Indirect evolution of hybrid lethality due to linkage with selected locus in Mimulus guttatus.

Most species are superbly and intricately adapted to the environments in which they live. Adaptive evolution by natural selection is the primary force shaping biological diversity. Differences between closely related species in ecologically selected characters such as habitat preference, reproductive timing, courtship behavior, or pollinator attraction may prevent interbreeding in nature, causing reproductive isolation. But does ecological adaptation cause reproductive incompatibilities such as hybrid sterility or lethality? Although several genes causing hybrid incompatibilities have been identified, there is intense debate over whether the genes that contribute to ecological adaptations also cause hybrid incompatibilities. Thirty years ago, a genetic study of local adaptation to copper mine soils in the wildflower Mimulus guttatus identified a locus that appeared to cause copper tolerance and hybrid lethality in crosses to other populations. But do copper tolerance and hybrid lethality have the same molecular genetic basis? Here we show, using high-resolution genome mapping, that copper tolerance and hybrid lethality are not caused by the same gene but are in fact separately controlled by two tightly linked loci. We further show that selection on the copper tolerance locus indirectly caused the hybrid incompatibility allele to go to high frequency in the copper mine population because of hitchhiking. Our results provide a new twist on Darwin's original supposition that hybrid incompatibilities evolve as an incidental by-product of ordinary adaptation to the environment.

Vertical distribution of acid-volatile sulfide and simultaneously extracted metals in mangrove sediments from the Jiulong River Estuary, Fujian, China.

BACKGROUND: Acid-volatile sulfide (AVS) is operationally defined as sulfides in sediment, which are soluble in cold acid, and is reported as the most active part of the total sulfur in aquatic sediments. It is a key partitioning phase controlling the activities of divalent cationic heavy metals in sediment. METHODS: In order to examine this in mangrove environments, six sites were selected along the Jiulong River Estuary in Fujian, China, which had previously been reported to be polluted by heavy metals. Sediments were sampled from 0-60 cm depth at each site, and the spatial distribution of AVS and SEM (simultaneously extracted metals: copper, cadmium, zinc, and lead) were determined. RESULTS AND DISCUSSION: The results indicate that the AVS concentrations had a spatial variation, ranging from 0.24 to 16.10 pmol g(-1) sediment dry weight. The AVS concentration in the surface layer is lower than that of the deeper sediment, with peak values in the 15-30 cm horizon. There was no correlation between the AVS value and organic matter content or total dissolved salts, but a significant positive correlation of AVS with surface sediment (0-5 cm) moisture content was found. This indicates that water logged sediments tend to have a high AVS value. The amount of SEM was within the range of 0.33-2.80 micromol g(-1) sediment dry weight and decreased with sediment depth. CONCLUSIONS: There was a marked variation in AVS and SEM among different sites studied. AVS concentrations were generally lower in the surface sediments, while SEM concentrations slightly decreased with the depth. Higher concentrations of SEM found in the upper layers of the sediments confirm the earlier suggestions that this study area may suffer from increasing heavy metal pollution. RECOMMENDATIONS AND PERSPECTIVES: When monitoring environmental impacts by using AVS, the micro and large-scale spatial variation as well as vertical distribution need to be estimated to avoid misleading results. Both AVS and SEM concentrations in different sediment layers should be taken into account in assessing the potential impact of heavy metals on the biotic environment,

Do rewardless orchids show a positive relationship between phenotypic diversity and reproductive success?

