Head from ventral and right side of Acipenser oxyrinchus 200 cm TL juvenile, from St. Lawrence River at Kamouraska, Quebec, which now resides at the Montreal Biodome (see the typical damage to the tip of the rostrum). Originals by Paul Vecsei. 1996.
Environmental Biology of Fishes 48: 385 398, 1997. 1997 Kluwer Academic Publishers. Printed in the Netherlands. Molecular analysis in the conservation of sturgeons and paddlefish Isaac I. Wirgin 1, Joseph E. Stabile 1 & John R. Waldman 2 1 Nelson Institute of Environmental Medicine, New York University Medical Center, Long Meadow Road, Tuxedo, NY 10987, U.S.A. 2 Hudson River Foundation, 40 West 20th Street, Ninth Floor, New York, NY 10011, U.S.A. Received 15.4.1995 Accepted 14.5.1996 Key words: mitochondrial DNA, nuclear DNA, Acipenser, Scaphirhynchus, Polyodon, stock identification, hybridization Synopsis Sturgeon and paddlefish populations worldwide have declined because of anthropogenic influences. The structure and magnitude of genetic diversity of natural populations serves to buffer these fishes against environmental variation and should be maintained. Modern molecular biological techniques provide the ability to sensitively characterize and quantify the extent of genetic variation in natural populations. We provide a summary of those problems in sturgeon population biology that are amenable to investigation with DNA approaches, and their applications to date. These have included genetic identification and discrimination of taxa, identification of hybrids, stock identification, mixed-stock analysis, and estimation of gene flow and homing fidelity. To date, almost all studies have been restricted to North American fauna. Improvements to these technologies, including nondestructive sampling, should permit more widespread application of molecular approaches to problems of acipenseriform conservation. We suggest that the use of more sensitive molecular tools such as analyses of hypervariable repetitive and non-coding single copy nuclear DNA may assist management even in those taxa which exhibit overall low levels of genetic diversity. Introduction The worldwide diversity of sturgeons and paddlefishes is currently threatened, and in fact, the extirpation of some forms in Europe and Asia has been reported (Rochard et al. 1990, Birstein 1993, Waldman 1995). At the same time, population abundances of most species are at historically low levels, including many North American taxa (e.g., Kynard 1997 this volume, Smith & Clugston 1997 this volume). Although molecular analysis has rapidly become a primary tool in the management and conservation of fishes (Hallerman & Beckmann 1988, Wirgin & Waldman 1994, Bernatchez 1995), its use for problems involving acipenseriforms has lagged, perhaps due to the scarcity of many of these species and the consequential difficulty of obtaining adequate numbers of tissue samples. However, improvements to DNA analysis technologies, including nonlethal sampling, slackening of tissue preservation requirements, and the ability of PCR to allow analysis of minute amounts of tissue have relaxed many of the constraints that challenged sample collection. Thus, we anticipate an expanding role for molecular analysis in the conservation of these highly threatened fishes. Wirgin & Waldman (1994) listed 12 areas of investigation in fisheries biology amenable to DNA analyses. Here, we review studies in these areas in which molecular techniques have been applied to problems in the conservation of sturgeons and paddlefishes. We also suggest other areas to which
386 these techniques may be applied. Additionally, we present a genetic comparison between two ambiguously differentiated, putative sturgeon species, Acipenser oxyrinchus (American Atlantic sturgeon) and A. sturio (European Atlantic sturgeon). Moreover, variation in the size of mtdna within individuals (heteroplasmy) was seen in A. transmontanus, but not A. oxyrinchus (Stabile et al. 1996). A thorough review of the acipenseriform genome appears in Birstein et al. (1997 this volume). Characteristics of the acipenseriform genome All acipenseriforms are polyploids and their chromosomes can be arranged as a series 4n 8n 16n (Birstein et al. 1993, Blacklidge & Bidwell 1993, Birstein et al. 1997 this volume). Chromosome numbers are very high: 120, 240, and possibly, 500. Two forms of chromosomes are found: normal-sized macrochromosomes and very small microchromosomes. A number of studies have indicated slow rates of DNA and protein evolution of sturgeons and paddlefishes (Birstein 1993); we believe that extended generation times in comparison with most other fishes may decelerate their molecular evolution. Acipenseriforms hybridize readily in the laboratory and in nature, at even the intergeneric level (Birstein et al. 1997 this volume). In most respects, the structure and evolutionary change of the