Illuminating the role of the calyptra in sporophyte development.

The study of moss calyptra form and function began almost 250 years ago, but calyptra research has remained a niche endeavor focusing on only a small number of species. Recent advances have focused on calyptra cuticular waxes, which function in dehydration protection of the immature sporophyte apex. The physical presence of the calyptra also plays a role in sporophyte development, potentially via its influence on auxin transport. Progress developing genomic resources for mosses beyond the model Physcomitrium patens, specifically for species with larger calyptrae and taller sporophytes, in combination with advances in CRISPR-Cas9 genome editing will enable the influence of the calyptra on gene expression and the production of RNAs and proteins that coordinate sporophyte development to be explored.

Shifts in flowering phenology in response to spring temperatures in eastern Tennessee.

PREMISE: Plant phenological shifts are among the clearest indicators of the effects of climate change. In North America, numerous studies in the northeastern United States have demonstrated earlier spring flowering compared to historical records. However, few studies have examined phenological shifts in the southeastern United States, a highly biodiverse region of North America characterized by dramatic variations in abiotic conditions over small geographic areas. METHODS: We examined 1000+ digitized herbarium records along with location-specific temperature data to analyze phenological shifts of 14 spring-flowering species in two adjacent ecoregions in eastern Tennessee. RESULTS: Spring-flowering plant communities in the Blue Ridge and the Ridge and Valley ecoregions differed in their sensitivity to temperature; plants in the Ridge and Valley flower 0.73 days earlier/°C on average compared to 1.09 days/°C for plants in the Blue Ridge. Additionally, for the majority of species in both ecoregions, flowering is sensitive to spring temperature; i.e., in warmer years, most species flowered earlier. Despite this sensitivity, we did not find support for community-level shifts in flowering within eastern Tennessee in recent decades, likely because increases in annual temperature in the southeast are driven primarily by warming summer (rather than spring) temperatures. CONCLUSIONS: These results highlight the importance of including ecoregion as a predictor in phenological models for capturing variation in sensitivity among populations and suggest that even small shifts in temperature can have dramatic effects on phenology in response to climate in the southeastern United States.

Genome size and endopolyploidy evolution across the moss phylogeny.

BACKGROUND AND AIMS: Compared with other plant lineages, bryophytes have very small genomes with little variation across species, and high levels of endopolyploid nuclei. This study is the first analysis of moss genome evolution over a broad taxonomic sampling using phylogenetic comparative methods. We aim to determine whether genome size evolution is unidirectional as well as examine whether genome size and endopolyploidy are correlated in mosses. METHODS: Genome size and endoreduplication index (EI) estimates were newly generated using flow cytometry from moss samples collected in Canada. Phylogenetic relationships between moss species were reconstructed using GenBank sequence data and maximum likelihood methods. Additional 1C-values were compiled from the literature and genome size and EI were mapped onto the phylogeny to reconstruct ancestral character states, test for phylogenetic signal and perform phylogenetic independent contrasts. KEY RESULTS: Genome size and EI were obtained for over 50 moss taxa. New genome size estimates are reported for 33 moss species and new EIs are reported for 20 species. In combination with data from the literature, genome sizes were mapped onto a phylogeny for 173 moss species with this analysis, indicating that genome size evolution in mosses does not appear to be unidirectional. Significant phylogenetic signal was detected for genome size when evaluated across the phylogeny, whereas phylogenetic signal was not detected for EI. Genome size and EI were not found to be significantly correlated when using phylogenetically corrected values. CONCLUSIONS: Significant phylogenetic signal indicates closely related mosses have more similar genome sizes and EI values. This study supports that DNA content in mosses is defined by small genomes that are highly endopolyploid, suggesting strong selective pressure to maintain these features. Further research is needed to understand the functional significance of DNA content evolution in mosses.

Stable isotope analyses reveal previously unknown trophic mode diversity in the Hymenochaetales.

