Extreme plastomes in holoparasitic Balanophoraceae are not the norm.

BACKGROUND: Balanophoraceae plastomes are known for their highly condensed and re-arranged nature alongside the most extreme nucleotide compositional bias known to date, culminating in two independent reconfigurations of their genetic code. Currently, a large portion of the Balanophoraceae diversity remains unexplored, hindering, among others, evolutionary pattern recognition. Here, we explored newly sequenced plastomes of Sarcophyte sanguinea and Thonningia sanguinea. The reconstructed plastomes were analyzed using various methods of comparative genomics based on a representative taxon sampling. RESULTS: Sarcophyte, recovered sister to the other sampled Balanophoraceae s. str., has plastomes up to 50% larger than those currently published. Its gene set contains five genes lost in any other species, including matK. Five cis-spliced introns are maintained. In contrast, the Thonningia plastome is similarly reduced to published Balanophoraceae and retains only a single cis-spliced intron. Its protein-coding genes show a more biased codon usage compared to Sarcophyte, with an accumulation of in-frame TAG stop codons. Structural plastome comparison revealed multiple, previously unknown, structural rearrangements within Balanophoraceae. CONCLUSIONS: For the "minimal plastomes" of Thonningia, we propose a genetic code change identical to sister genus Balanophora. Sarcophyte however differs drastically from our current understanding on Balanophoraceae plastomes. With a less-extreme nucleotide composition, there is no evidence for an altered genetic code. Using comparative genomics, we identified a hotspot for plastome reconfiguration in Balanophoraceae. Based on previously published and newly identified structural reconfigurations, we propose an updated model of evolutionary plastome trajectories for Balanophoraceae, illustrating a much greater plastome diversity than previously known.

Homoploid hybrids, allopolyploids, and high ploidy levels characterize the evolutionary history of a western North American quillwort (Isoëtes) complex.

Polyploidy and hybridization are important processes in seed-free plant evolution. However, a major gap lies in our understanding of how these processes affect the evolutionary history of high-ploidy systems. The heterosporous lycophyte genus Isoëtes is a lineage with many putative hybrids and high-level polyploid taxa (ranging from tetraploid to dodecaploid). Here, we use a complex of western North American Isoëtes, to understand the role of hybridization and high-level polyploidy in generating and maintaining novel diversity. To uncover these processes, we use restriction-site associated DNA sequencing (RADseq), multiple alleles of a single low-copy nuclear marker, whole plastomes, cytology (genome size estimates and chromosome counts), and reproductive status (fertile or sterile). With this dataset, we show that hybridization occurs easily between species in this complex and is bidirectional between identical, but not different, cytotypes. Furthermore, we show that fertile allopolyploids appear to have formed repeatedly from sterile homoploid and interploid hybrids. We propose that low prezygotic reproductive barriers and a high frequency of whole-genome duplication allow for high-level polyploid systems to generate novel lineages, and that these mechanisms may be important in shaping extant Isoëtes diversity.

Major trends in stem anatomy and growth forms in the perianth-bearing Piperales, with special focus on Aristolochia.