acipenseriform mitochondrial DNA (mtdna) genome is typical of vertebrates. Estimates of the size of mtdna in Acipenser transmontanus (white sturgeon, 16.1 16.7 kilobases, Brown et al. 1992) and Acipenser fulvescens (lake sturgeon, 16.6 16.9 kb, Guenette et al. 1993) are within the range of most other vertebrate taxa (16.5 ± 0.5 kb, Meyer 1993). Gene composition and gene order of acipenseriform mtdna have not been directly investigated; however, the mtdna control region of A. transmontanus is organized similarly to other vertebrates (Buroker et al. 1993). Differences in the size of the mtdna molecule have been observed among acipenserids. In A. transmontanus (Buroker et al. 1990) and A. fulvescens (Ferguson et al. 1993), the overall size of the mtdna molecule differs among individual specimens (both within and among populations) because of variable numbers of tandem repeat units within the control region. In contrast, no evidence of mtdna length variation was observed among specimens of A. oxyrinchus (Waldman et al. 1996b). Molecular studies of acipenseriforms directed toward management and conservation Identification and discrimination of taxa In the U.S., the taxonomic status of threatened or endangered populations of vertebrates is important in determining the degree of protection they may receive under the Endangered Species Act (O Brien & Mayr 1991). For Acipenser oxyrinchus, the Altantic (A. o. oxyrinchus) and Gulf forms (A. o. desotoi) were designated subspecies based on differentiation of morphological features, although only two Gulf specimens were examined (Vladykov 1955). Wooley (1985) reanalyzed these morphological features of a larger sample of Gulf sturgeon and concluded that only one, relative spleen length, was diagnostic. However, values for relative spleen length of the two forms were not discrete and it may be that differences in these values are ecophenotypic in nature. Ong et al. (1996) used direct sequencing of a hypervariable area (203 base pairs) of the control region of mtdna to quantify the extent of genetic differentiation between the two putative subspecies. Representatives of each subspecies from populations across their distributions were surveyed and three fixed differences were found among 15 polymorphic sites. An additional two nucleotides were nearly fixed. Although polymorphisms also were detected within populations across the distribution of each subspecies, no fixed differences among populations were found. Ong et al. (1996) concluded that these data strongly supported the designation of subspecies of A. oxyrinchus. The relationship between A. oxyrinchus and A. sturio has long been debated. These fish are found on opposite sides of the North Atlantic and are phenotypically very similar. Acipenser oxyrinchus occurs widely along the Atlantic coast of North Amer-
387 Figure 1. Comparison of mtdna control region sequence from Acipenser sturio and the Atlantic and Gulf of Mexico subspecies of Acipenser oxyrinchus. Alignment begins nine nucleotides upstream of the terminal association sequence (TAS 1) of trna proline. The position of the polymorphic sites observed among all 159 specimens of A. oxyrinchus are highlighted in bold. The position of the three fixed sites between the subspecies of A. oxyrinchus are noted by asterisks. ica. Acipenser sturio, which once had a similarly broad range in Europe and Asia, now occurs only in very low abundances in both the Gironde River, France, and the Black Sea (Rochard et al. 1990). Some workers have considered the Atlantic sturgeon to be synonymous with or a subspecies of A. sturio (Scott & Scott 1988, Birstein 1993), but Vladykov & Greeley (1963) and Magnin (1964) recommended they be given separate specific status, pending additional research. We obtained a tissue sample from one A. sturio specimen captured during 1994 in the Gironde River. We then compared a portion (203 bp) of the control region of mtdna between A. sturio and A. oxyrinchus. We found a minimum of 31, and a maximum of 33 nucleotide substitutions between the individual of A. sturio and 159 individuals of both subspecies of A. oxyrinchus (Figure 1). Three sites also exhibited nucleotide additions or deletions. In comparison, the number of nucleotide substitutions between any pair of specimens of the two subspecies of A. oxyrinchus ranged between five and eight, with no additions or deletions. Excluding additions or deletions, nucleotide divergence between A. oxyrinchus and A. sturio was approximately 15%, much higher than the maximum of 3.5% between two subspecies of A. oxyrinchus (Ong et al. 1996). There are no unambiguous criteria for the interpretation of molecular data in determining taxonomic status (e.g., O Brien & Mayr 1991, Wayne 1992). However, we believe that the level of differentiation observed argues strongly for full species status of each of the western and eastern Atlantic sturgeons. Scaphirhynchus suttkusi (Alabama sturgeon) has recently been described (Williams & Clemmer 1991); this exceedingly rare species is restricted to the Mobile River basin of Alabama and Mississippi. Scaphirhynchus suttkusi are morphologically similar to S. platorynchus (shovelnose sturgeon), but differ significantly in six meristic and at least eleven mensural variables. The extent of genetic similarity between S. suttkusi (one specimen) and S. platoryn-
