Developmental instability, hybridization and heterozygosity in stick insects of the genus Bacillus

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1 Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society The Linnean Society of London, 2006? ? Original Article DEVELOPMENTAL INSTABILITY, HYBRIDIZATION AND HETEROZYGOSITY IN BACILLUS D. H. ANDERSEN ET AL. Biological Journal of the Linnean Society, 2006, 87, With 2 figures Developmental instability, hybridization and heterozygosity in stick insects of the genus Bacillus (Insecta; Phasmatodea) with different modes of reproduction DITTE HOLM ANDERSEN 1,2 *, CINO PERTOLDI 2,3,4, VOLKER LOESCHCKE 2 and VALERIO SCALI 1 1 Dipartimento di Biologia Evoluzionistica Sperimentale, Via Selmi 3, I Bologna, Italy 2 Department of Ecology and Genetics, University of Aarhus, Ny Munkegade, Building 540, DK-8000 Aarhus C, Denmark 3 Department of Applied Biology, Estación Biológica Doñana, CSIC, Pabellón del Perú, Avda. Maria Luisa, s/n, S Seville, Spain 4 Department of Wildlife Ecology & Biodiversity, National Environmental Research Institute, Kalø Grenåvej 14, DK-8410 Rønde, Denmark Received 26 July 2004; accepted for publication 9 March 2005 Several genetic factors are assumed to influence developmental instability (DI). One is the level of heterozygosity, with higher levels often being associated with decreased DI; another is genetic incompatibility in hybrids, which in several cases has been shown to increase DI. The genus Bacillus includes species which have both amphigonic heterozygous reproducing populations and homozygous parthenogenetic reproducing populations (B. rossius rossius and ). Furthermore, Bacillus includes hybrid parthenogenetic species, which have very high levels of almost fixed heterozygosities (B. atticus, B. whitei, B. lynceorum). We investigated the phenotypic variance (σ 2 p) and the impact of hybridization and level of heterozygosity on DI in females from these populations and species of Bacillus. DI was estimated as fluctuating asymmetry (FA) for three bilateral traits: the labial palpus, the maxillary palpus and the antenna. For the labial palpus and maxillary palpus we found, in general, a lower level of DI in the amphigonic females compared with parthenogenetic counterparts from the same species and with parthenogenetic females from the three hybrid species. A higher DI of the antenna was found in the hybrid species when compared with both parthenogenetic and amphigonic populations of the nonhybrid species, suggesting that the genes controlling antenna development are located on the sex chromosomes. The development of the investigated bilateral characters in the hybrid species seemed to be affected more by factors relating to genetic incompatibilities as a consequence of hybridization than by the stabilizing force of increased heterozygosity. Only few differences in σ 2 p were observed, supporting the possibility that the observed differences in DI are related mainly to internal genetic factors The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, ADDITIONAL KEYWORDS: fluctuating asymmetry genetic incompatibility parthenogenesis phenotypic variance polyploids species hybrids. INTRODUCTION In nature interspecific hybridization is not a rare phenomenon. Hybrids can arise from crosses between *Corresponding author. ditte.andersen@biology.au.dk genetically well-differentiated bisexual species and may persist through unusual modes of reproduction (Dowling & Secor, 1997). Interspecific hybrids arise when heterospecific matings lead to the production of zygotes that occasionally can overcome developmental constraints. Most natural hybrid populations show female-biased or all-female progeny as male gameto- 249

2 250 D. H. ANDERSEN ET AL. genesis and/or development is more severely affected by hybridization compared with that of females (Turelli & Orr, 2000; Presgraves, 2003). Female oogenesis is also altered in hybrids, but some eggs may be functional and self-activating. A few hybrid progeny may thus hatch whose descendants can persist in time and become a hybrid all-female species if their modified gametogenesis stabilizes and their fertility increases in subsequent generations (Marescalchi, Pijnacker & Scali, 1991). Multiple hybridization events may create allopolyploid hybrids, and enhance the genetic variation in the hybrids (Marescalchi & Scali, 2003). Polyploidy is often associated with parthenogenetic reproduction and the hybrids may be able to combine the main advantage of sexual reproduction (genetic diversity) with the advantages of unisexual reproduction (faster population growth and the possibility of colonization by one individual) which would only be possible for a sexually reproducing female already mated (Bullini, 1994). Hybridization may affect the fecundity and development of hybrid individuals due to the accumulation of genes that cause recessive incompatible epistatic interactions between the parental species (Presgraves, 2003). Genetic incompatibilities in the hybrids may decrease their ability to develop an optimal phenotype due to increased developmental instability (DI). Developmental stability refers to the ability of an organism to buffer its developmental processes against environmental and genetic disturbance and ensures common developmental outcomes under specified conditions (Zakharov, 1992). DI results when developmental noise or stress affects the buffering capacity of the processes related to development that provide developmental stability (Lens et al., 2000). Fluctuating asymmetry (FA) is a very common measure of DI and is known