Cytonuclear Interactions and Subgenome Dominance Shape the Evolution of Organelle-Targeted Genes in the Brassica Triangle of U.

The interaction and coevolution between nuclear and cytoplasmic genomes are one of the fundamental hallmarks of eukaryotic genome evolution and, 2 billion yr later, are still major contributors to the formation of new species. Although many studies have investigated the role of cytonuclear interactions following allopolyploidization, the relative magnitude of the effect of subgenome dominance versus cytonuclear interaction on genome evolution remains unclear. The Brassica triangle of U features 3 diploid species that together have formed 3 separate allotetraploid species on similar evolutionary timescales, providing an ideal system for understanding the contribution of the cytoplasmic donor to hybrid polyploid. Here, we investigated the evolutionary pattern of organelle-targeted genes in Brassica carinata (BBCC) and 2 varieties of Brassica juncea (AABB) at the whole-genome level, with particular focus on cytonuclear enzyme complexes. We found partial evidence that plastid-targeted genes experience selection to match plastid genomes, but no obvious corresponding signal in mitochondria-targeted genes from these 2 separately formed allopolyploids. Interestingly, selection acting on plastid genomes always reduced the retention rate of plastid-targeted genes encoded by the B subgenome, regardless of whether the Brassica nigra (BB) subgenome was contributed by the paternal or maternal progenitor. More broadly, this study illustrates the distinct selective pressures experienced by plastid- and mitochondria-targeted genes, despite a shared pattern of inheritance and natural history. Our study also highlights an important role for subgenome dominance in allopolyploid genome evolution, even in genes whose function depends on separately inherited molecules.

Association Analysis Provides Insights into Plant Mitonuclear Interactions.

Cytonuclear interaction refers to the complex and ongoing process of coevolution between nuclear and organelle genomes, which are responsible for cellular respiration, photosynthesis, lipid metabolism, etc. and play a significant role in adaptation and speciation. There have been a large number of studies to detect signatures of cytonuclear interactions. However, identification of the specific nuclear and organelle genetic polymorphisms that are involved in these interactions within a species remains relatively rare. The recent surge in whole genome sequencing has provided us an opportunity to explore cytonuclear interaction from a population perspective. In this study, we analyzed a total of 3,439 genomes from 7 species to identify signals of cytonuclear interactions by association (linkage disequilibrium) analysis of variants in both the mitochondrial and nuclear genomes across flowering plants. We also investigated examples of nuclear loci identified based on these association signals using subcellular localization assays, gene editing, and transcriptome sequencing. Our study provides a novel perspective on the investigation of cytonuclear coevolution, thereby enriching our understanding of plant fitness and offspring sterility.

Extraordinary preservation of gene collinearity over three hundred million years revealed in homosporous lycophytes.

Homosporous lycophytes (Lycopodiaceae) are a deeply diverged lineage in the plant tree of life, having split from heterosporous lycophytes (Selaginella and Isoetes) ~400 Mya. Compared to the heterosporous lineage, Lycopodiaceae has markedly larger genome sizes and remains the last major plant clade for which no chromosome-level assembly has been available. Here, we present chromosomal genome assemblies for two homosporous lycophyte species, the allotetraploid Huperzia asiatica and the diploid Diphasiastrum complanatum. Remarkably, despite that the two species diverged ~350 Mya, around 30% of the genes are still in syntenic blocks. Furthermore, both genomes had undergone independent whole genome duplications, and the resulting intragenomic syntenies have likewise been preserved relatively well. Such slow genome evolution over deep time is in stark contrast to heterosporous lycophytes and is correlated with a decelerated rate of nucleotide substitution. Together, the genomes of H. asiatica and D. complanatum not only fill a crucial gap in the plant genomic landscape but also highlight a potentially meaningful genomic contrast between homosporous and heterosporous species.

Plant organellar genomes: much done, much more to do.