BACKGROUND AND AIMS: The order Piperales has the highest diversity of growth forms among the earliest angiosperm lineages, including trees, shrubs, climbers and herbs. However, within the perianth-bearing Piperales (Asarum, Saruma, Lactoris, Hydnora, Prosopanche, Thottea and Aristolochia), climbing species only occur in the most species-rich genus Aristolochia. This study traces anatomical and morphological traits among these lineages, to detect trends in growth form evolution and developmental processes. METHODS: Transverse stem sections of different developmental stages of representatives of Asarum, Saruma, Lactoris, Hydnora, Thottea and Aristolochia were compared and anatomical traits were linked to growth form evolution. Biomechanical properties of representative climbers were determined in three-point bending tests and are discussed based on the anatomical observations. Growth form evolution of the perianth-bearing Piperales was reconstructed by ancestral character state reconstruction using Mesquite. KEY RESULTS: While species of Asarum and Saruma are exclusively herbaceous, species of the remaining genera show a higher diversity of growth habit and anatomy. This growth form diversity is accompanied by a more complex stem anatomy and appropriate biomechanical properties. The ancestral growth form of the perianth-bearing Piperales is reconstructed with either a shrub-like or herbaceous character state, while the following three backbone nodes in the reconstruction show a shrub-like character state. Accordingly, the climbing habit most probably evolved in the ancestor of Aristolochia. CONCLUSIONS: Since the ancestor of the perianth-bearing Piperales has been reconstructed with a herb- or shrub-like habit, it is proposed that the climbing habit is a derived growth form, which evolved with the diversification of Aristolochia, and might have been a key feature for its diversification. Observed anatomical synapomorphies, such as the perivascular fibres in Lactoris, Thottea and Aristolochia, support the phylogenetic relationship of several lineages within the perianth-bearing Piperales. In addition, the hypothesis that the vegetative organs of the holoparasitic Hydnoraceae are most probably rhizomes is confirmed.

Structural Plastome Evolution in Holoparasitic Hydnoraceae with Special Focus on Inverted and Direct Repeats.

Plastome condensation during adaptation to a heterotrophic lifestyle is generally well understood and lineage-independent models have been derived. However, understanding the evolutionary trajectories of comparatively old heterotrophic lineages that are on the cusp of a minimal plastome, is essential to complement and expand current knowledge. We study Hydnoraceae, one of the oldest and least investigated parasitic angiosperm lineages. Plastome comparative genomics, using seven out of eight known species of the genus Hydnora and three species of Prosopanche, reveal a high degree of structural similarity and shared gene content; contrasted by striking dissimilarities with respect to repeat content [inverted and direct repeats (DRs)]. We identified varying inverted repeat contents and positions, likely resulting from multiple, independent evolutionary events, and a DR gain in Prosopanche. Considering different evolutionary trajectories and based on a fully resolved and supported species-level phylogenetic hypothesis, we describe three possible, distinct models to explain the Hydnoraceae plastome states. For comparative purposes, we also report the first plastid genomes for the closely related autotrophic genera Lactoris (Lactoridaceae) and Thottea (Aristolochiaceae).

Floral thermogenesis of three species of Hydnora (Hydnoraceae) in Africa.

BACKGROUND AND AIMS: Floral thermogenesis occurs in at least 12 families of ancient seed plants. Some species show very high rates of respiration through the alternative pathway, and some are thermoregulatory, with increasing respiration at decreasing ambient temperature. This study assesses the intensity and regulation of respiration in three species of African Hydnora that represent the Hydnoraceae, an unusual family of holoparasitic plants from arid environments. METHODS: Long-term respirometry (CO(2) production) and thermometry were carried out on intact flowers of H. africana, H. abyssinica and H. esculenta in the field, and short-term measurements were made on floral parts during the protogynous flowering sequence. KEY RESULTS: For H. africana, there was no temperature elevation in either the osmophores or the gynoecial chamber in any phase, and mass-specific respiration rates of the flower parts were low (maximum 8.3 nmol CO(2) g(-1) s(-1) in osmophore tissue). Respiration tracked ambient and floral temperatures, eliminating the possibility of the inverse relationship expected in thermoregulatory flowers. Hydnora abyssinica flowers had higher respiration (maximum 27.5 nmol g(-1) s(-1) in the osmophores) and a slight elevation of osmophore temperature (maximum 2.8 degrees C) in the female stage. Respiration by gynoecial tissue was similar to that of osmophores in both species, but there was no measurable elevation of gynoecial chamber temperature. Gynoecial chamber temperature of H. esculenta could reach 3.8 degrees C above ambient, but there are no respiration data available. Antheral tissue respiration was maximal in the male phase (4.8 nmol g(-1) s(-1) in H. africana and 10.3 nmol g(-1) s(-1) in H. abyssinica), but it did not raise the antheral ring temperature, which showed that thermogenesis is not a by-product of pollen maturation or release. CONCLUSIONS: The exceptionally low thermogenesis in Hydnora appears to be associated with scent production and possibly gynoecial development, but has little direct benefit to beetle pollinators.

