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(2004) 93, 51–61 & 2004 Nature Publishing Group All rights reserved 0018-067X/04 $30.00 www.nature.com/hdy Effect of captivity on genetic variance for five traits in the large milkweed bug (Oncopeltus fasciatus)

KM Rodrı´guez-Clark1 Department of Ecology and , Princeton University, Princeton, NJ 08544, USA

Understanding the changes in genetic variance which may generations in captivity. Analyses reveal significant heritable occur as move from nature into captivity has variation for some life history and morphological traits in been considered important when populations in captivity are both environments, with comparable absolute levels of used as models of wild ones. However, the inherent across all traits (0–30%). Randomization tests significance of these changes has not previously been show that while changes in and total phenotypic appreciated in a conservation context: are the methods variance were highly variable, additive genetic variance and aimed at founding captive populations with diversity evolvability remained stable across the environmental representative of natural populations likely also to capture transition in the three morphological traits (changing 1–2% representative quantitative ? Here, I investi- or less), while they declined significantly in the two life-history gate changes in heritability and a less traditional measure, traits (5–8%). Although it is unclear whether the declines evolvability, between nature and captivity for the large were due to selection or gene-by-environment interactions milkweed bug, Oncopeltus fasciatus, to address this ques- (or both), such declines do not appear inevitable: captive tion. Founders were collected from a 100-km transect across populations with small numbers of founders may contain the north-eastern US, and five traits (wing colour, pronotum substantial amounts of the evolvability found in nature, at colour, wing length, early fecundity and later fecundity) were least for some traits. recorded for founders and for their offspring during two Heredity (2004) 93, 51–61. doi:10.1038/sj.hdy.6800479

Keywords: evolvability; heritability; morphological; life-history; captivity vs nature; captive breeding

