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2 Maine Department of Marine Resources, 21 State House Station, Augusta, Maine 04333-0021 ... Oklahoma State University, Stillwater, Oklahoma 74078; tele:.
Estuaries

Vol. 24, No. 1, p. 41–48

February 2001

Morphological and Genetic Variation among Shortnose Sturgeon

Acipenser brevirostrum from Adjacent and Distant Rivers MAUREEN G. WALSH1,*, MARK B. BAIN1, THOMAS SQUIERS, JR.2, JOHN R. WALDMAN3, and ISAAC WIRGIN4 1

New York Cooperative Fish and Wildlife Research Unit, Fernow Hall—Cornell University, Ithaca, New York 14853-3001 2 Maine Department of Marine Resources, 21 State House Station, Augusta, Maine 04333-0021 3 Hudson River Foundation for Science and Environmental Research, 40 West 20th Street, Ninth Floor, New York City, New York 10011 4 Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, New York 10987

ABSTRACT: Shortnose sturgeon, Acipenser brevirostrum, is a small, endangered species which occurs in 19 estuary systems along the east coast of North America. These populations are considered as separate entities by the U.S. National Marine Fisheries Service although evidence of morphologic or genetic differentiation among populations has not been documented. The purpose of this study was to compare morphological and genetic attributes among shortnose sturgeon collected from the Kennebec and Androscoggin Rivers, Maine, and the Hudson River, New York. Six morphometric and five meristic characteristics were quantified. Multivariate and univariate analyses of covariance and variance were used to assess differences among populations. Our analyses provided evidence for distinct populations in the Androscoggin and Kennebec Rivers, but character differentiation was greater between fish from these two locations and the Hudson River. Analysis of morphometric characters indicated significant differences in fish shape among the three rivers, with Hudson River sturgeon differing from the Maine rivers for the characters of head length, snout length, and mouth width. Significant differences were observed for meristic characters, but pairwise comparisons did not reflect a clear pattern of variability. Sequencing of a portion of the mitochondrial DNA control region revealed 15 haplotypes among 73 total specimens from the three rivers. Shortnose sturgeon from the Kennebec and Androscoggin Rivers were different from each other (p ⴝ 0.0260); both differed significantly (p ⬍ 0.0001) from the Hudson River collection. Gene flow was estimated at approximately 7 female migrants per generation between the two Maine populations and about 1 per generation between each of the Maine populations and the Hudson River population. Such strong stock structuring among presumably recently established post-Pleistocene (⬍ 10,000 yr) populations suggests that this species occurs in highly discrete units. Morphological and genetic variation observed in this study combined with current knowledge of life history attributes of shortnose sturgeon indicate that conservative management decisions are necessary until the patterns and extent of differentiation among populations species-wide can be investigated further.

serving these fishes for aesthetic, ecological, and legislated reasons. Shortnose sturgeon were commercially fished along with Atlantic sturgeon (Acipenser oxyrinchus) in the late 1800s until exploitation, combined with factors such as habitat destruction and pollution, caused populations to crash by the early 1900s (U.S. National Marine Fisheries Service 1996a,b, 1998). As a result, shortnose sturgeon were granted federal protection by the U.S. Fish and Wildlife Service in 1967, and have been protected under the U.S. Endangered Species Act since its inception in 1973 (U.S. National Marine Fisheries Service 1998). Shortnose sturgeon are long-lived and do not reach sexual maturity until 7–10 yr (Dadswell et al. 1984), leaving them vulnerable to exploitation. Limited abundance and recruitment within most populations, combined with compli-

Introduction Shortnose sturgeon (Acipenser brevirostrum) is a small, amphidromous species that occupies freshwater and estuarine portions of rivers along the east coast of North America from New Brunswick to Florida (Vladykov and Greeley 1963; Dadswell et al. 1984). The basic body form of extant sturgeons, which includes a unique blend of primitive and derived characters, was established by end of the Cretaceous (Bemis et al. 1997). Aside from their evolutionary significance, the unusual appearance and life history of sturgeon has stimulated public and government-agency interest in pre* Corresponding author: current address: Oklahoma Cooperative Fish and Wildlife Research Unit, 404 Life Sciences West, Oklahoma State University, Stillwater, Oklahoma 74078; tele: 405/744-6342; fax: 405/744-5006; e-mail: [email protected]. 䊚 2001 Estuarine Research Federation

