Multiple-Locus Heterozygosity and the Physiological Energetics of Growth in the Coot Clam, Mulinia Lateralis, from a Natural Population

MULTIPLE-LOCUS HETEROZYGOSITY AND THE PHYSIOLOGICAL ENERGETICS OF GROWTH IN THE COOT CLAM, MULINIA LATERALIS, FROM A NATURAL POPULATION

DAVID W. CARTON, RICHARD K. KOEHN AND TIMOTHY M. SCOTT Department of Ecology and Evolution, State University of Nno York at Stony Brook, Stony Brook, New York 11 794 Manuscript received February 29, 1984 Revised copy accepted June 4, 1984

ABSTRACT The relationship between individual energy budgets and multiple-locus heterozygosity at six polymorphic enzyme loci was examined in Mulinia lateralis. Energy budgets were determined by measuring growth rates, rates of oxygen consumption, ammonia excretion and clearance rates. Enzyme genotypes were determined using starch gel electrophoresis. Growth rate and net growth efficiency (the ratio of energy available for growth to total energy absorbed) increased with individual heterozygosity. The positive relationship between observed growth and multiple-locus heterozygosity was associated with a negative relationship between routine metabolic costs and increasing heterozygosity. Re- duction in routine metabolic costs explained 60% of the observed increased growth of more heterozygous individuals. When routine metabolic costs were standardized for differences in feeding rates, these standard metabolic costs explained 97% of the differences in growth rate. Lower standard metabolic costs, associated with increasing heterozygosity, have been proposed as a physiological mechanism for the relationship between multiple-locus heterozygosity and growth rate that has been reported for a variety of organisms, ranging in diversity from aspens to humans. This study demonstrates that reduction of standard metabolic costs, at least in clams, accounts for virtually all of the differences in growth rate among individuals of differing heterozygosity.

VIDENCE of increasing growth rate and metabolic efficiency with greater E multiple-locus heterozygosity at electrophoretically detectable enzyme loci has been accumulating for a wide variety of plant and animal species (KOEHN and GAFFNEY1984). A positive relationship between multiple-locus heterozygosity and growth, or the energy available for growth, has recently been demonstrated in a variety of marine invertebrates, including oysters (SINGHand ZOUROS 1978; ZOUROS, SINCHand MILES 1980; KOEHN and SHUMWAY1982), mussels (KOEHN and GAFFNEY1984) and oyster drills (GARTON1984). The results of these studies on natural, outbred populations have been similar: growth rate increases with levels of heterozygosity. Except in one case (KOEHN and SHUMWAY1982), the underlying causes for these differences in growth have not been identified.

Genetics 108 445-455 October, 1984 446 D. W. GARTON, R. K. KOEHN AND T. M. SCOTT Scope for growth equals the energy available for growth and reproduction and is expressed as the difference between absorbed energy and energy lost through metabolic processes (WINBERG1956; BAYNEand NEWELL1983). En- ergy available for growth can be increased, in principle, by increasing metabolic efficiency (reducing weight-specific metabolic rates) and/or increasing absorption (by increasing feeding rate or absorption efficiency). KOEHN and SHUMWAY (1 982) demonstrated that weight-specific basal oxygen uptake declined significantly with increasing heterozygosity in starved oysters, Crassostrea wirginica. They hypothesized that the greater metabolic efficiency associated with heterozygosity would also be reflected in greater scope for growth. RODHOUSE and GAFFNEY(1 984) further demonstrated in starved oysters that the increased metabolic efficiency with increasing heterozygosity is reflected in reduced rates of dry weight loss (lower negative scope for growth). These studies demonstrate qualitatively and indirectly that relatively high scopes for growth can be due to increased metabolic efficiency. Growth, however, is also influenced by feeding rate and there are metabolic costs associated with feeding activities. In the oyster drills, Thais haemastoma and T. lamellosa, increased feeding rates were the most important factor associated with size and heterozygosity affecting scope for growth (GARTON 1984; CARTONand STICKLE1984). Although routine metabolic costs were inversely related to multiple-locus heterozygosity, increased metabolic eff- ciency could account for only 20% of the increased scope for growth associated with increased individual heterozygosity. The energetic cost of feeding was not determined in the oyster drill study (CARTON1984). To date there has been no investigation of the relative quantitative contributions of standard metabolic efficiency, feeding rate and the metabolic cost of feeding to the observed relationship between growth and multiple-locus heterozygosity. This study reports on the relative contributions of these components of the energy budget to the relationship between growth rate and multiple-locus heterozygosity in the clam, Mulinia lateralis.

