Genetic Diversity of Long-toed Salamanders
(Ambystoma macrodactylum) in High-Elevation Lakes
Seattle City Light
Barbara A. Shields, Co- Principal Investigator
William J. Liss, Principal Investigator
Department of Fisheries and Wildlife
Oregon State University
Corvallis, OR 97331
May 6, 2003
The genetic population structure of long-toed salamanders (Ambystoma macrodactylum)
from North Cascade Park was examined to investigate patterns of post-glacial colonization, population structure, gene flow, and genetic impacts from predation by introduced trout. Eight polymorphic microsatellite loci revealed surprising genetic diversity both within and among local populations. Genetic and geographic distances between populations were positively correlated, and estimated migration levels between populations were very low. These indicate genetic isolation of populations increases with geographic distance, and colonization may be a gradual process. Genetic diversity of salamander populations sympatric with non-reproducing trout was not significantly lower than genetic diversity of populations living without trout. Patterns of genetic diversity within the studied populations revealed two significantly distinct genetic groups representing higher elevation, east slope (Stehekin drainage) populations and lower elevation, west slope (Skagit drainage) populations. This divergence may represent colonization of NOCA‟s high-elevation lakes from separate glacial refugia.
Concern about the decline of amphibian species is particularly acute in areas such as high elevation locations in the western US that have been relatively undisturbed by human activities (Blaustein and Wake 1990). Fish are not indigenous to most high-elevation lakes in the west (Bahls 1992), and introduction of fishes including trout has been implicated as a possible cause of amphibian declines (e.g., Taylor 1983; Bradford 1989; Bradford et al. 1993; Fellers and Drost 1993; Blaustein et al. 1994; Tyler et al. 1998a).
To persist in high-elevation environments, species must maintain genotypes that are adapted to harsh and variable environmental conditions. The genetic diversity of a population provides its capacity to adapt to changes in the environment and it is a critical parameter in assessing the risks of human activities to indigenous populations (Reh and Seitz 1990; Olsen et al. 1996). In general, genetic diversity increases with increased population size (Hartl and Clark 1997a) so that the largest populations will have the greatest level of diversity.
Trout predation could reduce genetic diversity of salamanders both by reducing salamander population density and by selectively removing the phenotypes that behaviorally are the most vulnerable to predation (Storfer and Sih 1998). In addition, extirpation of populations could increase isolation among populations and reduce dispersal and gene flow. Reduced dispersal has been associated with increased probability of metapopulation extinction (Hanski 1991; Sjogren 1991; Sjogren-Gulve and Ray 1996). Of particular concern are the impacts of fish on large salamander populations, which may be analogous to core populations (sensu Harrison
1991, 1994) in a metapopulation. Core populations serve as repositories for genetic diversity and relatively stable sources of dispersers that can recolonize adjacent habitats where local population extinctions have occurred.
Since 1989, we have been conducting research on the impacts of introduced trout on native biota, including long-toed salamanders (Ambystoma macrodactylum), in North Cascades
National Park Service Complex (NOCA). Long-toed salamanders are the top carnivores in high-
elevation fishless NOCA lakes (Tyler et al. 1998a). Though naturally fishless, many lakes have been stocked with cutthroat (Oncorhynchus clarkii) and rainbow trout (Oncorhynchus mykiss
mykiss) to provide recreational opportunities. In some NOCA lakes, trout have established reproducing populations that often reach high densities (405-650 fish/ha; Gresswell et al. 1997), but in lakes where trout cannot reproduce, juvenile fish (fry) are periodically stocked (average interval between stocking >5 years) at low densities (average 179 fish/ha; Liss et al. 2002). Many anglers prefer to fish in lakes with non-reproducing trout because trout densities are low and individual fish reach a large size. Because trout can be eliminated in a few years simply through cessation of stocking, lakes with non-reproducing trout may offer the most options for future management for lake restoration if deleterious effects on native biota are demonstrated. In contrast, if fish impacts on native biota are not evident in non-reproducing fish lakes, NOCA could consider allowing stocking of these lakes to continue.