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cated spawning and migratory behavior (Bain 1997) have made the study and recovery of this species difficult. The U.S. National Marine Fisheries Service, the federal agency responsible for the protection and recovery of shortnose sturgeon, considers 19 remaining populations as distinct units under the Endangered Species Act (U.S. National Marine Fisheries Service 1996b, 1998). Two northern rivers supporting shortnose sturgeon populations are the Hudson River estuary, New York, and the lower portions of the Kennebec and Androscoggin Rivers, which in part form the Kennebec estuarine complex (Kennebec, Androscoggin, and Sheepscot Rivers), Maine. Both areas have been targeted for research on shortnose sturgeon due to the relatively large size of their populations; 52,898– 72,191 (95% confidence interval) fish were present in the Hudson River in 1997 (Bain et al. 1998) and 5,000–10,800 (95% confidence interval) were estimated to occupy the Kennebec estuarine complex in the early 1980s (Dadswell et al. 1984). The measurement of morphological features has been widely applied in the systematics and classification of fish, and research of this type has identified significant variation among and within acipenserid species. Morphometric variation has been used to distinguish among similar sturgeon species and to detect hybrids (Carlson et al. 1985; Keenlyne et al. 1994). Significant morphological differences have been documented among populations of some sturgeon species (Guenette et al. 1992; Keenlyne et al. 1994), and between wild and hatchery–reared sturgeon (Ruban and Sokolov 1986; Ruban 1989). No morphometric and meristic differentiation between populations of shortnose sturgeon inhabiting different rivers has been reported. Dadswell et al. (1984) briefly addressed geographic separation in their extensive compilation of biological data on shortnose sturgeon. The authors noted a lack of data concerning possible morphological or genetic separation among populations of shortnose sturgeon. Subsequent studies have focused on abundance, distribution, and life history attributes of the shortnose sturgeon, but analyses of either morphological or genetic variation among populations of this species have not occurred. Though the U.S. National Marine Fisheries Service manages each population as a distinct unit, they note that, ‘‘available genetic and morphometric data do not support any taxonomic splitting of the species’’ (U.S. National Marine Fisheries Service 1998, p. 4). Questions regarding population differentiation among populations of shortnose sturgeon were considered in response to a 1994 petition to delist

shortnose sturgeon in the Kennebec estuarine complex (U.S. National Marine Fisheries Service 1996a). The U.S. National Marine Fisheries Service reviewed the status of shortnose sturgeon in these rivers and concluded that the shortnose sturgeon populations in the Kennebec and Androscoggin Rivers were still endangered (U.S. National Marine Fisheries Service 1996a). An important question the U.S. National Marine Fisheries Service considered was the possibility that the two distinct populations occur in the Kennebec and Androscoggin Rivers, which would inform future delisting of discrete population segments. The purpose of this study was to evaluate morphological and genetic differentiation between shortnose sturgeon collected in the Kennebec and Androscoggin Rivers, using shortnose sturgeon from the Hudson River as an outgroup. The possibility of differentiation among populations has important ramifications for understanding the ecology of this species and for managing it at the stock level. Materials and Methods KENNEBEC

