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The
origin of introduced rainbow trout (Oncorhynchus
mykiss)
in the Santa Cruz River, Patagonia, Argentina, as inferred from
mitochondrial DNA
by Carla M. Riva Rossi, Enrique P. Lessa, and Miguel A.
Pascual. Centro Nacional Patagónico (CONICET), Uruguay
Salmon
and trout have been transplanted to habitats throughout the world and
self-sustaining populations have been successfully established
globally, with the exception of Antarctica (MacCrimmon 1971; Quinn et
al. 1996; Nielsen 1996). Rainbow trout (Oncorhynchus mykiss)
was first introduced into Argentinean Patagonia, the southernmost
region of South America, at the turn of the twentieth century and
eventually became the most conspicuous freshwater species in major
river basins of the region (Pascual et al. 2002b).
 |
| English: Oncorhynchus mykiss (rainbow trout) swimming in a display tank at the Olmsted County Fair in Rochester, Minnesota (Photo credit: Wikipedia) |
Like
all other known introduced rainbow trout around the world, typical
Patagonian fish remain in fresh water throughout their entire life
cycle, with a life history similar to that of resident populations in
rivers and head lakes in western North America (Wydosky and Whitney
1979). The Santa Cruz River in Patagonia (50°S) is the only drainage
in the world where introduced rainbow trout are known to have
developed partially migratory populations composed of individuals
exhibiting a marine migratory phase, so-called steelhead, and
strictly freshwater fish that remain resident in their native stream
(Pascual et al. 2001).
As
in many other salmonid populations with this dual
anadromous–nonanadromous life history, the way and extent to which
the two ecotypes intermingle in the Santa Cruz is uncertain. Genetic
analyses based on microsatellite loci revealed that the anadromous
form is genetically indistinguishable from main-stem resident trout
(Pascual et al. 2001), suggesting that significant gene flow occurs
between the two forms.
Whether
the introduced fish were in effect anadromous or anadromy arose in
situ remains unknown (Behnke 2002; Pascual et al. 2002a). We also
ignore the specific mechanisms underlying the expression of
alternative life histories in the Santa Cruz, i.e. a genetic
polymorphism, a genetically determined developmental threshold (i.e.
the link between individual growth performance and anadromy or
nonanadromy; Thorpe et al. 1998), or an entirely environmental
effect. At this point, there are critical aspects regarding the
environmental versus genetics bases of life history variation in
Santa Cruz River rainbow trout that we do not know.
A
logical first step to start elucidating the bases of life history
variation in Patagonian rainbow trout, in particular, the development
of anadromy, is to assess their genetic legacy through the
identification of the parental sources. Poor historical bookkeeping
and complex ancestry have made it difficult to address this issue
from transplant records alone.
The
Santa Cruz River, as well as all other rivers throughout Patagonia,
received rainbow trout from two main sources at different times.
Between 1904 and 1910, rainbow trout ova were imported from the
United States (US), most likely derived from rainbow trout and
steelhead from locations in northern California or southern Oregon
(Pascual et al. 2001,2002a; Behnke 2002). After the 1930s, and
particularly after the 1950s when fish transplants within the region
became more common, all rainbow trout plantings were based on new
stocks imported from Germany and Denmark (Baigún and Quirós 1985).
However, the Santa Cruz River has had a history largely independent
from that of more northerly Patagonia locations, with only occasional
introductions after 1920 (Pascual et al. 2001, 2002a). Thus,
presumably, wild populations in this river were mostly derived from
the early shipments from the United States.
Mitochondrial
DNA (mtDNA) has proven very successful for identifying the origins of
several introduced salmonid populations and for assessing genetic
differences between contemporary wild and introduced populations
(Quinn et al.1996; Burger et al. 2000). In this paper, we use mtDNA
sequence variation to identify the founding populations of Santa Cruz
River rainbow trout. We start by analysing mtDNA sequences of both
resident and migratory fish. We include in the analysis fish from a
local hatchery, which was founded with European trouts widely stocked
around the region after 1950.
