Hybridization between two cryptic filamentous brown seaweeds along the shore: analysing pre‐ and postzygotic barriers in populations of individuals with varying ploidy levels

We aimed to study the importance of hybridization between two cryptic species of the genus Ectocarpus, a group of filamentous algae with haploid–diploid life cycles that include the principal genetic model organism for the brown algae. In haploid–diploid species, the genetic structure of the two phases of the life cycle can be analysed separately in natural populations. Such life cycles provide a unique opportunity to estimate the frequency of hybrid genotypes in diploid sporophytes and meiotic recombinant genotypes in haploid gametophytes allowing the effects of reproductive barriers preventing fertilization or preventing meiosis to be untangle. The level of hybridization between E. siliculosus and E. crouaniorum was quantified along the European coast. Clonal cultures (568 diploid, 336 haploid) isolated from field samples were genotyped using cytoplasmic and nuclear markers to estimate the frequency of hybrid genotypes in diploids and recombinant haploids. We identified admixed individuals using microsatellite loci, classical assignment methods and a newly developed Bayesian method (XPloidAssignment), which allows the analysis of populations that exhibit variations in ploidy level. Over all populations, the level of hybridization was estimated at 8.7%. Hybrids were exclusively observed in sympatric populations. More than 98% of hybrids were diploids (40% of which showed signs of aneuploidy) with a high frequency of rare alleles. The near absence of haploid recombinant hybrids demonstrates that the reproductive barriers are mostly postzygotic and suggests that abnormal chromosome segregation during meiosis following hybridization of species with different genome sizes could be a major cause of interspecific incompatibility in this system.     

The Life Cycle of Cryptochlora perforans (Chlorarachniophyta)

Observations on the behaviour of different life cycle stages, gamete fusions, and measurements of nuclear DNA contents in Cryptochlora perforans resulted in a first concept concerning life histories in Chlorarachniophyta: the life cycle of Cr. perforans is diplohaplontic (gamete fusion with karyogamy - mitosis - meiosis - mitosis). In the haploid as well as in the diploid life cycle phases amoeboid and coccoid stages occur. The isomorphic gametes are modified amoebae frequently without filopodia. Only haploid flagellate stages are known representing mito- or meiozoospores. Diploid coccoid stages have a granular cytoplasmic structure and may be somewhat larger than haploid ones. Nevertheless, positive identification of haploid (gametophytic) and diploid (sporophytic) stages is only possible on the basis of nuclear DNA contents.     

Evolution and maintenance of haploid–diploid life cycles in natural populations: The case of the marine brown alga Ectocarpus

The evolutionary stability of haploid–diploid life cycles is still controversial. Mathematical models indicate that niche differences between ploidy phases may be a necessary condition for the evolution and maintenance of these life cycles. Nevertheless, experimental support for this prediction remains elusive. In the present work, we explored this hypothesis in natural populations of the brown alga Ectocarpus. Consistent with the life cycle described in culture, Ectocarpus crouaniorum in NW France and E. siliculosus in SW Italy exhibited an alternation between haploid gametophytes and diploid sporophytes. Our field data invalidated, however, the long‐standing view of an isomorphic alternation of generations. Gametophytes and sporophytes displayed marked differences in size and, conforming to theoretical predictions, occupied different spatiotemporal niches. Gametophytes were found almost exclusively on the alga Scytosiphon lomentaria during spring whereas sporophytes were present year‐round on abiotic substrata. Paradoxically, E. siliculosus in NW France exhibited similar habitat usage despite the absence of alternation of ploidy phases. Diploid sporophytes grew both epilithically and epiphytically, and this mainly asexual population gained the same ecological advantage postulated for haploid–diploid populations. Consequently, an ecological interpretation of the niche differences between haploid and diploid individuals does not seem to satisfactorily explain the evolution of the Ectocarpus life cycle.     