Introduction other things, determines a ’s ability to persist upon reintroduction (Bu¨rger and Lynch, 1995). The presence of heritable variation is a basic prerequisite Genetic variation available for response to selection for evolutionary response to on a trait. has been measured traditionally as narrow-senseÀÁ herit- Understanding how this variation changes when popu- 2 2 ability, h , the ratio of additive genetic variance sA to 2 lations are brought from nature to captivity is important total phenotypic variance (sP), itself composed of for two primary reasons. First, it is difficult, except in 2 additive genetic, nonadditive geneticÂÃ (sI ), and environ- rare circumstances (eg, Milner et al, 2000), to obtain the 2 2 2 2 2 mental (sE) components: sA= sA þ sI þ sE (Falconer information needed for quantitative genetic studies on and MacKay, 1996). Decomposed in this way, it is free-living populations. Thus, studies are commonly perhaps unsurprising that extensive study has failed to made on populations in captivity and then extrapolated find consistent patterns of change from nature to to make inferences about evolutionary forces in nature; captivity in this complex measure. Heritability may the circumstances under which this extrapolation may be increase upon captivity simply because environmental valid have been examined extensively (see Weigensberg components are reduced in more controlled conditions and Roff, 1996 for review). (eg, Simons and Roff, 1994) and may decrease in nature A second reason for examining changes across the due to seasonal or annual environmental variation (eg, nature-captivity transition, which has not been considered Sgro´ and Hoffmann, 1998). Similarly, it may remain previously, is that threatened and endangered species are stable if both environmental and genetic variance change increasingly recommended for captive breeding (Balm- proportionally (eg, Blanckenhorn, 2002). ford et al, 1996). Because such populations may remain in A less traditional but perhaps better measure for captivity for extended periods, and may become the only assessing changing capacity to respond to selection source for population recovery in the future, it is of across a variety of traits may instead be evolvability, inherent interest to understand how genetic variation the coefficientqffiffiffiffiffiffi of additive genetic variation, might change upon captivity, since such variation, among 2  CVA ¼ 100 Ã sA=X, which depends only on additive genetic variance, standardized by the trait mean, X (Houle, 1992). At least three nonexclusive processes may Correspondence: KM Rodrı´guez-Clark, Department of Ecology and influence changes in evolvability from nature to captiv- Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA. ity: drift, selection and gene-by-environment interaction. E-mail: [email protected] 2 1Current address: Centro de Ecologı´a, Instituto Venezolano de Investiga- Classic infinitesimal theory holds that sA should change ciones Cientı´ficas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela. in proportion to gene diversity, GD (equivalent to Received 21 March 2003; accepted 15 December 2003 expected heterozygosity; Falconer and MacKay, 1996), Effect of captivity on genetic variance KM Rodrı´guez-Clark 52 2 such that no change in sA due to drift is expected unless Asclepias (milkweed) species, O. fasciatus is highly the number of individuals used to found the captive dependent on this patchy and ephemeral resource for population is very small, and/or the population remains reproduction. It is not thought to overwinter in the small in subsequent generations. It is on the basis of this northern parts of its range, with these populations theory that it is often assumed that no more than 20 instead re-establishing every year with migrants from founders (N) are needed for a captive-bred population, continuously reproducing southern ones, leading to since they are expected to sample on average 1-1/(2N), likely panmixia within large geographic regions. This or 97.5% of wild GD, and thus also nearly all wild conclusion is supported by studies that have found additive genetic variance (Lacy, 1994). If additive quantitative and molecular genetic differentiation within 2 assumptions do not hold, however, and sI is large, the species only at the scale of thousands of kilometres sampling-induced changes in frequencies may in- (Leslie and Dingle, 1983; Leslie, 1990). Low temperatures, 2 flate sA, causing increases in evolvability (Willis and Orr, food limitation and short day lengths all induce flight 1993 and references therein). restlessness and reproductive diapause in northern A second process affecting changes in evolvability in populations, suggesting that some late-autumn northern nature vs captivity may be selection. Hedrick (1986) has residents may escape frosts by flying south (Rodrı´guez- argued that environmental heterogeneity is responsible Clark, 2002 and references therein). Wing length, the first for maintaining most polymorphic loci in natural trait examined in this study, appears to be an important 2 populations, such that higher sA is expected in the wild, character in this migratory syndrome, as it is genetically where it is maintained by heterogeneous selection. On correlated with flight behaviour in northern populations the other hand, if populations are under mostly (Dingle and Evans, 1987). 2 balancing selection in nature, sA may be expected to Another category of trait examined, fecundity, is also a increase in the permissive conditions of captivity, as part of this migratory syndrome. Unlike many insect selection-induced dissipates species, female O. fasciatus will produce large numbers of and/or accumulate which would have other- (infertile) eggs even if virgin, which is thought to allow wise been selectively removed (Prout and Barker, 1989). females to take advantage of patchily distributed mates However, both balancing and directional selection in and food as quickly as possible. Once females arrive at a captivity (‘domestication’) may also cause rapid and suitable host, they deposit clutches of B30 eggs per day 2 large losses of sA (Rodrı´guez-Clark, 2002). (up to 2000 or more total) in protected crevices on the Finally, gene-by-environment interactions have been plant (Rodrı´guez-Clark, 2002 and references therein). hypothesized to play an important role in determining Colour comprises the final category of trait examined. 2 immediate changes in sA from nature to captivity. As with many other milkweed specialists, O. fasciatus 2 Specifically, it has been suggested that greater sA may sequesters the plant’s poisonous cardiac glycosides and be expressed in more ‘stressful’ conditions, as would be has a contrasting black and coloured aposematic pattern characteristic of nature, allowing for rapid response to effective against both vertebrate and invertebrate pre- selection challenges there. Although the mechanisms dators (Evans, 1988). The gregariousness of this species underlying this stress-dependence hypothesis remain through the adult stage may be a further obscure, the findings of numerous studies appear enhancing the aposematic effect. Colouring of hard body consistent with this view. Nonadditive genetic varia- parts and the hemelytra (forewing) may vary consider- tion has similarly been hypothesized to be at higher ably, from nearly white through yellow and orange to levels in the stressful conditions of nature (reviewed in deep red. This study represents the first to explore the Hoffmann and Parsons, 1991; Bijlsma and Loeschcke, genetic basis of this variation. 1997). General conclusions about changes in evolvability from nature to captivity have been hampered in past Field collections studies by a focus on a few species, on only one trait in This experiment spanned three generations: Generation 0 any given study, or on heritability exclusively as the (wild caught), 1 (lab-reared) and 2 (lab-reared), each of parameter of interest. In the present paper, I address which contained 50 families. Individuals were collected these issues by combining minimum estimates of between 18 and 27 September 1998, from 29 locations heritability in nature and in captivity with estimates of along a 350 km transect within the north-eastern United phenotypic variability in both environments, to estimate States (Figure 1). changes in additive genetic variation and evolvability in The transect was constructed in order to sample five traits, in a well-characterized but underutilized within-subpopulation variation widely while ensuring experimental insect species. unrelatedness among founders, as is recommended for captive populations of endangered species (Lacy, 1994), taking into account the species’ long-distance dispersal Methods and likely panmixia within broad geographic regions. Distance between sampling locations was B15 km, as The study organism permitted by the patchy population distribution. Within The basic data collected consist of three categories of each location, individuals or clusters of bugs were observation – wing length, fecundity and colour – which collected from 2 to 10 common milkweed plants are of particular interest in the context of the natural (Asclepias syriaca), between 100 m–1 km apart. Migrating history of the large milkweed bug. Oncopeltus fasciatus adults tend to stay in a suitable patch once they find one, experiences great environmental variation across its and hatched broods usually stay together on a single natural range, which covers much of North America plant at least until the third instar, with subsequent and the Caribbean. A seed predator specializing on moves of usually less than a meter (Dingle, 1981). Thus,

