Grass Subfamily Research Articles

Ecological niche modelling and genetic diversity of Anomochloa marantoidea (Poaceae): filling the gaps for conservation in the earliest-diverging grass subfamily

AbstractAnomochlooideae (Poaceae) represent the earliest-diverging extant lineage of grasses. One of the two genera is the monotypic Anomochloa, which is extremely rare and restricted to the Atlantic Forest of southern Bahia state in Brazil, where only two natural populations have been recorded to date. Knowledge of A. marantoidea is considered crucial to understanding evolutionary and diversification patterns in Poaceae. Despite this, knowledge of the biology and distribution of A. marantoidea remain incomplete, and thus the conservation of this poorly known species is problematic. We used niche modelling to estimate its current distribution and assess potential ranges in situ to explore new occurrences. In addition, genetic diversity and the factors that disrupt gene flow between populations of this species were estimated using molecular markers. Two new populations were documented; the modelled ecological niche indicates high climatic restriction, but also revealed suitable sites for the establishment of new populations. Genetic diversity is correlated to population size, and genetic structure analysis suggests recent fragmentation and low gene flow among the remaining populations, which exhibit high levels of inbreeding. These levels also indicate the capacity of A. marantoidea to respond favourably to selection and, thus, that a conservation plan could be designed to maintain the current genetic diversity.

Bilobate phytolith size matters for taxonomical and ecological identification of Chad grasses: A case study on 15 species

The Poaceae family produces diagnostic phytoliths commonly called Grass Silica Short Cell Phytoliths (GSSCP), of which shapes and sizes are very distinguishable from those produced by other plant families. Grass Silica Short Cell Phytoliths are usually classified into four main categories: the rondels, bilobates, crosses, and saddles. Among them, the bilobates have in common a general "dumbbell" shape consisting of two lobes inter-connected by a shank. They represent the dominant morphotypes observed in the leaf tissues of many Panicoideae grass species. Besides, they tend to often be related to this subfamily in the fossil record, especially when they occur in large amounts. Several studies based on modern African grasses have however demonstrated that bilobates were also abundantly produced by a few members of the Aristidoideae, Chloridoideae, and Oryzoideae grass subfamilies which, like Panicoideae, are today mainly distributed in tropical low-altitude areas. The relationship between bilobates and Panicoideae is thus not so direct in the tropics. As a result, it can lead to significant misinterpretations when applied to paleoenvironmental and archaeological surveys.This study investigates variations in the size of bilobate phytoliths in 15 African grass species, including eight Panicoideae, three Aristidoideae, two Chloridoideae, and two Oryzoideae, with the intention to explore new alternatives for refining their identification in the past deposits of tropical Africa. Different statistical analyses (Principal Component Analysis, Mann–Whitney tests, decisional trees) applied to the previous dataset allowed testing whether: (i) bilobate length and width significantly vary between grass species; (ii) these variations (if so) have the potential to be used as taxonomical and/or ecological proxies.Our analyses confirm that bilobate length is significant in discriminating dry-adapted Aristidoideae and Chloridoideae species from the other sub-families. In particular, a bilobate base longer than 25 μm is almost exclusively observed for Aristidoideae and Chloridoideae, which is in agreement with previous studies. Although subject to less variations, the length/width of bilobate shanks and the average length of their lobes appear significantly useful to refine grass taxonomical identification and discriminate <120 cm-high grasses from taller grasses. Yet, it seems that the key towards a robust identification of bilobate phytoliths involves considering the size parameters.

Group-1 Grass Pollen Allergens with Near-Identical Sequences Identified in Species of Subtropical Grasses Commonly Found in Southeast Asia.

