Gametophyte Generation Research Articles

Functions and Regulation of HAM Family Genes in Meristems During Gametophyte and Sporophyte Generations.

A fascinating feature of land plants is their ability to continually initiate new tissues and organs throughout their lifespan, driven by a pool of pluripotent stem cells located in meristems. In seed plants, various types of meristems are initiated and maintained during the sporophyte generation, while their gametophytes lack meristems and rely on sporophyte tissues for growth. In contrast, seed-free vascular plants, such as ferns, develop meristems during both the sporophyte and gametophyte generations, allowing for the independent growth of both generations. Recent findings have highlighted both conserved and lineage-specific roles of the HAIRY MERISTEM (HAM) family of GRAS-domain transcriptional regulators in various meristems throughout the land plant lifecycle. Here, we review and discuss how HAM genes maintain meristem indeterminacy in both sporophytes and gametophytes, with a focus on studies performed in two model species: the flowering plant Arabidopsis thaliana and the fern Ceratopteris richardii. Additionally, we summarize the crucial and tightly regulated functions of the microRNA171 (miR171)-HAM regulatory modules, which define HAM spatial patterns and activities during meristem development across various meristem identities in land plants.

Rediscovery of the rare Cladochroa chnoosporiformis Skottsberg from the Falkland Islands and its merger with Utriculidium durvillei Skottsberg

Abstract The Subantarctic brown alga Cladochroa chnoosporiformis had been collected only on one occasion by Carl Skottsberg in 1907 from Port Philomel, West Falkland, Falkland Islands, resulting in its formal taxonomic description. Due to the lack of reports since then, doubts remained about its existence and identity. Within the framework of this study, Cladochroa was rediscovered 106 years after its original description at its type locality, confirming its existence and the morphological features described by Skottsberg. The re-collection enabled molecular studies on field material and isolated cultures. Sequences of nrDNA, rbcL, and 5′-COI suggest that C. chnoosporiformis is conspecific with Utriculidium durvillei, which has nomenclatural priority. However, the morphologies of C. chnoosporiformis and U. durvillei are significantly different. Utriculidium may be the gametophyte generation of Cladochroa, or hitherto unknown factors cause highly different morphologies without alternating generations.

Polyploidy and environmental stress response: a comparative study of fern gametophytes.

Climate change is rapidly altering natural habitats and generating complex patterns of environmental stress. Ferns are major components of many forest understories and, given their independent gametophyte generation, may experience unique pressures in emerging temperature and drought regimes. Polyploidy is widespread in ferns and may provide a selective advantage in these rapidly changing environments. This work aimed to understand whether the gametophytes of allopolyploid ferns respond differently to climate-related physiological stress than their diploid parents. The experimental approach involved a multifactorial design with 27 treatment combinations including exposure to multiple levels of drought and temperature over three treatment durations, with recovery measured at multiple timepoints. We measured Chl fluorescence from over 2000 gametophytes to evaluate stress avoidance and tolerance in diploid and polyploid species. Polyploids generally showed a greater ability to avoid and/or tolerate a range of stress conditions compared with their diploid counterparts, suggesting that polyploidy may confer enhanced flexibility and resilience under climate stress. Overall, these results suggest that polyploidy may provide some resilience to climate change in mixed ploidy populations. However, all species remain susceptible to the impacts of extreme drought and heat stress.

Characterization of the fiber-like cortical cells in moss gametophytes.

Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-β-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-β-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Male gametogenesis in flowering plants

The life cycles of plants are characterized by significant alternations between the diploid sporophytic and the haploid gametophytic generations. Meiosis and fertilization are the prerequisites for achieving the alternation of generations. Diploid sporophytes undergo meiosis to produce haploid gametes, and male–female gametes fuse (double fertilization) to revert to the diploid sporophyte. The formation and development of male gametophytes are crucial for the alternation of generations in higher plants. During the long evolution of plants from aquatic to terrestrial, the way of sexual reproduction has undergone subversive innovations. From sperm swimming for fertilization to relying on the precise delivery of pollen tubes to female gametes for the fusion of the male–female gametes, higher requirements are placed on the male gametophytes’ development and fertilization process. The formation of male gametophytes has undergone significant changes to meet the needs for delivery and transportation of the male gametes. With the emergence of more and more evidence, the molecular mechanism underlying male gametophytes’ development, especially the initiation and specialization of germline cells has been better understood. Focusing on the latest studies, we reviewed and elucidated the critical proteins and factors involved in male gametophyte formation, highlighting the decisive role of auxin in archesporial cell specialization and the importance of microspore maturation in pre-mitosis, and analyzed the molecular mechanisms underlying male gametogenesis, with a view to providing insights for further exploration of male gametophytes formation in the future.

TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis.

Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss-of-function mutants in Arabidopsis thaliana. pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

The pattern of natural variability of palynomorphological features with the example of some <i>Nierembergia</i> and <i>Bouchetia</i> species (Solanaceae) and natural system of bio-variety

On the example of pollen morphological features of 8 species of Nierembergia and 2 species of Bouchetia (family Solanaceae), the properties of individual variability are described. Most of the studied characters (structure of apertures, ultrastructure of the sporoderm, surface sculpture, dimensions) do not have significant differences at the individual and intraspecific levels; taxonomically significant variability of morphological features of pollen is manifested at the level of the genus and suprageneric groups. The genera differ significantly in the sculpture of the pollen grain surface – striate in Nierembergia and tuberculate in Bouchetia. Pollen contained in one bud, anther or tetrad (fully completed gametophytic generation, where there is no death – all descendants of one ancestor without exception), is considered as an extreme model (maximum completeness with minimum complexity) to study the properties of natural morphological variability and the causes of its occurrence. It was found that pollen characters (sculpture, number and location of apertures) have the same pattern of variability (continuous and transitively ordered series), which embodied at different taxonomic levels in different characters. The natural variability of pollen morphological features is ordered not into a genealogical clade, but into a cline – continuous, geometrically ordered and transitive series (taxon-nonspecific and rank-independent). In this system of parallelisms, homological series are inseparable from non-homological ones, and typical forms, from deviations. The origin of typical and deviant forms cannot be explained separately (typical – genealogically, and deviant – as parallelism, convergence, chance or regularity). The individual variability of pollen forms is geometrically ordered and is not the result of random disturbances, failures of the hereditary program, or pathology. The typical form turns out to be a harmonious part of a geometrically ordered series of pure forms, free from functional and historical connotations. The similarity of pollen forms in these series is determined by their geometry and does not depend on affinity, homology, functionality (improvement, exercise, adaptation). The natural system of pollen forms is formed not by the structure of supposed affinity of supposed taxa (universalia, general concepts, the result of speculation that requires confirmation and admits refutation), but the structure of the observed parallelisms of the variability of individual living bodies. Evolutionary novelty (the current state, the observed variability) arises initially ordered in a pre-established form.

Three-Dimensional Morphological Analysis Revealed the Cell Patterning Bases forthe Sexual Dimorphism Development inthe Liverwort Marchantia polymorpha.

In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana, those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator MpFGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha.

Single-nucleus sequencing deciphers developmental trajectories in rice pistils.

Angiosperms possess a life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here, we show a cell census of rice pistils before fertilization through the use of droplet-based single-nucleus RNA sequencing. The ab initio marker identification validated by in situ hybridization assists with cell-type annotation, revealing cell heterogeneity between ovule- and carpel-originated cells. A comparison of 1N (gametophyte) and 2N (sporophyte) nuclei identifies the developmental path of germ cells in ovules with typical resetting of pluripotency before the sporophyte-gametophyte transition, while trajectory analysis of carpel-originated cells suggests previously neglected features of epidermis specification and style function. These findings gain a systems-level view of cellular differentiation and development of rice pistils before flowering and lay a foundation for understanding female reproductive development in plants.

Pollen Mitosis is a Good Choice for Gametophytic Chromosome Study in Higher Plants—Investigation with <i>Allium cepa</i> L. and <i>Aloe vera</i> L.