Heredity Effect of captivity on genetic variance KM Rodrı´guez-Clark 53

Figure 1 Locations across the north-eastern USA from which 50 founding individuals were collected. Points indicate sampling locations with sex ratio of founders used from that location (males.females). Asterisks indicate number of possible pairs captured. bugs found together on a plant or in a patch of plants paintbrush, to provide an excess of individuals for the were conservatively considered to be a ‘sibship’. next generation. Labelled boxes were assigned random Very recently eclosed adults or 5th instars (just prior to locations within the rearing chamber. Upon reaching the sexual maturity) were preferentially collected, so that 5th instar, males and females in each family were sorted most development occurred under natural conditions, and redistributed among their four boxes, maintaining but females were still virgin. Of the hundred wild sexes separate and densities at no more than 10 bugs/ individuals used to form Generation 0, at collection, 25 box. A band of liquid Teflon (Northern Products Inc., were recently eclosed adults, 60 were 5th instar, 11 were Woonsocket, RI, USA) was painted on the inside rim of 4th instar, three were 3rd instar and two were 2nd instar. each box as an additional safeguard against escapes by Presumed sibships were maintained as groups upon smaller instars. Each box was provided with ad libitum capture, with males and females separate. Individuals deionized water in a disposable 37 ml vial with a cotton are easily sexed at both the 5th instar (when females have wick, secured to the base of the box with nontoxic poster two dark spots on their posterior segments, while males gum. have one) and as adults, when females have a caudal Individuals were reared on ripe milkweed seeds from median point on the posterior margin of the 4th a variety of wild sources. Ripened pods were collected abdominal sternite, as well as a triangular cleft pygi- from the vicinity of Princeton, New Jersey, in the dium, unlike the more rounded male structure (Rodrı´- autumns of 1997 and 1998, and seeds were removed guez-Clark, 2002). either by hand or according to methods described by Feir et al (1982). To randomize effects of seed age on nutrition, these fresh seeds were mixed periodically with 41-year- Culture techniques old seed purchased commercially (Valley Seed Service, In general, bugs were maintained in captive conditions Fresno, CA, USA), and older seeds obtained from as close as possible to those typical in the wild during the Missouri and Minnesota. Seeds were stored in airtight season of maximal reproduction: northern latitude containers with indicating silica gel to prevent mould midsummer. Groups were maintained in growth. 9.5 Â 9.5 Â 9.5 cm re-usable polystyrene boxes (Pioneer O. fasciatus from north-eastern populations fed ad Packaging, model 29-C), each with a sheer fabric aeration libitum are indistinguishable in size and survivorship window in the removable lid, which was secured with a from those given 12 seeds per bug from egg to eclosion rubber band. In Generation 0, groups larger than 10 were (Dingle, 1992), equivalent to 0.7 g for 10 bugs. To provide split so that density was never higher than 10 indivi- well in excess of this amount, B1.5 g seed was scattered duals/box, giving each bug at least 82 cm3, well in excess on the bottom of each box. Rearing boxes were kept at of the 35 cm3 minimum necessary to avoid density- 28711C with humidity maintained at 50–70%, on a dependent growth effects (Koerper and Jorgensen, 1984). schedule of 16 h light 8 h dark. These conditions maintain In Generations 1 and 2, once eggs were laid and optimal egg production and minimal probability of weighed (see below), sibships were divided among four diapause, with B27 days between hatch and adult boxes, with 10 eggs placed in each box using a sable metamorphosis, and 10 additional days to first

Heredity Effect of captivity on genetic variance KM Rodrı´guez-Clark 54 reproduction (Groeters and Dingle, 1988). Boxes were kept on clear Plexiglas trays on wire shelving, to maximize light penetration to all groups.

Husbandry The 50 mating pairs in Generation 0 were created by selecting one or two each of mature males and females captured at each sampling location, all from different presumed sibships where possible (never using indivi- duals of the same sex from the same sibship). These 100 individuals were then paired randomly, avoiding possi- ble sib matings. Two adult offspring (one of each sex) from each pair were again chosen arbitrarily to form Generation 1, and were paired randomly, avoiding sib matings, to give rise to Generation 2. If offspring reached maturity prior to separation, females found in a box with males were discarded. All pairs were made at least in quadruplicate to guard against family loss, with one replicate selected randomly to contribute to the next generation. Pairs were placed in plastic Petri dishes, 9 cm diameter  2.5 cm height, with a 14.8 ml disposable water vial with a cotton wick and B20 fresh seeds (0.12 g). The mouth of each dish was covered with 40x32-denier wrinkled cotton gauze secured with a rubber band and the dish lid. Dishes were kept entirely dark, inside Figure 2 (a) Cradle used to immobilize live bugs for microscope cardboard trays with cardboard coverings. Under these photography. (b) Morphological traits measured (WL ¼ wing circumstances, O. fasciatus from northern populations length; PC ¼ pronotum colour; WC ¼ wing colour). deposit eggs almost exclusively through wrinkled areas in the gauze onto the dish lid, allowing eggs to be easily removed (H Dingle, D Feir, personal communication). Repeatability was 94% for wing colour (50 individuals This set-up provided a total laying area of B64 cm2 per measured 10 times each, F ¼ 142, Po0.0001), and 66% for female (more than the 40 cm2 minimum required for pronotum colour (F ¼ 20, Po0.0001). A Meiji TechnoRZ optimal egg production; Koerper and Jorgensen, 1984). zoom stereo microscope (10  magnification) and V-Lux 1000 light source set to maximum were used throughout. Measurement of traits Values for colours and wing length were recorded for Just prior to creating mating pairs, wing length and both parents and eight randomly selected offspring per colour traits were assessed on the right-hand side of all family (four each males and females). individuals, at the same age (30–35 days posthatch) and For individual females, the rate of egg production at least 2 days posteclosion, when the cuticle hardens following mating has been found to be highly correlated and final adult colour and dimensions have stabilized. with the lifetime quantity of eggs produced, as well as Individuals were anaesthetized with carbon dioxide on a average clutch size and total fecundity by weight (Leslie, fritted glass microscope stage, and placed on a custom- 1990). Total mass of eggs laid during two time spans was designed brass cradle, using dental wax to hold wings thus recorded for each wild female, using a modification flat (Figure 2a). Bugs were photographed using a of Palmer and Dingle (1986) and Groeters and Dingle Polaroid Digital Microscope Camera with a glass-etched (1996): (1) from pair creation to 5–6 days afterward, and 2 mm scale bar adjusted to be exactly at the focal plane. (2) from that point to 9–12 days afterward. All eggs were For each picture, X-Y coordinates were recorded for both weighed on glassine paper using a Sartorious R200D ends of the scale bar as well as for the wing tip and the electronic semi-microbalance. Masses were converted proximal posterior wing process, using Scion Image into mg/day production rates (‘early fecundity’, EF and software (2000) on a PC (Figure 2b). Wing length (WL) ‘later fecundity’, LF) by dividing by the number of full was taken as the distance between these two points in days elapsed between mating and first weighing or mm. Repeatability for this trait was 99% (40 individuals between the first and second weighing dates, respec- measured 10 times each; F ¼ 638, Po0.0001). tively. Colours on parts of the pronotum (PC) and forewing Egg mass was considered to be a function of the (WC) (Figure 2b) were assessed by comparing indivi- maternal generation, following Palmer and Dingle duals to a colour bar created freshly from standard (1986), in recognition of the overwhelming likelihood of colour chips each generation. Although colour in this maternal effects in this and other egg characters (Lynch species varies nearly continuously, the range was and Walsh, 1998). Sperm storage in females prevents the divided into a 5-unit scale in order to have enough use of more than one male per female, so male influence classes to treat the data as continuous, but few enough to on the trait could not be separately assessed. Egg mass minimize classification error (1 ¼ light yellow #1215; was recorded for each dam and for four (Generation 1) or 2 ¼ yellow-orange #1225; 3 ¼ orange #116, 2X; 4 ¼ red- eight (Generation 2) of her female offspring at the same orange #130, 2X; 5 ¼ red # 165, 2X; Letraset Ltd., 1998). age.