Among rewardless orchids, pollinator sampling behavior has been suggested to drive a positive relationship between population phenotypic variability and absolute reproductive success, and hence population fitness. We tested this hypothesis by constructing experimental arrays using the rewardless orchid Dactylorhiza sambucina, which is dimorphic for corolla color. We found no evidence that polymorphic arrays had higher mean reproductive success than monomorphic arrays for pollinia removal, pollen deposition, or fruit set. For pollinia removal, monomorphic yellow arrays had significantly greater reproductive success, and monomorphic red the least. A tendency for yellow arrays to have higher pollen deposition was also found. We argue that differential population fitness was most likely to reflect differential numbers of pollinators attracted to arrays, through preferential long-distance attraction to arrays with yellow inflorescences. Correlative studies of absolute reproductive success in 52 populations of D. sambucina supported our experimental results. To our knowledge this is the first study to suggest that attraction of a greater number of pollinators to rewardless orchids may be of greater functional importance to population fitness, and thus ecology and conservation, than are the behavioral sequences of individual pollinators.

A quantitative trait loci analysis of zinc hyperaccumulation in Arabidopsis halleri.

The mechanisms of metal hyperaccumulation are still not understood, so we conducted a quantitative trait locus (QTL) analysis of zinc (Zn) hyperaccumulation in Arabidopsis halleri, in a cross between this and its sister species, A. petraea, in order to determine the number and approximate location of the genomic regions significantly contributing to this adaptation. An F2 cross between the two species was made, and the leaf Zn concentration of 92 individuals was measured at both low (10 microm) and high (100 microm) Zn concentrations. Twenty-five markers were established that were distributed on all of the eight chromosomes. Mapping of the markers established that they were essentially collinear with previous studies. QTLs exceeding a logarithm to the base 10 of the odds (LOD) value of 3 were found on chromosomes 4 (low Zn), 6 (high Zn) and 7 (both high and low Zn). Evidence for a QTL on chromosome 3 (low Zn) was also found. This analysis validates a previously used method of QTL analysis, based on microarray analysis of segregating families. Genes that have altered during the evolution of this character should also be QTL: this analysis calls into question a number of candidate genes from consideration as such primary genes because they do not appear to be associated with QTLs.

Comparison of gene expression in segregating families identifies genes and genomic regions involved in a novel adaptation, zinc hyperaccumulation.

One of the challenges of comparative genomics is to identify specific genetic changes associated with the evolution of a novel adaptation or trait. We need to be able to disassociate the genes involved with a particular character from all the other genetic changes that take place as lineages diverge. Here we show that by comparing the transcriptional profile of segregating families with that of parent species differing in a novel trait, it is possible to narrow down substantially the list of potential target genes. In addition, by assuming synteny with a related model organism for which the complete genome sequence is available, it is possible to use the cosegregation of markers differing in transcription level to identify regions of the genome which probably contain quantitative trait loci (QTLs) for the character. This novel combination of genomics and classical genetics provides a very powerful tool to identify candidate genes. We use this methodology to investigate zinc hyperaccumulation in Arabidopsis halleri, the sister species to the model plant, Arabidopsis thaliana. We compare the transcriptional profile of A. halleri with that of its sister nonaccumulator species, Arabidopsis petraea, and between accumulator and nonaccumulator F(3)s derived from the cross between the two species. We identify eight genes which consistently show greater expression in accumulator phenotypes in both roots and shoots, including two metal transporter genes (NRAMP3 and ZIP6), and cytoplasmic aconitase, a gene involved in iron homeostasis in mammals. We also show that there appear to be two QTLs for zinc accumulation, on chromosomes 3 and 7.

Within and between population variation for zinc and nickel accumulation in two species of Thlaspi (Brassicaceae).

In this study, the differences in zinc (Zn) and nickel (Ni) hyperaccumulation were investigated between three populations of Thlaspi pindicum together with genetic variation within populations of T. pindicum and Thlaspi alpinum var. sylvium, both serpentine endemics. Three experiments were conducted under standard conditions in hydroponic assay. Each experiment contained three treatments of metal: 100 microm Zn, 100 microm Ni, and combined 100/100 microm Zn/Ni. Genetic variation within populations was determined using maternal families. No genetic variation within populations was found for either Zn or Ni hyperaccumulation for both T. pindicum and T. alpinum var. sylvium, but differences were observed for both Zn and Ni hyperaccumulation between populations of T. pindicum. In combined Zn/Ni treatments, Zn inhibited Ni translocation in both species, which is unexpected considering that these species are serpentine endemics and well known Ni hyperaccumulators. The lack of genetic variation for metal hyperaccumulation is possibly due to inbreeding. Since Zn hyperaccumulation is not manifested in the field, inadvertent uptake of Zn is a plausible hypothesis for its preferential uptake.

