Capturing and managing genetic diversity in ex situ collections of threatened tropical trees: A case study in Karomia gigas.

Premise: Although ex situ collections of threatened plants are most useful when they contain maximal genetic variation, the conservation and maintenance of genetic diversity in collections are often poorly known. We present a case study using population genomic analyses of an ex situ collection of Karomia gigas, a critically endangered tropical tree from Tanzania. Only ~43 individuals are known in two wild populations, and ex situ collections containing 34 individuals were established in two sites from wild-collected seed. The study aimed to understand how much diversity is represented in the collection, analyze the parentage of ex situ individuals, and identify efficient strategies to capture and maintain genetic diversity. Methods: We genotyped all known individuals using a 2b-RADseq approach, compared genetic diversity in wild populations and ex situ collections, and conducted parentage analysis of the collections. Results: Wild populations were found to have greater levels of genetic diversity than ex situ populations as measured by number of private alleles, number of polymorphic sites, observed and expected heterozygosity, nucleotide diversity, and allelic richness. In addition, only 32.6% of wild individuals are represented ex situ and many individuals were found to be the product of selfing by a single wild individual. Discussion: Population genomic analyses provided important insights into the conservation of genetic diversity in K. gigas, identifying gaps and inefficiencies, but also highlighting strategies to conserve genetic diversity ex situ. Genomic analyses provide essential information to ensure that collections effectively conserve genetic diversity in threatened tropical trees.

Restoration age affects microbial-herbaceous plant interactions in an oak woodland.

In degraded ecosystems, soil microbial communities (SMCs) may influence the outcomes of ecological restoration. Restoration practices can affect SMCs, though it is unclear how variation in the onset of restoration activities in woodlands affects SMCs, how those SMCs influence the performance of hard-to-establish woodland forbs, and how different woodland forbs shape SMCs. In this study, we quantified soil properties and species abundances in an oak woodland restoration chronosequence (young, intermediate, and old restorations). We measured the growth of three woodland forb species when inoculated with live whole-soil from young, intermediate, or old restorations. We used DNA metabarcoding to characterize SMCs of each inoculum treatment and the soil after conditioning by each plant species. Our goals were to (1) understand how time since the onset of restoration affected soil abiotic properties, plant communities, and SMCs in a restoration chronosequence, (2) test growth responses of three forb species to whole-soil inoculum from restoration sites, and (3) characterize changes in SMCs before and after conditioning by each forb species. Younger restored woodlands had greater fire-sensitive tree species and lower concentrations of soil phosphorous than intermediate or older restored woodlands. Bacterial and fungal soil communities varied significantly among sites. Forbs exhibited the greatest growth in soil from the young restoration. Each forb species developed a unique soil microbial community. Our results highlight how restoration practices affect SMCs, which can in turn affect the growth of hard-to-establish forb species. Our results also highlight that the choice of forb species can alter SMCs, which could have long-term potential consequences for restoration success.

Harnessing the power of botanical gardens: Evaluating the costs and resources needed for exceptional plant conservation.

Premise: The effective ex situ conservation of exceptional plants, whether in living collections or cryo-collections, requires more resources than the conservation of other species. Because of their expertise with rare plants, botanical gardens are well positioned to lead this effort, but a well-developed strategy requires a clear understanding of the resources needed. Methods: Grant funding was obtained from the Institute of Museum and Library Services to support a three-year project on cryobanking, and to provide smaller grants to 10 other botanical gardens for one-year projects on either (1) seed behavior studies or (2) the development of protocols for in vitro propagation or cryopreservation. Results: Nine of the partner gardens worked on 19 species (one was unable to continue due to the COVID-19 pandemic), while the larger project focused on 14 species. A point system was developed for tasks accomplished, and the average costs per point of the larger and smaller projects were similar. Labor accounted for half the costs. Projects focused on species in the Asteraceae and Orchidaceae had lower costs per point than other species. Discussion: Both large and small projects can contribute to a strategy for exceptional plant conservation for similar costs. Prioritizing species with lower costs could help advance the field while allowing time for work on more difficult species to develop.