Plastids and mitochondria are the only organelles that possess genomes of endosymbiotic origin. In recent decades, advances in sequencing technologies have contributed to a meteoric rise in the number of published organellar genomes, and have revealed greatly divergent evolutionary trajectories. In this review, we quantify the abundance and distribution of sequenced plant organellar genomes across the plant tree of life. We compare numerous genomic features between the two organellar genomes, with an emphasis on evolutionary trajectories, transfers, the current state of organellar genome editing by transcriptional activator-like effector nucleases (TALENs), transcription activator-like effector (TALE)-mediated deaminase, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), as well as genetic transformation. Finally, we propose future research to understand these different evolutionary trajectories, and genome-editing strategies to promote functional studies and eventually improve organellar genomes.

Comparative Plastomes of Curcuma alismatifolia (Zingiberaceae) Reveal Diversified Patterns among 56 Different Cut-Flower Cultivars.

Curcuma alismatifolia (Zingiberaceae) is an ornamental species with high economic value due to its recent rise in popularity among floriculturists. Cultivars within this species have mixed genetic backgrounds from multiple hybridization events and can be difficult to distinguish via morphological and histological methods alone. Given the need to improve identification resources, we carried out the first systematic study using plastomic data wherein genomic evolution and phylogenetic relationships from 56 accessions of C. alismatifolia were analyzed. The newly assembled plastomes were highly conserved and ranged from 162,139 bp to 164,111 bp, including 79 genes that code for proteins, 30 tRNA genes, and 4 rRNA genes. The A/T motif was the most common of SSRs in the assembled genomes. The Ka/Ks values of most genes were less than 1, and only two genes had Ka/Ks values above 1, which were rps15 (1.15), and ndhl (1.13) with petA equal to 1. The sequence divergence between different varieties of C. alismatifolia was large, and the percentage of variation in coding regions was lower than that in the non-coding regions. Such data will improve cultivar identification, marker assisted breeding, and preservation of germplasm resources.

A high-quality Bougainvillea genome provides new insights into evolutionary history and pigment biosynthetic pathways in the Caryophyllales.

Bougainvillea is a perennial ornamental shrub that is highly regarded in ornamental horticulture around the world. However, the absence of genome data limits our understanding of the pathways involved in bract coloration and breeding. Here, we report a chromosome-level assembly of the giga-genome of Bougainvillea × buttiana 'Mrs Butt', a cultivar thought to be the origin of many other Bougainvillea cultivars. The assembled genome is ~5 Gb with a scaffold N50 of 151 756 278 bp and contains 86 572 genes which have undergone recent whole-genome duplication. We confirmed that multiple rounds of whole-genome multiplication have occurred in the evolutionary history of the Caryophyllales, reconstructed the relationship in the Caryophyllales at whole genome level, and found discordance between species and gene trees as the result of complex introgression events. We investigated betalain and anthocyanin biosynthetic pathways and found instances of independent evolutionary innovations in the nine different Caryophyllales species. To explore the potential formation mechanism of diverse bract colors in Bougainvillea, we analyzed the genes involved in betalain and anthocyanin biosynthesis and found extremely low expression of ANS and DFR genes in all cultivars, which may limit anthocyanin biosynthesis. Our findings indicate that the expression pattern of the betalain biosynthetic pathway did not directly correlate with bract color, and a higher expression level in the betalain biosynthetic pathway is required for colored bracts. This improved understanding of the correlation between gene expression and bract color allows plant breeding outcomes to be predicted with greater certainty.

Chromosome-level genome assemblies from two sandalwood species provide insights into the evolution of the Santalales.

