Comparative transcriptome analysis reveals candidate genes for cold stress response and early flowering in pineapple.

Pineapple originates from tropical regions in South America and is therefore significantly impacted by cold stress. Periodic cold events in the equatorial regions where pineapple is grown may induce early flowering, also known as precocious flowering, resulting in monetary losses due to small fruit size and the need to make multiple passes for harvesting a single field. Currently, pineapple is one of the most important tropical fruits in the world in terms of consumption, and production losses caused by weather can have major impacts on worldwide exportation potential and economics. To further our understanding of and identify mechanisms for low-temperature tolerance in pineapple, and to identify the relationship between low-temperature stress and flowering time, we report here a transcriptomic analysis of two pineapple genotypes in response to low-temperature stress. Using meristem tissue collected from precocious flowering-susceptible MD2 and precocious flowering-tolerant Dole-17, we performed pairwise comparisons and weighted gene co-expression network analysis (WGCNA) to identify cold stress, genotype, and floral organ development-specific modules. Dole-17 had a greater increase in expression of genes that confer cold tolerance. The results suggested that low temperature stress in Dole-17 plants induces transcriptional changes to adapt and maintain homeostasis. Comparative transcriptomic analysis revealed differences in cuticular wax biosynthesis, carbohydrate accumulation, and vernalization-related gene expression between genotypes. Cold stress induced changes in ethylene and abscisic acid-mediated pathways differentially between genotypes, suggesting that MD2 may be more susceptible to hormone-mediated early flowering. The differentially expressed genes and module hub genes identified in this study are potential candidates for engineering cold tolerance in pineapple to develop new varieties capable of maintaining normal reproduction cycles under cold stress. In addition, a total of 461 core genes involved in the development of reproductive tissues in pineapple were also identified in this study. This research provides an important genomic resource for understanding molecular networks underlying cold stress response and how cold stress affects flowering time in pineapple.

Identification of bromelain subfamily proteases encoded in the pineapple genome.

Papain (aka C1A) family proteases, including bromelain enzymes, are widespread across the plant kingdom and play critical regulatory functions in protein turnover during development. The proteolytic activity exhibited by papain family proteases has led to their increased usage for a wide range of cosmetic, therapeutic, and medicinal purposes. Bromelain enzymes, or bromelains in short, are members of the papain family that are specific to the bromeliad plant family. The only major commercial extraction source of bromelain is pineapple. The importance of C1A family and bromelain subfamily proteases in pineapple development and their increasing economic importance led several researchers to utilize available genomic resources to identify protease-encoding genes in the pineapple genome. To date, studies are lacking in screening bromelain genes for targeted use in applied science studies. In addition, the bromelain genes coding for the enzymes present in commercially available bromelain products have not been identified and their evolutionary origin has remained unclear. Here, using the newly developed MD2 v2 pineapple genome, we aimed to identify bromelain-encoding genes and elucidate their evolutionary origin. Orthologous and phylogenetic analyses of all papain-family proteases encoded in the pineapple genome revealed a single orthogroup (189) and phylogenetic clade (XIII) containing the bromelain subfamily. Duplication mode and synteny analyses provided insight into the origin and expansion of the bromelain subfamily in pineapple. Proteomic analysis identified four bromelain enzymes present in two commercially available bromelain products derived from pineapple stem, corresponding to products of four putative bromelain genes. Gene expression analysis using publicly available transcriptome data showed that 31 papain-family genes identified in this study were up-regulated in specific tissues, including stem, fruit, and floral tissues. Some of these genes had higher expression in earlier developmental stages of different tissues. Similar expression patterns were identified by RT-qPCR analysis with leaf, stem, and fruit. Our results provide a strong foundation for future applicable studies on bromelain, such as transgenic approaches to increase bromelain content in pineapple, development of bromelain-producing bioreactors, and studies that aim to determine the medicinal and/or therapeutic viability of individual bromelain enzymes.

Population Genomic Analyses Suggest a Hybrid Origin, Cryptic Sexuality, and Decay of Genes Regulating Seed Development for the Putatively Strictly Asexual Kingdonia uniflora (Circaeasteraceae, Ranunculales).