Background and objectives: Group-1 grass allergens or beta-expansins (EXPBs) are major allergens from pollen of all grass species. Previous studies showed that they are highly conserved (64–85%) in Pooideae species, which are found mostly in the temperate regions. However, the information about group-1 allergens from common grass species in subtropical areas is still lacking. This study aimed to assess the sequence diversity of group-1 grass pollen allergens in subtropical areas, especially in Southeast Asia. Materials and Methods: Group-1 allergens were cloned from pollen of eight grass species using a single set of primers. Sequences were analyzed and IgE and IgG4 binding regions were compared to the previously reported epitopes in homologous EXPBs. The phylogenetic analysis was used to assess the relationship between sequences of these species and previously characterized EXPBs. Moreover, three-dimensional structure of the EXPB was modeled based on homology to Zea m 1. Results: Sequences from eight grass species were nearly identical. It is conceivable that the primers used for cDNA amplification detected the same isoform in different species. In fact, the deduced amino acid sequences shared 97.79–100% identity with each other and 15/819 polymorphic nucleotide positions were identified. The predicted structure showed that the IgE and IgG4 epitopes and polymorphic residues were located in both domains 1 and 2. The dendrogram presents clustering of class A EXPBs into four groups corresponding to the grass subfamilies. Conclusions: This study identified the allergens with near-identical sequences from different grass species. This isoform could be the major cross-reacting allergenic protein from commonly found grass species.

The grass subfamily Pooideae: Cretaceous–Palaeocene origin and climate‐driven Cenozoic diversification

AimFrost is among the most dramatic stresses a plant can experience, and complex physiological adaptations are needed to endure long periods of sub‐zero temperatures. Owing to the need to evolve these complex adaptations, transitioning from tropical to temperate climates is regarded as difficult. Here, we study the transition from tropical to temperate climates in the grass subfamily Pooideae, which dominates cool temperate, continental and Arctic regions. We produce a dated phylogeny and investigate the role of climate cooling in diversification.LocationGlobal, temperate regions.Time periodCretaceous–Cenozoic.Major taxaPooideae.MethodsUsing newly available fossils and methods, we dated a comprehensive Pooideae phylogeny and tested for the impact of palaeoclimates on diversification rates. Using ancestral state reconstruction, we investigated whether Pooideae ancestors experienced frost and winter. To locate the ancestral distribution area of Pooideae, we performed biogeographical analyses.ResultsWe estimated a Late Cretaceous/early Palaeocene origin of the Pooideae (61–77 Ma), with all major clades already having diversified at the Eocene–Oligocene climate cooling (34 Ma). Climate cooling was a probable driving force of Pooideae diversification. Pooideae probably evolved in a temperate niche experiencing frost, but not long winters.Main conclusionPooideae probably originated in a temperate niche and experienced cold temperatures and frost long before expansion of temperate biomes after the Eocene–Oligocene transition. This suggests that the Pooideae ancestor had adaptations to temperate climate and that certain responses to low‐temperature stress are shared in extant Pooideae grasses. Throughout the Cenozoic, falling temperatures and expansion of temperate biomes were associated with an increase in diversification. However, complex mechanisms for enduring strongly seasonal climate with long, cold winters most probably evolved independently in daughter lineages. Our findings provide insight into how adaptations to historical changes in chill and frost exposure influence the distribution of plant diversity today.

Evolution of Cold Acclimation and Its Role in Niche Transition in the Temperate Grass Subfamily Pooideae.

The grass subfamily Pooideae dominates the grass floras in cold temperate regions and has evolved complex physiological adaptations to cope with extreme environmental conditions like frost, winter, and seasonality. One such adaptation is cold acclimation, wherein plants increase their frost tolerance in response to gradually falling temperatures and shorter days in the autumn. However, understanding how complex traits like cold acclimation evolve remains a major challenge in evolutionary biology. Here, we investigated the evolution of cold acclimation in Pooideae and found that a phylogenetically diverse set of Pooideae species displayed cold acclimation capacity. However, comparing differential gene expression after cold treatment in transcriptomes of five phylogenetically diverse species revealed widespread species-specific responses of genes with conserved sequences. Furthermore, we studied the correlation between gene family size and number of cold-responsive genes as well as between selection pressure on coding sequences of genes and their cold responsiveness. We saw evidence of protein-coding and regulatory sequence evolution as well as the origin of novel genes and functions contributing toward evolution of a cold response in Pooideae. Our results reflect that selection pressure resulting from global cooling must have acted on already diverged lineages. Nevertheless, conservation of cold-induced gene expression of certain genes indicates that the Pooideae ancestor may have possessed some molecular machinery to mitigate cold stress. Evolution of adaptations to seasonally cold climates is regarded as particularly difficult. How Pooideae evolved to transition from tropical to temperate biomes sheds light on how complex traits evolve in the light of climate changes.

