A linkage map reveals a complex basis for segregation distortion in an interpopulation cross in the moss Ceratodon purpureus

We report the construction of a linkage map for the moss Ceratodon purpureus (n = 13), based on a cross between geographically distant populations, and provide the first experimental confirmation of maternal chloroplast inheritance in bryophytes. From a mapping population of 288 recombinant haploid gametophytes, genotyped at 121 polymorphic AFLP loci, three gene-based nuclear loci, one chloroplast marker, and sex, we resolved 15 linkage groups resulting in a map length of approximately 730 cM. We estimate that the map covers more than three-quarters of the C. purpureus genome. Approximately 35% of the loci were sex linked, not including those in recombining pseudoautosomal regions. Nearly 45% of the loci exhibited significant segregation distortion (alpha = 0.05). Several pairs of unlinked distorted loci showed significant deviations from multiplicative genotypic frequencies, suggesting that distortion arises from genetic interactions among loci. The distorted autosomal loci all exhibited an excess of the maternal allele, suggesting that these interactions may involve nuclear-cytoplasmic factors. The sex ratio of the progeny was significantly male biased, and the pattern of nonrandom associations among loci indicates that this results from interactions between the sex chromosomes. These results suggest that even in interpopulation crosses, multiple mechanisms act to influence segregation ratios.     

Tip-growing cells of the moss Ceratodon purpureus Are gravitropic in high-density media

Gravity sensing in plants and algae is hypothesized to rely upon either the mass of the entire cell or that of sedimenting organelles (statoliths). Protonemata of the moss Ceratodon purpureus show upward gravitropism and contain amyloplasts that sediment. If moss sensing were whole-cell based, then media denser than the cell should prevent gravitropism or reverse its direction. Cells that were inverted or reoriented to the horizontal displayed distinct negative gravitropism in solutions of iodixanol with densities of 1.052 to 1.320 as well as in bovine serum albumin solutions with densities of 1.037 to 1.184 g cm(-3). Studies using tagged molecules of different sizes and calculations of diffusion times suggest that both types of media penetrate through the apical cell wall. Estimates of the density of the apical cell range from 1.004 to 1.085. Because protonemata grow upward when the cells have a density that is lower than the surrounding medium, gravitropic sensing probably utilizes an intracellular mass in moss protonemata. These data provide additional support for the idea that sedimenting amyloplasts function as statoliths in gravitropism.     

Amyloplasts as possible statoliths in gravitropic protonemata of the moss Ceratodon purpureus

The kinetics of gravitropism and of amyloplast sedimentation were studied in dark-grown protonemata of the moss Ceratodon purpureus (Hedw.) Brid. The protonemata grew straight up at a rate of 20–25 m·h^– in nutrient-supplemented agar. After they were oriented to the horizontal, upward curvature was first detected after 1–1.5 h and reached 84° by 24 h. The tip cells exhibited an amyloplast zonation, with a tip cluster of nonsedimenting amyloplasts, an amyloplast-free zone, and a zone with pronounced amyloplast sedimentation. This latter zone appears specialized more for lateral than for axial sedimentation since amyloplasts sediment to the lower wall in horizontal protonemata but do not fall to the basal wall in vertical protonemata. Amyloplast sedimentation started within 15 min of gravistimulation; this is within the 12–17-min presentation time. The data support the hypothesis that some amyloplasts function as statoliths in these cells.This work was supported by the National Aeronautics and Space Administration grant NAGW-780. We thank Professor E. Hartmann and J. Schwuchow for providing Ceratodon cultures, Dr. John Z. Kiss and Jeff Young for valuable discussions, and Professor Rainer Hertel (University of Freiburg, FRG) for bringing this material to our attention.     

Photoprotective pigment as an adaptive strategy in the antarctic moss Ceratodon purpureus

Summary Variation in leaf pigmentation from green to ginger is observed for Ceratodon purpureus (Hedw.) Brid. in Antarctica. Electron microscopy of ginger and green leaves reveals less thylakoid stacking, a response to greater light exposure, in the ginger leaves. In extremely exposed sites C. purpureus has low chlorophyll a/b ratios which correlate with decreased 77K chlorophyll fluorescence, indicating damage to chlorophyll a. Pigment analysis of ginger moss shows that even when the chlorophyll a/b ratio has not decreased the pigment composition differs from green moss. The increase in anthocyanin and decrease in chlorophyll concentrations largely account for the visual change from green to ginger. The ratio of total carotenoid to chlorophyll varies from 0.35 in green moss to 0.55 in the ginger moss, with violaxanthin increased preferentially. Since these changes in pigmentation are consistent with photoprotection and they are linked to light dependent variations in chloroplast structure, it appears that photoprotective pigments are a useful adaptation for the bright Antarctic environment.     

