Inbreeding depression and growth heterosis in larvae of the purple sea urchin Stronglyocentrotus purpuratus (Stimpson)

Journal of Experimental Marine Biology and Ecology 384 (2010) 68–75

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Inbreeding depression and growth heterosis in larvae of the purple sea urchin Stronglyocentrotus purpuratus (Stimpson) David Anderson, Dennis Hedgecock ⁎ Department of Biological Sciences, University of Southern California, 3616 Trousdale Pkwy, Los Angeles CA 90089-0371, USA

a r t i c l e

i n f o

Article history: Received 16 September 2009 Received in revised form 2 December 2009 Accepted 3 December 2009 Keywords: Cost of inbreeding Factorial cross Full-sib mating Inbreeding depression Larval growth Mutational load

a b s t r a c t Effects of inbreeding and crossbreeding in the purple sea urchin Stronglyocentrotus purpuratus were examined by means of a controlled factorial cross of adult urchins from two full-sib families, which produced inbred (f = 0.25) and crossbred offspring (f = 0). Larvae were reared in two different culture systems: static 20-l bucket cultures and replicated 8-l buckets in a shared flow-through water system. The square root of larval area, measured by image analysis, was taken as a measure of size at one and two weeks of age. Linear models explained 60–80% of size variance in these experiments. Inbred larvae were significantly smaller and had greater coefficients of size variance than crossbred larvae, in both systems and at both time points. At week 1, the worst-performing crossbred family in the 8-l system was 33 μm greater than the best inbred family (P ≪ 0.001); at week 2, the worst-performing crossbred family was 28 μm greater than the bestperforming inbred cross (P ≪ 0.001). The cost of inbreeding, δ/f, at week 1, was 1.0 and, at week 2, 0.8, suggesting severe inbreeding depression; the number of detrimental equivalents for larval size ranged from 0.89 to 1.32, with an average dominance of 0.06. These results, together with previous evidence for inbreeding depression of larval survival, suggest that the purple sea urchin has a large load of recessive deleterious mutations and that inbreeding and inbreeding depression could pose significant risks for hatchery-based stock enhancement or aquaculture programs, as well as for declining natural populations. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Inbreeding depression, the reduced fitness in inbred relative to random-bred individuals, and its counterpart, hybrid vigor (heterosis), the superiority of hybrids produced by crosses of inbred parent lines, have long been recognized in agriculture and evolutionary biology, yet the genetic bases of these phenomena remain poorly understood (Husband and Schemske, 1996; Charlesworth and Charlesworth, 1999). Furthermore, the extent of these phenomena in marine organisms, particularly marine invertebrates, is very poorly documented. Inbreeding depression may manifest early in the larval cycle and may have drastic effects on fitness in urchins and other highly fecund marine invertebrates, which display high early mortality or Type-III survivorship (Leahy et al., 1994; Reed and Frankham, 2003; Launey and Hedgecock, 2001). In populations of wild urchins, the potential for random inbreeding is thought to be reduced primarily by open population structure and dispersal capability (Knowlton and Jackson, 1993). There is little evidence that inbreeding exists in wild echinoderm populations, except where brooding or parthenogenesis is exhibited (Knowlton and Jackson, 1993). However, events that reduce effective

⁎ Corresponding author. Tel.: + 1 213 821 2091; fax: + 1 213 740 8123. E-mail address: [email protected] (D. Hedgecock). 0022-0981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2009.12.005

population size may have an effect on the potential for inbreeding of broadcast-spawning marine invertebrates (Hedgecock et al., 2007). Britten et al. (1978) provided evidence that the urchin genome contains an extraordinary degree of genetic variation. Thus, two wild urchins paired at random may be represented to be heterozygous at any given locus, ab and cd. The genotypes of their first-generation, G0 progeny is the set {ac, ad, bc, bd}. Random full-sib matings of the G0 generation creates homozygous G1 genotypes identical by descent in 12 of the possible 16 pairings. The inbreeding coefficient, f, of progeny from a full-sib mating, the probability that the two alleles in the offspring are identical by descent (IBD), is 0.25 (Falconer and Mackay, 1996). Homozygous IBD genotypes resulting from inbreeding may lead either to lethal or sub-lethal phenotypes. Launey and Hedgecock (2001) examined the departures at microsatellite DNA markers of observed from expected genotypic ratios in F2 families of the Pacific oyster Crassostrea gigas, and inferred a large load of recessive lethal alleles to be responsible for mortality of IBD genotypes. Although randomly bred sea urchins typically show 60–90% survival through the larval period, inbred larvae produced either by one or two generations of full-sib mating or by selfing (f = 0.25, 0.375, or 0.5) showed survival rates that ranged from b1% to 100% (average 59%) of paired non-inbred controls and elevated levels of developmental arrest, which would likely have led to mortality at metamorphosis (Leahy et al., 1994). In this study, we examine inbreeding depression for larval size rather than larval survival.