Sandalwood is one of the most expensive woods in the world and is well known for its long-lasting and distinctive aroma. In our study, chromosome-level genome assemblies for two sandalwood species (Santalum album and Santalum yasi) were constructed by integrating NGS short reads, RNA-seq, and Hi-C libraries with PacBio HiFi long reads. The S. album and S. yasi genomes were both assembled into 10 pseudochromosomes with a length of 229.59 Mb and 232.64 Mb, containing 21,673 and 22,816 predicted genes and a repeat content of 28.93% and 29.54% of the total genomes, respectively. Further analyses resolved a Santalum-specific whole-genome triplication event after divergence from ancestors of the Santalales lineage Malania, yet due to dramatic differences in transposon content, the Santalum genomes were only one-sixth the size of the Malania oleifera genome. Examination of RNA-seq data revealed a suite of genes that are differentially expressed in haustoria and might be involved in host hemiparasite interactions. The two genomes presented here not only provide an important comparative dataset for studying genome evolution in early diverging eudicots and hemiparasitic plants but will also hasten the application of conservation genomics for a lineage of trees recovering from decades of overexploitation.

The pan-plastome of tartary buckwheat (fagopyrum tataricum): key insights into genetic diversity and the history of lineage divergence.

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) is an important food and medicine crop plant, which has been cultivated for 4000 years. A nuclear genome has been generated for this species, while an intraspecific pan-plastome has yet to be produced. As such a detailed understanding of the maternal genealogy of Tartary buckwheat has not been thoroughly investigated. RESULTS: In this study, we de novo assembled 513 complete plastomes of Fagopyrum and compared with 8 complete plastomes of Fagopyrum downloaded from the NCBI database to construct a pan-plastome for F. tartaricum and resolve genomic variation. The complete plastomes of the 513 newly assembled Fagopyrum plastome sizes ranged from 159,253 bp to 159,576 bp with total GC contents ranged from 37.76 to 37.97%. These plastomes all maintained the typical quadripartite structure, consisting of a pair of inverted repeat regions (IRA and IRB) separated by a large single copy region (LSC) and a small single copy region (SSC). Although the structure and gene content of the Fagopyrum plastomes are conserved, numerous nucleotide variations were detected from which population structure could be resolved. The nucleotide variants were most abundant in the non-coding regions of the genome and of those the intergenic regions had the most. Mutational hotspots were primarily found in the LSC regions. The complete 521 Fagopyrum plastomes were divided into five genetic clusters, among which 509 Tartary buckwheat plastomes were divided into three genetic clusters (Ft-I/Ft-II/Ft-III). The genetic diversity in the Tartary buckwheat genetic clusters was the greatest in Ft-III, and the genetic distance between Ft-I and Ft-II was the largest. Based on the results of population structure and genetic diversity analysis, Ft-III was further subdivided into three subgroups Ft-IIIa, Ft-IIIb, and Ft-IIIc. Divergence time estimation indicated that the genera Fagopyrum and Rheum (rhubarb) shared a common ancestor about 48 million years ago (mya) and that intraspecies divergence in Tartary buckwheat began around 0.42 mya. CONCLUSIONS: The resolution of pan-plastome diversity in Tartary buckwheat provides an important resource for future projects such as marker-assisted breeding and germplasm preservation.

Variation in mitogenome structural conformation in wild and cultivated lineages of sorghum corresponds with domestication history and plastome evolution.

BACKGROUND: Mitochondria are organelles within eukaryotic cells that are central to the metabolic processes of cellular respiration and ATP production. However, the evolution of mitochondrial genomes (mitogenomes) in plants is virtually unknown compared to animal mitogenomes or plant plastids, due to complex structural variation and long stretches of repetitive DNA making accurate genome assembly more challenging. Comparing the structural and sequence differences of organellar genomes within and between sorghum species is an essential step in understanding evolutionary processes such as organellar sequence transfer to the nuclear genome as well as improving agronomic traits in sorghum related to cellular metabolism. RESULTS: Here, we assembled seven sorghum mitochondrial and plastid genomes and resolved reticulated mitogenome structures with multilinked relationships that could be grouped into three structural conformations that differ in the content of repeats and genes by contig. The grouping of these mitogenome structural types reflects the two domestication events for sorghum in east and west Africa. CONCLUSIONS: We report seven mitogenomes of sorghum from different cultivars and wild sources. The assembly method used here will be helpful in resolving complex genomic structures in other plant species. Our findings give new insights into the structure of sorghum mitogenomes that provides an important foundation for future research into the improvement of sorghum traits related to cellular respiration, cytonuclear incompatibly, and disease resistance.