RAPD profiling of genetic diversity in two populations of the moss Ceratodon purpureus in Victoria Land, Antarctica

Isolates of the moss Ceratodon purpureus were collected down a channel formed by a meltstream waterfall at Granite Harbour in Southern Victoria Land, Antarctica. The RAPD technique was used to analyse the extent of genetic variation within clumps, between clumps, and between this population and specimens of the same species from two other areas in Antarctica (one a few hundred metres away, the other at Edmonson Point, 300 km further north) and from Sydney, Australia. Genetic variation was detected within and among clumps, with some spatial structure to the population within the channel. Isolates from the nearby location were quite closely related, whereas those from Edmonson Point formed an outgroup on a phylogenetic tree of relatedness. Received: 10 June 1997 / Accepted: 30 September 1997     

Blue light- and genetically-reversed gravitropic response in protonemata of the moss Ceratodon purpureus

In darkness, protonemal filaments of Ceratodon purpureus (Brid.) grow negatively gravitropically (upwards). Red light induces a positive phototropic response mediated by the photoreceptor phytochrome. A red light treatment also has an inhibitory effect on the gravitropic response, an effect also mediated by phytochrome. In this study the effects of blue light on phototropism and on gravitropism were analysed. Unilateral blue light resulted in only a weak phototropic response, but markedly randomised growth direction. Blue light given together with a gravitropic stimulus reversed the gravitropism, changing it from negative to positive (filaments grow downward). The effect of blue light was also analysed with the mutant ptr116, which is defective in the biosynthesis of the phytochrome chromophore, and in a newly isolated mutant wwr2, which is positively gravitropic in darkness. Blue light induced the same reversal of gravitropism in ptr116 as in the wild type, indicating that phytochrome is not involved in this process. In wwr2 the direction of gravitropism was unaltered by the blue light treatment. Light also affects chlorophyll content and the size of plastids, potential statoliths for gravitropism. Red light induced an increase in plastid size and chlorophyll content in the wild type but not in ptr116. Blue light induced a similar change in wild type plastids. It seems as though light-induced alterations of gravitropism are not simply mediated by alterations in plastid properties, and that red light and blue light evoke fundamentally different responses. Received: 11 July 1997 / Accepted: 30 January 1998     

Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus

 In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one. Received: 8 March 1999 / Accepted: 30 July 1999     

A positively gravitropic mutant mirrors the wild-type protonemal response in the moss Ceratodon purpureus

Wild-type Ceratodon purpureus (Hedw.) Brid. protonemata grow up in the dark by negative gravitropism. When upright wild-type protonemata are reoriented 90°, they temporarily grow down soon after reorientation (“initial reversal”) and also prior to cytokinesis (“mitotic reversal”). A positively gravitropic mutant designated wrong-way response (wwr-1) has been isolated by screening ultraviolet light-mutagenized Ceratodon protonemata. Protonemata of wwr-1 reoriented from the vertical to the horizontal grow down with kinetics comparable to those of the wild-type. Protonemata of wwr-1 also show initial and mitotic reversals where they temporarily grow up. Thus, the direction of gravitropism, initial reversal, and mitotic reversal are coordinated though each are opposite in wwr-1 compared to the wild-type. Normal plastid zonation is still maintained in dark-grown wwr-1 apical cells, but the plastids are more numerous and plastid sedimentation is more pronounced. In addition, wwr-1 apical cells are wider and the tips greener than in the wild-type. These data suggest that a functional WWR gene product is not necessary for the establishment of some gravitropic polarity, for gravitropism, or for the coordination of the reversals. Thus, the WWR protein may normally transduce information about cell orientation. Received: 4 November 1996 / Accepted: 26 November 1996     

Irradiance-dependent regulation of gravitropism by red light in protonemata of the moss Ceratodon purpureus

