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

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

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.

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

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 with a frequency (*p*<sup>a</sup> ) of 5% is 36% (i.e., *p* = (1−*p*<sup>a</sup> ) *s* = 0.9520, [44]). Therefore, there is a non-zero likelihood that some populations characterized as "pure" southern Appalachian are of mixed genetic origin. Similarly, sample size also affects estimation of within-population diversity statistics such as *P* and *H*<sup>0</sup> . Sampling of a limited number of populations in a watershed also would affect estimates of between-population genetic variability.

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

The Yadkin (upper Pee Dee) River is an Atlantic-slope watershed. Despite the common presumption that Atlantic-slope drainages would contain native northern-form brook trout [8, 12, 15], two pure southern Appalachian populations (Pauls Creek, South Fork Stewarts Creek) were found in the Yadkin drainage. Although no early sampling efforts are known from the upper Pee Dee in Virginia [45], the section of the river that flows through North Carolina was excluded from the range of brook trout originally described by Smith [46]. However, several stream capture events have been inferred in this region, suggesting that these populations are descendants of brook trout captured from the New River [45]. Inspection of stocking records showed that both Pauls Creek and South Fork Stewarts Creek were stocked in the recent past, implying that the "native" southern strain persisted despite stocking.

Earlier genetic study [14] and geographic location suggest that the James River historically contained northern-form brook trout. Three populations from the James River screened in this study were characterized as northern form. This finding leaves little doubt that the New River is the boundary between northern and southern Appalachian brook trout populations.

In this study, 16 populations from the New River drainage (36%) were characterized as southern Appalachian brook trout. No geographic patterns of genetic variation were observed among the populations of putative pure southern origin. Interestingly, two of these "pure southern" populations (Crooked Creek and West Fork Dry Run) were stocked in the recent past with northern-derived hatchery fish. Crooked Creek is a "put-and-take" fishing area, and 5000 brook trout are stocked annually, yet it maintained an apparently pure southern population. Sixty-three percent of the populations from the New River drainage were of mixed origin, with the southern allele at frequencies ranging from 0.21 to 0.98. Although stocking records are limited, only two of these (Howell Creek and Little Indian Creek) are known to have been stocked with northern-derived hatchery fish. Only one population (Pearis Thompson Branch) in the New River was characterized as pure northern.

In addition to the 56 populations characterized in this study, we compiled data from all known genetic studies of brook trout populations in southwest Virginia [12, 14, 15]. Fortyseven percent (*n* = 39) of all 83 populations characterized in southwest Virginia were of mixed genetic origin (**Table 3**); however, many of these introgressed populations were largely southern. In addition, the "pure" southern populations (*n* = 26) that remain provide opportunities for restoration of southern Appalachian brook trout in Virginia.

### **4.4. Range-wide geographic distribution and genetic affinity of New River brook trout populations**

With the zone of contact between the northern and southern forms lying roughly at the New River watershed, it is unknown whether the New River historically contained the pure southern Appalachian form, or whether it was a zone of intergradation among southern and northern Appalachian lineages. Interpreting data across this study and the three studies noted above [12, 14, 15], the New River drainage contains 20 pure southern populations, suggesting that the presence of northern alleles could be due to either stocking or stream capture events. However, a large proportion (64%) of populations from the New

River are of mixed genetic origin, suggesting either that hatchery fish persisted in the New watershed or that the New River is a zone of natural intergradation. To gain a better understanding of the geographic distribution of southern Appalachian brook trout, we compiled

**Table 3.** Genetic characterization at the *CK-A2\** locus for southwest Virginia brook trout populations not sampled in this

*N* = number of individuals per sample.

study, compiled from both published and unpublished data sources.

