kinda complicated but alos shows what I have been claiming as true. Read carefully
http://www.nwfsc.noaa.gov/publications/techmemos/tm17/Papers/Nielsen.htm INTRAPOPULATION DIFFERENCES FOUND IN CALIFORNIA
STEELHEAD USING DIRECT SEQUENCE OF mtDNA
Jennifer L. Nielsen,1,2 W. Kelley Thomas,2 Christina Gan,1 and Douglas Tupper2
1USDA U. S. Forest Service
Pacific Southwest Research Station
P. O. Box 245, Berkeley, CA 94701
2Department of Molecular and Cell Biology
401 Barker Hall
University of California
Berkeley, CA 94720
Polymerase chain reaction (PCR) and direct sequencing of mitochondrial DNA (mtDNA) were used to depict putative wild and hatchery populations of steelhead (Oncorhynchus mykiss) in California. Intraspecific mtDNA types were derived from analysis of base pair (bp) differences found in two hypervariable regions of the salmonid mtDNA D-loop (360 bp). Nucleotide variation was found at 16 base pair sites (4.4%) and 13 different steelhead/rainbow haplotypes were identified in California. Distribution of steelhead mtDNA alleles showed a distinct biogeographic cline, with relict landlocked populations and anadromous fish retaining similar frequencies of mtDNA types. Hatchery populations showed no direct biogeographic cline and had higher numbers of mtDNA types than sympatric wild populations. The objective of this study was to test the hypothesis that "wild-type" California steelhead still exist as anadromous and resident stocks.
Steelhead are commonly found in their anadromous form in streams along the Pacific coast from Cook Inlet, Alaska, to Malibu Creek on Santa Monica Bay, California. Steelhead have demonstrated highly variable life histories (Shapolov and Taft 1954). Populations in California express classic characteristics of anadromy where individuals spend up to 4 years in fresh water and then migrate to sea, where they grow rapidly and return to spawn in their natal streams. Other steelhead remain small and mature in freshwater lakes or streams, never adopting the anadromous life style. In California, numerous dams constructed from 1875 to 1930 were reported to trap anadromous steelhead, with subsequent residualization of these populations in local reservoirs. Current genetic, morphological, and morphometric evidence suggests that resident trout and anadromous steelhead are simply different life history forms within the same species (Reisenbichler and Phelps 1985; Currens et al. 1988).
The identification and distribution of intraspecific steelhead stocks throughout California have been a subject of recent controversy. High-seas gillnet fisheries, continuous drought conditions in California, and increased sport pressure have combined to contribute to an exponential decline in wild steelhead spawning runs. Over the last 60 years, rainbow trout of hatchery origin have been extensively outplanted into lakes and streams throughout California, leaving wild stocks in jeopardy due to hybridization and introgression. Concern has been expressed for protection of wild steelhead stocks at the southern extent of their range, where fish may be selectively adapted to low levels of summer flow and higher stream temperatures. These concerns have led to speculation that relict wild stocks may remain in streams and reservoirs south of Monterey Bay. This study looked at mtDNA haplotypes in putative wild stocks from Usal Creek in Mendocino County to Malibu Creek near Los Angeles and explored the genetic relationships between resident and anadromous steelhead remaining in California and contemporary hatchery populations.
The ease of extraction, accelerated evolution, and non-recombination inherent in mtDNA make it a prime target for analysis of intrapopulation relationships in salmonids. Mitochondrial DNA was extracted from non-intrusive fin samples taken from live fish in the field and frozen immediately in liquid nitrogen. DNA was extracted using chelex resin and autoclave thermal acceleration. Approximately 50 ng of DNA was used as a template in the PCR amplification. Amplification was done in 25 ul volumes using homologue-specific primers designed to amplify the hypervariable regions of the salmonid mtDNA D-loop (W. K. Thomas and J. L. Nielsen, unpublished data). Asymmetrical amplification was done according to methods described in Thomas et al. (1990). The PCR product was directly sequenced on acrylimide gels using a radioactive marker (35S) and visualized on autoradiographs.
Tissues from 324 steelhead, from 25 streams and 6 hatcheries, were successfully sequenced. In California, 16 D-loop nucleotide sites (4.4%) were found to vary in steelhead. Wild samples pooled into broad geographic localities showed a geographic gradient from north to south, with a distinct change in the dominant mtDNA allele for each region (north coast = 70% mtDNA Type 1; central coast = 45% Type 3; south coast = 56% Type 5). An aquatic biogeographic species boundary running southwest from Point Conception into the Pacific Ocean (USGLOBEC 1992) parallels the division of steelhead mtDNA types found only in southern California (mtDNA Types 6 and 8). Putative wild steelhead found above reservoirs with no record of hatchery outplanting (St. Inez River near Santa Barbara, and Redwood Creek near Oakland) were found to have mtDNA alleles endemic to their geographic range. Malibu Creek, the southernmost anadromous population, maintained anadromous and resident spawners carrying the dominant southern "wild" haplotype.
Steelhead taken from hatchery populations (N = 60; mean number of mtDNA alleles per population = 4.17) carried significantly more mtDNA alleles than steelhead from sympatric wild populations sampled throughout their distribution (N = 64; mean number of mtDNA alleles = 2.5; two-tail t-test P(T < or = t) = 0.008). This unexpected result may reflect different selection factors at work in wild and hatchery populations tempered by recent drought conditions in California or stock mixing in hatcheries.
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