D. Anderson, D. Hedgecock / Journal of Experimental Marine Biology and Ecology 384 (2010) 68–75

Purple urchins have a larval cycle that is roughly 30 days long, progressing through a series of stages that precede metamorphosis and settlement. Urchin size and shape change continuously during this time as the juvenile urchin develops within the larva. The rudiment, as the developing juvenile is known, increases in size until the larva settles and begins its life as a benthic organism. Arm and body length are known to be inversely proportional to feed quality and availability during this time (Leighton et al., 1993). Overall body size and shape, however, is influenced by the fullness of the stomach and the size of the developing rudiment. Exactly how larval size may influence overall fitness is unknown, but it may be intermediate between life history traits and morphological traits, which do not exhibit as much inbreeding depression as traits directly related to fitness, such as fecundity or survivorship (Falconer and Mackay, 1996; DeRose and Roff, 1999). In urchins, it is reasonable to assume a relationship between larval size and larval health, but the connection has not yet been demonstrated experimentally. 2. Methods

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or 500 ml (20-l cultures), density of larvae was measured by counting three samples of known volume (0.1 ml or 1.0 ml). Samples taken for larval size measurements contained 96 larvae per cross, per time point. Larvae were placed on a Lovin Field Finder slide and a digital micrograph of each larva was recorded using an Olympus SZ11 microscope and Canon A95 camera. Every effort was made to orient larvae in the same position on the slide so that alternate perspectives would not affect the image analysis. Digital images were analyzed with ImageJ, using the slide markings to calibrate the measurement of each image. Although several measurements were recorded (including Feret's diameter, circularity, etc.), the area of larval image, in µm2, was used for this study, as trait Area. 2.4. Statistical analyses 2.4.1. Transformation of raw data Area was examined, by cross, culture volume, and week (16 cases total), for conformation to the normal distribution, using Kolmogorov– Smirnov (D) and Shapiro–Wilk (W) tests. Square root transformation of area (sqrtA), hereafter called Size (in μm), was made to improve fit to normal distributions.

2.1. G0 generation Mature Stronglyocentrotus purpuratus urchins were collected from Isthmus Reef, Catalina Island (33° 26′ 54″ N, 118° 29′27″ W) in January, 2005, and taken to the USC Wrigley Marine Science Center for the creation of G0 families. These wild urchins were induced to spawn with a 1.0 M KCl solution injected through the peristomal tissue into the gonads. To create each family, one male and one female were paired at random and their gametes were combined and distributed into separate 20-l polycarbonate buckets. Buckets were fitted with mechanical paddle agitators and placed in a temperature-controlled room at 15°–18 °C. Water changes were made every other day using 1-μm filtered, UV sterilized seawater. On day 3, algal feed (Isochrysis galbana) was introduced in concentrations of 30 × 103 cells/ml. Feed and water were replaced every other day until day 20, when benthic diatoms were introduced to encourage settling. At this time, classic indications of metamorphic competence were observed: squared-off bell, visible rudiment, ciliated bands, and noticeable increase in larval size. A large amount of variation in size and shape was noted among the families, but no measurements of size were made of the G0 larvae. All cultures settled by day 33. The G0 families were maintained through sexual maturity in isolated aquaria with flow-through seawater. 2.2. Inbred and crossbred larvae Of the nine families produced in 2005, families 2 and 3 were used in this study. The larval progeny in this study are referred to by the parental cross, dam × sire. In 2007, one female and one male urchin (identified after spawning induction) from each of two families were used in a factorial cross to produce the two G1 inbred (2 × 2, 3 × 3) and two crossbred (2 × 3, 3 × 2) families for this experiment. Gametes were mixed in equal proportions for all crosses and fertilized eggs were stocked into both 20-l and 8-l buckets. Cultures were maintained in 20-l buckets, as for the G0 cultures, without replication. The 8-l cultures were connected to a 150-l system, in which water was recirculated from a common sump into the replicate culture vessels. Algae and filtered seawater were added continuously into the sump, to ensure that each culture tank received the same culture conditions. Six replicates were used for each of the four families in this 8-l bucket system, although as few as two replicates remained for some families by the second week. 2.3. Measurements of body size Sampling of larvae proceeded by passing cultures through a screen for collection and concentration. Once concentrated into 40 ml (8-l cultures)