The potential for adaptive evolution of pollen grain size in Mimulus guttatus.

We tested whether pollen grain size (PGS) shows heritable variation in three independent populations of Mimulus guttatus by imposing artificial selection for this character. In addition, we looked for correlated responses to selection in a range of 15 other floral characters. Heritable variation in PGS was found in all three populations, with heritabilities of between 19 and 40% (average 30%). After three generations, upward and downward lines differed on average by 30% in pollen volume. No consistent patterns of correlated response were found in other characters, indicating that PGS can respond to selective forces acting on PGS alone. Possible selection mechanisms on PGS in this species could include intermale selection, if large pollen grains produce more competitive gametophytes; or optimization of patterns of resource allocation, if local mate competition varies.

Does zinc protect the zinc hyperaccumulator Arabidopsis halleri from herbivory by snails?

• Hyperaccumulation of metals has been proposed to be a defence against herbivores. Here we investigated whether snails discriminated between plants of Arabidopsis halleri, and the F2 of the cross between A. halleri and A. petraea, on the basis of their internal Zn concentration • A. halleri and F2 plants were grown in four different Zn concentrations. Snails preferred F2 plants to A. halleri plants, and preferred plants that had been grown under low external Zn concentrations, but there was no evidence that they discriminated on the basis of internal Zn concentration • F2 plants were germinated on Zn contaminated soil and snails were allowed to eat the seedlings for a range of different time periods. The survivors were grown on, and tested for, Zn accumulation under standard conditions. No difference was found between the different time periods, indicating that the snails had eaten seedlings randomly • The results do not support a hypothesis that high internal Zn concentration protects seedlings from predation or herbivory by snails.

Ecological and genetic correlates of long-term population trends in the park grass experiment.

The Park Grass Experiment (PGE) is the longest-observed set of experimental plant communities in existence. Although the gross composition of the vegetation was at equilibrium over the 60-yr period from 1920 to 1979, annual records show that individual species exhibited a range of dynamics. We tested two hypotheses to explain why some species initially increased and why subsequently some of these (the outbreak species) decreased again. The study was designed around eight phylogenetically independent contrasts (PICs), each containing related species with different dynamics. Our first hypothesis was that persistent increasers and outbreakers have higher intrinsic rates of natural increase than control species (species without trends), allowing them to spread when interspecific competition is reduced by drought. This was tested by measuring establishment and seed production of species in field experiments, with and without interspecific competition. Seed production in outbreak species responded more strongly to release from interspecific competition than it did in either of the other groups of species. Our second hypothesis was that outbreak species eventually declined because they lacked the genetic variation necessary to adapt to the novel habitats to which they had initially spread. We tested this by measuring mating systems and genetic diversity in persistent and outbreak species in the PGE. In seven out of seven PICs tested, the outbreak species was more selfing than its persistent relative. There was a significant positive correlation between outcrossing rate and gene diversity. These results support roles for both ecological and genetic traits in long-term dynamics.

Within and between population genetic variation for zinc accumulation in Arabidopsis halleri.

• Hyperaccumulator plants in the field show significant variation in the metal concentration in their aerial parts, but little is known of the causes of this variation. This paper investigates the role of soil zinc (Zn) concentration and genetic variation in causing between and within population variation in Zn accumulation in Arabidopsis halleri. • Seed from 17 populations of A. halleri collected in central Europe were grown under standard conditions at three external Zn concentrations and tested for Zn concentration in the leaves. • Between population variation was highest at low external zinc concentrations. At 10 µm Zn some plants had very low leaf Zn concentrations, and were indistinguishable from nonaccumulators. However, at higher Zn concentrations, all plants showed hyperaccumulation. There were no differences in the accumulating abilities of populations from sites with different degrees of contamination. • Heritability of accumulation, determined for individual families from three populations, was quite high (25-50%), indicating that selection for increased accumulating ability should be possible, although selection would be easier at low external Zn concentrations. The Zn concentration of field collected plants was affected partly by plant genotype but not by the total soil Zn around their roots.