Generationswechsel,Kernphasenwechsel und Sexualität der Braunalge Ectocarpus siliculosus im Kulturversuch

Zusammenfassung Der Entwicklungscyclus von Ectocarpus siliculosus wurde unter Kulturbedingungen untersucht. Gametophyten und Sporophyten können morphologisch und funktionell unterschieden werden. Die Gametophyten sind diöcisch, die Geschlechtsbestimmung erfolgt genotypisch. Sporophyten können in der haploiden, diploiden und tetraploiden Phase vorliegen. Sporophyten aller Kernphasen können unilokuläre Sporangien ausbilden, tetraploide und diploide Sporophyten führen dabei eine Reduktionsteilung durch. Schwärmer haploider Gametophyten und Sporophyten können sich spontan zu diploiden oder tetraploiden homozygoten Sporophyten entwickleln. Gametophyten können nur zusammen mit Sporophyten aus Schwärmern unilokulärer Sporangien entstehen (Heteroblastie). Aus der Reduktionsteilung tetraploider Sporophyten gingen diploide männliche Gametophyten hervor, deren Gameten mit normalen weiblichen Gameten kopulierten. Die verschiedenen Kernphasen und Wuchsformen stehen durch Reduktionsteilungen, Heteroblastie, Kopulation und spontane Aufregulierung der Chromosomenzahl miteinander in Verbindung.

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Culture experiments on life cycle, nuclear phases, and sexuality of the brown alga Ectocarpus siliculosus

Summary The life cycle of Ectocarpus siliculosus from Naples (Italy) was investigated, using well defined cultured material. Gametophytes and sporophytes differ morphologically and functionally. The gametophytes are dioecious, with genotypic determination of their sex. Sporophytes exist in the haploid, diploid and tetraploid phase. All sporophytes can form unilocular sporangia. In tetraploid and diploid sporophytes the formation of unilocular sporangia is connected with meiosis. Certain motile cells of haploid plants may spontaneously give rise to diploid or tetraploid sporophytes which are homozygous. Gametophytes can only be formed together with sporophytes form the swarmers of unilocular sporangia (heteroblasty). Meiosis in tetraploid sporophytes resulted in diploid gametophytes, the gametes of which fused with haploid female gametes. All observed nuclear phases and growth forms are connected with each other by meiosis, heteroblasty, fusion of gametes and spontaneous increase in chromosome number.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

ALTERNATION OF GENERATIONS IN ALGAE: ITS COMPLEXITY, MAINTENANCE AND EVOLUTION

Life histories of photosynthetic eukaryotes traditionally-termed algae exhibit a considerably greater degree of complexity than those of ‘higher cryptogams.’ Some algae have a so-called ‘obligate’alternation between spore-producing and gamete-producing phases, but the majority seem capable of following other pathways depending upon environmental conditions. In only four algal classes do life histories show a change in morphological and/or nuclear phases. The following basic life histories are recognized in the Chlorophyceae, Phaeophyceae and Rhodophyceae:(a) monophasic, a diploid or haploid phase, (b) two or more phases, most commonly an alternation of an isomorphic or heteromorphic haploid gametangial phase and a diploid sporangial phase, and (c) three phases (unique to florideophyte Rhodophyceae), with a diploid spore-producing phase (carposporophyte) developing on the gametangial phase, a diploid phase (tetrasporophyte if meiosis is sporic) and a haploid gametangial phase. Evidence from recent research indicates that in many algae there is an uncoupling of the morphological and nuclear phases. The dominance of one phase and suppression of another has been suggested to be due to the common occurrence in algae of apogamy, apomeiosis and parthenogenesis. Free-living morphs in heteromorphic life histories may be morphologically so dissimilar that formerly they were attributed to different genera. Evolution of the carposporangial phase in red algae is speculated to be a means of achieving zygotic amplification to compensate for the infrequency of syngamy. Such amplification allows the production of a large number of dispersible products from a single fertilization. The direct development of a free-living tetrasporangial phase is considered another mechanism for achieving amplification. In freshwater red algae the growth of an upright phase from a perennial microscopic one is considered an adaptation for maintaining their upstream position. Life history pathways in algae are controlled by subtle environmental influences (e.g. photoperiodism, temperature, light quality, nutrients). Experimental evidence is lacking to support the contention that spatial and/or temporal partitioning of the environment is a mechanism favouring the maintenance of heteromorphy. Herbivory is known to be an important selective force suppressing some morphs and accentuating the seasonal dominance of others. Differential resistance of morphs to herbivory in environments where grazing intensity is predictable may lead to the selective maintenance of heteromorphy. Algal life history patterns are unexplored in terms of evolutionary processes. Various models for the evolution of biphasic or polyphasic life histories stress the importance of the capacity for both asexual dispersal of successful genotypes and for the generation of new genotypes via meiosis and syngamy. All evidence points to the fact that many life history processes operative in algae differ significantly from those described for other cryptogams.     

