Evolutionary history of the grass gynoecium.

The grass family (Poaceae) includes cereal crops that provide a key food source for the human population. The food industry uses the starch deposited in the cereal grain, which develops directly from the gynoecium. Morphological interpretation of the grass gynoecium remains controversial. We re-examine earlier hypotheses and studies of morphology and development in the context of more recent analyses of grass phylogenetics and developmental genetics. Taken in isolation, data on gynoecium development in bistigmatic grasses do not contradict its interpretation as a solitary ascidiate carpel. Nevertheless, in the context of other data, this interpretation is untenable. Broad comparative analysis in a modern phylogenetic context clearly demonstrates that the grass gynoecium is pseudomonomerous. A bistigmatic grass gynoecium has two sterile carpels, each producing a stigma, and a fertile carpel that lacks a stigma. To date, studies of grass developmental genetics and developmental morphology have failed to fully demonstrate the composite nature of the grass gynoecium be-cause its complex evolutionary history is hidden by extreme organ integration. It is problematic to interpret the gynoecium of grasses in terms of normal angiosperm gynoecium typology. Even the concept of a carpel becomes misleading in grasses; instead, we recommend the term pistil for descriptive purposes.

Pollen in water of unstable salinity: Evolution and function of dynamic apertures in monocot aquatics.

PREMISE: The sporoderm of seed-plant pollen grains typically has apertures in which the outer sporopollenin-bearing layer is relatively sparse. The apertures allow regulation of the internal volume of the pollen grain during desiccation and rehydration (harmomegathy) and also serve as sites of pollen germination. A small fraction of angiosperms undergo pollination in water or at the water surface, where desiccation is unlikely. Their pollen grains commonly lack apertures, though with some notable exceptions. We tested a hypothesis that in some angiosperm aquatics that inhabit water of unstable salinity, the pollen apertures accommodate osmotic effects that occur during pollination in such conditions. METHODS: Pollen grains of the tepaloid clade of the monocot order Alismatales, which contains ecologically diverse aquatic and marshy plants, were examined using light microscopy and scanning electron microscopy. We used Ruppia as a model to test pollen grain response in water of various salinities. Pollen aperture evolution was also analyzed using molecular tree topologies. RESULTS: Phylogenetic optimizations demonstrated an evolutionary loss and two subsequent regains of the aperturate condition in the tepaloid clade of Alismatales. Both of the taxa that have reverted to aperturate pollen (Ruppia, Ruppiaceae; Althenia, Potamogetonaceae) are adapted to changeable water salinity. Direct experiments with Ruppia showed that the pollen apertures have a role in a harmomegathic response to differences in water salinity. CONCLUSIONS: Our results showed that the inferred regain of pollen apertures represents an adaptation to changeable water salinity. We invoke a loss-and-regain scenario, prompting questions that are testable using developmental genetics and plant physiology.

Two centuries from species discovery to diagnostic characters: molecular and morphological evidence for narrower species limits in the widespread SW Australian Anarthria gracilis complex (Restionaceae s.l./Anarthriaceae, Poales).

BACKGROUND: The extreme southwest of Australia is a biodiversity hotspot region that has a Mediterranean-type climate and numerous endemic plant and animal species, many of which remain to be properly delimited. We refine species limits in Anarthria, a Western Australian endemic genus characterised by the occurrence of the greatest number of plesiomorphic character states in the restiid clade of Poales. In contrast to many other groups of wind-pollinated Australian Poales, Anarthria was traditionally viewed as having well-established species limits. All six currently recognised species, which are conspicuous members of some Western Australian plant communities, were described in the first half of the 19th century. They are traditionally distinguished from each other mainly using quantitative characters. METHODS: We examined extensive existing herbarium specimens and made new collections of Anarthria in nature. Scanning electron microscopy and light microscopy were used to study leaf micromorphology. Molecular diversity of Anarthria was examined using a plastid (trnL-F) and a low-copy nuclear marker (at103). This is the first study of species-level molecular diversity in the restiid clade using a nuclear marker. RESULTS: Material historically classified as Anarthria gracilis R.Br. actually belongs to three distinct species, A. gracilis s.str., A. grandiflora Nees and A. dioica (Steud.) C.I.Fomichev, each of which forms a well-supported clade in phylogenetic analyses. Both segregate species were described in the first half of the 19th century but not recognised as such in subsequent taxonomic accounts. Anarthria dioica was first collected in 1826, then wrongly interpreted as a species of Juncus (Juncaceae) and described as Juncus dioicus. We provide a formal transfer of the name to Anarthria and for the first time report its clear and qualitative diagnostic characters: an extremely short leaf ligule and distinctive pattern of leaf epidermal micromorphology. A long ligule is present in A. gracilis s.str. and A. grandiflora. These species differ from each other in leaf lamina morphology and anatomy and have mostly non-overlapping distribution ranges. The narrower definition of species provides a basis for future phylogeographic analyses in Anarthria. Our study highlights a need for more extensive use of nuclear DNA markers in Restionaceae. The use of the low copy nuclear marker at103 allowed a clade comprising all three ligulate species of Anarthria to be recognised. The ligule character is used here for the first time in the taxonomy of Anarthria and merits special attention in studies of other restiids. In general, our study uncovered a superficially hidden but, in reality, conspicuous diversity in a common group of wind-pollinated plants in the southwest of Western Australia.