Can an increased copper requirement in copper-tolerant Mimulus guttatus explain the cost of tolerance? II. Reproductive phase.

Circumstantial evidence suggests that plants that have evolved metal tolerance are at a disadvantage on normal soil, i.e. there is a cost of tolerance. One hypothesis for the cause of this cost is that individuals have a greater requirement for copper, and so suffer micronutrient deficiency on normal soils, as a result of a reduced uptake, distribution and/or utilization of copper. We provided highly and less copper-tolerant plants of Mimulus guttatus Fischer ex DC. (the common monkey flower) with sub-optimal copper, and demonstrated the importance of copper as an essential micronutrient during the reproductive phase, both in the production of viable pollen and in seed set. We also looked at the effect of sub-optimal copper supply on the growth of the microgametophyte, and the efficiency with which seed was set. No evidence was found that highly tolerant plants have an increased copper requirement during the reproductive phase. This is in agreement with earlier work on Mimulus guttatus, which investigated the copper requirement of highly tolerant plants during vegetative growth and found that any differences in copper requirement were small. The 'metal requirement hypothesis' is, therefore, not the sole explanation for the cost of copper tolerance in M. guttatus.

NEGATIVE FREQUENCY-DEPENDENT SELECTION BY POLLINATORS ON ARTIFICIAL FLOWERS WITHOUT REWARDS.

Many species of nonmodel deceptively pollinated orchids are polymorphic for corolla color. These species are pollinated by naive insects searching for nectar, and are not mimics. It has been suggested that the foraging behavior of insect pollinators during the avoidance learning process results in these stable corolla color polymorphisms; for this to occur pollinators must induce negative frequency-dependent selection on corolla color. Therefore the hypothesis that pollinator behavior results in a preference for rare color morphs of deceptive species was tested experimentally. Bumblebees (Bombus terrestris) foraged in the laboratory on arrays of artificial flowers with different corolla color morphs. Morphs were varied in frequency, and bumblebee preferences were recorded on arrays where morphs did and did not contain sucrose solution rewards. Bumblebees preferred the most common color morph when flowers contained sucrose solution rewards, but overvisited rare morphs when sampling flowers that contained no rewards. Bumblebees also tended to move between unlike color morphs when these were unrewarding, suggesting that a probabilistic sampling strategy was adopted. Thus experiments demonstrated that pollinator behavior could result in a selective advantage for rare color morphs of plant species that are pollinated by deception without mimicry, which would induce negative frequency-dependent selection on corolla color. The observed pollinator behavior could allow stable corolla color polymorphisms to be maintained by selection in nonmodel deceptively pollinated species.

Can an increased copper requirement in copper-tolerant Mimulus guttatus explain the cost of tolerance?: I. Vegetative growth.

If metal tolerant plants, by virtue of their tolerance mechanism, are less efficient at the uptake, distribution or utilization of metals then essential micronutrient deficiency may occur at the low levels of metal supply found on non-mine soils. This argument forms the basis of the metal requirement hypothesis put forward to explain the lower fitness of tolerant individuals on uncontaminated soil, the so called'cost of tolerance'. In this paper, copper balance was investigated in Mimulus guttatus Fischer ex. DC (the yellow monkey flower) for plants with or without the major tolerance gene which confers primary tolerance, and plants with few or many modifier genes which control degree of tolerance. No conclusive evidence to support an increased copper requirement in plants with the major tolerance gene, and/or many modifier genes was shown. Any differences in copper requirement found during vegetative growth were small, and were deemed insufficient to explain the apparent cost of tolerance.