Detecting and Characterizing the Highly Divergent Plastid Genome of the Nonphotosynthetic Parasitic Plant Hydnora visseri (Hydnoraceae).

Plastid genomes of photosynthetic flowering plants are usually highly conserved in both structure and gene content. However, the plastomes of parasitic and mycoheterotrophic plants may be released from selective constraint due to the reduction or loss of photosynthetic ability. Here we present the greatly reduced and highly divergent, yet functional, plastome of the nonphotosynthetic holoparasite Hydnora visseri (Hydnoraceae, Piperales). The plastome is 27 kb in length, with 24 genes encoding ribosomal proteins, ribosomal RNAs, tRNAs, and a few nonbioenergetic genes, but no genes related to photosynthesis. The inverted repeat and the small single copy region are only approximately 1.5 kb, and intergenic regions have been drastically reduced. Despite extreme reduction, gene order and orientation are highly similar to the plastome of Piper cenocladum, a related photosynthetic plant in Piperales. Gene sequences in Hydnora are highly divergent and several complementary approaches using the highest possible sensitivity were required for identification and annotation of this plastome. Active transcription is detected for all of the protein-coding genes in the plastid genome, and one of two introns is appropriately spliced out of rps12 transcripts. The whole-genome shotgun read depth is 1,400× coverage for the plastome, whereas the mitochondrial genome is covered at 40× and the nuclear genome at 2×. Despite the extreme reduction of the genome and high sequence divergence, the presence of syntenic, long transcriptionally active open-reading frames with distant similarity to other plastid genomes and a high plastome stoichiometry relative to the mitochondrial and nuclear genomes suggests that the plastome remains functional in H. visseri. A four-stage model of gene reduction, including the potential for complete plastome loss, is proposed to account for the range of plastid genomes in nonphotosynthetic plants.

Single-copy nuclear genes place haustorial Hydnoraceae within piperales and reveal a cretaceous origin of multiple parasitic angiosperm lineages.

Extreme haustorial parasites have long captured the interest of naturalists and scientists with their greatly reduced and highly specialized morphology. Along with the reduction or loss of photosynthesis, the plastid genome often decays as photosynthetic genes are released from selective constraint. This makes it challenging to use traditional plastid genes for parasitic plant phylogenetics, and has driven the search for alternative phylogenetic and molecular evolutionary markers. Thus, evolutionary studies, such as molecular clock-based age estimates, are not yet available for all parasitic lineages. In the present study, we extracted 14 nuclear single copy genes (nSCG) from Illumina transcriptome data from one of the "strangest plants in the world", Hydnora visseri (Hydnoraceae). A ~15,000 character molecular dataset, based on all three genomic compartments, shows the utility of nSCG for reconstructing phylogenetic relationships in parasitic lineages. A relaxed molecular clock approach with the same multi-locus dataset, revealed an ancient age of ~91 MYA for Hydnoraceae. We then estimated the stem ages of all independently originated parasitic angiosperm lineages using a published dataset, which also revealed a Cretaceous origin for Balanophoraceae, Cynomoriaceae and Apodanthaceae. With the exception of Santalales, older parasite lineages tend to be more specialized with respect to trophic level and have lower species diversity. We thus propose the "temporal specialization hypothesis" (TSH) implementing multiple independent specialization processes over time during parasitic angiosperm evolution.

Nutritional and isotopic relationships of selected Bornean tropical mistletoe-host associations in Brunei Darussalam.

