The Influence of Cone Age and Urbanisation on the Diversity and Community Composition of Culturable Seed Fungal Endophytes within Native Australian Banksia ericifolia L.f. subsp. ericifolia.

Seed fungal endophytes play a crucial role in assisting the overall health and success of their host plant; however, little is known about the factors that influence the diversity and composition of these endophytes, particularly with respect to how they change over time and within urban environments. Using culturing techniques, morphological analyses, and Sanger sequencing, we identified the culturable seed fungal endophytes of Banksia ericifolia at two urban and two natural sites in Sydney, New South Wales, Australia. A total of 27 Operational Taxonomic Units were obtained from 1200 seeds. Older cones were found to contain, on average, more colonised endophytes than younger cones. Species richness was also significantly influenced by cone age, with older cones being more speciose. Between urban and natural sites, the overall community composition did not change, although species richness and diversity were greatest at urban sites. Understanding how these endophytes vary in time and space may help provide an insight into the transmission pathways used and the potential role they play within the development and survival of the seed. This knowledge may also be crucial for restoration purposes, especially regarding the need to consider endophyte viability in ex situ seed collection and storage in seed-banking practices.

Capturing Genetic Diversity in Seed Collections: An Empirical Study of Two Congeners with Contrasting Mating Systems.

Plant mating systems shape patterns of genetic diversity and impact the long-term success of populations. As such, they are relevant to the design of seed collections aiming to maximise genetic diversity (e.g., germplasm conservation, ecological restoration). However, for most species, little is known empirically about how variation in mating systems and genetic diversity is distributed. We investigated the relationship between genetic diversity and mating systems in two functionally similar, co-occurring species of Hakea (Proteaceae), and evaluated the extent to which genetic diversity was captured in seeds. We genotyped hundreds of seedlings and mother plants via DArTseq, and developed novel implementations of two approaches to inferring the mating system from SNP data. A striking contrast in patterns of genetic diversity between H. sericea and H. teretifolia was revealed, consistent with a contrast in their mating systems. While both species had mixed mating systems, H. sericea was found to be habitually selfing, while H. teretifolia more evenly employed both selfing and outcrossing. In both species, seed collection schemes maximised genetic diversity by increasing the number of maternal lines and sites sampled, but twice as many sites were needed for the selfing species to capture equivalent levels of genetic variation at a regional scale.

Plant collections for conservation and restoration: Can they be adapted and adaptable?

Plant collections are important for the conservation of threatened species, and can provide material for ecological restoration. Typically we want collections to have high genetic diversity so populations founded from it are adaptable to future challenges. Sometimes, we have additional objectives for collections, such as enrichment for desirable traits controlled by adaptive alleles. We used landscape genomic data sets for two plants, Westringia fruticosa and Wilkiea huegeliana, to design collections that are genetically diverse, and that are adapted to warming climates. We characterized temperature adaptation by: (i) using the mean annual temperature of the sites of origin of the plants, and (ii) using the representation of alleles that are associated with warm temperatures. In Westringia fruticosa, there was a negative correlation, or tradeoff, between designing a collection that was both genetically diverse and adapted to warm temperatures. This tradeoff was weaker in Wilkiea huegeliana. We hypothesized this was because neutral genetic variation was strongly correlated with temperature in Westringia fruticosa, and not in Wilkiea huegeliana. Accordingly, when we shuffled the temperature data, breaking up the covariance between Westringia fruticosa genetic variation and temperature, there was a relaxation of the observed tradeoff. In summary, we explore tradeoffs between promoting genetic diversity and selecting for a specific trait in plant collections, and show that the strength of this tradeoff varies between two species. This represents a useful step towards understanding when selection will have a large cost in genetic diversity, and when it will be possible to design a collection that is both adapted and adaptable.

Chromosome-level de novo genome assembly of Telopea speciosissima (New South Wales waratah) using long-reads, linked-reads and Hi-C.

