Morphometric analysis of salamanders of the plethodon vandykei species group.
The Plethodon vandykei species group (Highton and Larson 1979) includes Van Dyke's salamander (P. vandykei), a Washington State endemic occurring in the coast ranges and Cascade Mountains and the Coeur d'Alene salamander (P. idahoensis), the sole lungless salamander (Plethodontidae) of the northern Rocky Mountains. These terrestrial amphibians inhabit temperate mesophytic forests broadly separated by the Columbia Basin, an arid shrub-steppe province in the rainshadow of the Cascades (Franklin and Dyrness, 1988; Wilson et al., 1995; Wilson et al., 1997).
Morphological variation within the Plethodon vandykei group has not been thoroughly examined. In describing the holotype for P idahoensis, Slater and Slipp (1940) suggested that this species has a more elongate body, more angular snout and a wider head than P. vandykei. A subsequent comparison of western Plethodon (Brodie, 1970) revealed that P. vandykei and P. idahoensis differ in pigmentation and tail length, but have similar costal groove and tooth numbers.
Plethodon vandykei and P idahoensis were originally described as separate species (Slater and Slipp, 1940) and substantial biochemical distance has since been demonstrated between them (Highton and Larson, 1979; Howard et al., 1993). However, the validity of these species has been debated because of incongruence between previous molecular and morphological studies (Highton and Larson, 1979; Nussbaum et al., 1983; Highton, 1990). Howard et al. (1993) proposed that a detailed morphological analysis might reveal inter-specific and intraspecific differentiation within the species group and help resolve the taxonomic status of P.. vandykei and P. idahoensis.
We conducted a multivariate analysis to investigate these possibilities. Multivariate techniques are commonly used in studies of amphibian morphological variation (Lynch and Wake, 1975; Lynch et al., 1977; Lynch, 1981; Lynch et al., 1983; Chippindale et al., 1993; Good and Wake, 1992; Green et al., 1997; Irschick and Shaffer, 1997). An underlying assumption in such analyses is that absence of gene flow between populations results in a general increase in phenotypic differences between them (Good and Wake, 1993).
Because terrestrial plethodontids are closely associated with mesophytic forests, the presence of forest corridors is often cited as requisite for dispersal and gene flow in these salamanders (Lowe, 1950; Wake, 1966; Larson, 1984; Welsh, 1990; Howard et al., 1993; Highton, 1997). In the present study we compare our results with previous systematic analyses, and relate patterns of phenotypic divergence within the P. vandykei group to forest discontinuities in the Pacific Northwest.
MATERIALS AND METHODS
We collected 270 salamanders between 1990 and 1992 for our analysis. Nine samples delimited by major river drainages were treated as operational taxonomic units (Sneath and Sokal, 1973). Males and females were included in all samples; sample sizes ranged from 23 to 49 (Fig. 1, Table 1). To eliminate distortion due to preservation (Lee, 1982) salamanders were measured alive after being anesthetized in a bath of 0.1% Tricaine Methanesulfonate (MS222).
Dial calipers and an ocular micrometer were used to estimate the following 16 body dimensions to 0.1 mm [ILLUSTRATION FOR FIGURE 2 OMITTED]: snout to posterior angle of vent (SVL); head width at widest span of jaw (HEAW); head depth, posterior side of orbit to ventral margin of mandible (HEAD); snout to posterior margin of parotoid gland (SPL), snout to posterior margin of orbit (SOL); eye length, posterior to anterior margins of orbit (EYEL), pectoral depth (PECD); pectoral width (PECW); forelimb length, carpal fold to body wall (FORL); manus width, tip of first foredigit to tip of fourth foredigit (MANW); metacarpal length, carpal fold to the margin of webbing midway between second and third foredigits (MECL); length of third foredigit from tip to a point in line with the margin of webbing midway between second and third foredigits (FDIL); hindlimb length, tarsal fold to body wall (HINL); pes width, tip of first hinddigit to tip of fifth hinddigit (PESW); metatarsal length, tarsal fold to the margin of webbing midway between third and fourth hinddigits (METL); length of fourth hinddigit from tip to a point in line with the margin of webbing midway between third and fourth hinddigits (HDIL). Manus and pes measurements were taken with digits spread on a flat surface so that no folds were present in the interdigital webbing.