The genomic and bulked segregant analysis of Curcuma alismatifolia revealed its diverse bract pigmentation.

Compared with most flowers where the showy part comprises specialized leaves (petals) directly subtending the reproductive structures, most Zingiberaceae species produce showy "flowers" through modifications of leaves (bracts) subtending the true flowers throughout an inflorescence. Curcuma alismatifolia, belonging to the Zingiberaceae family, a plant species originating from Southeast Asia, has become increasingly popular in the flower market worldwide because of its varied and esthetically pleasing bracts produced in different cultivars. Here, we present the chromosome-scale genome assembly of C. alismatifolia "Chiang Mai Pink" and explore the underlying mechanisms of bract pigmentation. Comparative genomic analysis revealed C. alismatifolia contains a residual signal of whole-genome duplication. Duplicated genes, including pigment-related genes, exhibit functional and structural differentiation resulting in diverse bract colors among C. alismatifolia cultivars. In addition, we identified the key genes that produce different colored bracts in C. alismatifolia, such as F3'5'H, DFR, ANS and several transcription factors for anthocyanin synthesis, as well as chlH and CAO in the chlorophyll synthesis pathway by conducting transcriptomic analysis, bulked segregant analysis using both DNA and RNA data, and population genomic analysis. This work provides data for understanding the mechanism of bract pigmentation and will accelerate breeding in developing novel cultivars with richly colored bracts in C. alismatifolia and related species. It is also important to understand the variation in the evolution of the Zingiberaceae family. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00081-6.

Evaluation of Intracellular Gene Transfers from Plastome to Nuclear Genome across Progressively Improved Assemblies for Arabidopsis thaliana and Oryza sativa.

DNA originating from organellar genomes are regularly discovered in nuclear sequences during genome assembly. Nevertheless, such insertions are sometimes omitted during the process of nuclear genome assembly because the inserted DNA is assigned to organellar genomes, leading to a systematic underestimation of their frequency. With the rapid development of high-throughput sequencing technology, more inserted fragments from organelle genomes can now be detected. Therefore, it is necessary to be aware of the insertion events from organellar genomes during nuclear genome assembly to properly attribute the impact and rate of such insertions in the evolution of nuclear genomes. Here, we investigated the impact of intracellular gene transfer (IGT) from the plastome to the nuclear genome using genome assemblies that were refined through time with technological improvements from two model species, Arabidopsis thaliana and Oryza sativa. We found that IGT from the plastome to the nuclear genome is a dynamic and ongoing process in both A. thaliana and O. sativa, and mostly occurred recently, as the majority of transferred sequences showed over 95% sequence similarity with plastome sequences of origin. Differences in the plastome-to-nuclear genome IGT between A. thaliana and O. sativa varied among the different assembly versions and were associated with the quality of the nuclear genome assembly. IGTs from the plastome to nuclear genome occurred more frequently in intergenic regions, which were often associated with transposable elements (TEs). This study provides new insights into intracellular genome evolution and nuclear genome assembly by characterizing and comparing IGT from the plastome into the nuclear genome for two model plant species.

The Roles of Mutation and Selection Acting on Mitochondrial Genomes Inferred from Intraspecific Variation in Seed Plants.