Heredity Effect of captivity on genetic variance KM Rodrı´guez-Clark 55 Statistical analyses Quantitative genetic parameters based on family 2 2 2 Heritability in captivity, hC ¼ sAC=sPC, is estimated by relationships may be unreliable if there are significant bðMOcÁMPcÞ, the regression of mid-offspring on mid-parent gender differences in the means or variances of traits values, or 2 Ã bðMDcÁDcÞ, twice the regression of mid- under consideration, or if data do not fit the assumptions daughter on dam values, with both generations reared of tests performed (Falconer and MacKay, 1996). Thus, in captivity (Generations 1 and 2) (Falconer and MacKay, prior to conducting the above analyses, all traits were 1996). VPC, an estimate of the total phenotypic variance examined for normality of residuals, with sexes both 2 (sPC), was obtained from the total variance within dams apart and combined, as well as for nonrandom associa- or sires for single-sex estimates, or pooled across sexes tions between means and variances, and were trans- for two-sex estimates. Additive genetic variance in formed as needed (see Results). Differences in mean 2 2 across sexes and generations in colours and length were captivity (sAC) was estimated by VAC ¼ hC Ã VPC. Resi- 2 examined using a 2-way ANOVA, while differences dual variation in captivity (sRC, nonadditive genetic and environmental variance combined) was estimated as across generations in egg mass were examined with a VRC ¼ VPC À VAC, and the coefficient of additive genetic Kruskal–Wallis test. Differences in variance were tested variation (CVA) was estimated as defined in the using Levene’s test (SAS Institute Inc., 2001). Where the Introduction. sexes differed systematically in mean, this was corrected Two consecutive generations were not available in by adding to each member of the smaller sex the average nature, so estimation in this environment is less difference between female and male means (Norry et al, straightforward. Coyne and Beecham (1987), Prout and 1997). Following transformation, all analyses were Barker (1989), and Riska et al (1989) together develop the conducted on both sexes separately to look for sex- relationships among three different estimates of herit- specific effects, before combining sexes for final analyses 2 2 if no differences were found. ability in nature, hN. The first of these, hNV, assumes 2 Because of the complex, composite, nature of many of constancy of sA across environments, and is given by 2 2 the estimated in this study, parametric standard hNV ¼ VAC=VPN, in which VPN estimates sPN, the total phenotypic variance in parents from nature (Generation errors may be either unreliable, cumbersome to calculate, 2 2 and/or simply unavailable (Lynch and Walsh, 1998, p. 0). When sA is lower in captivity than in nature, hNVwill be an underestimate of the true heritability in nature. 560). The distributions of all statistics were thus Using the fact that the regression of captive offspring estimated empirically using the bootstrap, following b Riska et al (1989) and Manly (1997). In all, 50 family on parents from nature ðMOcÁMPNÞ is defined as 2 units (keeping parents with their associated offspring) gsACsAN=sPN (Lande in Coyne and Beecham, 1987), with some rearrangement, the second estimate of heritability were sampled independently with replacement from Generations 0–1 and 1–2 for 2000 replicates. All statistics in nature, g2h2 , is given by N  were calculated for each replicate; bias-corrected para- V g2 2 ¼ 2 PN meter estimates were produced for the original data set hN bðMOcÁMPNÞ VAC following Efron and Tibshirani (1986), while the standard 2 deviations of the empirical distributions were used to in which b is the squared estimate of bð Á Þ, ðMOcÁMPNÞ MOc MPN estimate standard errors of all statistics (Manly, 1997). and g is the additive between the trait Hypothesis testing is similarly complicated when in nature and in captivity. g is high when the rank composite statistics are the subject of comparison. ordering of performances in both environ- Although individual regression coefficients could be ments is the same, and low when it is not, when norms of tested for difference from 0 and from each other using reaction cross (Coyne and Beecham, 1987), so that this standard F and homogeneity-of-slopes methods, direct g estimate is biased when j jo1. comparison of estimates from nature and captivity 2 (when the two are estimated using different methods The final estimate of heritability in nature, hNb is defined or are themselves combinations of other estimates) is as b and is estimated by b . It contains ðMOcÁMPNÞ ðMOcÁMPNÞ cumbersome or not possible with traditional large- both sources of bias: it assumes both that jgj¼1 and that sample approximations (Lynch and Walsh, 1998, pp. s2 A is constant across environments. 561, 807). Hence, cross-environment differences were Based on the relationships among these three estimates tested using randomization, using the ratio of the larger of heritability in nature, Riska et al (1989) develop a to smaller estimate as the test for both variance parameter k, which is defined as components and the additive coefficient of variation h2 b s (Manly, 1997). For each comparison, the test statistic was k ¼ NV ¼ ðMOcÁMPNÞ ¼ AC 2 2 calculated for the original data, then parents were b g h gsAN ðMOcÁMPNÞ N randomized with respect to offspring, leaving each in 2 2 and is estimated in this study by k ¼ hNV=hNb. Because of their respective generations, and the test statistic was the conditions in which these estimates are biased, when recalculated for each of 5000 replicates. This ‘restricted 2 2 2 2 ko1, it follows that hNVohNbog hN and that additive randomization’ tests for changes in variance across genetic variance is greater in nature than in captivity – environments while holding mean effects constant (that 2 2 and furthermore, that g hN is the best approximation of is, correcting for any overall mean difference in offspring the lower bound of heritability in nature. Conversely, across generations; Petraitis et al, 2001). The probability 2 2 2 2 when k41, hNV4hNb4g hN , and k provides no of the observed test statistic was then taken as its location information about the relationship between additive to the nearest percentile in the empirical distribution of variance in the two environments, nor which of the 5000 test statistics. All analyses were conducted on a PC estimates of heritability in nature is the best (though using Statistical Analysis System procedures (SAS 2 2 g hN, as the smallest, is most conservative). Institute Inc., 2001).