Understanding how an amphicarpic species with a mixed mating system responds to fire: a population genetic approach.

Amphicarpic plants produce both above-ground and below-ground seeds. Because below-ground seeds are protected in the soil and may maintain viability when above-ground conditions are stressful, they were proposed as an adaptation to recolonize a site after disturbance. However, whether below-ground seeds are the main colonizers after a disturbance remains unknown. Our goal was to understand whether recolonization by an amphicarpic species after fire was accomplished primarily through germination of seeds produced above-ground or below-ground. We investigated Polygala lewtonii, an amphicarpic, perennial species endemic to fire-prone Florida sandhill and scrub, where fire kills plants but subsequently increases recruitment and population sizes. Polygala lewtonii produces three flower types: above-ground chasmogamous flowers and above-ground and below-ground cleistogamous flowers, with previous research demonstrating chasmogamous flowers produce a much greater proportion of seeds than cleistogamous flowers. We quantified outcrossing in seeds produced by chasmogamous flowers to determine whether it differed from the 100 % self-fertilized below-ground seeds. Approximately 25 % of seeds from chasmogamous flowers showed evidence of cross-pollination. Assuming that chasmogamous flowers produce the majority of the above-ground seeds, as was shown previously, this indicates it is possible to differentiate between germination by above-ground versus below-ground seeds in post-fire colonization. We next compared genetic diversity, admixture, inbreeding and population genetic structure pre- and post-fire. If fire promoted germination of chasmogamous seeds, heterozygosity and admixture would increase, and genetic structure and inbreeding would decrease. Instead, inbreeding and genetic structure increased and admixture decreased, suggesting that the below-ground selfed seeds (with limited dispersal ability) increased their contribution to the population after fire, possibly because fire reduced above-ground seed viability. Additionally, new alleles not found previously in range-wide analyses emerged from the seed bank post-fire. These results suggest that amphicarpy is a powerful adaptation to preserve genetic variation, maintain adaptive potential and promote rapid post-fire colonization.

Conservation genetics of the threatened plant species Physaria filiformis (Missouri bladderpod) reveals strong genetic structure and a possible cryptic species.

Understanding genetic diversity and structure in a rare species is critical for prioritizing both in situ and ex situ conservation efforts. One such rare species is Physaria filiformis (Brassicaceae), a threatened, winter annual plant species. The species has a naturally fragmented distribution, occupying three different soil types spread across four disjunct geographical locations in Missouri and Arkansas. The goals of this study were to understand: (1) whether factors associated with fragmentation and small population size (i.e., inbreeding, genetic drift or genetic bottlenecks) have reduced levels of genetic diversity, (2) how genetic variation is structured and which factors have influenced genetic structure, and (3) how much extant genetic variation of P. filiformis is currently publicly protected and the implications for the development of conservation strategies to protect its genetic diversity. Using 16 microsatellite markers, we genotyped individuals from 20 populations of P. filiformis from across its geographical range and one population of Physaria gracilis for comparison and analyzed genetic diversity and structure. Populations of P. filiformis showed comparable levels of genetic diversity to its congener, except a single population in northwest Arkansas showed evidence of a genetic bottleneck and two populations in the Ouachita Mountains of Arkansas showed lower genetic variation, consistent with genetic drift. Populations showed isolation by distance, indicating that migration is geographically limited, and analyses of genetic structure grouped individuals into seven geographically structured genetic clusters, with geographic location/spatial separation showing a strong influence on genetic structure. At least one population is protected for all genetic clusters except one in north-central Arkansas, which should therefore be prioritized for protection. Populations in the Ouachita Mountains were genetically divergent from the rest of P. filiformis; future morphological analyses are needed to identify whether it merits recognition as a new, extremely rare species.

C2 photosynthesis generates about 3-fold elevated leaf CO2 levels in the C3-C4 intermediate species Flaveria pubescens.

