Evolution in the Cycles of Life.

The life cycles of eukaryotes alternate between haploid and diploid phases, which are initiated by meiosis and gamete fusion, respectively. In both ascomycete and basidiomycete fungi and chlorophyte algae, the haploid-to-diploid transition is regulated by a pair of paralogous homeodomain protein encoding genes. That a common genetic program controls the haploid-to-diploid transition in phylogenetically disparate eukaryotic lineages suggests this may be the ancestral function for homeodomain proteins. Multicellularity has evolved independently in many eukaryotic lineages in either one or both phases of the life cycle. Organisms, such as land plants, exhibiting a life cycle whereby multicellular bodies develop in both the haploid and diploid phases are often referred to as possessing an alternation of generations. We review recent progress on understanding the genetic basis for the land plant alternation of generations and highlight the roles that homeodomain-encoding genes may have played in the evolution of complex multicellularity in this lineage.

Transcriptomic analyses in the gametophytes of the apomictic fern Dryopteris affinis.

MAIN CONCLUSION: A novel genomic map of the apogamous gametophyte of the fern Dryopteris affinis unlocks oldest hindrance with this complex plant group, to gain insight into evo-devo approaches. The gametophyte of the fern Dryopteris affinis ssp. affinis represents a good model to explore the molecular basis of vegetative and reproductive development, as well as stress responses. Specifically, this fern reproduces asexually by apogamy, a peculiar case of apomixis whereby a sporophyte forms directly from a gametophytic cell without fertilization. Using RNA-sequencing approach, we have previously annotated more than 6000 transcripts. Here, we selected 100 of the inferred proteins homolog to those of Arabidopsis thaliana, which were particularly interesting for a detailed study of their potential functions, protein-protein interactions, and distance trees. As expected, a plethora of proteins associated with gametogenesis and embryogenesis in angiosperms, such as FERONIA (FER) and CHROMATING REMODELING 11 (CHR11) were identified, and more than a dozen candidates potentially involved in apomixis, such as ARGONAUTE family (AGO4, AGO9, and AGO 10), BABY BOOM (BBM), FASCIATED STEM4 (FAS4), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MATERNAL EFFECT EMBRYO ARREST29 (MEE29). In addition, proteins involved in the response to biotic and abiotic stresses were widely represented, as shown by the enrichment of heat-shock proteins. Using the String platform, the interactome revealed that most of the protein-protein interactions were predicted based on experimental, database, and text mining datasets, with MULTICOPY SUPPRESSOR OF IRA4 (MSI4) showing the highest number of interactions: 16. Lastly, some proteins were studied through distance trees by comparing alignments with respect to more distantly or closely related plant groups. This analysis identified DCL4 as the most distant protein to the predicted common ancestor. New genomic information in relation to gametophyte development, including apomictic reproduction, could expand our current vision of evo-devo approaches.

Apomixis and the paradox of sex in plants.

BACKGROUND: The predominance of sex in eukaryotes, despite the high costs of meiosis and mating, remains an evolutionary enigma. Many theories have been proposed, none of them being conclusive on its own, and they are, in part, not well applicable to land plants. Sexual reproduction is obligate in embryophytes for the great majority of species. SCOPE: This review compares the main forms of sexual and asexual reproduction in ferns and angiosperms, based on the generation cycling of sporophyte and gametophyte (leaving vegetative propagation aside). The benefits of sexual reproduction for maintenance of genomic integrity in comparison to asexuality are discussed in the light of developmental, evolutionary, genetic and phylogenetic studies. CONCLUSIONS: Asexual reproduction represents modifications of the sexual pathway, with various forms of facultative sexuality. For sexual land plants, meiosis provides direct DNA repair mechanisms for oxidative damage in reproductive tissues. The ploidy alternations of meiosis-syngamy cycles and prolonged multicellular stages in the haploid phase in the gametophytes provide a high efficiency of purifying selection against recessive deleterious mutations. Asexual lineages might buffer effects of such mutations via polyploidy and can purge the mutational load via facultative sexuality. The role of organelle-nuclear genome compatibility for maintenance of genome integrity is not well understood. In plants in general, the costs of mating are low because of predominant hermaphroditism. Phylogenetic patterns in the archaeplastid clade suggest that high frequencies of sexuality in land plants are concomitant with a stepwise increase of intrinsic and extrinsic stress factors. Furthermore, expansion of genome size in land plants would increase the potential mutational load. Sexual reproduction appears to be essential for keeping long-term genomic integrity, and only rare combinations of extrinsic and intrinsic factors allow for shifts to asexuality.

