**2. Methods**

This species was once abundant in coldwater lakes and streams throughout its range, but environmental disturbances such as deforestation, development, and pollution: and the introduction of non-native rainbow trout (*Oncorhynchus mykiss*) and brown trout (*Salmo trutta*)

Beginning in the mid-1800s, fishery managers began stocking hatchery-reared brook trout extensively. However, hatchery-reared brook trout often exhibit lower growth, yield, survival, and natural reproduction than locally adapted wild populations [3, 4]. Further, the hybridization of hatchery-derived fish with wild populations can compromise the genetic integrity and fitness of receiving populations by introducing foreign alleles and breaking up locally adapted gene complexes [5, 6]. The stocking of northern-derived hatchery brook trout is of particular concern in the southern part of its range due to significant population genetic differentiation between southern and northern lineages of brook trout. Genetic differences between the two lineages may be large enough to justify distinction at the subspecies level [7, 8]. In addition, screening of allozyme [7–16], mitochondrial DNA [17–19], and microsatellite nuclear DNA [20, 21] markers has uncovered smaller scale genetic variation throughout the geographic range of brook trout. Differentiation at smaller geographic scales may reflect different colonization histories, as well as differential effects of selective and non-selective population genetic

Native southern Appalachian brook trout (SABT) populations share several biological characteristics [22]. Food availability being a limiting factor in these systems, adult fish are typically small (<229 mm total length) and life span seldom exceeds 3 years [23, 24]. Native SABT and introduced northern-lineage brook trout differed in terms of survival in the laboratory and diet in a natural stream [25]. Comparison of external microbial assemblages suggested that SABT exhibit greater ability to inhibit microbial growth in their epidermal mucus than do northern brook trout of hatchery ancestry [26]. Demonstration that SABT are genetically distinct from northern-origin hatchery stocks led management agencies to assess the heritage of populations within their jurisdiction, for example, in the Great Smoky Mountains National Park [8, 13], Tennessee [11], North Carolina [12, 16, 27], and Georgia [10]. Molecular and adaptive differentiation may warrant management of brook trout populations or groups of populations as evolutionary significant units [28], although some of their population genetic differentiation may reflect stocking

The zone of contact between the southern and northern lineages of Appalachian brook trout is roughly at the New River watershed [14, 15, 29]. Against the background of decline of the southern form and history of stocking with non-native strains, genetic characterization of brook trout populations at the zone of contact is needed to support informed management decisions and conserve the native form of the species. The objective of this study was to use established allozyme markers to wild Appalachian brook trout populations at the zone of contact in southwest Virginia as southern or northern

have drastically reduced the number and sizes of wild populations [2].

processes.

56 Biological Resources of Water

history.

lineages or introgressed.

#### **2.1. Sampling**

Seventy-eight historic wild brook trout streams from the New, James, Holston, and Yadkin river drainages [30] were sampled by backpack electrofishing. Brook trout tissue samples were collected from 916 individuals from 56 streams (**Table 1**). Sample sizes ranged from 8 to 26 individuals per stream. Fish were anesthetized, and two samples of dorsal muscle tissue (from fish greater than 120 mm TL) were collected non-lethally using an 18-gauge Monopty Biopsy Instrument (C.R. Bard, Inc., Covington, GA) and immediately placed on dry ice. Anesthetized fish were fully revived in fresh water prior to release. A limited number of fish of <120 mm total length were sacrificed to sample streams from which few adults were collected. Samples were stored at −80°C.

## **2.2. Protein analysis**

Genetic analysis was performed using cellulose acetate gel electrophoresis to observe variability at nine loci encoding five polymorphic enzymes: creatine kinase (*CK-A2*\*), aspartate aminotransferase (*sAAT-1,2*\*), glycerol-3-phosphate dehydrogenase (*G3PDH*\*), glucose-6-phosphate isomerase (*GPI-A*\*, *GPI-B1,2*\*), and malate dehydrogenase (*sMDH-B1,2*\*). Muscle tissue was homogenized in 200 μl of 0.09 M tris-HCl (pH 8.0), and subjected to electrophoresis in tris-glycine buffer (pH 7.5 or 8.0) for 45 min, followed by staining for enzyme activity. Electrophoretic conditions and histochemical staining procedures were modified from those described by Hebert and Beaton [31] and Galbreath et al. [16]. Individuals from the Paint Bank Hatchery in Virginia were included in the analysis as a northern reference population because the hatchery is known to culture the northern lineage. The North Carolina Wildlife Resource Commission provided tissue samples from individuals from Charles Creek of the North Toe River drainage, a known SABT population, for use as a reference population.