388 chus and S. albus (pallid sturgeon) was compared using PCR products (amplicons) (Genetic Analyses 1 ). Acipenser fulvescens and the two subspecies of A. oxyrinchus were analyzed to provide additional points of reference. PCR primers were designed based on conserved sequences (among mammals) for eight ndna genes and these were shown to amplify sturgeon DNA and to produce amplicons. These amplicons were then digested with a battery of different restriction enzymes and DNA band sharing was compared between two putative taxa. The level of genetic differentiation between S. suttkusi and the other two species of Scaphirhynchus was greater than that between subspecies of A. oxyrinchus but less than that between the two species of Acipenser. The authors also concluded that distinct species designation for S. albus and S. platorynchus may not be valid, but instead, that the two forms may represent morphological variants of a single species. A later, expanded mtdna study (Campton et al. 2 ) also found very low levels of divergence between S. suttkusi (N = 3) and the other two Scaphirhynchus species. A unique haplotype distinguished the three specimens of S. suttkusi from all individuals of the other two species of Scaphirhynchus collected from the upper Missouri River, but this haplotype differed from the most common haplotype found among S. albus and S. platorynchus by only a single nucleotide substitution. Campton et al. 2 concluded that the mtdna data support the morphological and biogeographic arguments for recognizing S. suttkusi as an endangered species of distinct population segment as defined by the U.S. Endangered Species Act. Analysis of additional specimens of S. albus and S. platorynchus from the lower Mississippi River (nearer the range of S. suttkusi) is 1 Genetic Analyses, Inc. 1994. Genetic studies of Scaphirhynchus spp. Report to the U.S. Army Corps of Engineers, Omaha District; U.S. Fish and Wildlife Service, Bismarck, North Dakota; U.S. Army Corps of Engineers, Mobile District. 41 pp. 2 Campton, D.E., A.I. Garcia, B.W. Bowen & F.A. Chapman. 1995. Genetic evaluation of pallid, shovelnose, and Alabama sturgeon (Scaphirhynchus albus, S. platorynchus, and S. suttkusi) based on control region (D-loop) sequences of mitochondrial DNA. Final Report to the U.S. Fish and Wildlife Service, Bismarck, North Dakota. 35 pp. needed to confirm the genetic discreteness of S. suttkusi from its congeners. Analyses of hybridization and introgression Scaphirhynchus albus and S. platorynchus have been recognized as similar, but distinct species of river sturgeon within the Mississippi River drainage (Forbes & Richardson 1905). Significant differences in meristic, morphometric, and life history characteristics support this taxonomic division. In recent years, a small but increasing number of fish collected from the Missouri and Mississippi rivers exhibited intermediacy in the expression of these discriminatory characters, suggesting the existence of interspecific hybrids (Carlson et al. 1985). Populations of both species, but particularly of S. albus, currently are severely depleted, probably due to habitat alterations, and S. albus is listed as endangered by the U.S. Fish and Wildlife Service (Dryer & Sandoval 3 ). Hybridization between two sturgeon species under these circumstances would not be unexpected; Hubbs (1955) concluded that anthropogenic impacts and sharp imbalances in the abundances of potentially hybridizing species significantly increase the frequency of hybridization events in fishes. Genetic studies were conducted to quantify the relatedness of S. albus and S. platorynchus and to identify markers which could be used to identify F 1 and later generation hybrids. Protein electrophoresis studies revealed only low levels of polymorphism within and between these taxa and those loci (3 of 37) which were polymorphic did not display significant interspecific allelic differences (Phelps & Allendorf 1983). Thus, protein electrophoresis proved insensitive in distinguishing these species, perhaps due to its focus on gene products that are essential for survival and that tend to be conservative across taxa. Recently, direct analyses of nuclear DNA (ndna) and mtdna were used to further discrimi- 3 Dryer, M.P. & A.J. Sandoval. 1993. Recovery plan for the pallid sturgeon (Scaphirhynchus albus). U.S. Fish and Wildlife Service, Denver, 55 pp.