There is a paradox in the plant mitochondrial genome, that is, the genic region evolves slowly while the intergenic region evolves rapidly. Thus, the intergenic regions of the plant mitochondrial genome are difficult to align across different species, even in closely related species. Here, to character the mechanism of this paradox, we identified interspecific variations in the Ginkgo biloba, Oryza sativa, and Arabidopsis thaliana mitochondrial and plastid genome at a genome-wide level. The substitution rate of synonymous sites in genic regions was similar to the substitution rate of intergenic regions, while the substitution rate of nonsynonymous sites in genic regions was lower than that in intergenic regions, suggesting the mutation inputs were the same among different categories within the organelle genome, but the selection pressure varied. The substitution rate of single-copy regions was higher than that of IR (inverted repeats) in the plastid genome at an intraspecific level. The substitution rate of single-copy regions was higher than that of repeats in the G. biloba and A. thaliana mitochondrial genomes, but lower in that of O. sativa. This difference may be related to the length and distribution of repeats. Copy number variations that existed in the G. biloba and O. sativa mitochondrial genomes were confirmed. This study reveals the intraspecific variation pattern of organelle genomes at a genome-wide level, and that copy number variations were common in plant mitochondrial genomes.

A Chromosome-Level Genome of the Camphor Tree and the Underlying Genetic and Climatic Factors for Its Top-Geoherbalism.

Camphor tree [Cinnamomum camphora (L.) J. Presl], a species in the magnoliid family Lauraceae, is known for its rich volatile oils and is used as a medical cardiotonic and as a scent in many perfumed hygiene products. Here, we present a high-quality chromosome-scale genome of C. camphora with a scaffold N50 of 64.34 Mb and an assembled genome size of 755.41 Mb. Phylogenetic inference revealed that the magnoliids are a sister group to the clade of eudicots and monocots. Comparative genomic analyses identified two rounds of ancient whole-genome duplication (WGD). Tandem duplicated genes exhibited a higher evolutionary rate, a more recent evolutionary history and a more clustered distribution on chromosomes, contributing to the production of secondary metabolites, especially monoterpenes and sesquiterpenes, which are the principal essential oil components. Three-dimensional analyses of the volatile metabolites, gene expression and climate data of samples with the same genotype grown in different locations showed that low temperature and low precipitation during the cold season modulate the expression of genes in the terpenoid biosynthesis pathways, especially TPS genes, which facilitates the accumulation of volatile compounds. Our study lays a theoretical foundation for policy-making regarding the agroforestry applications of camphor tree.

Integrated Metabolome and Transcriptome Analyses Reveal Dissimilarities in the Anthocyanin Synthesis Pathway Between Different Developmental Leaf Color Transitions in Hopea hainanensis (Dipterocarpaceae).

Changes in plant leaf color during development are directly related to the accumulation or degradation of certain phytochemicals such as anthocyanins. Since some anthocyanins can be beneficial to human health and provide insights into the biology of leaves, the underlying processes and timing by which plants produce these molecules has been the focus of numerous studies. The tree species Hopea hainanensis generally produces green leaves at all growth stages; however, a few explored individuals have been identified possessing red leaves on the top of the seedlings at a young stage. While the phenomenon of leaf color varying with age has been studied in several species, the underlying mechanisms are largely unknown in H. hainanensis. Using a metabolomics approach, the young red leaves in H. hainanensis were found to contain higher levels of anthocyanins and flavonoids than the young green-leaved individuals. Among anthocyanins, pelargonidin and cyanidin were the most likely candidates contributing to the red color of the young leaves. Transcriptome results indicated the genes related to the production of these anthocyanins were significantly upregulated, leading to greater accumulation of red pigments. Specifically, the expression of several MYB and bHLH genes in young red leaf lines was significantly higher than that in the young green leaf lines, especially HhMYB66, HhMYB91, HhMYB6, and HhbHLH70. As such these four transcription factors are probably the main regulatory genes resulting in young red leaves in H. hainanensis. From these results, comparative analyses with other species can be made to better understand the evolution of pigment biosynthesis and how anthocyanins function in plant metabolism and evolution/adaptation.

Integrated multi-omic data and analyses reveal the pathways underlying key ornamental traits in carnation flowers.