Alternate life history phases of a common seaweed have distinct microbial surface communities

Macroalgal life histories are complex, often involving the alternation of distinct free‐living life history phases that differ in morphology, longevity and ploidy. The surfaces of marine macroalgae support diverse microbial biofilms, yet the degree of microbial variation between alternate phases is unknown. We quantified bacterial (16S rRNA gene) and microeukaryote (18S rRNA gene) communities on the surface of the common intertidal seaweed, Mastocarpus spp., which alternates between gametophyte (foliose, haploid) and sporophyte (encrusting, diploid) life history phases. A large portion (97%) of bacterial taxa on the surface Mastocarpus was also present in samples from the environment, indicating that macroalgal surface communities are largely assembled from the surrounding seawater. Still, changes in the relative abundance of bacterial taxa result in significantly different communities on alternate Mastocarpus life history phases, rocky substrate and seawater at all intertidal elevations. For microeukaryote assemblages, only high intertidal samples had significant differences between life history phases although sporophytes were not different from the rocky substrate at this elevation; gametophytes and sporophytes did not differ in microeukaryote communities in the mid and low zones. By sequencing three host genes, we identified three cryptic species of Mastocarpus in our data set, which co‐occur in the mid‐to‐low intertidal zone. In these samples, M. alaskensis sporophytes harboured distinct bacterial communities compared to M. agardhii and M. intermedius sporophytes, which were not distinguishable. Conversely, microeukaryote communities did not differ among species.     

Alternation of generations – unravelling the underlying molecular mechanism of a 165‐year‐old botanical observation

Characteristically, land plants exhibit a life cycle with an ‘alternation of generations’ and thus alternate between a haploid gametophyte and a diploid sporophyte. At meiosis and fertilisation the transitions between these two ontogenies take place in distinct single stem cells. The evolutionary invention of an embryo, and thus an upright multicellular sporophyte, in the ancestor of land plants formed the basis for the evolution of increasingly complex plant morphologies shaping Earth's ecosystems. Recent research employing the moss Physcomitrella patens revealed the homeotic gene BELL1 as a master regulator of the gametophyte‐to‐sporophyte transition. Here, we discuss these findings in the context of classical botanical observations.     

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin‐celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6‐1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)‐dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6‐1 during male meiosis and pollen mitosis I using fluorescent MT‐markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6‐1 mutants as a valuable tool for the investigation of augmin‐dependent MT nucleation and dynamics in plant cells.     

ON THE LIFE CYCLE,CYTOLOGY AND TAXONOMY OF CLADOPHORA CALLICOMA FROM INDIA

The present investigation deals with the cultural observations on the morphology, reproduction, life cycle, cytology and taxonomy of a freshwater Cladophora, C. callicoma (L.) Kütz, from India. There is a regular isomorphic alternation of generations between quadriflagellate zoospore-producing diploid plants and biflagellate isogamete-producing haploid plants, coupled with heterothallism in the latter. Chromosome numbers of n = 12 and 2n = 24 were determined respectively for gametophytes and sporophytes. Twelve bivalents were counted in meiosis where chiasmata varied from one to three. In light of above observations, the affinity of C. callicoma with C. glomerata, to which the former has often been assigned, has been discussed. It is proposed that the C. glomerata complex, which consists of intraspecific polyploid races, might have two distinct lines of evolution with regard to life cycle; one with forms having higher ploidy levels and lacking an alternation of generations, and the other with forms having low ploidy levels and alternation of generations.     