An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

Unreduced spore formation in a spontaneous chimeric pinnule in an artificially produced haploid Anisocampium niponicum (Athyriaceae, Polypodiales).

Haploid sporophytes of Anisocampium niponicum with 2n = 40, were produced artificially by induced apogamy in vitro. They were subsequently transplanted into pots and two of them have been cultivated for the investigation of sporogenesis and/or production of chimera for more than 20 years. Haploid A. niponicum is sterile, but an abnormal chimeric pinnule that developed spontaneously in a single frond produced sporangia with spores. Each sporangium bore approximately 32 spores that were almost uniform in size. Sowing of these spores resulted in 50 gametophytes. Of 20 gametophytes cultured individually, five produced sporophytes apogamously after eight months. Both the gametophytes and subsequent apogamous sporophytes showed a chromosome number of 2n = 40. Our study demonstrates that a haploid sporophyte offspring can be produced from a haploid mother sporophyte via haploid spores. Since asexual reproduction is a prominent evolutionary process in ferns, the reproduction of a haploid A. niponicum sporophyte by unreduced spore formation might help to elucidate how apogamous ferns occur and evolve.

Apomictic fern fathers: an experimental approach to the reproductive characteristics of sexual, apomict, and hybrid fern gametophytes.

PREMISE: Apomixis and hybridization are two essential and complementary factors in the evolution of plants, including ferns. Hybridization combines characteristics from different species, while apomixis conserves features within a lineage. When combined, these two processes result in apo-sex hybrids. The conditions leading to the formation of these hybrids are poorly understood in ferns. METHODS: We cultivated spores from 66 fern samples (43 apomicts, 7 apo-sex hybrids, and 16 sexuals), and measured their development in vitro over 16 weeks. We evaluated germination, lateral meristem formation rates, sexual expression, and production of sporophytes and then compared ontogenetic patterns among the three groups. RESULTS: The three examined groups formed antheridia (male gametangia) but differed in overall gametophyte development. Sexual species created archegonia (female, 86% of viable samples), but no sporophytes. Apomicts rarely created nonfunctional archegonia (8%) but usually produced apogamous sporophytes (75%). Surprisingly, apomictic and sexual species showed similar development speed. The sexually reproducing parents of viable studied hybrids formed about twice as many meristic gametophytes as the apomictic parents (39% vs. 20%, respectively). CONCLUSIONS: We present the most thorough comparison of gametangial development of sexual and apomictic ferns, to date. Despite expectations, apomictic reproduction might not lead to earlier sporophyte formation. Apomicts produce functional sperm and thus can contribute this type of gamete to their hybrids. The development patterns found in the parents of hybrids indicate a possible increase of hybridization rates by antheridiogens. The apo-sex hybrids always inherit the apomictic reproductive strategy and are thus capable of self-perpetuation.

The Shared Proteome of the Apomictic Fern Dryopteris affinis ssp. affinis and Its Sexual Relative Dryopteris oreades.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3'-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein-protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species.

A drought-driven model for the evolution of obligate apomixis in ferns: evidence from pellaeids (Pteridaceae).

PREMISE: Xeric environments impose major constraints on the fern life cycle, yet many lineages overcome these limitations by evolving apomixis. Here, we synthesize studies of apomixis in ferns and present an evidence-based model for the evolution and establishment of this reproductive strategy, focusing on genetic and environmental factors associated with its two defining traits: the production of "unreduced" spores (n = 2n) and the initiation of sporophytes from gametophyte tissue (i.e., diplospory and apogamy, respectively). METHODS: We evaluated existing literature in light of the hypothesis that abiotic characteristics of desert environments (e.g., extreme diurnal temperature fluctuations, high light intensity, and water limitation) drive the evolution of obligate apomixis. Pellaeid ferns (Cheilanthoideae: Pteridaceae) were examined in detail, as an illustrative example. We reconstructed a plastid (rbcL, trnG-trnR, atpA) phylogeny for the clade and mapped reproductive mode (sexual versus apomictic) and ploidy across the resulting tree. RESULTS: Our six-stage model for the evolution of obligate apomixis in ferns emphasizes the role played by drought and associated abiotic conditions in the establishment of this reproductive approach. Furthermore, our updated phylogeny of pellaeid ferns reveals repeated origins of obligate apomixis and shows an increase in the frequency of apomixis, and rarity of sexual reproduction, among taxa inhabiting increasingly dry North American deserts. CONCLUSIONS: Our findings reinforce aspects of other evolutionary, physiological, developmental, and omics-based studies, indicating a strong association between abiotic factors and the establishment of obligate apomixis in ferns. Water limitation, in particular, appears critical to establishment of this reproductive mode.