MATERIALS AND METHODS

Clams: Individuals of M. lateralis were collected by dredging from Great South Bay, Long Island, New York, in July 1983. Clams were brought to the laboratory and placed in 5.5-liter plastic trays filled with filtered, aerated seawater at 23” and 26”/00 salinity. The seawater was changed every 2-4 days. Clams were fed a diet of mixed culture algae, Isochrysis galbana and Monochrysis sp., reared by the methods of GUILLARD(1975). Clams were fed an average of twice per day during the 30-day experimental period for determining growth rates. Individual clams (N = 150) were tagged with numbered discs, or “bee dots” (Chr. Graze AG, West Germany) and polyacrylate glue. Shell lengths and heights were measured to the nearest 0.01 mm using vernier calipers. Soft tissue dry weight was estimated using the allometric relationship between dry weight and shell height: flesh dry weight (milligrams) = 0.006 (shell height in millimeters)3-495,r2 = 0.777, n = 494 (S. E. SHUMWAYand T. M. SCOTT,unpublished data). Energy budgets: Equations describing the flow of energy through organisms have been developed (WINBERG1956; CRISP 1971) and applied to marine bivalves (reviewed by BAYNEand NEWELL 1983). “Scope for growth” represents the energy remaining after the metabolic demands of the organism have been met and can be positive or negative. Scope for growth (SG) was determined using the following form of the energy budget:

SG = Ab - (R + U), HETEROZYGOSITY AND GROWTH IN CLAMS 44 7 where Ab (absorption) is that part of consumed energy that is not eliminated as feces, R (respiration) is that part of the assimilated energy that is converted directly to heat or to mechanical work performed by the organism and U (excretion) is that part of the assimilated energy that is passed out of the organism as excretory material, usually as urine. Direct measurement of growth rates were made over 4 wk of the experiment. Following the measurement of growth rate, the oxygen uptake, ammonia excretion and feeding rate of individual clams were determined. Respiration was measured using a Gilson differential respirometer and standard manometric techniques. Oxygen uptake was converted to energy equivalents by applying the oxycaloric coefficient of 4.73 cal/ml of oxygen consumed at standard temperature and pressure (STP) (CRISP 197 1). Ammonia excretion was determined by GRASHOFFand JOHANNSEN’S (1 972) modification of the phenol-hypo- chlorite method. ELLIOTand DAVISON(1975) give the energy equivalent of ammonia excretion at 5.94 cal fmg of ammonia nitrogen excreted. Feeding rates were determined for each clam (independent of oxygen uptake) by measuring the rate of disappearance of an algal suspension in a static system using a model ZB Coulter Counter. Therefore, routine metabolic costs (R + U) were measured directly and scope for growth (SG) was estimated from the change in dry weight over time, assuming a caloric equivalent of 4 calfmg of dry weight. Total energy absorption (Ab) was calculated from the difference. “Routine metabolism” refers to oxygen consumption measured with uncontrolled but minimum motor activity (PROSSER1973). As activity varies among individuals, oxygen uptake was extrapo- lated to zero activity. Since the primary activity of filter-feeding bivalves is feeding (clearance of algae from water) (BAYNEand NEWELL1983), oxygen uptake was standardized using the linear relationship between oxygen uptake and clearance rate: Oxygen uptake rate (VOs) = a + b (clearance rate) and adjusted intercepts were calculated for each individual as a = observed VOn - b (observed clearance rate). The intercept, a, represents oxygen uptake at zero activity and is termed “standard metabolism.” Similarly, growth rate is also related positively to feeding rate. Clearance rate-specific growth rates were calculated from the linear relationship: Growth rate = a + b (clearance rate), and the adjusted intercept was calculated for each individual as a = observed growth rate - b (observed clearance rate). This provides an estimate of growth rate on a “standard ration.” Therefore, both energetic costs (oxygen consumption) and energetic gains (growth rate) were standardized on a common variable, feeding rate. Weight-specific rate functions were calculated from the whole animal rate functions using the general form:

V, = aWb where V, is the whole animal rate variable and W is the flesh dry weight; a and b are fitted constants calculated from the regression of the log of the rate variable on the log of dry weight, and a represents the intercept and b the slope of the regression equation. Weight-specific rate functions were finally calculated as v, = VJWb where Vx is the weight-specific rate variable in units per milligram of dry weight of a “standard” (ie., constant weight) animal. In this study, the “standard sized” animal was the mean final weight, 6.13 mg of flesh dry weight. Starch gel electrophoresis: Samples were prepared by homogenizing whole animals in 0.05 M Tris- HCI, pH 8.0, buffer, followed by centrifugation at 0” for 10-15 min at 7000 X g. The supernatant was used as the enzyme source. Enzymes scored were those coding for an a-amino acyl peptide hydrolase (LAP, EC 3.4.1.2).phosphoglucomutase (PGM, EC 2.7.5. l), esterase (EST, EC 3.1.1.-), 448 D. W. CARTON, R. K. KOEHN AND T. M. SCOTT strombine dehydrogenase (STDH, no EC number assigned), peptidase (PEP, EC 3.4.1 .-) and glu- cosephosphate isomerase (GPI, EC 5.3.1.9). Electrophoresis for all six enzymes was done in hori- zontal starch gels (KOEHN,MILKMAN and MITTON 1976). LAP, EST, PGM and GPI were run with a Tris-maleate gel and buffer system, PEP with an LiOH system and STDH with Tris-borate- EDTA (KOEHN, MILKMANand MIITON 1976). PGM, GPI and EST activities were demonstrated by the techniques of HARRISand HOPKINSON(1976), STDH as described by DANLWet al. (1981) and LAP as described by KOEHN, MILKMANand MITTON (1976) except that ~-leucyl-4-methoxy-& naphthylamide was substituted for [email protected] PEP-staining procedure was similar to that for LAP, but at pH 5.0 and with ~-alanyl-4-methoxy-P-naphthylamideas the substrate.

RESULTS Mortality: Of the 150 clams used to initiate the experiment, 112 survived the 30-day experimental period and the manipulations necessary for determining energy budgets, a mortality rate of 25%. Accidental sealing of clam valves while applying numbered tags, particularly in individuals less than 5 mm in length, was the probable cause of most deaths. Natural populations of M. lateralis are characterized by high growth rates and a high individual mortality rate (CALABRESE1969). Growth rates: Mean shell length, height and estimated dry weight for each measurement date are presented in Table 1. Shell length and height increased linearly during the experiment. All energy budget parameters (oxygen consumption, ammonia excretion, total energy losses, clearance rate and growth rate) were positively related to estimated dry weight (Table 2 and Figure 1). Oxygen uptake (R) accounted for 94% of the total energy losses (R + U). Hence, nitrogen excretion via ammonia is not a significant component of the energy budget under constant environmental conditions. Net growth efficiency (SGIAb), was independent of clam dry weight and was constant between 45 and 49% over the size range of clams used in this study (calculated from the data in Figure 1). Growth rates were positively correlated with clearance rates and routine metabolic costs, but only the relationship with feeding rate was statistically significant (Table 3). Growth rate and oxygen consumption were significantly related to clearance rate (Table 3, equations 3 and 4). The equations used to calculate “standard metabolic rate” and “standard growth rate” are presented in Table 3. Starch gel electrophoresis: The six studied loci were selected because they are each highly polymorphic. The number of heterozygotes and homozygotes observed at each locus is presented in Table 4. The most polymorphic locus was PGM (88.4% heterozygotes), whereas the least was EST (15.2% heterozygotes). Five of the six loci were at Hardy-Weinberg equilibrium; PGM differed significantly from Hardy-Weinberg expectations. There was a greater than ex- pected frequency of heterozygotes at the PGM locus. Energy budget:heterozygosity relationships: Weight-specific growth rates were positively and significantly related to multiple-locus heterozygosity (Table 5 and Figure 2). Weight-specific clearance rates were also positively correlated with heterozygosity but not significantly (Table 5 and Figure 2). Hence, the observed differences in growth are most likely to be explained by differences in metabolic efficiency and not differences in feeding rate. Weight-specific HETEROZYGOSITY AND GROWTH IN CLAMS 449

TABLE 1

Length, height and estimated dry weight for experimental animals at three measurement points during 29-day growth experiment

Day Length (mm) Height (mm) Flesh dry weight (mg)

0 6.86 f 0.10 5.29 f 0.07 2.02 f 0.05 14 7.75 f 0.12 6.07 f 0.09 3.27 f 0.08 29 9.16 f 0.14 7.27 f 0.12 6.13 f 0.12 Values are means f SE. N = 112.