DEVELOPMENTAL SELECTION IN RESPONSE TO ENVIRONMENTAL CONDITIONS OF THE MATERNAL PARENT IN MIMULUS GUTTATUS.

Copper tolerance is expressed in the diploid sporophyte as well as the microgametophyte of Mimulus guttatus. Previous studies, based on reproductive output, suggested that selection for copper tolerance could occur within the pistil. The objective of this study was to determine if selection within the pistil can increase sporophytic tolerance to copper and to determine whether this selection occurs pre- or postzygotically. Mixtures of pollen from copper tolerant or sensitive sources or from plants heterozygous for tolerance to copper were applied at two intensities to plants cloned and grown in control or copper supplemented solutions. The proportion of copper tolerant progeny showed a small, 7%, but significant increase when pollen recipients were grown with added copper. Comparisons of the numbers of tolerant progeny, as well as various components of reproduction, following light and heavy pollinations suggested that microgametophytic selection was unlikely to account for this increase. However, the 8 to 10% decrease in the seed/zygote ratio, compared to control values, was sufficient to account for the difference in proportion of copper tolerant progeny from control and copper treated plants. Thus, it appeared likely that selection for copper tolerance could occur within the pistil, and that much of this selection occurred postzygotically through the early failure of developing seeds.

The genetics of metal tolerance in vascular plants.

In many parts of the world soils are detrimental to plant growth owing to elevated levels of metal ions, caused either by natural processes or by the result of man's activities. Many plants have evolved ecotypes or varieties that are able to grow more-or-less normally on these soils. This paper reviews our knowledge of the genetics of this phenomenon. The nature of tolerance and the problems of its measurement are discussed. Tolerance is frequently measured by an index produced by comparing growth in a contaminated environment with growth in a control environment. It is argued that this measurement is inappropriate for many genetical studies, and that it is frequently more useful to use growth at a single critical level of metal as a measure of tolerance. Polygenic inheritance provides a null hypothesis that has to be tested in a genetical analysis. Examples of major genes for tolerance to aluminium, arsenic, boron, cadmium, copper and manganese are discussed. Even where major genes have been demonstrated, it is probable that other minor genes, 'modifiers', are present as well. Because of the nature of tolerance as a character, dominance and epistasis are likely to vary with the level of metal at which an analysis is performed. Tolerance is generally found to be dominant at some levels of the metal. Studies which have mapped tolerance genes, particularly to aluminium and salt, are discussed. The specificity of tolerance is a matter of some confusion. Some studies indicate that tolerances evolve independently to different metals, but others have suggested that tolerance to one metal may often confer a degree of tolerance to some other metals. Very little is known about the molecular genetics of tolerance, and the mechanisms of tolerance to most metals. The possible role of phytochelatins and metallothionein-like proteins in metal tolerance is discussed. The distribution of tolerance in natural populations suggests that tolerance is a disadvantage in uncontaminated environments, but how this 'cost' arises is not known. There is some evidence that the disadvantage to tolerance may be associated more with the modifiers of tolerance than with the primary tolerance gene. The study of the genetics of tolerance is of importance in planning breeding programmes to produce tolerant crops for use in areas where metal contamination is a limiting factor in productivity. It can also assist in understanding the mechanisms of tolerance, as exemplified by the study of the mechanism of arsenic tolerance in Holcus lanatus. Important areas for further research are discussed. Contents Summary 541 I. Introduction 542 II. Introduction 542 III. Transmission genetics of tolerance 544 IV. Specificity of tolerance 550 V. Molecular genetics of tolerance 552 VI. Ecological genetics of tolerance 553 VII. Conclusions 555 Acknowledgements 556 References 556.

The mechanisms of arsenate tolerance in Deschampsia cespitosa (L.) Beauv. and Agrostis capillaris L.: Adaptation of the arsenate uptake system.