#### **2.3. Data analysis**

Allele frequencies for *CK-A2*\*, *G3PDH*\*, *GPI-A*\*, and *MDH-B1,2*\* were calculated for all populations using the Excel Microsatellite Toolkit [32]. Allele frequencies could not be calculated for *sAAT-1,2*\* and *GPI-B1,2*\* using that program because both enzymes are encoded by isoloci (i.e., duplicated loci with alleles of overlapping mobility). Since genotypes among heterozygous individuals could not be determined with certainty for *sAAT-1,2*\*, phenotype frequencies were calculated using the program FDASH [33]. The *GPI-B1,2*\* isoloci contain multiple alleles that could not be assigned to either locus with confidence; hence, they were treated as a single tetraploid locus and allele frequencies were estimated using the program AUTOTET [34]. Initially, allele frequency data from all nine marker loci were used to calculate genetic distance, population differentiation, contingency-table analysis of heterogeneity among populations, and hierarchical cluster analysis using the program BIOSYS-1 [35]. The same statistics then were calculated using only the five marker loci with unambiguous interpretation of allelic expression (i.e., omitting data from *sAAT-1,2*\* and *GPI-B1,2*\*), to determine any effect of


*CK-A2\* G3PDH\* GPI-A\* MDH-B1,2\**

East Fork Cove Creek

East Fork Crooked Creek

East Fork Little Reed Island

Little Indian Creek

Little Snake Creek

Little Stony Creek

Little Wilson Creek

Middle Fox Creek

NF Stony Creek

No Business Creek

Opossum Creek

Pearis Thompson Branch

East Fork Dry Run

*N \*78 \*100 \*45 \*100 \*87 \*100 \*115 \*100 \*145 P A HO HE*

14 0.11 0.89 1.00 0.93 0.07 1.00 0.75 1.8 0.145 0.133

Genetic Characteristics of Southern and Northern Brook Trout (*Salvelinus fontinalis*) Populations...

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20 0.03 0.98 1.00 0.98 0.02 1.00 0.50 1.5 0.089 0.084

20 1.00 1.00 1.00 1.00 0.50 1.5 0.025 0.025

10 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000

19 0.79 0.21 1.00 1.00 0.95 0.05 0.50 1.5 0.125 0.101

8 1.00 1.00 1.00 1.00 0.50 1.5 0.132 0.111

14 0.11 0.89 1.00 0.96 0.04 1.00 0.00 1.0 0.000 0.000

19 0.21 0.79 0.03 0.97 1.00 0.82 0.18 0.50 1.5 0.071 0.067

12 0.04 0.96 1.00 0.04 0.96 0.58 0.42 0.00 1.0 0.000 0.000

21 0.02 0.98 1.00 0.98 0.02 1.00 0.50 1.5 0.147 0.128

20 0.20 0.80 0.03 0.98 1.00 0.90 0.10 0.50 1.5 0.024 0.024

17 0.03 0.97 1.00 1.00 0.72 0.28 0.00 1.0 0.000 0.000

17 1.00 0.15 0.85 1.00 0.91 0.09 0.50 1.5 0.155 0.119

Mill Creek 17 0.12 0.88 1.00 1.00 0.82 0.18 0.75 1.8 0.184 0.176 NB Elk Creek 14 0.25 0.75 1.00 1.00 1.00 0.75 1.8 0.250 0.168

