042    -0.658    accept
                                                     9. CHO       0.031     0.000     0.018    -1.738    accept
                                                     10. SAN      0.126     0.113     0.047    -0.271    accept
                                                     11. SAV      0.103     0.092     0.043    -0.252    accept
                                                     12. STJ      0.062     0.052     0.033    -0.296    accept
























                                 Table 5. Estimated stock composition of groups of American shad.
                                          A. That portion of Virginia's ocean harvest which shared haplotypes
                                             with the baseline populations.
                                          B. That portion of Susquehanna River shad Lifted over Conowingo Dam
                                             in 1990-91 which shared hapLotypes with the baseline populations.
                                          C. That portion of Susquehanna Flats shad which shared haptotypes
                                             with the baseline populations.
                                          D. That portion of Rudee Inlet, Chincoteague, and Quinby Landed shad
                                             which shared haplotypes with the baseline populations.






                                     A. Estimated compositon of 81% of Virginia's 1991 intercept fishery-
                                         Nineteen percent of intercept shad could not be characterized by
                                         the current baseline data set.


                                                       Source       Contribution          SD


                                                       CT             0.0000             0.0000
                                                       DEL            0.0000             0.0000
                                                       HUD            0.14T7             0.0958
                                                       SF             0.0000             0.0000
                                                       NAN            0.0000             0.0000
                                                       PAM            0.0721             0.0636
                                                       JAM            0.0118             0.1309
                                                       CHO            0.0000             0.0000
                                                       SAN            0.0731             0.1422
                                                       SAV            0.0000             0.0000
                                                       STJ            0.0421             0.0408
                                                       SUS            0.6531             0.1189


                                    B. Estimated composition of 41% of Susquehanna River shad stock (1990-91).
                                         Fifty-nine percent of Susquehanna shad could not be characterized
                                         by the current baseline data set.



                                                       Source      Contribution           SD


                                                       COL            0.0000             0.0000
                                                       CT             0.0001             0.0001
                                                       DEL            0.0359             0.6163
                                                       HUD            0.0000             0.0000
                                                       SF             0.0000             0.0000
                                                       NAN            0.3105             0.1314
                                                       PAM            0.1008             0.0945
                                                       JAM            0.0000             0.0000
                                                       CHO            0.0000             0.0000
                                                       SAN            0.3640             0.1364
                                                       SAV            0.0000             0.0000
                                                       STJ            0.1887             0.5097


                                    C. Estimated composition of 91% of Susquehanna      Flats shad in 1991.
                                         Wine percent of Susquehanna Fiats shad were   unique and could not be
                                         characterized by the current baseline data set.



                                                       Source      Contribution           SO


                                                       COL            0.0000             0.0000
                                                       CT             0.0000             0.0000
                                                       DEL            0.0000             0.0000
                                                       HUD            0.5294             0.3342
                                                       NAN            0.0000             0.0000
                                                       PAM            0.0000             0.0000
                                                       JAM            0.0000             0.0000
                                                       CHO            0.0206             0.3800
                                                       SAN            0.4500             0.2843
                                                       SAV            0.0000             0.0000
                                                       STJ            0.0000             0.0000


















                                    D. Estimated composition of intercept harvests Landed at Rudee InLet,
                                        Chincoteague, and Quinby Virginia in 1991 (83%, 93%, and 91% of totaL samptes,
                                        respectiveLy). Unique individuaLs from each Location couLd not be characterized
                                        by the current data set.