Mitosis in the coenocytic green algaBoergesenia forbesii (Harvey) Feldmann (Siphonocladales,Ulvophyceae)

Mitosis in vegetative cells of the siphonocladalean algaBoergesenia forbesii (Harvey) Feldmann was investigated mainly by electron microscopy. The mitotic spindle was centric and closed. The interphase nucleus contained a spherical nucleolus. The nucleolus was slightly dispersed at prophase, but nucleolar materials remained during nearly all stages of mitosis. Kinetochores were evident on chromosomes. The polar regions of nuclear envelope had no fenestrae during mitosis. Anaphase separation of the chromosomes was asynchronous. Elongation of interzonal spindle at telophase separated the two daughter nuclei widely. The ultrastructural features of mitosis inB. forbesii revealed by the present investigation are compared with those of other siphonous and siphonocladous algae in the Ulvophyceae.     

STUDIES ON DIPLOID STRAINS OF DICTYOSTELIUM DISCOIDEUM

Ross, Ian K. (Yale U., New Haven, Conn.) Studies on diploid strains of Dictyostelium discoideum. Amer. Jour. Bot. 47 (1) : 54—59. Illus. 1960.–Three strains of Dictyostelium discoideum having the diploid number of chromosomes (14) at all stages of their life cycle were examined. No evidence of sexuality as shown by syngamy or meiosis was found in the diploid strains. Two of the diploid strains were unstable and reverted to the haploid type with 7 chromosomes. These haploid strains had a sexual phase in which syngamy and meiosis were observed. The nuclear behavior of both the diploid and haploid strains differed from that reported in previous papers.     

A COMPREHENSIVE STUDY OF THE LIFE CYCLE OF A SOUTH AMERICAN POPULATION OF STIGEOCLONIUM TENUE (CHAETOPHORALES,CHLOROPHYTA)1

The diplobiontic–haplodiplontic life cycle with alternating isomorphic generations in Stigeoclonium tenue (C. Agardh) Kütz. is described for the first time. Sporophytes (2n = 10) arise from tetraflagellate zoospores that are produced by meiosis. Sporic meiosis might be inferred from the cruciform divisions formed during zoosporogenesis and is confirmed through observations of prophase I substages. Zoospores do not germinate directly but produce a haploid cyst that germinates to give rise to a gametophyte (n = 5). Gametophytes produce biflagellate isogametes, which fuse to produce zygotes that germinate by mitosis into the sporophytic stage. Gametophytes and sporophytes reproduce asexually both via mitotic tetraflagellate zoospores and by thallus fragmentation. Results from this study indicate that both the cosmopolitan distribution and dominance of S. tenue in many periphytic communities might be due to its multiple reproductive strategies.     

Morphological changes of giant mitochondria in the unicellular to multicellular phase during parthenogenesis of Ulva partita (Ulvophyceae) revealed by expression of mitochondrial targeting GFP and PEG transformation

A giant mitochondrion that branches and connects as a single mitochondrion in a cell has been observed during specific phases of the cell cycle of unicellular green algae, but has not been observed in multicellular algae. The genus Ulva is a green macroalga in which the haploid and diploid phases are isomorphic and its gametes develop parthenogenetically. The existence or absence of the giant mitochondrion, and its behavior in Ulva partita, were investigated using a parthenogenesis system. To observe the parthenogenesis of gametes and the dynamics of mitochondria by fluorescence microscopy, we developed an experimental system for culturing and observing U. partita on cover slips: gametes were suspended in 6‐well plates filled with artificial seawater, and cover slips were placed on the well bottoms. The gametes settled on the cover slips as spherical cells (1‐cell S phase). These cells grew into larger cells, losing their eyespot (1‐cell L phase), and developed into multicellular thalli. Gene introduction using the polyethylene glycol (PEG) method is available with transformation efficiencies of 9.0–15.1%. Transformation was performed using a plasmid encoding green fluorescent protein (GFP) fused to the mitochondrial targeting sequence, and mitochondria were labeled by GFP fluorescence. This revealed a string‐shaped giant mitochondrion in a cell of the 1‐cell S phase. In the 1‐cell L phase, a reticular mitochondrion was observed. After the initiation of cell division, the reticular mitochondrion was fragmented, and small oval mitochondria were observed in the 5‐cell phase.     