Our research has shown that, in fishless lakes, there was a significant positive relationship between total Kjeldahl nitrogen (TKN), an indicator of lake productivity (Lambou et al. 1983; Paleheimo and Fulthorpe 1987), and larval salamander density (Tyler et al. 1998a). Increased larval density was related to increased availability of cladocerans, an important food resource for larval salamanders. The effect of trout on larval salamander density appears to
0.05 depend primarily on the concentration of TKN in a given lake. In lakes with low TKN (< ~
mg/L), there was no significant difference in larval densities between fishless lakes and lakes with trout (Tyler et al. 1998a, Liss et al. 2002), as salamander densities were very low regardless
of whether trout where present or absent. In contrast, at high TKN (> ~ 0.05 mg/L), conditions
where salamander larvae likely would have been abundant, larval abundance was significantly higher in fishless lakes than in lakes with fish (Tyler et al. 1998a), including lakes with non-reproducing trout (Liss et al. 2002). Larval salamanders were absent in nearly all lakes with reproducing trout within this TKN range.
The principle goal of this project was to test the hypothesis that introduced trout have had no significant effect on within-and among-population genetic diversity of high-elevation (>800 meters) A. macrodactylum populations in NOCA. Four primary objectives were involved:
1. Determine the relationship between within-population genetic diversity of salamanders and larval salamander population size.
2. Determine within-population genetic diversity of larval salamanders in lakes with non-reproducing trout and compare it to genetic diversity in fishless lakes.
3. Determine genetic relationships among salamander populations in fishless lakes and lakes with non-reproducing fish.
4. Determine genetic relationships among populations within a watershed (the Skagit and Stehekin drainages) and between watersheds.
Accurate assessment of these genetic parameters within and among populations on the fine scale of local populations in NOCA requires the study of genetic markers that have a bi-parental mode of inheritance. Previous studies have examined allozymes, which require lethal sampling, to document genetic variation and gene flow in Ambystomatid salamanders (Howard and Wallace 1981; Jones 1989; Titus 1990; Shaffer et al. 1991; Larson and Dimmick 1993; Storfer and Sih 1998, Tallmon et al. 2000). This would be unacceptable for studies of A. macrodactylum
in NOCA, since the removal of ten to twenty individuals for genetic studies may translate into
the loss of a significant proportion of the animals in many of the small, local populations. Genetic investigations for this study utilized the polymerase chain reaction (PCR) (Avise 1994; Bruford and Wayne 1993; Burke et. al. 1991) to amplify large quantities of specific targeted DNA sequences from minute amounts of tissue, eliminating the need for lethal sampling.
Tissue Collection and Estimation of Population Abundance
Two species of Ambystomatid salamanders (A. macrodactylum and A. gracile) occur in
NOCA, but they were not observed together in any of the study lakes. In NOCA, A. gracile is
generally restricted to lower-elevation lakes (Liss et al 1995b), and most collections of larval tissue for this study were made in higher-elevation lakes where A. gracile rarely occurs
Roughskinned newts (Taricha granulosa) were observed in one of the study lakes (RD3), so
care was taken in field identification at the time of tissue excision. Larvae in sampled lakes were determined to be A. macrodactylum based on larval characteristics (Leonard et al. 1993; Corkran and Thoms 1996), absence of large larvae (>60mm total length) or neotenes and egg masses
, and the presence of premetamorphic individuals with adult coloration. characteristic of A. gracile
A hand-held sonar gun was used to determine maximum depth of each lake. Lake elevations were derived from 7.5 min USGS topographical maps, and lake surface areas were determined by digitization of lake shorelines outlined on these maps (Table 1). During each sampling visit, water samples were collected from one meter below the lake surface with a 1.5 L van Dorn-style sampling bottle. Water samples were gathered over each lake‟s deepest point to
standardize sampling between lakes. One liter of collected water was filtered in the field using a Whatman GF/C mesh glass fiber filter, and water samples were frozen immediately upon return
from the field prior to analysis. Frozen filtered and unfiltered water samples were analyzed for total Kjeldahl-N (Table 1) and other chemical parameters at the Cooperative Chemical Analytical Laboratory at Oregon State University, Corvallis, Oregon.
