Early Discrimination of Atlantic Salmon Smolt Age - Wiley Online Library

Transactions of the American Fisheries Society 136:1622–1632, 2007 American Fisheries Society 2007 DOI: 10.1577/T07-010.1

[Article]

Early Discrimination of Atlantic Salmon Smolt Age: Time Course of the Relative Effectiveness of Body Size and Shape JAMIE H. PEARLSTEIN U.S. Geological Survey, Leetown Science Center, S. O. Conte Anadromous Fish Research Center, Post Office Box 796, One Migratory Way, Turners Falls, Massachusetts 01376, USA; and Department of Natural Resources Conservation, University of Massachusetts, Amherst, Massachusetts 01003, USA

BENJAMIN H. LETCHER* U.S. Geological Survey, Leetown Science Center, S. O. Conte Anadromous Fish Research Center, Post Office Box 796, One Migratory Way, Turners Falls, Massachusetts 01376, USA

MARISKA OBEDZINSKI U.S. Geological Survey, Leetown Science Center, S. O. Conte Anadromous Fish Research Center, Post Office Box 796, One Migratory Way, Turners Falls, Massachusetts 01376, USA; Department of Natural Resources Conservation, University of Massachusetts, Amherst, Massachusetts 01003, USA; and University of California Cooperative Extension, 133 Aviation Boulevard, Suite 109, Santa Rosa, California 95403, USA Abstract.—The goal of this study was to test the relative effectiveness of morphological measurements and body size in predicting the smolt age of Atlantic salmon Salmo salar and to determine the time course of body size and shape differences between smolt ages. Analyses were conducted on age-0 to age-2 fish that were stocked as fry in the West Brook, Massachusetts and on laboratory-raised age-0 to age-1 fish. Using both body size and shape, we could partition the age-0 fish collected during fall into future early or late smolts, although the predictive ability of body shape was somewhat weaker than that of body size, especially in the laboratory. Classification success averaged 81% (size) and 79% (shape) in the field and 85% (size) and 73% (shape) in the laboratory. Despite differences in smolt age between the field and the laboratory, the relative timing of growth rate differences between future early and late smolts was similar in the field and the laboratory and peaked at 50–60% of development from fry to smolt. While body shape differed between early and late smolts well before smoltification, it did not improve classification based on size alone.

Smoltification in salmon is a seasonal process marked by changes in physiology, biochemistry, morphology, and behavior that prepare anadromous fish for migration and saltwater residence (Hoar 1976; McCormick and Saunders 1987). These changes include spring peaks in salinity tolerance, silvering of the body, fin darkening, decreased condition factor (Vanstone and Markert 1968), increased sensitivity to external stimuli (Saunders and Henderson 1970), changes in swimming, agonistic behaviors, and social structure (Schreck 1981), and increased plasma growth hormone and gill Naþ,Kþ-ATPase (enzyme number 3.6.1.36; IUBMB 1992) activity (McCormick et al. 1995). Although the changes that occur during the parr– smolt transformation are well documented, the specific timing of the developmental decision to smolt remains unclear (Nicieza et al. 1991). This timing is difficult to determine because it is influenced both by endogenous * Corresponding author: [email protected] Received January 11, 2007; accepted June 6, 2007 Published online November 15, 2007

and environmental factors that direct future smolts to adopt the smolt developmental trajectory well in advance of the life history event (Thorpe et al. 1998). There is evidence that to migrate in the spring, juvenile salmon must meet population-specific thresholds (size or nutritional requirements), and a fish’s ability to meet such thresholds depends on the environmental (e.g., temperature and feeding conditions) and genetic (inherited standard metabolic rate) conditions that determine its opportunity for resource acquisition and growth (Thorpe et al. 1998). Thus, these factors and their interaction cause variation within and among populations in the timing of the developmental decision to smolt and, consequently, the age at smoltification (Metcalfe and Thorpe 1990; Metcalfe 1998). The number and size of fish smolting in a particular year can be related to the number of returning adults and therefore can be used in understanding the longterm survival of local populations (Lundqvist et al. 1994). Smolt size is also positively correlated with smolt age (Økland et al. 1993) and survival during migration (Marschall et al. 1998). In addition, the differential survival and reproduction of early smolts

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ATLANTIC SALMON SMOLT AGE PREDICTION