Telopea speciosissima, the New South Wales waratah, is an Australian endemic woody shrub in the family Proteaceae. Waratahs have great potential as a model clade to better understand processes of speciation, introgression and adaptation, and are significant from a horticultural perspective. Here, we report the first chromosome-level genome for T. speciosissima. Combining Oxford Nanopore long-reads, 10x Genomics Chromium linked-reads and Hi-C data, the assembly spans 823 Mb (scaffold N50 of 69.0 Mb) with 97.8% of Embryophyta BUSCOs "Complete". We present a new method in Diploidocus (https://github.com/slimsuite/diploidocus) for classifying, curating and QC-filtering scaffolds, which combines read depths, k-mer frequencies and BUSCO predictions. We also present a new tool, DepthSizer (https://github.com/slimsuite/depthsizer), for genome size estimation from the read depth of single-copy orthologues and estimate the genome size to be approximately 900 Mb. The largest 11 scaffolds contained 94.1% of the assembly, conforming to the expected number of chromosomes (2n = 22). Genome annotation predicted 40,158 protein-coding genes, 351 rRNAs and 728 tRNAs. We investigated CYCLOIDEA (CYC) genes, which have a role in determination of floral symmetry, and confirm the presence of two copies in the genome. Read depth analysis of 180 "Duplicated" BUSCO genes using a new tool, DepthKopy (https://github.com/slimsuite/depthkopy), suggests almost all are real duplications, increasing confidence in the annotation and highlighting a possible need to revise the BUSCO set for this lineage. The chromosome-level T. speciosissima reference genome (Tspe_v1) provides an important new genomic resource of Proteaceae to support the conservation of flora in Australia and further afield.

Miocene diversification of a golden-thread nanmu tree species (Phoebe zhennan, Lauraceae) around the Sichuan Basin shaped by the East Asian monsoon.

Understanding the role of climate changes and geography as drivers of population divergence and speciation is a long-standing goal of evolutionary biology and can inform conservation. In this study, we used restriction site-associated DNA sequencing (RAD-seq) to evaluate genetic diversity, population structure, and infer demographic history of the endangered tree, Phoebe zhennan which is distributed around the Sichuan Basin. Genomic patterns revealed two distinct clusters, each largely confined to the West and East. Despite sympatry of the two genomic clusters at some sites, individuals show little or no evidence of genomic introgression. Demographic modeling supported an initial divergence time between the West and East lineages at ~15.08 Ma with further diversification within the West lineage at ~7.12 Ma. These times largely coincide with the two independent intensifications of the East Asian monsoon that were initiated during the middle (Langhian) and late Miocene (Messinian), respectively. These results suggest that the Miocene intensification phases of the East Asian monsoon played a pivotal role in shaping the current landscape-level patterns of genetic diversity within P. zhennan, as has been found for the interspecific divergence of other subtropical Chinese plants. Based on isolation-by-distance and species distribution modeling, we hypothesize that P. zhennan followed a ring diversification which was facilitated by the Sichuan Basin acting as barrier to gene flow. In situ and ex situ conservation management plans should consider the results obtained in this study to help secure the future of this beautiful and culturally significant endangered tree.

Evaluating the Impact of Genomic Data and Priors on Bayesian Estimates of the Angiosperm Evolutionary Timescale.

The evolutionary timescale of angiosperms has long been a key question in biology. Molecular estimates of this timescale have shown considerable variation, being influenced by differences in taxon sampling, gene sampling, fossil calibrations, evolutionary models, and choices of priors. Here, we analyze a data set comprising 76 protein-coding genes from the chloroplast genomes of 195 taxa spanning 86 families, including novel genome sequences for 11 taxa, to evaluate the impact of models, priors, and gene sampling on Bayesian estimates of the angiosperm evolutionary timescale. Using a Bayesian relaxed molecular-clock method, with a core set of 35 minimum and two maximum fossil constraints, we estimated that crown angiosperms arose 221 (251-192) Ma during the Triassic. Based on a range of additional sensitivity and subsampling analyses, we found that our date estimates were generally robust to large changes in the parameters of the birth-death tree prior and of the model of rate variation across branches. We found an exception to this when we implemented fossil calibrations in the form of highly informative gamma priors rather than as uniform priors on node ages. Under all other calibration schemes, including trials of seven maximum age constraints, we consistently found that the earliest divergences of angiosperm clades substantially predate the oldest fossils that can be assigned unequivocally to their crown group. Overall, our results and experiments with genome-scale data suggest that reliable estimates of the angiosperm crown age will require increased taxon sampling, significant methodological changes, and new information from the fossil record. [Angiospermae, chloroplast, genome, molecular dating, Triassic.].