Heredity Effect of captivity on genetic variance KM Rodrı´guez-Clark 56 Results , variance components and errors Heritabilities were significantly different from zero in captivity for all three morphological traits, and in nature Trait distributions for the two colour traits and for early fecundity (Table 2). The distributions of wing colour (WC) and pronotum Although differences in single-sex estimates indicate the colour (PC) were remarkably constant across different possibility of slight maternal effects for wing colour in rearing environments, although the pronotum was on captivity, differences were not significant, so midparent– average B1 unit darker than the wing, and less variable midoffspring estimates were used in all subsequent (Table 1). There was a slight and significant trend toward analyses. Similarly, in estimates from nature, there was a females being darker than males in pronotum colour trend toward a paternal effect on pronotum colour (PC) (F ¼ 10.8, Po0.001), and a similar nonsignificant trend in and a on wing length (WL), but, in the wing colour, so values for males were corrected by absence of significant differences, sexes were combined adding the average difference between males and in further analyses. females to each male value. The phenotypic correlation Analysis of changes in heritability in wing colour (WC) between WC and PC was moderate (Spearman’s rank across environments is the most straightforward of all 2 coefficient ¼ 0.56, Po0.0001), such that individuals with traits; all three estimates of hN were high (0.90–0.92) and darker wings tended to have darker pronota; however, virtually identical to each other, as well as to the estimate 2 B neither was correlated with other traits considered. of h (0.92) (Table 2). kWC thus was 1, indicating that g E C Average wing lengths (WL) in both environments 1, and that VA for WC remained constant between (Table 1) were comparable to those recorded in previous nature and captivity. Pronotum colour (PC), despite its studies (Groeters and Dingle, 1996). As with colour traits, phenotypic correlation with WC, displayed a slightly 2 WL differed consistently between sexes, with females different pattern. All three estimates of hN (0.76–1.00) 2 larger (F ¼ 1326, Po0.0001) and more variable (F ¼ 8.5, exceeded hC (0.59), indicating a decline in heritability P ¼ 0.004). This correlation of mean and variance held upon captivity. k was just slightly larger than 1, across generations as well, so wing lengths were ln- indicating that despite the decline in heritability, additive transformed, and the mean difference between female variance remained essentially unchanged. Although in and male lengths was then added to each male length. these circumstances, the data do not indicate which of WL was not phenotypically correlated with any other the three estimates in nature is most accurate, the 2 2 trait in the study. estimate of g hN was used in later analyses, as it is the In contrast to morphological traits, distributions of egg most conservative. production rates (early fecundity, EF and later fecundity, In the case of wing length (WL), the three estimates for LF) changed greatly across rearing environments heritability in nature range from considerably more to (Table 1). Mean production rates increased significantly considerably less (0.55–0.02) than that in captivity (0.34), each generation (H ¼ 203, Po0.0001 for EF, 362.5 and k is 6.05 (much larger than 1), indicating that no Po0.0001 for LF), and variability increased with mean information is available from this level of analysis production (F ¼ 35.2 for EF, 59.8 for LF, Po0.0001). regarding changes in heritability or additive genetic Simple ln-transformation did not remove the depen- variance from nature to captivity in this trait. dence of variance on mean in these traits, so EF and LF However, both fecundity traits (EF and LF) were were both transformed using Kleczkowski’s method associated with a k below 1 (0.29 and 0.21, respectively), (Lynch and Walsh, 1998, p. 300). The phenotypic indicating a decline in additive genetic variance in correlation between the two measures was moderate captivity in both cases (Table 2). Although Riska et al 2 2 (Spearman’s rank coefficient ¼ 0.54, Po0.0001), and (1989) assert that in cases where ko 1, g hN is the best 2 neither was phenotypically correlated with any other estimator of hN , the low estimate of VA in captivity for trait. both of these traits in this case makes the estimator

Table 1 Summary statistics for five traits in wild-caught and laboratory reared O. fasciatus (original measurement scales) Trait Sex Generation

0 (nature) 1 (captivity) 2 (captivity)