389 nate between S. albus and S. platorynchus with the expectation that higher levels of diversity would be detected with these more sensitive approaches. Nuclear DNA sequences were PCR amplified with conserved primers and these PCR products were then digested with a battery of different restriction enzymes and DNA band sharing was compared between the two putative taxa. Variability among individuals in the digestion patterns of amplicons was seen only at a prealbumin-related locus (Genetic Analyses, Inc. 1 ). A comparison of allelic frequencies between S. albus and S. platorynchus at the prealbumin-related locus showed no significant differences. Campton et al. 2 sequenced more than 400 bp of mtdna from the control regions of S. albus (N = 18) and S. platorynchus (N = 20) and found 8 haplotypes from an area of sympatry in the upper Missouri River. However, these haplotypes overlapped between the two species to the extent that maximum parsimony analysis did not reveal two distinct species-congruent branches, but χ 2 analysis did show significant haplotypic frequency differences (p < 0.001) between the species. Control region of other acipenserids were similarly sequenced to serve as references; mean nucleotide diversity among specimens of the three scaphirhynchids was 0.58%, compared to 1.20% for A. transmontanus. Also, the mean nucleotide diversity was 0.62% between S. albus and S. platorynchus, compared with 14.1% between A. transmontanus and A. medirostris (green sturgeon). Campton et al. 2 were unable to dismiss hybridization as a factor in the differentiation of S. albus and S. platorynchus, but they favored the hypothesis that the two species maintain some degree of reproductive isolation. Identification of stocks and assessment of genetic variability Several studies have assessed genetic differentiation among hypothesized stocks of sturgeons and paddlefishes. Identification of genetic stocks requires that individuals from hypothesized stocks be surveyed for genetic variability, usually at rapidly evolving sites. If statistically significant genetic differences exist among two or more spawning populations, they may be considered separate stocks and managed as distinct units (Waldman & Wirgin 1994). Acipenseriforms investigated for stock differences include two freshwater species, Polyodon spathula (American paddlefish) and A. fulvescens; and three anadromous sturgeons, S. stellatus (stellate sturgeon), A. transmontanus, and A. oxyrinchus. Genetic studies on populations of P. spathula have been conducted on both the protein and DNA levels. Carlson et al. (1982) observed extremely low levels of protein variation in P. spathula and did not detect evidence of differentiation among populations. In contrast, using both protein analysis and restriction fragment length polymorphism (RFLP) analysis of mtdna, Epifanio et al. 4 found greater levels of genetic polymorphisms among P. spathula collected from 21 populations, including the Mississippi, Pearl, Alabama, and Neches drainages. However, the majority of protein variation was observed within populations and was of little use in elucidating population structure. Mitochondrial DNA genotypes showed greater geographic partitioning with both northern (mid-missouri River) and southern haplotypes evident, suggesting the existence of some population structure within their overall distribution. Acipenser fulvescens also is widely distributed in North America. Porter et al. 5 used protein electrophoresis to compare the genetic status of A. fulvescens from Lake Erie with other populations from the Laurentian Great Lakes. Only the lactate dehydrogenase enzyme system (LDH) revealed useful population-level variation. Although the Lake Erie population was the least variable of those surveyed, one of its three LDH phenotypes was not found among other populations. Guenette & Fortin (1993) found low levels of mtdna variation in A. fulvescens from the St. Lawrence River and James 4 Epifanio, J., M. Nedbal & D.P. Phillipp. 1989. A population genetic analysis of paddlefish (Polyodon spathula). Report to Missouri Department of Conservation. 63 pp. 5 Porter, B., T. Cavender, P. Fuerst & T. Nickell. 1995. The genetic status of lake sturgeon in Lake Erie and other populations from the Laurentian Great Lakes. Report to Ohio Department of Natural Resources. 47 pp.
390 Bay drainages. No differences in mtdna haplotypes were detected in sturgeon from different locations in the St. Lawrence River; however, haplotypic frequencies differed from those in the Waswanipi River in the James Bay basin. The lower mtdna heterogeneity in fish from the St. Lawrence River was interpreted to reflect greater anthropogenic influences on population abundance. Ferguson et al. (1993, 1997 this volume) used RFLP analysis of mtdna and direct sequencing of the control region of mtdna to investigate stock structure of A. fulvescens from Canadian systems, with an emphasis on the Moose River basin in Ontario. Little differentiation of mtdna haplotypes was found among major tributaries of the Moose River basin, suggesting an absence of discrete stocks. However, sturgeon from the Moose River basin exhibited significantly higher levels of mtdna diversity and were genetically differentiated from fish from the Great Lakes-St. Lawrence drainage and other Hudson Bay-James Bay populations. These workers hypothesized that mtdna differentiation in sturgeon from Canadian waters resulted from post-pleistocene colonization from separate refugia in the Mississippi and St. Lawrence River drainages. Further investigation of mtdna diversity in extant stocks of A. fulvescens in eastern and western U.S. drainages should resolve this question. Acipenser stellatus is an abundant sturgeon species in the Caspian Sea. Stock structure of this species in the southern portion of the Caspian Sea was investigated by restriction enzyme digestion of the PCR amplified ND5/ND6 region of mtdna. Although polymorphic haplotypes were identified, no significant differences in haplotype frequencies were detected among fish from four regions, which led to the conclusion that A. stellatus in the southern Caspian Sea probably represent a single stock (M. Pourkazemi personal communication). Acipenser transmontanus occurs from California to Alaska with viable fisheries currently centered in the Columbia River, Washington and the Fraser River, British Columbia. Both sequencing of the rapidly evolving control region of mtdna and RFLP analyses of the entire mtdna molecule were used to examine the extent of differentiation between populations of A. transmontanus from the Fraser and Columbia rivers (Brown et al. 1992, 1993). In the most recent glacial advance, the lower Columbia River was believed to remain an ice-free refugium, whereas the Fraser River was completely glaciated. Sequence variation of the control region was consistent with the hypothesis that the Columbia River was the founder source for the Fraser River population (Brown et al. 1993). However, levels of overall mtdna diversity were significantly higher among fish from the Fraser River than the Columbia River, despite the greater recency of the Fraser River population (Brown et al. 1992). It was hypothesized that lower genetic diversity in the Columbia River population resulted from reduced access to historic spawning areas due to the construction of dams. Brown et al. (1992) also found significant differences in mtdna haplotype frequencies between samples from the Fraser River and Columbia River, indicating that these two rivers support genetically distinct populations of sturgeon. Because haplotype differences were not fixed between the two populations, the authors suggested that low levels of gene flow between systems may be occurring. Alternatively, we suggest that given the brief time since divergence of these populations, the accumulation of significant differences in genotype frequencies indicates effective reproductive isolation of these systems. Populations of A. oxyrinchus occur in drainages of the Gulf of Mexico and along the Atlantic coast of North America. Acipenser oxyrinchus desotoi (Gulf sturgeon) are considered threatened by the U.S. Fish and Wildlife Service. Historically, A. o. desotoi were found in major river systems extending from central Florida to the Mississippi River; many of these drainages still host depleted populations (USFWS and GSMFC 6 ). Efforts are being considered to restore depressed populations through hatchery supplementation. However, knowledge of the stock composition of A. o. desotoi was federally mandated prior to the initiation of restorative efforts, so that native gene pools are pre- 6 U.S. Fish and Wildlife Service and Gulf States Marine Fisheries Commission. 1995. Gulf sturgeon recovery plan. Atlanta, Georgia. 170 pp.