**Stream River drainage County** *N* **% Southern allele Source** Green Cove Creek Holston Washington 19 95 [15] Grindstone Branch Holston Smyth 16 97 [14] Houndshell Branch Holston Smyth 12 100 [14] Jerry Creek Holston Smyth 11 100 [14] Little Laurel Creek Holston Smyth 16 100 [14] Johns Creek James Giles 23 0 [14] Shawvers Run James Giles 23 0 [14] Spy Run James Augusta 21 0 [14] Valley Branch James Craig 15 0 [14] Burks Fork New Floyd 15 67 [15] Cox Branch New Tazewell 15 53 [15] Dry Creek New Smyth 24 100 [14] Hanks/EF Chestnut Creek New Grayson 10 70 [14] Helton Creek New Grayson 21 79 [15] Jerry Creek New Grayson 15 67 [15] Killinger Creek New Smyth 12 88 [14] Laurel Branch New Floyd 15 97 [14] Laurel Fork New Floyd 7 79 [12] Lewis Fork New Grayson 21 79 [15] Middle Fork Helton New Grayson 20 100 [14] NF Elk Creek New Grayson 19 100 [14] NP Buckhorn Creek New Carroll 25 100 [14] Wilburn Branch New Grayson 21 75 [15] Big Stony Creek Roanoke Bedford 10 0 [12] Little Stony Creek Roanoke Bedford 6 0 [12] Rock Castle Creek Roanoke Patrick 25 36 [14] Turkey Creek Yadkin Carroll 15 47 [15]

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65

Genetic Characteristics of Southern and Northern Brook Trout (*Salvelinus fontinalis*) Populations... http://dx.doi.org/10.5772/intechopen.70719 65


The Yadkin (upper Pee Dee) River is an Atlantic-slope watershed. Despite the common presumption that Atlantic-slope drainages would contain native northern-form brook trout [8, 12, 15], two pure southern Appalachian populations (Pauls Creek, South Fork Stewarts Creek) were found in the Yadkin drainage. Although no early sampling efforts are known from the upper Pee Dee in Virginia [45], the section of the river that flows through North Carolina was excluded from the range of brook trout originally described by Smith [46]. However, several stream capture events have been inferred in this region, suggesting that these populations are descendants of brook trout captured from the New River [45]. Inspection of stocking records showed that both Pauls Creek and South Fork Stewarts Creek were stocked in the recent past, implying that the "native" southern strain persisted despite stocking.

Earlier genetic study [14] and geographic location suggest that the James River historically contained northern-form brook trout. Three populations from the James River screened in this study were characterized as northern form. This finding leaves little doubt that the New River is the boundary between northern and southern Appalachian brook trout populations.

In this study, 16 populations from the New River drainage (36%) were characterized as southern Appalachian brook trout. No geographic patterns of genetic variation were observed among the populations of putative pure southern origin. Interestingly, two of these "pure southern" populations (Crooked Creek and West Fork Dry Run) were stocked in the recent past with northern-derived hatchery fish. Crooked Creek is a "put-and-take" fishing area, and 5000 brook trout are stocked annually, yet it maintained an apparently pure southern population. Sixty-three percent of the populations from the New River drainage were of mixed origin, with the southern allele at frequencies ranging from 0.21 to 0.98. Although stocking records are limited, only two of these (Howell Creek and Little Indian Creek) are known to have been stocked with northern-derived hatchery fish. Only one population (Pearis Thompson Branch)

In addition to the 56 populations characterized in this study, we compiled data from all known genetic studies of brook trout populations in southwest Virginia [12, 14, 15]. Fortyseven percent (*n* = 39) of all 83 populations characterized in southwest Virginia were of mixed genetic origin (**Table 3**); however, many of these introgressed populations were largely southern. In addition, the "pure" southern populations (*n* = 26) that remain provide opportunities

**4.4. Range-wide geographic distribution and genetic affinity of New River brook trout** 

With the zone of contact between the northern and southern forms lying roughly at the New River watershed, it is unknown whether the New River historically contained the pure southern Appalachian form, or whether it was a zone of intergradation among southern and northern Appalachian lineages. Interpreting data across this study and the three studies noted above [12, 14, 15], the New River drainage contains 20 pure southern populations, suggesting that the presence of northern alleles could be due to either stocking or stream capture events. However, a large proportion (64%) of populations from the New

in the New River was characterized as pure northern.