(in most streams, those that smolt in spring at age 2) and late smolts (those that smolt in spring at age 3) affect the ability to predict adult returns to natal streams and identify sources of freshwater and marine mortality. A technique that could be implemented earlier in the year (e.g., the fall) to indicate an individual’s future smolt status would refine smolt estimates and decrease dependence on spring sampling, when water levels are high and sampling methods are inefficient (Johnsson et al. 1996). In addition, the timing of smolting is a vital aspect of the study of the evolution of conditional strategies and the means of strategy choice in salmon (Bohlin et al. 1994). Numerous studies have focused on predicting smolt age, but predictive methods are inconsistent among systems, unsuitable for field studies, or unable to predict the timing of smolting and the number of smolts to a level of accuracy useful for management purposes. One common method for predicting smolting, for instance, is an individual’s position in the autumn bimodal size frequency distribution, which usually is initiated by late summer, about 9 months prior to smolting. Although bimodality has been shown to be distinct 6 months prior to smolting (Thorpe 1977), other data show that entrance into either mode can occur as late as winter or can be a continuous process (Kristinsson et al. 1985). Bimodality also may be unclear, especially in rivers where fish grow slower and smolt at an older age (Heggenes and Metcalfe 1991), or may be obscured by overlapping sizes of upper- and lower-mode fish (Saunders et al. 1994) or by other biotic and abiotic factors influencing growth (Heggenes and Metcalfe 1991). Physiological and endocrinological changes that occur during smolt transformation, such as increased gill Naþ,Kþ-ATPase activity (McCormick 1993), plasma thyroxine levels, salinity tolerance, and decreased lipid content (Fessler and Wagner 1969) also have been used as markers for smolting. Although not all of these methods require sacrificing the animal to obtain samples, they do involve complex laboratory assays or experiments and typically are not diagnostic until late March or early April, just prior to smolting. Some investigators have used behavioral means to predict future smolting (Metcalfe et al. 1989; Thorpe et al. 1992). Social status, along with length (Metcalfe et al. 1989) or growth rate (Thorpe et al. 1992), has been used to predict smolt age. It also has been suggested that decreased swimming proficiency (as measured by increased tail beat frequency) could be a predictor of smolt status (Flagg and Smith 1982). Behavioral measurements, however, are better suited for laboratory than field settings.

Morphological techniques, which are well suited for field research and studies with threatened or endangered species (because sacrificing specimens is not necessary) are a potential alternative. Numerous studies have measured the morphological changes correlated with smolting (Fessler and Wagner 1969; Riddell and Leggett 1981; Winans 1984; Beeman et al. 1995) and found that smolts develop longer caudal regions, shallower bodies, and larger heads than fish that do not emigrate (Beeman et al. 1995; Nicieza 1995). Morphological differences between salmon of hatchery and natural origin have also been observed (von Cramon-Taubadel et al. 2005). Other studies identified morphological changes in early development before the perceived onset of the parr–smolt transformation. A study of the early morphometric development in hatchery-reared Chinook salmon (Oncorhynchus tshawytscha) detected changes in body slenderness during smoltification and suggested the use of these changes for predicting the release time of hatchery-reared fish and maximizing survival and return (Winans 1984). Similarly, morphometric variables had an average success rate of 67% in classifying Atlantic salmon Salmo salar as smolts from their age-1 summer until smolting at age 2 (Letcher 2003), but the morphometric approach was not compared to predictions based on size alone. Although shape variation has the potential to help discriminate between early and late smolts, the relative effectiveness of shape and the traditionally used variable—body size—has not been evaluated. The present analysis examines the use of morphometric measurements created from a truss network to predict timing of smolting. Body measurements were collected from digital photographs taken seasonally of tagged Atlantic salmon reared in field and laboratory settings, and their ability to predict smolt age at various times throughout ontogeny was tested. The predictive ability of these morphometric variables was then compared with that of fork length. Methods Field and laboratory studies were used to compare the ability of shape and size to predict Atlantic salmon smolt age (age of smolting in the spring). Both groups were derived from adult sea-run Atlantic salmon in the Connecticut River. Owing to differing growth conditions in the field and laboratory, fish in the field usually first smolt at age 2, while those in the laboratory first develop smolt characteristics at age 1. Early smolts are defined here as age-2 smolts (field) or age-1 smolts (laboratory), and late smolts are defined as fish that did not smolt at age 2 (field) or age 1 (laboratory). Different criteria were used to assign smolt age in the