PREMISE OF THE STUDY: The Hymenochaetales are dominated by lignicolous saprotrophic fungi involved in wood decay. However, the group also includes bryophilous and terricolous taxa, but their modes of nutrition are not clear. Here, we investigate patterns of carbon and nitrogen utilization in numerous non-lignicolous Hymenochaetales and provide a phylogenetic context in which these non-canonical ecological guilds arose. METHODS: We combined stable isotope analyses of δ13 C and δ15 N and phylogenetic analyses to explore assignment and evolution of nutritional modes. Clustering procedures and statistical tests were performed to assign trophic modes to Hymenochaetales and test for differences between varying ecologies. Genomes of Hymenochaetales were mined for presence of enzymes involved in plant cell wall and lignin degradation and sucrolytic activity. KEY RESULTS: Three different trophic clusters were detected - biotrophic, saprotrophic, and a second biotrophic cluster including many bryophilous Hymenochaetales and mosses. Non-lignicolous Hymenochaetales are generally biotrophic. All lignicolous Hymenochaetales clustered as saprotrophic and most terricolous Hymenochaetales clustered as ectomycorrhizal. Overall, at least 15 species of Hymenochaetales are inferred as biotrophic. Bryophilous species of Rickenella can degrade plant cell walls and lignin, and cleave sucrose to glucose consistent with a parasitic or endophytic life style. CONCLUSIONS: Most non-lignicolous Hymenochaetales are biotrophic. Stable isotope values of many bryophilous Hymenochaetales cluster as ectomycorrhizal or in a biotrophic cluster indicative of parasitism or an endophytic life style. Overall, trophic mode diversity in the Hymenochaetales is greater than anticipated, and non-lignicolous ecological traits and biotrophic modes of nutrition are evolutionarily derived features.

The moss Funaria hygrometrica has cuticular wax similar to vascular plants, with distinct composition on leafy gametophyte, calyptra and sporophyte capsule surfaces.

BACKGROUND AND AIMS: Aerial surfaces of land plants are covered with a waxy cuticle to protect against water loss. The amount and composition of cuticular waxes on moss surfaces had rarely been investigated. Accordingly, the degree of similarity between moss and vascular plant waxes, and between maternal and offspring moss structure waxes is unknown. To resolve these issues, this study aimed at providing a comprehensive analysis of the waxes on the leafy gametophyte, gametophyte calyptra and sporophyte capsule of the moss Funaria hygrometrica METHODS: Waxes were extracted from the surfaces of leafy gametophytes, gametophyte calyptrae and sporophyte capsules, separated by gas chromatography, identified qualitatively with mass spectrometry, and quantified with flame ionization detection. Diagnostic mass spectral peaks were used to determine the isomer composition of wax esters. KEY RESULTS: The surfaces of the leafy gametophyte, calyptra and sporophyte capsule of F. hygrometrica were covered with 0·94, 2·0 and 0·44 µg cm(-2) wax, respectively. While each wax mixture was composed of mainly fatty acid alkyl esters, the waxes from maternal and offspring structures had unique compositional markers. ß-Hydroxy fatty acid alkyl esters were limited to the leafy gametophyte and calyptra, while alkanes, aldehydes and diol esters were restricted to the sporophyte capsule. Ubiquitous fatty acids, alcohols, fatty acid alkyl esters, aldehydes and alkanes were all found on at least one surface. CONCLUSIONS: This is the first study to determine wax coverage (µg cm(-2)) on a moss surface, enabling direct comparisons with vascular plants, which were shown to have an equal amount or more wax than F. hygrometrica Wax ester biosynthesis is of particular importance in this species, and the ester-forming enzyme(s) in different parts of the moss may have different substrate preferences. Furthermore, the alkane-forming wax biosynthesis pathway, found widely in vascular plants, is active in the sporophyte capsule, but not in the leafy gametophyte or calyptra. Overall, wax composition and coverage on F. hygrometrica were similar to those reported for some vascular plant species, suggesting that the underlying biosynthetic processes in plants of both lineages were inherited from a common ancestor.

eQTL Regulating Transcript Levels Associated with Diverse Biological Processes in Tomato.

Variation in gene expression, in addition to sequence polymorphisms, is known to influence developmental, physiological, and metabolic traits in plants. Genetic mapping populations have facilitated identification of expression quantitative trait loci (eQTL), the genetic determinants of variation in gene expression patterns. We used an introgression population developed from the wild desert-adapted Solanum pennellii and domesticated tomato (Solanum lycopersicum) to identify the genetic basis of transcript level variation. We established the effect of each introgression on the transcriptome and identified approximately 7,200 eQTL regulating the steady-state transcript levels of 5,300 genes. Barnes-Hut t-distributed stochastic neighbor embedding clustering identified 42 modules revealing novel associations between transcript level patterns and biological processes. The results showed a complex genetic architecture of global transcript abundance pattern in tomato. Several genetic hot spots regulating a large number of transcript level patterns relating to diverse biological processes such as plant defense and photosynthesis were identified. Important eQTL regulating transcript level patterns were related to leaf number and complexity as well as hypocotyl length. Genes associated with leaf development showed an inverse correlation with photosynthetic gene expression, but eQTL regulating genes associated with leaf development and photosynthesis were dispersed across the genome. This comprehensive eQTL analysis details the influence of these loci on plant phenotypes and will be a valuable community resource for investigations on the genetic effects of eQTL on phenotypic traits in tomato.