**populations**

64 Biological Resources of Water

for restoration of southern Appalachian brook trout in Virginia.

**Table 3.** Genetic characterization at the *CK-A2\** locus for southwest Virginia brook trout populations not sampled in this study, compiled from both published and unpublished data sources.

River are of mixed genetic origin, suggesting either that hatchery fish persisted in the New watershed or that the New River is a zone of natural intergradation. To gain a better understanding of the geographic distribution of southern Appalachian brook trout, we compiled allele frequency data from all known genetic studies of brook trout populations throughout the native range (**Table 4**). Frequencies of the *CK-A2*\*100 (i.e., southern) allele were weighted based on sample size and averaged across all populations in each river drainage. **Figure 2** shows the frequency of the southern allele in each of the major river drainages from which data were collected.


All river drainages north of the New River were characterized as pure northern, with the exception of the Roanoke River drainage that contained a single population with a low frequency of the southern allele, likely due to the transfer of individuals from another location or stream capture. The frequency of the southern allele in river drainages south of the New River ranges from 29% in the Broad River of North Carolina to 100% in the Coosa River of Georgia. Genetic characterization of individuals from 111 populations in the New River drainage showed an 85% frequency of the southern-form allele. **Figure 2** shows that the south/north break is sharp and that this break occurs at the New/Roanoke-James watershed divide. This weakens the hypothesis that the New River is a zone of natural intergradation between the southern and northern forms of brook trout, and supports the hypothesis that the presence of northern alleles is due to either stocking or stream capture. However, it is important to qualify this inference by noting that genetic characterization is based on variation at a single locus. Ongoing screening of New River populations using microsatellite DNA markers will provide further insights into patterns of population genetic differentiation, shedding light on the native character of New River brook trout populations. In particular, microsatellite variation may clarify whether northern alleles observed in populations examined are characteristic

**Figure 2.** Genetic characterization of brook trout populations in major river drainages, based on the *CK-A2*\* locus, using data compiled from all known genetic studies of brook trout populations throughout the native range. See **Table 4** for

Northern Southern Introgressed

Genetic Characterization

WV

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

**9**

**14**

**10**

**25**

**13**

River Drainage 1 Susquehanna 15 Savannah 2 Ohio 16 Nolichucky 3 Gunpowder 17 Nantahala 4 Patapsco 18 Tuckasegee 5 Potomac 19 French Broad 6 James 20 Hiwassee 7 Rappahannock 21 Cheoah 8 Roanoke 22 Little 9 New 23 Pigeon 10 Broad 24 Little Tennessee 11 Chattahoochee 25 Watauga 12 Tellico 26 Holston 13 Catawba 27 Tennessee 14 Yadkin 28 Coosa

MD

**4**

**1**

67

PA

**2 3**

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

**5**

VA

**7**

**6**

**8**

of particular hatchery stocks or of native regional variation.

0 50 0 50000 100 150 100000 150000200 25 200000 2500000 kilometers

OH

NC TN

**19**

**18**

**16**

**26**

**15**

GA SC

**17**

**22 23**

**11**

**20**

**12**

**21**

**24**

KY

**28**

details.

**27**

N

1 Relative to eastern continental divide.

2 Allele frequency based on number of individuals analyzed per stream and averaged across all populations in each drainage.

**Table 4.** Genetic characterization of brook trout populations in regional river drainages, based on frequency of the diagnostic *CK-A2\*100* allele using data gathered from all available published and unpublished studies.