AND

ANDROSCOGGIN RIVERS

Morphometric and meristic data were collected from shortnose sturgeon captured in the spring (April and May) of 1980 and 1981 at the spawning grounds in the Kennebec and Androscoggin Rivers. Shortnose sturgeon spawning sites are located below the Augusta Dam (44⬚19⬘N, 69⬚46⬘W) on the Kennebec River and below the Brunswick Dam (43⬚55⬘N, 69⬚58⬘W) on the Androscoggin River (U.S. National Marine Fisheries Service 1996b). Both the Kennebec and Androscoggin Rivers flow into Merrymeeting Bay, a large freshwater tidal bay where shortnose sturgeon from both river systems commingle during summer months. Below the outlet of Merrymeeting Bay, the Kennebec River estuary forms a complex with the Sheepscot River estuary. Water is indirectly exchanged between the Kennebec and Sheepscot Rivers through a series of brackish water bays and channels. The drainage area of the Kennebec River includes the Androscoggin and Sheepscot Rivers, as well as smaller tributaries to the system, and is often referred to as the Kennebec estuarine complex. For the morphological portion of the study, 24 shortnose sturgeon were captured from the Kennebec River and 267 shortnose sturgeon were captured from the Androscoggin River. Experimental gillnets with stretch mesh sizes ranging from 152 to 203 mm were set overnight and left in the water for approximately 20–24 h. Six morphometric and five meristic features were measured according to Vladykov and Greeley

Variation among Shortnose Sturgeon

(1963): total length (TL), fork length (FL), head length (HL, anterior tip of snout to the rearmost point of the opercle excluding the opercular membrane), snout length (SNL, anterior tip of snout to the anterior margin of the orbit with the membranous rim excluded), mouth width (MW, greatest transverse distance across the mouth slit with the lips excluded and mouth closed), interorbital width (IOW, maximum distance across top of head between the bony edges of the orbit), dorsal scute count (DSC), left lateral scute count (LLSC), right lateral scute count (RLSC), left ventral scute count (LVSC), and right ventral scute count (RVSC). All tissue samples for mtDNA analysis were fin clips which were preserved in 95% EtOH. Tissue samples from 22 Kennebec River and 23 Androscoggin shortnose sturgeon specimens were obtained via gillnetting during May 1999. HUDSON RIVER Shortnose sturgeon in the Hudson River usually range from the brackish waters of the lower estuary to the Troy Dam near Albany, New York (Bain 1997). A total of 68 shortnose sturgeon were collected from the Hudson River in 1996, and all fish were returned to the river unharmed after the counts and measurements were completed. We collected fish in November and March at Esopus Meadows (river kilometer 140; 41⬚52⬘N, 73⬚51⬘W) near Kingston, New York; a known overwintering site for most or all shortnose sturgeon in this river (Dovel et al. 1992; Bain et al. 1998). Monofilament gill nets (152 mm stretch) were set during slack tide for approximately 30 min. Tissue samples for DNA analysis from 28 Hudson River individuals were collected from Esopus Meadows during November 1996 and 1997 and June 1998. MORPHOLOGICAL ANALYSES Pearson product-moment correlations were computed between fork length and all other morphological and meristic features to identify features that varied by fish length. For features related to length, multivariate analysis of covariance (MANCOVA) and analyses of covariance (ANCOVA) were used to determine significant differences among river fish groups using fork length as the covariate to adjust for size. Multivariate analysis of variance (MANOVA) and analyses of variance (ANOVA) were used to test for significant variation among the three river groups for features unrelated to fish size. Least significant difference post-hoc tests were used to identify pairwise differences among rivers for all significant ANCOVA and ANOVA test results (main effects, p ⱕ 0.05).

