Karolina Heyduk

Resolving phylogenetic relationships of the recently radiated carnivorous plant genus Sarracenia using target enrichment

The North American carnivorous pitcher plant genus Sarracenia (Sarraceniaceae) is a relatively young clade (<3 million years ago) displaying a wide range of morphological diversity in complex trapping structures. This recently radiated group is a promising system to examine the structural evolution and diversification of carnivorous plants; however, little is known regarding evolutionary relationships within the genus. Previous attempts at resolving the phylogeny have been unsuccessful, most likely due to few parsimony-informative sites compounded by incomplete lineage sorting. Here, we applied a target enrichment approach using multiple accessions to assess the relationships of Sarracenia species. This resulted in 199 nuclear genes from 75 accessions covering the putative 8-11 species and 8 subspecies/varieties. In addition, we recovered 42kb of plastome sequence from each accession to estimate a cpDNA-derived phylogeny. Unsurprisingly, the cpDNA had few parsimony-informative sites (0.5%) and provided little information on species relationships. In contrast, use of the targeted nuclear loci in concatenation and coalescent frameworks elucidated many relationships within Sarracenia even with high heterogeneity among gene trees. Results were largely consistent for both concatenation and coalescent approaches. The only major disagreement was with the placement of the purpurea complex. Moreover, results suggest an Appalachian massif biogeographic origin of the genus. Overall, this study highlights the utility of target enrichment using multiple accessions to resolve relationships in recently radiated taxa.

Resolving relationships within the palm subfamily Arecoideae (Arecaceae) using plastid sequences derived from next-generation sequencing.

PREMISE OF THE STUDY Several studies have incorporated molecular and morphological data to study the phylogeny of the palms (Arecaceae), but some relationships within the family remain ambiguous-particularly those within Arecoideae, the most diverse subfamily including coconut and oil palm. Here, two next-generation, targeted plastid-enrichment methods were compared and used to elucidate Arecoideae phylogeny. METHODS Next-generation sequencing techniques were used to generate a plastid genome data set. Long range PCR and hybrid gene capture were used to enrich for chloroplast targets. Ten taxa were enriched using both methods for comparison. Chloroplast sequence data were generated for 31 representatives of the 14 Arecoideae tribes and five outgroup taxa. The phylogeny was reconstructed using maximum likelihood, maximum parsimony, and Bayesian analyses. KEY RESULTS Long range PCR and hybrid gene capture both enriched the plastid genome and provided similar sequencing coverage. Subfamily Arecoideae was resolved as monophyletic with tribe Chamaedoreeae as the earliest-diverging lineage, implying that the development of flowers in triads defines a synapomorphy for the Arecoideae clade excluding Chamaedoreeae. Three major clades within this group were recovered: Roystoneeae/Reinhardtieae/Cocoseae (RRC), Areceae/Euterpeae/Geonomateae/Leopoldinieae/Manicarieae/Pelagodoxeae (core arecoids), and Podococceae/Oranieae/Sclerospermeae (POS). An Areceae + Euterpeae clade was resolved within the core arecoids. The POS clade was sister to a RRC + core arecoids clade, implying a shared ancestral area in South America for these three clades. CONCLUSIONS The plastome phylogeny recovered here provides robust resolution of previously ambiguous studies and new insights into palm evolution.

Evolution of a CAM anatomy predates the origins of Crassulacean acid metabolism in the Agavoideae (Asparagaceae)