IDENTIFICATION OF GREEN ALGAL ENDOPHYTES AS THE ALTERNATE PHASE OF ACROSIPHONIA (CODIOLALES, CHLOROPHYTA) USING ITS1 AND ITS2 RIBOSOMAL DNA SEQUENCE DATA

The internal transcribed spacer regions of the nuclear ribosomal DNA cistron of green algal endophytes, previously identified as Chlorochytrium inclusum Kjellman and Codiolum petrocelidis Kuckuck, were sequenced. Culture studies have implicated marine Chlorochytrium and Codiolum -like cells in the life histories of a number of macrophytic green algae. The identity of the endophytes was resolved by comparing DNA sequences from unicells endophytic in the foliose red alga Mazzaella splendens (Setchell et Gardner) Hommersand and in the crustose phase of Mastocarpus papillatus (J. Agardh) Kylin, with sequences from species of Acrosiphonia, Urospora, Ulothrix, and Monostroma. All endophyte isolates were more closely related to species of Acrosiphonia than to those of Urospora, Ulothrix, or Monostroma. The results support previous culture studies, and conclusively identify the endophytes obtained in the field as alternate life history phases of one or more Acrosiphonia species in southern British Columbia, Canada.     

Life history of Chnoospora implexa (Chnoosporaceae, Phaeophyceae) in culture

The life history of the brown alga Chnoospora implexa J. Agardh (Chnoosporaceae, Scytosiphonales) from Japan was studied in laboratory cultures. This species showed a heteromorphic and diphasic life history, alternating between erect gametophytes and discoid sporophytes. The gametophytes were dioecious and produced isogametes. The zygotes developed into sporophytes at 20°C under long‐day conditions, which formed plurilocular zoidangia. Zoids released from the plurilocular zoidangia developed again into sporophytes that always formed plurilocular zoidangia at 20°C and 25°C in long‐day conditions, and mainly unilocular zoidangia at 25°C in short‐day conditions. Zoids released from unilocular zoidangia developed into dioecious gametophytes. At 15°C zygotic erect thalli were formed and were revealed to be diploid by microspectrofluorometric measurements of nuclear DNA contents. The development and reproduction of unfused gametes were similar to those of zygotes. Some strains showed a direct‐type life history; gametophytic thalli were produced, but not via a sporophytic phase.     

The life history ofAcrochaete wittrockii (Ulvellaceae,Chlorophyta)

Acrochaete wittrockii (Wille) Nielsen is a heteromorphic diplohaplont. The haplophase consists of isomorphic, dioecious filamentous epiphytes on brown algae. Several generations follow each other by triflagellate zoospores from spring to early summer. By late summer and throughout autumn, quadriflagellate zoopores are produced by the epiphytic thalli; they give rise to male and female gametophytes of a globular, pseudoparenchymatic appearance in culture. The gametophytes produce anisogamic biflagellate gametes which, after gametic union, develop into diploid unicellular sporophytes. After 6–7 days, the sporophyte produces triflagellate zoospores, repeating the life history when germinating on brown algal hosts. Alternatively, triflagellate zoospores which settle on the bottom of petri dishes, develop into unicellular, autonomous sporangial plants. Their triflagellate spores repeat the epiphytic stage on brown algal hosts, or the sporangial plant cycle on non-living substrate, respectively.     