Evolution of Lomandroideae: Multiple origins of polyploidy and biome occupancy in Australia.

Asparagaceae: Lomandroideae are a species-rich and economically important subfamily in the monocot order Asparagales, with a center of diversity in Australia. Lomandroideae are ecologically diverse, occupying mesic and arid biomes in Australia and possessing an array of key traits, including sexual dimorphism, storage organs and polyploidy that are potentially adaptive for survival in seasonally arid and fire-dependent habitats. The Lomandroideae phylogeny was reconstructed using maximum likelihood and Bayesian inference criteria, based on plastome data from genome-skimming to infer relationships. A fossil-calibrated chronogram provided a temporal framework for understanding trait transitions. Ancestral state reconstructions and phylogenetic comparative trait correlation analyses provided insights into the evolutionary and ecological drivers associated with Lomandroideae diversification. Lomandroideae diverged from the other Asparagaceae ca. 56.61 million years ago (95% highest posterior density values 70.31-45.34 million years) and the major lineages diversified since the Oligocene. The most recent common ancestor of the clade likely occupied the mesic biome, was hermaphroditic and geophytic. Biome occupancy transitions were correlated with polyploidy and the presence of storage roots. Polyploidy potentially serves as an "enabler" trait, generating novel phenotypes, which may confer tolerance to climatic ranges and soil conditions putatively required for expansion into and occupation of new arid biomes. Storage roots, as a key factor driving biome transitions, may have been associated with fire rather than with aridification events in the Australian flora. This study contributes significantly to our understanding of biome evolution by identifying polyploidy and storage organs as key factors associated with transitions in biome occupancy in this lineage.

The Evolutionary Origin of C4 Photosynthesis in the Grass Subtribe Neurachninae.

The Australian grass subtribe Neurachninae contains closely related species that use C3, C4, and C2 photosynthesis. To gain insight into the evolution of C4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C3-C4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO2) compensation point in the absence of day respiration (C * ) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO2 is concentrated in C2 and C4 Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C3 Neurachninae species with C * values of 56-61 µmol mol-1, whereas two-thirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C2 phenotype with C * of 44 µmol mol-1 Increased investment of GLDP in MS tissue of the C2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO2 in MS tissues.

Transcriptome-derived evidence supports recent polyploidization and a major phylogeographic division in Trithuria submersa (Hydatellaceae, Nymphaeales).

Relatively little is known about species-level genetic diversity in flowering plants outside the eudicots and monocots, and it is often unclear how to interpret genetic patterns in lineages with whole-genome duplications. We addressed these issues in a polyploid representative of Hydatellaceae, part of the water-lily order Nymphaeales. We examined a transcriptome of Trithuria submersa for evidence of recent whole-genome duplication, and applied transcriptome-derived microsatellite (expressed-sequence tag simple-sequence repeat (EST-SSR)) primers to survey genetic variation in populations across its range in mainland Australia. A transcriptome-based Ks plot revealed at least one recent polyploidization event, consistent with fixed heterozygous genotypes representing underlying sets of homeologous loci. A strong genetic division coincides with a trans-Nullarbor biogeographic boundary. Patterns of 'allelic' variation (no more than two variants per EST-SSR genotype) and recently published chromosomal evidence are consistent with the predicted polyploidization event and substantial homozygosity underlying fixed heterozygote SSR genotypes, which in turn reflect a selfing mating system. The Nullarbor Plain is a barrier to gene flow between two deep lineages of T. submersa that may represent cryptic species. The markers developed here should also be useful for further disentangling species relationships, and provide a first step towards future genomic studies in Trithuria.

