Global Brassicaceae phylogeny based on filtering of 1,000-gene dataset.

The mustard family (Brassicaceae) is a scientifically and economically important family, containing the model plant Arabidopsis thaliana and numerous crop species that feed billions worldwide. Despite its relevance, most phylogenetic trees of the family are incompletely sampled and often contain poorly supported branches. Here, we present the most complete Brassicaceae genus-level family phylogenies to date (Brassicaceae Tree of Life or BrassiToL) based on nuclear (1,081 genes, 319 of the 349 genera; 57 of the 58 tribes) and plastome (60 genes, 265 genera; all tribes) data. We found cytonuclear discordance between the two, which is likely a result of rampant hybridization among closely and more distantly related lineages. To evaluate the impact of such hybridization on the nuclear phylogeny reconstruction, we performed five different gene sampling routines, which increasingly removed putatively paralog genes. Our cleaned subset of 297 genes revealed high support for the tribes, whereas support for the main lineages (supertribes) was moderate. Calibration based on the 20 most clock-like nuclear genes suggests a late Eocene to late Oligocene origin of the family. Finally, our results strongly support a recently published new family classification, dividing the family into two subfamilies (one with five supertribes), together representing 58 tribes. This includes five recently described or re-established tribes, including Arabidopsideae, a monogeneric tribe accommodating Arabidopsis without any close relatives. With a worldwide community of thousands of researchers working on Brassicaceae and its diverse members, our new genus-level family phylogeny will be an indispensable tool for studies on biodiversity and plant biology.

Phylogenomic analyses of Sapindales support new family relationships, rapid Mid-Cretaceous Hothouse diversification, and heterogeneous histories of gene duplication.

Sapindales is an angiosperm order of high economic and ecological value comprising nine families, c. 479 genera, and c. 6570 species. However, family and subfamily relationships in Sapindales remain unclear, making reconstruction of the order's spatio-temporal and morphological evolution difficult. In this study, we used Angiosperms353 target capture data to generate the most densely sampled phylogenetic trees of Sapindales to date, with 448 samples and c. 85% of genera represented. The percentage of paralogous loci and allele divergence was characterized across the phylogeny, which was time-calibrated using 29 rigorously assessed fossil calibrations. All families were supported as monophyletic. Two core family clades subdivide the order, the first comprising Kirkiaceae, Burseraceae, and Anacardiaceae, the second comprising Simaroubaceae, Meliaceae, and Rutaceae. Kirkiaceae is sister to Burseraceae and Anacardiaceae, and, contrary to current understanding, Simaroubaceae is sister to Meliaceae and Rutaceae. Sapindaceae is placed with Nitrariaceae and Biebersteiniaceae as sister to the core Sapindales families, but the relationships between these families remain unclear, likely due to their rapid and ancient diversification. Sapindales families emerged in rapid succession, coincident with the climatic change of the Mid-Cretaceous Hothouse event. Subfamily and tribal relationships within the major families need revision, particularly in Sapindaceae, Rutaceae and Meliaceae. Much of the difficulty in reconstructing relationships at this level may be caused by the prevalence of paralogous loci, particularly in Meliaceae and Rutaceae, that are likely indicative of ancient gene duplication events such as hybridization and polyploidization playing a role in the evolutionary history of these families. This study provides key insights into factors that may affect phylogenetic reconstructions in Sapindales across multiple scales, and provides a state-of-the-art phylogenetic framework for further research.

Evolutionary Relationships and Range Evolution of Greenhood Orchids (Subtribe Pterostylidinae): Insights From Plastid Phylogenomics.

Australia harbours a rich and highly endemic orchid flora with over 90% of native species found nowhere else. However, little is known about the assembly and evolution of Australia's orchid flora. Here, we used a phylogenomic approach to infer evolutionary relationships, divergence times and range evolution in Pterostylidinae (Orchidoideae), the second largest subtribe in the Australian orchid flora, comprising the genera Pterostylis and Achlydosa. Phylogenetic analysis of 75 plastid genes provided well-resolved and supported phylogenies. Intrageneric relationships in Pterostylis were clarified and monophyly of eight of 10 sections supported. Achlydosa was found to not form part of Pterostylidinae and instead merits recognition at subtribal level, as Achlydosinae. Pterostylidinae were inferred to have originated in eastern Australia in the early Oligocene, coinciding with the complete separation of Australia from Antarctica and the onset of the Antarctic Circumpolar Current, which led to profound changes in the world's climate. Divergence of all major lineages occurred during the Miocene, accompanied by increased aridification and seasonality of the Australian continent, resulting in strong vegetational changes from rainforest to more open sclerophyllous vegetation. The majority of extant species were inferred to have originated in the Quaternary, from the Pleistocene onwards. The rapid climatic oscillations during the Pleistocene may have acted as important driver of speciation in Pterostylidinae. The subtribe underwent lineage diversification mainly within its ancestral range, in eastern Australia. Long-distance dispersals to southwest Australia commenced from the late Miocene onwards, after the establishment of the Nullarbor Plain, which constitutes a strong edaphic barrier to mesic plants. Range expansions from the mesic into the arid zone of eastern Australia (Eremaean region) commenced from the early Pleistocene onwards. Extant distributions of Pterostylidinae in other Australasian regions, such as New Zealand and New Caledonia, are of more recent origin, resulting from long-distance dispersals from the Pliocene onwards. Temperate eastern Australia was identified as key source area for dispersals to other Australasian regions.