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GENETIC ANALYSES Genetic analysis focused on a rapid-evolving marker, the control region of mitochondrial DNA (mtDNA). Total DNA was extracted from tissues with CTAB buffer and proteinase K as described by Wirgin et al. (1990, 2000). DNAs were purified following incubations by standard phenol-chloroform extractions and alcohol (isopropanol and 70% EtOH) precipitations. DNAs were then air dried and resuspended in TE buffer. The mtDNA control region was amplified initially using the conser vative oligonucleotide primers L15926 and H00651 described by Kocher et al. (1989). Sequences were then derived from a 1.2-kb mtDNA control region product using these same primers. New shortnose sturgeonspecific primers (ABL1–5⬘-CAATCTCTCACCCTTAATCCC-3⬘ and ABH1–5⬘-GCTTTAGTTAAGCTACGCTAGC-3⬘) were synthesized based on the derived sequence, which allowed for a more reliable amplification of a 1.1-kb fragment. Two additional primers (ABL2–5⬘-TAGCCACCATATCACAATGT-3⬘ and ABH2–5⬘-GGCATCTTGATTATTATGTGG-3⬘) were synthesized based on derived sequence data. To identify hypervariable areas within the shortnose sturgeon mtDNA control region, mtDNA from 12 geographically-widespread specimens was amplified with ABH1 and ABL1 and sequenced with ABH1, ABL2, ABH2, and ABL2. For all subsequent population analysis, ABH1 and ABL1 were used as PCR primers and ABH2 and ABL2 were used as sequencing primers. The PCR reactions were performed as reported in Wirgin et al. (2000); cycling parameters were 94⬚C for 5 min and 35 cycles of denaturation at 94⬚C for 1 min, annealing at 53⬚C for 1 min, and extension at 72⬚C for 1 min, followed by a final extension for 10 min at 72⬚C. The PCR products were purified in 2.0% low melting point agarose gels (Agarose II, Amresco). The 1.1-kb control region fragment was excised in an agarose plug and sequenced directly (Kretz and O’Brien 1993) with 32P using the Cyclist Taq DNA Sequencing Kit (Stratagene) and primers ABH2 and ABL2. Sequencing profiles were as follows: one cycle at 94⬚C for 5 min and 40 cycles of denaturation at 94⬚C for 30 s, annealing at 60⬚C for 30 s, and extension at 72⬚C for 1 min, followed by a final extension at 72⬚C for 7 min. DNA sequences were resolved in 6% polyacrylamide/7M urea denaturing gels. Gels were air-dried on glass plates and exposed to X-ray film for 1–8 d. The mtDNA haplotypes of specimens were assigned on the basis of discrete combinations of nucleotides. Population-level differences based on

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TABLE 1. Body measurements (mm) and meristic counts for three populations of shortnose sturgeon with the mean values and the 95% confidence intervals (CI) of the means. Androscoggin River Measurement

Total length Fork length Head length Snout length Mouth width Interorbital width Dorsal scute count Left lateral scute count Right lateral scute count Left ventral scute count Right ventral scute count

Range

616–1,085 525–962 124–206 47–82 28–63 38–71 6.0–16.0 20.0–39.0 21.0–39.0 5.0–14.0 6.0–13.0

Mean (CI)

807 (10.5) 705 (9.9) 157 (2.0) 63 (0.8) 40 (0.7) 52 (0.8) 10.1 (0.8) 26.6 (0.3) 26.6 (0.3) 8.1 (0.1) 8.1 (0.1)

haplotype frequencies were assessed using the Monte Carlo-based chi-square approach of Roff and Bentzen (1989) with the software program REAP (McElroy et al. 1992). Haplotype frequency differences were considered significant at p ⬍ 0.05. Haplotypic diversity was estimated using the method of Nei and Tajima (1981). Gene flow (Nm) between populations was estimated using analysis of molecular variance (AMOVA) software (Excoffier et al. 1992). Results MORPHOLOGICAL Shortnose sturgeon captured in the Kennebec River were the longest (mean total length ⫽ 910 mm; Table 1) of the three sample groups, while Hudson River fish were the shortest (mean length ⫽ 784 mm). The differences among rivers in fish size (fork length) were significant (ANOVA, F2,356 ⫽ 22.22, p ⱕ 0.0001) and each river was distinct from the others in post-hoc tests (p ⱕ 0.028 in all pairwise comparisons). All shortnose sturgeon measured in this portion of the study were larger than 616 mm total length and are considered adults (Dadswell et al. 1984; Bain 1997). It is likely that shortnose sturgeon captured represent ages spanning the known age range of this species (over 60 years, Dadswell et al. 1984). Similar to length differences, mean values for the four remaining morphometric features (head length, snout length, mouth width, interorbital width) were largest for the Kennebec River population, and smallest for the Hudson River population (Table 1). The morphometric features were well correlated with fork length (8 of 12 correlation coefficients ⱖ 0.80, range ⫽ 0.41–0.96), so further analyses of morphometric features were adjusted by fork length. Meristic features (4 scute counts; Table 1) were similar among shortnose sturgeon from the three rivers; these features were not related to fish size (10 of 12 fork length cor-