Asexual lineages are perceived to be short-lived on evolutionary timescales. Hence, reports for exceptional cases of putative 'ancient asexuals' usually raise questions about the persistence of such species. So far, there have been few studies to solve the mystery in plants. The monotypic Kingdonia dating to the early Eocene, contains only K. uniflora that has no known definitive evidence for sexual reproduction nor records for having congeneric sexual species, raising the possibility that the species has persisted under strict asexuality for a long period of time. Here, we analyze whole genome polymorphism and divergence in K. uniflora. Our results show that K. uniflora is characterized by high allelic heterozygosity and elevated πN/πS ratio, in line with theoretical expectations under asexual evolution. Allele frequency spectrum analysis reveals the origin of asexuality in K. uniflora occurred prior to lineage differentiation of the species. Although divergence within K. uniflora individuals exceeds that between populations, the topologies of the two haplotype trees, however, fail to match each other, indicating long-term asexuality is unlikely to account for the high allele divergence and K. uniflora may have a recent hybrid origin. Phi-test shows a statistical probability of recombination for the conflicting phylogenetic signals revealed by the split network, suggesting K. uniflora engages in undetected sexual reproduction. Detection of elevated genetic differentiation and premature stop codons (in some populations) in genes regulating seed development indicates mutational degradation of sexuality-specific genes in K. uniflora. This study unfolds the origin and persistence mechanism of a plant lineage that has been known to reproduce asexually and presents the genomic consequences of lack of sexuality.

An updated phylogeny, biogeography, and PhyloCode-based classification of Cornaceae based on three sets of genomic data.

PREMISE: A major goal of systematic biology is to uncover the evolutionary history of organisms and translate that knowledge into stable classification systems. Here, we integrate three sets of genome-wide data to resolve phylogenetic relationships in Cornaceae (containing only Cornus s.l.), reconstruct the biogeographic history of the clade, and provide a revised classification using the PhyloCode to stabilize names for this taxonomically controversial group. METHODS: We conducted phylogenetic analyses using 312 single-copy nuclear genes and 70 plastid genes from Angiosperms353 Hyb-Seq, plus numerous loci from RAD-Seq. We integrated fossils using morphological data and produced a dated phylogeny for biogeographical analysis. RESULTS: A well-resolved, strongly supported, comprehensive phylogeny was obtained. Biogeographic analyses support an origin and rapid diversification of Cornus into four morphologically distinct major clades in the Northern Hemisphere (with an eastern Asian ancestor) during the late Cretaceous. Dispersal into Africa from eastern Asia likely occurred along the Tethys Seaway during the Paleogene, whereas dispersal into South America likely occurred during the Neogene. Diversification within the northern hemisphere likely involved repeated independent colonization of new areas during the Paleogene and Neogene along the Bering Land Bridge, the North Atlantic Land Bridge, and the Tethys Seaway. Thirteen strongly supported clades were named following rules of the PhyloCode. CONCLUSIONS: Our study provides an example of integrating genomic and morphological data to produce a robust, explicit species phylogeny that includes fossil taxa, which we translate into an updated classification scheme using the PhyloCode to stabilize names.

Foliar endophyte diversity in Eastern Asian-Eastern North American disjunct tree species - influences of host identity, environment, phylogeny, and geographic isolation.

Introduction: The well-known eastern Asian (EA) and eastern North American (ENA) floristic disjunction provides a unique system for biogeographic and evolutionary studies. Despite considerable interest in the disjunction, few studies have investigated the patterns and their underlying drivers of allopatric divergence in sister species or lineages isolated in the two areas. Endophyte diversity and assembly in disjunct sister taxa, as an ecological trait, may have played an important role in the processes of allopatric evolution, but no studies have examined endophytes in these lineages. Here we compared foliar endophytic fungi and bacteria-archaea (FEF and FEB) in 17 EA-ENA disjunct species or clade pairs from genera representing conifers and 10 orders of five major groups of angiosperms and 23 species of Cornus from EA and North America. Methods: Metagenomic sequencing of fungal ITS and bacterial-archaeal 16S rDNA was used to capture the foliar endophytic communities. Alpha and beta diversity of fungi and bacteria were compared at multiple scales and dimensions to gain insights into the relative roles of historical geographic isolation, host identity, phylogeny, and environment from samples at different sites in shaping endophytic diversity patterns. Results: We found that beta diversity of endophytes varied greatly among plant individuals within species and between species among genera at the same sampling site, and among three sampling sites, but little variation between region-of-origin of all plant species (EA vs ENA) and between EA-ENA disjunct counterparts within genera. Various numbers of indicator fungal species differing in abundance were identified for each plant genus and Cornus species. An overall significant correlation between endophyte community dissimilarity and phylogenetic distance of plants was detected among the disjunct genera but not among species of Cornus. However, significant correlations between beta diversities at different taxonomic scales of endophytes and phylogenetic distances of Cornus species were observed. Discussion: Our results suggest important roles of host identity and environment (sampling sites), and a likely minor role of phylogenetic divergence and historical biogeographic isolation in shaping the pattern of foliar endophyte diversity and assembly in the EA-ENA disjunct genera and Cornus. The results lead to a hypothesis that the sister taxa in EA and ENA likely differ in FEF and FEB when growing in native habitats due to differences in local environments, which may potentially drive allopatric divergence of the functional features of species.