391 Figure 2. Gulf of Mexico rivers from which specimens of Acipenser oxyrinchus were obtained. served. The Suwannee River population has been extensively studied over the past decade and probably contains the largest population of sturgeon along the Gulf of Mexico. Miracle & Campton (1995) examined the extent of genetic variation in sturgeon from the Suwannee River to determine if its population constituted a single homogeneous unit. Sequence analysis of 268 base pairs of a highly variable area of the mtdna control region did not reveal significant genetic heterogeneity among sturgeon from different sampling locations. We used RFLP and sequencing analysis of mtdna to assess the stock structuring of A. o. desotoi populations among eight drainages (Figure 2) extending from the Mississippi River to the Suwannee River (Stabile et al. 1996). RFLP analysis using four diagnostic restriction enzymes yielded eight composite haplotypes; genotypic diversity indices (Nei & Tajima 1981) ranged between 0.173 for the Choctawhatchee River sample to 0.732 for the Yellow River collection. Significant differences (p < 0.05) in haplotype frequencies indicated substantial geographic structuring of sturgeon populations; results from the RFLP (N = 164) and sequence (N = 141) analyses were largely congruent (Figure 3). Five regional or river-specific stocks were identified (from west to east): (1) Lake Ponchartrain and Pearl River, (2) Pascagoula River, (3) Escambia and Yellow rivers, (4) Choctawhatchee River, and (5) Apalachicola, Ochlockonee, and Suwannee rivers. These results suggest strong reproductive isolation of A. o. desotoi stocks on at least a regional basis, and point to the inadvisibility of mixing of hatchery-reared progeny of broodstock from different Gulf rivers. Also, we used RFLP analysis of mtdna with five diagnostic restriction enzymes to characterize the stock structure of populations of A. o. oxyrinchus along the Atlantic coast, including the St. Lawrence River, Quebec; St. John River, New Brunswick; Hudson River, New York; Edisto River, South Carolina; and four rivers in Georgia; the Altamaha, Ogeechee, Savannah, and Satilla (Waldman et al. 1996a, b). Chi-square analysis showed the eight populations could be grouped as three highly differentiated (p < 0.0001) stocks: (1) Canadian (St. Lawrence and St. John rivers); (2) Hudson River; and (3) southeastern (Edisto, Savannah, Ogeechee, Altamaha, and Satilla). Composite haplotypes showed a clear cline in genotypic diversity indices (Nei & Tajima 1981) among populations that ranged from complete monomorphism (0.0) of the two Canadian populations to considerable polymorphism among southeastern populations (e.g., Edisto River: 0.646; Ogeechee River: 0.750). A latitudinal cline in genotypic diversity along the Atlantic coast is consistent with founder effects among
392 Figure 3. UPGMA phenograms of the interpopulation diversity indices for the (a) RFLP data, and (b) control region sequence data for Acipenser oxyrinchus from the Gulf of Mexico. northern populations that recolonized glaciated drainages from more genotypically diverse populations in southern, nonglaciated regions. Mixed-stock analysis For some wide ranging species, fisheries have developed distant from spawning and nursery areas, and these fisheries may harvest individuals from more than one stock. For management purposes, it is important to quantitatively estimate the relative contributions of individual stocks to mixed fisheries to allow managers to protect threatened stocks at sites distant from their natal rivers. Successful application of genetic approaches to mixed-stock analysis is dependent on the existence of significant differentiation of genetic characters among spawning stocks which contribute to the mixed fishery (Utter & Ryman 1993, Xu et al. 1994). To conduct mixed
393 stock analysis, frequencies of genotypes must be characterized in reference spawning stocks and in the mixed fishery. A targeted coastal fishery for A. o. oxyrinchus has developed in recent years along the mid-atlantic coast of New Jersey and New York (New York Bight) and bycatch fisheries have been reported off the southeastern coast of the U.S. (Collins et al. 1996). Waldman et al. (1966a) performed RFLP analysis of specimens of A. o. oxyrinchus from eight populations from Canada to Georgia and concluded, based on haplotype frequency differences, that three statistically discrete (p < 0.0001) stocks exist: (1) Canadian, (2) Hudson River, and (3) southeastern. Haplotypic frequency data of these stocks were then used in a mixture model (constrained least squares; Xu et al., 1994) to estimate the relative contributions of each of these stocks to a sample of Atlantic sturgeon (N = 112) from the fishery in the New York Bight off New Jersey. This analysis showed a 97% to 99% contribution from the Hudson River stock, with the remainder from the southeastern stock. The overwhelming contribution of the Hudson River stock was attributed both to (1) a hypothesized tendency for marine migrating Atlantic sturgeon to remain within the geographic provinces of their natal rivers (the Hudson River is within the Virginian province), and (2) to the absence of other