Comparative Cuticle Development Reveals Taller Sporophytes Are Covered by Thicker Calyptra Cuticles in Mosses.

The calyptra is a maternal structure that protects the sporophyte offspring from dehydration, and positively impacts sporophyte survival and fitness in mosses. We explore the relationship between cuticle protection and sporophyte height as a proxy for dehydration stress in Funariaceae species with sporophytes across a range of sizes. Calyptrae and sporophytes from four species were collected from laboratory-grown populations at two developmental stages. Tissues were embedded, sectioned, and examined using transmission electron microscopy. Cuticle thickness was measured from three epidermal cells per organ for each individual and compared statistically. All four species have cuticles consisting of a cuticle proper and a cuticular layer on the calyptra and sporophyte at both developmental stages. Across species, shorter sporophytes are associated with smaller calyptra and thinner calyptra cuticles, whereas taller sporophytes are associated with larger calyptra and thicker calyptra cuticles. Independent of size, young sporophytes have a thin cuticle that thickens later during development, while calyptrae have a mature cuticle produced early during development that persists throughout development. This study adds to our knowledge of maternal effects influencing offspring survival in plants. Released from the pressures to invest in protection for their sporophyte offspring, maternal resources can be allocated to other processes that support sporophyte reproductive success. Using a comparative developmental framework enables us to broaden our understanding of cuticle development across species and provides structural evidence supporting the waterproofing role of the moss calyptra.

Identification of ß-hydroxy fatty acid esters and primary, secondary-alkanediol esters in cuticular waxes of the moss Funaria hygrometrica.

The plant cuticle, a multi-layered membrane that covers plant aerial surfaces to prevent desiccation, consists of the structural polymer cutin and surface-sealing waxes. Cuticular waxes are complex mixtures of ubiquitous, typically monofunctional fatty acid derivatives and taxon-specific, frequently bifunctional specialty compounds. To further our understanding of the chemical diversity of specialty compounds, the waxes on the aerial structures of the leafy gametophyte, sporophyte capsule, and calyptra of the moss Funaria hygrometrica were surveyed. Respective moss surfaces were extracted, and resulting lipid mixtures were analyzed by gas chromatography-mass spectrometry (GC-MS). The extracts contained ubiquitous wax compound classes along with two prominent, unidentified classes of compounds that exhibited some characteristics of bifunctional structures. Microscale transformations led to derivatives with characteristic MS fragmentation patterns suggesting possible structures for these compounds. To confirm the tentative structure assignments, one compound in each of the suspected homologous series was synthesized. Based on GC-MS comparison with the authentic standards, the first series of compounds was identified as containing esters formed by ß-hydroxy fatty acids and wax alcohols, with ester chain lengths varying from C42 to C50 and the most prominent homolog being C46. The second series consisted of fatty acid esters of 1,7-alkanediols, linked via the primary hydroxyl group, with ester chain lengths C40-C52 also dominated by the C46 homolog. The ß-hydroxy acid esters were restricted to the sporophyte capsule, and the diol esters to the leafy gametophyte and calyptra. Based on their homolog and isomer distributions, and the presence of free 1,7-triacontanediol, possible biosynthetic reactions leading to these compounds are discussed.

Dehydration protection provided by a maternal cuticle improves offspring fitness in the moss Funaria hygrometrica.

BACKGROUND AND AIMS: In bryophytes the sporophyte offspring are in contact with, nourished from, and partially surrounded by the maternal gametophyte throughout their lifespan. During early development, the moss sporophyte is covered by the calyptra, a cap of maternal gametophyte tissue that has a multilayered cuticle. In this study the effects on sporophyte offspring fitness of removing the maternal calyptra cuticle, in combination with dehydration stress, is experimentally determined. METHODS: Using the moss Funaria hygrometrica, calyptra cuticle waxes were removed by chemical extraction and individuals were exposed to a short-term dehydration event. Sporophytes were returned to high humidity to complete development and then aspects of sporophyte survival, development, functional morphology, and reproductive output were measured. KEY RESULTS: It was found that removal of calyptra cuticle under low humidity results in significant negative impacts to moss sporophyte fitness, resulting in decreased survival, increased tissue damage, incomplete sporophyte development, more peristome malformations, and decreased reproductive output. CONCLUSIONS: This study represents the strongest evidence to date that the structure of the calyptra cuticle functions in dehydration protection of the immature moss sporophyte. The investment in a maternal calyptra with a multilayered cuticle increases offspring fitness and provides a functional explanation for calyptra retention across mosses. The moss calyptra may represent the earliest occurance of maternal protection via structural provisioning of a cuticle in green plants.