The molecular allergology of subtropical grass pollen

Grass pollens are amongst the most important aeroallergen sources world-wide triggering allergic rhinoconjunctivitis and asthma in sensitised patients. Much of what we know about the allergen components of grasses is informed by research on pollen of temperate (Pooideae) species that are abundant in the temperate climate zones. However, climate changes are altering the biogeographical distribution as well as timing and allergenicity of grass pollens. This provides an impetus for better understanding of the contribution of subtropical subfamilies of grasses to pollen allergy globally. Pollen of Chloridoideae (e.g. Cynodon dactylon; Bermuda grass) and Panicoideae (e.g. Paspalum notatum; Bahia grass or Sorghum halepense; Johnson grass) subfamilies are clinically important in subtropical zones of Australia, Asia, India, Africa, and America. These grasses differ ecologically and phylogenetically from temperate grasses and, importantly their allergen composition is qualitatively different. For example, subtropical grass pollens appear to lack the major group 5 grass pollen allergen family. In this review we summarize current knowledge of the epidemiology and immunology of subtropical Chloridoideae and Pancoideae pollen allergens, describe the biochemical characteristics of known isoforms and variants as well as properties and structures of subtropical pollen allergen components. Whilst only one subtropical allergen component; Cyn d 1 of Bermuda grass pollen, is available commercially for diagnostic use, in a natural purified form, a number of allergens of Panicoideae grass pollen; Zea m 1, Zea m 3 and Zea m 13 of maize, Pas n 1 and Pas n 13 of Bahia, as well as Sor h 1, Sor h 2, Sor h 13 and Sor h 23 of Johnson grass, have been discovered. Research effort is directed towards making available subtropical grass pollen allergen components as innovative treatment and diagnostic options that more specifically address the needs of patients from warmer regions of the globe.

New biomolecular tools for aerobiological monitoring: Identification of major allergenic Poaceae species through fast real-time PCR.

Grasses (Poaceae) are very common plants, which are widespread in all environments and urban areas. Despite their economical importance, they can represent a problem to humans due to their abundant production of allergenic pollen. Detailed information about the pollen season for these species is needed in order to plan adequate therapies and to warn allergic people about the risks they take in certain areas at certain moments. Moreover, precise identification of the causative species and their allergens is necessary when the patient is treated with allergen‐specific immunotherapy. The intrafamily morphological similarity of grass pollen grains makes it impossible to distinguish which particular species is present in the atmosphere at a given moment. This study aimed at developing new biomolecular tools to analyze aerobiological samples and identifying major allergenic Poaceae taxa at subfamily or species level, exploiting fast real‐time PCR. Protocols were tested for DNA extraction from pollen sampled with volumetric and gravimetric methods. A fragment of the matK plastidial gene was amplified and sequenced in Poaceae species known to have high allergological impact. Species‐ and subfamily‐specific primer–probe systems were designed and tested in fast real‐time PCRs to evaluate the presence of these taxa in aerobiological pollen samples. Species‐specific systems were obtained for four of five studied species. A primer–probe set was also proposed for the detection of Pooideae (a grass subfamily that includes also major cereal grains) in aerobiological samples, as this subfamily includes species carrying both grass allergens from groups 1 and 5. These, among the 11 groups in which grass pollen allergens are classified, are considered responsible for the most frequent and severe symptoms.

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis

BackgroundQuantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data.ResultsEleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5′-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues.ConclusionsThis approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C4 crop species.

Variability of Pyrrolizidine Alkaloid Occurrence in Species of the Grass Subfamily Pooideae (Poaceae).

Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites found in various unrelated angiosperm lineages including cool-season grasses (Poaceae, subfamily Pooideae). Thesinine conjugates, saturated forms of PA that are regarded as non-toxic, have been described to occur in the two grass species Lolium perenne and Festuca arundinacea (Poaceae, subfamily Pooideae). In a wider screen, we tested various species of the Pooideae lineage, grown under controlled conditions, for their ability to produce thesinine conjugates or related structures. Using an LC-MS based targeted metabolomics approach we were able to show that PA biosynthesis in grasses is limited to a group of very closely related Pooideae species that produce a limited diversity of PA structures. High variability in PA levels was observed even between individuals of the same species. These individual accumulation patterns are discussed with respect to a possible function and evolution of this type of alkaloid.