Formation of a photorespiration-based CO2-concentrating mechanism in C3-C4 intermediate plants is seen as a prerequisite for the evolution of C4 photosynthesis, but it is not known how efficient this mechanism is. Here, using in vivo Rubisco carboxylation-to-oxygenation ratios as a proxy to assess relative intraplastidial CO2 levels is suggested. Such ratios were determined for the C3-C4 intermediate species Flaveria pubescens compared with the closely related C3 plant F. cronquistii and the C4 plant F. trinervia. To this end, a model was developed to describe the major carbon fluxes and metabolite pools involved in photosynthetic-photorespiratory carbon metabolism and used quantitatively to evaluate the labelling kinetics during short-term (14)CO2 incorporation. Our data suggest that the photorespiratory CO2 pump elevates the intraplastidial CO2 concentration about 3-fold in leaves of the C3-C4 intermediate species F. pubescens relative to the C3 species F. cronquistii.

Detection of toxic monofluoroacetate in Palicourea species.

Numerous plant species worldwide including some Palicourea (Rubiaceae), Tanaecium (Bignoniaceae), and Amorimia (Malpighiaceae) species in Brazil cause sudden death and are known to contain monofluoroacetate (MFA). Two species of Palicourea, Palicourea aenofusca and Palicourea marcgravii, cause sudden death and are reported to contain MFA while other Palicourea species are reported to cause sudden death in livestock and are suspected to contain MFA due to the similarity in clinical signs. Using an HPLC-APCI-MS method to detect MFA, herbarium specimens representing 46 Palicourea taxa were screened for the presence of MFA. Additionally we screened five Psychotria taxa that are closely related to Palicourea species. Ten species of Palicourea were identified that contained MFA, two previously reported and eight newly reported here; these are closely related to each other, though some other related species did not contain MFA.

Novel plant transformation vectors containing the superpromoter.

We developed novel plasmids and T-DNA binary vectors that incorporate a modified and more useful form of the superpromoter. The superpromoter consists of a trimer of the octopine synthase transcriptional activating element affixed to the mannopine synthase2' (mas2') transcriptional activating element plus minimal promoter. We tested a superpromoter-beta-glucuronidaseA fusion gene in stably transformed tobacco (Nicotiana tabacum) and maize (Zea mays) plants and in transiently transformed maize Black Mexican Sweet protoplasts. In both tobacco and maize, superpromoter activity was much greater in roots than in leaves. In tobacco, superpromoter activity was greater in mature leaves than in young leaves, whereas in maize activity differed little among the tested aerial portions of the plant. When compared with other commonly used promoters (cauliflower mosaic virus 35S, mas2', and maize ubiquitin), superpromoter activity was approximately equivalent to those of the other promoters in both maize Black Mexican Sweet suspension cells and in stably transformed maize plants. The addition of a maize ubiquitin intron downstream of the superpromoter did not enhance activity in stably transformed maize.

Auxin and root initiation in somatic embryos of Arabidopsis.

Somatic embryos of Arabidopsis thaliana can be produced from explants of developing zygotic embryos. Cultivation of explants on maturation medium results in development of three main classes of regenerative structures: adventitious shoots, fused shoots, and complete somatic embryos. The ontogeny and anatomy of these structures was examined using serial plastic sections. Furthermore, two molecular markers were assayed to monitor transcriptional auxin responses and formation of a root meristem in this process: the LENNY allele of PIN4, a transposon insertion creating a fusion to the reporter gene GUS; and DR5::GUS, a synthetic reporter of auxin-induced transcription. In zygotic embryogenesis, PIN4 expression is confined to the center of the root meristem and begins to be detectable by the globular stage of embryogenesis, while DR5::GUS expression marks an "auxin perception maximum" in the more distal regions of the root. Adventitious and fused shoots develop no anatomically recognizable root meristem and do not express either of the two markers at their basal pole. Instead, the vasculature of their axis is directly connected to the vasculature of the explant. By contrast, complete somatic embryos were only loosely attached to the explant, had an anatomically defined root meristem and showed expression of both markers at their root pole. Our results suggest that the establishment of a root meristem in somatic embryos required appropriate auxin levels during the course of their development.