We
then build and apply a probabilistic model of random survival and
reproduction of individual fish to calculate the likelihood that wild
Santa Cruz fish had originated from a collection of candidate North
American stocks. Finally, we discuss the merits of the techniques
applied to evaluate the relative contribution of pre-1950 transplants
from US stocks and post-1950 transplants from Danish stocks to wild
populations of rainbow trout throughout Patagonia.
Transplant
history
From
1904 to 1910, several consignments of rainbow trout embryos arrived
in Argentina, mainly from the United States, with only occasional
imports from European countries, such as France and Germany (Tulian
1908; Marini and Mastrarrigo 1963; Behnke 2002). Between 1906 and
1910, a total of 105,000 rainbow trout ova collected in the United
States were shipped to the Santa Cruz River. 25,000 in 1906, 30,000
in 1908, and 50,000 in 1909. The 1908 shipment was completely lost,
but the other two consignments were successfully hatched and planted
in the river, with comparable losses throughout (about 65 percent;
Tulian 1908; Marini and Mastrarrigo 1963). For practical purposes,
the number of eggs from the parental populations giving rise to the
Santa Cruz stock was 75,000.
The
most likely origin of these eggs was the Baird Station on the McCloud
River, California (Pascual et al. 2001). However, they may as well
have come from steelhead and rainbow trout in alternative northern
California and southern Oregon locations (Behnke 2002; Pascual et al.
2002a). Rainbow trout introductions into Argentina intensified after
1950, this time based on stocks from Denmark (Pillay 1969; MacCrimmon
1971) and maintained by Bariloche.
Northern
Patagonia hatchery
By
that time, Bariloche became the main center of salmonid propagation
in Argentinean waters, contributing to the distribution of these new
stocks throughout the 1950s, 1960s, and 1970s. Danish stocks of
rainbow trout have a complex ancestry; multiple lineages from
California, Michigan, Canada, New Zealand, and France appear to have
contributed to their foundation (MacCrimmon 1971). Small consignments
of these fish(<2000 embryos) arrived at the Santa Cruz River from
Bariloche in the 1970s and were planted in second- to-third order
tributaries flowing into the upper basin (Fig. 1).
Finally,
in 1991, the Piedra Buena Hatchery was built on the lower Santa Cruz
River (Fig. 1). The fish used to found this hatchery’s broodstock
also came from Danish fish, as those kept by the Bariloche Hatchery.
Although
fish of this hatchery are not used for stocking the river, escapes
are likely, so that some introgression with wild fish might occur
(Pascual et al. 2001). In any case, these fish provide a
representative group of known Danish origin with which to contrast
the genetic structure of Santa Cruz
River
wild fish.
Study
localities
The
upper Santa Cruz basin is dominated by two large glacial-fed lakes,
Viedma and Argentino, that form the Santa Cruz River. The main stem
river has an average flow of 690 m3·s–1 and extends for 382 km
across the Patagonian plateau draining into the Atlantic Ocean (Fig.
1). Landlocked populations of rainbow trout inhabit most of the
second- to third-order tributaries that feed the head lakes; few
springs and small tributaries enter the main-stem river, none of them
significant from the point of view of their trout populations.
We
restricted our analysis to the main-stem river populations, which, as
revealed by a telemetry study, is the domain of the anadromous
rainbow trout and of the resident fish to whom they are most likely
related (Riva Rossi et al. 2003). Adult anadromous and resident
rainbow trout were caught by hook and line and by gill nets between
2000 and 2002 in April and September along the main-stem Santa Cruz
River (Fig. 1).
Sampling
locations were based on spawning and fishing abundances documented in
previous surveys and consisted of two river reaches located in the
upper course ('Primer Laberinto' and 'Segundo Laberinto'), one in the
middle course ('Ea. Rincón Grande'), and one in the lower course
('Cte. L. Piedra Buena' City).
At
each locality, tissue samples were obtained from fish of each
ecotype. From a total of 182 wild fish captured, 20 were successfully
sequenced: five individuals of each ecotype from the upper course,
three resident fish from the middle course, and three anadromous and
five resident fish from the lower course. Direct inspection of
external characteristics and scale pattern analysis were used to
distinguish anadromous from nonanadromous fish (Pascual et al. 2001).