TABLE 2

Mean rate values for energy budget parameters and regression of energy budget components on estimated jlesh dry weight

Parameter Rate Regression equation r VOZ (PI. hr-' .mg-') 1.61 f 0.03 0.488 + 0.657 (W) 0.70 VNHl (nmol. hr-'. mg-') 6.1 f 1.5 0.939 + 0.840 (W) 0.75 Total energy loss (cal. day-'. 0.20 f 0.01 -0.459 + 0.698 (W) 0.76 mg-') Clearance rate (mI. min-' . 1.22 f 0.03 0.368 + 0.660 (W) 0.64 mg-I) Growth rate (mg. mg-' initial 1.58 f 0.12 0.323 + 0.762 (W) 0.42 size). 30 day-' Energy budget components are expressed as cal .day-', except for clearance rate which is expressed in ml'min-'; dry weight (W) is in mg. Data for the regression were log transformed. N = 112. All regressions are significant at the P < 0.01 level.

54 -15 T 4- s A 1 -E3- -I 0 5 I 0 - U 2- -05 I-

I I I 5 IO 15 Dry Weight (mg) FIGURE1 .-Relationship between energy budget components and clam dry weight. SG, Scope for growth, estimated from observed growth and assuming 1 mg of dry weight equals 4 cal; R + U, routine metabolic costs, measured directly; C.R., clearance rate in m1.min-I and Ab, caloric absorption, calculated from the sum of R + U and SG. See also Table 2. routine metabolic cost (R + U) were negatively and significantly related to multiple-locus heterozygosity (Table 5 and Figure 2). The net contribution of increased routine metabolic efficiency to explaining increased growth associated with heterozygosity was approximately 60% (Table 6). Net growth efficiency increased from 38.4 to 60.8% with increasing degree of heterozygosity (Table 6). 450 D. W. GARTON, R. K. KOEHN AND T. M. SCOTT

TABLE 3

Regression of growth (cal. day-' .mg-'; equations 1-3) on energy budget components and regression of oxygen uptake on feeding rate (equation 4)

Equation Component Regression equation r Significance 1. Oxygen uptake (PI. hr-'. mg-') 1.968 + 0.1 16 (VOp) 0.080 NS

2. Total energy losses (cal . day-' .mg-') 1.900 + 1.210 (R + U) 0.084 NS

3. Clearance rate (mI . min-' .mg-') 1.408 + 1.298 (CR) 0.330 **

4. Oxygen uptake regressed on feeding 2.866 + 0.615 (CR) 0.221 * rate (pl Oz.hr-' . (ml . min-')-' . mg-I) Equations 3 and 4 were used to calculate the adjusted intercepts discussed in the text. NS, not significant. * P < 0.05. ** P < 0.01.

TABLE 4

Frequency of single-locus heterozygotes as determined by starch gel electrophoresis

Locus

PGM STDH EST PEP LAP GPI Heterozygotes 99 54 17 66 72 77 Homozygotes 13 58 95 46 40 35 % Heterozygotes 88.4 48.2 15.2 58.9 64.3 68.8

TABLE 5

Regression of weight-specajic energy budget components on multiple-locus heterozygosity (H)

Parameter Regression equation r P Oxygen uptake (PI. hr-I) 4.016 - 0.206 (H) -0.214 * Ammonia excretion (nmol . 8.876 + 0.274 (H) 0.060 NS hr-') Total energy losses (cal' 0.459 - 0.025 (H) -0.252 ** day-') Clearance rate (ml. min-I) 0.570 + 0.047 (H) 0.131 NS Growth rate (mg. 30 1.655 + 0.304 (H) 0.200 * days-') All components are expressed as units.mg-' of a standard clam (=6.13 mg). N = 112. NS, not significant. P < 0.05. ** P c 0.01. Following correction for differences in clearance rate there were significant relationships between total energy losses, and growth rate regressed on multiple-locus heterozygosity (Table 7 and Figure 3). The subtraction of the metabolic costs of feeding and individual variation in feeding rates provides an HETEROZYGOSITY AND GROWTH IN CLAMS 451

- 0 0

0.2 *- 0.4 -&

t 1 I I 1 I I I 234 56 NO. OF HETEROZYGOUS LOCI. CLAM-' FIGURE2.-Regression of weight-specific growth (SG, open circles), routine metabolic costs (R + U, closed circles) and clearance rates (CR, dashed line) on multiple-locus heterozygosity. See also Table 5. Values are means zk standard error; sample sizes in parentheses.