HybPhaser identifies hybrid evolution in Australian Thelypteridaceae.

Hybridisation has been proposed to play an important role in fern evolution, but has been difficult to investigate. This study explores the utility of target sequence capture and read-to-reference phasing of putative hybrids to investigate the role of evolutionary reticulation in ferns using Australian Thelypteridaceae as a model. The bioinformatics workflow HybPhaser was used to assess divergence between alleles, phase sequence reads to references and construct accessions resembling parental haplotypes. These accessions were included in phylogenetic and network analyses to detect hybrids and infer their parentage. This approach identified two novel hybrid lineages in Thelypteridaceae, one occurring between two different genera (Abacopteris and Christella), and another as part of a complex of Christella. In addition, hybrid phasing successfully reduced conflicting data and improved overall resolution in the Thelypteridaceae phylogeny, highlighting the power of this approach for reconstructing evolutionary history in reticulated lineages.

New targets acquired: Improving locus recovery from the Angiosperms353 probe set.

PREMISE: Universal target enrichment kits maximize utility across wide evolutionary breadth while minimizing the number of baits required to create a cost-efficient kit. The Angiosperms353 kit has been successfully used to capture loci throughout the angiosperms, but the default target reference file includes sequence information from only 6-18 taxa per locus. Consequently, reads sequenced from on-target DNA molecules may fail to map to references, resulting in fewer on-target reads for assembly, and reducing locus recovery. METHODS: We expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a 'mega353' target file, with each locus represented by 17-373 taxa. This mega353 file is a drop-in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user-selected taxon groups, and to incorporate other transcriptome or protein-coding gene data sets. RESULTS: Compared to the default Angiosperms353 file, the mega353 file increased the percentage of on-target reads by an average of 32%, increased locus recovery at 75% length by 49%, and increased the total length of the concatenated loci by 29%. DISCUSSION: Increasing the phylogenetic density of the target reference file results in improved recovery of target capture loci. The mega353 file and associated scripts are available at: https://github.com/chrisjackson-pellicle/NewTargets.

HybPhaser: A workflow for the detection and phasing of hybrids in target capture data sets.

PREMISE: Hybrids contain divergent alleles that can confound phylogenetic analyses but can provide insights into reticulated evolution when identified and phased. We developed a workflow to detect hybrids in target capture data sets and phase reads into parental lineages using a similarity and phylogenetic framework. METHODS: We used Angiosperms353 target capture data for Nepenthes, including known hybrids to test the novel workflow. Reference mapping was used to assess heterozygous sites across the data set and to detect hybrid accessions and paralogous genes. Hybrid samples were phased by mapping reads to multiple references and sorting reads according to similarity. Phased accessions were included in the phylogenetic framework. RESULTS: All known Nepenthes hybrids and nine additional samples had high levels of heterozygous sites, had reads associated with multiple divergent clades, and were phased into accessions resembling divergent haplotypes. Phylogenetic analysis including phased accessions increased clade support and confirmed parental lineages of hybrids. DISCUSSION: HybPhaser provides a novel approach to detect and phase hybrids in target capture data sets, which can provide insights into reticulations by revealing origins of hybrids and reduce conflicting signal, leading to more robust phylogenetic analyses.

Comparative Analysis of Plastid Genomes in the Non-photosynthetic Genus Thismia Reveals Ongoing Gene Set Reduction.