                                                     Rudee                Chincoteague                 Quinby
                                    Source     Contrib.        SD      Contrib.        SD      Contrib.         SD


                                    CT          0.0000      0.0000      0.0000      0.0000       0.0000     0.0000
                                    DEL         0.0000      0.0000      0.0000      0.0000       0.0000     0.0000
                                    HUD         0.0000      0.0000      0.1740      0.1624       0.1516     0.1414
                                    SF          0.0000      0.0000      0.0000      0.0000       0.0009     0.0022
                                    NAN         0.0000      0.0000      0.0000      0.0000       0.0000     0.0000
                                    PAM         0.6601      0.5244      0.0000      0.0000       0.0000     0.0000
                                    JAM         0.0468      1.6115      0.0000      0.0000       0.0000     0.0000
                                    CHO         0.0000      0.0000      0.0000      0.0000       0.0000     0.0000
                                    SAN         0.0000      0.0000      0.3544      0.2568       0.0640     0.1541
                                    SAV         0.0979      1.2387      0.0077      0.2004       0.0011     0.0040
                                    STJ         0.0000      0.0000      0.1047      0.0999       0.0000     0.0000
                                    90          0.0000      0.0000      0.3006      0.1718       0.7824     0.1013
                                    91          0.1952      0.1689      0.0585      0.0564       0.0000     0.0000






                         Appendix A. Genotypes of American shad taken in the coastal intercept fishery
                                     outside the mouth of Chesapeake Bay during the Spring of 1991. Enzyme
                                     titles are abbreviated versions of those listed in the text on p.7.


                                                                        Enzyme Genotype
                             Location       ID      Aat Apa BcI Bgt Dra Ecl EcV Hin Kpn Pst Pvu  Sal Sma Sst Xba


                             Rudee Intet      I     D   A   A   A   A   A   B   A   A   A   A    A/C A   A   B
                                              2     D   A   A   A   A   A   A/C A   A   A/C A    A   A   A   A
                                              3         B   A   A   A   A   A   A   A   0   A    A   -   -   A
                                              4         A   A   -   A   A   A   A   -   A   A    - - - -
                                              5     A   A   A   F   A   A   A   A   A   A   A    A   A   A   A
                                              6         6   -   A   -   A       -   A   6   -    B   A   A   8
                                              7         A   - - -       A       A   A   -   A    - - -       A
                                              8     . . . .         A A A           A A A        - - -       A
                                              9         A   -   A       A           A   - - - - -            A
                                            10          .   A   -       A           A   -   A    A   - - -
                                            11      A A A A A A A A A A A A A A A
                                            12      -   A   A   A   -   A   -   A   A   A   A    A   - - -
                                            13      - - - - -           A   -   A   A   A   A    A   - - -
                                            14      A   -   A   -   A   A   -   A   A   A   A    A   -   -   A
                                            15      A   -   A   A   A   A   A   A   -   A   A    -   -   A   A
                                            16      A   A   A   A   A   A   A   A   8   A   A    B   A   A   A
                                            17      -   A   A   A   -   A   -   A   A   A   A    A   -   A   A
                                            18      A   A   A   A   A   A   A   A   B   A   A    A   A   A   A
                                            19      .   A   A   -   -   A   -   -   A   A   A    A   -   A   A
                                            20      . . . . .           A   -   -   A   A   A    - - - -
                                            21      . . . . .           A   - - - - - - - - -
                                            22      -   A   A   A   -   A   -   A   A   A   A    A   -   A   A
                                            23      . . .       A   -   A   -   A   A   - - - - -            A
                                            24      .   A   -   A   -   8   -   A   A   -   -    -   .-  -   A
                                            25      A   A   A   A   A   A   C   A   A   A   A    C   A   A   A
                                            26      A   A   -   A   A               A   - - - - -            A
                                            27      A   -   -   A   A   A   -       A   A   A    - - - -
                                            28      A   -   -   A   A   A   -   A   A   - - - - -            A
                                            29      A   A   A   A   A   A   C   A   A   A   A    A/C A   A   B
                                            30      A   -   -   A   A   A   -       A                        A
                                            31      D   -   A   A   A   A   -       A   A        A           A
                                            32      A   A   A   A   A   A   A   A   8   A   A    A   A   8   A
                                            33      - - - - -           A   - - - - - - - - -
                                            34      A   A   A   A   A   A   C   A   8   A   A    A   A   A   A
                                            35      A   -   -   A   A   A   -   -   A   - - - - -            A
                                            36      A   A   A   A   A   -   C   A   8   A   A    A/C -   A   B
                                            37      A   A   A   A   A   A       -   A   A   A    A   -   -   A
                                            38      A   -   -   A   A   -       A   A   A   A    - - -       A
                                            39      -   A   -   A   A   A       A   B   A   A    A   -   A
                                            40      A   A   A   A   A   A   C   A   6   A   A    A   A   A   B
                                            41      A A A A A A A A 6 E A A A A A
                                            42      A   A   A   A   A   -   A   A   B   A   A    A   -   A   8
                                            43      -   A   A   A   A   -   A   A   B   A   A    B   -   A   -
                                            44      -   A   A   A   A   A/B A   A   8   A   A    A   -   A   -
                                            45      A   A   A   A   A   A   A   A   6   A   A    A   A   A   B
                                            46      - - - - - - -               A   - - - - - - -
                                            47      .   A   A   A   -   -   A   A   B   -   A    A   - - -
                                            48      - - -       A   - - -       A   - - - - - - -
                                            49      A   -   A   A   A   -   A   -   A   A   - - -        A   -
                                            50      - - - - - - - - - - - - - - -
                                            51      - - - - - - - - - - - - - - -
                                            52      A   A   A   A   A   A   A   A   A   A   A    A   A   A   A







