Chemical and biological characterization of hazardous industrial waste. I. Prokaryotic bioassays and chemical analysis of a wood-preserving bottom-sediment waste

Prokaryotic bioassays, capable of detecting point mutations and lethal damage to DNA, and a GC/MS/Data System analysis were employed to evaluate the genotoxic characteristics of wood-preserving bottom sediment. Organic compounds in the waste were initially extracted with dichloromethane and then fractionated by liquid-liquid extraction into acid, base and neutral fractions. The crude extract and each of 3 subfractions were tested in 4 strains of S. typhimurium to detect point mutations and 6 strains of B subtilis to detect lethal damage to DNA. The assay using S. typhimurium responded to indirect-acting mutagens in the crude extract and all 3 primary fractions, with the maximum mutagenic response of 181 net revertants induced by the base fraction at a dose of 500 micrograms/plate. In the DNA-repair assay, the survival ratio for the repair-deficient strain recE4 when compared to the repair-proficient strain 168 wt was 0.17 and 0.09 in the acid and base fractions, respectively, at a dose of 100 micrograms/plate. Potentially genotoxic compounds identified in the waste fractions by GG/MS/DS analysis include acenaphthylene, pentachlorophenol, methyl phenanthrene, fluoranthene and pyrene. However, it appears that these identified chemicals did not contribute significantly to the observed mutagenic activity of the sample extracts.     

Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program

The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

II. Haploid assay systems and overall response of all systems A report of the U.S. EPA gene-tox program

The status of the Aspergillus systems, with respect to their usefulness in screening chemicals for genotoxic effects, was evaluated using information available in the open literature. A total of 179 references were evaluated; 58 contained relevant information for the purpose of determining the genotoxic status of a chemical. To simplify presentation, these papers were divided into two groups. The first group of papers analyzed the effect of chemicals on mitotic segregation in Aspergillus (reported in Käfer et al., this issue). The second group of papers, reporting on the response of the haploid Aspergillus systems to various agents, are reviewed in this paper which also contains an overall summary of the responses of an Aspergillus system and compares these results with those obtained from in vivo carcinogenicity studies.The publications reporting on haploid mutational systems fall into one of three main systems. The methionine suppressor system, the 2-thioxanthine system, and the arginine system. The first two systems are multiallelic forward-mutational systems that respond to agents inducing base-pair changes and small deletions. No report on the nature of the reverse mutations in the arginine system could be found in the literature, although the spontaneous reversion rate would indicate intra-locus reversion rather than forward mutation at suppressor loci as is the case for the methionine assay.Adequate numerical information for 124 chemicals is available from all the acceptable Aspergillus papers listed at the Environmental Mutagen Information Center, Oak Ridge National Laboratory (1965–1978). For 27 of these compounds, corresponding animal bioassays for carcinogenesis were available. The same response in 95% of the chemicals was obtained with both eukaryotic bioassays. 2 of these responses were negative; the remaining 23 were positive for both types of tests. Of the 23 positive responses, 2 required metabolic activation. 5 of the compounds giving positive responses in Aspergillus systems and the carcinogenicity bioassays were not mutagenic for Salmonella.The general conclusion of the working group was that a combination of the simple haploid mutational system, coupled with the uniqueness of diploid analysis, depicts the true value of Aspergillus nidulans for both screening and in-depth analysis of genotoxic chemicals (effects on genes, chromosomes, and “machinery” associated with their segregation).     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.     

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.     

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.     

Frequency of induced mutations at the haploid and diploid levels inNicotiana tabacum L.

The frequencies of chlorophyll mutants were investigated in anther cultures derived from mutagen-treated plants ofN. tabacum cv. Samsun (haploid level) and in the seed offspring from the same treated plants (diploid level). Comparison of the induced mutation frequencies at the haploid and diploid levels demonstrated that selection existed against the haploid embryoids with induced chlorophyll deficient mutations. The diploid vegetative stage with phenotypic expression of the chlorophyll mutation was more vital than the haploid one. The suitability of anther cultures for studying induced mutagenesis is discussed.     

Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms

The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells.     

Comparative evaluation of the genotoxic properties of potassium bromate and potassium superoxide in V79 Chinese hamster cells

The genotoxic potential of two oxidizing compounds, potassium bromate and potassium superoxide, was comparatively tested in various genotoxicity tests with V79 Chinese hamster cells. Both substances clearly induced cytotoxicity, chromosome aberrations and increased DNA migration in the alkaline comet assay. Using a modified comet assay protocol with FPG protein, a DNA repair enzyme which specifically nicks DNA at sites of 8-oxoguanines and formamidopyrimidines, we detected oxidative DNA base damage only after potassium bromate treatment. HPLC analysis also revealed significantly increased levels of 8-oxodeoxyguanosine after potassium bromate treatment but not after potassium superoxide treatment. Furthermore, potassium bromate clearly induced gene mutations at the HPRT locus while potassium superoxide only had a small effect on HPRT mutant frequencies. Molecular analysis of potassium bromate-induced mutations indicated a high portion of deletion mutations. Three out of four point mutations were G to T transversions which typically arise after replication of 8-oxoguanine. Our results suggest that the two oxidizing compounds induce specific patterns of genotoxic effects that reflect the types of DNA alterations induced by different reactive oxygen species (ROS).     

Mutability of the LYS2 gene in diploid saccharomycete yeasts. I. The occurrence of spontaneous mutants and mutants induced by x-ray and ultraviolet radiation

More than 3000 spontaneous and induced lys2 mutants were obtained in haploid and diploid strains of yeast Saccharomyces. The ability to utilize alpha-aminoadipate was used for lys2 mutant screening. The spontaneous and induced mutation rates were measured in haploid and diploid strains. Mitotic segregation of pho1 marker linked to LYS2 was studied in lys2 mutants obtained in diploid strains. Fertility of diploid lys2 mutants was tested. The conclusion to be drawn from the data presented is that mutations appeared in one of two homologous chromosomes and then segregated by mitotic homozygotization.     

Ploidy controls the success of mutators and nature of mutations during budding yeast evolution

BACKGROUND: We used the budding yeast Saccharomyces cerevisiae to ask how elevated mutation rates affect the evolution of asexual eukaryotic populations. Mismatch repair defective and nonmutator strains were competed during adaptation to four laboratory environments (rich medium, low glucose, high salt, and a nonfermentable carbon source). RESULTS: In diploids, mutators have an advantage over nonmutators in all conditions, and mutators that win competitions are on average fitter than nonmutator winners. In contrast, haploid mutators have no advantage when competed against haploid nonmutators, and haploid mutator winners are less fit than nonmutator winners. The diploid mutator winners were all superior to their ancestors both in the condition they had adapted to, and in two of the other conditions. This phenotype was due to a mutation or class of mutations that confers a large growth advantage during the respiratory phase of yeast cultures that precedes stationary phase. This generalist mutation(s) was not selected in diploid nonmutator strains or in haploid strains, which adapt primarily by fixing specialist (condition-specific) mutations. In diploid mutators, such mutations also occur, and the majority accumulates after the fixation of the generalist mutation. CONCLUSIONS: We conclude that the advantage of mutators depends on ploidy and that diploid mutators can give rise to beneficial mutations that are inaccessible to nonmutators and haploid mutators.     

The comet assay: a method to measure DNA damage in individual cells

We present a procedure for the comet assay, a gel electrophoresis-based method that can be used to measure DNA damage in individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell. DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring (e.g., DNA damage in earthworms crawling through toxic waste sites) to measurement of DNA damage in specific genomic sequences. This protocol can be completed in fewer than 24 h.     