Genetic Characteristics of Southern and Northern Brook Trout (*Salvelinus fontinalis*) Populations... http://dx.doi.org/10.5772/intechopen.70719 67

allele frequency data from all known genetic studies of brook trout populations throughout the native range (**Table 4**). Frequencies of the *CK-A2*\*100 (i.e., southern) allele were weighted based on sample size and averaged across all populations in each river drainage. **Figure 2** shows the frequency of the southern allele in each of the major river drainages

**River drainage State Position1 # of streams # of individuals % Southern2 Source(s)** Susquehanna PA/MD East 4 145 0 [7, 9] Ohio MD West 3 110 0 [9] Gunpowder MD East 1 40 0 [9] Patapsco MD East 1 40 0 [9] Potomac MD/VA East 6 190 0 [9, 14] James VA East 7 142 0 [14, current]

Rappahannock VA East 1 25 0 [14] Roanoke VA East 3 41 22 [12, 14] New VA/NC West 101 1999 85 [14,

Yadkin VA/NC East 37 691 58 [8, 12,

Nolichucky NC/TN West 51 1058 64 [7, 8, 11] French Broad NC/TN West 80 1281 73 [8, 11, 16] Little Tennessee NC/TN West 49 886 82 [8, 13] Watauga NC/TN West 44 691 88 [8, 11] Broad NC East 3 41 29 [8, 11] Hiwassee NC West 6 146 76 [8, 11] Cheoah NC West 10 210 80 [8, 11] Little TN West 8 90 80 [8, 11] Tellico TN West 5 64 42 [11] Savannah NC/GA East 27 533 63 [10, 16] Chattahoochee GA West 1 21 31 [10] Tennessee GA West 7 145 93 [10] Coosa GA West 1 12 100 [10]

Holston VA/TN West 24 320 91 [8, 11, 14, current]

Allele frequency based on number of individuals analyzed per stream and averaged across all populations in each

**Table 4.** Genetic characterization of brook trout populations in regional river drainages, based on frequency of the

diagnostic *CK-A2\*100* allele using data gathered from all available published and unpublished studies.

15, current]

15, current]

from which data were collected.

66 Biological Resources of Water

1

2

drainage.

Relative to eastern continental divide.

**Figure 2.** Genetic characterization of brook trout populations in major river drainages, based on the *CK-A2*\* locus, using data compiled from all known genetic studies of brook trout populations throughout the native range. See **Table 4** for details.

All river drainages north of the New River were characterized as pure northern, with the exception of the Roanoke River drainage that contained a single population with a low frequency of the southern allele, likely due to the transfer of individuals from another location or stream capture. The frequency of the southern allele in river drainages south of the New River ranges from 29% in the Broad River of North Carolina to 100% in the Coosa River of Georgia. Genetic characterization of individuals from 111 populations in the New River drainage showed an 85% frequency of the southern-form allele. **Figure 2** shows that the south/north break is sharp and that this break occurs at the New/Roanoke-James watershed divide. This weakens the hypothesis that the New River is a zone of natural intergradation between the southern and northern forms of brook trout, and supports the hypothesis that the presence of northern alleles is due to either stocking or stream capture. However, it is important to qualify this inference by noting that genetic characterization is based on variation at a single locus. Ongoing screening of New River populations using microsatellite DNA markers will provide further insights into patterns of population genetic differentiation, shedding light on the native character of New River brook trout populations. In particular, microsatellite variation may clarify whether northern alleles observed in populations examined are characteristic of particular hatchery stocks or of native regional variation.

#### **4.5. Management implications**

Brook trout is the only salmonid native to the southern Appalachian region. The American Fisheries Society Southern Division Trout Committee developed a position statement [22] expressing the importance of SABT and presenting recommendations for conservation-oriented management of this regional resource. Our results contribute to the recommended completion of genetic inventory of critical populations using non-lethal sampling methods. In this context, we frame the management implications for management of SABT populations.

knoxnews.com/news/aquarium-helping-to-restore-native-trout-ep-510367109-355447741. html). SABT can be stocked to re-establish populations in streams where they have been extirpated. Also, while we do not recommend eradicating non-native or introgressed populations in watersheds where brook trout are native, we recommend stocking southern-strain hatchery fish into these populations to shift allele frequencies toward those of native populations. Progress in re-establishing native brook trout populations should be monitored using genetic