Table 1. Physical variables of eight NOCA lakes from which salamander
larvae were sampled
Elevation, Maximum Depth, Surface Area, TKN,
Lake (m) (m) (ha) (mg/L)
DD1 1496 2.4 0.5 0.09
LS3FS 1375 6.7 1.2 0.02
MC7 1556 27.2 6.7 0.02
MR12-93,98 1981 4.0 0.6 0.125, 0.06
MR13-1 1800 2.0 0.3 0.06
MR2 1873 1.5 0.3 0.09
MR3-93,99 1873 2.5 0.2 0.14, 0.06
RD3-94,99 802 8.8 0.3 0.08, 0.07
We determined the density of larval salamanders and processed tissue samples for genetic analysis from eight lakes in NOCA sampled between 1993 and 1999 (Table 2). To assess temporal changes in genetic diversity within populations, tissue samples were processed from archived and recent collections from lakes RD3 (1994, 99), MR3 (1993, 99) and MR12 (1993, 98). Although MR 12 was fishless in 1993, fish were present in the lake in 1998.
It is very difficult to assess accurately the abundance of adult A. macrodactylum in high-
elevation areas in NOCA, so it was assumed that larval abundance was related to adult population sizes. Larval salamander density was estimated by snorkel surveys (Tyler et al. 1998a). Because of the remoteness of lakes (they could be reached only by hiking or helicopter), snorkeling provided the best estimates of larval densities given constraints of time and equipment. Four, 25-meter segments of shoreline were chosen randomly along the perimeter of
Table 2. Summary of data on NOCA long-toed salamander populations.
Estimated Individuals Population
Population Density Lake Total Used for Used For
East/ Fish Sample Estimate PerimeterLarval Genetic Genetic Lake West +/- Date (Number/100m) (m) Population Analysis Analysis DD1 W + Jul-98 2 280 5.6 5 89% LS3FS W + Sep-99 4 479 19.16 4 21% MC7 W + Sep-99 11 992 109.12 6 5% MR 12-93 E - Jul-93 110 284 312.4 5 2% MR12-98 E + Aug-98 6 284 17.04 4 23% MR13-1 E - Aug-93 20.3 194 39.38 4 10% MR2 E - Aug-99 65 214 139.1 20 14% MR3-93 E - Aug-93 38.7 144 55.73 3 5% MR3-99 E - Aug-99 14 144 20.16 20 99% RD3-94 W - Jun-94 81 146 118.26 7 6% RD3-99 W - Aug-99 41 146 59.86 18 30% Average 35.7 300.6 81.4 8.7 28%
each lake (all study lakes were < 7 ha in area, and all but two were less than one ha). During mid-afternoon, a snorkeler carefully searched through substrate material (talus, woody debris, fine organic material, and aquatic vegetation) within two meters of the shoreline and recorded the number of larvae observed. We found no statistically significant difference in larval A.
macrodactylum density between surveys conducted during the day and at night (Tyler et al. 1998a).
We extrapolated total larval population sizes (n) from estimates of salamander densities using the following equation:
n = d x p, where
d = density of salamanders (number of larvae per 100 linear meters of sampled shoreline)
p = perimeter of lake (meters).
The presence of trout in lakes was determined from stocking records and confirmed by gill netting, angling, and snorkeling.
Small quantities of tail tissue from larval salamanders were collected concurrently with estimation of population density. Tissue was excised from non-vascularized caudal finfold areas, without incursion into muscular tissue. Minimal or no bleeding was observed in all specimens, and no mortalities were documented. All tissue samples were preserved in either 70% ethanol or Modified Queen‟s Buffer (10mM Tris pH 7.5, 100mM EDTA pH 8, 5% v/v DMSO, saturated with NaCl) and stored at ambient temperature in screw-top 1.5 ml polypropylene tubes with silicone gaskets. Though sample sizes were small, they often represented a significant proportion of the estimated total number of larvae present in the population in each lake at the time of sampling (range 2-99% of the estimated larval population; Table 2).