Kennebec River Range

673–1,165 585–1,030 136–225 56–81 32–63 43–81 8.0–11.0 22.0–31.0 23.0–33.0 6.0–10.0 7.0–10.0

Hudson River

Mean (CI)

Range

Mean (CI)

910 (49.4) 808 (45.6) 176 (8.8) 67 (2.4) 48 (3.3) 61 (4.3) 9.9 (0.3) 27.5 (0.9) 27.8 (0.9) 8.0 (0.4) 8.0 (0.3)

634–962 548–895 119–176 37–76 22–42 44–65 9.0–15.0 23.0–33.0 23.0–33.0 7.0–10.0 6.0–10.0

784 (16.4) 680 (15.8) 145 (12.7) 58 (1.5) 35 (0.9) 52 (1.2) 11.3 (1.2) 27.5 (0.5) 27.5 (0.6) 8.1 (0.2) 8.1 (0.2)

relation coefficients ⬍ 0.20, range ⫽ 0.18–0.29). Further analyses of the meristic feature differences among rivers were not adjusted for fish size. Overall, the morphological features reflected significant differences in fish shape among the three rivers (MANCOVA, p ⱕ 0.0001; Table 2). Each of the four individual morphometric features also differed among rivers (ANCOVA, p ⱕ 0.0001; Table 2). For three of the features (head length, snout length, mouth width), the Kennebec and Androscoggin populations did not appear different while the Hudson population was distinct from the two Maine rivers (Table 2). This pattern was the same as that seen in Fig. 1 where head length is plotted against fork length by river group. The head length versus fork length relations (Fig. 1) for the Androscoggin and Kennebec Rivers overlapped and had nearly identical slopes, while the Hudson group has a similar slope but lower y-intercept, indicating that the Hudson River specimens were consistently smaller in dimensions at all sizes. The interorbital width of Androscoggin River fish was distinct from the interorbital width of shortnose sturgeon from the Kennebec and Hudson Rivers (Table 2). The plot of interorbital width versus fork length suggests inconsistency in the slopes, indicating that statistical results should be interpreted cautiously for this feature. The five scute counts (dorsal, lateral and ventral, left and right sides of the fish) indicated significant meristic feature differences among shortnose sturgeon collected in the three rivers (MANCOVA, p ⱕ 0.0001; Table 2). The two ventral scute counts did not differ among the rivers (ANOVA, p ⱕ 0.8012; Table 2) but dorsal scute counts clearly varied among rivers (p ⱕ 0.0001; Fig. 2). Hudson River sturgeon had the highest mean dorsal scute count (Table 2). Lateral scute counts varied significantly among rivers (p ⱕ 0.0051) but the results of specific between river comparisons were mixed (indicated in Table 2). Right lateral scute counts

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Variation among Shortnose Sturgeon

TABLE 2. Statistical results of tests for differences in morphological and meristic characters among shortnose sturgeon collected in the three study rivers (AND ⫽ Androscoggin River, KEN ⫽ Kennebec River, HUD ⫽ Hudson River). Multivariate analyses of covariance and variance were used for tests involving groups of characters, analyses of covariance and variance were used for single characters, and the least significant difference post-hoc tests were used to identify specific differences between rivers in univariate cases. Similar superscripts indicate no significant difference between means in post-hoc tests (p ⱕ 0.05). Means and Adjusted Means1 by River