Our understanding of mineral nutrition and carbon heterotrophy in mistletoes is derived largely from arid and temperate plant communities. Sharp differences between the tropical, temperate and arid communities, such as seasonality, water availability and mean temperature may influence basic assumptions regarding mistletoe physiology. Thus, we present mineral nutrition profiles and natural abundance carbon and nitrogen stable isotope data for tropical mistletoes and their hosts. Parasite-host mineral nutrition profiles were estimated for three Loranthaceous mistletoes: Scurrula ferruginea Danser, Macrosolen cochinchinensis Blume, and Dendrophthoe curvata Blume and 12 unique mistletoe-host associations. δ13C and δ15N values were estimated for 12 parasite-host associations. Differences between host and parasite δ13C values were small but showed significant depletion in mistletoe leaves compared with the distal branch and proximal branch host leaves. Host and mistletoe δ13C values were uncorrelated whereas δ15N values were significantly correlated, demonstrating mistletoe N dependence. Concentrations of K were higher in mistletoes relative to hosts and significantly higher for Dendrophthoe host associations. For Scurrula and Macrosolen, mean mistletoe-host concentrations of major and minor elements did not differ significantly.

Isotopic evidence of partial mycoheterotrophy in the Gentianaceae: Bartonia virginica and Obolaria virginica as case studies.

PREMISE OF THE STUDY: An estimated 10% of plant species have evolved to steal C from their symbiotic fungal partners (mycoheterotrophy), and while physiological evidence for full and partial mycoheterotrophy is well developed in the Orchidaceae and Ericaceae, it is lacking for the majority of other mycoheterotrophic taxa. The family Gentianaceae not only contains several lineages of achlorophyllous mycoheterotrophs, but also contains species that are putative partially mycoheterotrophic. The North American genera Bartonia and Obolaria (Gentianaceae) are green but have leaves reduced to scales or foliose bracts and so have ambiguous mycoheterotrophic status. • METHODS: We investigated the natural abundance (13)C and (15)N profiles of both genera along with total N and chlorophyll content and investigated mycorrhizal infection using light microscopy. • KEY RESULTS: The shoots of B. virginica were significantly more enriched in (15)N than the surrounding vegetation but not in (13)C. In contrast, the shoots of O. virginica are not enriched in (15)N compared to the surrounding vegetation but were significantly enriched in (13)C. Total N concentrations were significantly higher than the surrounding vegetation in B. virginica, while the collaroid roots of both species were infected by arbuscular mycorrhizal fungi. • CONCLUSIONS: This microscopic evidence coupled with the natural abundance stable isotope profiles strongly suggests that both species are partially mycoheterotrophic. However, differences in the root-shoot stable isotopic patterns relative to surrounding vegetation between B. virginica and O. virginica are suggestive of the utilization of different physiological pathways or extent of commitment to mycoheterotrophic C gain.

Structural attributes of the hypogeous holoparasite Hydnora triceps Drege & Meyer (Hydnoraceae).

The morphology of the hypogeous root holoparasite Hydnora triceps is highly reduced, and as with many holoparasites, the vegetative body is difficult to interpret. The vegetative body of H. triceps has been historically considered a "pilot root" studded with lateral appendages known as "haustorial roots." We found the vegetative body of H. triceps to consist of a rhizome with a thickened root-cap-like structure that covered a vegetative shoot apical meristem. From the apical meristem, procambial strands originated and developed into endarch collateral vascular bundles arranged radially around a pith without an interfascicular cambium. Xylem vessels had scalariform pitting and simple perforation plates. A continuous periderm without root hairs was observed. Increase in girth was attributed to cork and fascicular cambia. "Haustorial roots" or bumps on the surface of the vegetative body were exogenous, contained meristems and were the origins of vegetative branching, budding, and haustoria. The haustoria of H. triceps were cylindrical and penetrated the host root stele. Phloem and xylem elements were observed within the endophyte, and direct xylem to host-xylem contacts were observed. The arrangement of vascular tissues and xylem anatomy of H. triceps are likely plesiomorphic features in light of Hydnoraceae's placement in the Piperales.