Carnation (Dianthus caryophyllus) is one of the most popular ornamental flowers in the world. Although numerous studies on carnations exist, the underlying mechanisms of flower color, fragrance, and the formation of double flowers remain unknown. Here, we employed an integrated multi-omics approach to elucidate the genetic and biochemical pathways underlying the most important ornamental features of carnation flowers. First, we assembled a high-quality chromosome-scale genome (636 Mb with contig N50 as 14.67 Mb) of D. caryophyllus, the 'Scarlet Queen'. Next, a series of metabolomic datasets was generated with a variety of instrumentation types from different parts of the flower at multiple stages of development to assess spatial and temporal differences in the accumulation of pigment and volatile compounds. Finally, transcriptomic data were generated to link genomic, biochemical, and morphological patterns to propose a set of pathways by which ornamental traits such as petal coloration, double flowers, and fragrance production are formed. Among them, the transcription factors bHLHs, MYBs, and a WRKY44 homolog are proposed to be important in controlling petal color patterning and genes such as coniferyl alcohol acetyltransferase and eugenol synthase are involved in the synthesis of eugenol. The integrated dataset of genomics, transcriptomics, and metabolomics presented herein provides an important foundation for understanding the underlying pathways of flower development and coloration, which in turn can be used for selective breeding and gene editing for the development of novel carnation cultivars.

A chromosome-scale genome and transcriptomic analysis of the endangered tropical tree Vatica mangachapoi (Dipterocarpaceae).

Vatica mangachapoi is a tropical tree species native to Southeast Asia. It has long been valued as a timber species because the wood resists decay, but it is now considered vulnerable to extinction due to habitat loss and overexploitation. Here, we present the first chromosome-level genome assembly of V. mangachapoi that we created by combining data from PacBio long read sequencing with Hi-C proximity ligation and Illumina short-read sequencing. The assembled genome was 456.21 Mb, containing 11 chromosome and a BUSCO score of 93.4%. From the newly assembled genome, 46,811 protein-coding genes were predicted. Repetitive DNA accounted for 53% of the genome. Phylogenomic and gene family analyses showed that V. mangachapoi diverged from a common ancestor of Gossypium raimondii 70 million years ago. Transcriptome analyses found 227 genes that were differentially expressed in the leaves of plants grown in normal soil relative to plants grown in dry, coastal, sandy soil. For these genes, we identified three significantly enriched with GO terms: responses to organonitrogen compounds, chitin-triggered immunity, and wound response. This genome provides an important comparative benchmark not only for future conservation work on V. mangachapoi but also for phylogenomics work on Dipterocarpaceae.

A complete mitochondrial genome for fragrant Chinese rosewood (Dalbergia odorifera, Fabaceae) with comparative analyses of genome structure and intergenomic sequence transfers.

BACKGROUND: Dalbergia odorifera is an economically and culturally important species in the Fabaceae because of the high-quality lumber and traditional Chinese medicines made from this plant, however, overexploitation has increased the scarcity of D. odorifera. Given the rarity and the multiple uses of this species, it is important to expand the genomic resources for utilizing in applications such as tracking illegal logging, determining effective population size of wild stands, delineating pedigrees in marker assisted breeding programs, and resolving gene networks in functional genomics studies. Even the nuclear and chloroplast genomes have been published for D. odorifera, the complete mitochondrial genome has not been assembled or assessed for sequence transfer to other genomic compartments until now. Such work is essential in understanding structural and functional genome evolution in a lineage (Fabaceae) with frequent intergenomic sequence transfers. RESULTS: We integrated Illumina short-reads and PacBio CLR long-reads to assemble and annotate the complete mitochondrial genome of D. odorifera. The mitochondrial genome was organized as a single circular structure of 435 Kb in length containing 33 protein coding genes, 4 rRNA and 17 tRNA genes. Nearly 4.0% (17,386 bp) of the genome was annotated as repetitive DNA. From the sequence transfer analysis, it was found that 114 Kb of DNA originating from the mitochondrial genome has been transferred to the nuclear genome, with most of the transfer events having taken place relatively recently. The high frequency of sequence transfers from the mitochondria to the nuclear genome was similar to that of sequence transfer from the chloroplast to the nuclear genome. CONCLUSION: For the first-time, the complete mitochondrial genome of D. odorifera was assembled in this study, which will provide a baseline resource in understanding genomic evolution in the highly specious Fabaceae. In particular, the assessment of intergenomic sequence transfer suggests that transfers have been common and recent indicating a possible role in environmental adaptation as has been found in other lineages. The high turnover rate of genomic colinearly and large differences in mitochondrial genome size found in the comparative analyses herein providing evidence for the rapid evolution of mitochondrial genome structure compared to chloroplasts in Faboideae. While phylogenetic analyses using functional genes indicate that mitochondrial genes are very slowly evolving compared to chloroplast genes.