Comparative transcriptome analysis of two reproductive modes in Adiantum reniforme var. sinense targeted to explore possible mechanism of apogamy.

BACKGROUND: Apogamy is a unique asexual reproduction in the ferns, in which somatic cells of gametophytes go through dedifferentiation and then differentiate into haploid sporophytes bypassing fertilization. Restricted to the lack of genomic information, molecular mechanisms of apogamy have remained unclear. Comparative transcriptome analysis was conducted at six stages between sexual reproduction and apogamy in the fern Adiantum reniforme var. sinense, in an effort to identify genes and pathways that might initiate the asexual reproduction. RESULTS: Approximately 928 million high-quality clean reads were assembled into 264,791 unigenes with an average length of 615 bp. A total of 147,865 (55.84%) unigenes were successfully annotated. Differential genes expression analysis indicated that transcriptional regulation was more active in the early stage of apogamy compared to sexual reproduction. Further comparative analysis of the enriched pathways between the early stages of the two reproductive modes demonstrated that starch and sucrose metabolism pathway responsible for cell wall was only significantly enriched in asexual embryonic cell initiation. Furthermore, regulation of plant hormone related genes was more vigorous in apogamy initiation. CONCLUSION: These findings would be useful for revealing the initiation of apogamy and further understanding of the mechanisms related to asexual reproduction.

A fern AINTEGUMENTA gene mirrors BABY BOOM in promoting apogamy in Ceratopteris richardii.

Asexual reproduction is widespread in land plants, including ferns where 10% of all species are obligate asexuals. In these ferns, apogamous sporophytes are generated directly from gametophytes, bypassing fertilization. In the model fern Ceratopteris richardii, a sexual species, apogamy can be induced by culture on high sugar media. BABY BOOM (BBM) genes in angiosperms are known to promote somatic embryogenesis, which like apogamy produce sporophytes without fertilization. Here, a Brassica napus BBM (BnBBM) was used to investigate genetic similarity between apogamy in ferns and somatic embryogenesis in angiosperms. A C. richardii transcriptome was constructed from which one AINTEGUMENTA-LIKE unigene, CrANT, was identified. Whole mount in situ hybridization showed that CrANT is expressed in sperm and fertilized eggs. Phylogenetic analysis grouped CrANT with other non-seed-plant ANT genes to the euANT clade but in a branch separate from BBM genes. Overexpression of CrANT or BnBBM promotes apogamy in C. richardii without sugar supplement. CrANT knockdown gametophytes responded weakly to sugar for apogamy promotion. Theses results suggest some genetic conservation between apogamy and somatic embryogenesis and that such asexual reproduction may be ancient.

Between sexual and apomictic: unexpectedly variable sporogenesis and production of viable polyhaploids in the pentaploid fern of the Dryopteris affinis agg. (Dryopteridaceae).

