Conservation applications of niche modeling: Native and naturalized ferns may compete for limited Hawaiian dryland habitat.

Premise: Competition from naturalized species and habitat loss are common threats to native biodiversity and may act synergistically to increase competition for decreasing habitat availability. We use Hawaiian dryland ferns as a model for the interactions between land-use change and competition from naturalized species in determining habitat availability. Methods: We used fine-resolution climatic variables and carefully curated occurrence data from herbaria and community science repositories to estimate the distributions of Hawaiian dryland ferns. We quantified the degree to which naturalized ferns tend to occupy areas suitable for native species and mapped the remaining available habitat given land-use change. Results: Of all native species, Doryopteris angelica had the lowest percentage of occurrences of naturalized species in its suitable area while D. decora had the highest. However, all Doryopteris spp. had a higher percentage overlap, while Pellaea ternifolia had a lower percentage overlap, than expected by chance. Doryopteris decora and D. decipiens had the lowest proportions (<20%) of suitable area covering native habitat. Discussion: Areas characterized by shared environmental preferences of native and naturalized ferns may decrease due to human development and fallowed agricultural lands. Our study demonstrates the value of place-based application of a recently developed correlative ecological niche modeling approach for conservation risk assessment in a rapidly changing and urbanized island ecosystem.

CAM evolution is associated with gene family expansion in an explosive bromeliad radiation.

The subgenus Tillandsia (Bromeliaceae) belongs to one of the fastest radiating clades in the plant kingdom and is characterised by the repeated evolution of Crassulacean acid metabolism (CAM). Despite its complex genetic basis, this water-conserving trait has evolved independently across many plant families and is regarded as a key innovation trait and driver of ecological diversification in Bromeliaceae. By producing high-quality genome assemblies of a Tillandsia species pair displaying divergent photosynthetic phenotypes, and combining genome-wide investigations of synteny, transposable element (TE) dynamics, sequence evolution, gene family evolution and temporal differential expression, we were able to pinpoint the genomic drivers of CAM evolution in Tillandsia. Several large-scale rearrangements associated with karyotype changes between the two genomes and a highly dynamic TE landscape shaped the genomes of Tillandsia. However, our analyses show that rewiring of photosynthetic metabolism is mainly obtained through regulatory evolution rather than coding sequence evolution, as CAM-related genes are differentially expressed across a 24-hour cycle between the two species but are not candidates of positive selection. Gene orthology analyses reveal that CAM-related gene families manifesting differential expression underwent accelerated gene family expansion in the constitutive CAM species, further supporting the view of gene family evolution as a driver of CAM evolution.

Predicting photosynthetic pathway from anatomy using machine learning.

Plants with Crassulacean acid metabolism (CAM) have long been associated with a specialized anatomy, including succulence and thick photosynthetic tissues. Firm, quantitative boundaries between non-CAM and CAM plants have yet to be established - if they indeed exist. Using novel computer vision software to measure anatomy, we combined new measurements with published data across flowering plants. We then used machine learning and phylogenetic comparative methods to investigate relationships between CAM and anatomy. We found significant differences in photosynthetic tissue anatomy between plants with differing CAM phenotypes. Machine learning-based classification was over 95% accurate in differentiating CAM from non-CAM anatomy, and had over 70% recall of distinct CAM phenotypes. Phylogenetic least squares regression and threshold analyses revealed that CAM evolution was significantly correlated with increased mesophyll cell size, thicker leaves, and decreased intercellular airspace. Our findings suggest that machine learning may be used to aid the discovery of new CAM species and that the evolutionary trajectory from non-CAM to strong, obligate CAM requires continual anatomical specialization.

A comparison of freezer-stored DNA and herbarium tissue samples for chloroplast assembly and genome skimming.