2.4.2. ANOVA of Size Analyses of variance were conducted to test for differences in mean Size among the four crosses, in the two culture systems, at the two time points sampled. Separate tests were performed on week-1 and week-2 data. Analyses were conducted with SAS statistical software (version 9.1). The model for measurements in the 8-l culture system was Size = Cross + Rep(Cross) + e, where Size is the square root of the area of the larval image in μm2, Cross is the family (2 × 2, 2 × 3, 3 × 2, or 3 × 3), and Rep(Cross) is the replicate culture vessel, considered as nested within the family, since these were randomized within the system. Cross and Rep were considered as random variables and the Rep(Cross) mean square was used to test the significance of Cross. For the 8-l cultures, causal components of variance were calculated for week 1 as Rep (Cross) = Var(Error) + 16 × Var(Rep(Cross)) and Cross = Var(Error) + 16 × Var(Rep(Cross)) + 96 × Var(Cross) and for week 2 as Rep(Cross) = Var(Error) + 24.8 × Var(Rep(Cross)) and Cross = Var(Error) + 34 × Var (Rep(Cross)) + 96 × Var(Cross). The model for measurements taken in the 20-l culture system was simply Size = Cross + e. For both weeks, causal components of variance were calculated as Cross = Var (Error) + 96 × Var(Cross). Potence (Griffing, 1990), hp = Q/L, where L is the linear contrast in Size between the larger and smaller of the inbred parent lines and Q is the quadratic contrast, twice the deviation of crossbred Size from the mean of parental values (the mid-parent value), was calculated from estimated family means. The significance of heterosis, defined as hp N 1.0, was determined by a post hoc contrast of each crossbred with the best parent; for the 8-l data, Rep(Cross) mean square was used to test this contrast. 2.4.3. Inbred vs. crossbred comparisons Estimates of mean and variance of larval size at week 1 for crossbred (inbreeding coefficient, f = 0) and inbred (f = 0.25) families were used to compare phenotypic variability (Coefficient of Variance) and to calculate an index of inbreeding depression, δ = 1 − (Si /So) (Lande and Schemske, 1985), where Si is the mean Size of inbred larvae and So is the mean Size of crossbred larvae. The standardized cost of inbreeding, δ/f, which is the inbreeding depression expected of a completely inbred population (f = 1) was also calculated for comparative purposes (Crnokrak and Roff, 1999). Data on the sizes of inbred and crossbred larvae were also used to estimate the “detrimental equivalents” per gamete, following Lynch and Walsh (1998; equations 10.21a and b, p. 278), as a number between B and A +B, where A = −lnS0 is the sum of probabilities of reduced growth in random-mating populations and B = −ln(Sf /S0) /f is the excess sum of probabilities for reduced growth in a completely inbred population. Here, S0 and Sf are Size for crossbred and

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inbred larvae re-scaled as a proportion of maximum size in either culture system, by Si = 1 − [(Max(Size) −Size) / Max(Size)]. Variance of B was calculated according to Lynch and Walsh (1998; equation 10.22). The harmonic mean of the dominance coefficients among newly arising detrimental mutations was estimated as ≤A / 2(A +B) (Lynch and Walsh, 1998, equation 10.27).

Larvae from these families were reared in two culture systems, one comprising replicate 8-l vessels sharing the same water system and the other a set of four separate, non-replicated 20-l containers, and sampled at one and two weeks post-fertilization for measurement of size. Density was not controlled in these culture systems and ranged, at week 1, from 0.1 to 4.85 larvae per ml over 8-l cultures and from 9.1 to 11 larvae per ml over 20-l cultures.