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TABLE 1.—Number of Atlantic salmon parr and smolts photographed digitally for morphometric analysis to predict early or late smoltification. Sampling environments were West Brook, Massachusetts (field) and the laboratory. Sample date

Age

Smolts

Parr

Total

Field 1999 Sep 2000 Mar May Sep Dec 2001 Mar May

0

13

58

71

1 1 1 1

20 24 24 21

57 75 74 76

77 99 98 55

2 2

19 42

39 52

58 94

Laboratory 2002 Oct Nov Dec 2003 Jan Feb Mar Apr May Jun

0 0 0

71 70 71

48 49 49

119 119 120

1 1 1 1 1 1

71 71 71 71 71 66

49 49 49 49 49 49

120 120 120 120 120 115

field and laboratory. In the field, fish that were caught in the smolt trap were considered early smolts and those captured in the study area after the smolt run were coded as late smolts. In the laboratory, smolt age was assessed via a series of factors (see below), one of which was fork length at the final sampling occasion. Because the difference in fork length between early and late smolts is well established at the time of smolting, we assumed that the use of fork length to assess smolt status at the last sampling occasion did not conflict with the use of fork length in the present analysis as a predictor of future smolt age. Study area.—This study was conducted as part of a long-term mark–recapture study of a small third-order stream (West Brook) in western Massachusetts within the Connecticut River basin (42825 0 N, 72839 0 W). The 1-km long study reach is stocked with Atlantic salmon fry (26–28 mm fork length) each spring at a density of 50 fish/100 m2 and sampled four times per year, beginning in autumn. Stocked fry were F1 offspring of adult returns to the Connecticut River. Individual tagging combined with multiple sampling provided a means to follow a fish throughout its freshwater residence and to retrospectively classify it as a parr or smolt for each sampling occasion. Atlantic salmon were collected via electroshocking (400-V unpulsed DC). Fish were also sampled via a smolt trap (picket weir) assembled 3 km downstream of

the study site in the spring of 2001. Each fish was anesthetized with NaHCO3-buffered tricaine methanesulfonate (MS-222; 100 mg/L) and measured for fork length (61 mm) and wet mass (60.1 mg). All untagged fish greater than 60 mm fork length and 2 g were tagged with passive integrated transponder tags (12 mm; Destron-Fearing Corp.) through a small incision between the pectoral fins (Gries and Letcher 2002), allowing for individual identification. Each fish was then aligned on a straight line, and a digital photograph of the left side was taken at a fixed distance. After recovery, each fish was returned to its location of capture. Further details of the study stream and fish capture are described by Letcher et al. (2002) and Letcher and Gries (2003). The 573 field photographs used in this study were of Atlantic salmon from the 1999 stocking year and were taken on eight sampling occasions between September 1999 (age 0) and May 2001 (age 2; Table 1). For analyses, the data from the 2001 smolt trap sampling occasion (containing only early smolts) and the subsequent sampling occasion that occurred 1 month later (containing only future late smolts) were combined. Laboratory methods.—The photographs of laboratory fish were taken during a study conducted by Obedzinski and Letcher (2004) in which Atlantic salmon from the Connecticut River stock were raised from eyed eggs to age-1 smolts in a controlled environment. Eggs were obtained from the U.S. Fish and Wildlife Service’s White River National Fish Hatchery (White River, Vermont). Broodstock were F1 offspring of sea-run adult returns to the Connecticut River. Eggs were kept in darkness except during sampling periods, and artificial light was timed based on natural photoperiod after hatching. Once hatched, fish were fed a combination of commercial dry feed and nauplii of the brine shrimp Artemia franciscana. Fish were sampled monthly from October 2002 (age 0) to June 2003 (age 1; Table 1). On every sampling occasion, each fish was anesthetized with MS-222, measured for fork length (6 1 mm) and wet mass (6 1 mg), and photographed on its left side at a fixed distance. Individuals were assigned to life history group (early or late smolt) based on size, condition factor, and a morphological rating of silvering and darkening of fin margins at the last sample. We analyzed 953 pictures taken over the nine sampling occasions (Table 1). Morphometric and statistical analyses.—Linear distance measurements taken from digital photographs served as variables in two multivariate analyses, principal components analysis (PCA) and discriminant analysis (DA). The aim of PCA was to identify and