Wing colour f 3.0271.06 (n ¼ 50) 3.2470.99 (n ¼ 192) 3.3170.93 (n ¼ 200) (units) m 3.0671.08 (n ¼ 50) 3.1471.09 (n ¼ 201) 3.1970.95 (n ¼ 200)

Pronotum colour f 4.0870.40 (n ¼ 50) 4.0270.52 (n ¼ 192) 4.0570.48 (n ¼ 200) (units) m 3.9670.45 (n ¼ 50) 3.9070.54 (n ¼ 201) 3.9670.53 (n ¼ 200)

Wing length f 13.1370.54 (n ¼ 50) 12.7770.66 (n ¼ 198) 12.9770.50 (n ¼ 200) (mm) m 11.7470.48 (n ¼ 50) 11.5170.55 (n ¼ 206) 11.6170.45 (n ¼ 200)

Early fecundity (mg/day) f 0.6871.84 (n ¼ 183) 3.6874.07 (n ¼ 208) 5.3573.77 (n ¼ 399)

Later fecundity (mg/day) f 1.1972.58 (n ¼ 183) 1.0171.71 (n ¼ 208) 7.6574.28 (n ¼ 399) Results presented as mean7SD (sample size).

Heredity Effect of captivity on genetic variance KM Rodrı´guez-Clark 57 Table 2 Heritability analysis for five traits in O. fasciatus in nature and captivity Trait 2 k 2 DNtoC Estimates of hN hC (SE)

2 2 2 2 hVN hNb g hN

Wing colour MO 0.90 ¼ 0.91*** ¼ 0.92 0.98 0.92 (0.06)*** No change VA SS 0.93 1.12*** 1.34 0.77 (0.13)*** No change h2 DD 0.80 0.99*** 1.22 1.07 (0.19)***

Pronotum colour MO 1.00 4 0.87*** 4 0.76 1.15 0.59 (0.10)*** No change VA? SS 1.13 0.87*** 0.68 0.58 (0.19)*** Decrease h2? DD 0.82 0.56* 0.39 0.58 (0.20)***

Wing length MO 0.55 4 0.09 4 0.02 6.05 0.34 (0.07) *** No info VA SS 0.48 0.04 0.002 0.34 (0.15)* No info h2 DD 0.62 0.20 0.06 0.35 (0.12)**

Fecundity (E) DD 0.13 o 0.44* o 1.50 0.29 0.20 (0.18) Decrease VA Decrease h2?

Fecundity (L) DD 0.018 o 0.08 o 0.40 0.21 0.07 (0.22) Decrease VA No info h2 MO ¼ mid-parent/offspring; SS ¼ sons on sires; DD ¼ daughters on dams. See text for detailed definitions of heritability estimates. Asterisks 2 2 2 2 indicate that hNb differs significantly from 0 at Po0.05, 0.01 and 0.001, respectively (hVN and g hN not tested). Changes in heritability from nature to captivity (Dh2 N to C) are determined either by k statistic (WC, WL, LF), or by relationship between all estimates of heritability in nature vs captivity (PC, EF). Changes in VA determined by k as described in text.

Table 3 Information about additive genetic variance (VA) and decreased significantly for EF and LF (Table 4). No residual variance (VR) that may be derived from known changes in significant changes were found in heritability for any 2 heritability (h ) and phenotypic variance (VP) from nature to trait using randomization tests, in the case of PC because captivity the estimate used for lower bound of heritability in 2 2 2 g Pattern h VP Implied D in VA and VR from nature nature, hN, is associated with wide standard errors. to captivity Despite their disparate changes across traits when 2 considered singly, estimates of VP and h combine to reveal a striking pattern: CVA was relatively stable in 1 D D Decrease VA, with no change in V (or smaller than DV ) WC, PC and WL (with very small but sometimes R A significant increases or decreases, around 1–2%), but 2 D I Increase VR, with no change in VA (or smaller than DVR) declined significantly in EF and LF, between 5 and 7%. 3 D N Not theoretically possible (very These changes appear to be independent of initial slight decrease in VA) evolvabilities, which though highest in wing colour, are 4 I D Decrease VR, with no change comparable (0–12%) in all other traits, regardless of trait VA (or smaller than DVR) 5 I I Increase V , with no change type. A Hypothesis testing on variance components in this VR (or smaller than DVA) 6 I N Not theoretically possible (very study presents something of a challenge in terms of slight increase in VA) multiple comparisons. If all traits and statistics consid- 7 N D Decrease VA and VR ered were independent, Table 4 would contain a total of 5 8 N I Increase VA and VR traits *5 statistics ¼ 25 independent comparisons. Manly 9 N N No change VA and VR (1997) suggests that in randomization tests, one method of guarding against Type 1 errors is to divide the desired D, I, and N indicate decrease, increase, and no change, respectively. significance level (typically 0.05) by the number of independent comparisons made, which would leave Pp0.002 as ‘significant’ in this case. However, he further 2 unstable, and hNb was used as a (more conservative) points out that any correlations in the data will render lower bound of heritability instead. There was a trend this overly conservative. Indeed, the statistics in these 25 toward decline in heritability in EF, as it changed from comparisons are not independent; within a trait, VA is 2 significantly above zero to no different from zero. positively correlated with CVA and h , and VA and VR are A more detailed picture of changes in variance constrained to sum to VP. Furthermore, some traits in this components can be gleaned by combining the above study are correlated phenotypically, though presence or heritability estimates with estimates of total phenotypic absence of phenotypic correlations may not provide any variance (VP) for the traits in question (Table 3). Patterns information about genetic correlations (Roff, 1995). For in VP were clearer than those in heritability, due to example, Palmer and Dingle (1986) found a strong and relatively smaller standard errors: VP remained constant significant negative genetic correlation between fecund- for WC, increased significantly for PC and WL, and ity and wing length in O. fasciatus, despite finding a