to be elevated due to both environmental stress factors (Kristensen et al., 2003, 2004) and genetic factors, such as hybridization (Andersen et al., 2002), loss of genetic variation (Lens et al., 2000; Pertoldi et al., 2003), degree of protein heterozygosity (Leary et al., 1985), episodes of directional selection (Manning & Chamberlain, 1993) and mutations (Clarke & McKenzie, 1987, 1992). DI also contributes to the phenotypic variance (σ 2 p) of individuals. σ 2 p in a sexually reproducing population is given by: σ 2 p = σ 2 g + σ 2 e + (GxE) + cov(ge) + DI, where σ 2 g and σ 2 e are the genetic and environmental variance, respectively, (GxE) is the genotype environmental interaction and cov(ge) is the genotype environmental covariance (Pertoldi, Kristensen & Loeschcke, 2001a). Since both genetic and environmental factors affect σ 2 p, σ 2 p of a quantitative character can only be used as a measure of DI in populations with no genetic variance and no environmental variance among the individuals. Another phenomenon that can be observed in some hybrids is increased fitness in the hybrids compared with their parental populations, referred to as hybrid vigour (Rieseberg & Carney, 1998). The underlying genetic mechanisms are not clearly understood but the increased fitness in some hybrids is generally ascribed to an increase in heterozygosity (Alibert et al., 1997). The advantages of being more heterozygous could be due to dominant alleles which mask deleterious recessive alleles or to overdominance, i.e. generally heterozygous individuals are more fit than are more homozygous individuals (Rieseberg & Carney, 1998). This could be due to increased flexibility in the production of biochemical products and increased metabolic flexibility (Mitton, 1993). The expression of hybrid vigour is often restricted to the F1 generation in sexually reproducing populations since meiotic recombination and segregation in the F1 individuals split epistatic and additive coadapted interactions among loci derived from the two parents. This may result in decreased fitness and increased DI in the F2 progeny or in later generations (Ross & Robertson, 1990; Andersen et al., 2002). Polyploidy is also known to affect FA. Some investigations have found that an increased level of ploidy and therefore very high levels of heterozygosity might decrease FA (Leary et al., 1985; Scheerer, Thorgaard & Seeb, 1987; Mesaros, Tucic & Tucic, 1994). However, in most cases elevated ploidy is disadvantageous as ploidy can significantly alter the developmental pattern and physiology of cells, which might result in an increase in FA. Elevated ploidy prolongs celldivision time and consequently slows down the developmental rate, which might make allopolyploids inferior to their diploid ancestors (Keller & Gerhardt, 2000). GENUS BACILLUS The genus Bacillus Latreille, is a good experimental set of taxa for investigating the effects of hybridization, different levels of heterozygosity, and polyploidy on DI because of its highly variable reproductive systems and variation in ploidy levels. The genus is known to include the bisexual B. grandii Nascetti & Bullini, the bisexual B. rossius (Rossi) which also has all-female facultative parthenogenetic populations, the automictic parthenogen B. atticus Brunner, and their related hybrids: the apomictic B. whitei (B. rossius/grandii grandii) Nascetti & Bullini, and the apomictic triploid B. lynceorum (B. rossius/ grandii grandii/atticus) Bullini, Nascetti & Bianchi Bullini.

3 DEVELOPMENTAL INSTABILITY, HYBRIDIZATION AND HETEROZYGOSITY IN BACILLUS 251 BACILLUS GRANDII GRANDII B. grandii is endemic to three tiny Sicilian areas. The species is very rare and recent observations (V. Scali, pers. observ.) indicate that the habitat of B. g. grandii is decreasing. Furthermore, its area of distribution has been invaded by B. whitei, which may threat the persistence of B. g. grandii in the future. BACILLUS ROSSIUS B. rossius spreads over most of the western Mediterranean basin with two Italian subspecies B. r. rossius and. B. r. rossius is found on the Thyrrenian and Ligurian coasts of the Italian mainland, on Sicily and on the north-western part of Sardinia. is found along the Adriatic coast, on Sicily and on south-eastern Sardinia (Fig. 1). Previous studies of protein heterozygosity from samples of the two subspecies B. r. rossius and (which also included populations collected close to our collecting sites), gave heterozygosity values of H = and H = 0.033, respectively (Mantovani & Scali, 1991). The heterozygosity in the parthenogenetic reproducing populations of the two species is zero. This is due to an automictic mechanism that can be viewed as gamete multiplication, which, after diploidization, generates a female offspring homozygous at all loci in one generation (Scali et al., 2003). BACILLUS ATTICUS B. atticus is a thelytokous obligate parthenogen with very complex population genetics. This can be explained by its reproduction mechanism, in which automixis occurs before the second meiotic division. This leads to a slow but cumulative change from heterozygous combinations to homozygous ones (Mareschalchi, Pijnacker & Scali, 1993). Allozyme heterozygosity of B. atticus populations from Sicily has been estimated to be 0.1 < H < 0.2 (Mantovani, Scali & Tinti, 1990). The origin of B. atticus is not fully understood but it is known that it has a rather close relationship with B. grandii (Marescalchi & Scali, 2003) and investigations have suggested that B. atticus arose not as a result of interspecific hybridization but more likely through a complex of interracial hybridization events (Marescalchi & Scali, 1997). BACILLUS WHITEI AND BACILLUS LYNCEORUM The apomictic hybrids B. whitei and the triploid B. lynceorum reproduce by a process that allows invariant transmission of fixed heterozygous loci of the maternal genome to the thelytokous progeny, with very few exceptions (Marescalchi et al., 1991). The Figure 1. Map showing the collecting sites of Bacillus populations. 