Crassulacean acid metabolism (CAM) is a modified form of photosynthesis that has arisen independently at least 35 times in flowering plants. The occurrence of CAM is often correlated with shifts to arid, semiarid, or epiphytic habits, as well as transitions in leaf morphology (e.g. increased leaf thickness) and anatomy (e.g. increased cell size and packing). We assess shifts between C3 and CAM photosynthesis in the subfamily Agavoideae (Asparagaceae) through phylogenetic analysis of targeted loci captured from the nuclear and chloroplast genomes of over 60 species. Carbon isotope data was used as a proxy for mode of photosynthesis in extant species and ancestral states were estimated on the phylogeny. Ancestral character state mapping suggests three independent origins of CAM in the Agavoideae. CAM species differ from C3 species in climate space and are found to have thicker leaves with densely packed cells. C3 ancestors of CAM species show a predisposition toward CAM-like morphology. Leaf characteristics in the ancestral C3 species may have enabled the repeated evolution of CAM in the Agavoideae subfamily. Anatomical changes, including a tendency toward 3D venation, may have initially arisen in C3 ancestors in response to aridity as a way to increase leaf succulence for water storage.

The Kalanchoë genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops.Crassulacean acid metabolism (CAM) is a metabolic adaptation of photosynthesis that enhances water use efficiency. Here, via genomic analysis of Kalanchoë, the authors provide evidence for convergent evolution of protein sequence and temporal gene expression underpinning the multiple independent emergences of CAM.

Nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae)

Palms (Arecaceae) include economically important species such as coconut, date palm, and oil palm. Resolution of the palm phylogeny has been problematic due to rapid diversification and slow rates of molecular evolution. The focus of this study is on relationships of the 14 tribes of subfamily Arecoideae and their inferred ancestral areas. A targeted sequencing approach was used to generate a data set of 168 single/low copy nuclear genes for 34 species representing the Arecoideae tribes and the other palm subfamilies. Species trees from the concatenated and coalescent based analyses recovered largely congruent topologies. Three major tribal clades were recovered: the POS clade (Podococceae, Oranieae, Sclerospermeae), the RRC clade (Roystoneeae, Reinhardtieae, Cocoseae), and the core arecoid clade (Areceae, Euterpeae, Geonomateae, Leopoldinieae, Manicarieae, Pelagodoxeae). Leopoldinieae was sister to the rest of the core arecoids (Geonomateae, Manicarieae+Pelagodoxeae, and Areceae+Euterpeae). The nuclear phylogeny supported a North American origin for subfamily Arecoideae, with most tribal progenitors diversifying within the Americas. The POS clade may have dispersed from the Americas into Africa, with tribe Oranieae subsequently spreading into the Indo-Pacific. Two independent dispersals into the Indo-Pacific were inferred for two tribes within the core arecoids (tribes Areceae and Pelagodoxeae).

Horizontal Transfer of Non-LTR Retrotransposons from Arthropods to Flowering Plants

&NA; Even though lateral movements of transposons across families and even phyla within multicellular eukaryotic kingdoms have been found, little is known about transposon transfer between the kingdoms Animalia and Plantae. We discovered a novel non‐LTR retrotransposon, AdLINE3, in a wild peanut species. Sequence comparisons and phylogenetic analyses indicated that AdLINE3 is a member of the RTE clade, originally identified in a nematode and rarely reported in plants. We identified RTE elements in 82 plants, spanning angiosperms to algae, including recently active elements in some flowering plants. RTE elements in flowering plants were likely derived from a single family we refer to as An‐RTE. Interestingly, An‐RTEs show significant DNA sequence identity with non‐LTR retroelements from 42 animals belonging to four phyla. Moreover, the sequence identity of RTEs between two arthropods and two plants was higher than that of homologous genes. Phylogenetic and evolutionary analyses of RTEs from both animals and plants suggest that the An‐RTE family was likely transferred horizontally into angiosperms from an ancient aphid(s) or ancestral arthropod(s). Notably, some An‐RTEs were recruited as coding sequences of functional genes participating in metabolic or other biochemical processes in plants. This is the first potential example of horizontal transfer of transposons between animals and flowering plants. Our findings help to understand exchanges of genetic material between the kingdom Animalia and Plantae and suggest arthropods likely impacted on plant genome evolution.