             Appendix A. Continued


                                        Enzyme Genotype
               Location  ID Aat Aps Scl BgL Dra Ecl EcV Hin Kpn Pst Pvu Sat Sma Sst Xba

               Chincoteague1A A  A A 8  A A A  A A A  A A A  A
                         2  D A  A A A  A A A  A A A  A A A  A
                         3  A A  A A A  A A A  A A A  A A A  A
                         4  D A  A A A  A A A  A A A  A A A  A
                         5  A A  A A A  A A A  A A A  8 A A  A
                         6  A A  A A A  A A A  8 A A  A A A  A
                         7  0 A  A A A  A A A  A A A  A A A  A
                         8       A A A A A A     A A A A A
                         9       A A A  A A A    A A  A A A
                         10   A  A A A  A A A    A -  8 - A
                         11 A A A A A A A A A A A A A A A
                         12 A A  A A A  A A A  A A A  A A A  A/B
                         13 A A  A A A  A A A  A A A  A A A  A
                         14 A A  A A A  A A A  A A A  A A A  A
                         15 A A A A A A A A A A A A A A 8
                         16   .  A A A  A A A  - A A  A - A  -
                         17 D A  A A A  A A A  A A A  A A A  A
                         18 - A A A A A A A A A    - - -  A -
                         19 . A  A A A  A A A  A A -  - A A  -
                         20 D A  A A A  A A A  A A A  A A A  A
                         21 A A A A A A A A A A A A A A A
                         22 A A  A A A  A A A  A A A  A A A  A
                         23 A A  A A A  A A A  A A A  A A A  A
                         24 A A  A A A  A A A  A A 8  A A A  A
                         25 A A A A A A A A A A A A A A A
                         26 A A  A A A  A A A  A A A  A A A  A
                         27 A A  A A A  A A A  A A A  A A A  A
                         28 A A  A A A  B A A  A A A  A A A  A
                         29 A A  A A A  A A A  A A A  A A A  A
                         30 A A  A A A  - - -  A - A  A A A  -
                         31 - -  A - - - - - - - - - - - -
                         32 . A  A - - - - - - -   A  A - A  -
                         33 - A  A - - - - - - -   A  B - A  -
                         34 A G  A A A  A A A  A A A  6 A A  A
                         35 A B  A A A  A A A  A A A  A A A  A
                         36 A A  A A A  A A A  A A A  A A A  A
                         37 A A  A A A  A A A  B A A  A A A  A
                         38 A A A A A A A A B A A A A A A
                         39 - - - - - - - -    A - A  A - - -
                         40 - -  A - - - - - - -   A  A - A  -
                         41 A A A A A   - - -  8 A A B A A A
                         42 A A  A A A  A A A  A A A  A A A  A
                         43 A A  A A A  A A A  B A A  B A A  A
                         44 A A  A A A  A A A  8 A A  B A A  A
                         45 A A  A A A  A A A  B A A  B A A  A
                         46 A A  A A A  A A A  A A A  A A A  A
                         47 A A  A A A  A A A  8 A A  A A A  A
                         48 A A  A A A  A A A  B A A  B A A  A
                         49 A A  A A A  A A A  8 A A  8 A A  A
                         50 A A  A A A  A A A  A A A  A A A  A
                         51 A A A A A A A A 8 A A 6 A A A
                         52 A A  A A A  A A A  B A A  B A A  A
                         53 A A  A A A  A A A  A A A  A A A  A
                         54 - A  A A A  - A A  - A -  A A A  -