Adaptive radiations should not be simplified: The case of the danthonioid grasses

Although much of extant diversity is probably the product of evolutionary radiations, the special case of adaptive radiations has not yet been thoroughly explored. Adaptive radiations are postulated to occur when a lineage is exposed to new ecological opportunities, where it can diversify ecologically. We argue that adaptive radiations have two characteristics. Firstly, the diversification rate accelerates initially, and is then followed by a density-dependent slow-down. Secondly, traits relevant to the new ecological opportunity should evolve at or just before the radiation. We also argue that a correct identification of adaptive radiations is dependent on the phylogenies underlying the diversification dynamics being sampled adequately (i.e. comprehensive species sampling), and that the traits should be treated continuously if they exhibit a biological continuum and not be over-simplified into binary traits. Here, we test the hypothesis that much of the extant diversity of the south-temperate grass subfamily Danthonioideae is the result of two geographically separated but contemporaneous adaptive radiations, in response to Late-Miocene—Pliocene aridification and increasingly seasonal climates. We show that both Pentameris (83 African species) and Rytidosperma (73 Australasian-South American species) exhibit accelerations in diversification rates followed by linear density-dependent declines. We also show that two selected traits show differential evolutionary regimes with different evolutionary optima, and that these are linked to changes in the diversification rate. These results are consistent with these being adaptive, and putatively parallel, radiations. However, by mapping traits over the whole danthonioid phylogeny, it is evident that no identified trait or trait combination is sufficient and necessary for adaptive radiations. Furthermore, we show that simplifying the traits to binary gives a strong but potentially erroneous link between trait shift and diversification rate shift.

Are diversification rates and chromosome evolution in the temperate grasses (Pooideae) associated with major environmental changes in the Oligocene-Miocene?

The Pooideae are a highly diverse C3 grass subfamily that includes some of the most economically important crops, nested within the highly speciose core-pooid clade. Here, we build and explore the phylogeny of the Pooideae within a temporal framework, assessing its patterns of diversification and its chromosomal evolutionary changes in the light of past environmental transformations. We sequenced five plastid DNA loci, two coding (ndhF, matk) and three non-coding (trnH-psbA, trnT-L and trnL-F), in 163 Poaceae taxa, including representatives for all subfamilies of the grasses and all but four ingroup Pooideae tribes. Parsimony and Bayesian phylogenetic analyses were conducted and divergence times were inferred in BEAST using a relaxed molecular clock. Diversification rates were assessed using the MEDUSA approach, and chromosome evolution was analyzed using the chromEvol software. Diversification of the Pooideae started in the Late-Eocene and was especially intense during the Oligocene-Miocene. The background diversification rate increased significantly at the time of the origin of the Poodae + Triticodae clade. This shift in diversification occurred in a context of falling temperatures that potentially increased ecological opportunities for grasses adapted to open areas around the world. The base haploid chromosome number n = 7 has remained stable throughout the phylogenetic history of the core pooids and we found no link between chromosome transitions and major diversification events in the Pooideae.

Late Holocene palaeoenvironmental reconstruction from Mpumalanga Province (South Africa) inferred from geochemical and biogenic proxies

Here we present a palaecological reconstruction covering the last 1700yr from Lydenburg fen, located in the north-eastern grassland biome, Mpumalange, South Africa. A 300cm peat sequence was analysed for biogenic (grass phytoliths, diatoms) and geochemical proxies (δ13C, δ15N, carbon/nitrogen content) to infer past grassland dynamics and hydro-climatic changes. The Lydenburg record reports a C4 dominated grassland throughout the studied period, with more or less pronounced fluxes between C4-Chloridoideae and C4-Panicoideae grass subfamilies. The record reflects moderate to dry conditions from AD 400 to 1000; more mesic conditions until around AD 1250; followed by a significantly drier period between c. AD 1250 and c. AD 1350, when Chloridoideae grasses expand at the expense of Panicoideae grasses. During this phase, the δ13C-record reports more enriched values indicating higher influx of C4 grasses. Furthermore, lithological evidence indicates highly erosive conditions, with significant gravel input from the surrounding hills. After AD 1350, proxy indications suggest a shift towards more mesic conditions. During this increasingly mesic but also unstable period, farming communities using specialized agricultural practices (e.g. the people in Bokoni) expanded their settlements into new regions (Delius et al., 2008). This expansion was also coupled to population growth, suggesting these communities applied techniques that enabled improved food production under environmentally challenging conditions. Over the last century, Lydenburg δ13C-values indicate increased input of C3 taxa. The phytolith record shows that this increase is not coupled to an increase in Pooideae (C3) grasses, suggesting that the C3 input may be related to woody encroachment.