Oldfield Creek 12 1.00 1.00 1.00 1.00 0.75 1.8 0.163 0.141

Elkhorn Creek 10 1.00 1.00 1.00 0.95 0.05 0.25 1.3 0.125 0.097 Fox Creek 20 0.18 0.83 1.00 0.95 0.05 0.88 0.12 0.25 1.3 0.025 0.025 Grassy Creek 9 1.00 1.00 1.00 1.00 0.75 1.8 0.150 0.154 Howell Creek 20 0.05 0.95 1.00 1.00 0.98 0.02 0.00 1.0 0.000 0.000 Laurel Branch 22 0.23 0.77 1.00 1.00 0.98 0.02 0.50 1.5 0.038 0.037 Laurel Creek 10 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000 Laurel Creek 20 0.10 0.90 1.00 0.98 0.02 1.00 0.50 1.5 0.063 0.059 *CK-A2\* G3PDH\* GPI-A\* MDH-B1,2\**

Grassy Branch 12 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000

Parks Creek 10 0.05 0.95 1.00 1.00 1.00 0.25 1.3 0.025 0.025

Roaring Fork 8 0.56 0.44 1.00 0.69 0.31 1.00 0.50 1.5 0.188 0.246 Sturgill Branch 16 0.19 0.81 1.00 1.00 0.75 0.25 0.50 1.5 0.219 0.175

Ewins Run 20 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000 Pickles Branch 20 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000

Bear Creek 23 0.02 0.98 1.00 1.00 1.00 0.25 1.3 0.016 0.016

Buffalo Branch 16 0.06 0.94 1.00 0.97 0.03 1.00 0.75 1.8 0.125 0.111 Cabin Creek 20 0.05 0.95 1.00 1.00 1.00 0.50 1.5 0.047 0.046

Ding Branch 26 0.25 0.75 0.02 0.98 1.00 0.94 0.06 0.00 1.0 0.000 0.000

5 1.00 1.00 1.00 1.00

16 1.00 0.44 0.56 1.00 1.00

Controls Charles Creek,

Paint Bank Hatchery

Henshew Branch

Pennington Branch

Barbours Creek

Big Horse Creek

Big Laurel Creek

Big Reed Island Creek

Bournes Branch

Chestnut Creek

Chisholm Creek

Crooked Creek

James River drainage

New River drainage

Holston River drainage

58 Biological Resources of Water

NC

*N \*78 \*100 \*45 \*100 \*87 \*100 \*115 \*100 \*145 P A HO HE*

20 1.00 0.45 0.55 1.00 1.00 0.25 1.3 0.125 0.127

12 0.08 0.92 1.00 1.00 1.00 0.25 1.3 0.042 0.040

20 1.00 0.08 0.93 1.00 1.00 0.25 1.3 0.021 0.036

18 1.00 1.00 1.00 1.00 0.25 1.3 0.011 0.011

11 0.05 0.95 0.09 0.91 1.00 1.00 0.00 1.0 0.000 0.000

20 0.08 0.93 1.00 1.00 0.95 0.05 0.50 1.5 0.068 0.066

16 0.03 0.97 1.00 1.00 1.00 0.50 1.5 0.063 0.061

17 0.12 0.88 1.00 1.00 1.00 0.25 1.3 0.000 0.024

12 1.00 1.00 1.00 0.96 0.04 0.25 1.3 0.021 0.021

15 1.00 1.00 1.00 1.00 0.25 1.3 0.059 0.053



Allele frequency data from previous studies of brook trout population genetics were compiled and combined with the results from this study to gain a better understanding of the geographic distribution of SABT in Virginia, as well as the genetic composition of brook trout

Genetic Characteristics of Southern and Northern Brook Trout (*Salvelinus fontinalis*) Populations...