Heterotrophic plants provide intriguing examples of reductive evolution. This is especially evident in the reduction of their plastid genomes, which can potentially proceed toward complete genome loss. Several milestones at the beginning of this path of degradation have been described; however, little is known about the latest stages of plastome reduction. Here we analyze a diversity of plastid genomes in a set of closely related non-photosynthetic plants. We demonstrate how a gradual loss of genes shapes the miniaturized plastomes of these plants. The subject of our study, the genus Thismia, represents the mycoheterotrophic monocot family Thismiaceae, a group that may have experienced a very ancient (60-80 mya) transition to heterotrophy. In all 18 species examined, the plastome is reduced to 14-18 kb and is highly AT-biased. The most complete observed gene set includes accD, seven ribosomal protein genes, three rRNA, and two tRNA genes. Different clades of Thismia have undergone further gene loss (complete absence or pseudogenization) compared to this set: in particular, we report two independent losses of rps2 and rps18.

Australasian orchid biogeography at continental scale: Molecular phylogenetic insights from the Sun Orchids (Thelymitra, Orchidaceae).

Australia harbours a rich and highly endemic orchid flora, with c. 90% of species endemic to the country. Despite that, the biogeographic history of Australasian orchid lineages is only poorly understood. Here we examined evolutionary relationships and the spatio-temporal evolution of the sun orchids (Thelymitra, 119 species), which display disjunct distribution patterns frequently found in Australasian orchid lineages. Phylogenetic analyses were conducted based on one nuclear (ITS) and three plastid markers (matK, psbJ-petA, ycf1) using Maximum Likelihood and Bayesian inference. Divergence time estimations were carried out with a relaxed molecular clock in a Bayesian framework. Ancestral ranges were estimated using the dispersal-extinction-cladogenesis model and an area coding based on major disjunctions. The phylogenetic analyses clarified intergeneric relationships within Thelymitrinae, with Epiblema being sister to Thelymitra plus Calochilus, both of which were well-supported. Within Thelymitra, eight major and several minor clades were retrieved in the nuclear and plastid phylogenetic reconstructions. Five major clades corresponded to species complexes previously recognized based on morphological characters, whereas other previously recognized species groups were found to be paraphyletic. Conflicting signals between the nuclear and plastid phylogenetic reconstructions provided support for hybridization and plastid capture events both in the deeper evolutionary history of the genus and more recently. Divergence time estimation placed the origin of Thelymitra in the late Miocene (c. 10.8 Ma) and the origin of the majority of the main clades within Thelymitra during the late Pliocene and early Pleistocene, with the majority of extant species arising during the Pleistocene. Ancestral range reconstruction revealed that the early diversification of the genus in the late Miocene and Pliocene took place predominantly in southwest Australia, where most species with highly restricted distributional ranges occur. Several long-distance dispersal events eastwards across the Nullarbor Plain were inferred, recurrently resulting in lineage divergence within the genus. The predominant eastwards direction of long-distance dispersal events in Thelymitra highlights the importance of the prevailing westerly winds in the Southern Hemisphere for the present-day distribution of the genus, giving rise to the Thelymitra floras of Tasmania, New Zealand and New Caledonia, which were inferred to be of comparatively recent origin.

Evolutionary diversification of the African achyranthoid clade (Amaranthaceae) in the context of sterile flower evolution and epizoochory.

Background and Aims: Many African genera of the Amaranthaceae exhibit unique inflorescences that include sterile flowers modified to hooks or spines. Considering that the abundance of large terrestrial herbivores increased on the African continent with the expansion of grassland and savannah ecosystems, modified sterile flowers could have been an innovation that boosted the diversification of an African achyranthoid clade of Amaranthaceae, with large animals serving dispersal. Methods: We generated an extensively sampled phylogeny comprising 26 of the 31 achyranthoid genera as well as representatives of all other lineages of Amaranthaceae. Phylogenetic tree inference employed four genomic regions, using parsimony, likelihood and Bayesian inference methods. We estimated divergence times, evaluated trait-dependant changes and species diversification rates using state-dependent speciation and extinction models, and reconstructed ancestral character states for modified sterile flowers. Key Results: The achyranthoids were found to be a major clade of the Amaranthaceae, comprising mostly African members. Phylogenetic relationships within this clade were well resolved and supported two main subclades. Several genera were found to be polyphyletic. Our results indicate that the achyranthoids started to diversify ~28 million years ago, and that modified sterile flowers evolved multiple times. An asymmetry in transition rates towards the gain of sterile flowers was observed, whereas no trait-dependent increase in species diversification rates was detected. Bayesian rate heterogeneity analyses indicated that the achyranthoids diversified without significant rate shifts. Conclusions: The accumulation of modified sterile flowers within achyranthoids appears to result from the higher transition rates in favour of modified sterile flowers. Multiple gains suggest an adaptive value for this trait. However, epizoochory does not appear to fuel species diversification, possibly due to extensive gene flow through regularly migrating mammals, which limits the possibility of speciation by isolation.