3. Results 3.2. Measurements of body size and normality of data 3.1. Production of inbred and crossbred families Full-sib families of the sea urchin S. purpuratus were successfully established by crossing pairs of wild males and females. These sea urchin families were reared to maturity in isolation, and two families, 2 and 3, provided parents at two years of age for a factorial cross, which yielded first-generation inbred families, 2 ×2 and 3 ×3, having an expected inbreeding coefficient of 0.25, and reciprocal crossbred families, 2 ×3 and 3 ×2, which have an inbreeding coefficient of zero and are no different than any randomly bred sea urchin in the natural population.

Area was significantly non-normal in nine cases, by the Kolmogorov– Smirnov (D) test, and in four cases, by the Shapiro–Wilk (W) test. Square root transformation of area (sqrtA), hereafter called Size (in μm), improved fit to normal distributions (example of 2 ×2 in Fig. 1A and Table 1). Size was significantly non-normal in only four of 16 cases, by the D statistic, and in only two cases, by W. Density plots of Area and Size for the two cases significant by both tests—family 2 ×3, at week 1 in the 8-l culture system, and family 3 ×3, at week 1 in the 20-l culture system—revealed multimodality as the cause of poor fit to the normal

Fig. 1. Density curves (solid lines) of Area (left column) and Size (right column), for larvae of the sea urchin Stronglyocentrotus purpuratus, together with fitted normal distribution (dotted lines), in (A) the 2 × 2 family in 8-l cultures at week 1, (B) the 2 × 3 family in 8-l cultures at week 1, and (C) the 3 × 3 family in 20-l culture at week 1. Size is a better fit to the normal distribution than Area in (A) but not in (B) or (C), because of multimodality, owing to underlying variance among replicate cultures (B) or developmental stages (C, see Fig. 2).

D. Anderson, D. Hedgecock / Journal of Experimental Marine Biology and Ecology 384 (2010) 68–75

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Table 1 Fit to normal distributions of Area and Size (square-root Area). Cross

Vol.

Trait

Mean

SD

Sk

Ks

D

W

PD

PW

2×2 2×2 2×3 2×3 3×3 3×3

8 8 8 8 20 20

Area Size Area Size Area Size

15,997.1 125.135 29,926.2 172.527 27,338.9 164.634

4787 18.49 4290 12.74 4915 15.40

0.61 0.31 − 0.28 − 0.76 − 0.44 − 0.71

− 0.18 − 0.49 1.75 3.19 0.17 0.38

0.088 0.064 0.092 0.107 0.087 0.107

0.963 0.983 0.972 0.952 0.971 0.953

0.064 N 0.15 0.046 b 0.01 0.071 b 0.01

0.009 0.243 0.040 0.002 0.029 0.002

Measurements at week 1 of larval culture give one example (2 × 2), in which transformation succeeded in normalizing the data, and the two cases, in which it did not, by both Kolmogorov–Smirnov (D) and Shapiro–Wilk (W) test statistics (see Fig. 1). Skewness and kurtosis are also given. PD is the probability of observing a more extreme D statistic; PW is the probability of observing a more extreme statistic W statistic.

distribution (Fig. 1B, C; statistics in Table 1). For 2 ×3 in the 8-l culture system at week 1, this multimodality could be attributed to differences in growth among replicate cultures; seven of 14 individuals, with Size b 160 μm, were from one replicate. Replicate is an explicit source of variance in the ANOVA model (see below), permitting separation of variance among replicates from variance among families. For the one 20-l culture of 3 ×3, multimodality could be attributed, in the week-1 sample, to the presence of prism stages, 4-arm, pluteus stages and abnormally developing larvae, which vary in shape and size (Fig. 2). Cross classification of this sample, by size (b=150, n = 17 and N150, n = 79) and by stage (prism, 4-arm, abnormal), yields {10, 2, 5}, for the smaller larvae, and {0, 78, 1}, for the larger larvae, a highly significant contingency of stage on size (χ2 = 76.9, P = 0.0, 2 df). Bimodality caused by the mixture of development stages, however, does not obscure the greater differences in Size among families. Thus, the variable Size was used to examine variance in size-at-age among the inbred and crossbred families produced by the experimental cross.