Chloroplast genomes in Populus (Salicaceae): comparisons from an intensively sampled genus reveal dynamic patterns of evolution.

The chloroplast is one of two organelles containing a separate genome that codes for essential and distinct cellular functions such as photosynthesis. Given the importance of chloroplasts in plant metabolism, the genomic architecture and gene content have been strongly conserved through long periods of time and as such are useful molecular tools for evolutionary inferences. At present, complete chloroplast genomes from over 4000 species have been deposited into publicly accessible databases. Despite the large number of complete chloroplast genomes, comprehensive analyses regarding genome architecture and gene content have not been conducted for many lineages with complete species sampling. In this study, we employed the genus Populus to assess how more comprehensively sampled chloroplast genome analyses can be used in understanding chloroplast evolution in a broadly studied lineage of angiosperms. We conducted comparative analyses across Populus in order to elucidate variation in key genome features such as genome size, gene number, gene content, repeat type and number, SSR (Simple Sequence Repeat) abundance, and boundary positioning between the four main units of the genome. We found that some genome annotations were variable across the genus owing in part from errors in assembly or data checking and from this provided corrected annotations. We also employed complete chloroplast genomes for phylogenetic analyses including the dating of divergence times throughout the genus. Lastly, we utilized re-sequencing data to describe the variations of pan-chloroplast genomes at the population level for P. euphratica. The analyses used in this paper provide a blueprint for the types of analyses that can be conducted with publicly available chloroplast genomes as well as methods for building upon existing datasets to improve evolutionary inference.

Comparative Plastome Analysis of Root- and Stem-Feeding Parasites of Santalales Untangle the Footprints of Feeding Mode and Lifestyle Transitions.

In plants, parasitism triggers the reductive evolution of plastid genomes (plastomes). To disentangle the molecular evolutionary associations between feeding on other plants below- or aboveground and general transitions from facultative to obligate parasitism, we analyzed 34 complete plastomes of autotrophic, root- and stem-feeding hemiparasitic, and holoparasitic Santalales. We observed inexplicable losses of housekeeping genes and tRNAs in hemiparasites and dramatic genomic reconfiguration in holoparasitic Balanophoraceae, whose plastomes have exceptionally low GC contents. Genomic changes are related primarily to the evolution of hemi- or holoparasitism, whereas the transition from a root- to a stem-feeding mode plays no major role. In contrast, the rate of molecular evolution accelerates in a stepwise manner from autotrophs to root- and then stem-feeding parasites. Already the ancestral transition to root-parasitism coincides with a relaxation of selection in plastomes. Another significant selectional shift in plastid genes occurs as stem-feeders evolve, suggesting that this derived form coincides with trophic specialization despite the retention of photosynthetic capacity. Parasitic Santalales fill a gap in our understanding of parasitism-associated plastome degeneration. We reveal that lifestyle-genome associations unfold interdependently over trophic specialization and feeding mode transitions, where holoparasitic Balanophoraceae provide a system for exploring the functional realms of plastomes.