              Appendix A. Continued
                                         Enzyme Genotype
                 Location ID  Aat Apa BcL 89L Dra Ecl EcV Hin Kpn Pst Pvu SaL Sma Sst xba


                 Quinby   I   A A A  A A A  A A A  A A A  A A  A
                          2     A               A  - A A
                          3     A A  A - - -  A A  A A A  A A  A
                          4     A
                          5   A A A  A A A  A A B  A A B  A A  A
                          6   - - - - - - -   A 8  A A A  A A  -
                          7   A A A  A A A  B A B  A A A  A A  A
                          8   A A - - - - -   A A  A - A  A A  A
                          9   - - - - - - - - - - - - - - -
                         10   A A A  A A A  A A A/BA A A/BA A  A
                         11   A A A A A A A A B A A B A A A
                         12   A A A  A A A  6 A 8  A A B  A A  A
                         13   . . . . . . . . . . . . . . .
                         14   . . . . . . . . . . .    A  - - -
                         15   . A - - -  A  - - - - -  A  - -  A
                         16   - - - - - - - - -    A - - - - -
                         17   A A A  A A A  A A A  A A A  A A  A
                         is   A A A A A A A A B A A A A A A
                         19   D A A  A A A  A A A  A B A  A A  A
                         20   . A - - - - -   A A  A A A  A A  A
                         21   - A A A A  - - -  A A A A - - A
                         22   A A A  A A -  A A C  A A A  A A  A
                         23   A A A  A A A  A A 8  A A A  A A  A
                         24   D A A  A A A  A A A  A A A  A A  A
                         25   D A A  A A A  A A A  A A A  A A  A
                         26   D A A  A A A  A A B  A A B  A A  A
                         27   0 A A  A A A  B A A  A A A  A A  A
                         28   D A A  A A A  A A A  A B A  A A  A
                         29   A A A  A A A  A A A  A A A  A A  A
                         30   D A A  A A A  A A 8  A A A  A A  A
                         31   A A A A A A A A 8 A A A A A A
                         32   D A A  A A A  A A A  A A A  A A  A
                         33   A A A  A A -  A A A  A A A  - A  -
                         34   D A A  A A A  A A A  A A A  A A  A
                         35   D A A  A A A  A A A  A A A  A A  A
                         36   . . .  A A -  - A -  A A A  A A  A
                         37   D A A  A A A  A A A  A A A  A A  A
                         38   D A A A A A A A A A A A A A A
                         39   D A A  A A A  A A A  A A A  A A  A
                         40   A - A  A - A  A - -  A A A  - A  -
                         41   A A A A A A A A A A A A A A A
                         42   A A A  A A A  A A A  A A A  A A  A
                         43   A A A  A A A  A A B  A A B  A A  A
                         44   A A A  A A A  A A B  A A A  A A  A
                         45   8 A B  A A A  A A E  A A A  A A  A
                         46   A A A  A A A  A A A  A A A  A A  A
                         47   A A A  A A A  A A A  A A B  A A  A
                         48   A A A  A A A  A A A  A A A  A A  A
                         49   A A A  A A A  A A A  A A A  A A  A
                         50   A A A  A A A  A A A  A B A  A A  A
                         51   A A A A A A A A A A A A A A A
                         52   A A A  A A A  A A B  A A A  A A  A
                         53   A A A  A A A  A A A  A A A  A A  A
                         54   A A A  A A A  A A A  A A A  A A  A
                         55   A A A  A A A  A A A  A A A  A A  A
                         56   A A A  A A A  A A A  A A A  A A  A



