1, parthenogenetic B. rossius rossius; 2, amphigonic B. r. rossius; 3, parthenogenetic ; 4, amphigonic ; 5, B. whitei; 6, B. lynceorum; 7, B. atticus; 8, B. grandii grandii. hybrid species B. whitei and B. lynceorum reflect fully the genetic structure of their two and three ancestral species, respectively, with heterozygosity estimates around H = 0.7 due to the great differentiation of B. rossius on one side and B. grandii/b. atticus on the other (Scali et al., 2003). AIMS OF THE STUDY As described above, the reproductive mode in the genus Bacillus ranges from sexual in B. r. rossius and to automictic in the parthenogenetic populations of B. r. rossius, and B. atticus and apomictic in B. whitei and B. lynceorum. Furthermore, the level of heterozygosity

4 252 D. H. ANDERSEN ET AL. ranges from zero in the parthenogenetic populations of B. r. rossius and to high in the interspecific hybrids B. whitei and B. lynceorum. All these factors make the genus very suitable for our investigation of factors affecting DI. The first aim of this study was to investigate if there exists in Bacillus a general relationship between the level of heterozygosity and measures of DI. Another aim was to investigate the main factors affecting DI in the hybrids. The hybrid species of Bacillus have successfully established large and thriving populations and the origins of the hybrid species have been estimated to more than 1 Mya (Scali et al., 2003). A factor that may contribute to the success of the hybrids could be their increased heterozygosity, which could also have an antagonistic effect on DI. On the other hand, the genetic structure of the hybrids still reflects their parental genomes since no or very little recombination occurs in B. whitei and B. lynceorum, and recombination rates in the race hybrids B. atticus are rather low. This means that all hybrids may still suffer from the established genetic incompatibilities between their parental taxa, since selection cannot act to reestablish more fit hybrids. It was therefore possible to evaluate whether it is mainly the stabilizing force of increased heterozygosity or the destabilizing force of genetic incompatibilities that affect DI in the hybrids. Lastly, we compared the sexually reproducing females with the parthenogenetic females within the same species to evaluate whether higher levels of heterozygosity without genetic incompatibilities affect the DI level. MATERIAL AND METHODS Specimens from populations of B. r. rossius and were collected during October B. r. rossius were collected on the Thyrrenian coast of Tuscany; the parthenogenetic individuals of B. r. rossius came from just north of the Ombrone river and the sexually reproducing individuals from just south of the same river (Fig. 1). Specimens of parthenogenetic and amphigonic were collected on the Adriatic coast at Grottamare and Torino di Sangro Marina, respectively. The Sicilian hybrid parthenogenetic individuals were collected during October B. atticus were collected on the Iblean area at Scoglitti, B. lynceorum at Fontane Bianche and Cassibile, and B. whitei at Canicattini Bagni (Fig. 1). Sicilian collections also included Ponte Manghisi, which is the locality of the very rare B. g. grandii. Unfortunately it was only possible to obtain five specimens of pure B. g. grandii since the habitat of this endemic species has been invaded by the parthenogenetic B. whitei. We were therefore unable to include an analysis of B. g. grandii in our investigation. With the exception of B. atticus, which feeds on lentisk, all populations of Bacillus were collected on bramble. Collected specimens were preserved in 95% alcohol before being measured. We measured three bilateral traits and three unilateral traits. The bilateral traits considered were the antenna, the labial palpus, and the maxillary palpus, and were measured using a binocular microscope with a digital filar eyepiece (Los Angeles Scientific Instrument Company, Inc., USA). The unilateral traits, which were the mesonotum, metanotum and abdomen, were measured with a digital calliper to the nearest 0.1 mm. For all statistical analyses each population was analysed separately and from the sexually reproducing B. r. rossius and only female populations were analysed. Due to the large number of tests, a Bonferroni correction (Rice, 1989) was applied. Following Miller (1981), we made a separate probability statement for each trait. STATISTICAL PROPERTIES OF FA FA was estimated as the difference in length between each bilateral pair of traits (right left side). To determine whether the obtained data displayed the statistical properties of FA, which is characterized by a normal distribution with a mean of zero (Palmer & Strobeck, 1986), a t-test was applied to test for deviations of the mean values from zero, and Shapiro Wilk s test was conducted to check for normality (Zar, 1984). MEASUREMENT ERROR To estimate measurement error for the bilateral traits a subsample of 30 B. whitei individuals was measured twice, the second set of measurements being made without reference to the first set. A two-way ANOVA was conducted to test for significance of FA relative to measurement error (the difference between the two independent measures of FA) following Palmer & Strobeck (1986). To quantify possible errors associated with measuring the unilateral traits, 20 parthenogenetic individuals were taken arbitrarily and for each individual the three traits were measured ten times. The within-individual coefficient of variation for each mean was taken as an estimate of the measurement error, adding Haldane s (1955) correction for small sample size. CORRELATION OF FA WITH BODY SIZE AND AMONG TRAITS A t-test for possible associations between body size and FA was conducted because such correlations may affect the interpretation of DI (Palmer, 1994). Spearman s rank correlation test (Zar, 1984) was applied to