Reconstructing the age and historical biogeography of the ancient flowering-plant family Hydatellaceae (Nymphaeales).

BACKGROUND: The aquatic flowering-plant family Hydatellaceae has a classic Gondwanan distribution, as it is found in Australia, India and New Zealand. To shed light on the biogeographic history of this apparently ancient branch of angiosperm phylogeny, we dated the family in the context of other seed-plant divergences, and evaluated its biogeography using parsimony and likelihood methods. We also explicitly tested the effect of different extinction rates on biogeographic inferences. RESULTS: We infer that the stem lineage of Hydatellaceae originated in the Lower Cretaceous; in contrast, its crown originated much more recently, in the early Miocene, with the bulk of its diversification after the onset of the Pliocene. Biogeographic reconstructions predict a mix of dispersal and vicariance events, but considerations of geological history preclude most vicariance events, besides a split at the root of the family between southern and northern clades. High extinction rates are plausible in the family, and when these are taken into account there is greater uncertainty in biogeographic inferences. CONCLUSIONS: A stem origin for Hydatellaceae in the Lower Cretaceous is consistent with the initial appearance of fossils attributed to its sister clade, the water lilies. In contrast, the crown clade is young, indicating that vicariant explanations for species outside Australia are improbable. Although long-distance dispersal is likely the primary driver of biogeographic distribution in Hydatellaceae, we infer that the recent drying out of central Australia divided the family into tropical vs. subtropical/temperate clades around the beginning of the Miocene.

Impact of spatial constraints during seed germination on the evolution of angiosperm cotyledons: a case study from tropical Hydatellaceae (Nymphaeales).

PREMISE OF THE STUDY: A bipolar embryo with cotyledons is a characteristic feature that appeared early in the evolution of seed plants. Cotyledon number is an important character in angiosperm classification. We explore the links between functional aspects of seed germination and the number and location of the cotyledons, using as a model the early-divergent angiosperm family Hydatellaceae, in which seedlings are superficially monocot-like. • METHODS: Seedlings of two species of tropical Hydatellaceae were studied using light and scanning electron microscopy. • KEY RESULTS: Seedlings of Trithuria cowieana bear two free cotyledons. Each cotyledon possesses a green, filiform, vascularized blade that resembles subsequent leaves, and a basal, nonvascularized, haustorial outgrowth that remains in close contact with the endosperm. Seedlings of Trithuria konkanensis have two free cotyledonary haustoria inserted close to each other and a leaf blade probably belonging to one of the cotyledons. The cotyledonary node elongates between the haustoria and the leaf blade to form a mesocotyl. • CONCLUSIONS: To date, the absence or presence of a cotyledonary tube represents the only known qualitative morphological difference between the two major clades of Hydatellaceae. Cotyledons with a haustorium and leaf blade are unusual at the scale of seed plants and probably evolved due to homeosis. The mesocotyl of T. konkanensis resembles that of grasses and sedges. Seedling diversity in Hydatellaceae and other seed plants is linked with the principal physical and spatial constraint of their embryo structure, with the primary root and shoot apical meristems located at opposite poles, and haustorial cotyledon tips.

Multiple photosynthetic transitions, polyploidy, and lateral gene transfer in the grass subtribe Neurachninae.

The Neurachninae is the only grass lineage known to contain C(3), C(4), and C(3)-C(4) intermediate species, and as such has been suggested as a model system for studies of photosynthetic pathway evolution in the Poaceae; however, a lack of a robust phylogenetic framework has hindered this possibility. In this study, plastid and nuclear markers were used to reconstruct evolutionary relationships among Neurachninae species. In addition, photosynthetic types were determined with carbon isotope ratios, and genome sizes with flow cytometry. A high frequency of autopolyploidy was found in the Neurachninae, including in Neurachne munroi F.Muell. and Paraneurachne muelleri S.T.Blake, which independently evolved C(4) photosynthesis. Phylogenetic analyses also showed that following their separate C(4) origins, these two taxa exchanged a gene encoding the C(4) form of phosphoenolpyruvate carboxylase. The C(3)-C(4) intermediate Neurachne minor S.T.Blake is phylogenetically distinct from the two C(4) lineages, indicating that intermediacy in this species evolved separately from transitional stages preceding C(4) origins. The Neurachninae shows a substantial capacity to evolve new photosynthetic pathways repeatedly. Enablers of these transitions might include anatomical pre-conditions in the C(3) ancestor, and frequent autopolyploidization. Transfer of key C(4) genetic elements between independently evolved C(4) taxa may have also facilitated a rapid adaptation of photosynthesis in these grasses that had to survive in the harsh climate appearing during the late Pliocene in Australia.