robust American Atlantic sturgeon populations within the Virginian province. Gene flow and homing fidelity Most populations of sturgeons are anadromous or potamadromous and thus, migrate from marine or lake waters to rivers to spawn (Bemis & Kynard 1997 this volume). However, almost nothing is known of the degree of homing fidelity shown by acipenseriforms. Although homing fidelity of fishes may be studied directly by means of capture-markrecapture (e.g., Melvin et al. 1986), the relative scarcity and high value of sturgeons precludes such an approach. An alternative is to assess homing fidelity indirectly through genetic analysis (Tallman & Healey 1994). Homing fidelity of sturgeons through genetic analyses would best be assessed among populations in rivers that drain to a common water body and that historically have a stable geographic history to avoid confoundment by founder effects as a consequence of recolonizations. Stabile et al. (1996) used both RFLP and sequencing analysis of mtdna to estimate gene flow among five stocks of A. o. desotoi that occur in eight drainages that feed the Gulf of Mexico between Mississippi and Florida. The five stocks were defined based on χ 2 analyses (p < 0.05) of haplotype frequencies; some stocks were equivalent to single populations, whereas others were regional stocks made up to two or more populations. Pairwise gene flow estimates (N m ) between stocks were derived from F st values (Wright, 1943) obtained via AMOVA analysis (Excoffier et al. 1992). Pairwise estimates of gene flow (Table 1) among the Gulf stocks based on sequencing analysis ranged from 0.15 between the western (Lake Ponchartrain and Pearl River) stock and the Escambia River-Yellow River stock, to 1.2 between the Escambia River-Yellow River stock and the eastern stock Table 1. Estimates of gene flow among populations of Acipenser oxyrinchus desotoi. Values above diagonal are based on data from restriction fragment length polymorphism analysis of mitochondrial DNA (mtdna); values below diagonal are based on data from sequence analysis of 203 base pairs of mtdna control region. Western Pascagoula Escambia- Yellow Choctawhatchee Eastern Western 0.26 0.11 0.23 Pascagoula 249.75 Escambia-Yellow 0.15 0.23 0.45 0.66 Choctawhatchee 0.22 0.35 0.79 0.09 Eastern 0.17 0.27 1.20 0.97
394 (Apalachicola, Ochlockonee, and Suwannee rivers). Gene flow estimates derived from RFLP analysis were even lower on average, and ranged from 0.09 between the western and Choctawhatchee River stocks to 0.66 between the western and Escambia River-Yellow River stock. These gene flow values are very low in comparison with estimates for other anadromous fishes. Estimated annual straying rates among populations of Pacific salmons have ranged between about 1% and 27% (reviewed in Adkindson 1996). Laughlin & Turner (1996) used two statistical methods to estimate N m of Morone saxatilis (striped bass) among three Virginia tributaries of Chesapeake Bay; the private allele approach of Barton & Slatkin (1986) yielded an estimate of N m = 14.2, whereas the F st approach yielded an estimate of N m = 2.7. In a capturemark-recapture study, Melvin et al. (1986) estimated an annual straying rate of 3% among Canadian populations of Alosa sapidissima (American shad). Moreover, the low gene flow estimates for A. o. desotoi were obtained across populations that occur in eight rivers, the mouths of which are arrayed across little more than 500 km of coastline. Sturgeon from these rivers have the opportunity to mix in the Gulf of Mexico during winter. These mtdna data show that despite the geographic proximity of these rivers, stocks of A. o. oxyrinchus generally exchange less than one female per generation, a level sufficient to permit differentiation at the stock level (Adkinson 1996). Gene flow estimates also were generally higher among proximal stocks, suggesting that what straying occurs does so in stepping stone fashion (Kimura & Weiss 1964) in which migrants among semi-isolated populations are exchanged chiefly with neighboring populations. If this is true for A. o. desotoi, then such spatially restricted straying should have contributed to the geographic structuring observed among these populations (Adkinson 1996). Stabile et al. (1996) hypothesized that the homing imperative of A. o. desotoi for spawning purposes is strong, but that it may be reinforced by metabolic constraints. Acipenser oxyrinchus desotoi returns to rivers from the Gulf of Mexico to summer near cold water springs; tagging has shown that individuals are recaptured at the same cool water refuges in which they were first tagged (Clugston et al. 1995). Conclusions and recommendations The comparatively few molecular analyses directed to conservation of acipenseriforms have yielded unique and important information. Some of these studies have refined notions concerning species and subspecies status; our analysis of mtdna control region differences between the American and European Atlantic sturgeons shows that should relict populations of the latter become extinct, restocking with A. oxyrinchus would constitute introduction of a foreign species. Likewise, the study by Ong et al. (1996) supporting subspecies designations