TABLE 6

Contribution of reduced routine metabolic costs to explaining increased growth associated with increasing multiple-locus heterozygosity

(3) (1) (2) Routine metabolic (4) (5) (6) Scope for growth Difference costs (~al.~day-'. Difference % Net growth H (caI.day-'.mg-') from H = 1 mg- ) from H = 1 explained' efficiency (%)* 1 0.270 0.434 38.4 2 0.312 0.042 0.409 0.025 59.5 43.3 3 0.354 0.084 0.384 0.050 59.5 48.0 4 0.396 0.126 0.359 0.075 59.5 52.5 5 0.438 0.168 0.334 0.100 59.5 56.7 6 0.480 0.210 0.309 0.125 59.5 60.8 Components for each heterozygote class, H, estimated from regressions presented in Table 5. a Percent of (2) explained by (4); i.e., percent of observed growth increase over most homo- zy ous class explained by decreased metabolic costs with increasing heterozygosity. 'Net growth efficiency equals (SG/Ab); in this cask SG/((R + U) + SG). estimate of the contribution of standard metabolic efficiency in explaining differences in growth rates of active, well-fed clams. Approximately 97% of the observed differences in growth rate was explained by standard metabolic efficiency (Table 8). Stated differently, approximately two-thirds of the variation in growth rate with heterozygosity is explained by differences in standard metabolic efficiency and approximately one-third by variation in feeding rates. There were no strong single-locus effects on the relationship between metabolic costs or growth rates and heterozygosity (Table 9). The effects at any 452 D. W. GARTON, R. K. KOEHN AND T. M. SCOTT