5 DEVELOPMENTAL INSTABILITY, HYBRIDIZATION AND HETEROZYGOSITY IN BACILLUS 253 determine whether the absolute value of FA of each of the bilateral traits was correlated with the sum of the mesonotum, metanotum and abdomen, which was taken as an estimate of insect body size. Finally we used Spearman s rank correlation test to check for correlations of the absolute value of FA among the different traits. COMPARISON OF FA AND σ 2 P AMONG POPULATIONS FA was estimated as FA 1 (the mean value of the absolute FA) and as FA 4 [the variance of (r l)] (following Palmer & Strobeck, 1986). Some individuals showed very strong asymmetries exceeding three standard deviations of the signed values of (r l) of the three bilateral traits. These individuals were considered phenodeviants and FA values of their traits were not included in further analyses. A permutation t-test (1000 permutations) was applied to test for significant differences in FA 1 among the populations. This test should be more accurate for small sample sizes and non-normal distributions like the distribution of the absolute value of FA, which was half normal. An F-test was used to test for significant differences in FA 4. Due to a highly significant correlation between the size of the right side of the bilateral traits and the unilateral traits (matrix available from D.H.A. on request), we estimated the σ 2 p as the variance of body size (mesonotum + metanotum + abdomen). An F-test was applied to test for differences in σ 2 p among populations. RESULTS STATISTICAL PROPERTIES OF FA There was one deviation from zero of the mean of the trait (r l) distributions for the labial palpus (one-sample t-test: < P < 0.94, 24 < d.f. < 76) and there were two significant deviations from zero for the maxillary palpus (one-sample t-test: 0.01 < P < 0.056, 24 < d.f. < 78). There were two significant deviations for the antenna (one-sample t-test: < P < 0.91, 20 < d.f. < 76). We determined that it would not affect our results to include the samples in which significant deviations from zero were found, as the deviations were probably due to small sample size and the significance disappeared when we removed one or two extreme values. There were two deviations from normality out of 21 tests (Shapiro Wilk s test: < P < 0.96, 20 < d.f. < 76); these were in both cases due to leptokurtic distributions. MEASUREMENT ERROR The interaction mean square (MS) containing information about FA was tested against error MS (reflecting measurement error), showing that FA was significantly larger than measurement error for all three traits (P < 0.001). The measurement error for the three unilateral traits was low, with a mean value of 0.13%. CORRELATION OF FA WITH BODY SIZE AND AMONG TRAITS There was a significant correlation between body size and absolute FA in 14% of the cases, with all significant results showing a negative relationship. There was no correlation among absolute FA for the three bilateral traits for B. atticus B. whitei, and B. lynceorum (0.05 < r s < 0.44, 19 < N < 23, 0.05 < P < 0.83). In the parthenogenetic population of B. r. rossius, there were highly significant positive correlations among absolute FA of all three traits (0.36 < r s < 0.69, N = 77, P < 0.001). In the parthenogenetic population of, there was a highly significant postive correlation between absolute FA of the maxillary palpus and the labial palpus (r s = 0.87, N = 73, P < 0.001) but there was no correlation with the absolute FA of the antenna (0.04 < r s < 0.01, N = 73, 0.42 < P < ). In the amphigonic individuals of B. r. rossius and B. redtenbacheri, there were highly significant positive correlations between FA of the maxillary palpus and the antenna (0.53 < r s < 0.68, 48 < N < 77, P < 0.001), but there was no correlation with the labial palpus (0.08 < r s < 0.29, 48 < N < 77, 0.5 < P < 0.57). COMPARISON OF FA AND σ 2 P AMONG POPULATIONS There were several significant differences in both FA 1 and FA 4 among the individuals from the seven populations. There was no significant difference in FA between the two sexually reproducing populations and they always exhibited the lowest levels of both FA 1 and FA 4 (Table 1, Fig. 2). The highest levels of FA were in B. lynceorum in almost all comparisons (Table 1, Fig. 2). For the labial palpus and the maxillary palpus the permutation t-test showed no significant differences in FA 1 among the five parthenogenetic reproducing populations. However, for almost all comparisons between the amphigonic females of B. r. rossius and and the parthenogenetic populations, FA 1 was lower in the amphigonic individuals (except for the maxillary palpus of B. whitei) (Table 1, Fig. 2). In general, for the antenna of the parthenogenetic hybrids B. whitei, B. lynceorum and B. atticus, there was a significantly higher FA 1 when compared with the amphigonic and parthenogenetic individuals of B. r. rossius and (Table 1, Fig. 1). There was no difference in antenna FA 1 among