TABLE 1. - Samples used in the morphometric analysis of the Plethodon vandykei species group Sample Number Location Township/Range size 1 Willapa Hills, T12N/R8W 49 Pacific Co., WA 2 Olympic Mountains, T23N/R8W 17 Grays Harbor Co., WA T23N/R7W 6 3 Mount St. Helens, T10N/R6W 27 Skamania Co., WA 4 Tilton River, T13N/R3W 27 Lewis Co., WA 5 St. Joe River, T45N/R4E 25 Shoshone Co., ID T46N/R5E 8 6 Kootenai River, T33N/R29W 12 Lincoln Co., MT T34N/R29W 16 7 Lochsa River, T34N/R8E 27 Idaho Co., ID 8 Clark Fork River, T18N/R25W 19 Sanders Co., MT T19N/R25W 10 9 Elk Creek, T39N/R2E 27 Clearwater Co., ID
Using data from all 270 salamanders, a linear regression was calculated for each dimension against SVL. The residuals of each of the nine samples were used as variables in statistical procedures to eliminate the effect of size differences between individual salamanders (Lamb, 1983; Grudzien et al., 1992). To avoid nonlinearity associated with allometric growth, adult salamanders in a 20mm size range (45-65 mm SVL) were used exclusively.
We applied discriminant analysis as outlined by Pimentel (1979). MANOVA, using Wilk's Criterion, was employed to test for significant phenetic differences among samples. Discriminant function analysis was used to calculate the probability of correctly classifying each salamander relative to its sample of origin. Canonical discriminant analysis enabled assessment of each variables' contribution to phenetic differentiation. Such differentiation was graphically summarized by plotting canonical discriminant scores and by cluster analysis, using unweighted pair-group average linkage (UPGMA), conducted on a matrix of Mahalanobis' distances. The Washington State University IBM mainframe computer version of SAS (SAS Institute, 1985) was used for most analyses: CANDISC provided descriptive statistics and performed MANOVA, canonical discriminant analysis and calculation of Mahalanobis' distances; DISCRIM was used to produce the classification matrix. All other operations were performed with SYSTAT (Wilkinson, 1990) on an IBM personal computer.
MANOVA revealed significant phenetic differentiation among samples (F = 8.68, P [less than] 0.01) and all Mahalanobis' distances among samples were significant (greatest P = 0.02). Discriminant function analysis (Table 2) correctly assigned 94.4% of the individual salamanders to their home populations, and 97.8% to either Plethodon vandykei or P. idahoensis.
Canonical discriminant score clustering is apparent in a plot of the first and second canonical axes [ILLUSTRATION FOR FIGURE 3 OMITTED]. Segregation of Plethodon vandykei and P. idahoensis occurs along the first axis, which accounts for 56.0% of the morphological variation among samples. Standardized canonical coefficients for this axis (Table 3) show that P. idahoensis has a more flattened form, wider head and larger eyes than P vandykei.
UPGMA analysis of Mahalanobis' distances among samples (Fig. 4, Table 4) shows the deepest morphological division to be between Plethodon vandykei and P. idahoensis populations. The largest intraspecific distances occur in P. vandykei between Cascade and coastal samples. Cascade samples are more differentiated than either coastal or P. idahoensis samples.
TABLE 2. - Classification matrix for nine samples of salamanders in the Plethodon vandykei species group From Number of salamanders classified into sample: sample: 1 2 3 4 5 6 7 8 9 Total 1 46 2 0 0 0 0 0 0 1 49 2 0 22 0 0 0 1 0 0 0 23 3 0 0 27 0 0 0 0 0 0 27 4 0 0 0 26 1 0 0 0 0 27 5 2 0 0 0 28 0 1 1 1 33 6 0 0 0 0 0 27 0 0 1 28 7 0 0 0 0 0 1 26 0 0 27 8 1 0 0 0 0 0 0 28 0 29 9 0 0 0 0 0 0 1 1 25 27
MANOVA and discriminant function analysis indicate considerable morphological differentiation among samples from the Plethodon vandykei species group. Similar results have been found in multivariate morphometric studies of other terrestrial plethodontids (Lynch, 1981; Lynch et al., 1983; Good and Wake, 1993) and are predictable in these salamanders, given their slow dispersal and low gene flow between populations (Larson, 1984). The morphometric variation revealed by canonical discriminant analysis and UPGMA cluster analysis in this study is concordant with molecular and pigmentary variation within the species group (Brodie, 1970; Highton and Larson, 1979; Howard et al., 1995). Overall differentiation between P. vandykei and P. idahoensis indicates an old separation of the two forms.