Organellar genome, nuclear ribosomal DNA repeat unit, and microsatellites isolated from a small-scale of 454 GS FLX sequencing on two mosses.

Recent innovations in high-throughput DNA sequencing methodology (next generation sequencing technologies [NGS]) allow for the generation of large amounts of high quality data that may be particularly critical for resolving ambiguous relationships such as those resulting from rapid radiations. Application of NGS technology to bryology is limited to assembling entire nuclear or organellar genomes of selected exemplars of major lineages (e.g., classes). Here we outline how organellar genomes and the entire nuclear ribosomal DNA repeat can be obtained from minimal amounts of moss tissue via small-scale 454 GS FLX sequencing. We sampled two Funariaceae species, Funaria hygrometrica and Entosthodon obtusus, and assembled nearly complete organellar genomes and the whole nuclear ribosomal DNA repeat unit (18S-ITS1-5.8S-ITS2-26S-IGS1-5S-IGS2) for both taxa. Sequence data from these species were compared to sequences from another Funariaceae species, Physcomitrella patens, revealing low overall degrees of divergence of the organellar genomes and nrDNA genes with substitutions spread rather evenly across their length, and high divergence within the external spacers of the nrDNA repeat. Furthermore, we detected numerous microsatellites among the 454 assemblies. This study demonstrates that NGS methodology can be applied to mosses to target large genomic regions and identify microsatellites.

The cuticle on the gametophyte calyptra matures before the sporophyte cuticle in the moss Funaria hygrometrica (Funariaceae).

PREMISE OF THE STUDY: In vascular plants, leaf primordia prevent desiccation of the shoot apical meristem. Lacking leaves, the undifferentiated moss sporophyte apex is covered by the calyptra, a cap of maternal gametophyte tissue that is hypothesized to function in desiccation protection. Herein, we compare cuticle development on the calyptra and sporophyte to assess the calyptra's potential to protect the sporophyte from desiccation. As the first comprehensive study of moss sporophyte cuticle development, this research broadens our perspectives on cuticle development and evolution across embryophytes. METHODS: Calyptrae and sporophytes at nine developmental stages were collected from a laboratory-grown population of the moss Funaria hygrometrica. Tissues were embedded, sectioned, then examined using transmission electron microscopy. Epidermal cells were measured for thickness of the cuticle layers, cell wall thickness, and lumen size. KEY RESULTS: The calyptra cuticle develops precociously and reaches maturity before the sporophyte cuticle. Calyptrae are covered by a four-layered cuticle at all stages, whereas sporophyte cuticle maturation is delayed until sporangium formation. The development and thickening of the sporophyte cuticle occurs in an acropetal wave. CONCLUSIONS: A multilayered calyptra cuticle at the earliest developmental stages is consistent with its ability to protect the immature sporophyte from desiccation. Young sporophytes lack a complex cuticle and thus may require protection, whereas in older sporophytes a mature cuticle develops. The moss calyptra is not a vestigial structure, but rather the calyptra's role in preventing desiccation offers a functional explanation for calyptra retention during the 450 Myr of moss evolution.

Phylogenetic inference rejects sporophyte based classification of the Funariaceae (Bryophyta): rapid radiation suggests rampant homoplasy in sporophyte evolution.

The moss family Funariaceae, which includes the model systems Funaria hygrometrica and Physcomitrella patens, comprises 15 genera, of which three accommodate approximately 95% of the 250-400 species. Generic concepts are drawn primarily from patterns in the diversity of morphological complexity of the sporophyte. Phylogenetic inferences from ten loci sampled across the three genomic compartments yield a hypothesis that is incompatible with the current circumscription of two of the speciose genera of the Funariaceae. The single clade, comprising exemplars of Funaria with a compound annulus, is congruent with the systematic concept proposed by Fife (1985). By contrast, Entosthodon and Physcomitrium are resolved as polyphyletic entities, and even the three species of Physcomitrella are confirmed to have diverged from distinct ancestors. Although the backbone relationships within the core clade of the Funariaceae remain unresolved, the polyphyly of these genera withstands alternative hypothesis testing. Consequently, the sporophytic characters that define these lineages are clearly homoplasious suggesting that selective pressures (or their relaxation) are in fact driving the diversification rather than the conservation of sporophytic architecture in the Funariaceae.