6 254 D. H. ANDERSEN ET AL. Table 1. Upper half of each trait analysis presents t-test for differences in FA 1 and lower half F-test for differences in FA 4 for the labial palpus, maxillary palpus and antenna between the hybrid parthenogenetic populations of Bacillus whitei, B. lynceorum and B. atticus and the parthenogenetic and amphigonic populations of B. rossis rossius and labial palpus (n) FA 4 \(n) FA 1 ± SE B. whitei (26) ± B. lynceorum (24) ± B. atticus (25) ± B. r. rossius parth. (79) ± parth. (73) ± B. r. rossius amph. (48) ± amph. (35) ± B. whitei (26) n.s n.s n.s n.s n.s. 2.82** B. lynceorum (24) n.s n.s n.s n.s. 2.92*** 3.19*** B. atticus (25) n.s n.s n.s n.s. 3.34*** 3.66*** B. r. rossius parth. (76) n.s n.s n.s n.s n.s n.s. parth. (73) n.s n.s n.s n.s. 5.13*** 5.50*** B. r. rossius amph. (48) *** 6.25*** 5.37*** 4.28*** 6.66*** 0.82 n.s. (35) *** 7.62*** 6.55*** 5.21*** 8.11*** 1.22 n.s amph. maxillary palpus (n) FA 4 \(n) FA 1 ± SE (29) ± (25) ± (25) ± (77) ± (73) ± (48) ± (35) ± B. whitei (29) ** 1.15 n.s n.s n.s n.s n.s. B. lynceorum (26) *** 2.08 n.s n.s n.s. 4.46*** 4.16*** B. atticus (25) n.s. 5.37*** 0.26 n.s n.s. 3.36*** 2.85 n.s B. r. rossius parth. (79) ** 2.79*** 1.92 n.s n.s. 4.77** 3.94 n.s parth. (73) *** 2.31 n.s n.s n.s. 6.37*** 5.66*** B. r. rossius amph. (48) *** 31.68*** 5.90*** 11.36*** 13.71*** 0.70 n.s (35) n.s *** 4.51*** 8.69*** 10.48*** 1.31 n.s amph. antenna (n) FA 4 \(n) FA 1 ± SE (22) ± (23) ± (19) ± (77) ± (73) ± (48) ± (35) ± B. whitei (23) n.s n.s n.s. 3.07*** 3.66*** 3.56*** B. lynceorum (24) *** 1.34 n.s. 4.18*** 4.35*** 4.62*** 4.58*** B. atticus (21) *** 1.31 n.s. 3.69*** 3.95*** 4.35*** 4.29*** B. r. rossius parth. (77) n.s *** 20.18*** 0.57 n.s n.s. 1.5 n.s. (73) *** 21.46*** 28.19*** 1.40 n.s n.s n.s. parth. B. r. rossius amph. (48) *** 45.65*** 59.97*** 2.97*** 2.13 n.s n.s. amph. (35) *** 43.89*** 57.66*** 2.86*** 2.05 n.s n.s. Due to the large number of tests conducted a Bonferroni test (Rice, 1989) was applied. Following Miller (1981) we made a separate probability statement for each trait (k = 21). The values are the t and F-values, respectively. **P < 0.01; ***P < 0.001; n.s., not significant.

7 DEVELOPMENTAL INSTABILITY, HYBRIDIZATION AND HETEROZYGOSITY IN BACILLUS FA labial palpus maxillary palpus antenna B. whitei B. lynceorum B. atticus B. r. rossius parth Population B. r. redtenbacheri parth B. r. rossius sex B. r. redtenbacheri sex Figure 2. FA 1 of the labial palpus, maxillary palpus and antenna in the hybrid parthenogenetic populations Bacillus whitei, B. lynceorum, B. atticus and the parthenogenetic and amphigonic populations of B. rossius rossius and (see Table 1 for significant differences among populations). Error bars represent the standard error. the amphigonic and parthenogenetic individuals of B. r. rossius and as well as among the hybrid B. whitei, B. lynceorum and B. atticus. The results for FA 4 showed more or less the same trends as those for FA 1. There were always highly significantly lower levels of FA 4 of the labial palpus in the amphigonic females compared with the parthenogenetic populations (Table 1). For the maxillary palpus, sexual populations showed in almost all cases significantly lower levels of FA 4 compared with parthenogenetic ones (Table 1). For the antenna, B. lynceorum and B. atticus showed significantly higher levels of FA 4 than did all the other populations. There were significantly lower levels of FA 4 in the amphigonic females compared with all the other populations except for the parthenogenetic (Table 1). There were only two significant differences in σ 2 p of insect body size: σ 2 p was highly significantly increased in the amphigonic B. r. rossius compared with both the parthenogenetic hybrid B. whitei and the amphigonic (Table 2). pathways differently (Parson, 1990). Unlike many other studies of FA (e.g. Pertoldi, Scali & Loeschcke, 2001b; Andersen et al., 2002), in several cases we found a highly significant correlation of FA among traits at the individual level in the amphigonic and parthenogenetic reproducing populations of B. r. rossius and B. r. redtenbacher. The high correlation of FA among the three investigated traits cannot be explained by the same (group of) genes controlling the development of all three bilateral traits since the observed pattern for antenna FA indicates the genes that control antenna development to have a distinct chromosomal location on the sex chromosomes (discussed below). The high correlation of FA among these traits could arise if the three traits reflect the underlying organism-wide developmental stability (Lerner, 1954). There was no correlation among the FA of traits in the hybrid populations. This could be due to the hybrids possession of genes that have not undergone selection for optimal development together. Hybridization may therefore lead to different levels of DI or vulnerability of the three traits. DISCUSSION CORRELATION OF FA AMONG TRAITS Differences in FA between traits can be explained by different levels of DI; these might vary depending on differences in selection and canalization, where traits closely related to fitness are believed to be subject to stronger selection and canalization for optimal development (Palmer & Strobeck, 1986). Furthermore, different traits may have trait-specific developmental windows; in such a developmental window, one trait may be more vulnerable to stress factors than are other traits. Differences in DI among traits may also arise if different (groups of) genes control the development of distinct traits, influencing developmental OVERALL RELATIONSHIP BETWEEN HETEROZYGOSITY AND FA Our results clearly indicate that the amphigonic females are less developmentally instable than are both the homozygous parthenogenetic females of B. r. rossius and and the highly fixed heterozygous hybrid parthenogenetic reproducing females. This investigation did not, therefore, find any evidence for a general relationship between heterozygosity and FA. The factors determining the higher level of FA in the hybrid parthenogenetic females compared with the amphigonic females are probably very different from the factors leading to a higher FA in the parthenoge-