Metagenomic study reveals hidden relationships among fungal diversity, variation of plant disease, and genetic distance in Cornus florida (Cornaceae).

Introduction: Understanding patterns of plant-microbe interactions across plant species and populations is a critical yet poorly characterized aspect in the field of plant pathology. Microbial DNA sequences present as contaminants in omics data of plants obtained using next-generation sequencing methods provide a valuable source to explore the relationships among endophytic microbial diversity, disease and genetic differentiation of host plants, and environmental variation, but few such studies have been conducted. The flowering dogwood tree (Cornus florida L.), an ecologically important species in North America, is threatened by powdery mildew and dogwood anthracnose diseases, and knowledge of the microbial diversity harbored within genetically and environmental distinct populations of this species remains largely unknown. Methods: We conducted a metagenomics study utilizing the sequences of RAD-tag/genotype-by-sequence libraries from leaf tissues of C. florida to examine such host-fungus interactions across the dogwood's US range. We performed various combinations of alignments to both host and pathogen genomes to obtain filtered sets sequences for metagenomics analysis. Taxonomic assignments were determined on each filtered set of sequences, followed by estimation of microbial diversity and correlation to environment and host-genetic variation. Results: Our data showed that microbial community composition significantly differed between visually healthy and diseased sites. Several microbial taxa known to interact with dogwood were identified from these sequences. We found no correlation between microbial diversity and relative abundances of sequences aligning to draft genomes of either pathogen causing powdery mildew or dogwood anthracnose. We found a significant relationship between differences of fungal communities and geographic distances of plant populations, suggesting roles of environments in shaping fungal communities in leaf tissues. Significant correlations between the genetic differentiation of plant samples and fungal community dissimilarity (beta diversity) were also observed in certain sets of our analyses-suggesting the possibility of a relationship between microbial community composition and plant genetic distance. This relationship persisted in significance even after controlling for significant effects of geographic-bioclimatic variation of microbial diversity. Discussion: Our results suggest that both genetics and the environment play a significant role in shaping foliar fungal communities. Our findings underscore the power of leveraging hidden microbial sequences within datasets originally collected for plant genetic studies to understand plant-pathogen interactions.

Phylogenomics AND biogeography of Castanea (chestnut) and Hamamelis (witch-hazel) - Choosing between RAD-seq and Hyb-Seq approaches.