Frequent pseudogenization and loss of the plastid-encoded sulfate-transport gene cysA throughout the evolution of liverworts.

PREMISE OF THE STUDY: The presence or absence of a functional copy of a plastid gene may reflect relaxed selection, and may be phylogenetically significant, reflecting shared ancestry. In some liverworts, the plastid gene cysA is a pseudogene (inferred to be nonfunctional). We surveyed 63 liverworts from all major clades to determine whether the loss of cysA is phylogenetically significant, whether intact copies of cysA are under selective constraints, and whether rates of nucleotide substitution differ in other plastid genes from taxa with and without a functional copy of cysA. METHODS: Primers annealing to flanking and internal regions were used to amplify and sequence cysA from 61 liverworts. Two additional cysA sequences were downloaded from NCBI. The ancestral states of cysA were reconstructed on a phylogenetic hypothesis inferred from seven markers. Rates of nucleotide substitution were estimated for three plastid loci to reflect the intrinsic mutation rate in the plastid genome. The ratio of nonsynonymous to synonymous substitutions was estimated for intact copies of cysA to infer selective constraints. KEY RESULTS: Throughout liverworts, cysA has been lost up to 29 times, yet intact copies of cysA are evolving under selective constraints. Gene loss is more frequent in groups with an increased substitution rate in the plastid genome. CONCLUSIONS: The number of inferred losses of cysA in liverworts exceeds any other reported plastid gene. Despite frequent losses, cysA is evolving under purifying selection in liverworts that retain the gene. It appears that cysA is lost more frequently in lineages characterized by a higher rate of nucleotide substitutions in the plastid.

A hundred-year-old question: is the moss calyptra covered by a cuticle? A case study of Funaria hygrometrica.

BACKGROUND AND AIMS: The maternal gametophytic calyptra is critical for moss sporophyte development and ultimately sporogenesis. The calyptra has been predicted to protect the sporophyte apex, including the undifferentiated sporogenous region and seta meristem, from desiccation. We investigate the hypothesis that this waterproofing ability is due to a waxy cuticle. The idea that moss calyptrae are covered by a cuticle has been present in the literature for over a century, but, until now, neither the presence nor the absence of a cuticle has been documented for any calyptra. METHODS: The epidermis of the calyptra, leafy gametophyte and sporophyte sporangia of the moss Funaria hygrometrica were examined using scanning and transmission electron microscopy. Thicknesses of individual cuticle layers were quantified and compared statistically. The immunochemistry antibody (LM19) specific for pectins was used to locate cell wall material within the cuticle. KEY RESULTS: A multi-layered cuticle is present on the calyptra of F. hygrometrica, including layers analogous to the cuticular layer, cell wall projections, electron-lucent and electron-dense cuticle proper observed in vascular plants. The calyptra rostrum has a cuticle that is significantly thicker than the other tissues examined and differs by specialized thickenings of the cuticular layer (cuticular pegs) at the regions of the anticlinal cell walls. This is the first documentation of cuticular pegs in a moss. CONCLUSIONS: The calyptra and its associated cuticle represent a unique form of maternal care in embryophytes. This organ has the potential to play a critical role in preventing desiccation of immature sporophytes and thereby may have been essential for the evolution of the moss sporophyte.

Development of the enigmatic peristome of Timmia megapolitana (Timmiaceae; Bryophyta).

The Timmiaceae (Bryophyta) have been traditionally classified within the Bryales based on peristome architecture. Phylogenetic analyses of nucleotide sequences have revealed relationships that are incongruent with this hypothesis and have implicated an origin for this lineage early in the radiation of arthrodontous mosses (Bryopsida). This unexpected phylogenetic placement raises important questions about the evolutionary significance of the Timmia peristome, which differs from all other mosses by 64 isomorphic filaments topping the endostomial membrane. A developmental study of the peristome in Timmia megapolitana was undertaken to examine alignments of anticlinal cell walls in the inner peristomial layer (IPL) with those of the primary peristomial layer (PPL), a character that has been used to define major arthrodontous lineages. Criteria were established for assessing longitudinally homologous regions that contribute to the peristome-forming region. Young sporophytes were examined using histological techniques, and the alignment of the cell wall divisions was quantified. Critical divisions in the IPL of T. megapolitana were determined to be symmetrical, similar to patterns in the Funariales. This research provides novel developmental observations for a putative ancestral lineage of arthrodontous mosses, reevaluates criteria used to compare developmental studies of different lineages and discusses the phylogenetic implications of these observations.