                                 Appendix B. Haptotypes of American shad harvested in Virginia's coastal intercept
                                            fishery and of shad from fourteen baseline rivers. Abbreviations
                                            are as follows: R-Rudee Inlet, C-Chincoteague, a-uiriby, 90-Susuehanna
                                            River at Conowingo Dam in 1990, 91-Susuehanna River at Conowingo Dam in 1991,
                                            COL-CoLumbia, CT-Connecticut, DEL-DeLaware, HLID-Hudson, SF-Susuehanna
                                            Flats, NAN-Nanticoke, PAM-Pamunkey, JAM-James, CHO-Chowan, SAN-Santee,
                                            SAV-SAvannah, STJ-St. Johns.






                                                                                            Poputation
                                     HapLotypes            R    C   0 90 91 COL CT DEL HUD SF NAN PAN JAN CHO SAN SAV STJ


                                     1AAAAAAAAAAAAAAA      2 16     14 17           15 22 14      6    3   5    5  9 14      3   6
                                     2AAAAAAAAAAAAAAB           I
                                     3AAAAAAAAAAAAAAA/B         1
                                     4AAAAAAAAAAAAAAD,
                                     5AAAAAAAAAAAAARA                    3
                                     6AAAAAAAAAAAAABA/B                  1
                                     7 AAAAAAAAAAAAASD                                                 1
                                     8AAAAAAAAAAAABAA                                                  1        1            1   3
                                     9AAAAAAAAAAAABBA                                                           3            9
                                     10 AAAAAAAAAAAACAA                                                                      1
                                     11 AAAAAAAAAAABAAA         1   1    2                2            1            1    4.
                                     12 AAAAAAAAAAABABA                      1
                                     13 AAAAAAAAAAACBBA                                                                      1
                                     14 AAAAAAAAAAADAAA                                                                      1
                                     15 AAAAAAAAAARAAAA         I   1                     1   4
                                     16 AAAAAAAAAABAARA                                                         1
                                     17 AAAAAAAAAABABBA                                                                      1
                                     18 AAAAAAAAAA/BAAAAA                1
                                     19 AAAAAAAAAA/CAAAAA                1
                                     20 AAAAAAAAAA/CABAAA/B              1
                                     21 AAAAAAAAACAAAAA                                   1
                                     22 AAAAAAAAACAAAAA                                                         8                1
                                     23 AAAAAAAAADAAAAA                              1
                                     24 AAAAAAAAAEAAAAA                                                                          1
                                     25 AAAAAAAAAFAAAAA                                                                  11
                                     26 AAAAAAAAA/BAAA/BAAA         1
                                     27 AAAAAAAABAAAAAA    1    4   5    5          6     a   2            1    3        2   2   2
                                     28 AAAAAAAAsAAAAAs    1                                               2    1  2
                                     29 AAAAAAAABAAAABA    1                 3
                                     30 AAAAAAAABAAABBA                                   1                                  6
                                     31 AAAAAAAABAAACBA                                                                      1
                                     32 AAAAAAAABAABAAA    1    7   3        2      2     2   5  2         2    2        7   1
                                     33 AAAAAAAABAABABA                      1                         1
                                     34 AAAAAAAABAABACA                                                                  1
                                     35 AAAAAAAABAABBAA                                                                          1
                                     36 AAAAAAAABAABBBA                                   1                                  1
                                     37 AAAAAAAABCAAAAA                                                         1
                                     38 AAAAAAAABCAABBA                                                         1
                                     39 AAAAAAAAscBBAAA                                                         1
                                     40 AAAAAAAABC/AABAAA                                                       1
                                     41 AAAAAAAABFAABBA                                                                      1
                                     42 AAAAAAAACAABAAA                                                         1
                                     43 AAAAAAAACAABBBA                                                                      I
                                     44 AAAAAAAADAAAAAA
                                     45 AAAAAAAADAAAABA
                                     46 AAAAAAAAECAAAAA                  1
                                     47 AAAAAAABAAAAABA
                                     48 AAAAAAABAAAABBA                                                                      1
                                     49 AAAAAAABEAAAAAA    1             1
                                     50 AAAAAAACAAAAAAA             1                         1
                                     51 AAAAAAA/CAAAAABAA/B              1
                                     52 AAAAAAA/CABAAAAAA                1
                                     53 AAAAAAA/CABEAAAAA                I
                                     54 AAAAAABAAAAAAAA                  3                    4        3   1
                                     55 AAAAAABAAAABAAB                                                1
                                     56 AAAAAABAAACAAAA                                       2
                                     57 AAAAAABAAA/CAAABA                1