Hyb-Seq and RAD-seq are well-established high throughput sequencing technologies that have been increasingly used for plant phylogenomic studies. Each method has its own pros and cons. The choice between them is a practical issue for plant systematists studying the evolutionary histories of biodiversity of relatively recent origins. However, few studies have compared the congruence and conflict between results from the two methods within the same group of organisms in plants. In this study, we employed RAD-seq and Hyb-Seq of Angiosperms353 genes in phylogenomic and biogeographic studies of Hamamelis (the witch-hazels) and Castanea (chestnuts), two classic examples exhibiting the well-known eastern Asian (EA) -eastern North American (ENA) disjunct distribution, and compared them side by side. Our results showed congruences in phylogenetic inference and divergence time dating between the two data sets obtained through our customized procedures of library preparation and sequence trimming, although they differed in the number of loci and informative sites, the amount of missing data, and sampling within species. We provide recommendations regarding the selection of the two methods for phylogenomic study at generic level based on fund availability and sampling scale. If funds and time are not constrained, we recommend Hyb-Seq. If funds and time are somewhat limited and sampling is large, we recommend RAD-seq. However, we found greater conflict among gene trees from the RAD-seq data due to the short sequences per locus. Therefore, species tree building and network detecting with the RAD-seq data with short RAD-seq loci (e.g., <150 bp) should avoid using analytical methods relying on gene trees of individual locus, but using site-based methods such as SVDQuartets and D-statistic method. Our phylogenetic analyses of RAD-seq and Hyb-Seq data resulted in well-resolved species relationships. Analyses of the data using the D-statistic test and PhyloNet revealed ancient introgressions in both genera. Biogeographic analyses including fossil data using total evidence-based dated tree and DEC model applying specific inter-area dispersal probabilities revealed a complicated history for each genus, indicating multiple interareal dispersals and local extinctions within and outside areas of the taxa's modern ranges in both the Paleogene and Neogene. The study demonstrates the importance of including fossil taxa for a more accurate reconstruction of biogeographic histories of taxa to understand the EA and ENA floristic disjunction. Our results support a widespread ancestral range in EA-western North America (WNA) followed by early diversification in EA and expansion to North America (NA) and Europe for Castanea and a more widespread ancestral range in EA-ENA-WNA for Hamamelis. The origins of the modern EA-ENA disjunction in both genera were suggested to be the result of vicariance from widespread ancestors in Eurasia-ENA of the mid-Miocene and in EA-NA of the late Oligocene, respectively.

Next Steps in Integrative Biology: Mapping Interactive Processes Across Levels of Biological Organization.

Emergent biological processes result from complex interactions within and across levels of biological organization, ranging from molecular to environmental dynamics. Powerful theories, database tools, and modeling methods have been designed to characterize network connections within levels, such as those among genes, proteins, biochemicals, cells, organisms, and species. Here, we propose that developing integrative models of organismal function in complex environments can be facilitated by taking advantage of these methods to identify key nodes of communication across levels of organization. Mapping key drivers or connections among levels of organization will provide data and leverage to model potential rule-sets by which organisms respond and adjust to perturbations at any level of biological organization.

A New Pipeline for Removing Paralogs in Target Enrichment Data.

Target enrichment (such as Hyb-Seq) is a well-established high throughput sequencing method that has been increasingly used for phylogenomic studies. Unfortunately, current widely used pipelines for analysis of target enrichment data do not have a vigorous procedure to remove paralogs in target enrichment data. In this study, we develop a pipeline we call Putative Paralogs Detection (PPD) to better address putative paralogs from enrichment data. The new pipeline is an add-on to the existing HybPiper pipeline, and the entire pipeline applies criteria in both sequence similarity and heterozygous sites at each locus in the identification of paralogs. Users may adjust the thresholds of sequence identity and heterozygous sites to identify and remove paralogs according to the level of phylogenetic divergence of their group of interest. The new pipeline also removes highly polymorphic sites attributed to errors in sequence assembly and gappy regions in the alignment. We demonstrated the value of the new pipeline using empirical data generated from Hyb-Seq and the Angiosperms353 kit for two woody genera Castanea (Fagaceae, Fagales) and Hamamelis (Hamamelidaceae, Saxifragales). Comparisons of data sets showed that the PPD identified many more putative paralogs than the popular method HybPiper. Comparisons of tree topologies and divergence times showed evident differences between data from HybPiper and data from our new PPD pipeline. We further evaluated the accuracy and error rates of PPD by BLAST mapping of putative paralogous and orthologous sequences to a reference genome sequence of Castanea mollissima. Compared to HybPiper alone, PPD identified substantially more paralogous gene sequences that mapped to multiple regions of the reference genome (31 genes for PPD compared with 4 genes for HybPiper alone). In conjunction with HybPiper, paralogous genes identified by both pipelines can be removed resulting in the construction of more robust orthologous gene data sets for phylogenomic and divergence time analyses. Our study demonstrates the value of Hyb-Seq with data derived from the Angiosperms353 probe set for elucidating species relationships within a genus, and argues for the importance of additional steps to filter paralogous genes and poorly aligned regions (e.g., as occur through assembly errors), such as our new PPD pipeline described in this study. [Angiosperms353; Castanea; divergence time; Hamamelis; Hyb-Seq, paralogs, phylogenomics.].

