**3. Results**

omitting these data from analysis. Similar conclusions were drawn from analysis of both data

**Table 1.** Allele frequencies and genetic diversity at four polymorphic loci (*CK-A2\*, G3PDH\*, GPI-A\*, sMDH-B1,2\**) in

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

Ripshin Creek 10 0.15 0.85 1.00 1.00 0.75 0.25 0.50 1.5 0.200 0.166 Roads Creek 11 1.00 1.00 0.95 0.05 1.00 0.25 1.3 0.023 0.023 Snake Creek 20 1.00 1.00 1.00 0.98 0.02 0.50 1.5 0.200 0.166

Stony Creek 20 0.18 0.83 0.03 0.98 1.00 0.95 0.05 0.25 1.3 0.100 0.111

Tory Creek 19 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000

Pauls Creek 20 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000

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

), and observed heterozygosity (*H*<sup>e</sup>

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

20 1.00 1.00 1.00 1.00 0.25 1.3 0.013 0.013

10 0.30 0.70 1.00 1.00 1.00 0.00 1.0 0.000 0.000

10 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000

19 1.00 1.00 1.00 1.00 0.25 1.3 0.063 0.057

12 0.13 0.88 1.00 1.00 1.00 0.00 1.0 0.000 0.000

17 0.12 0.88 1.00 1.00 0.97 0.03 0.50 1.5 0.044 0.068

24 1.00 1.00 1.00 1.00 0.00 1.0 0.000 0.000

).

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

sets. Here, we report results based on analysis of the reduced dataset only.

wild brook trout populations in 56 southwest Virginia streams, grouped by drainage.

based on Nei's unbiased genetic distance [38].

Standrock Branch

60 Biological Resources of Water

Sulfur Springs Branch

Upper West Fork Dry Run

West Fork Dry

Run

Whitetop Creek

West Fork Furnace Creek

South Fork Stewarts Creek

Yadkin River drainage

locus (*A*), expected heterozygosity (*H*<sup>o</sup>

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

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 (*H*) for each population are listed in **Table 1**. Observed mean *P* and *H*<sup>0</sup> values were lowest in 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 heterozygosity (*Ho*), and observed heterozygosity (*He*).

**Table 2.** Genetic diversity of brook trout populations, variously grouped by drainage, lineage, and geographic location relative to the eastern continental divide.

(*P* = 0.08, *H*<sup>0</sup> = 0.007), New River (*P* = 0.34, *H*<sup>0</sup> = 0.064), and Holston River (*P* = 0.29, *H*<sup>0</sup> = 0.100) populations. Atlantic-slope populations exhibited lower mean percent polymorphic loci and heterozygosity values (*P* = 0.05, *H*<sup>0</sup> = 0.004) than Gulf of Mexico drainage populations (*P* = 0.33, *H*<sup>0</sup> = 0.068). Analysis of molecular variance showed that approximately 34% of the total genetic diversity resulted from variation within populations, 18% among populations within drainages, and 48% among drainages. Most of the total limiting variance was attributed to the *CK-A2*\* locus, meaning that most of the variance that we measured with allozyme markers was due to differentiation among northern and southern lineages of the species.

**4. Discussion**

with a frequency (*p*<sup>a</sup>

statistics such as *P* and *H*<sup>0</sup>

**4.1. Decline of brook trout**

**4.2. Duplicated isozyme loci in brook trout**

We sampled 78 streams that historically contained brook trout populations, but found the species in only 56 of them [30]. The range of brook trout is shrinking [39] for several reasons, including habitat alteration, overexploitation, competition with introduced rainbow trout

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

http://dx.doi.org/10.5772/intechopen.70719

63

Certain allozyme markers posed complications to interpretation of underlying genotype. Brook trout show a high incidence of duplicated enzyme loci due to the tetraploid ancestry of salmonids [40]. Duplicated loci (termed isoloci) are genetically independent, but exhibit alleles of similar electrophoretic mobility that cannot be unambiguously assigned to either locus. Three of the five enzymes that we screened were encoded by isoloci (i.e., *MDH-B1,2*\*, *sAAT-1,2*\*, and *GPI-B1,2*\*). Ambiguous interpretation of the banding patterns of two of these isoloci, *sAAT-1,2*\* and *GPI-B1,2*\*, led us to eliminate them from statistical analysis [30]. Precise estimation of genetic diversity and differentiation metrics require data from many loci [41, 42]. Information from only four markers clearly limited the power of statistical analysis of genetic differentiation, especially with small sample sizes for some of the populations [43]. Genotypic data from more markers likely would reveal genetic differentiation not detected with only four loci. Ongoing screening of additional, more highly polymorphic markers, such as microsatellite DNA markers, will increase the ability to quantify population genetic differentiation.

Based on fixation for the diagnostic allele at the *CK-A2*\* locus and allele frequency differences at three other marker loci, 34% (*n* = 19) of the brook trout populations analyzed in this study were of southern Appalachian origin, 9% (*n* = 5) were of northern origin, and 57% (*n* = 32) were of mixed genetic origin (**Tables 1** and **2**). The level of certainty for precise characterization of a population is directly related to sample size. That is, any population observed to be fixed for the common allele actually may harbor the alternate allele at a low, undetected frequency. For example, with a sample size (*s*) of 20, our likelihood (*p*) of detecting an allele

likelihood that some populations characterized as "pure" southern Appalachian are of mixed genetic origin. Similarly, sample size also affects estimation of within-population diversity

Of the six populations from the Holston drainage, four were of mixed genetic origin, with the southern allele at frequencies ranging from 0.44 to 0.95. The Grassy Branch population was characterized as southern Appalachian, and the Henshew Branch population was characterized as pure northern. Results from earlier genetic studies [8, 11, 14] and its geographic location suggest that the Holston River historically contained the southern Appalachian lineage,

) *s* = 0.9520, [44]). Therefore, there is a non-zero

. Sampling of a limited number of populations in a watershed also

(*O. mykiss*) and brown trout (*S. trutta*) and more recently, climate change.

**4.3. Geographic distribution of SABT in southwest Virginia**

) of 5% is 36% (i.e., *p* = (1−*p*<sup>a</sup>

would affect estimates of between-population genetic variability.

so the presence of the northern allele is likely due to stocking.

There was no apparent pattern regarding where populations characterized as southern, northern, or introgressed were located geographically within the New, Holston, Yadkin, and James drainages (**Figure 1**). Cluster analysis of unbiased genetic distances [38] among all populations showed that all populations of northern origin or with a high frequency of the *CK-A2*\*78 allele clustered together; these included populations from the James River drainage (Barbours Creek, Ewin Run, and Pickles Branch), the Holston drainage (Henshew Creek), the New River drainage (Pearis Thompson and Little Indian Creek), and Paint Bank Hatchery. The Roaring Fork population in the Holston drainage had a high frequency of the northern allele, but did not cluster closely with the other northern populations due to a high frequency of a rare allele at the *GPI-A*\* locus. Cluster analysis of unbiased genetic distances [38] among populations showed no geographic patterns of genetic variation among the populations of putative southern Appalachian origin.

**Figure 1.** Genetic characterization at the *CK-A2*\* locus for 83 wild brook trout populations in southwest Virginia, including 56 populations characterized in this study and 27 populations characterized previously.