Molecular phylogenetics of Hydatellaceae (Nymphaeales): sexual-system homoplasy and a new sectional classification.

PREMISE OF THE STUDY: Species relationships are unknown in Hydatellaceae, a small family of dwarf aquatics related to water lilies that arose near the base of angiosperm phylogeny. Here we use molecular evidence to infer a species tree for the family and apply this to reconstructing major transitions in morphology and sexual system in this early branch of angiosperms. METHODS: We assembled plastid (atpB, matK, ndhF, rbcL) and nuclear (ribosomal ITS) data for 50 samples (including outgroups) and estimated a species tree for Hydatellaceae using a Bayesian multispecies coalescent approach. We reconstructed the evolution of several morphological characters, then tested for associations between sexual system and reproductive morphology using phylogenetic ANOVA. KEY RESULTS: Dioecious species of Hydatellaceae have significantly greater stamen number and anther length than do cosexual species, suggesting changes in male function. The perennial habit that defines one subclade likely represents a reversion from annuality. Species relationships do not fall along traditional morphological divisions, but new sections proposed here are supported by fruit and seed synapomorphies. The earliest split in the family is reflected in geography and climate (i.e., tropical vs. subtropical/temperate clades). We found limited evidence of incongruence between plastid and nuclear trees, with one exception involving gene-tree nonmonophyly for two close relatives (Trithuria submersa, T. bibracteata). CONCLUSIONS: While the direction of sexual-system evolution is ambiguous, transitions are significantly associated with changes in involucral phyllome length and proxies of pollen production. We propose a new sectional circumscription based on fruit, seed, and DNA evidence.

Reproductive ecology of the basal angiosperm Trithuria submersa (Hydatellaceae).

BACKGROUND AND AIMS: Trithuria, the sole genus in the family Hydatellaceae, is an important group for understanding early angiosperm evolution because of its sister relationship to the ancient lineage, Nymphaeales (water lilies). Although also aquatic, Trithuria differs from water lilies in that all species are extremely small, and most have an annual life form and grow in seasonal wetlands. Very little is known about their reproductive ecology. This paper reports on reproductive timing, mode of pollination and characteristics of the breeding system of Trithuria submersa in Western Australia. METHODS: Mass collections of open-pollinated plants from different ecological settings were used to characterize the reproductive developmental sequence and natural pollen reception. Hand-pollination, caging and emasculation experiments were used to measure outcross + geitonogamous pollen reception versus autonomous self-pollination in two populations over two field seasons. KEY RESULTS: Natural outcross or geitonogamous pollination was by wind, not by water or insects, but pollen reception was extremely low. Pollen production was very low and pollen release was non-synchronous within populations. The pollen to ovule (P/O) ratio was 23·9, compared with 1569·1 in dioecious Trithuria austinensis. Stigmas became receptive before male phase and remained so until anthers dehisced and autonomous self-pollination occurred. Natural pollen loads are composed primarily of self pollen. Self- and open-pollinated plants had equivalent seed set (both >70 %). Self-pollinated plants produced seed within 17 d. CONCLUSIONS: Autonomous self-pollination and self-fertilization are predominant in T. submersa. The low P/O ratio is not an artefact of small plant size and is inconsistent with long-term pollination by wind. It indicates that T. submersa has evolved a primarily autogamous breeding system. Selfing, along with the effect of small plant size on the speed of reproduction, has enabled T. submersa to colonize marginal ephemeral wetlands in the face of unpredictable pollination.

Comparative ovule and megagametophyte development in Hydatellaceae and water lilies reveal a mosaic of features among the earliest angiosperms.