Premise: The use of DNA from herbarium specimens is an increasingly important source for evolutionary studies in plant biology, particularly in cases where species are rare or difficult to obtain. Here we compare the utility of DNA from herbarium tissues to their freezer-stored DNA counterparts via the Hawaiian Plant DNA Library. Methods: Plants collected for the Hawaiian Plant DNA Library were simultaneously accessioned as herbarium specimens at the time of collection, from 1994-2019. Paired samples were sequenced using short-read sequencing and assessed for chloroplast assembly and nuclear gene recovery. Results: Herbarium specimen-derived DNA was statistically more fragmented than freezer-stored DNA derived from fresh tissue, leading to poorer chloroplast assembly and overall lower coverage. The number of nuclear targets recovered varied mostly by total sequencing reads per library and age of specimen, but not by storage method (herbarium or long-term freezer). Although there was evidence of DNA damage in the samples, there was no evidence that it was related to the length of time in storage, whether frozen or as herbarium specimens. Discussion: DNA extracted from herbarium tissues will continue to be invaluable, despite being highly fragmented and degraded. Rare floras would benefit from both traditional herbarium storage methods and extracted DNA freezer banks.

The Agavoideae: an emergent model clade for CAM evolutionary biology.

Crassulacean acid metabolism - or CAM photosynthesis - was described in the early to mid-20th century, and our understanding of this metabolic pathway was later expanded upon through detailed biochemical analyses of carbon balance. Soon after, scientists began to study the ecophysiological implications of CAM, and a large part of this early work was conducted in the genus Agave, in the subfamily Agavoideae of the family Asparagaceae. Today, the Agavoideae continues to be important for the study of CAM photosynthesis, from the ecophysiology of CAM species, to the evolution of the CAM phenotype and to the genomics underlying CAM traits. Here we review past and current work on CAM in the Agavoideae, in particular highlighting the work of Park Nobel in Agave, and focusing on the powerful comparative system the Agavoideae has become for studying the origins of CAM. We also highlight new genomics research and the potential for studying intraspecific variation within species of the Agavoideae, particularly species in the genus Yucca. The Agavoideae has served as an important model clade for CAM research for decades, and undoubtedly will continue to help push our understanding of CAM biology and evolution in the future.

Phylogenomic resolution of order- and family-level monocot relationships using 602 single-copy nuclear genes and 1375 BUSCO genes.

We assess relationships among 192 species in all 12 monocot orders and 72 of 77 families, using 602 conserved single-copy (CSC) genes and 1375 benchmarking single-copy ortholog (BUSCO) genes extracted from genomic and transcriptomic datasets. Phylogenomic inferences based on these data, using both coalescent-based and supermatrix analyses, are largely congruent with the most comprehensive plastome-based analysis, and nuclear-gene phylogenomic analyses with less comprehensive taxon sampling. The strongest discordance between the plastome and nuclear gene analyses is the monophyly of a clade comprising Asparagales and Liliales in our nuclear gene analyses, versus the placement of Asparagales and Liliales as successive sister clades to the commelinids in the plastome tree. Within orders, around six of 72 families shifted positions relative to the recent plastome analysis, but four of these involve poorly supported inferred relationships in the plastome-based tree. In Poales, the nuclear data place a clade comprising Ecdeiocoleaceae+Joinvilleaceae as sister to the grasses (Poaceae); Typhaceae, (rather than Bromeliaceae) are resolved as sister to all other Poales. In Commelinales, nuclear data place Philydraceae sister to all other families rather than to a clade comprising Haemodoraceae+Pontederiaceae as seen in the plastome tree. In Liliales, nuclear data place Liliaceae sister to Smilacaceae, and Melanthiaceae are placed sister to all other Liliales except Campynemataceae. Finally, in Alismatales, nuclear data strongly place Tofieldiaceae, rather than Araceae, as sister to all the other families, providing an alternative resolution of what has been the most problematic node to resolve using plastid data, outside of those involving achlorophyllous mycoheterotrophs. As seen in numerous prior studies, the placement of orders Acorales and Alismatales as successive sister lineages to all other extant monocots. Only 21.2% of BUSCO genes were demonstrably single-copy, yet phylogenomic inferences based on BUSCO and CSC genes did not differ, and overall functional annotations of the two sets were very similar. Our analyses also reveal significant gene tree-species tree discordance despite high support values, as expected given incomplete lineage sorting (ILS) related to rapid diversification. Our study advances understanding of monocot relationships and the robustness of phylogenetic inferences based on large numbers of nuclear single-copy genes that can be obtained from transcriptomes and genomes.