In higher groups of plants, the somatic cells are conventionally used for karyomorphological studies, which contain a diploid (2n) set of chromosomes. Similar to diploid sporophytic cells, a chromosomal study in haploid (n) gametophytic cells is also necessary as it unfolds the karyomorphological status of germ cells. The mature male gametophytic plant body (i.e., pollen grain) is only two- or three-celled structures. This study focused on the first mitotic division in a microspore, popularly known as ‘pollen mitosis-I.’ Allium cepa L. and Aloe vera L. are two model species for karyomorphological study. Microspores of these species have been stained with 2.0% acetocarmine. The present finding reveals that the cell division in microspores of these two species is completely normal without any significant irregularities. As expected, the chromosome number of the microspores of Allium cepa is n=8 and Aloe vera is n=7, exactly half the number of their sporophytic plants. The haploid karyotype of Allium cepa is the symmetric type with karyotype formula ‘6m+2sm,’ whereas that of Aloe vera is between symmetric and asymmetric types with karyotype formula ‘3sm+4st.’ This study not only enriches the chromosomal database of gametophytes of Allium cepa and Aloe vera, but also will encourage further research on this extremely neglected topic—chromosome study in the gametophytic generation of flowering plants. As Allium cepa and Aloe vera are easily available and model species for karyomorphological study, and with two contrast karyotypes (i.e., symmetric and asymmetric), this article also helps numerous school/college teachers to teach their students about the structure and behavior of gametophytic chromosomes.

Evaluation of Crossover Number, Distribution, and Interference Using Cytological Assays in Arabidopsis.

Meiosis involves the replication of nuclear chromosomes in a parent cell, followed by two successive nuclear divisions to produce haploid spores, which differentiate into the gametophyte generations that produce the egg and sperm in plants. Meiotic recombination or crossover (CO) is a hallmark of meiosis that allows shuffling of genetic information between homologous chromosomes (homologs), thereby giving rise to genetically diverse progeny cells and, ultimately, individuals in the progeny; this opens vast opportunities for genetic differentiation and hence speciation. Meiotic COs also ensure the formation of bivalents and fidelity of their equal segregation. Therefore, mechanisms that regulate meiotic recombination have been extensively studied in multiple species. Several approaches have been developed to observe or estimate the frequency of CO, in which CO can be visualized and analyzed cytologically by estimating the number of chiasma (plural chiasmata), a position where non-sister chromatids exchange genetic material between homologs. Furthermore, one CO event might influence the occurrence of another one nearby, along a chromosome; this is known as CO interference. Over the past decades, visualizing CO events and measuring CO interference have contributed greatly to our understanding of the regulatory mechanisms of meiotic recombination. Here, we describe protocols to estimate the number of chiasmata and CO interference in Arabidopsis using cytological methods involving chromosome spreads and immunostaining. Specifically, we describe how chromosome spreads can be used to estimate the number of chiasmata based on the conformations of metaphase I bivalents and provide a revised acid-based quick immunostaining assay that permits high-throughput and quantitative digital estimation of the relative distance between adjacent interference-sensitive CO foci at diakinesis. These methods can be easily established or modified, if necessary, for studying meiotic recombination in other plants and crops. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Estimation of the number of chiasmata per nucleus based on metaphase I bivalent conformations Basic Protocol 2: A chromosome spread-based immunostaining method for relative distance analysis of adjacent interference-sensitive CO foci at diakinesis in Arabidopsis meiocytes.

Population level variation in reproductive development and output in the golden kelp Laminaria ochroleuca under marine heat wave scenarios

Thermal tolerance is often interpreted as a species-wide thermal niche in the absence of studies focusing on the adaptive potential of populations to exhibit differential thermal tolerance. Thus, considering intraspecific thermal plasticity, local adaptation or both between populations along distributional gradients when interpreting and predicting species responses to warming is imperative. Removing the effect of environmental histories by raising kelp gametophyte generations in vitro under common garden conditions allows unbiased comparison between population-specific adaptive variation under different environmental conditions. Following this approach, this study aims to detect (potentially) adaptive differentiation in microscopic life-stages (gametophytes) between populations of a temperate forest forming kelp, Laminaria ochroleuca from locations with distinct thermal conditions. Gametophytes from four geographically distinct populations were subjected to different temperature treatments (17, marine heat waves of 23, 25 and 27°C) and gametophyte survival during thermal stress as well as reproductive success and photosynthetic responses during recovery were investigated. Intraspecific variation in resilience and reproductive output to thermal stress was found in L. ochroleuca; gametophytes from the most northern population (Brittany, France) were the most thermally sensitive, with mortality onset at 23°C, whereas mortality in the remaining populations was only apparent at 27°C. Gametophytes from northern Spain and Morocco exhibited very low reproductive success during recovery from 23 and 25°C. However, when recovering from the highest thermal treatment (27°C) the reproductive development and sporophyte output was higher than in the gametophytes from France and Italy (Mediterranean). The population-specific responses of gametophyte resilience and reproductive success to temperature stress suggest genetic differentiation in response to variation in local thermal regimes.