Polyphyly of Arundinoideae (Poaceae) and evolution of the twisted geniculate lemma awn.

Subfamily Arundinoideae represents one of the last unsolved taxonomic mysteries in the grass family (Poaceae) due to the narrow and remote distributions of many of its 19 morphologically and ecologically heterogeneous genera. Resolving the phylogenetic relationships of these genera could have substantial implications for understanding character evolution in the grasses, for example the twisted geniculate awn - a hygroscopic awn that has been shown to be important in seed germination for some grass species. In this study, the phylogenetic positions of most arundinoid genera were determined using DNA from herbarium specimens, and their placement affects interpretation of this ecologically important trait. A phylogenetic analysis was conducted on a matrix of full-plastome sequences from 123 species in 107 genera representing all grass subfamilies, with 15 of the 19 genera in subfamily Arundinoideae. Parsimony and maximum likelihood mapping approaches were used to estimate ancestral states for presence of a geniculate lemma awn with a twisted column across Poaceae. Lastly, anatomical characters were examined for former arundinoid taxa using light microscopy and scanning electron microscopy. Four genera traditionally included in Arundinoideae fell outside the subfamily in the plastome phylogeny, with the remaining 11 genera forming Arundinoideae sensu stricto . The twisted geniculate awn has originated independently at least five times in the PACMAD grasses, in the subfamilies Panicoideae, Danthonioideae/Chloridoideae and Arundinoideae. Morphological and anatomical characters support the new positions of the misplaced arundinoid genera in the phylogeny, but also highlight convergent and parallel evolution in the grasses. In placing the majority of arundinoid genera in a phylogenetic framework, our study answers one of the last remaining big questions in grass taxonomy while highlighting examples of convergent evolution in an ecologically important trait, the hygroscopic, twisted geniculate awn.

Evolution of the miR5200-FLOWERING LOCUS T flowering time regulon in the temperate grass subfamily Pooideae

Flowering time is a carefully regulated trait controlled primarily through the action of the central genetic regulator, FLOWERING LOCUS T (FT). Recently it was demonstrated that a microRNA, miR5200, targets the end of the second exon of FT under short-day photoperiods in the grass subfamily Pooideae, thus preventing FT transcripts from reaching threshold levels under non-inductive conditions. Pooideae are an interesting group in that they rapidly diversified from the tropics into the northern temperate region during a major global cooling event spanning the Eocene-Oligocene transition. We hypothesize that miR5200 photoperiod-sensitive regulation of Pooideae flowering time networks assisted their transition into northern seasonal environments. Here, we test predictions derived from this hypothesis that miR5200, originally found in bread wheat and later identified in Brachypodium distachyon, (1) was present in the genome of the Pooideae common ancestor, (2) is transcriptionally regulated by photoperiod, and (3) is negatively correlated with FT transcript abundance, indicative of miR5200 regulating FT. Our results demonstrate that miR5200 did evolve at or around the base of Pooideae, but only acquired photoperiod-regulated transcription within the Brachypodium lineage. Based on expression profiles and previous data, we posit that the progenitor of miR5200 was co-regulated with FT by an unknown mechanism.

The global distribution of bamboos: assessing correlates of introduction and invasion.