BACKGROUND AND AIMS: In ferns, apomixis is an important mode of asexual reproduction. Although the mechanisms of fern reproduction have been studied thoroughly, most previous work has focused on cases in which ferns reproduce either exclusively sexually or exclusively asexually. Reproduction of ferns with potentially mixed systems and inheritance of apomixis remains largely unknown. This study addresses reproduction of the pentaploid Dryopteris × critica, a hybrid of triploid apomictic D. borreri and tetraploid sexual D. filix-mas. METHODS: Spore size, abortion percentage and number of spores per sporangium were examined in pentaploid plants of D. × critica grown in an experimental garden. The sporangial content of leaf segments was cultivated on an agar medium, and DNA ploidy levels were estimated by DAPI flow cytometry in 259 gametophytes or sporophytes arising from the F2 generation of the pentaploid hybrid. KEY RESULTS: The hybrid is partly fertile (89-94% of aborted spores) and shows unstable sporogenesis with sexual and apomictic reproduction combined. The number of spores per sporangium varied from approx. 31 to 64. Within a single sporangium it was possible to detect formation of either only aborted spores or various mixtures of aborted and well-developed reduced spores and unreduced diplospores. The spores germinated in viable gametophytes with two ploidy levels: pentaploid (5x, from unreduced spores) and half of that (approx. 2·5x, from reduced spores). Moreover, 2-15% of gametophytes (both 2·5x and 5x) formed a viable sporophyte of the same ploidy level due to apogamy. CONCLUSIONS: This study documents the mixed reproductive mode of a hybrid between apomictic and sexual ferns. Both sexual reduced and apomictic unreduced spores can be produced by a single individual, and even within a single sporangium. Both types of spores give rise to viable F2 generation gametophytes and sporophytes.

Extreme multiple reticulate origins of the Pteris cadieri complex (Pteridaceae).

The Pteris cadieri complex displays extensive morphological variation and seems to have originated through hybridization. However, the members of this complex reproduce by apogamy, which usually limits genetic variation. To evaluate the hypotheses of hybrid origins, the pattern of evolution in this species complex is reconstructed. Multiple methodologies were used. Diploids, triploids, and tetraploids were identified by chromosome counts and flow cytometry. Nuclear DNA markers (cytosolic phosphoglucose isomerase gene, PgiC) were used, together with chloroplast DNA markers (atpB-rbcL spacer and rbcL gene) to infer the biparental and maternal lineages of the Pteris cadieri complex. The three cpDNA haplotype groups and five PgiC alleles found in this study indicate that the evolution of the Pteris cadieri complex has been extremely reticulate. Up to 11 taxa belonging to eight morphs were identified. By comparing genetic variation in the Pteris cadieri in two independent areas, Hainan and Taiwan, we inferred that hybridization has occurred independently in different areas. Furthermore, we found evidence for phenological divergence (evergreen and deciduous) within Taiwan. We propose that the Pteris cadieri complex originated from different genetic lineages through multiple hybridizations in different geographical areas, leading to its present morphological diversity.

Sexual and Apogamous Species of Woodferns Show Different Protein and Phytohormone Profiles.

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

Differential gene expression profiling of one- and two-dimensional apogamous gametophytes of the fern Dryopteris affinis ssp. affinis.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.

Gamma radiation-induced in vitro hormetic apogamy in the fern Pityrogramma calomelanos (L.) link.

Pityrogramma calomelanos (L.) Link, popularly known as "Silver fern" has significant importance as a medicinal plant used traditionally for its astringent, analgesic, anti-haemorrhagic, anti-hypertensive, anti-pyretic and anthelminthic properties. This fern demonstrates an increased morphogenetic potential towards sporophyte formation, upon exposure to low doses of gamma radiation. Young sporophytic leaf crosier cultures were established in vitro on agar based Knop's media with and without 20 g/l sucrose. The cultures were subjected to 60Co radiations in the range of 2.5-100 Gy. Apospory (production of gametophytes on sporophytic tissue without spores) was observed on leaf tissue cultured on Knops media with and without sucrose in P. calomelanos, at the end of 60 days. 5 Gy treated explants showed high number of aposporous gametophytes and was comparable to the control. Other tested doses reduced the aposporous gametophyte production significantly. In the second phase of the experimentation, the cultures were retained on the gametophyte induction media for a period of 4 weeks. Aposporous gametophytes were observed to proliferate with occasional development of antheridia. At the end of 4 weeks, morphogenetic development on the gametophytic tissue resulted in a significantly higher number of apogamous sporophytes (production of sporophytes without fusion of gametes) were obtained on 5 Gy treated tissue as compared to control and all the other treated explants. Apogamous sporophytes thus produced were successfully grown in the greenhouse and transferred to the field. Thus the use of gamma radiation in vitro not only reduced the need for sucrose for induction of apospory in P.calomelanos, it also exhibited hormesis at 5 Gy for improved sporophyte production.

Oxidative Stress Promotes Asexual Reproduction and Apogamy in the Red Seaweed Pyropia yezoensis.