Heredity Effect of captivity on genetic variance KM Rodrı´guez-Clark 58 Table 4 Heritabilities, variance components, and evolvabilities for five traits in O. fasciatus using bootstrap bias-corrected estimates (standard errors in parentheseis)

Trait Location (Table 2) VP (SE) (Table 3) VAmin or VRmax or CVAmin or Dmin 2 2 hmin or h Pattern VA (SE) VR (SE) CVA (SE) NtoC

Wing colour Nature 0.91 (0.07) 113.55 (12.66) 103.20 (14.19) 32.9% (2.8) À2.3% Captivity 0.92 (0.06) 109.90 (11.75) 9 100.93 (13.04) 8.97 (6.50) 30.6% (2.6) (P) 0.97 0.64 0.14 0.39 0.13

Pronotum colour Nature 0.70 (0.45) 17.81 (3.56) 12.01 (9.47) 5.80 (8.06) 8.9% (3.0) +1.7% Captivity 0.58 (0.10) 30.15 (4.09) 2 or 8 17.47 (3.65) 12.68 (3.44) 10.6% (1.1) (P) 0.26 0.01 0.20 0.04 0.20

Wing length Nature 0.06 (0.13) 17.49 (2.60) 1.03 (2.16) 16.46 (3.20) 0.5% (0.4) +0.7% Captivity 0.34 (0.07) 27.79 (3.05) 5 or 8 9.56 (2.03) 18.22 (2.89) 1.2% (0.1) (P) 0.23 0.01 0.04 0.30 0.004

Early fecundity Nature 0.44 (0.20) 115.18 (19.34) 50.17 (23.88) 65.01 (24.37) 12.3% (3.2) À7.7% Captivity 0.20 (0.18) 73.8 (11.33) 7 14.95 (12.34) 58.74 (16.46) 4.8% (1.8) (P) 0.44 0.009 0.10* 0.38 0.008

Later fecundity Nature 0.08 (0.08) 149.69 (18.01) 11.62 (11.79) 138.07 (19.44) 5.7% (3.0) À4.7% Captivity 0.07 (0.22) 45.35 (9.23) 7 2.51 (9.41) 42.84 (9.92) 1.0% (0.2) (P) 0.91 o 0.001 0.05* o0.001 0.007

2 2 2 2 Estimates for first three traits derived from both sexes. g hN used in bootstrap estimate of minimum hN for PC, since k41. hNb used in 2 2 2 bootstrap estimate of minimum hN for WC, EF, and LF, since kp1, and in WL, since bias-corrected estimate of g hN less than 0. Probability levels derived from parametric tests for all h2 estimates except PC, or otherwise by randomization (see text for details); significant differences (nature vs captivity) by these tests indicated in bold. Significant differences by k statistic indicated with asterisk. Variance components are in transformed measurement units squared (see Table 2), and multiplied by 102 (WC, PC), 103 (EF, LF) or 104 (WL) for convenience of presentation.

weaker and positive phenotypic correlation. Unfortu- due not to small changes in the location of estimates, but nately, the number of families available in this study do to the large errors around those estimates. This is not provide sufficient power for examining genetic particularly so in the fecundity traits examined, and correlations. Other authors have suggested that hetero- underscores the shortcomings of studies on single traits geneity in phenotypic variability among traits within in isolation. Had only wing colour been considered, the populations indicates that they may be treated statisti- conclusion that estimates in captivity can readily be cally as independent estimates of shifts across environ- extrapolated to nature might seem reasonable, as ments (Bryant and Meffert, 1998). Combining these estimates in both environments are very similar, with considerations, I take probabilities of 0.01 or less to be small errors. However, when the additional traits are ‘significant’ for randomization tests. considered, this pattern is much less clear, in keeping with findings summarized across a large number of single-trait studies (Weigensberg and Roff, 1996). Discussion Indeed, large errors in heritability estimates suggest that the other measure of heritable variation used in this Previous studies have compared heritabilities in nature study, evolvability, may be a valuable additional metric and in captivity in order to examine the question of the in comparisons of genetic variance. Although significant validity of extrapolating more easily obtained laboratory heritable variation was detected in a life history and two results to natural populations of interest. In the present morphological traits in nature and in three morphologi- study, I have used techniques developed in this context cal traits in captivity, comparison of these values with to examine an assumption often made in forming captive other studies emphasizes the variability in heritability in populations for conservation purposes, that a small species even within the same environment, across number of founders can capture a large proportion of different studies. While in agreement with a previous the genetic variation of wild populations. Although my study finding moderate heritability for early fecundity in estimates in nature are less than ideal, since they rely on captivity (0.44 in the present study vs 0.25–0.31 in data derived in part from captive individuals, if anything Groeters and Dingle, 1988) and a low minimum herit- they will be conservative, as they are minima which may ability for wing length in nature (0.06 compared with be biased downward if additive genetic correlations 0.10–0.20 in Groeters and Dingle, 1996), the present across environments are not perfect. estimate of heritability of wing length in captivity (0.34) is In the context of the first question, no significant considerably lower than the range of values uncovered in changes in heritability were observed in any trait in the previous work (0.6–0.87: Palmer and Dingle, 1986; 0.45– transition from nature to captivity. However, this cannot 0.53: Dingle et al, 1988; 0.80: Groeters and Dingle, 1996). be taken as straightforward support for previous claims The comparisons of evolvability across environments of a predictable relationship between heritability in in this study provide additional evidence that the lack of nature and captivity (eg, Bitner-Mathe´ and Klaczko, significant change observed in heritability from nature to 1998): the lack of significant differences appears to be captivity is likely to stem from large errors, rather than