Variability among the most rapidly evolving plastid genomic regions is lineage-specific: implications of pairwise genome comparisons in Pyrus (Rosaceae) and other angiosperms for marker choice.

Plastid genomes exhibit different levels of variability in their sequences, depending on the respective kinds of genomic regions. Genes are usually more conserved while noncoding introns and spacers evolve at a faster pace. While a set of about thirty maximum variable noncoding genomic regions has been suggested to provide universally promising phylogenetic markers throughout angiosperms, applications often require several regions to be sequenced for many individuals. Our project aims to illuminate evolutionary relationships and species-limits in the genus Pyrus (Rosaceae)-a typical case with very low genetic distances between taxa. In this study, we have sequenced the plastid genome of Pyrus spinosa and aligned it to the already available P. pyrifolia sequence. The overall p-distance of the two Pyrus genomes was 0.00145. The intergenic spacers between ndhC-trnV, trnR-atpA, ndhF-rpl32, psbM-trnD, and trnQ-rps16 were the most variable regions, also comprising the highest total numbers of substitutions, indels and inversions (potentially informative characters). Our comparative analysis of further plastid genome pairs with similar low p-distances from Oenothera (representing another rosid), Olea (asterids) and Cymbidium (monocots) showed in each case a different ranking of genomic regions in terms of variability and potentially informative characters. Only two intergenic spacers (ndhF-rpl32 and trnK-rps16) were consistently found among the 30 top-ranked regions. We have mapped the occurrence of substitutions and microstructural mutations in the four genome pairs. High AT content in specific sequence elements seems to foster frequent mutations. We conclude that the variability among the fastest evolving plastid genomic regions is lineage-specific and thus cannot be precisely predicted across angiosperms. The often lineage-specific occurrence of stem-loop elements in the sequences of introns and spacers also governs lineage-specific mutations. Sequencing whole plastid genomes to find markers for evolutionary analyses is therefore particularly useful when overall genetic distances are low.

Global history of the ancient monocot family Araceae inferred with models accounting for past continental positions and previous ranges based on fossils.

The family Araceae (3790 species, 117 genera) has one of the oldest fossil records among angiosperms. Ecologically, members of this family range from free-floating aquatics (Pistia and Lemna) to tropical epiphytes. Here, we infer some of the macroevolutionary processes that have led to the worldwide range of this family and test how the inclusion of fossil (formerly occupied) geographical ranges affects biogeographical reconstructions. Using a complete genus-level phylogeny from plastid sequences and outgroups representing the 13 other Alismatales families, we estimate divergence times by applying different clock models and reconstruct range shifts under different models of past continental connectivity, with or without the incorporation of fossil locations. Araceae began to diversify in the Early Cretaceous (when the breakup of Pangea was in its final stages), and all eight subfamilies existed before the K/T boundary. Early lineages persist in Laurasia, with several relatively recent entries into Africa, South America, South-East Asia and Australia. Water-associated habitats appear to be ancestral in the family, and DNA substitution rates are especially high in free-floating Araceae. Past distributions inferred when fossils are included differ in nontrivial ways from those without fossils. Our complete genus-level time-scale for the Araceae may prove to be useful for ecological and physiological studies.

Giant taro and its relatives: a phylogeny of the large genus Alocasia (Araceae) sheds light on Miocene floristic exchange in the Malesian region.

Alocasia comprises over 113 species of rainforest understorey plants in Southeast Asia, the Malesian region, and Australia. Several species, including giant taro, Alocasia macrorrhizos, and Chinese taro, Alocasia cucullata, are important food plants or ornamentals. We investigated the biogeography of this genus using plastid and nuclear DNA sequences (5200 nucleotides) from 78 accessions representing 71 species, plus 25 species representing 16 genera of the Pistia clade to which Alocasia belongs. Divergence times were inferred under strict and relaxed clock models, and ancestral areas with Bayesian and maximum likelihood approaches. Alocasia is monophyletic and sister to Colocasiagigantea from the SE Asian mainland, whereas the type species of Colocasia groups with Steudnera and Remusatia, requiring taxonomic realignments. Nuclear and plastid trees show topological conflict, with the nuclear tree reflecting morphological similarities, the plastid tree species' geographic proximity, suggesting chloroplast capture. The ancestor of Alocasia diverged from its mainland sister group c. 24 million years ago, and Borneo then played a central role in the expansion of Alocasia: 11-13 of 18-19 inferred dispersal events originated on Borneo. The Philippines were reached from Borneo 4-5 times in the Late Miocene and Early Pliocene, and the Asian mainland 6-7 times in the Pliocene. Domesticated giant taro originated on the Philippines, Chinese taro on the Asian mainland.