3.3. Growth of inbred and crossbred sea urchin larvae in experimental culture systems Crossbred larvae were larger than inbred larvae in both culture systems and at both time points (Fig. 3). Indeed, crossbreds were larger than the larger of the parental inbred lines in all cases. The rank order of families by size was 3 ×2 N 2 ×3 N 3 ×3 N 2 ×2 in all but the 20-l cultures at week 2, in which the ranks of both parental lines and crossbred families reversed relative to each other, though preserving crossbred superiority. Larval size was clearly influenced by genotype in our experiment. Larval size was also affected by the culture system. At week 1, larvae in the 20-l containers were larger than those in the 8-l containers (grand means of 181 vs. 154 μm, respectively), despite greater mean densities in the 20-l vessels (9.7 vs. 1.5 larvae per ml, respectively). In the following week, however, growth in the 20-l vessels was slower than that in the 8-l vessels, so that grand mean larval size at week 2 was 192 in the 20-l vs. 198 μm in the 8-l cultures. A higher mortality rate in the

Fig. 2. Photographs of one-week old larvae from the 20-l culture of family 3 × 3. (A) A 4-arm stage, 178 μm Size; (B) a prism stage, 134 μm Size; (C) an abnormally developing larva, 147 μm Size; and (D) a 4-arm stage, 148 μm Size. No prism stages were observed in larvae N150 μm Size.

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Fig. 3. Vertical box and whisker plots of Size (in μm) of larvae from two inbred (2 ×2 and 3 ×3) and two crossbred families (2 ×3 and 3 ×2) of the sea urchin Stronglyocentrotus purpuratus. Samples of 96 larvae were taken and measured at weeks 1 and 2, in 8-l and 20-l culture systems. The top and bottom of each box are the 75th and 25th quantiles, respectively; the horizontal solid line bisecting the box is the 50th quantile or median, and the dotted horizontal line is the mean. The whisker caps denote the 10th and 90th percentiles and data points lying outside of that interval are shown by filled circles. Crossbred mean size is significantly above the mean of the largest parent line (heterosis) in all conditions.

20-l compared to the 8-l vessels during the second week of larval culture was evident in declines of average densities between weeks one and two (54% in 8-l vessels; 31% in 20-l vessels). 3.4. Analysis of larval size in 8-l cultures Families were reared in replicate 8-l larval culture vessels, allowing error among replicates within cross to be used to test the significance of differences among the crosses (Table 2). The linear models of Size = Cross + Rep(Cross) are highly significant and explain 80% and 74% of variance in larval size at weeks 1 and 2, respectively. At both time points, Size varies significantly among replicates (see Fig. 1B; multimodality in the pooled density curve of Size for the 2 × 3 family is caused by variation among replicates), but the differences among families is much greater (cf. 2 × 2 with 2 × 3 in Fig. 1A and B, respectively; Fig. 3). Indeed, all crossbred replicates are larger than all inbred replicates at week 1, and at week 2, there is only a single overlap, of less than a micrometer, between the mean sizes of the largest replicate of 3 × 3 and the smallest replicate of 2 × 3. Crossbred Table 2 ANOVA tables for Size (square root of larval area, in μm) in 8-l cultures of Stronglyocentrotus purpuratus larvae. Source

df

a. Size at week 1 Cross 3 Rep(Cross) 20 Error 360 2

r 0.797 b. Size at week 2 Cross 3 Rep(Cross) 10 Error 370 r2 0.743

SS

MS

F

P

%V

201,584 26,817 58,342

67,195 1340.83 162.06

50.11 8.27

b0.0001 b0.0001

74.4 8.0 17.6

CV 8.24

RMSE 12.73

409,812 46,072 157,826

136,604 4607.23 426.56

CV 10.45

RMSE 20.65

Mean 154.46 22.18 10.8

0.0001 b.0001

69.5 8.6 21.8

Mean 197.68

The factor Cross is tested with MS Rep(Cross). The percentage contribution of each factor to summed causal components of variance is given in the last column.

and inbred larvae differ on average by 44.3 μm and 58.0 μm at weeks 1 and 2, respectively, which represent differences of 3.48 and 2.80 standard deviations (see root mean square errors, Table 2). Cross accounts for 74.4% and 69.5% of variance, at weeks 1 and 2, respectively (Table 2a, b).