                                                                                     PopuLation
                              HapLotypes               R   C   0 90   91 COL CT DEL HLID SF WAN PAN JAM CHO SAN SAV STJ


                              58 AAAAAABASAAAAAA               1    1
                              59 AAAAAASABAABAAA               1
                              60 AAAAAACAAAAAAAA                    4              1       2   4    3
                              61 AAAAAACAAAAAAAA/B                  1
                              62 AAAAAACAAAAAAAD                    1
                              63 AAAAAACAAAAABBA                                               1
                              64 AAAAAACAAAABAAA                                                    1
                              65 AAAAAACAAAACAAA       1
                              66 AAAAAACAAAAA/CAAB     1
                              67 AAAAAACABAAAAAA       1                                            2
                              68 AAAAAACABAAAAAB       1
                              69 AAAAAACABAAAABA                                                    1
                              70 AAAAAACABAASAAA                                                    2
                              71 AAAAAACABAABAAA                                                    1
                              72 AAAAAACACAABAAA                                                    1
                              73 AAAAAACABAAA/CAAS     1
                              74 AAAAAACBAAAAAAA                                                       1
                              75 AAAAAACBAAAAABD                    1
                              76 AAAAAACBAAAABBA
                              77 AAAAAACBAAACBBA
                              78 AAAAAACBACAAASA
                              79 AAAAAACBBAAAABA
                              80 AAAAAACBBAAC/BBBA
                              81 AAAAAACSDAAAAAA
                              82 AAAAAACCAAAAAAA
                              83 AAAAAACCAAAAARA
                              84 AAAAAACCAAAABBA
                              85 AAAAAACCCAACHBA
                              86 AAAAAADBBAAAABA
                              87 AAAAABAAAAAAAAA          1                                                             2
                              88 AAAAABAAAA/CAAAAA                  1
                              89 AAAAABAABAAAAAA                                                                1
                              90 AAAABAAAAAAAAAA          1
                              91 AAAABBAAAAAAAAA                                                       1
                              92 AAAA/EAAAAAAAAAAA                         26          2
                              93 AAAA/EAAAASAAAAAA                                     3
                              94 AAAA/EAARAAAAAAAA                          3
                              95 AAAA/EAACAAAAAAAA                          5
                              96 AAAAA/EACAAAAAAAA                  1
                              97 AAABAAAAAAAAAAA                                                    1
                              98 AAABAAAAAAAABAA                                   1
                              99 AAASAACAAAAAAAA                                                    1
                              100 AAACAAAAAAABBBA
                              101 AAACAAAABAAABBA
                              102 AAAEAAAAAAAAAAA                           1
                              103 AAAEAACCABAAAAA                           1
                              104 AAAFAAAAAAAAAAA      1
                              105 AABAAAAABAAAAAA
                              106 AACAAAAAAAAAAAA
                              107 AACAAAAAAABAAAA
                              108 AADAAAAAAAAAAAA                                                               2
                              109 AADAAAAABAAAABA
                              110 AADAAAAAAAAABBA
                              111 AADAAAAACCABAAC
                              112 AAEAAAAAAAAAAAA                                                               1
                              113 AAEAAAAAACABBAA