describe patterns of variation between early and late smolts present at each sampling occasion. To determine the influence of size and shape on defining the principal components (PCs), we calculated Pearson’s product-moment correlations between the first three PCs and fork length or wet mass at each sampling occasion. The PCs that had a strong correlation with length, mass, or both were characterized by size, whereas those that lacked a correlation were considered nearly free of its influence and were characterized by shape (Haas and McPhail 2001). The purpose of DA was to identify the variables that best described the differences between early and late smolts at each sampling occasion and to test the variables’ ability to predict group membership (early or late smolt). Three different DAs (described below) were conducted to determine the relative discriminatory power of size and shape. Morphometric measurements.—To analyze morphometric variation, 20 linear distance measurements were used based on the box truss protocol (Bookstein et al. 1985; Figure 1). These network truss distances consisted of a series of measurements computed between 10 landmarks (Figure 1) that form an array of contiguous quadrilaterals across the body form (Strauss and Bookstein 1982). The landmarks were digitized on each photograph using tpsDig software (Rohlf 2001). To reduce skewness and kurtosis, loge transformation was applied to all 20 distance measurements. The distance measurements were divided into head, body, and tail regions (Figure 1). Fork length and growth rate analyses.—The mean fork lengths and mean growth rates of early and late smolts at each sampling occasion were calculated to aid in explaining the trends in the discriminatory power of shape and size over time. To determine the growth rates for early and late smolts, we first performed a linear regression to obtain the residual length between time intervals for every fish caught on consecutive sampling occasions. The mean residual of early and late smolts for each time interval was then calculated and plotted. We also calculated standardized lengths (observed length minus average length at each sampling occasion) to allow comparison between laboratory and field studies. Paired t-tests and the nonparametric alternative, the Mann–Whitney U-test, were used to evaluate the differences in early and late smolt mean standardized fork lengths and mean growth rate residuals for each sampling occasion. The normality and homogeneity of variance assumptions of the t-test were evaluated via the Shapiro–Wilk W statistic and Levene’s test (Statistica 6.1).

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FIGURE 1.—Landmark locations and morphometric distances (lines) used in shape analysis for the purpose of predicting smolt age among Atlantic salmon.

Principal components analysis.—Body form variability between early and late smolts was assessed using 16 (7 field and 9 laboratory) pooled-group PCAs, one per sampling occasion. The PCA was performed on the variance–covariance matrix of the 20 logtransformed truss distances. The significance of the eigenvalues and eigenvector coefficients was evaluated using the jackknifed standard errors and coefficient error ratios (Gibson et al. 1984). Discriminant analysis.—Discriminant analysis was used to classify fish as either early or late smolts based on morphometric variables, fork length, growth rate, or a combination of these. The null hypothesis was that the truss measurements or fork length for early and late smolts at each sampling occasion were equal (i.e., the mean discriminant scores did not differ between groups within any sampling occasion). All DAs were performed using the Statistical Analysis System version 8.02. Prior probabilities were set proportional to group sample sizes, and morphometric variables were selected at each sampling occasion by the forward stepwise procedure. The cross-validation (jackknife) procedure was used to test the stability of the discrimination functions. To compare the relative importance of size and shape variables as discriminators between life histories, classification success was compared among three different DAs at each sampling occasion. The first set of DAs classified fish at each sampling occasion based on their shape only. This was achieved by applying Burnaby’s (1966) size adjustment procedure to the logtransformed truss variables, which allowed for discrimination based on size- or age-related shape differences alone (Klingenberg 1996). The second set of DAs was performed using only the fork length of each individual at each sampling occasion. Fork length is used here as a measure of size. The third set of DAs was conducted using the log-transformed truss variables without Burnaby’s size-adjustment; this allowed a comparison of discrimination based on both shape and size. For each sampling occasion, a one-way analysis of variance (ANOVA) was used to test for differences in classification success rate among the three sets of DAs.

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FIGURE 2.—Mean fork lengths for early (solid lines) and late (dashed lines) Atlantic salmon smolts (a) collected in the field (West Brook, Massachusetts; 1999–2001) and (b) held in a laboratory (2002–2003). The vertical lines indicate 95% confidence intervals.