TABLE 3. - Standardized canonical coefficients for the first two canonical variables used in the morphometric analysis of the Plethodon vandykei species group. Percent of among-sample variation contributed by each canonical variable is indicated. See Materials and Methods for abbreviations Body Canonical Canonical dimension variable 1 variable 2 HEAW -0.464 0.065 HEAD 0.143 0.013 SPL -0.013 -0.143 SOL -0.000 0.409 EYEL -0.336 -0.124 PECD 0.738 -0.027 PECW 0.004 -0.153 FORL 0.230 0.336 MANW 0.010 0.053 MECL -0.016 0.406 FDIL 0.199 0.039 HINL -0.034 -0.095 PESW -0.190 0.388 METL 0.036 0.247 HDIL 0.247 0.045 % Variation explained: 56.0 20.7 TABLE 4. - Mahalanobis' distances among nine samples of salamanders in the Plethodon vandykei species group Sample Sample 1 2 3 4 5 6 7 8 9 1 0.0 2 5.0 0.0 3 14.8 9.8 0.0 4 9.9 10.1 10.6 0.0 5 13.0 10.1 15.6 15.3 0.0 6 17.7 9.8 11.2 18.6 5.6 0.0 7 15.1 9.2 17.1 18.0 2.0 5.1 0.0 8 22.6 13.2 18.4 24.9 5.0 3.1 3.1 0.0 9 18.7 12.6 21.4 22.9 4.5 5.6 2.8 2.8 0.0
We suggest this separation must be at least as old as the Cascade Mountains rainshadow on the Columbia Basin. The rainshadow resulted from Cascade orogeny during the Pliocene and eliminated formerly widespread mesophytic communities from the Basin (Smiley, 1963; Daubenmire, 1975). These communities, which included Plethodon and other amphibians such as the Pacific Northwest giant salamanders (Dicamptodon) (Welsh, 1990), were restricted to the west slope of the Cascades and northern Rocky Mountains by the beginning of the Pleistocene (Detling, 1968; Daubenmire, 1975; Mack et al., 1976; Barnosky et al., 1987). A pre-Pleistocene vicariance for the Plethodon vandykei species group is consistent with molecular estimates of the timing of speciation in Plethodon and Dicamptodon (Highton and Larson, 1979; Daugherty et al., 1983).
Congruent morphological and molecular differentiation, coupled with paleoecological evidence of an ancient vicariance, support the view (Howard et al., 1993) that Plethodon vandykei and P. idahoensis are separate evolutionary species (Frost and Hillis, 1990). Comparing morphological and molecular data is commonly used to reinforce systematic interpretations (Kluge, 1989; Larson and Dimmick, 1993; Good and Wake, 1992, 1993; Green et al., 1997; Jackman et al., 1997). This approach, when augmented with historical and ecological information, results in robust taxonomic decisions regarding allopatric populations such as those in the P. vandykei group (Good and Wake, 1993).
The phenotypic divergence between coastal and Cascade Mountains Plethodon vandykei supports the argument of Howard et al. (1993) that these population centers have long been separated. This is in agreement with the fossil pollen record of the Pacific slope, which indicates xerophytic habitats in lowlands separating the coast ranges and Cascades through the late Pleistocene and much of the Holocene (Baker, 1983; Barnosky et al., 1987). Additionally, these lowlands are underlain by alluvial and glacial deposits that limit dispersal by Plethodon (Wilson et al., 1995).
A comparable geology and holocene fossil record (Barnosky, 1981; Wilson et al., 1995) exists for regions separating Cascade populations of Plethodon vandykei. The effect of these dispersal barriers is apparent in the molecular (Howard et al., 1993) and morphological differentiation of Cascade populations. Conversely, the relative homogeneity between coastal samples of P vandykei and among P. idahoensis samples is consistent with recent Holocene presence of mesophytic dispersal corridors in coastal Washington and the northern Rocky Mountains (Heusser, 1974; Baker, 1983; Mack et al., 1983; Wilson et al., 1995; Wilson and Larsen, 1998).
The variation we encountered in the Plethodon vandykei species group is interesting, given that Plethodon is widely regarded as an extreme example of morphological conservatism in which such variation is only weakly expressed (Wake et al., 1983; Larson, 1984). This view of the genus may stem, in part, from the results of studies in which only a few "standard" characters (Wake, 1981) were used in comparisons. Detailed multivariate morphometric analyses of Plethodon have not been widely performed (Hanlin, 1979; Wake, 1981; Highton, 1989). In the present study, characters and methods were chosen to identify differences in shape (Pimentel, 1979), a feature that may be more variable in the genus than is widely appreciated. Our results suggest that this approach will be useful in future evolutionary studies of other Plethodon species.
Acknowledgments. - This study received financial support from the Washington State Department of Wildlife and the Northwest Scientific Association. We are grateful for advice from Amy Jean Gilmartin and Ralph Granner, and for technical assistance from Daniel Bivens, Marc Evans and Joan Follwell. Thanks to Evelyn Wilson for help in collecting specimens. The manuscript was improved by reviews from Richard Johnson, Don Miller and Richard Wallace.
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|Author:||Wilson, Albert G., Jr.; Larsen, John H., Jr.|
|Publication:||The American Midland Naturalist|
|Date:||Apr 1, 1999|
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