Heredity Effect of captivity on genetic variance KM Rodrı´guez-Clark 59 from true stability in variance components. Although It is tempting to conclude that the stability of evolvability remained unchanged in the three morpho- evolvability in WC, PC and WL across the nature– logical traits examined, shifting 1–2% or less, it declined captivity transition vs its decline in EF and LF represents 5–8%, and significantly, in the fecundity traits. This large a fundamental difference between morphological and and significant decline is striking for a number of life-history traits in general. Such a difference could reasons. First, changes are inherently more difficult to result from the consistent directional selection traits detect in statistics with the decreased precision imposed closely correlated with fitness are assumed to experience by single-sex estimation. Second, estimates of VAN and in nature (Mousseau and Roff, 1987) or from enhanced CVAN for these traits are lower bounds of these developmental noise in life-history traits, which may be parameters, such that if anything, true declines in under the influence of a greater number of loci (Houle, captivity were greater than observed. Finally, the ratio 1992 and references therein), both of which might test used to detect declines in evolvability loses power increase the opportunity for gene-by-environment inter- when means between groups differ (Manly, 1997, p. 103), actions. However, with so small a sample size (just two as they did in the case of both EF and LF (Table 1). These ‘life-history’ traits and three ‘morphological’ traits, same factors also make it less surprising that a change of themselves phenotypically correlated), such conclusions less than 1% in evolvability in wing length, which has would be premature. fewer of these limitations, was significant. Regardless of the reasons behind the difference, an The significant decline in evolvability for EF and LF important result is that, though selection or gene-by- may be the result of several factors, as they differed from environment interactions may generate significant the other traits examined in several respects. Although changes in evolvability in some circumstances, this does generation 0 completed its development in nature, these not always have to be the case. The conservation two traits, unlike others, were not measured in fully implications of stability in evolvability for some traits natural conditions: pairs were mated and eggs were in the first generation following capture are not trivial: in collected in a captive context. However, this would tend line with classic theory, it appears possible, at least for to make the estimates from different generations more some traits, to capture the full compliment of potentially similar rather than more different. Second, selection may adaptive genetic variation from nature, with even a have been another factor depleting additive genetic limited number of founders. If gene-by-environment variation in these traits. Every effort was made to interactions or selective losses were the rule, fundamental minimize selection for improved captive performance genetic constraints on all traits at the moment of sampling by ensuring that every family was represented in every would have to be added to the already considerable list of generation and also randomly selecting individuals limitations on the usefulness of captive breeding as a within families to be used as parents (Falconer and conservation tool (Kleiman et al, 1996). MacKay, 1996). However, unlike other traits examined, However, it is sobering to note that the 50 pairs of individuals on the low end (or at 0) on the fecundity founders in this study is still greater than the 10 pairs scale could not, by definition, be represented in the currently considered adequate in conservation contexts next generation, so any genetic factors associated with (eg, Clark et al, 1997). Given recent advances in applying poor reproductive performance in captivity were techniques used in agricultural production to estimate necessarily lost. Finally, although care was taken to variance components in nondomestic species with sample wild individuals widely and yet within a single complex pedigrees (eg, Milner et al, 2000) it would be subpopulation, it is conceivable that founders came of great interest to apply the above methods to from more than one subpopulation. However, the endangered species currently in captivity in which wild inflation of genetic variance possible from such mixing founders are still alive, to determine whether evolva- would be expected to influence natural and captive bility can remain stable in nonmodel organisms, even estimates equally, and thus is not likely to be a factor when founder numbers are smaller and drift might be contributing to the declines observed in evolvability for expected to have a more pronounced effect. Such fecundity. methods also might help identify which among many Instead, other factors seem more likely to be the cause traits would be useful to monitor in such populations of the significant declines in evolvability in EF and LF. over successive generations. It seems likely that such ‘Domesticating’ selection reducing additive genetic efforts would be of greater value than simply monitoring variance for fecundity could have been generated in heritability, which has been advocated in the past the first generation of captivity by gene-by-environment (Cheverud et al, 1994; Lynch, 1996). interactions, such that variants were lost who produce well in nature but poorly in captivity. Such interactions can cause changes in additive genetic variance even in Acknowledgements the absence of selection, when genetic variants them- selves are not lost, but their effects change. Extensive Crucial assistance with field collections came from Jon interactions of fecundity with photoperiod (Groeters and Paul Rodrı´guez, David Smith and Amy Whorlton, while Dingle, 1987), temperature (Groeters and Dingle, 1988), essential husbandry advice came from Hugh Dingle. I and flight activity (Slansky, 1980) have been observed in thank Dorothy Feir and Sonia Alteiser for their help this species and in closely related ones (Kasule, 1991), obtaining seeds, and many in Princeton’s Department of consistent with the sharp change in fecundity measures Ecology and Evolutionary Biology (EEB) for help with across the two environments seen here. However, seed collection and processing. I am grateful to Wolf whether observed declines may also be due to lower Blanckenhorn, John Brookfield, Andy Dobson, Peter levels of ‘stress’ in captivity remains unclear (Bijlsma and Grant, Hope Hollocher, Isabel Oliveri, Don Stratton and Loeschcke, 1997). one anonymous reviewer for their insightful comments

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