8 256 D. H. ANDERSEN ET AL. Table 2. F-test of the variance of body size among the hybrid parthenogenetic populations of Bacillus whitei, B. lynceorum, B. atticus and the parthenogenetic and amphigonic females of B. rossis rossius and B. whitei B. lynceorum B. atticus B. r. rossius parth. parth. B. r. rossius amph. amph. (n) σ 2 p body size (43) (25) (25) (78) (73) (48) (35) B. whitei 2.86 n.s n.s n.s n.s *** 1.06 n.s. B. lynceorum 1.42 n.s n.s n.s n.s 3.04 n.s. B. atticus 1.08 n.s n.s n.s n.s. B. r. rossius parth n.s n.s n.s n.s n.s. parth. B. r. rossius amph *** Due to the large number of tests conducted a Bonferroni test (Rice, 1989) was applied. Following Miller (1981) we made a separate probability statement for each trait (k = 21). The values in the table are F-values.***P < 0.001; n.s., not significant. netic populations of B. r. rossius and compared with their amphigonic counterparts. HETEROZYGOSITY AND FA IN PARTHENOGENETIC AND AMPHIGONIC BACILLUS ROSSIUS The higher FA in the parthenogenetic populations of B. r. rossius and compared with the sexually reproducing females could be due to the almost complete homozygous genome in these females. The complete homozygosity could make the parthenogenetic B. r. rossius and less able to control their development against external and internal disturbances, compared with their amphigonic counterparts, which possess a more heterozygous genome. This observation is also in agreement with other investigations finding that homozygous individuals are less stable compared with more heterozygous ones (Leary et al., 1985; Hutchison & Cheverud, 1995). The increased FA in parthenogenetic females of B. r. rossius and compared with the amphigonic females of the same species, was however, found only in the labial palpus and the maxillary palpus, but not in the antenna. The reason for the lack of differences in FA of the antenna between the parthenogenetic B. r. rossius and and the amphigonic females could be explained by the location of the genes controlling antennal development on the sex chromosomes. The males are hemizygous and therefore the genes controlling antennal development being located on the sex chromosomes would expose them to selection against recessive sublethal mutations in the males. The parthenogenetic mechanism in B. r. rossius and generates a female offspring homozygous at all loci in one generation (Scali et al., 2003). The genes controlling the development of the labial and maxillary palpus may therefore be homozygous for recessive sublethal alleles. This is because these genes have not been subject to the strong kind of selection against these alleles as the genes responsible for development of the antenna. This may explain why FA was increased in these two traits but not in the antenna. The location of the genes controlling development of the antenna on the sex chromosomes has also been suggested by Pertoldi et al. (2001b) due to the finding of significantly higher FA in the antenna of the males compared with the amphigonic females of both B. r. rossius and. HETEROZYGOSITY AND FA IN BACILLUS HYBRIDS The factors leading to the observed higher FA in the hybrid parthenogenetic populations of Bacillus compared with the amphigonic females probably have a very different cause than have the factors responsible

9 DEVELOPMENTAL INSTABILITY, HYBRIDIZATION AND HETEROZYGOSITY IN BACILLUS 257 for the higher FA in parthenogenetic B. r. rossius and compared with the amphigonic females. All hybrids were found to have considerably higher levels of heterozygosity compared with the amphigonic females, but the higher heterozygosity was not associated with lower levels of FA. The increased FA could arise from genetic incompatibilities of the heterospecific genomes affecting development in the hybrid individuals. These incompatibilities may evolve in the parental species by substitutions in one species of advantageous or neutral mutations that have never occurred in combination with those of other species and therefore have harmful effects when brought together in hybrids. On average, the alleles involved in these hybrid incompatibilities are thought to be partially recessive (Turelli & Orr, 2000; Presgraves, 2003). The trend seems to be that B. lynceorum has the highest levels of FA, which could be explained by the fact that B. lynceorum is affected by incompatibilities from three different genomes. Furthermore, the genetic incompatibilities may affect the development of all three traits, explaining why the antenna FA was increased in the hybrid parthenogenetic females but not in the parthenogenetic populations of B. r. rossius and. It could be argued that the observed differences in FA among the populations are due not to genetic factors (homozygosity on one side and hybridization on the other) but to differences in external environmental factors since the populations are allopatric. In a previous study of FA in parthenogenetic and