Genetic insights into the evolution of genera with the eastern Asia-eastern North America floristic disjunction: a transcriptomics analysis.

PREMISE: Large disjunctions in species distributions provide excellent opportunities to study processes that shape biogeographic patterns. One such disjunction is the eastern Asia-eastern North America (EA-ENA) floristic disjunction. For many genera with this disjunction, species richness is greater in EA than in ENA; this pattern has been attributed, in part, to higher rates of molecular evolution and speciation in EA. Longer branch lengths have been found in some EA clades, relative to their ENA sister clades, suggesting that the EA lineages have evolved at a higher rate, possibly due to environmental heterogeneity, potentially contributing to the species richness anomaly. METHODS: To evaluate whether rates of molecular evolution are elevated in EA relative to ENA, we used transcriptomes from species in 11 genera displaying this disjunction. Rates of molecular evolution were estimated for up to 385 orthologous nuclear loci per genus. RESULTS: No statistically significant differences were identified in pairwise comparisons between EA and ENA sister species, suggesting equal rates of molecular evolution for both species; the data also suggest similar selection pressures in both regions. For larger genera, evidence likewise argues against more species-rich clades having higher molecular evolutionary rates, regardless of region. Our results suggest that genes across multiple gene ontology categories are evolving at similar rates under purifying selection in species in both regions. CONCLUSIONS: Our data support the hypothesis that greater species richness in EA than ENA is due to factors other than an overall increase in rates of molecular evolution in EA.

Phylogenomics, co-evolution of ecological niche and morphology, and historical biogeography of buckeyes, horsechestnuts, and their relatives (Hippocastaneae, Sapindaceae) and the value of RAD-Seq for deep evolutionary inferences back to the Late Cretaceous.

In this study, we used RAD-seq data to resolve the phylogeny of the tribe Hippocastaneae (Sapindaceae) and conducted comparative analyses to gain insights into the evolution and biogeography of the group that had fossils dating back to the late Cretaceous. Hippocastaneae, including the horsechestnuts and buckeyes, is a well-supported clade in Sapindaceae that comprises 12-14 species in Aesculus, two in Billia, and one in Handeliodendron. Most species in the tribe are distributed in Eurasia and North America and exhibit a classic pattern of intercontinental disjunction in the Northern Hemisphere, while Billia occurs from southern Mexico to northern South America. The earliest fossils of Aesculus date back to at least the earliest Paleocene of eastern Asia and western North America, where there are also putative occurrences from the latest Cretaceous. The group provides an excellent system for understanding floristic disjunction in the Northern Hemisphere extending to the Neotropics. However, a strongly supported and well resolved phylogeny is presently lacking for the tribe. Previous phylogenetic studies using several gene regions revealed five well-supported clades in Aesculus, largely corresponding to five recognized taxonomic sections, but relationships among these clades and among Aesculus, Billia, and Handeliodendron were not well supported. In this study, we used RAD-seq data from 68 samples representing all clades and species of Hippocastaneae except Billia, for which we used one of two species, to further resolve relationships within the tribe. Our phylogenomic analyses showed strong support for a sister relationship between Aesculus and Handeliodendron, in contrast to previous findings which supported Billia as sister to Aesculus. Within Aesculus, relationships among sections were strongly supported as (sect. Calothyrsus, (sect. Aesculus, (sect. Macrothyrsus, (sect. Parryana, sect. Pavia)))). We found that the traditionally recognized section Calothyrsus was monophyletic, with all eastern Asian species sister to the western North American species, A. californica. Analyses of divergence times combined with biogeographic analyses suggested a Late Cretaceous origin of Hippocastaneae, in eastern Asia, western North America, and Central America (including southern Mexico), followed by isolation of Billia in Central America, extinction of the tribe ancestor in western North America, and divergence of Aesculus from Handeliodendron in eastern Asia. A Late Cretaceous origin of the common ancestor of Aesculus in eastern Asia was followed by dispersals into western North America, Europe, and eastern North America during the Late Cretaceous and the Paleogene. Our results support Aesculus as a relic of the boreotropical flora and subsequent intercontinental spread of the genus through the Bering and North Atlantic land bridges. We performed character mapping analyses, which revealed that biogeographic isolation and niche divergence may have played important roles in driving morphological evolution and lineage divergence in Aesculus. Our study demonstrates the value of RAD-seq data for reconstructing phylogeny back to the Late Cretaceous.