Also, fin clips were obtained from five spawners from the Piedra
Buena Hatchery broodstock.
DNA
techniques
Whole
genomic DNA was extracted from alcohol-preserved fin tissue by means
of a sodium chloride extraction of proteins followed by ispropylic
alcohol precipitation of DNA (Miller et al. 1988). The polymerase
chain reaction (PCR) was used to amplify a segment of the
mitochondrial genome containing 188 base pairs (bp) of the O. mykiss
control region and 5 bp of the adjacent phenylalanine tRNA gene using
primers S-phe (5′-TAGTTAAGCTACG-3′) and P2
(5′-TGTTAAACCCCTAAACCAG-3′) (Nielsen et al. 1994).
Nomenclature
for mtDNA control region haplotypes follow those given in Nielsen et
al. (1997a, 1998). Amplifications were conducted in a total volume of
50 µL containing 1× retype (ST1, Nielsen et al. 1994; details in
Results), suggesting either that they descended from a monomorphic
population, that the population became fixed for haplotype ST1 during
establishment and colonization, or that not all population haplotypes
were represented in our sample. We thus developed an ad hoc model to
evaluate the likelihood of ending with an all-ST1 sample given that
the stock of origin was nonmonomorphic.
We
consider three processes that, starting with a nonmonomorphic
maternal stock, could lead to an all-ST1 sample: the sampling of
females from the donor population that produced the eggs imported
(founder effect), the mortality between eggs and reproductive fish
contributing to establish the new stock (postfounding drift), and the
chance of missing population haplotypes during our sampling process
(sampling effect).
Each
of these three processes can be viewed as sampling from a finite
population, which is most properly modeled by a hypergeometric
distribution. For the sample sizes and probabilities used in our
analysis, the binomial distribution approximates the hypergeometric
well. We therefore opted for computational simplicity and modeled the
foundation of Santa Cruz populations as a chain of three binomial
processes.
The
number of donor females, different females that could have
contributed to the Santa Cruz River stock, F, is calculated as
(1)
F=F/fec
Where
E is the number of eggs imported and “fec” are putative values
for average female fecundity. Assuming that the maternal females were
randomly drawn from a particular population, we modeled the number of
ST1 eggs effectively extracted from it and imported into Argentina, E
ST1, as a binomial process:
(2)
E ST1
≈ fec · Bin(F, φ)
Where
φ is the frequency of the ST1 haplotype in
the original population. The post-introduction mortality from eggs to
founding fish, W, i.e. fish that effectively contributed to the Santa
Cruz stock, is simply modeled as (3)
W = surv ·E
Where
“surv” are putative values of survival rate from eggs to founding
fish. The number of ST1 fish in this founding stock is (4)
Where
EST1/E is the proportion of ST1 eggs effectively imported as modeled
in eq. 2. The number of ST1 fish in the sample taken from the present
population (SST1) is (5)
Where
n is the sample size and WST1/W is the proportion of ST1 individuals
among the founding fish.
It
is assumed that the frequency of ST1 currently observed in the
population is well represented by that of the founding fish. In other
words, we assumed that there was a single, primeval bottleneck
associated with initial establishment, after which the population
expanded rapidly enough for the frequency of ST1 to remain reasonably
unchanged.
The
probability of obtaining and all-ST1 sample from the present
population is (6)
Finally,
for given founding stock (φ is the
frequency of ST1 in the maternal population), average fecundity
(i.e., or number of donor females (eq. 1)), egg to founding fish
survival (i.e., or number of founding wild fish (eq. 3), and sample
size (n), the probability of obtaining an all-ST1 sample is given by
integrating eq. 6 over all possible outcomes of eqs. 4 and 2: (7)
The
number of eggs imported, E, was set to 75 000. We used an array of
values for “fec” between 500 (low fecundity) and 4500 (high
fecundity), considering 2800 to be an average fecundity for typical
Sacramento River rainbow trout stocks (Carlander 1969). These values
correspond to a range of 17–150 donor females. We used values of φ
consistent with the frequency of haplotype ST1 in different candidate
donor populations of Santa Cruz River fish (Table 1).