To test for the violation of assumptions of parametric analysis, the Shapiro–Wilk W-test (normality) and Levene’s test (homogeneity of variances) were used. Contrasts for least-squares means were used as a post hoc test. Due to the violation of assumptions, the Kruskal–Wallis test was used as a nonparametric alternative to the one-way ANOVA. Differences with P-values less than 0.05 were considered significant. Analyses were performed using Statistica version 6.1. Results Fork Lengths and Growth Rates The average fork lengths of early smolts were significantly larger (P , 0.001) than those of late smolts in the laboratory and field on all but the first field sampling occasion (September 1999; P ¼ 0.469; Figure 2a, b). Growth rates were significantly higher (P , 0.05) for early smolts than for late smolts at all field sampling intervals except for March–May 2000 and

FIGURE 3.—Mean growth rate residuals of early (solid lines) and late (dashed lines) Atlantic salmon smolts (a) collected in the field (1999–2001) and (b) held in a laboratory (2002– 2003). The vertical lines indicate 95% confidence intervals.

2001 (Figure 3a). Early smolts also grew significantly faster (P , 0.05) than late smolts in the laboratory at all but the last two intervals (April–May and May– June; Figure 3b). In the field, growth rates started to diverge between early and late smolts in April 2000 (age 1; Figure 3a), 11 months before the largest difference in lengths (March 2001; age 2; Figure 2a) and 13 months prior to smolt migration (May 2001). In the laboratory, growth rates started to diverge in October 2002 (age 0; Figure 3b), 7 months before the largest difference in lengths (May 2003; age 1; Figure 2b) and 8 months prior to smolting (June 2003). Although the peak difference in growth rates occurred in November for laboratory fish (Figure 3b) and midsummer in the field, the peak occurred at about the same percentage of days to smolting (51% in laboratory, 61% in the field) as did the largest difference in size (85% in the laboratory, 90.5% in the field; Figure 4).

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TABLE 3.—Proportion of variance between Atlantic salmon exhibiting early and late smoltification in the field (West Brook, Massachusetts), as explained by principal components (PCs) 1–3, which represent size and shape variation. Sample date 1999 Sep 2000 Mar May Sep Dec 2001 Mar May Average

FIGURE 4.—Mean growth rate residual and standardized fork length ratios in relation to the percent time to smolting of Atlantic salmon collected in the field (1999–2001) and held in a laboratory (2002–2003).

Principal Components Analysis Because size effects were not removed prior to conducting the PCAs, the first PC (PC1) contained a combination of size and size-related shape variation, as reflected by the relatively large and positive loadings and large eigenvalues. The identification of the PC1 values as size components and PC2 and PC3 as shape components was reinforced by the highly significant (P , 0.01) correlations of Atlantic salmon fork length TABLE 2.—Correlations between Atlantic salmon fork length and principal components (PCs) 1–3, representing size and shape variation. All correlations for PC1 were significant (P , 0.01); none were significant PC2 or PC3 (P . 0.05). Sample date