There is a long history of species being moved around the world by humans. These introduced species can provide substantial benefits, but they can also have undesirable consequences. We explore the importance of human activities on the processes of species dissemination and potential invasions using the grass subfamily Bambusoideae ("bamboos"), a group that contains taxa that are widely utilised and that are often perceived as weedy. We (1) compiled an inventory of bamboo species and their current distributions; (2) determined which species have been introduced and become invasive outside their native ranges; and (3) explored correlates of introduction and invasion. Distribution data were collated from Kew's GrassBase, the Global Biodiversity Information Facility and other online herbarium information sources. Our list comprised 1662 species in 121 genera, of which 232 (14%) have been introduced beyond their native ranges. Twelve species were found to be invasive. A non-random selection of bamboos have been introduced and become invasive. Asiatic species in particular have been widely introduced. There was a clear over-representation of introduced species in the genera Bambusa and Phyllostachys which also contain most of the listed invasive species. The introduction of species also correlated with certain traits: taxa with larger culm dimensions were significantly more likely to have been moved to new areas; and those with many cultivars had a higher rate of dissemination and invasion. It is difficult to determine whether the patterns of introduction and invasion are due simply to differences in propagule pressure, or whether humans have deliberately selected inherently invasive taxa. In general, we suggest that human usage is a stronger driver of introductions and invasions in bamboos than in other taxa that have been well studied. It is likely that as bamboos are used more widely, the number and impact of invasions will increase unless environmental risks are carefully managed.

Evolutionary history of chloridoid grasses estimated from 122 nuclear loci

Chloridoideae (chloridoid grasses) are a subfamily of ca. 1700 species with high diversity in arid habitats. Until now, their evolutionary relationships have primarily been studied with DNA sequences from the chloroplast, a maternally inherited organelle. Next-generation sequencing is able to efficiently recover large numbers of nuclear loci that can then be used to estimate the species phylogeny based upon bi-parentally inherited data. We sought to test our chloroplast-based hypotheses of relationships among chloridoid species with 122 nuclear loci generated through targeted-enrichment next-generation sequencing, sometimes referred to as hyb-seq. We targeted putative single-copy housekeeping genes, as well as genes that have been implicated in traits characteristic of, or particularly labile in, chloridoids: e.g., drought and salt tolerance. We recovered ca. 70% of the targeted loci (122 of 177 loci) in all 47 species sequenced using hyb-seq. We then analyzed the nuclear loci with Bayesian and coalescent methods and the resulting phylogeny resolves relationships between the four chloridoid tribes. Several novel findings with this data were: the sister lineage to Chloridoideae is unresolved; Centropodia+Ellisochloa are excluded from Chloridoideae in phylogenetic estimates using a coalescent model; Sporobolus subtilis is more closely related to Eragrostis than to other species of Sporobolus; and Tragus is more closely related to Chloris and relatives than to a lineage of mainly New World species. Relationships in Cynodonteae in the nuclear phylogeny are quite different from chloroplast estimates, but were not robust to changes in the method of phylogenetic analysis. We tested the data signal with several partition schemes, a concatenation analysis, and tests of alternative hypotheses to assess our confidence in this new, nuclear estimate of evolutionary relationships. Our work provides markers and a framework for additional phylogenetic studies that sample more densely within chloridoid tribes. These results represent progress towards a robust classification of this important subfamily of grasses, as well as proof-of-concept for hyb-seq next-generation sequencing as a method to generate sequences for phylogenetic analyses in grasses and other plant families.

Bronze Age crop processing evidence in the phytolith assemblages from the ditch and fen around Fondo Paviani, northern Italy

The Terramare civilization (ca. 1650–1150 cal bce) on the Po plain in northern Italy is considered to have been an agrarian society typical of the European Bronze Age, with a subsistence economy based on arable and livestock farming, and which showed some innovations such as the introduction of millets as cultivars. Some questions are still open concerning the agricultural system, the food and non-food uses of plant resources and the organization of labour at these sites. In this paper, for the first time, phytolith analysis has been integrated with more standard archaeobotanical methods applied to material from the long-lasting settlement of Fondo Paviani, Verona. The aim of the study was to use phytoliths as a tool to investigate the cereal economy in order to detect different grass subfamilies and possibly provide hints about local crop processing activities. For this purpose, two contexts, a shallow ditch at the edge of the site that had been filled with domestic waste and a near-site fen with natural infilling, have been the objects of a multi-proxy inter-disciplinary investigation. This includes the analyses of phytoliths, pollen, NPP, sediment texture and micromorphology. The phytolith record shows remains of panicoid as well as pooid grasses, including chaff material with frequent traces of threshing that indicate the processing of cereals at the site and the possible use of chopped straw as fodder. The comparison of different kinds of evidence strengthens the interpretation and offers a new perspective on the application of phytolith analysis to Bronze Age northern Italy.