3.5. Analysis of larval size in the 20-l cultures Families were not replicated in the 20-l culture system, so this experiment had less power to detect differences in Size among families. One-way ANOVAs nevertheless yield highly significant F statistics for Cross (P b 0.0001), both at week 1 and week 2, and explain 71% and 59% of variance in Size, respectively, for those two time points (Table 3). The pattern of crossbred size exceeding that of inbred size is again evident, despite the reversal in the rank orders of parent inbred lines and reciprocal crossbreds at week 2. In this culture system, the differences between crossbred and inbred larvae, 40.1 μm and 37.7 μm at weeks 1 and 2, respectively, are less than in the 8-l system but still amount to differences of 2.95 and 2.17 standard deviations (see root

Table 3 ANOVA tables for Size (square root of larval area, in μm) in 20-l cultures of Stronglyocentrotus purpuratus larvae. Source

df

a. Size at week 1 Cross 3 Error 380 r2 0.706 b. Size at week 2 Cross 3 Error 380 r2 0.590597

SS

MS

F

P

%V

169,138.3 70,322.96

56,379.45 185.06

304.65

b 0.0001

76.0 24.0

b 0.0001

65.4 34.6

CV 7.51 164,867 114,286.3 CV 9.028223

RMSE 13.60 54,955.68 300.75 RMSE 17.34224

Mean 181.12 182.73

Mean 192.0892

The percentage contribution of each factor to summed causal components of variance is given in the last column.

D. Anderson, D. Hedgecock / Journal of Experimental Marine Biology and Ecology 384 (2010) 68–75

mean square errors, Table 3). Cross accounts for 76.0% and 65.4% of variance, at weeks 1 and 2, respectively (Table 3a, b). 3.6. Post hoc size-contrasts among inbred and crossbred families Although our experimental crosses were between G0 siblings of wild parents rather than divergent inbred lines, we use potence to express and test differences between inbred and crossbred families. Potence for Size, the ratio of the quadratic to linear contrasts among inbred and crossbred larvae, ranged from 2.82 to 13.43, over the four comparisons afforded by measurements from two culture systems, at two time points (Table 4). In crosses between inbred lines, heterosis is defined by hp N 1.0, which occurs when the hybrid is better than the better parent; the contrast between a hybrid and the better parent thus provides a statistical test of heterosis. All potence values for larval size are very highly significant (P b 0.0001), except for h2 × 3 at week 2 in 8-l cultures, for which the associated contrast is significant at the P = 0.017 level. The linear contrast between inbred parent lines is highly significant (P b 0.001) in all four cases (Table 4). Reciprocal crossbreds 2 × 3 and 3 × 2 were significantly different from each other in all comparisons (P b 0.0001; Table 4). The absolute differences between reciprocal crossbreds, 8.2 μm and 29.3 μm in the 8-l system, at weeks 1 and 2, respectively, represent a difference of 0.6 and 1.41 standard deviations at these two time points (Table 2). In the 20-l system, differences between reciprocal crossbreds, 15.9 μm and 20.4 μm, are 1.17 standard deviations (Table 3). 3.7. Comparison of inbred vs. crossbred larvae and the number of detrimental equivalents Grouping families into crossbred (f = 0) and inbred (f = 0.25) types, we compare the variability of size, expressed as the coefficient of variation (CV, Table 5A). The CV for Size of inbred larvae is nearly twice as large as that for crossbred larvae in the 8-l cultures and is 70% larger than that for crossbred larvae in the 20-l cultures. The greater variation in inbred compared to crossbred larval size is also evident in the box plots, as greater inter-quartile ranges (box height) and greater numbers and ranges of outliers (Fig. 3). Finally, data on larval size for experimental crossbred and inbred populations of sea urchin larvae permit calculations of the cost of inbreeding, δ/f = 0.25, where δ measures inbreeding depression, and the detrimental equivalent number of alleles per gamete that affect larval size or growth in the sea urchin. The cost of inbreeding estimated from 8-l cultures is 1.0 and from 20-l cultures, 0.8 (Table 5A). To Table 4 Means, contrasts, and potence values for larval Size in inbred and crossbred families of the sea urchin Stronglyocentrotus purpuratus reared in 8-l and 20-l cultures and sampled at one and two weeks postfertilization. Estimatea