Evolution of Crassulacean acid metabolism in response to the environment: past, present, and future.

Crassulacean acid metabolism (CAM) is a mode of photosynthesis that evolved in response to decreasing CO2 levels in the atmosphere some 20 million years ago. An elevated ratio of O2 relative to CO2 caused many plants to face increasing stress from photorespiration, a process exacerbated for plants living under high temperatures or in water-limited environments. Today, our climate is again rapidly changing and plants' ability to cope with and adapt to these novel environments is critical for their success. This review focuses on CAM plant responses to abiotic stressors likely to dominate in our changing climate: increasing CO2 levels, increasing temperatures, and greater variability in drought. Empirical studies that have assessed CAM responses are reviewed, though notably these are concentrated in relatively few CAM lineages. Other aspects of CAM biology, including the effects of abiotic stress on the light reactions and the role of leaf succulence, are also considered in the context of climate change. Finally, more recent studies using genomic techniques are discussed to link physiological changes in CAM plants with the underlying molecular mechanism. Together, the body of work reviewed suggests that CAM plants will continue to thrive in certain environments under elevated CO2. However, how CO2 interacts with other environmental factors, how those interactions affect CAM plants, and whether all CAM plants will be equally affected remain outstanding questions regarding the evolution of CAM on a changing planet.

Differential timing of gene expression and recruitment in independent origins of CAM in the Agavoideae (Asparagaceae).

Crassulacean acid metabolism (CAM) photosynthesis has evolved repeatedly across the plant tree of life, however our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well watered and drought-stressed treatments. We further assessed gene expression and the molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. By contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2, a copy of PPC that has never been shown to be recruited for CAM in angiosperms. Together the physiological and transcriptomic comparison of closely related C3 and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.

The genetic control of succulent leaf development.

Succulent leaves have long intrigued biologists; much research has been done to define succulence, understand the evolutionary trajectory and implications of leaf succulence, and contextualize the ecological importance of water storage for plants inhabiting dry habitats, particularly those using CAM photosynthesis. Surprisingly little is understood about the molecular regulation of leaf succulence, despite advances in our understanding of the molecular foundation of leaf architecture in model systems. Moreover, leaf succulence is a drought avoidance trait, one that has yet to be fully used for crop improvement. Here, connections between disparate literatures are highlighted: research on the regulation of cell size, the determination of vascular patterning, and water transport between cells have direct implications for our understanding of leaf succulence. Connecting functional genomics of leaf patterning with knowledge of the evolution and ecology of succulent species will guide future research on the determination and maintenance of leaf succulence.

Leaf anatomy is not correlated to CAM function in a C3+CAM hybrid species, Yucca gloriosa.

BACKGROUND AND AIMS: Crassulacean acid metabolism (CAM) is often considered to be a complex trait, requiring orchestration of leaf anatomy and physiology for optimal performance. However, the observation of trait correlations is based largely on comparisons between C3 and strong CAM species, resulting in a lack of understanding as to how such traits evolve and the level of intraspecific variability for CAM and associated traits. METHODS: To understand intraspecific variation for traits underlying CAM and how these traits might assemble over evolutionary time, we conducted detailed time course physiological screens and measured aspects of leaf anatomy in 24 genotypes of a C3+CAM hybrid species, Yucca gloriosa (Asparagaceae). Comparisons were made to Y. gloriosa's progenitor species, Y. filamentosa (C3) and Y. aloifolia (CAM). KEY RESULTS: Based on gas exchange and measurement of leaf acids, Y. gloriosa appears to use both C3 and CAM, and varies across genotypes in the degree to which CAM can be upregulated under drought stress. While correlations between leaf anatomy and physiology exist when testing across all three Yucca species, such correlations break down at the species level in Y. gloriosa. CONCLUSIONS: The variation in CAM upregulation in Y. gloriosa is a result of its relatively recent hybrid origin. The lack of trait correlations between anatomy and physiology within Y. gloriosa indicate that the evolution of CAM, at least initially, can proceed through a wide combination of anatomical traits, and more favourable combinations are eventually selected for in strong CAM plants.