Degrees of Freedom Main Effect

Morphological characters Head length Snout length Mouth width Interorbital width Meristic characters Dorsal scute count Left lateral scute count Right lateral scute count Left ventral scute count Right ventral scute count

Error

Test Statistic2

Probability Level

8 2 2 2 2

704 355 355 355 355

0.65 26.72 25.53 43.53 9.84

ⱕ0.0001 ⱕ0.0001 ⱕ0.0001 ⱕ0.0001 ⱕ0.0001

10 2 2 2 2 2

704 356 356 356 356 356

0.84 26.99 5.17 5.35 0.22 0.08

ⱕ0.0001 ⱕ0.0001 0.0061 0.0051 0.8012 0.9272

AND

158a 63a 41a 52a 10.1a 26.6a 26.6a 8.1a 8.1a

KEN

159a 64a 42a 54b 9.9a 27.5ab 27.8b 8.0a 8.0a

HUD

150b 59b 37b 54b 11.3b 27.5b 27.5b 8.1a 8.1a

1 Adjusted means are reported for univariate morphological characters because the absolute values were related to fish lengths. The adjustment removes length differences among rivers and it was the same adjustment used in the analysis of covariance tests. 2 The test statistic for multivariate tests were the Wilks Lambda criteria, and an F-ratio is reported for univariate tests.

differed between the two Maine rivers (p ⫽ 0.0184; Fig. 2) while the Kennebec and Hudson sturgeon had similar right lateral scute counts (p ⫽ 0.5261). The two Maine rivers did not differ significantly in the number of left lateral scutes (p ⫽ 0.0698, not indicated as different with boxes in Table 2), while the Androscoggin and Hudson River groups of sturgeon had distinct differences in the number of left lateral scutes (p ⫽ 0.0043; Fig. 2). A relatively high variance in lateral scute counts for the Ken-

Fig. 1. Head length and interorbital width versus fork length relations for shortnose sturgeon collected by study river. The pattern of measurement and size relations and among river differences shown for head length was very similar to the patterns found for snout length and mouth width. Interorbital width changes by fish size were inconsistent among rivers suggesting that analysis of covariance test results should be interpreted with caution.

nebec River (Fig. 2) largely explains these between river differences. In general, scute counts distinguished Hudson River shortnose sturgeon from the populations of the two Maine rivers. GENETIC Sequencing of a portion of the mtDNA control region revealed 15 haplotypes among shortnose sturgeon from the three rivers: 6 among 22 Kennebec specimens, 5 among 23 Androscoggin specimens, and 9 among 28 Hudson specimens (Table 3). Genotypic diversity indices (which can range between zero and one) were high for all three collections: 0.746 for the Kennebec, 0.775 for the Androscoggin, and 0.810 for the Hudson. Specimens from the two Maine rivers shared a modal haplotype (P: 37.7%) which was absent among Hudson River individuals. Seven of the haplotypes observed

Fig. 2. Select results of scute count differences among rivers. Means are plotted as filled circles with lines corresponding to the 95% confidence intervals for the means.

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TABLE 3. Mitochondrial DNA haplotype frequencies of shortnose sturgeon by collection location. Haplotype Designation Collection Location