Natural selection and repeated patterns of molecular evolution following allopatric divergence.

Although geographic isolation is a leading driver of speciation, the tempo and pattern of divergence at the genomic level remain unclear. We examine genome-wide divergence of putatively single-copy orthologous genes (POGs) in 20 allopatric species/variety pairs from diverse angiosperm clades, with 16 pairs reflecting the classic eastern Asia-eastern North America floristic disjunction. In each pair, >90% of POGs are under purifying selection, and <10% are under positive selection. A set of POGs are under strong positive selection, 14 of which are shared by 10-15 pairs, and one shared by all pairs; 15 POGs are annotated to biological processes responding to various stimuli. The relative abundance of POGs under different selective forces exhibits a repeated pattern among pairs despite an ~10 million-year difference in divergence time. Species divergence times are positively correlated with abundance of POGs under moderate purifying selection, but negatively correlated with abundance of POGs under strong purifying selection.

Plastid phylogenomics and biogeographic analysis support a trans-Tethyan origin and rapid early radiation of Cornales in the Mid-Cretaceous.

The Cornales is a relatively small but morphologically diverse order in the basal position of the Asterids clade. Previous study hypothesized that the order might have undergone ancient rapid radiation during the Cretaceous when major angiosperm lineages were established. We conducted the phylogenomic analysis of Cornales using 81 plastid genome sequences with 67 newly generated in this study to test the hypothesis. This sampling represents all the families and 31 out of 48 genera in the order. Phylogenetic analyses were conducted using different datasets to examine the effects of different coding positions and character coding methods. We further conducted divergence time, diversification rate, and biogeographic analyses to understand the early evolutionary history of Cornales in space and time. Our phylogenetic analyses of four datasets (the amino acid characters, the 1st and 2nd codon positions of protein coding genes, nucleotide characters with degenerated coding method, and noncoding regions) resulted in a robust phylogeny congruent with results of previous studies, showing (((Cornaceae-Alangiaceae)-(Curtisiaceae-Grubbiaceae))-(((Nyssaceae-Davidiaceae)-Mastixiaceae)-((Hydrostachyaceae-(Hydrangeaceae-Loasaceae)))). Phylogenetic relationships within families were also well resolved. Conflicts in the placement of Hydrostachyaceae were found from analyses of two datasets, the nucleotide characters of all codon position and the 3rd codon positions, where the family was united with Loasaceae, but not strongly supported. Results from divergence time analyses suggested a mid-Cretaceous origin of Cornales followed by rapid early diversification into major clades/families within 10 million years. The early diversification of Cornales may have been facilitated by divergence in habitat and morphology following geographic dispersals. The ancestral distribution of the order was inferred as a widespread range covering Asia, Europe, North America, and Africa when including fossils in the analyses, suggesting an origin of the order likely along the Tethys Seaway where the areas were connected in the mid-Cretaceous. Inferred geographic origins of each family differed to some extent between analyses including fossils vs excluding fossils. In the analysis with extant and fossil species, the origins of the African Hydrostachyaceae and Grubbiaceae-Curtisiaceae clade were inferred to have involved two independent events, an intercontinental dispersal from the northern hemisphere to Africa and an intercontinental vicariance between the northern hemisphere and Africa, respectively. Other families were inferred to have evolved in the northern hemisphere with subsequent intercontinental dispersal(s) to other areas including to Central and South America, during their subsequent diversification. Net diversification rate analysis based on treePL dated phylogeny using MEDUSA detected a nearly 5-fold decrease in the African endemic Curtisiaceae-Grubbiaceae (CuG) clade and an increase of rate in the Hydrangeaceae-Loasaceae (HL) clade. Within HL, a decrease in the Fendlera-Jamesia clade and an increase in the Philadelphus clade were also detected. The findings are also consistent with the level of present species diversity in these lineages. Our study demonstrated the value of plastid genome in phylogenomic study, but posed an old challenge of biogeographic study with fossil data and raised caution for the synonymous substitution sites of plastid genome in phylogenomics studies.