                              114 ASAAAAAAAAAAAAA








                                                                                         Population
                               HapLotypes                 R   C    Q 90 91 COL CT DEL HLID SF NAN PAM JAN CHO SAN SAV STJ

                               115 ABAAAAAAAAAABAA                                     2
                               116 ABAAAAAAAAAABAA                                                                              3
                               117 ABAAAAAAAAACAAA                                     1
                               118 ABAAAAAAACAAAAA                                                           2
                               119 ABAAAAAA88AAABA
                               120 ABAAAACAAABAAAA
                               121 ABAAAACABBAAAAA
                               122 ABAAABAAAAAABAA                                                                              1
                               123 ABACAAAAACAAAAA
                               124 ACAAAAAAAAAAAAA                                                                         1    3
                               125 ACAAAAAAAAAABAA                                                                              1
                               126 ACAAAAAAACAAAAA                                                                         2
                               127 ACABAAAAAAAACBA                                                                         2
                               128 ADDAAACBAAAAAAA
                               129 AEAAAAAAAAAAAAA
                               130 AEAAAACAAAAAAAA
                               131 AEAAAACBBBAAAAA
                               132 AA/EAAAACBAAAABBA                       1
                               133 AFAAAkAAAA/BABAAA                   1
                               134 AGAAAAAAAAABAAA            1
                               135 BAAAAABAAAAAAAA/B                   1
                               136 BAAAAACCAAAAAAA,                    I
                               137 BABAAAAAEAAAAAA
                               138 BBAAAAAAAEAABAA
                               139 BBAABACABAAAABA                     1
                               140 CAAAAAAABBA8AAA                     1
                               141 CAAAAAAACAAABBA
                               142 CAAAAAABBA/CAAABA                   1
                               143 CAAAAACAAAAAAAA                     I
                               144 CAAAAAA/CAAAAAAAA                   2
                               145 CAAAAACAAAAAAAA/B                   I
                               146 CAAAAACBAAAARBA                                                                         1
                               147 CAAAAACBAAABBBA                                                                         1
                               148 CAAAAACBAA/CAAAAA                   1
                               149 CAAAABCBAAAAAAA                                                                              1
                               150 DAAAAAAAAAAAAAA            5    8   4
                               151 DAAAAAAAAAAABAA                                                                         1
                               152 DAAAAAAABAABAAA                                              1
                               153 DAAAAAAAAABAAAA                 2   1
                               154 DAAAAAAAAA/CAAAAA                   1
                               155 DAAAAAAAABAAAAA                     1
                               156 DAAAAAAABAAAAAA                 1   1
                               157 DAAAAAAABAABAAA                 1   1
                               158 DAAAAAA/CBAAAAABA                   1
                               159 DAAAAAA/CAAA/CAAAAA 1
                               160 DAAAAABAAAAAAAA                 1
                               161 DAAAAABAAAAA/CAAB       1
                               162 DAAAAACAAAAAAAA                     1

                                                         15 40 42 75 10 36 24 48 39 11 24 31 38 12 38 45 26



                                                                                                                              JOAA COASTAL SERVICES CTR LIBRARY
                                                                                                                               3 6668 14111266 6