We
used values of “surv” between 0.00006 and 0.0029, corresponding
to founding population sizes of 5 (very low survival) to 215 fish
(high survival). Finally, we used a sample size n of 20, the number
of wild fish sequenced in this study.
We
did not consider in our model the chance of missing low-frequency
population haplotypes during our sampling process. While this
probability may not be unimportant for sample sizes of less than 10
individuals and frequencies of 0.85, it becomes low for sample sizes
of 20 individuals. We therefore preferred to accept a small bias and
avoid the need for the much more intensive calculations demanded by
including three nested conditional probabilities in our model.
Results
Sequence
data revealed that all Santa Cruz River fish, both anadromous and
resident, had the ST1 haplotype described by Nielsen et al. (1994).
Hatchery fish, on the other hand, were genetically different from
wild fish. Only one of the five fish examined had haplotype ST1,
while the remaining four fish had haplotypes ST3 and ST9 in similar
proportions. Each of these haplotypes differed by only a single
transitional base change from haplotype ST1 (G → A) at positions
1109 (ST3) and 1147 (ST9). All of these mtDNA haplotypes were
previously reported by Nielsen et al. (1994, 1997b, 1998) and Bagley
and Gall (1998) in rainbow trout populations from California and by
McCusker et al. (2000) in populations from British Columbia.
Mitochondrial
DNA haplotype ST1 is dominant in steel-head populations from the
Sacramento and Eel rivers in northern California but among the
putative parental stocks was found to be monomorphic only in the
McCloud River rainbow trout (Table 1) and in the Río Santo Domingo
rainbow trout populations from Baja California (Nielsen et al.1997b,
1998, 1999). We discard this last stock as a candidate source of
Patagonian fish because Baja California trout did not contribute to
fish culture at the time of the introductions.
Haplotype
ST3 is rare in steelhead populations from northern California but is
common in coastal populations from the San Francisco Bay area and
dominant in resident populations from the upper Sacramento River and
the Kern and Little Kern rivers (Nielsen et al. 1997b, 1998; Bagley
and Gall 1998). Haplotypes ST1 and ST3 were found inequal frequencies
in steelhead populations from central California (Table 1) (Nielsen
1996). Haplotype ST9 is rare (<2 percent) in coastal populations
from California (J. Nielsen, US Geological Survey, Alaska Biological
Science Center, 1011 East Tudor Road, Anchorage, AK 99503, USA,
personal communication), but it is more common in inland steelhead
populations from the Snake River in Idaho (Kucera andArmstrong 2001)
and in inland populations from the upper Columbia River in Canada
(McCusker et al. 2000).
To
explore the hypothesis that a monomorphic sample of Santa Cruz wild
fish could have originated through haplotype loss and sampling bias,
as opposed to a truly monomorphic origin in the McCloud River, we
applied our probabilistic model to two extreme alternative scenarios:
a central California type parental stock, with a minimum 40 percent
frequency of the ST1 haplotype, and a northern California type stock,
with a maximum of 83 percent frequency of ST1 (Figs. 2a and 2b,
respectively).
As
expected from first principles of a binomial sampling process, the
probability of haplotype fixation increases as the number of donor
females and founding fish considered, indicating that it is highly
unlikely that Santa Cruz River fish originated from such a stock
(Fig. 2a).
When
a northern California type parental stock is considered, results are
less clearcut, with probabilities ranging between 3 percent and 45
percent depending on the values chosen for decreases (lower left in
Figs. 2a and 2b).
When
a central California type parental stock is considered, the
probability of an all-ST1 sample remains very low (<1 percent) for
practically all values of donor females and founding fish average
fecundity and initial survival (Fig. 2b). This led us to scrutinize
these parameters in more detail. Individual rainbow trout can have
fecundities as low as 500 and as high as 13,000 eggs (Carlander
1969).