8-l, week 1

8-l, week 2

20-l, week 1

20-l, week 2

2×2 2×3 3×2 3×3 L Q2× 3 Q3× 2 h2× 3 h3× 2 R

125.13 172.53 180.71 139.49 14.35 80.43 96.81 5.60 6.75 8.19

153.26 212.05 241.32 184.08 30.82 86.76 145.30 2.82 4.71 29.27

157.47 193.24 209.12 164.63 7.16 64.37 96.13 8.99 13.43 15.88

179.86 221.15 200.72 166.62 13.23 95.83 54.97 7.24 4.15 20.43

a L is the difference between larger and smaller inbred parent lines; Q is twice the deviation of the crossbred indicated by the subscript from the average or mid-parent value for the two parental lines; h is the potence, Q/L, for the crossbred indicated by the subscript (significance determined by contrast with best parent and tested with the Rep (Cross) mean square for the 8-l cultures and with error for the 20-l cultures. All h are significant at P b 0.0001, except h2 × 3 at week 2 in the 8-l cultures (P = 0.017). R is the difference between reciprocal crossbreds (all significantly different than zero, at P b 0.0001).

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Table 5 Comparisons of size variability, cost of inbreeding, and detrimental equivalents in crossbred and inbred larvae of the sea urchin Stronglyocentrotus purpuratus. A. Means and coefficients of variance in Size Culture system

Size

Crossbred f = 0

Inbred f = 0.25

8-l

n Mean CV n Mean CV

192 176.62 7.27 192 201.18 6.21

192 132.31 14.13 192 161.05 10.54

20-l

δ/0.25 1.0

0.8

B. Detrimental equivalentsa Culture system

A

B

Var(B)

h

8-l 20-l

0.16 0.13

1.16 0.89

0.06 0.05

0.06 0.07

a A is the sum of probabilities of alleles detrimental to larval growth in the randommating population; B is the excess sum of probabilities of alleles detrimental to larval growth in a completely inbred population and Var(B) is its variance; h is the harmonic mean dominance of newly arising mutations affecting larval growth (see Methods).

estimate detrimental equivalents, size is scaled relative to maximum Size and the sum of probabilities of detrimental alleles in non-inbred and completely inbred populations is calculated (Table 5B). In 8l cultures, the detrimental equivalent is estimated to be between 1.16 (B) and 1.32 (A +B), and in the 20-l cultures, the detrimental equivalent is between 0.89 and 1.02. The average dominance of newly arising mutations affecting larval growth is quite low, 0.06, suggesting that most detrimental mutations are recessive. 4. Discussion Inbred and crossbred larvae of the purple sea urchin S. purpuratus were created by a factorial cross of two, full-sib parent families, which were themselves created by random pair-crosses of wild sea urchins. The larval families were reared in 8-l and 20-l culture systems over the first two weeks post-fertilization. Inbred larvae were significantly smaller than crossbred larvae in both systems and at both time points; indeed, there was very little overlap in size between inbred and crossbred larvae. Linear models of larval size, in which cross and, for 8-l cultures, replicate culture vessels are factors, explain a remarkable 60–80% of size variance in these experiments. Cross comprises 65– 76% of the summed causal components of variance in these experiments, suggesting that most of the inbred vs. crossbred sizedifference has a genetic basis. The sharp decline in larval size, following one generation of full-sib mating, implies strong inbreeding depression for larval growth. The substantial increase in the coefficient of variation in the sizes of inbred compared to crossbred larvae, as observed in comparisons of inbred and hybrid larval growth in the Pacific oyster (Curole and Hedgecock, 2007), also suggests a classical breakdown in homeostasis of inbred growth physiology (Lerner, 1954; Pace et al., 2006). Calculations of the cost of inbreeding suggest that larval growth in a completely inbred population would be reduced by 80–100% of normal growth in randomly bred larvae. Gametes from wild sea urchins are estimated to carry about one detrimental equivalent, which could be one mutation with highly detrimental effects on larval growth or any number of less deleterious mutations, having the same cumulative effect on larval growth. These mutations are nearly completely recessive, with an estimated dominance of only 0.06. This study thus provides the first evidence that the purple sea urchin carries a large load of recessive detrimental mutations affecting larval growth. The magnitude of inbreeding depression observed in our experiment agrees generally with inbreeding depression estimated for captive and wild populations of plants and animals, including two marine invertebrates. Leahy et al. (1994) measured larval survival and