amphigonic populations of B. r. rossius and, Pertoldi et al. (2001b) found a higher FA in the parthenogenetic females compared with the amphigonic females and the investigated populations were allopatric. In our study the investigated females of the parthenogenetic and amphigonic B. r. rossius were living in sympatry. Our results confirm the results found for the nonsympatric populations, which justifies our ascribing the observed differences in FA mainly to internal genetic factors rather than to external environmental factors. COMPARISON OF σ 2 P AMONG POPULATIONS Contrary to the study of Pertoldi et al. (2001b) we did not find significantly higher levels of σ 2 p in the amphigonic females compared with their parthenogenetic counterparts in both B. r. rossius and. In the amphigonic females, σ 2 p was of both genetic and environmental origin, whereas σ 2 p in the parthenogenetic B. r. rossius and was due only to σ 2 e and DI due to the lack of σ 2 g. The reason for the lack of increased σ 2 p in the amphigonic females, even though the genetic component contributes to σ 2 p, could therefore be the increased contribution of DI to σ 2 p in the parthenogenetic females. The homozygosity of the parthenogenetic females could also make them less able to buffer their development against environmental factors, increasing the contribution from σ 2 e. In fact, only two significant differences in σ 2 p were found (Table 2). Higher σ 2 g is one factor that could contribute to the observed higher σ 2 p in the amphigonic B. r. rossius compared with the amphigonic females of and the hybrid parthenogenetic B. whitei, since σ 2 g has been shown to be lower in amphigonic (Mantovani & Scali, 1991) and not present in B. whitei (Marescalchi et al., 1991). The three hybrid parthenogenetic populations are characterized by high heterozygosity but low genetic variability, and σ 2 p should therefore reflect mainly DI and σ 2 e. The pattern observed for σ 2 p reflected very well the pattern found for FA, with B. lynceorum having the highest σ 2 p, followed by B. atticus and then B. whitei; this could indicate that the environmental component contributes equally to the σ 2 p observed in the three hybrid populations (Table 2). The equal contribution from σ 2 e to σ 2 p in these three populations also supports our ascribing the observed differences in FA mainly to internal genetic factors rather than to differences in external environmental factors. CONCLUSION This study found no general relationship between heterozygosity and DI. In the nonhybrid species of B. rossius the homozygous parthenogenetic females were more asymmetric than were their amphigonic counterparts, which could be due to differences in heterozygosity. The level of FA was also considerably higher in the ten times more heterozygous hybrid species than it was in the amphigonic females. In these cases the increased FA probably arose due to genetic incompatibilities between the genomes of the parental species, leading to decreased DI in the hybrids. The interest in studying FA has very often been due to its relation to fitness. Our study found that hybridization might lead to increased levels of FA, but it should be stressed that this relation does not seem to be correlated with fitness. All three hybrid species have existed for more than 1 Myr (Scali et al., 2003) and they have in several cases been able to invade or even out-compete their parental species, for instance on Sicily, where B. whitei has invaded the habitat of B. g. grandii, and B. lynceorum has invaded the habitat of both B. atticus and B. whitei. In the hybrid species there seems, therefore, to be an uncoupling of the negative impact of hybridization on development from the impact on fitness. This could be due to the very

10 258 D. H. ANDERSEN ET AL. strong selection acting on the modified gametogenesis in the hybrid species during the first reproduction event securing optimal fitness. Further parthenogenetic reproducing individuals have a two-fold fitness advantage compared with sexually reproducing individuals, under stable environmental conditions (Barton & Charlesworth, 1998). It could also be speculated that the lack of long-distance dispersal in adult stick insects and larval instars, due to their strong addiction to their food plants (Mantovani, Passamonti & Scali, 2001), could result in inbreeding depression in some sexual populations, and this could give the parthenogenetic hybrid species an advantage. ACKNOWLEDGEMENTS We would like to thank two anonymous reviewers for valuable and useful comments on the manuscript. The work was supported by grants from the Danish Natural Sciences Research Council ( ) to Ditte Holm Andersen, and by grants from the Danish Natural Sciences Research Council ( and ) and the Marie Curies Fellowship of the European Community Host Development program under contract number HPMD-CT to Cino Pertoldi. REFERENCES Alibert P, Fel-Clair F, Manolakou K, Britton-Davidian J, Auffray J Developmental stability, fitness and trait size in laboratory hybrids between European subspecies of the house mouse. Evolution 51: Andersen DH, Pertoldi C, Scali V, Loeschcke V Intraspecific hybridization, developmental stability and fitness in Drosophila mercatorum. Evolutionary Ecology Research 4: Barton NH, Charlesworth B Why sex and recombination? Science 281: Bullini L Origin and evolution of animal hybrid species. Trends in Ecology and Evolution 9: Clarke GM, McKenzie JA Developmental stability of insecticide resistant phenotypes in blowfly; a result of canalising natural selection. Nature 325: Clarke GM, McKenzie JA