for Atlantic and Gulf forms of A. oxyrinchus reinforces the current status of the latter as a threatened subspecies under the U.S. Endangered Species Act. Unfortunately, to date, conservation directed molecular studies of acipenseriforms at the species level and below have almost exclusively been restricted to North American species. Highly sensitive determination of the genetic relationships among extant species, stocks, and between extinct and closely related extant taxa are possible. Much of the latter analyses will use archived museum samples as sources of DNA. Birstein (1993) provided many examples of Eurasian sturgeons that are phenotypically differentiated (i.e., large and small forms ) below the species level but that have not received genetic analysis. Even if financial resources are currently not available, samples can be collected and archived for future analyses. Population surveys also have been limited largely to North America. Genetic analyses of P. spathula and A. fulvescens paddlefish and sturgeon species with similar and broad North American distributions have revealed relatively little genetic variation and only minor substructuring among populations. In comparison, coastal species such as A. oxyrinchus and A. transmontanus exhibit higher levels of polymorphism and greater geographic population structuring. The reasons for this disparity are not apparent, and are contrary to comparisons among other freshwater and anadromous fishes (Waldman & Wirgin 1994). We cannot be sure if present levels of genetic diversity among these four species reflects prebottleneck levels, given the widespread anthropogenically-imposed bottle-
395 necks on their populations. However, it is likely that the paucity of genetic diversity and shallow genetic differentiation among populations of the two freshwater species is at least partly due to a combination of Pleistocene bottlenecks and recent recolonization of northern waters, prolonged generation times leading to low mutation rates, and continued gene flow among populations that remain largely linked through their inhabitation of few discrete drainages. Although the anadromous sturgeons of North America were not exempt from glacial influences, post-pleistocene genetic diversity was maintained, except in far northern drainages recolonized by A. oxyrinchus. Genetic analyses to date of the freshwater scaphirhynchids show very limited genetic variation, consistent with P. spathula and A. fulvescens species that are largely sympatric with S. albus and S. platorynchus. Low genetic variation within Scaphirhynchus is in accordance with pre-existing biogeographic theories suggesting recent speciation with Wisconsinian glacial events (70 000 to 10 000 years before present). However, the taxonomic boundaries among the three putative scaphirhynchid species remain unclear, but of great importance to their conservation status under the U.S. Endangered Species Act (ESA). Future molecular analyses of Scaphirhynchus spp. and other North American acipenseriforms will not only attempt to assess traditional taxonomic divisions such as species and subspecies, but also their Evolutionarily Significant Units as mandated under the Endangered Species Act (Waples 1995). Molecular analyses to date also are relevant to conservation efforts involving hatchery-based stocking (St. Pierre 7 ). Anadromous sturgeons show stock structure, implying that interpopulation transfers will have genetic repercussions; to maximize the likelihood that stocked fish will have high fitness for a particular environment, broodstock from the same environment as wild fish should be 7 St. Pierre, R.A. 1996. Breeding and stocking protocol for cultured Atlantic sturgeon. Final Report from the Atlantic Sturgeon Aquaculture and Stocking Committee to the Atlantic States Marine Fisheries Commission Atlantic Sturgeon Management Board. 17 pp. used (Krueger et al. 1981). The single study of gene flow in an acipenserid suggests that hatchery-based restocking may be necessary to reestablish sturgeon populations in a reasonable length of time in drainages where they are extinct. That is, if gene flow estimates among populations of A. o. desotoi are indicative of general levels of straying for acipenserids, then straying rates less than 1.0 per generation, combined with the long generation times of sturgeons, means that natural restocking may require decades before it even is initiated, notwithstanding additional decades necessary for population growth (Boreman 1997 this volume). Circumstantial evidence for extremely slow rates of natural restocking through straying is provided by the example of the Maryland tributaries of Chesapeake Bay, where there has been no indication of recolonization by A. oxyrinchus over many decades in rivers that once supported large populations (David Secor personal communication). However, a generic problem with the hatchery production of sturgeons is the need to acquire sufficient broodstock to prevent inbreeding (Nelson & Soulé 1987). Commonly accepted guidelines for fish production are for an effective population size of 100 or more individuals (Kincaid 1983, Kapuscinski & Lannan 1986, Allendorf & Ryman 1987). But, given