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

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69

This work was funded through the Federal Aid in Sport Fish Restoration Project F-128-R, administered by the Virginia Department of Game and Inland Fisheries, and is based on the Master's degree research of Joanne (Davis) Printz. We thank George Palmer and Cliff Kirk for assistance with fieldwork and collection of genetic samples. Ray Morgan of the Maryland Department of Natural Resources kindly provided unpublished data from Maryland populations, and Doug Besler of the NCWRC generously provided tissue samples. Finally, we thank Chris Printz of ATS International, Inc. of Christiansburg, VA for his assistance in the design and production of the maps. Funding for EH's participation in this work was provided in part by the Virginia Agricultural Experiment Station and the Hatch Program of the National

and Eric M. Hallerman1

1 Department of Fish and Wildlife Conservation, Virginia Polytechnic Institute and State

[1] MacCrimmon HR, Campbell JS. World distribution of brook trout, *Salvelinus fontinalis*. Journal of the Fisheries Research Board of Canada. 1969;**26**:1699-1723. DOI: 10.1139/

[2] Hudy M, Thieling TM, Gillespie N, Smith EP. Distribution, status, and land use characteristics of subwatersheds within the native range of brook trout in the eastern United States. North American Journal of Fisheries Management. 2008;28:1069-1085. DOI: http://

2 Virginia Department of Game and Inland Fisheries, Blacksburg, VA, USA

\*

Institute of Food and Agriculture, U.S. Department of Agriculture.

, Joseph Williams2

\*Address all correspondence to: ehallerm@vt.edu

University, Blacksburg, VA, USA

dx.doi.org/10.1577/M07-017.1.

markers every few generations.

**Acknowledgements**

**Author details**

Joanne E. Printz1

**References**

f69-159

Results from this and other studies demonstrate that stocking of non-native genotypes poses long-term genetic impacts and interferes with efforts to conserve southern Appalachian brook trout. Although negative effects of stocking have become well known, some fisheries management agencies maintain imprecise stocking records. Further, hatchery personnel often substitute one brook trout stock for another based on availability. We recommend that all stocking and transfers of brook trout be well planned with cognizance of genetic conservation objectives and thoroughly and accurately documented.

Management units—that is, populations that are demographically independent of one another—may be defined functionally as populations that have substantially divergent allele frequencies at many loci [47]. We had but limited ability to estimate levels of genetic diversity and differentiation among regional brook trout populations using allozyme markers. The results of ongoing screening of microsatellite DNA markers will be used to quantify differentiation among native populations, providing the basis for defining defensible management units. Results to date support the view that southern Appalachian brook trout populations should be managed on a stream-by-stream basis.

Those populations characterized as pure SABT should be given conservation priority. The stocking and transfer of non-native genotypes into these populations should be prohibited. Harvest should be allowed only in those populations that are demographically able to sustain themselves. We recommend that introgressed populations that contain less than 5% admixture from northern-strain brook trout be treated as 'pure' southern. However, we caution that the level of introgression in these populations may be higher than allozyme frequencies suggest; hence, individuals from these streams should not be transferred into streams that contain pure SABT populations. Hatchery brook trout should be stocked only into those streams that contain pure northern-strain populations and those with greater than 5% admixture.

We caution that any negative consequences of stocking also would apply to native northernstrain populations (i.e., in the James and Roanoke river drainages). Allozyme markers do not provide enough resolution to differentiate between native northern and hatchery populations, and so we recommend that all brook trout populations should be screened and characterized using microsatellite or single nucleotide polymorphism markers. Until we know more about the genetic composition of these populations, it may be wise to stock only infertile triploid brook trout [48].

Southern Appalachian brook trout hatchery stocks are being established in conservationoriented hatchery programs ([49], https://brooktrouthatchery.wordpress.com/, http://archive. knoxnews.com/news/aquarium-helping-to-restore-native-trout-ep-510367109-355447741. html). SABT can be stocked to re-establish populations in streams where they have been extirpated. Also, while we do not recommend eradicating non-native or introgressed populations in watersheds where brook trout are native, we recommend stocking southern-strain hatchery fish into these populations to shift allele frequencies toward those of native populations. Progress in re-establishing native brook trout populations should be monitored using genetic markers every few generations.