Apical cells of protonemata of the moss Ceratodon purpureus (Hedw.) Brid. are negatively gravitropic in the dark and positively phototropic in red light. Various fluence rates of unilateral red light were tested to determine whether both tropisms operate simultaneously. At irradiances ≥140 nmol m⁻² s⁻¹ no gravitropism could be detected and phototropism predominated, despite the presence of amyloplast sedimentation. Gravitropism occurred at irradiances lower than 140 nmol m⁻² s⁻¹ with most cells oriented above the horizontal but not upright. At these low fluence rates, phototropism was indistinct at 1 g but apparent in microgravity, indicating that gravitropism and phototropism compete at 1 g. The frequency of protonemata that were negatively phototropic varied with the fluence rate and the duration of illumination, as well as with the position of the apical cell before illumination. These data show that the fluence rate of red light regulates whether gravitropism is allowed or completely repressed, and that it influences the polarity of phototropism and the extent to which apical cells are aligned in the light path. Received: 19 January 1999 / Accepted: 19 March 1999     

Life history variation in gametophyte populations of the moss Ceratodon purpureus (Ditrichaceae)

The life cycles of mosses and other bryophytes are unique among land plants in that the haploid gametophyte stage is free-living and the diploid sporophyte stage is ephemeral and completes its development attached to the maternal gametophyte. Despite predictions that populations of haploids might contain low levels of genetic variation, moss populations are characterized by substantial variation at isozyme loci. The extent to which this is indicative of ecologically important life history variation is, however, largely unknown. Gametophyte plants from two populations of the moss Ceratodon purpureus were grown from single-spore isolates in order to assess variation in growth rates, biomass accumulation, and reproductive output. The data were analyzed using a nested analysis of variance, with haploid sib families (gametophytes derived from the same sporophyte) nested within populations. High levels of life history variation were observed within both populations, and the populations differed significantly in both growth and reproductive characteristics. Overall gametophytic sex ratios did not depart significantly from 1:1 within either population, but there was significant variation among families in both populations for progeny sex ratio. Some families produced predominantly male gametophytes, while others yielded predominantly females. Because C. purpureus has a chromosomal mechanism of sex determination, these observations suggest differential (but unpredictable) germination of male and female spores. Life history observations showed that male and female gametophytes are dimorphic in size, maturation rates, and reproductive output.     

Amyloplasts that sediment in protonemata of the moss Ceratodon purpureus are nonrandomly distributed in microgravity

Little is known about whether or how plant cells regulate the position of heavy organelles that sediment toward gravity. Dark-grown protonemata of the moss Ceratodon purpureus displays a complex plastid zonation in that only some amyloplasts sediment along the length of the tip cell. If gravity is the major force determining the position of amyloplasts that sediment, then these plastids should be randomly distributed in space. Instead, amyloplasts were clustered in the subapical region in microgravity. Cells rotated on a clinostat on earth had a roughly similar non-random plastid distribution. Subapical clusters were also found in ground controls that were inverted and kept stationary, but the distribution profile differed considerably due to amyloplast sedimentation. These findings indicate the existence of as yet unknown endogenous forces and mechanisms that influence amyloplast position and that are normally masked in stationary cells grown on earth. It is hypothesized that a microtubule-based mechanism normally compensates for g-induced drag while still allowing for regulated amyloplast sedimentation.     

Genetic correlations do not constrain the evolution of sexual dimorphism in the moss Ceratodon purpureus

The trajectory of phenotypic evolution is constrained in the short term by genetic correlations among traits. However, the extent to which genetic correlations impose a lasting constraint is generally unknown. Here, I examine the genetic architecture of life-history variation in male and female gametophytes from two populations of the moss Ceratodon purpureus, focusing on genetic correlations within and between the sexes. A significant negative correlation between allocation to vegetative and reproductive tissue was evident in males of both populations, but not females. All traits showed between-sex correlations of significantly less than one, indicating additive genetic variance for sexual dimorphism. The degree of dimorphism for traits was significantly negatively associated with the strength of the between-sex correlation. The structure of genetic correlations among life-history traits was more divergent between the two populations in females than in males. Collectively, these results suggest that genetic correlations do not impose a lasting constraint on the evolution of life-history variation in the species.     

Selective sweeps and intercontinental migration in the cosmopolitan moss Ceratodon purpureus (Hedw.) Brid

The moss Ceratodon purpureus has long been used as a model system in plant development and physiology. However, the molecular population genetics of the species remains virtually unexplored. In this study, we used population genetic analyses of DNA sequence data from three unlinked loci (atpB-rbcL spacer, adk, and phy2) to examine biogeographical patterns in a global sample of this species. The three loci differed significantly in mutation frequency spectra and implied population structure. Pairs of haplotypes from single populations were frequently more divergent than haplotypes sampled from widely disjunct populations. In the atpB-rbcL spacer and adk samples, Australasian haplotypes were more closely related to Northern Hemisphere haplotypes than to haplotypes found in the equatorial regions. In contrast, the phy2 sample showed that the north and south temperate regions were genetically divergent, with the equatorial regions intermediate. Maximum-likelihood estimates (MLE) of the rates of migration between the two hemispheres were significantly different for the two nuclear genes. The frequency spectra of mutations indicated that differences in implied population structure among the three loci resulted from directional selection on the chloroplast genome and on the chromosomal segment containing adk. Collectively, these data suggest that long-distance migration within the Northern Hemisphere and Australasian regions is common (relative to the mutation rate) and that migration between these two regions, potentially via equatorial populations, is more frequent than migration among equatorial populations.     