The marine red seaweed Pyropia yezoensis has a haploid-diploid life cycle wherein two heteromorphic generations, a haploid gametophyte and a diploid sporophyte, are reciprocally generated from conchospores and carpospores, respectively. When we treated gametophytic blades of P. yezoensis with H2O2, discharge of asexual monospores was accelerated, resulting in increased numbers of gametophytic clones. Production of sporophytes without fertilization of male and female gametes was also observed. These findings indicate that oxidative stress can induce vegetative cells to develop into monospores that produce gametophytes asexually and can sometimes prompt carpospores to develop into sporophytes. The discovery of oxidative stress-triggered asexual reproduction and -apogamy will stimulate progress in studies of life-cycle regulation in P. yezoensis.

Proteogenomic Analysis Greatly Expands the Identification of Proteins Related to Reproduction in the Apogamous Fern Dryopteris affinis ssp. affinis.

Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis. For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a "species-specific transcriptome database" (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non-model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models.

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.

In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture.

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500-1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns.

Presencia, abundancia y estrategias reproductivas de helechos en áreas alteradas de la Sierra Nevada, México

Presence, abundance and reproductive strategies of ferns in disturbed areas of Sierra Nevada, México. Diverse reproductive strategies shown by ferns and lycophytes allow them to colonize a variety of habitats, particularly after the incidence of natural or anthropogenic disturbances. This study assessed the presence, abundance and reproductive strategies of ferns growing in soils of temperate forests with different levels of disturbance at the Sierra Nevada mountain range in the State of Mexico. Vegetation, soil and environmental variables were recorded in 200m² permanent plots located in four forest stands. One-Way ANOVA and Canonical Correspondence Analysis resulted in the recognition of three landscape types defined by the degree of environmental alteration: low, moderate and severe. Also, from five soil samples collected in each stand, germination of ferns and lycophytes was induced. A positive relationship was found between the alteration degree and Cheilanthes abundance. Under a low landscape alteration regime, species richness is restricted to Cheilanthes bonariensis, C. marginata and Pellaea ternifolia subsp. ternifolia. The soil is a reservoir of spores of Cheilanthes and Pellaea ternifolia subsp. ternifolia because their spores can remain viable for different time intervals. Apogamy is the usual reproductive strategy of Cheilanthes species in disturbed ecosystems, although these species also show sexual reproduction in natural ecosystems with adequate water availability. Apogamy may be related to a shorter generation time in comparison with a low disturbed ecosystem. On the other hand, Pellaea ternifolia subsp. ternifolia only has sexual reproduction. Apogamy might be related to a faster generation in comparison with a low disturbed ecosystem. Rev. Biol. Trop. 59 (1): 417-433. Epub 2011 March 01.

Los helechos y licófitas tienen diversas estrategias reproductivas que las habilitan para invadir ambientes perturbados y áreas sin vegetación. En este trabajo se estudió la presencia, abundancia y estrategias reproductivas de individuos que crecen en suelos de bosques templados, en la Sierra Nevada, Estado de México. Se eligieron cuatro zonas en las que se instaló un cuadrante permanente de 200m² para cada una, con el fin de registrar variables edáficas, ambientales y de vegetación. La relación entre variables se pudo explorar con análisis de varianza y Análisis Canónico de Correspondencia dando como resultado tres paisajes con base en el grado de alteración ambiental: baja, moderada y severa. Fueron recolectadas cinco muestras de suelo en cada zona para inducir la germinación de helechos y licofitas, obteniendo resultados que revelan una relación positiva entre el grado de alteración y la abundancia de Cheilanthes. En paisajes con baja alteración, la riqueza de especies fue mayor comparada con paisajes de alteración severa, en la que la presencia de especies se redujo a Cheilanthes bonariensis, C. marginata y Pellaea ternifolia subsp. ternifolia. El suelo fue un banco de esporas de Cheilanthes y Pellaea ternifolia subsp. ternifolia ya que las esporas permanecieron viables por diferentes intervalos de tiempo. La apogamia fue la estrategia reproductiva común para especies de Cheilanthes en ecosistemas alterados, aunque estas especies tienen reproducción sexual en ecosistemas naturales con suficiente agua. Por otro lado, Pellaea ternifolia subsp. ternifolia solo presentó reproducción sexual. La apogamia puede estar relacionada con que la alternancia de generaciones se realice en menos tiempo, comparado con un ecosistema de baja alteración.