The ovulation and activation of primary and secondary oocytes in LT/Sv strain mice

Eggs were isolated from the oviducts or ovaries of LT/Sv strain mice in order to investigate the pathways taken by them following spontaneous or induced parthenogenetic activation. The chromosome preparations from the ovarian oocytes that matured in vitro to metaphase I were all morphologically normal. Of 42 recently ovulated eggs that failed to activate parthenogenetically in culture, 57% on nuclear densitometric analysis were found to have the normal 2C amount of DNA, and 1N (haploid) number of chromosomes present, and were arrested at metaphase II. Somewhat unexpectedly, 43% had a 4C amount of DNA, and 2N (diploid) number of chromosomes present, had been arrested at metaphase I, and were evidently ovulated as primary oocytes. Following parthenogenetic activation, the majority of oocytes extruded a polar body and developed a single pronucleus. The activated eggs could be divided into two sub-populations according to the diameter (and therefore volume) of the pronucleus—in one group this was about one-third greater than in the other. The chromosome constitution of the two groups was determined separately at the first cleavage mitosis. Those with a normal-sized pronucleus were invariably haploid, while those with an enlarged pronuclear volume were invariably found to be diploid. The chromosomes in the diploid spreads often appeared to be associated in homologous pairs. We conclude that almost uniquely in LT/Sv strain females eggs may be activated parthenogenetically at either stage of meiotic maturation giving rise to diploid or haploid embryos, respectively.     

The immunosuppressive drug leflunomide affects mating-pheromone response and sporulation by different mechanisms in Saccharomyces cerevisiae

Leflunomide (LFM) is a novel anti-inflammatory and immunosuppressive drug, and inhibits the growth of cytokine-stimulated lymphoid cells in vitro. The effect of LFM on haploid and diploid cells of Saccharomyces cerevisiae was investigated to elucidate the molecular mechanism of action of the drug. Using a halo assay, LFM was shown to enhance the cell cycle arrest of haploid cells induced by mating pheromone alpha-factor. LFM also inhibited sporulation of diploid cells completely. S. cerevisiae genes which were cloned to suppress the anti-proliferative effect when present in increased copy number were introduced and examined for their activity to suppress the effect of LFM. Out of them, MLF4/SSH4, was found to suppress the sporulation-inhibitory effect of LFM. However, MLF4 failed to suppress the enhancing effect of LFM on pheromone response. Thus, LFM is suggested to act on haploid and diploid cells by different mechanisms.     

The capacity of Drosophila for detecting relevant genetic damage

For the detection and study of mutagenic agents, Drosophila offers many advantages. It is a higher organism with a short generation time that is cheap and easy to breed in large numbers. The simple genetic testing methods provide unequivocal answers about the whole spectrum of relevant genetic damage. A comparison of the detection capacity of assays sampling different kinds of genetic damage revealed that various substances are highly effective in inducing mutations, but do not produce chromosome breakage effects at all, or only at much higher concentrations than those required for mutation induction. Of the different assay systems available, the classical sex-linked recessive lethal test thus deserves priority, in view of its superior capacity to detect mutagens. Of practical importance is also its high sensitivity, because a large number of loci in one-fifth of the genome is tested for newly induced forward mutations, including small deletions.Drosophila is capable of carrying out the same metabolic activation reactions as the mammalian liver. An additional advantage, in this respect, is the capacity of Drosphila for detecting short-lived activation products, because intracellular activation occurs within the spermatids and spermatocytes. These properties make the test for recessive sex-linked lethals a useful tool for verifying results obtained in the pre-screening of potential mutagens with fast microbial assay systems.In studies on non-disjunction, detailed genetic analysis of the induced changes is possible, and these may shed light on the mechanisms involved.A new adaptation of the bithorax transvection method by Mendelson permits the recovery of high yields of chromosome aberrations in a fast one-generation test.     

Ribosomal DNA magnification in Saccharomyces cerevisiae.