Age

PC1

PC2

PC3

Field 1999 Sep 2000 Mar May Sep Dec 2001 Mar May

0

0.94

0.13

0.01

1 1 1 1

0.93 0.96 0.94 0.97

0.13 0.05 0.10 0.07

0.02 0.06 0.05 0.03

2 2

0.76 0.93

0.19 0.13

0.04 0.04

Laboratory 2002 Oct Nov Dec 2003 Jan Feb Mar Apr May Jun

0 0 0

0.88 0.93 0.94

0.15 0.06 0.06

0.04 0.02 0.08

1 1 1 1 1 1

0.99 0.98 0.99 0.98 0.98 0.96

0.03 0.01 0.02 0.02 0.06 0.01

0.04 0.01 0.04 0.03 0.01 0.07

Age

PC1

PC2

PC3

0

0.474

0.125

0.087

1 1 1 1

0.573 0.675 0.690 0.710

0.093 0.085 0.051 0.068

0.084 0.051 0.048 0.053

2 2

0.740 0.621 0.640

0.072 0.115 0.087

0.046 0.069 0.063

(Table 2) and wet mass (data not shown, but similar to length data) with PC1 and their lack of correlation with PC2 and PC3. Field.—Averaged over the seven sampling occasions, the first three PCs accounted for 79% of the total variation in the data set. Principal component 1 explained the maximum amount of the variation (64.0%), and PC2 (8.7%) and PC3 (6.3%) explained considerably less of the remaining variation (Table 3). The large amount of variation accounted for by PC1 and the lack of variation accounted for by PC2 are reflected in the average PC scores for early and late smolts (Figures 5a, 6a). Among these three PCs, size (64.0%) accounted for four times more variation than did shape (14.9%). The results of the jackknife procedure for PC1 showed that, on average, 19.8 (99%) of the 20 coefficients of PC1 had significant coefficient error ratios (using the conservative ratio of 3.0; 0.01 , a , 0.001). For PC2, an average of only 3.8 (19%) of the coefficients had coefficient error ratios greater than 3.0. The characterization of PC2 in general was based on the number and type of significant characters for PC2 at all sampling occasions combined. The number of significant characters was fairly balanced among head (10, or 20%), body (10, or 24%), and tail (7, or 14%) characters, but almost all significant characters varied over time. The three earliest sampling occasions contained 74% of the significant characters and all of the significant body characters. For PC3, an average of only two (11%) of the coefficients had coefficient error ratios greater than 3.0. These results indicate that most of the shape variation was described by PC2 and that shape change occurred in all three body regions and occurred relatively early in development (but see comment below on PC stability). The jackknife estimates of the eigenvalues and their errors indicated that PC1 was a stable vector but that

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FIGURE 5.—Mean principal component 1 (PC1) scores for early (solid lines) and late (dashed lines) Atlantic salmon smolts (a) collected in the field (1999–2001) and (b) held in a laboratory (2002–2003). The vertical lines indicate 95% confidence intervals.

FIGURE 6.—Mean principal component 2 (PC2) scores for early (solid lines) and late (dashed lines) Atlantic salmon smolts (a) collected in the field (1999–2001) and (b) held in a laboratory (2002–2003). The vertical lines indicate 95% confidence intervals.

PC2 and PC3 were not. The first eigenvalue was separate from the second and third at all sampling occasions; except for September 1999 and May 2000, there was overlap between the second and third eigenvalues, which suggests that PC2 and PC3 were unstable (Gibson et al. 1984) and thus should be interpreted cautiously. Laboratory.—As with the field samples, the majority of the variation in the laboratory data set was explained by PC1 (73.5%) and less by PC2 (7.5%) and PC3 (5.0%; Table 4). The large amount of variance accounted for by PC1 and the lack of variation accounted for by PC2 are reflected in the average PC scores for early and late smolts (Figures 5b, 6b). Among these three PCs, size (73.5%) accounted for four times more variation than did shape (12.5%). The results of the jackknife procedure showed that, on average, 19.8 (99%) of the 20 coefficients of PC1 had significant coefficient error ratios (using the conservative ratio of 3.0; 0.01 , a , 0.001). For

PC2, 30% of the jackknifed coefficients were significantly different from zero and 81% of those were tail characters. The pattern of significance was almost the same at all times except for the second sampling occasion, when only head characters were significant. The jackknife procedure showed that the average number of significant coefficients found in PC3 was half that of PC2 (15.6%) and that significance was split between head (50%) and tail (43%) characters. The jackknife estimates of eigenvalues and their errors indicated that PC1 was a stable vector but that PC2 and PC3 were not. The first eigenvalue was separate from the second and third at all samples and, except in March and May of 2003, there was overlap between the second and third eigenvalues. Discriminant Analysis Field.—The average classification success rates of the three predictors in the field were 81% for fork length, 79% for shape, and 84% for shape and size.

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TABLE 4.—Proportion of variance between Atlantic salmon exhibiting early and late smoltification in the laboratory, as explained by principal components (PCs) 1–3, which represent size and shape variation. Sample date 2002 Oct Nov Dec 2003 Jan Feb Mar Apr May Jun Average