the generally large size and scarcity of sturgeons, annual hatchery reproduction at these effective population levels are unrealistic for most restoration efforts aimed toward single stocks. If lower than recommended numbers of broodstock must be used, one way to reduce inbreeding would be to use genetic screening of individuals. With the data from these analyses, hatchery crosses can be optimized to maximize diversity and yet still maintain stock-specific gene frequencies. We envision that a priori knowledge of the genetic composition of broodstock can help ease the burden of rigorous demands for large numbers of broodstock. Rapid technical advances in the development of molecular biological approaches will allow for their routine application in the future to problems of the conservation of sturgeons and paddlefishes. It is now possible to obtain DNA sequence information from non-destructively obtained tissues such as barbels, fin clips, or blood, from early life intervals
396 such as single eggs, embryo or larva, and even from archived museum specimens. Additionally, a variety of DNA level approaches have been developed which allow for investigations which focus on characters whose rate of change varies from extremely slow to exceedingly rapid (Wirgin & Waldman 1994). This permits quantification of genetic relationships extending from the interspecific to interindividual levels. We strongly encourage the use of ndna-based approaches to resolve management questions concerning taxa such as Scaphirhynchus in which low levels of genetic diversity have been reported. Because of the polyploid character of the acipenserid genome, it is highly likely that many duplicated gene loci have been relieved of functional constraints and were free to rapidly evolve. Thus, the nuclear genome of sturgeon species should offer a wealth of rapidly evolving single copy or repetitive DNA sequences for analysis. Several other avenues for future molecular research of sturgeons and paddlefishes are apparent. Poaching of these fishes across North America and Eurasia is a major threat to their existence; much of this illegal harvest is sold in world markets (Birstein 1993, Bemis & Findeis 1994). A study is now underway (DeSalle & Birstein 1996) to develop forensic molecular markers to help identify illegally procured and mislabeled acipenserid products. Also, most acipenseriforms are difficult to sex except at spawning. Nonlethal molecular gender determination, as has been developed for Oncorhynchus tshawytscha (chinook salmon, Devlin et al. 1994), would be useful for many management purposes. Additionally, contaminant exposure has been proposed as a major factor in the decline of certain sturgeon populations (Birstein 1993). However, quantitative data on comparative exposure histories or possible biological effects are largely lacking. We suggest that a molecular biomarker approach, in which structural alterations at anonymous genetic loci or expression levels of xenobiotically responsive genes are quantified, can help fill this void. The extent of DNA sequence variation at these loci or levels of expression of inducible genes such as cytochrome P4501A or metallothionein can allow for a comparison of the exposure histories of sturgeons from environments with differing degrees of pollution. This approach has been used successfully to quantify the exposure histories of species such as Parophrys vetulus (English sole, Stein et al. 1992) and Microgadus tomcod (Atlantic tomcod, Wirgin et al. 1994) from North American estuaries. Furthermore, increased levels of gene expression in fish from contaminated environments have been correlated with higher level biological effects at the population level (Wirgin & Garte 1994). Acknowledgements We thank Patrick Williot for the tissue sample of European Atlantic sturgeon. Work was funded by the National Oceanic and Atmospheric Administration award # NA46RG0090 to the Research Foundation of the State University of New York for the New York Sea Grant Institute. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon. Views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies. This work also was supported by the U.S. Fish and Wildlife Service and the Hudson River Foundation and NIEHS center grant ES00260. References cited Adkinson, M.D. 1996. Population differentiation in Pacific salmon: local adaptation, genetic drift, or the environment? Can. J. Fish. Aquat. Sci. 52: 2762 2777. Allendorf, F.W. & N. Ryman. 1987. Genetic management of hatchery stocks. pp. 141 159. In: N. Ryman & F. Utter (ed.) Population Genetics and Fishery Management, University of Washington, Seattle. Barton, N.H. & M. Slatkin. 1986. A quasi-equilibrium theory of the distributions of rare alleles in a subdivided population. Heredity 56: 409 415. Bemis, W.E. & E.K. Findeis. 1994. The sturgeon s plight. Nature 370: 602. Bemis, W.E. & B. Kynard. 1997. Sturgeon rivers: an introduction to acipenseriform biogeography and life history. Env. Biol. Fish. (this volume). Bernatchez, L. 1995. A role for molecular systematics in defining evolutionarily significant units in fishes. Amer. Fish. Soc. Symp. 17: 114 132.
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