A

B

F

I

J

L

M

P

S

T

U

V

X

Y

Z

⌺

Kennebec River Androscoggin River Hudson River

0 0 1

0 0 1

0 0 11

0 2 0

0 0 1

2 2 5

0 0 3

10 7 0

1 0 0

5 0 0

0 0 3

0 0 1

1 4 0

0 0 2

3 8 0

22 23 28

among Hudson specimens were not found among individuals from the two Maine rivers, including the modal haplotype (F: 39.3%) for the Hudson River population. Haplotype frequencies for the Kennebec and Androscoggin River collections were significantly different from each other (␹2 ⫽ 12.59; p ⫽ 0.0260). Both the Kennebec River (␹2 ⫽ 44.20; p ⫽ 0.0000) and the Androscoggin River (␹2 ⫽ 45.23; p ⫽ 0.0000) collections were highly significantly different from the Hudson River collection. Estimated gene flow (Nm) between the Kennebec and Androscoggin River populations was 6.89. Gene flow between the Kennebec and Hudson populations was estimated at Nm ⫽ 0.78 and between the Androscoggin and Hudson at Nm ⫽ 1.09. Discussion The degree of morphological differentiation among shortnose sturgeon from the three rivers varied with their proximity to each other. Whereas the Hudson River’s shortnose sturgeon population differed markedly from the other two river groups for most morphological features, significant differences were found between fish from the Androscoggin and Kennebec Rivers for one morphometric (interorbital width) feature and one set of meristic features (lateral scute counts). Although the degree of differentiation between the collections from the two Maine rivers was markedly lower than either was to the Hudson River population, the differences were significant and they suggest that the Kennebec and Androscoggin Rivers support largely discrete populations of shortnose sturgeon. The lack of clear and persistent feature differentiation, as seen in comparison with the Hudson River fish, raises the question of what degree of morphological variation between populations would justify their treatment as separate management units. The results of mtDNA analysis correspond well with the morphological analysis. The degree of differentiation between haplotypic frequencies of either of the collections from the Maine rivers and the Hudson River collection was considerably greater than between specimens from the two Maine rivers. As in the morphological analysis, mtDNA haplotype frequencies between the Kennebec and Androscoggin collections were significantly different. Congruence of results between ap-

proaches solidifies notions of stock structure (Begg and Waldman 1999). Multiple approaches (particularly the pairing of morphologically and genetically-based techniques) also may detect different environmental and evolutionary signals that permit a deeper understanding of differentiation among population units (Begg and Waldman 1999; Waldman 1999). The gene flow rates estimated between the Hudson River population and the Kennebec (Nm ⫽ 0.78) and the Androscoggin River (Nm ⫽ 1.09) populations were low and consistent with well-differentiated, distant populations. The two Maine river populations only overlapped with the Hudson River population in 3 of the 15 haplotypes revealed among them. Although it would be advantageous to sequence more individuals from all three populations, these data suggest that there may be substantially discrete sets of haplotypes between the two Maine river and the Hudson River populations. The genetic distinctiveness of the two populations in Maine compared with the Hudson’s is especially noteworthy in that it presumably has evolved within a short geological time frame. River systems from the Hudson northward were glaciated during the Pleistocene and probably were not recolonized by anadromous fishes until within the past 10,000 years (Schmidt 1986). The gene flow rate estimated between the Kennebec and Androscoggin River populations (Nm ⫽ 6.89) was considerably higher than either relative to the Hudson River. This is not surprising given that the two rivers in Maine are contiguous and share the same estuary mouth and that straying of anadromous fishes is often greatest between neighboring populations (e.g., Pacific salmon: Quinn et al. 1991; Pascual and Quinn 1994). Four of the seven haplotypes revealed between these populations were shared, which, together with the higher estimated gene flow rate, indicates a closer genetic relationship between these two populations than between either of these and the Hudson River population. However, the apparently high estimated gene flow rate is not inconsistent with statistically significant haplotype frequencies. Allendorf and Phelps (1981) have shown that substantial divergence in allele frequencies can occur despite many migrants per population. Estimation of gene flow rates infer migration