Age

PC1

PC2

PC3

0 0 0

0.381 0.496 0.695

0.178 0.143 0.086

0.118 0.112 0.055

1 1 1 1 1 1

0.842 0.854 0.877 0.884 0.846 0.738 0.735

0.052 0.039 0.036 0.033 0.040 0.068 0.075

0.033 0.024 0.019 0.022 0.024 0.045 0.050

Length’s predictive ability decreased on the last sampling occasion from 91% to 73% classification success (Figure 7a). This is probably because the final sampling occasion contained fish that were captured on two separate occasions and combined for the DA. Future (age 3 in the subsequent spring) late smolts were sampled in June, 1 month after sampling of the early age-2 smolts and therefore had more opportunity to grow, thus artificially decreasing the difference in size between early and late smolts. In contrast, shape variables performed well on the last sampling occasion (96% classification success) because morphological differences between early and late smolts were greatest at this time and were apparent by visual inspection. Fork length (82% classification success), shape (80%), and shape and size (82%) could all be used to distinguish between early and late smolts early in development (September 1999; age 0; Figure 7a). The prior probabilities were set proportional (rather than equal) to group sample sizes so that classification would be weighted by early and late smolt numbers in the field and laboratory. However, because late smolts, on average, were three times more numerous than early smolts in the field (except for in May 2001), classification error rates were much lower for late smolts (4.5%) than for early smolts (62%). The ANOVAs revealed that there were no significant differences among classification success rates until the last sampling occasion (P , 0.00001; age 2), when the discrimination by fork length was significantly lower (73%) than that by either shape (96%) or the combination of shape and size (97%; Figure 7a). Laboratory.—The average classification success rates of the three predictors in the laboratory were 85% for fork length, 73% for shape, and 86% for shape and size. As with the field data, all three DAs could

FIGURE 7.—Rates of jackknife classification success in predicting early or late smoltification based on size, shape, or both in Atlantic salmon (a) collected in the field (1999–2001) and (b) held in a laboratory (2002–2003). Asterisks denote significant differences within a sampling occasion (0.001 , P , 0.05*; P , 0.0001**).

discriminate between early and late smolts early in development (October 2002; age 0; Figure 7b). Using ANOVA, significant differences in classification rates were found at all but the first two sampling occasions. The planned comparisons revealed that from December 2002 (age 1) to May 2003 (age 2), the classification success rates were significantly lower when shape was used as a predictor then when either fork length or shape and size were used as predictors. In June 2003 (age 2), however, fork length resulted in a significantly lower classification success rate than the other two discriminant functions (Figure 7b). Discussion In this study, we have shown that both body size and shape can be used to discriminate early from late smolts in the fall at age 0. Size and shape together performed as well as size alone, indicating that a large portion of the early differences between early and late smolts could be attributed to size differences, but shape

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by itself also allowed correct classification. In fact, in the field there was no difference in classification success between shape alone and size alone. In the laboratory, however, size alone consistently outperformed shape alone. Interestingly, the relative timing of early differences in size and growth were very similar between early and late smolts for both the field and laboratory, despite different smolt ages (age 1 in the laboratory; age 2 in the field). Overall, our results indicate that size and shape differences are expressed long before smoltification and that shape accounted for relatively more of the difference between early and late smolts in the field than in the laboratory. The timing of the growth rate differences between early and late smolts in this study suggests that some fish increased their energy intake in spring, which may have resulted in their attainment of a summer emigration threshold (Metcalfe and Thorpe 1990; Thorpe et al. 1998). Differences in growth rate among field fish increased in April (age 1) and peaked in July, perhaps because appetite was declining in some individuals (parr that would not smolt at age 2) but was sustained in others (age-2 smolts; Metcalfe 1998). Laboratory fish also may have met an early emigration threshold because the differences in growth rate peaked at a similar percentage of time to smolting for laboratory fish and field fish, despite faster average growth rates in laboratory fish and a different smolt age. The similarity in the relative timing of growth rate and size patterns suggests that fish in the laboratory and field are following similar developmental courses. Although the timing of the peaks in growth rate in both settings points to a summer emigration threshold, the significant size differences that occurred early in development in the field (age 1 in March, at 42% of time to smolting) and laboratory (age 0 in October, at 40% of time to smolting) suggest that a developmental decision was made prior to midsummer. Similarly, Letcher and Gries (2003) found early differences in mass (at age 0 in December onward) between West Brook parr that smolted at age 2 and those that did not. Likewise, the early size differences in the present study imply that fish entered the early smolt trajectory before the summer prior to smolting. If an emigration switch does occur earlier in development, then perhaps a second threshold during the summer before smolting either prevents or promotes the loss of freshwater adaptation (Thorpe et al. 1988). In contrast to the relatively early peaks in growth rate that resulted in size differences early on, differences in shape did not peak until spring. One reason shape differences were not as evident until the parr–smolt transformation, rather than earlier in stream residence, may be the influence of seasonal physiolog-