Pivotal role of STIP in ovule pattern formation and female germline development in Arabidopsis thaliana.

In spermatophytes the sporophytic (diploid) and the gametophytic (haploid) generations co-exist in ovules, and the coordination of their developmental programs is of pivotal importance for plant reproduction. To achieve efficient fertilization, the haploid female gametophyte and the diploid ovule structures must coordinate their development to form a functional and correctly shaped ovule. WUSCHEL-RELATED HOMEOBOX (WOX) genes encode a family of transcription factors that share important roles in a wide range of processes throughout plant development. Here, we show that STIP is required for the correct patterning and curvature of the ovule in Arabidopsis thaliana. The knockout mutant stip-2 is characterized by a radialized ovule phenotype due to severe defects in outer integument development. In addition, alteration of STIP expression affects the correct differentiation and progression of the female germline. Finally, our results reveal that STIP is required to tightly regulate the key ovule factors INNER NO OUTER, PHABULOSA and WUSCHEL, and they define a novel genetic interplay in the regulatory networks determining ovule development.

MS1/MMD1 homologues in the moss Physcomitrium patens are required for male and female gametogenesis.

The Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. This pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins. We addressed the function of two MS1/MMD1-homologues in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines. The two genes are together essential for both male and female fertility by providing functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms. We propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis, and possibly for sporogenesis, represent a heritage from ancestral land plants.

Arsenic accumulation in Pteris vittata: Time course, distribution, and arsenic-related gene expression in fronds and whole plantlets

In this work, arsenic (As) accumulation and distribution over time in Pteris vittata young fronds from adult plants and in whole plantlets, grown on a highly contaminated As-soil, was determined by μ-XRF. A linear increase in As content up to 60 days was found in young fronds at different times, and a progressive distribution from the apex to the base of the fronds was observed. In whole plantlets, As signal was detectable from 9 to 20 days in the apex of a few fronds and fiddleheads. Later, up to 60 days, As was localized in all fronds, in the rhizome and in basal part of the roots. The dynamics of expression of As-related genes revealed a good correlation between As content and the level of the As (III)-antiporter PvACR3 transcript in plantlets roots and fronds and in young fronds. Moreover, the transcription of As (V)-related gametophytic genes PvGAPC1, PvOCT4 increases over time during As accumulation while PvGSTF1 is expressed only in roots. Here, we demonstrate the suitability of the μ-XRF technique to monitor As accumulation, which allowed us to propose that As is initially directly transported to fiddleheads and apex of fronds, is later distributed to the whole fronds and simultaneously accumulated in the rhizome and roots. We also provide indications on the expression of candidate genes possibly involved in As (hyper)accumulation.

How was apical growth regulated in the ancestral land plant? Insights from the development of non-seed plants.

Land plant life cycles are separated into distinct haploid gametophyte and diploid sporophyte stages. Indeterminate apical growth evolved independently in bryophyte (moss, liverwort, and hornwort) and fern gametophytes, and tracheophyte (vascular plant) sporophytes. The extent to which apical growth in tracheophytes co-opted conserved gametophytic gene networks, or exploited ancestral sporophytic networks, is a long-standing question in plant evolution. The recent phylogenetic confirmation of bryophytes and tracheophytes as sister groups has led to a reassessment of the nature of the ancestral land plant. Here, we review developmental genetic studies of apical regulators and speculate on their likely evolutionary history.

Cold Thermal Priming of Laminaria digitata (Laminariales, Phaeophyceae) Gametophytes Enhances Gametogenesis and Thermal Performance of Sporophytes