Holococcolithophore‐heterococcolithophore (Haptophyta) life cycles: Flow cytometric analysis of relative ploidy levels

Several coccolithophore species are known to exhibit heteromorphic life cycles. In certain species, notably Emiliania huxleyi, the heterococcolith‐bearing phase alternates with a non‐calcifying stage, whereas in others the heterococcolith‐bearing phase alternates with a holococcolith‐bearing phase. Heterococcolithophore‐holococcolithophore life cycles have previously been observed for only one species in culture, but have also been inferred from an increasing number of observations of combination coccospheres. 18S rDNA sequences from pure cultures of both the heterococcolith‐bearing and holococcolith‐bearing phases of Coccolithus pelagicus were identical, providing an additional indication of their identity as different life cycle stages of the same species. Flow cytometric analyses have been undertaken on SybrGreen‐stained nuclei isolated from pure cultures of the two phases of four coccolithophore species (Coccolithus pelagicus, Calcidiscus leptoporus, Coronosphaera mediterranea and Emiliania huxleyi) in order to determine relative DNA content. Results confirm the hypothesis that holococcolithophore‐heterococcolithophore life cycles are haplo‐diploid in nature. Light microscope observations of the processes of sexual fusion and meiosis are reported for two of the experimental species. The results are discussed in the context of the evolution of bio‐mineralization in the coccolithophores and the possible ubiquity of haplo‐diploidy in the haptophytes.     

Homologous Versus Antithetic Alternation of Generations and the Origin of Sporophytes

The late-nineteenth/early-twentieth century debate over homologous versus antithetic alternation of generations is reviewed. Supporters of both theories, at first, used Coleochaete as a model for the origin of land-plant life cycles. The early debate focused on the morphological interpretation of the sporophyte and on whether vascular cryptogams had bryophyte-like ancestors. The terms of the debate shifted after the discovery that the alternation of morphological generations was accompanied by an alternation of chromosome number. Supporters of homologous alternation now promoted a model in which land plants had been derived from an algal ancestor with an isomorphic alternation of haploid and diploid generations whereas supporters of antithetic alternation favored a model in which land plants were derived from a haploid algal ancestor with zygotic meiosis. Modern evidence that embryophytes are derived from charophycean green algae is more compatible with an updated version of the antithetic theory.     

Life cycle and nuclear behavior in three rust fungi (Uredinales)

Kuehneola japonica has a microcyclic life cycle with a regular alternation of generations. Single basidiospore inoculations onto Rosa wichuraiana resulted in teliospore production, indicating its homothallic nature. Dikaryotization in a vegetative mycelium in the host seemed to occur through nuclear division that was not followed by septum formation. Karyogamy and meiosis took place through teliospore and metabasidium development; this fungus was considered to reproduce genetically homogeneous progenies. Puccinia lantanae and P. patriniae were also microcyclic in their life cycle; however, these fungi differed from K. japonica in the mode of nuclear behavior. In the former two fungi, both vegetative and reproductive cells were uninucleate. No karyogamy was observed, and nuclear division in the metabasidium development was thought to be mitotic. In P. lantanae, a basidiospore was formed on a sterigma, whereas a whiplike hypha emerged from each metabasidium cell in P. patriniae. Inoculations of Justicia procumbens with a single basidiospore of P. lantanae resulted in teliospore production. The fungus seemed to remain uninucleate, either haploid or diploid, throughout the life cycle. Thus, reproduction was considered to be apomictic. Received: August 16, 2001 / Accepted: October 1, 2001     

Evolution of the alternation of haploid and diploid phases in life cycles. II. Maintenance of the haplo-diplontic cycle

The relative duration of the haploid and the diploid phases during the reproductive cycle varies greatly between organisms. This paper addresses the question of the evolution of haploid, diploid, and haplo-diplontic life cycles. When the life span of haploid and diploid individuals is constant whatever their cycle, we show that the haplo-diplontic cycle has an advantage, which depends on the sex-ratio in anisogamous species and on the probability of fertilization in isogamous species. This is because meiosis and fertilization occur half as often in the haplo-diplontic cycle as in haploid or diploid cycles, for the same number of generations of individuals. This argument is demonstrated using a model which considers a genetic determination of the cycle, and fixed haploid and diploid fitnesses. The relevance of measures of fitness of haploid and diploid individuals in predicting the evolution of life cycles is discussed. Measures obtained in algae are compared with theoretical predictions.