ical changes on growth and, hence, shape. After an increase in day length, circulating salmon growth hormone (GH) usually increases in smolting salmon (McCormick et al. 1995), resulting in an increase in metabolism and skeletal (length) growth (for review of salmon GH, see Bjo¨rnsson 1997). The decrease in total lipids (Vanstone and Markert 1968; Fessler and Wagner 1969) and increase in skeletal growth probably results in a leaner and more streamlined body, which is reflected in the decrease in condition factor (100 3 [weight/length3]; Vanstone and Markert 1968; Fessler and Wagner 1969; Saunders and Henderson 1970) and disproportionately fast growth in caudal peduncle length (Winans 1984; Winans and Nishioka 1987) during the parr–smolt transformation. Hence, changes in body morphology may be tightly linked to the hormonal changes affecting growth during the parr– smolt transformation. There is also evidence that the initiation of some smolt characteristics (weight–length relationships, silvering, and salinity tolerance) is size dependent, which may influence survival probabilities. Treatment with GH, for instance, has been shown to reduce antipredator behavior in brown trout S. trutta (Johnsson et al. 1996); thus, the requirement that fish reach a certain size before GH levels increase would not only limit the amount of time they might be behaviorally compromised but also restrict the release of GH to a period when the fish are larger and less susceptible to predation. Furthermore, the GH-induced increase in skeletal growth that occurs during smolting may be of benefit later in life, since vertebrae are an important source of calcium during the spawning migration (Kacem et al. 1998). Larger smolts have greater osmoregulatory ability in saltwater (reviewed in McCormick and Saunders 1987), experience less predation, and have higher return rates than smaller smolts (reviewed in Marschall et al. 1998). The presence of size-dependent and seasonal physiological changes helps explain why shape changes during smolt metamorphosis, but it does not explain the shape differences that occur earlier in development. Even though the shape variables had the most predictive success during smolt metamorphosis, they had an average of 76% (field) and 70% (laboratory) classification success prior to peak discrimination. Based on our results, it appears that shape changes occur throughout freshwater residence, but these are smaller in magnitude than those present during smoltification. We observed that the strength of shape differences between early and late smolts throughout the course of our tagging studies was stronger in the field than in the laboratory. The primary differences between the field


and laboratory studies were habitat, food source, and developmental rate (age-2 versus age-1 smolts). The similarity of differences in time-adjusted growth between field and laboratory suggests that the compressed smolt development cannot account for weaker shape differences in the laboratory. It is possible that food differences (stream drift and benthos versus dry pellet) could influence shape variation. Fish fed pelleted food tend to have a higher fat content (Fleming et al. 1994), but it is unknown whether this could mediate shape differences between smolt ages. Habitat seems the most likely explanation for stronger smolt age differences in the field. Shape differences are common between hatchery and wild populations (Swain et al. 1991; Fleming et al. 1994; von CramonTaubadel et al. 2005). Based on common environment studies, shape differences between habitats seem to arise mainly from phenotypic plasticity (e.g., Swain et al. 1991; von Cramon-Taubadel et al. 2005). From a practical standpoint, size is much easier to measure than shape. Our results suggest that shape adds little to the discriminatory power of size alone; therefore, shape measurements will not add to predictive ability except at the time of smolting. Despite this, it is intriguing that shape differences were expressed well before smoltification. It will be interesting to determine with future studies whether early shape differences reflect variation resulting from growth rate differences alone or from early expression of future life history differences. Finally, if early shape differences are adaptive in the field, the less-distinct shape differences in the laboratory may argue against smolt stocking and for stocking at an earlier life stage. Acknowledgments We thank Steven Cadrin of the National Marine Fisheries Service, whose generosity with his time and expertise helped to move this project forward. We wish to extend thanks to all the people who worked many hours in the laboratory and field to collect these data, especially Aimee Varady, Todd Dubreuil, Gabe Gries, and Matt O’Donnell. References Beeman, J. W., D. W. Rondorf, M. E. Tilson, and D. A. Venditti. 1995. A nonlethal measure of smolt status of juvenile steelhead based on body morphology. Transactions of the American Fisheries Society 124:764–769. Bjo¨rnsson, B. T. 1997. The biology of salmon growth hormone: from daylight to dominance. Fish Physiology and Biochemistry 17:9–17. Bohlin, T., C. Dellefors, and U. Faremo. 1994. Probability of first sexual maturation of male parr in wild sea-run brown trout (Salmo trutta) depends on condition factor 1 year in

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