Variation among Shortnose Sturgeon

only indirectly. Although shortnose sturgeon are endangered, they were once present in greater numbers and occupied more river systems than populations currently in existence (U.S. National Marine Fisheries Service 1998). It is possible that at one time, these larger populations commingled through marine migrations, and marine captures in locations along the Atlantic coast are still occasionally reported (reviewed in Dadswell et al. 1984). In 1997, an adult shortnose sturgeon tagged in the Hudson River in 1995 was recaptured in the Connecticut River (Savoy personal communication). Kynard (1997) associated the presence of marine captures of shortnose sturgeon to exchange of individuals among separate river populations. He also linked population size to the frequency of marine migrations, hypothesizing that emigration by shortnose sturgeon is density dependent. As the number of shortnose sturgeon decreased early in this century, the species may have become fragmented into small, isolated, river–restricted populations. The relatively large populations of shortnose sturgeon in the Hudson and Kennebec Rivers would be most likely to contribute individuals to other populations through density-dependent marine migrations. Species–wide genetic analyses could elucidate stock structure and gene flow rates among existing populations of shortnose sturgeon. Analyses of both morphometric and meristic data clearly indicate morphological variation among shortnose sturgeon in the New York and Maine rivers. One possible explanation for this morphological variation is that it is environmental in nature. Environmental conditions and growth rates during larval development affect the relative sizes of body parts and the development of serial features (Martin 1949; Wimberger 1992). Guenette et al. (1992) suggest that differences in growth rates among separate sturgeon populations could result in differing head proportions. Since meristic features have been observed to vary with developmental rate in sturgeon and other fishes (Martin 1949; Stouracova et al. 1988; Ruban 1989), when growth is retarded during larval development, for example by low temperature, serial features are allowed more time to form, resulting in a higher number (Martin 1949). While this trend has been primarily studied for features such as fin rays and vertebrae, Ruban and Sokolov (1986) suggested that the same pattern may apply to sturgeon scutes. It is reasonable to assume that the latitudinally-distant environments of the Hudson River and the Maine rivers differ more than the two proximal Maine rivers do from each other, a pattern which would correspond with the observed pattern of morphological variation.

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Even though the particular genetic marker analyzed in our study is unrelated to the elaboration of the morphological features analyzed, the finding of significant genetic differences among all three populations indicates substantial reproductive isolation among them and that the observed morphological differences may be partly or wholly genetic. Common garden experiments among specimens from different populations raised under identical conditions would be particularly revealing to this end. Whether the genetic differentiation we document is a result of genetic drift, or selection due to ecological differences between the rivers cannot be resolved without additional study. Casselman et al. (1981) argued that spatially separated fish populations with varying degrees of differentiation should be managed separately to preserve the integrity of each population. However, it was recommended that lake sturgeon in the St. Lawrence system be managed as one integrated unit despite significant differences among geographic subunits (Guenette et al. 1992). Intraspecific variation, whether genetic or morphological, must be interpreted in conjunction with each specific species and management environment, and particular consideration of possible ramifications must be given in the case of an endangered species, such as the shortnose sturgeon. Quattro et al. (1996) stressed the need to understand the historical and evolutionary connections between extant populations of endangered species, and also recommended using a variety of biological information when making management decisions. Variation observed in this study, combined with current knowledge about the life history attributes of shortnose sturgeon indicate that conservative decision making is necessary. Possibly distinct genetic lines of shortnose sturgeon should be protected to the fullest extent until further morphological and genetic studies on shortnose sturgeon populations can be completed to determine the extent and meaning of differentiation among populations. ACKNOWLEDGMENTS The authors thank C. Adams, D. Ladanyi, and S. Nack for assistance in the collection of the Hudson River data, I. B. Wirgin for DNA isolations, and N. Haley, K. Arend, D. Secor, and an anonymous reviewer for providing comments that improved this manuscript. This work is a contribution from research sponsored by the Hudson River Foundation with supplemental support provided by the U.S. Army Corps of Engineers, the New York Sea Grant Institute, the New York Department of Environmental Conservation, the Atlantic Center for the Environment, National Institute of Environmental Health Sciences Center Grant ES00260, and the U.S. National Marine Fisheries Service.

LITERATURE CITED ALLENDORF, F. W. AND S. R. PHELPS. 1981. Use of allele frequencies to describe population structure. Canadian Journal of Fisheries and Aquatic Sciences 38:1507–1514.

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UNPUBLISHED MATERIALS

SAVOY, T. personal communication. Connecticut Department of Environmental Protection, Marine Fisheries Office, P. O. Box 719, Old Lyme, Connecticut 06371. Received for consideration, March 24, 2000 Accepted for publication, September 8, 2000