Alpine grassland palaeoecology of the Virunga Volcanoes, East Africa: A new phytolith record from Mt. Muhavura

Abstract This study presents a first attempt at using phytoliths from a high-altitude crater lake as a palaeoecological tool at a tropical montane site. Mt. Muhavura (4127 m asl) at the border of Uganda and Rwanda is part of the Virunga volcanoes located in the Albertine Rift, one of the most biodiverse areas in Africa. To investigate the potential of phytoliths to record grassland history in an Afroalpine environment, a unique and little studied ecosystem, we analysed the phytolith content of 37 sediment samples from a 159 cm sediment core from the crater lake at the summit of Mt. Muhavura representing the last ca. 2400 years BP. Ten plant species from the Alpine belt were also analysed for their main phytolith morphotypes. Phytoliths from the sediments were grouped into 12 main categories and the most frequently occurring individual morphotypes were bilobate (33%), oblong (16%), elongate (17%) and globular psilate (13%). The phytolith assemblage consists predominantly of phytoliths characteristic of grasses (70%). A literal interpretation of the results would be that Panicoideae grasses (mainly C 4 ) that are characteristic of warm and wet climate and C 3 Pooideae subfamily grasses characteristic of cold climate/high altitude have co-existed throughout the late Holocene in this belt. As only C 3 cold climate grass subfamilies are known to occur at this altitude today, the abundance of short cell phytoliths (bilobates) typical of Panicoideae grasses in the lake sediments at this altitude presents a new perspective to the phytolith discussion. The C 3 Pooideae grasses analysed did not produce bilobates. In this study, the phytolith index, Ic that is used as a proxy of past grass subfamily dominance is not representative of the taxonomic significance for which it is known. This study suggests that caution needs to be taken when using bilobate morphotypes and the Ic as indicators of grass subfamily dominance at high altitude on East African mountains as the bilobates may not be taxonomically representative of the grasses for which they are typically known. This highlights the inherent problem of phytolith redundancy even at an altitude where C 3 /C 4 grass distribution is better delineated.

Can grass phytoliths and indices be relied on during vegetation and climate interpretations in the eastern Himalayas? Studies from Darjeeling and Arunachal Pradesh, India

While documenting the vegetation response to climatic changes in mountains, the use of grass phytolith data relies on the ability of phytolith assemblages or indices to differentiate the elevationally stratified vegetation zones. To infer the potential and limitations of grass phytolith assemblages and indices to reconstruct vegetation vis-à-vis climate in the Himalayan mountain regions, we analyzed phytolith assemblages from 66 dominant grasses and 153 surface soils from four different forest types along the c. 130–4000 m a.s.l. elevation gradients in the Darjeeling and Arunachal Himalayas. Grass short cell phytolith assemblages from modern grasses show significant variability with rising elevation. To test the reliability of the above observation, phytoliths from the soil samples were subjected to linear discriminant analysis (DA). DA classified 85.3% and 92.3% of the sites to their correct forest zones in the Darjeeling and Arunachal Himalayas respectively. Relative abundance of bilobate, cross, short saddle, plateau saddle, rondel and trapeziform types allow discrimination of the phytolith assemblage along the elevation gradient. Canonical correspondence analysis (CCA) on the soil phytolith data further revealed their relationships with the climatic variables. Temperature and evapotranspiration were found to be the most influential for differential distribution of grass phytolith assemblages with rising elevation in the eastern Himalayas. We also tested the reliability of phytolith indices (Ic, Iph and Fs) for tracing the dominance of different grass subfamilies in the eastern Himalayas. Ic proved to be most reliable in discriminating C3/C4 grass along the elevation gradient while Iph and Fs proved to be less reliable. We observed that in the monsoon dominated eastern Himalayas, a little adjustment in Ic index may enhance the accuracy of interpretations. In future studies more precise identification of phytolith sub-types from additional sites in the eastern Himalayas may further improve our interpretations related to past climate condition.

Lectotypification of Arundo roraimensis, the basionym of Cortaderia roraimensis (Poaceae—Danthonioideae)

The genus Cortaderia Stapf (1897: 378, 396) is a member of the grass subfamily Danthonioideae and includes, in its current delimitation, 20 species restricted to South America (Linder et al. 2010, Kellogg 2015). The individual plants in this genus are gynodioecious, either entirely pistillate or with bisexual flowers (Kellogg 2015).