Functional characterization of Terminal Flower1 homolog in Cornus canadensis by genetic transformation.

KEY MESSAGE: TFL1homologCorcanTFL1suppresses the initiation of inflorescence development and regulates the inflorescence morphology inCornus canadensis. In flowering plants, there is a wide range of variation of inflorescence morphology. Despite the ecological and evolutionary importance, efforts devoted to the evolutionary study of the genetic basis of inflorescence morphology are far fewer compared to those on flower development. Our previous study on gene expression patterns suggested a CorTFL1-CorAP1 based model for the evolution of determinate umbels, heads, and mini dichasia from elongated inflorescences in Cornus. Here, we tested the function of CorcanTFL1 in regulating inflorescence development in Cornus canadensis through Agrobacterium-mediated transformation. We showed that transgenic plants overexpressing CorcanTFL1 displayed delayed or suppressed inflorescence initiation and development and extended periods of vegetative growth. Transgenic plants within which CorcanTFL1 had been down-regulated displayed earlier emergence of inflorescence and a reduction of bract and inflorescence sizes, conversions of leaves to bracts and axillary leaf buds to small inflorescences at the uppermost node bearing the inflorescence, or phyllotaxy changes of inflorescence branches and leaves from decussate opposite to spirally alternate. These observations support an important role of CorcanTFL1 in determining flowering time and the morphological destinies of leaves and buds at the node bearing the inflorescence. The evidence is in agreement with the predicted function of CorTFL1 from the gene expression model, supporting a key role of CorTFL1 in the evolutionary divergence of inflorescence forms in Cornus.

Haplotyping of Cornus florida and C. kousa chloroplasts: Insights into species-level differences and patterns of plastic DNA variation in cultivars.

Chloroplast DNA is a part of plant non-nuclear genome, and is of particular interest for lineage studies. Moreover, the non-coding regions of cpDNA display higher mutation rates than the conserved coding cpDNA, which has been employed for phylogenetic and population research. We analyzed the cpDNA of 332 gDNA samples from collections of Cornus florida and C. kousa (commercial cultivars, breeding selections, and wild kousa accessions from Asia), using the chlorotyping system developed on North America-native, wild accessions of C. florida. Our results indicated significant differences in chlorotype frequencies between the two species. Cornus florida samples were represented by all major chlorotypes previously described, whereas all C. kousa samples analyzed had only one of the chlorotype patterns shown by C. florida. The chlorotyping analytic panel was then expanded by sequencing the targeted three non-coding cpDNA regions. Results indicated a major difference in the maternally-inherited cpDNA between the two closely related Big-Bracted Cornus species. Chlorotype diversity and differences in the proportion of informative sites in the cpDNA regions of focus emphasized the importance of proper loci choice for cpDNA-based comparative studies between the closely related dogwood species. Phylogenetic analyses of the retrieved sequences for the other species of Cornus provided information on the relative utility of the cpDNA regions studied and helped delineate the groups (Big-Bracted, Cornelian Cherries, Blue/White-Fruited) within the genus. Genealogical relationships based on the cpDNA sequences and the inferred chlorotype networks indicated the need for continued analyses across further non-coding cpDNA regions to improve the phylogenetic resolution of dogwoods.

Discovering variation of secondary metabolite diversity and its relationship with disease resistance in Cornus florida L.

Understanding intraspecific relationships between genetic and functional diversity is a major goal in the field of evolutionary biology and is important for conserving biodiversity. Linking intraspecific molecular patterns of plants to ecological pressures and trait variation remains difficult due to environment-driven plasticity. Next-generation sequencing, untargeted liquid chromatography-mass spectrometry (LC-MS) profiling, and interdisciplinary approaches integrating population genomics, metabolomics, and community ecology permit novel strategies to tackle this problem. We analyzed six natural populations of the disease-threatened Cornus florida L. from distinct ecological regions using genotype-by-sequencing markers and LC-MS-based untargeted metabolite profiling. We tested the hypothesis that higher genetic diversity in C. florida yielded higher chemical diversity and less disease susceptibility (screening hypothesis), and we also determined whether genetically similar subpopulations were similar in chemical composition. Most importantly, we identified metabolites that were associated with candidate loci or were predictive biomarkers of healthy or diseased plants after controlling for environment. Subpopulation clustering patterns based on genetic or chemical distances were largely congruent. While differences in genetic diversity were small among subpopulations, we did observe notable similarities in patterns between subpopulation averages of rarefied-allelic and chemical richness. More specifically, we found that the most abundant compound of a correlated group of putative terpenoid glycosides and derivatives was correlated with tree health when considering chemodiversity. Random forest biomarker and genomewide association tests suggested that this putative iridoid glucoside and other closely associated chemical features were correlated to SNPs under selection.