We
used a range of 500–4500 to accommodate probable values for
individual mothers of Santa Cruz river fish, but average fecundities
reported for Californian wild populations are closer to the lower
half of this range. For example, Hallock et al. (1961) reported a
mean number of 2808 eggs for larger Sacramento River stripped
females. Perhaps more relevant to our case, Wales (1939) reported
that female rainbow trout trapped at Greens Creek, the trapping site
of the first egg-taking station of rainbow trout on the McCloud
River, weighed 2lb on average, with a mean fecundity between 1000 and
2000. Average fecundities lower than 2500 (at least 30 donor females)
result in a probability of sampling only ST1 fish of less than 10
percent (Fig. 2), unless the survival from eggs to founding fish was
very low (<0.00033 or <25 founding fish), in which case this
probability becomes greater.
In
summary, unless a small number of particularly large females (<17)
had been used to produce the eggs shipped to the Santa Cruz River and
(or) a very small proportion of the imported eggs survived to become
founding fish (<25 individuals), the probability of obtaining an
all-ST1 sample of 20 individuals from a northern California type
parental stock is less than 10 percent (Fig. 2).
Discussion
Our
analyses allowed us to establish the most likely origin of Santa Cruz
River main-stem fish as well as to advance our general knowledge on
the relative contribution of different parental stocks to rainbow
trout in Patagonia. As previously suggested by microsatellite
analyses (Pascual et al.2001), mtDNA data reinforce the idea that
anadromous and nonanadromous Santa Cruz fish do not constitute
independent lineages but have a common ancestry.
Wild
fish are clearly differentiated from hatchery Danish stocks widely
propagated in the region after 1950, providing strong evidence for an
origin of Santa Cruz populations in Californian rainbow trout
imported to Argentina during the first decade of the twentieth
century. Additionally, these results indicate that the introgression
from hatchery fish into the wild population has not been significant.
Although
it has been widely accepted that early transplants of rainbow trout
from the United States to locations around the world, including
Argentina, came from the McCloud River (Scott et al. 1978; Busack and
Gall 1980; Pascual et al. 2002a), historical records alone were
insufficient to verify this, conferring some credence to the idea
that other locations in northern California and southern Oregon could
have potentially contributed fish to these early transplants (Behnke
2002). The fact that Santa Cruz River wild fish analyzed were
monomorphic for haplotype ST1, together with the results from our
probabilistic model, provides additional support to the view that the
source of these populations was indeed the McCloud River.
It
must be noted, however, that our approach does not consider some
complex scenarios that could muddle the identification of parental
stocks. First, Santa Cruz fish could have been derived from a mixture
of fish from the McCloud and other northern California locations,
leading to a larger probability of haplotype fixation than purported
by our scenarios. Second, the founding population could have
experienced multiple bottleneck events instead of the single event at
the onset of the introduction that we modeled, increasing as well the
probability of haplotype fixation. Since there are no conceivable
bounds on the exercise of conjecturing combinations of parental
stocks or bottleneck sequences, we did not attempt additional
analyses, leaving it simply as a precautionary note.
The
clear differences found between wild and hatchery Santa Cruz fish
point at mtDNA analysis as a powerful tool to elucidate the ancestral
genetic makeup of rainbow trout in Patagonia at a regional scale and
to determine the relative contribution of stocks used before and
after 1950. The occurrence of ST3 in hatchery stocks may suggest a
Californian origin, most likely a genetic heritage derived from the
Sacramento River rainbow trout, while haplotype ST9,which is rare in
Californian wild stocks, hints at a complex ancestry of stocks
imported from Europe, with probable contributions of non-Californian
fish (e.g., British Columbia, where ST9 is more frequent). However,
we analyzed only a handful of hatchery fish and larger samples from
different hatchery stocks will be required to fully characterize the
genetic makeup of this stock.
At
present, these data make no suggestion as to what extent anadromy and
residency in the Santa Cruz River are merely recreating the
preexistent variation or have been modified in response to the
specific selective pressures of the novel environment. Nevertheless,
the identification of the genetic roots of Santa Cruz fish provides
relevant background information to guide future research about the
origin of life history variation in this river.
Regardless
of the specific processes underlying life history variation, our
results indicate that the Santa Cruz River may well constitute a
unique, secluded reservoir of those ancestral McCloud fish widely
distributed around the world during the late nineteenth and early
twentieth century, which in their native range have been
substantially affected by habitat modification and introgression from
hatchery stocks (Busby et al. 1996; Nielsen et al. 1999; McEwan
2001).
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