BACKGROUND AND AIMS: The embryo sac, nucellus and integuments of the early-divergent angiosperms Hydatellaceae and other Nymphaeales are compared with those of other seed plants, in order to evaluate the evolutionary origin of these characters in the angiosperms. METHODS: Using light microscopy, ovule and embryo sac development are described in five (of 12) species of Trithuria, the sole genus of Hydatellaceae, and compared with those of Cabombaceae and Nymphaeaceae. KEY RESULTS: The ovule of Trithuria is bitegmic and tenuinucellate, rather than bitegmic and crassinucellate as in most other Nymphaeales. The seed is operculate and possesses a perisperm that develops precociously, which are both key features of Nymphaeales. However, in the Indian species T. konkanensis, perisperm is relatively poorly developed by the time of fertilization. Perisperm cells in Trithuria become multinucleate during development, a feature observed also in other Nymphaeales. The outer integument is semi-annular ('hood-shaped'), as in Cabombaceae and some Nymphaeaceae, in contrast to the annular ('cap-shaped') outer integument of some other Nymphaeaceae (e.g. Barclaya) and Amborella. The megagametophyte in Trithuria is monosporic and four-nucleate; at the two-nucleate stage both nuclei occur in the micropylar domain. Double megagametophytes were frequently observed, probably developed from different megaspores of the same tetrad. Indirect, but strong evidence is presented for apomictic embryo development in T. filamentosa. CONCLUSIONS: Most features of the ovule and embryo sac of Trithuria are consistent with a close relationship with other Nymphaeales, especially Cabombaceae. The frequent occurrence of double megagametophytes in the same ovule indicates a high degree of developmental flexibility, and could provide a clue to the evolutionary origin of the Polygonum-type of angiosperm embryo sac.

Seedling diversity in Hydatellaceae: implications for the evolution of angiosperm cotyledons.

BACKGROUND AND AIMS: Cotyledon number has long been a primary morphological feature distinguishing monocots from other angiosperms. Recent placement of Hydatellaceae near the early-divergent angiosperm order Nymphaeales, rather than in the monocot order Poales, has prompted reassessment of seedling morphology in this poorly known family. METHODS: Seedlings of six species representing all eco-geographical groups of Hydatellaceae are described using light and scanning electron microscopy. KEY RESULTS: Two seedling types were discovered. Material examined of Trithuria submersa, T. bibracteata, T. austinensis and T. filamentosa possess a transparent bilobed sheathing structure that surrounds the main axis below the first foliage leaf. The seed coat is attached to the sheathing structure. Seedlings of Trithuria lanterna and T. konkanensis lack a sheathing structure, and the seed coat is attached to a short, narrow lateral outgrowth on the main axis of the seedling. CONCLUSIONS: The sheathing structure that is present in seedlings of some Hydatellaceae could be homologized with the two united cotyledons of water lilies. It also resembles the single cotyledon of some monocots, and hence demonstrates a possible pathway of the origin of a monocot-like embryo, though no homology is implied. The sheathing structure is reduced in Trithuria lanterna and T. konkanensis, and the short, narrow outgrowth of its seedling could represent a single cotyledon. This synapomorphy suggests that the only Indian species of Hydatellaceae, T. konkanensis, is closer to the northern Australian T. lanterna than to the south-western Australian T. bibracteata.

Morphology of Hydatellaceae, an anomalous aquatic family recently recognized as an early-divergent angiosperm lineage.

The family Hydatellaceae was recently reassigned to the early-divergent angiosperm order Nymphaeales rather than the monocot order Poales. This dramatic taxonomic adjustment allows comparison with other early-divergent angiosperms, both extant and extinct. Hydatellaceae possess some monocot-like features that could represent adaptations to an aquatic habit. Ecophysiological parallels can also be drawn from fossil taxa that are known from small achene-like diaspores, as in Hydatellaceae. Reproductive units of Hydatellaceae consist of perianthlike bracts enclosing several pistils and/or stamens. In species with bisexual reproductive units, a single unit resembles an "inside-out" flower, in which stamens are surrounded by carpels that are initiated centrifugally. Furthermore, involucre development in Trithuria submersa, with delayed growth of second whorl bracts, resembles similar delayed development of the second perianth whorl in Cabomba. Several hypotheses on the homologies of reproductive units in Hydatellaceae are explored. Currently, the most plausible interpretation is that each reproductive unit represents an aggregation of reduced unisexual apetalous flowers, which are thus very different from flowers of Nymphaeales. Each pistil in Hydatellaceae is morphologically and developmentally consistent with a solitary ascidiate carpel. However, ascidiate carpel development, consistent with placement in Nymphaeales, is closely similar to pseudomonomerous pistil development as in Poaes.