Thermal characteristics of kelp species have been studied in many ways, but potentially persistent effects of temperature across generations are yet poorly understood. In this context, the effect of thermal priming on fertility and growth of the N-Atlantic kelp speciesLaminaria digitatawas investigated within and across life cycle generations in a two-step common garden experiment. Using vegetative clonal gametophytes from cold (5°C) and warm (15°C) pre-experimental cultivation (3 years), we first quantified gametogenesis and recruitment over two weeks at a common temperature of 10°C. Then, recruited sporophytes were transferred to a temperature gradient spanning the tolerance range of the species from 0°C to 20°C. We hypothesized that a warm gametophyte pre-experimental cultivation promotes performance of sporophytes at warm temperatures and vice versa. Interestingly, gametogenesis speed and sporophyte recruitment were higher in gametophytes following cold compared to warm pre-experimental cultivation, which indicates carry-over effects of temperature within the gametophyte generation. Compared to warm pre-experimental cultivation of gametophytes, a cold pre-experimental cultivation enhanced growth of juvenileLaminaria digitatasporophytes by more than 69% at the extreme low and high temperatures of 0 and 20°C. This is the first evidence for a cross-generational effect between gametophyte parents and offspring sporophytes. As cold gametophyte cultivation increased the trait performance of gametogenesis, recruitment and thermal tolerance of juvenile sporophytes, priming of early life cycle stages may be used to increase resilience and productivity of kelps in marine forest restoration efforts and kelp mariculture.

KANADI promotes thallus differentiation and FR-induced gametangiophore formation in the liverwort Marchantia.

Summary In angiosperms, KANADI transcription factors have roles in the sporophyte generation regulating tissue polarity, organogenesis and shade avoidance responses, but are not required during the gametophyte generation. Whether these roles are conserved in the gametophyte‐dominant bryophyte lineages is unknown, which we examined by characterising the sole KANADI ortholog, MpKAN, in the liverwort Marchantia polymorpha.In contrast to angiosperm orthologs, MpKAN functions in the gametophyte generation in Marchantia, where it regulates apical branching and tissue differentiation, but does not influence tissue polarity in either generation. MpKAN can partially rescue the sporophyte polarity defects of kanadi mutants in Arabidopsis, indicating that MpKAN has conserved biochemical activity to its angiosperm counterparts.Mpkan loss‐of‐function plants display defects in far‐red (FR) light responses. Mpkan plants have reduced FR‐induced growth tropisms, have a delayed transition to sexual reproduction and fail to correctly form gametangiophores.Our results indicate that MpKAN is a modulator of FR responses, which may reflect a conserved role for KANADI across land plants. Under FR, MpKAN negatively regulates MpDELLA expression, suggesting that MpKAN and MpDELLA act in a pathway regulating FR responses, placing MpKAN in a gene regulatory network exhibiting similarities with those of angiosperms.

The ecology and physiology of fern gametophytes: A methodological synthesis.

All green plants alternate between the gametophyte and sporophyte life stages, but only seed‐free vascular plants (ferns and lycophytes) have independent, free‐living gametophytes. Fern and lycophyte gametophytes are significantly reduced in size and morphological complexity relative to their sporophytic counterparts and have often been overlooked in ecological and physiological studies. Understanding the ecological and physiological factors that directly impact this life stage is of critical importance because the ultimate existence of a sporophyte is dependent upon successful fertilization in the gametophyte generation. Furthermore, previous research has shown that the dual nature of the life cycle and the high dispersibility of spores can result in different geographic patterns between gametophytes and their respective sporophytes. This variation in distribution patterns likely exacerbates the separation of selective pressures acting on gametophyte and sporophyte generations, and can uniquely impact a species’ ecology and physiology. Here, we provide a review of historical and contemporary methodologies used to examine ecological and physiological aspects of fern gametophytes, as well as those that allow for comparisons between the two generations. We conclude by suggesting methodological approaches to answer currently outstanding questions. We hope that the information covered herein will serve as a guide to current researchers and stimulate future discoveries in fern gametophyte ecology and physiology.

Identifying cryptic fern gametophytes using DNA barcoding: A review.

Ferns and lycophytes are unique among land plants in having sporophyte (diploid) and gametophyte (haploid) generations that can grow independently of each other. While most studies of fern ecology focus on the more visible sporophytic stage, the gametophyte is critically important, as it is the sexual phase of the life cycle. Yet, fern gametophytes have long been neglected in field studies due to their small size and cryptic morphology. DNA barcoding is a powerful method that can be used to identify field‐collected gametophytes to species and allow for detailed study of their ecology. Here, we review the state of DNA barcoding as applied to fern gametophytes. First, we trace the history of DNA barcoding and how it has come to be applied to fern gametophytes. Next, we summarize case studies that show how DNA barcoding has been used to better understand fern species distributions, gametophyte ecology, and community ecology. Finally, we propose avenues for future research using this powerful tool, including next‐generation DNA sequencing for in‐field identification of cryptic gametophytes.