Aphototropic mutants of the moss Ceratodon purpureus with spectrally normal and with spectrally dysfunctional phytochrome

Following UV mutagenesis of protonemal tissue of the moss Ceratodon purpureus we have isolated different aphototropic mutant lines that can be divided into two distinct classes. One class, represented by the line ptr1, shows characteristic features of phytochrome chromophore deficiency. ptrl shows negligible photoreversibility (<5% of wild type), whereas immunoblots show normal apoprotein levels. The aphototropic phenotype could be partially restored with biliverdin, a precursor of the phytochrome chromophore. It was found that, whereas in wild type formation of Pfr leads to suppression of gravitropism, there is no such suppression ptrl. In addition, ptr1 shows lower chlorophyll levels than the wild type. These findings indicate that, as expected for a chromophore-deficient mutant, multiple phytochrome effects are lost. The other class of mutants, represented by the line ptr103, shows more specific effects. In this mutant, only phototropism is affected. Suppression of gravitropism, the content of chlorophyll and photoreversibility of phytochrome were similar to those of the wild type.     

Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response

New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.     

Cell wall-bound ultraviolet-screening compounds explain the high ultraviolet tolerance of the Antarctic moss, Ceratodon purpureus

* Studies of ultraviolet (UV) light-induced DNA damage in three Antarctic moss species have shown Ceratodon purpureus to be the most UV tolerant, despite containing lower concentrations of methanol-soluble UV-screening compounds than the co-occurring Bryum pseudotriquetrum. * In this study, alkali extraction of cell wall-bound phenolics, combined with methanol extraction of soluble phenolics, was used to determine whether cell wall-bound UV screens explain the greater UV tolerance of C. purpureus. * The combined pool of UV screens was similar in B. pseudotriquetrum and C. purpureus, but whilst B. pseudotriquetrum had almost equal concentrations of MeOH-soluble and alkali-extractable cell wall-bound UV-screening compounds, in C. purpureus the concentration of cell wall-bound screening compounds was six times higher than the concentration of MeOH-soluble UV screens. The Antarctic endemic Schistidium antarctici possessed half the combined pool of UV screens of the other species but, as in C. purpureus, these were predominantly cell wall bound. Confocal microscopy confirmed the localization of UV screens in each species. * Greater investment in cell wall-bound UV screens offers C. purpureus a more spatially uniform, and potentially more effective, UV screen. Schistidium antarctici has the lowest UV-screening potential, indicating that this species may be disadvantaged under continuing springtime ozone depletion. Cell wall compounds have not previously been quantified in bryophytes but may be an important component of the UV defences of lower plants.     

High levels of genetic variability in the moss Ceratodon purpureus from continental Antarctica, subantarctic Heard and Macquarie Islands, and Australasia

The random amplified polymorphic DNA (RAPD) technique, and DNA sequencing of the conserved nuclear ribosomal DNA internal transcribed spacer region (ITS1-5.8S-ITS2), have been used to assess levels of genetic diversity in the moss Ceratodon purpureus from several locations in Australasia, subantarctic Heard and Macquarie Islands, and continental Antarctica. Populations from Heard and Macquarie Islands and from Antarctica maintain high levels of genetic variation. Both within- and among-colony variation were observed at these locations. DNA sequence analysis showed that samples from the Ross Sea region of Antarctica were most closely related to colonies from Casey and Macquarie Island, and that one colony from Heard Island was most closely related to one from Europe. DNA sequence data separated two Australian populations from the Antarctic and subantarctic group on a dendrogram. Detailed RAPD analysis of a single colony from continental Antarctica demonstrated that mutation probably causes the high variability observed in this moss. DNA sequencing and RAPD analysis are complementary techniques for genetic investigation of Antarctic moss populations.Jenny Ninham was an integral part of this research team for several years. Unfortunately she did not live to see these results published.