Gas exchange and leaf anatomy of a C3–CAM hybrid, Yucca gloriosa (Asparagaceae)

Highlight Physiological and anatomical study of a C3–CAM hybrid species, which has the ability to use CAM under drought and has significant anatomical differences from both C3 and CAM parental species.

Shifts in gene expression profiles are associated with weak and strong Crassulacean acid metabolism

PREMISE OF THE STUDY The relative ease of high throughput sequencing is facilitating comprehensive phylogenomic and gene expression studies, even for nonmodel groups. To date, however, these two approaches have not been merged; while phylogenomic methods might use transcriptome sequences to resolve relationships, assessment of gene expression patterns in a phylogenetic context is less common. Here we analyzed both carbon assimilation and gene expression patterns of closely related species within the Agavoideae (Asparagaceae) to elucidate changes in gene expression across weak and strong phenotypes for Crassulacean acid metabolism (CAM). METHODS Gene expression patterns were compared across four genera: Agave (CAM), which is paraphyletic with Polianthes (weak CAM) and Manfreda (CAM), and Beschorneria (weak CAM). RNA-sequencing was paired with measures of gas exchange and titratable acidity. Climate niche space was compared across the four lineages to examine abiotic factors and their correlation to CAM. KEY RESULTS Expression of homologous genes showed both shared and variable patterns in weak and strong CAM species. Network analysis highlights that despite shared expression patterns, highly connected genes differ between weak and strong CAM, implicating shifts in regulatory gene function as key for the evolution of CAM. Variation in carbohydrate metabolism between weak and strong CAM supports the importance of sugar turnovers for CAM physiology. CONCLUSIONS Integration of phylogenetics and RNA-sequencing provides a powerful tool to study the evolution of CAM photosynthesis across closely related but photosynthetically variable species. Our findings regarding shared or shifted gene expression and regulation of CAM via carbohydrate metabolism have important implications for efforts to engineer the CAM pathway into C3 food and biofuel crops.

Targeted DNA Region Re-sequencing

Although massively parallel sequencing (MPS) allows researchers to obtain huge amounts of data at low cost compared to Sanger sequencing, current costs and computational constraints for whole genome sequencing and analysis generally prohibit phylogenomic and population genomic studies of non-model organisms and species with large, complex genomes. Therefore, new methods have been developed to select specific genomic regions for re-sequencing. Many of these methods can be applied to non-model organisms or species with very few genetic resources. Choosing which method to use for the study system of interest often depends on a variety of factors. In this chapter, we describe various re-sequencing methods with a focus on target enrichment. Additionally, we lay out experimental design considerations, bioinformatics pipelines, and proper reporting of results for target enrichment.

Altered Gene Regulatory Networks are Associated with the Transition from C3 to Crassulacean Acid Metabolism in Erycina (Oncidiinae: Orchidaceae)

Crassulacean acid metabolism (CAM) photosynthesis is a modification of the core C3 photosynthetic pathway that improves the ability of plants to assimilate carbon in water-limited environments. CAM plants fix CO2 mostly at night, when transpiration rates are low. All of the CAM pathway genes exist in ancestral C3 species, but the timing and magnitude of expression are greatly altered between C3 and CAM species. Understanding these regulatory changes is key to elucidating the mechanism by which CAM evolved from C3. Here we use two closely related species in the Orchidaceae, Erycina pusilla (CAM) and Erycina crista-galli (C3), to conduct comparative transcriptomic analyses across multiple time points. Clustering of genes with expression variation across the diel cycle revealed some canonical CAM pathway genes similarly expressed in both species, regardless of photosynthetic pathway. However, gene network construction indicated that 149 gene families had significant differences in network connectivity and were further explored for these functional enrichments. Genes involved in light sensing and ABA signaling were some of the most differently connected genes between the C3 and CAM Erycina species, in agreement with the contrasting diel patterns of stomatal conductance in C3 and CAM plants. Our results suggest changes to transcriptional cascades are important for the transition from C3 to CAM photosynthesis in Erycina.