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61

Of 56 wild brook trout populations from 4 major river drainages analyzed in this study, 19 were fixed for the diagnostic *CK-A2*\*100 allele, and were designated as pure SABT populations (**Table 1**). Five populations fixed for the *CK-A2*\*78 allele were designated as northern, and 32 populations exhibiting variation at the *CK-A2*\* locus were designated as introgressed. The three James watershed populations exhibited alleles characteristic of northern-form brook trout. Populations in other watersheds were characterized as southern (*n* = 19), north-

Only the Cabin Creek population (New River drainage, Grayson County) deviated significantly (*p* < 0.05) from Hardy-Weinberg equilibrium at the *CK-A2*\* locus. No other deviations from Hardy-Weinberg equilibrium were detected, indicating that the respective populations were in reasonable conformance with assumptions underlying the model. The proportions of polymorphic loci (*P*), the mean number of alleles per locus (*A*), and mean heterozygosities

the putative southern populations (*P* = 0.05, *H*<sup>0</sup> = 0.004; **Table 2**). The introgressed populations exhibited the highest means for metrics of genetic variability (*P* = 0.48, *H*<sup>0</sup> = 0.099), and the northern populations exhibited intermediate means (*P* = 0.20, *H*<sup>0</sup> = 0.053). Grouped by drainage, Yadkin River populations had the lowest means (*P* = 0, *H*<sup>0</sup> = 0), followed by James River

Based on analysis at four polymorphic allozyme loci (*CK-A2\*, G3PDH\*, GPI-A\*, sMDH-B1,2\**). Abbreviations: number of populations per group (*N*), proportion of polymorphic loci (*P*), mean number of alleles per locus (*A*), expected

**Table 2.** Genetic diversity of brook trout populations, variously grouped by drainage, lineage, and geographic location

**Group** *N P A Ho He* Holston River drainage 6 0.29 1.3 0.100 0.102 James River drainage 3 0.08 1.1 0.007 0.012 New River drainage 45 0.34 1.4 0.064 0.058 Yadkin River drainage 2 0.00 1.0 0.000 0.000 Southern lineage 19 0.05 1.1 0.004 0.004 Northern lineage 5 0.20 1.2 0.053 0.036 Introgressed 32 0.48 1.5 0.099 0.091 Atlantic Ocean drainages 5 0.05 1.1 0.004 0.007 Gulf of Mexico drainages 51 0.33 1.4 0.068 0.063

values were lowest in

populations throughout the Appalachian portions of the native range.

(*H*) for each population are listed in **Table 1**. Observed mean *P* and *H*<sup>0</sup>

**3. Results**

ern (*n* = 2), or introgressed (*n* = 32).

heterozygosity (*Ho*), and observed heterozygosity (*He*).

relative to the eastern continental divide.

Charles Creek, a known southern-strain population, was included as a southern-strain reference group. Individuals from Paint Bank Hatchery, which cultures the northern strain, were included as a northern-strain reference group. Abbreviations: number of individuals analyzed (N), proportion of polymorphic loci (*P*), mean number of alleles per locus (*A*), expected heterozygosity (*H*<sup>o</sup> ), and observed heterozygosity (*H*<sup>e</sup> ).

**Table 1.** Allele frequencies and genetic diversity at four polymorphic loci (*CK-A2\*, G3PDH\*, GPI-A\*, sMDH-B1,2\**) in wild brook trout populations in 56 southwest Virginia streams, grouped by drainage.

omitting these data from analysis. Similar conclusions were drawn from analysis of both data sets. Here, we report results based on analysis of the reduced dataset only.

Initial characterization of the genetic origin of each population was based on allele frequencies at the diagnostic *CK-A2*\* locus. Allele frequencies at the other markers were compared to those observed in northern and SABT populations characterized in previous studies [7–16]. Individual heterozygosity and polymorphism were calculated across five loci to assess levels of genetic diversity within each population [32]. Arlequin [36] was used to test for departures from Hardy-Weinberg equilibrium and to perform analysis of molecular variance (AMOVA) to characterize the distribution of the genetic diversity within and among populations and river basins. Cluster analysis using the unweighted pair-group with arithmetic averaging algorithm (UPGMA, [37]) was performed using BIOSYS-1 [35], and a dendrogram was built based on Nei's unbiased genetic distance [38].

Allele frequency data from previous studies of brook trout population genetics were compiled and combined with the results from this study to gain a better understanding of the geographic distribution of SABT in Virginia, as well as the genetic composition of brook trout populations throughout the Appalachian portions of the native range.