Origins and evolution of cinnamon and camphor: A phylogenetic and historical biogeographical analysis of the Cinnamomum group (Lauraceae).

Tropical and subtropical amphi-Pacific disjunction is among the most fascinating distribution patterns, but received little attention. Here we use the fossil-rich Cinnamomum group, a primarily tropical and subtropical Asian lineage with some species distributed in Neotropics, Australasia and Africa to shed light upon this disjunction pattern. Phylogenetic and biogeographic analyses were carried out using sequences of three nuclear loci from 94 Cinnamomum group and 13 outgroup samples. Results show that although there are three clades within a monophyletic Cinnamomum group, Cinnamomum and previously recognized subdivisions within this genus were all rejected as natural groups. The Cinnamomum group appears to have originated in the widespread boreotropical paleoflora of Laurasia during the early Eocene (ca. 55Ma). The formation and breakup of the boreotropics seems to have then played a key role in the formation of intercontinental disjunctions within the Cinnamomum group. The first cooling interval (50-48Ma) in the late early Eocene resulted in a floristic discontinuity between Eurasia and North America causing the tropical and subtropical amphi-Pacific disjunction. The second cooling interval in the mid-Eocene (42-38Ma) resulted in the fragmentation of the boreotropics within Eurasia, leading to an African-Asian disjunction. Multiple dispersal events from North into South America occurred from the early Eocene to late Miocene and a single migration event from Asia into Australia appears to have occurred in the early Miocene.

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution.

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.

The host bias of three epiphytic Aeridinae orchid species is reflected, but not explained, by mycorrhizal fungal associations.

PREMISE OF THE STUDY: The three co-occurring epiphytic orchid species, Sarcochilus hillii, Plectorrhiza tridentata, and Sarcochilus parviflorus vary in host specificity; all are found predominantly on the tree Backhousia myrtifolia but some also associate with a broad range of species. Despite this specialization, no fitness advantage has been detected for adult orchid plants growing on the preferred host. Therefore, we predicted that the host specialization of these orchid species is a consequence of a bias toward particular orchid mycorrhizal fungi, which are in turn biased toward particular woody plant species. METHODS: To test this hypothesis, we sampled representatives of each orchid species on B. myrtifolia and other host species across sites. Rhizoctonia-like fungi were isolated from orchid roots and identified using molecular markers. KEY RESULTS: Three groups of fungi were identified, and the orchid species varied in their specificity for these. All fungal groups were found on the host B. myrtifolia; yet at all sites, only one orchid species, S. hillii, associated with all three groups. CONCLUSIONS: Our results demonstrate that these orchid species did vary in their mycorrhizal specificity; however, the distribution of their mycorrhizal associates did not directly explain their host associations. Rather, we propose that the mycorrhizal relationship of these orchid species is complex and have suggested future avenues of research.

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree.

BACKGROUND: With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. RESULTS: The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. CONCLUSIONS: Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology.

Are the microcyclic rust species Puccinia melampodii and Puccinia xanthii conspecific?

The taxonomic relationship between two microcyclic rust species, Puccinia melampodii and Puccinia xanthii, recorded from a number of Asteraceae hosts, was explored by comparing teliospore morphology, host specificity and sequence data. Teliospore morphology varied between and within individual rust accessions but, in general, teliospores of P. xanthii were larger than those of P. melampodii. Field observations and cross-inoculation greenhouse studies showed that P. melampodii accessions were highly specific to their original host species and unable to establish compatible interactions with related Asteraceae species. Though small amounts of genetic variation were detected between P. melampodii and P. xanthii accessions, the ITS and TEF phylogenetic analyses comprising several other rust species grouped these accessions within a well supported clade. Our data indicate that despite differences between P. xanthii and P. melampodii accessions these rusts cannot readily be separated at the species level, supporting earlier taxonomic conclusions and the recent proposal to group them into a morphospecies. Based on host-specificity data, the name Puccinia xanthii var. parthenii-hysterophorae is proposed to accommodate all Mesoamerican records of P. melampodii associated with the host Parthenium hysterophorus.