Strains monosomic for chromosome I of Saccharomyces cerevisiae contain 25 to 35% fewer rRNA genes than do normal diploid strains. When these strains are repeatedly subcultured, colonies are isolated that have magnified their number of rRNA genes to the diploid amount while remaining monosomic for chromosome I. We have determined the amount of DNA complementary to rRNA in viable haploid spores derived from a magnified monosomic strain. Some of these haploids contained 24 to 48% more rRNA genes than a normal euploid strain. These extra genes may be responsible for the increased number of rRNA genes in the strain monosomic for chromosome I. Genetic analysis of the haploids containing extra rRNA genes suggested that these genes are linked to chromosomal DNA and are heterozygous. They were not closely linked to any centromere and were not located on chromosome I. Furthermore, all the DNA complementary to rRNA in one of these haploid strains with magnified rRNA genes sedimented at a chromosomal molecular weight, consistent with chromosomal linkage. In addition, several new mutations mapping on chromosome I were used to show that ribosomal DNA magnification was not due to a chromosome I duplication.     

Masking and purging mutations following EMS treatment in haploid, diploid and tetraploid yeast (Saccharomyces cerevisiae)

The yeast, Saccharomyces cerevisiae, was used as a model to investigate theories of ploidy evolution. Mutagenesis experiments using the alkylating agent EMS (ethane methyl sulphonate) were conducted to assess the relative importance that masking of deleterious mutations has on response to and recovery from DNA damage. In particular, we tested whether cells with higher ploidy levels have relatively higher fitnesses after mutagenesis, whether the advantages of masking are more pronounced in tetraploids than in diploids, and whether purging of mutations allows more rapid recovery of haploid cells than cells with higher ploidy levels. Separate experiments were performed on asexually propagating stationary phase cells using (1) prototrophic haploid (MAT alpha) and diploid (MATa/alpha) strains and (2) isogenic haploid, diploid and tetraploid strains lacking a functional mating type locus. In both sets of experiments, haploids showed a more pronounced decrease in apparent growth rate than diploids, but both haploids and diploids appeared to recover very rapidly. Tetraploids did not show increased benefits of masking compared with diploids but volume measurements and FACScan analyses on the auxotrophic strains indicated that all treated tetraploid strains decreased in ploidy level and that some of the treated haploid lines increased in ploidy level. Results from these experiments confirm that while masking deleterious mutations provides an immediate advantage to higher ploidy levels in the presence of mutagens, selection is extremely efficient at removing induced mutations, leading growth rates to increase rapidly over time at all ploidy levels. Furthermore, ploidy level is itself a mutable trait in the presence of EMS, with both haploids and tetraploids often evolving towards diploidy (the ancestral state of S. cerevisiae) during the course of the experiment.     

The evolution of sex chromosomes in organisms with separate haploid sexes

The evolution of dimorphic sex chromosomes is driven largely by the evolution of reduced recombination and the subsequent accumulation of deleterious mutations. Although these processes are increasingly well understood in diploid organisms, the evolution of dimorphic sex chromosomes in haploid organisms (U/V) has been virtually unstudied theoretically. We analyze a model to investigate the evolution of linkage between fitness loci and the sex‐determining region in U/V species. In a second step, we test how prone nonrecombining regions are to degeneration due to accumulation of deleterious mutations. Our modeling predicts that the decay of recombination on the sex chromosomes and the addition of strata via fusions will be just as much a part of the evolution of haploid sex chromosomes as in diploid sex chromosome systems. Reduced recombination is broadly favored, as long as there is some fitness difference between haploid males and females. The degeneration of the sex‐determining region due to the accumulation of deleterious mutations is expected to be slower in haploid organisms because of the absence of masking. Nevertheless, balancing selection often drives greater differentiation between the U/V sex chromosomes than in X/Y and Z/W systems. We summarize empirical evidence for haploid sex chromosome evolution and discuss our predictions in light of these findings.