Coadaptation, developmental stability, and fitness of insecticide resistance genotypes in the Australian sheep blowfly, Lucilia cuprina: a review. Acta Zoologica Fennica 191: Dowling TE, Secor C The role of hybridisation and introgression in the diversification of animals. Annual Reviews of Ecology and Systematics 28: Haldane JBS The measurement of variation. Evolution 9: 484. Hutchison DW, Cheverud JM Fluctuating asymmetry in tamarin (Saguinus) cranial morphology: intra- and interspecific comparisons between taxa with varying levels of genetic heterozygosity. Journal of Heredity 86: Keller MJ, Gerhardt HC Polyploidy alters advertisment call structure in gray treefrogs. Proceedings of the Royal Society of London. Series B, Biology Sciences 268: Kristensen TN, Pertoldi C, Andersen DH, Loeschcke V The use of fluctuating asymmetry and phenotypic variability as indicators of developmental instability: a test of a new method employing clonal organisms and high temperature stress. Evolutionary Ecology Research 5: Kristensen TN, Pertoldi C, Pedersen LD, Andersen DH, Bach L, Loeschcke V The increase of fluctuating asymmetry in a monoclonal strain of collombolans after chemical exposure discussing a new method for estimating the environmental variance. Ecological Indicators 4: Leary RF, Allendorf FW, Knudsen KL, Thorgaard GH Heterozygosity and developmental stability in gynogenetic diploid and triploid rainbow trout. Heredity 54: Lens L, Van Dongen S, Galbusera P, Schenck T, Matthysen E, Van De Casteele T Developmental instability and inbreeding in natural bird populations exposed to different levels of habitat disturbance. Journal of Evolutionary Biology 13: Lerner IM Genetic homeostasis. Edinburgh, UK: Oliver and Boyd. Manning JT, Chamberlain AT Fluctuating asymmetry, sexual selection and canine teeth in primates. Proceedings of the Royal Society of London. Series B, Biology Sciences 251: Mantovani B, Passamonti M, Scali V The mitochondrial cytochrome oxidase II gene in Bacillus stick insects: Ancestry of hybrids, androgenesis, and phylogenetic relationships. Molecular Phylogenetics and Evolution 19: Mantovani B, Scali V Allozyme characterization of Sardinian Bacillus rossius (Rossi) and B. atticus Brunner (Insecta Phasmatodea). Genetica 83: Mantovani B, Scali V, Tinti F Allozymatic characterisation and taxonomy of Sicilian Bacillus atticus (Insecta Phasmatodea). Biologisches Zentralblatt 109: Marescalchi O, Pijnacker LP, Scali V Cytology of parthenogenesis in Bacillus whitei and Bacillus lynceorum (Insecta Phasmatodea). Invertebrate Reproduction and Development 20: Marescalchi O, Scali V Chromosomal and NOR patterns in the polyclonal stick insect Bacillus atticus atticus (Insecta; Phasmatodea). Genome 40: Marescalchi O, Scali V Automictic parthenogenesis in the diploid-triploid stick insect Bacillus atticus and its flexibility leading to heterospecific diploid hybrids. Invertebrate Reproduction and Development 43: Mareschalchi O, Pijnacker LP, Scali V Automictic parthenogenesis and its genetic consequence in Bacillus atticus atticus (Insecta Phasmatodea). Invertebrate Reproduction and Development 24: Mesaros GB, Tucic B, Tucic N Directional and fluctuating asymmetry in sexual and asexual Otiorhynchus alpicola populations. Journal of Zoological Systematics and Evolutionary Research 32:

11 DEVELOPMENTAL INSTABILITY, HYBRIDIZATION AND HETEROZYGOSITY IN BACILLUS 259 Miller RG Simultaneous statistical inference. New York: McGraw-Hill. Mitton JB Enzyme heterozygosity, metabolism, and developmental stability. Genetica 89: Palmer AR Fluctuating asymmetry analyses: a primer. In: Markov TA, ed. Developmental instability: its origins and evolutionary implications. Dordrecht: Kluwer, Palmer AR, Strobeck C Fluctuating asymmetry: measurement, analysis, patterns. Annual Review of Ecological Systems 17: Parson PA Fluctuating asymmetry: an epigenetic measure of stress. Biological Review 65: Pertoldi C, Andersen DH, Kristensen TN, Loeschcke V Consequences of a reduction in genetic variance on developmental instability estimators. Evolutionary Ecology Research 5: Pertoldi C, Kristensen TN, Loeschcke V. 2001a. A new method for estimating environmental variability for clonal organisms, and the use of fluctuating asymmetry as an indicator of developmental instability. Journal of Theoretical Biology 210: Pertoldi C, Scali V, Loeschcke V. 2001b. Developmental instability in sexual reproducing and parthenogenetic populations of Bacillus rossius rossius and Bacillus rossius redtenbacheri. Evolutionary Ecology Research 3: Presgraves DC A fine-scale genetic analysis of hybrid incompatibilities in Drosophila. Genetics 163: Rice WR Analysing tables of statistical tests. Evolution 43: Rieseberg LH, Carney SE Transley review no Plant hybridisation. New Phytology 140: Ross KG, Robertson JL Developmental stability, heterozygosity, and fitness in two introduced fire ants (Solenopsis invicta and S. richteri) and their hybrid. Heredity 64: Scali V, Passamonti M, Mareschalchi O, Mantovani B Linkage between sexual and asexual lineages: genome evolution in Bacillus stick insects. Biological Journal of the Linnean Society 79: Scheerer PD, Thorgaard GH, Seeb JE Performance and developmental stability of triploid tiger trout (Brown trout FF Brook trout MM). Transactions of the American Fisheries Society 116: Turelli M, Orr HA Dominance, epistasis and the genetics of postzygotic isolation. Genetics 154: Zakharov VM Population phenogenetics: analysis of developmental stability in natural populations. Acta Zoologica Fennica 191: Zar JH Biostatistical analysis. New Jersey: Prentice Hall.