Arsenate influx isotherms in Deschampsia cespitosa (L.) Beauv. and Agrostis capillaris L. showed that tolerance to arsenate in these grasses is achieved at least in part by adaptation of the arsenate uptake system, which leads to reduced influx of arsenate in arsenate-tolerant plants. In D. cespitosa, this reduction in arsenate ioflux was by suppression of the Vmax of the high-affinity uptake system and by an increase in the Km of the low-affinity uptake system. In A. capillaris the reduction in arsenate uptake in arsenate-tolerant plants was by a decrease in the Vmax of both uptake systems and by an increase in the Km of the high-affinity uptake system. The differences in the rates of arsenate influx between tolerant and non-tolerant plants was much greater for D. cespitosa than for A. capillaris. The mechanism of arsenate tolerance in D. cespitosa and A. capillaris was the same mechanism of tolerance that had been demonstrated in arsenate-tolerant Holcus lanatus L. Reduced arsenate influx by adaptation of the arsenate uptake mechanism is a mechanism of tolerance that has evolved in three separate species.

DIFFERENTIAL SEED PRODUCTION IN MIMULUS GUTTATUS IN RESPONSE TO INCREASING CONCENTRATIONS OF COPPER IN THE PISTIL BY POLLEN FROM COPPER TOLERANT AND SENSITIVE SOURCES.

Selection can occur in the pistil, during a series of stages that include both pre-zygotic (pollen germination, pollen tube growth, and fertilization) and post-zygotic events. This study explores the extent to which selection, at this level, could be due to the environmental conditions under which the maternal parent is growing. Five plants of Mimulus guttatus, tolerant to copper, were vegetatively cloned and each clone was grown in control and in solutions to which copper was added. The maternal plants received pollen from plants either tolerant or sensitive to copper. Seeds and ovules were counted to estimate the number of seeds/capsule, the seed/ovule ratio, the percent fertilization, and the proportion of zygotes aborting for each clone, treatment and pollen source combination. There were large differences among the pollen recipients for each of the measurements. However, there was a consistent pattern to seed production depending on the pollen source and copper treatment. The seed/ovule ratio was unaffected if pollen came from tolerant sources but was reduced by an average of 24% for both copper supplemented treatments if pollen came from copper sensitive sources. Thus, the data indicated that selection due to environmental factors could occur within the pistil. Differences in percent fertilization were not statistically significant, but the seed/zygote ratio showed a pattern that was similar to seed production suggesting that abortion of immature seeds was responsible for most of the difference in seed production.

THE DISTRIBUTION OF POSTMATING REPRODUCTIVE ISOLATING GENES IN POPULATIONS OF THE YELLOW MONKEY FLOWER, MIMULUS GUTTATUS.

Postmating reproductive isolating barriers are generally believed to arise as the chance by-product of genetic differentiation. The classical view is that these barriers normally involve differentiation at many loci, and therefore require long periods of allopatric isolation. The formal genetics of, and the distribution of genes responsible for, such barriers are known in very few cases. This paper examines the distribution of the genes responsible for two different postmating barriers in 18 populations of the yellow monkey flower, Mimulus guttatus. The genetic relatedness of the populations was measured by a morphometrical analysis. Widespread polymorphism was found for three of the four components of the two genetic systems responsible for the two barriers, with at least 13 populations possessing genes for one or both of the barriers. In one system (the C7/U8 system; Christie and Macnair, 1984), the distribution of the two component genes was correlated with the morphometrical similarity and geographical location of the populations. This pattern could be produced by a historical association or by an adaptive response. In the other system (the Cerig/C10 system; Macnair and Christie, 1983), the genes were more widely dispersed, and there was no obvious morphometrical or geographical association. Populations possessing the complementary factors causing partial reproductive isolation are not always widely separated geographically. These results indicate that the spread of postmating reproductive isolating genes through drift, selection, or hitchhiking could readily cause reproductive isolation to evolve in this species.>.