TABLE 7

Regression of clearance rate corrected (adjusted intercepts) energy budget components on multiple-locus heterozygosity, H

~~~~ Parameter Regression equation r Significance Total energy losses 3.621 - 0.243 (H) -0.270 ** Growth rate 0.172 + 0.251 (H) 0.219 * Components expressed as cal.day-'.standard clam (6.13 mg). N = 112. * P < 0.05. ** P < 0.01.

I I I 23456 NO. OF HETEROZYGOUS LOCI FIGURE3.-Regression of clearance rate corrected metabolic costs (R + U, closed circles) and growth rate (SC, open circles) on heterozygosity. See also Table 7. Values are means f standard error; sample sizes in parentheses. one locus are small, usually statistically nonsignificant, but cumulative, resulting in a statistically significant relationship with increasing multiple-locus heterozygosity.

DISCUSSION Growth rates of M. lateralis increase significantly with individual multiple- locus heterozygosity. Increased routine metabolic efficiency (standard metabolic costs plus costs of routine activity) can explain 60% of the observed increase in growth associated with heterozygosity. The amount of increased growth explained increases to 97% after variation of the metabolic costs of feeding activity and growth were accounted for (Table 8 and Figure 3). Indi- vidual variability of the metabolic costs of routine activity tend to mask the physiological relationship between standard metabolic efficiency and multiple- locus heterozygosity. These results in M. lateralis justify the interpretation by KOEHN and SHUMWAY(1 982) that the increased metabolic efficiency associated with heterozygosity in starved oysters would result in a higher scope for growth. This assumption holds because routine metabolic costs for feeding and growth are similar in each multiple-locus heterozygote class. The fact that HETEROZYGOSITY AND GROWTH IN CLAMS 453

TABLE 8 Contribution of reduced standard metabolic costs to explaining increased growth, after correcting for variation in clearance rates

Scope for growth Standard metabolic (cal .day-'. Difference costs (cal .day-'. Difference % H standard clam) from H = 1 standard clam) from H = 1 explained" 1 0.423 3.378 2 0.475 0.251 3.135 0.243 96.8 3 0.925 0.502 2.892 0.486 96.8 4 1.176 0.753 2.649 0.729 96.8 5 1.427 1.004 2.406 0.972 96.8 6 1.678 1.255 2.163 1.215 96.8 Components for each heterozygote class, H, estimated from regression presented in Table 7. Percent of increased growth explained by increased metabolic efficiency. routine metabolic costs did not contribute significantly to growth efficiency in T. haemastoma may be the result of metabolic costs of feeding as feeding rate increased with heterozygosity (GARTON1984). Although considerable infor- mation exists on the relationship between oxygen uptake and filter feeding in bivalves, little is known on the metabolic costs of predation in oyster drills (GARTONand STICKLE1980; BAYNEand NEWELL1983). It would be of interest to determine the relative costs of standard and routine (feeding and growth) metabolism as a function of multiple-locus heterozygosity in a carnivorous gastropod, compared with those demonstrated here in M. lateralis. The statistical relationships between two of the components of the energy budget, scope for growth and routine metabolic costs, are significant but have very low coefficients of determination (5-10%). This high degree of variability has been noted in other studies (GARTON1984; KOEHN and GAFFNEY1984). Sources of this variability could include nongenetic (i.e., environmental) effects on growth such as different age classes in the oyster drill study (GARTON1984) or physical location within a clump of mussels (KOEHN and GAFFNEY1984). Another possibility, however, is that the studied loci do not include all loci associated with growth rate or are not linked to all of the loci actually respon- sible for metabolic efficiency variation (genetic background). The lack of heterozygosity-linked effects on growth rate in limited-parent crosses also suggests genetic background effects (GAFFNEYand SCOTT1984; KOEHN and GAFFNEY 1984). The hypothesis that other loci are involved in the relationship between growth and heterozygosity is also supported by the general lack of strong single-locus effects on metabolic efficiency and growth (Table 9). In general, there are few significant differences between means for heterozygotes and homozygotes for oxygen uptake and growth. Rather, the effect of heterozygosity is additive across loci, resulting in an overall significant relationship. Overdominance for growth rate has been reported in other mollusks (SINGH and ZOUROS 1978; GARTON1984; Zou~oset a1 1983; KOEHN and GAFFNEY 1984). 454 D. W. CARTON, R. K. KOEHN AND T. M. SCOTT

TABLE 9

Energy budget components for single-locus heterozygotes and homozygotes

Component PGM STDH EST PEP LAP GPI

~ VOn (NI. hr-') Heterozygotes 9.99 9.36 9.3 1 9.49 10.05 9.36 Homozygotes 9.20 10.42 10.00 10.50 9.62 11.17 Significance NS NS NS NS NS *

VN"+ (nmol.hr-') Heterozygotes 38.4 38.6 36.7 35.7 38.3 38.7 Homozygotes 34.7 36.5 37.7 40.2 36.1 35.0 Significance NS NS NS NS NS NS

Energy losses (cal .day-') Heterozygotes 1.22 1.15 1.14 1.18 1.23 1.14 Homozygotes 1.13 1.26 1.22 1.24 1.16 1.37 Significance NS NS NS NS NS **

Clearance rate (mI. min-l) Heterozygotes 0.74 0.76 0.79 0.74 0.73 0.75 Homozygotes 0.78 0.74 0.74 0.76 0.78 0.74 Significance NS NS NS NS NS NS

Total growth (nip) Heterozygotes 4.84 4.93 6.32 5.37 4.85 4.7 1 Homozygotes 4.84 4.76 4.62 4.17 4.83 5.15 Significance NS NS * * NS NS Means and significance tests are from analysis of covariance using dry weight as the covariable. All rate functions are standardized to a 6.13-mg clam. NS, not significant. * P < 0.05. ** P < 0.01. The average superiority of heterozygotes relative to homozygotes for growth, reproduction and survival has now been demonstrated in several species of marine invertebrates (KOEHN and GAFFNEY1984). These studies provide a mechanism for the maintenance of stable genetic polymorphisms within populations and also provide valuable insight into the factors controlling the genetic structure of natural populations. However, the actual biochemical mechanism of how heterozygosity at loci coding for soluble proteins is trans- lated into greater metabolic efficiency is unknown. Further study is necessary to elucidate the connection between protein polymorphism and efficiency of metabolic pathways.

We are grateful to LEWISDEATON, who assisted in collecting specimens, and RITA SICKLES,who typed the manuscript. The work was supported by United States Public Health Service grant GM 21 133. This paper is contribution 491 in Ecology and Evolution at the State University of New York at Stony Brook.

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