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developmental arrest at two to three weeks post-fertilization for inbred and paired random-bred purple sea urchin larvae. From their data, we calculate, for larvae produced by one generation of full-sib matings (f = 0.25), that average costs of inbreeding (δ/f) for survival and developmental arrest are 0.78 and 0.98, respectively. For larvae resulting from two generations of full-sib mating (f = 0.375), δ is nearly the same as that for one-generation sib-mated larvae but costs of inbreeding are lower—0.54 and 0.62, respectively—because of the greater level of inbreeding. Costs of inbreeding based on survival of larvae from three self-fertilizations (Leahy et al., Table 2) range from 0.94 to 1.25. Evans et al. (2004) reported data on inbreeding depression in the Pacific oyster C. gigas for yield and its components, survival and growth, at 157 and 570 days of age; from their data, we calculate costs of inbreeding on yield of 0.74 and 1.21 for these two time points. In a meta-analysis of the literature on inbreeding depression in wild populations, Crnokrak and Roff (1999) found that mean inbreeding depression, δ, “ranged from 0.2 in poikilotherms to 0.51 in homeotherms” and that, “when corrected for F [i.e. the cost of inbreeding, δ/f], mean inbreeding depression for all estimates ranged from 0.55 in plants to 0.82 in homeotherms.” These estimates for natural populations exceed those reported for captive populations (e.g. Ralls et al., 1988; Roff, 1997) and, collectively, lend support to the hypothesis that inbreeding depression is more severe in nature than in captivity. Our estimates of detrimental equivalents, 0.89 to 1.32 are also within the range of 0.5 to 3 lethal equivalents per gamete reported for vertebrates (Lynch and Walsh, 1998, Table 10.4). Our study may have over- or underestimated inbreeding depression in the purple sea urchin. First, inbreeding depression tends to be larger for traits closely related to fitness, such as survival and fecundity or fertility, and smaller for traits not closely related to fitness, such as morphological traits (Falconer and MacKay, 1996; DeRose and Roff, 1999). The trait measured in this study, larval size, is indirectly related to fitness, so that our estimate of inbreeding depression for this trait may underestimate inbreeding depression and mutational load for larval survival, for example. Indeed, if one considers the developmental arrest observed by Leahy et al. (1994) as resulting in eventual mortality, the combined cost of inbreeding for survival plus arrest is 1.55, which is 55–94% greater than the costs of inbreeding that we estimate for larval size. Recent marker-based studies in oysters have revealed a large mutational load for early survival, with lethal equivalents per gamete ranging as high as 12 or more (Bierne et al., 1998; Launey and Hedgecock, 2001). If these large mutational loads are a by-product of high fecundity, as hypothesized by Williams (1975), then we might expect to find still higher genetic loads for early survival in the highly fecund sea urchin. On the other hand, we have measured inbreeding depression under artificial, not wild culture conditions. Although the literature suggests that inbreeding depression is ameliorated by captive conditions optimized for survival and growth, especially compared to harsh natural conditions, inbreeding depression in artificial environments can be biased upwards by poor husbandry (Crnokrak and Roff, 1999). That average densities declined in week 2 to 54% of week 1 densities in 8-l cultures and 31% of week 1 densities in 20-l cultures suggests that the rearing conditions used in this study may not have been ideal (Leahy et al., 1994). Finally, we have measured inbreeding depression in only two families, which is not sufficient for determining the average inbreeding depression and detrimental equivalents for the purple sea urchin. Nevertheless, our study has likely underestimated the extent of mutational load in the sea urchin and, consequently, the severity of inbreeding depression that might be expected in cultured populations or natural populations subject to decline from overfishing or changing ocean conditions. Certainly, the precautionary principle (UNEP, 1992) would lead one to factor inbreeding depression into calculations of extinction risk. Sea urchin populations worldwide are heavily exploited and sea urchin culture, either for stock enhancement or for aquaculture, is expanding (Andrews et al., 2002; Micael et al., 2009). The results of

this study suggest that inbreeding and inbreeding depressions could pose significant risks in hatchery-based stock enhancement programs (Hedgecock and Coykendall, 2007) or in the development of closed aquaculture stocks (Hedgecock et al., 1992). On the other hand, there may be considerable potential, as in oysters (Hedgecock et al., 1995; Hedgecock and Davis, 2007), for exploiting growth heterosis in the domestication and improvement of cultured sea urchin stocks. Although growth potence values measured in this study, ranging from 2.82 to 13.43, were obtained in a comparison of crossbred and inbred lines and are, strictly speaking, more a reflection of inbreeding depression than heterosis, strong heterosis is likely to be obtained in crosses among inbred lines. 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