Diversity and evolution of effector loci in natural populations of the plant pathogen Melampsora lini.

Genetic variation for pathogen infectivity is an important driver of disease incidence and prevalence in both natural and managed systems. Here, we use the interaction between the rust pathogen, Melampsora lini, and two host plants, Linum marginale and Linum usitatissimum, to examine how host-pathogen interactions influence the maintenance of polymorphism in genes underlying pathogen virulence. Extensive sequence variation at two effector loci (AvrP123, AvrP4) was found in M. lini isolates collected from across the native range of L. marginale in Australia, as well as in isolates collected from a second host, the cultivated species L. usitatissimum. A highly significant excess of nonsynonymous compared with synonymous polymorphism was found at both loci, suggesting that diversifying selection is important for the maintenance of the observed sequence diversity. Agrobacterium-mediated transient transformation assays were used to demonstrate that variants of both the AvrP123 and AvrP4 genes are differentially recognized by resistance genes in L. marginale. We further characterized patterns of nucleotide variation at AvrP123 and AvrP4 in 10 local populations of M. lini infecting the wild host L. marginale. Populations were significantly differentiated with respect to allelic representation at the Avr loci, suggesting the possibility of local selection maintaining distinct genetic structures between pathogen populations, whereas limited diversity may be explained via selective sweeps and demographic bottlenecks. Together, these results imply that interacting selective and nonselective factors, acting across a broad range of scales, are important for the generation and maintenance of adaptively significant variation in populations of M. lini.

Positive selection in AvrP4 avirulence gene homologues across the genus Melampsora.

Pathogen genes involved in interactions with their plant hosts are expected to evolve under positive Darwinian selection or balancing selection. In this study a single copy avirulence gene, AvrP4, in the plant pathogen Melampsora lini, was used to investigate the evolution of such a gene across species. Partial translation elongation factor 1-alpha sequences were obtained to establish phylogenetic relationships among the Melampsora species. We amplified AvrP4 homologues from species pathogenic on hosts from different plant families and orders, across the inferred phylogeny. Translations of the AvrP4 sequences revealed a predicted signal peptide and towards the C-terminus of the protein, six identically spaced cysteines were identified in all sequences. Maximum likelihood analysis of synonymous versus non-synonymous substitution rates indicated that positive selection played a role in the evolution of the gene during the diversification of the genus. Fourteen codons under significant positive selection reside in the C-terminal 28 amino acid region, suggesting that this region interacts with host molecules in most sequenced accessions. Selection pressures on the gene may be either due to the pathogenicity or avirulence function of the gene or both.

Putative natural hybrid between Puccinia lagenophorae and an unknown rust fungus on Senecio madagascariensis in KwaZulu-Natal, South Africa.

Several specimens of an aecial rust fungus were collected on Senecio madagascariensis during a field survey carried out in KwaZulu-Natal, South Africa. As telia were not present in the specimens collected, DNA sequence analyses were undertaken to determine the identity of the rust species. ITS and beta-tub1 sequencing confirmed that one of the isolates recovered is Puccinia lagenophorae sensu lato. On the other hand, sequencing and RFLP analysis revealed the presence of two divergent copies of ITS and beta-tub1 in all the other six isolates investigated. In both phylogenetic trees, one copy of the gene region grouped within a well supported clade with sequences of P. lagenophorae accessions from different geographical origins and hosts, and the Australian rusts Puccinia saccardoi and Puccinia stylidii. The other copy of these gene regions grouped within a separate clade comprising European accessions of Puccinia dioicae (ITS) and Uromyces sommerfeltii (beta-tub1) that occur on Asteraceae hosts. Multiple copies of these gene regions were not observed in Australian isolates of P. lagenophorae. Our study provides some evidence that an interspecific hybrid rust fungus, with P. lagenophorae as one of its parents, may occur on S. madagascariensis in South Africa. The identity of the other parent remains unknown.