Hybridization History and Repetitive Element Content in the Genome of a Homoploid Hybrid, Yucca gloriosa (Asparagaceae).

Hybridization in plants results in phenotypic and genotypic perturbations that can have dramatic effects on hybrid physiology, ecology, and overall fitness. Hybridization can also perturb epigenetic control of transposable elements, resulting in their proliferation. Understanding the mechanisms that maintain genomic integrity after hybridization is often confounded by changes in ploidy that occur in hybrid plant species. Homoploid hybrid species, which have no change in chromosome number relative to their parents, offer an opportunity to study the genomic consequences of hybridization in the absence of change in ploidy. Yucca gloriosa (Asparagaceae) is a young homoploid hybrid species, resulting from a cross between Yucca aloifolia and Yucca filamentosa. Previous analyses of ∼11 kb of the chloroplast genome and nuclear-encoded microsatellites implicated a single Y. aloifolia genotype as the maternal parent of Y. gloriosa. Using whole genome resequencing, we assembled chloroplast genomes from 41 accessions of all three species to re-assess the hybrid origins of Y. gloriosa. We further used re-sequencing data to annotate transposon abundance in the three species and mRNA-seq to analyze transcription of transposons. The chloroplast phylogeny and haplotype analysis suggest multiple hybridization events contributing to the origin of Y. gloriosa, with both parental species acting as the maternal donor. Transposon abundance at the superfamily level was significantly different between the three species; the hybrid was frequently intermediate to the parental species in TE superfamily abundance or appeared more similar to one or the other parent. In only one case-Copia LTR transposons-did Y. gloriosa have a significantly higher abundance relative to either parent. Expression patterns across the three species showed little increased transcriptional activity of transposons, suggesting that either no transposon release occurred in Y. gloriosa upon hybridization, or that any transposons that were activated via hybridization were rapidly silenced. The identification and quantification of transposon families paired with expression evidence paves the way for additional work seeking to link epigenetics with the important trait variation seen in this homoploid hybrid system.

Shared expression of crassulacean acid metabolism (CAM) genes pre-dates the origin of CAM in the genus Yucca.

Crassulacean acid metabolism (CAM) is a carbon-concentrating mechanism that has evolved numerous times across flowering plants and is thought to be an adaptation to water-limited environments. CAM has been investigated from physiological and biochemical perspectives, but little is known about how plants evolve from C3 to CAM at the genetic or metabolic level. Here we take a comparative approach in analyzing time-course data of C3, CAM, and C3+CAM intermediate Yucca (Asparagaceae) species. RNA samples were collected over a 24 h period from both well-watered and drought-stressed plants, and were clustered based on time-dependent expression patterns. Metabolomic data reveal differences in carbohydrate metabolism and antioxidant response between the CAM and C3 species, suggesting that changes to metabolic pathways are important for CAM evolution and function. However, all three species share expression profiles of canonical CAM pathway genes, regardless of photosynthetic pathway. Despite differences in transcript and metabolite profiles between the C3 and CAM species, shared time-structured expression of CAM genes in both CAM and C3Yucca species suggests that ancestral expression patterns required for CAM may have pre-dated its origin in Yucca.

The genetics of convergent evolution: insights from plant photosynthesis.

The tree of life is resplendent with examples of convergent evolution, whereby distinct species evolve the same trait independently. Many highly convergent adaptations are also complex, which makes their repeated emergence surprising. In plants, the evolutionary history of two carbon concentrating mechanisms (CCMs) - C4 and crassulacean acid metabolism (CAM) photosynthesis - presents such a paradox. Both of these modifications of ancestral C3 photosynthesis require the integration of multiple anatomical and biochemical components, yet together they have evolved more than one hundred times. The presence of CCM enzymes in all plants suggests that a rudimentary CCM might emerge via relatively few genetic changes in potentiated lineages. Here, we propose that many of the complexities often associated with C4 and CAM photosynthesis may have evolved during a post-emergence optimization phase. The ongoing development of new model clades for young, emerging CCMs is enabling the comparative studies needed to test these ideas.

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.

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.

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

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.