Resolving relationships and phylogeographic history of the Nyssa sylvatica complex using data from RAD-seq and species distribution modeling.

Nyssa sylvatica complex consists of several woody taxa occurring in eastern North America. These taxa were recognized as two or three species including three or four varieties by different authors. Due to high morphological similarities and complexity of morphological variation, classification and delineation of taxa in the group have been difficult and controversial. Here we employ data from RAD-seq to elucidate the genetic structure and phylogenetic relationships within the group. Using the genetic evidence, we evaluate previous classifications and delineate species. We also employ Species Distribution Modeling (SDM) to evaluate impacts of climatic changes on the ranges of the taxa and to gain insights into the relevant refugia in eastern North America. Results from Molecular Variance Analysis (AMOVA), STRUCTURE, phylogenetic analyses using Maximum likelihood, Bayesian Inference, and Splittree methods of RAD-seq data strongly support a two-clade pattern, largely separating samples of N. sylvatica from those of N. biflora-N. ursina mix. Divergence time analysis with BEAST suggests the two clades diverged in the mid Miocene. The ancestor of the present trees of N. sylvatica was suggested to be in the Pliocene and that of N. biflora-N. ursina mix in the end of the Miocene. Results from SDM predicted a smaller range in the southern part of the species present range of each clade during the Last Glacial Maximum (LGM). A northward expansion of the ranges during interglacial period and a northward shift of the ranges in the future under a model of global warming were also predicted. Our results support the recognition of two species in the complex, N. sylvatica and N. biflora, following the phylogenetic species concept. We found no genetic evidence supporting recognitions of intraspecific taxa. However, we propose subsp. ursina and subsp. biflora within N. biflora due to their distinction in habits, distributions, and habitats. Our results further support movements of trees in eastern North America in response to climatic changes. Finally, our study demonstrates that RAD-seq data and a combination of population genomics and SDM are valuable in resolving relationship and biogeographic history of closely related species that are taxonomically difficult.

Ecological genomics of local adaptation in Cornus florida L. by genotyping by sequencing.

Discovering local adaptation, its genetic underpinnings, and environmental drivers is important for conserving forest species. Ecological genomic approaches coupled with next-generation sequencing are useful means to detect local adaptation and uncover its underlying genetic basis in nonmodel species. We report results from a study on flowering dogwood trees (Cornus florida L.) using genotyping by sequencing (GBS). This species is ecologically important to eastern US forests but is severely threatened by fungal diseases. We analyzed subpopulations in divergent ecological habitats within North Carolina to uncover loci under local selection and associated with environmental-functional traits or disease infection. At this scale, we tested the effect of incorporating additional sequencing before scaling for a broader examination of the entire range. To test for biases of GBS, we sequenced two similarly sampled libraries independently from six populations of three ecological habitats. We obtained environmental-functional traits for each subpopulation to identify associations with genotypes via latent factor mixed modeling (LFMM) and gradient forests analysis. To test whether heterogeneity of abiotic pressures resulted in genetic differentiation indicative of local adaptation, we evaluated Fst per locus while accounting for genetic differentiation between coastal subpopulations and Piedmont-Mountain subpopulations. Of the 54 candidate loci with sufficient evidence of being under selection among both libraries, 28-39 were Arlequin-BayeScan Fst outliers. For LFMM, 45 candidates were associated with climate (of 54), 30 were associated with soil properties, and four were associated with plant health. Reanalysis of combined libraries showed that 42 candidate loci still showed evidence of being under selection. We conclude environment-driven selection on specific loci has resulted in local adaptation in response to potassium deficiencies, temperature, precipitation, and (to a marginal extent) disease. High allele turnover along ecological gradients further supports the adaptive significance of loci speculated to be under selection.