Coevolution with higher taxonomic host groups within the Puccinia/Uromyces rust lineage obscured by host jumps.

Partial beta-tubulin 1 sequence data were obtained for 80 taxa of Pucciniaceae, with hosts from 33 angiosperm families, covering all major ordinal groups in the Angiosperm Phylogeny Group classification. As in previous studies, most species of Puccinia and Uromyces fell into two main clades (I and II), with P. glechomatis and P. psidii excluded from Pucciniaceae. Results suggest two processes; a coevolution of and hosts in each clade, as well as associated frequent jumps to ecologically close, but taxonomically distant, hosts. Clade I contained all rusts on Cyperaceae and Juncaceae, and most rusts on host orders from rosids to euasterids II. Clade II contained all rusts on Poaceae, and most on host orders from monocots to core eudicots. In both main clades, several well-supported subclades were identified. The grouping in clade I, subclade E of rusts of Cyperaceae and Asteraceae and, in particular, of an Australian isolate of P. dioicae with rusts on Australian families of Asterales, suggested a local radiation, and supported the coevolutionary relationship between rusts on these two families seen with a different range of asteraceous rusts in the Northern Hemisphere. In clade I, two clades contained only rusts of Asteraceae and Fabaceae, respectively, and in clade II, subclade F contained only rusts of pooid hosts. Rusts on non-pooid hosts were separated from them in subclade G. Other subclades contained a range of rusts on distantly related angiosperm families. Urediniospore morphology was often, but not always, correlated with the molecular phylogeny. Most rusts with urediniospores having few (1-5) equatorial germ pores were in clade I, whereas most with spores having several (5-14) scattered pores were in clade II. The distribution of telial host families on the beta-tubulin rust phylogeny was not random. Aecial hosts of heteroecious rusts played an important role in the evolutionary process. Possible examples of host jumps were seen in rusts on Geraniaceae, Polygonaceae, and Apiaceae. Despite such jumps obscuring past host associations, possible ancestral hosts were identified by the pattern of host distribution at higher taxonomic levels along the ss-tubulin phylogeny. Results suggest that clade I diverged with Cyperaceae, Juncaceae, and the more advanced core eudicot orders (rosids and asterids), whereas clade II diversified with earlier angiosperm groups, such as monocots, Poaceae, and Ranunculales. Qualified support was given to the hypothesis that rusts can reveal taxonomic relationships between their hosts, at genus, family, and ordinal levels.

Evolutionary relationships among species of Puccinia and Uromyces (Pucciniaceae, Uredinales) inferred from partial protein coding gene phylogenies.

The two rust genera with the largest number of species are Puccinia Pers. ex Pers. and Uromyces (Link) Unger in the family Pucciniaceae (Uredinales). The hosts of these pathogens include representatives from almost all major angiosperm orders. Despite their ecological and economic importance, the status of Puccinia and Uromyces as distinct genera has been disputed, and little is known about relationships within and among these groups. Here we present phylogenetic analyses based on sequence data from the translation elongation factor 1alpha gene for over 60 species in the family Pucciniaceae. In particular, we investigate evolutionary relationships between Puccinia and Uromyces. A relatively smaller phylogeny using the beta-tubulin 1 gene was generated to test support for this phylogeny. Two main phylogenetic clades were identified and indicate at least two radiations within the Pucciniaceae. As expected neither Puccinia s. lat. nor Uromyces s. lat. are supported as monophyletic groups by either of the protein coding genes. However, both Puccinia sensu stricto (type P. graminis), and Uromyces sensu stricto (type U. appendiculatus) constitute distinct clades. In general, members of Uromyces spp. occurred scattered throughout the phylogeny suggesting that they represent more recent radiations. Several host families are found in both of the two main clades while two families, Poaceae and Cyperaceae, are separated, with one in each of the two main clades.