A chromosome level reference genome of Diviner's sage (Salvia divinorum) provides insight into salvinorin A biosynthesis.

BACKGROUND: Diviner's sage (Salvia divinorum; Lamiaceae) is the source of the powerful hallucinogen salvinorin A (SalA). This neoclerodane diterpenoid is an agonist of the human Κ-opioid receptor with potential medical applications in the treatment of chronic pain, addiction, and post-traumatic stress disorder. Only two steps of the approximately twelve step biosynthetic sequence leading to SalA have been resolved to date. RESULTS: To facilitate pathway elucidation in this ethnomedicinal plant species, here we report a chromosome level genome assembly. A high-quality genome sequence was assembled with an N50 value of 41.4 Mb and a BUSCO completeness score of 98.4%. The diploid (2n = 22) genome of ~ 541 Mb is comparable in size and ploidy to most other members of this genus. Two diterpene biosynthetic gene clusters were identified and are highly enriched in previously unidentified cytochrome P450s as well as crotonolide G synthase, which forms the dihydrofuran ring early in the SalA pathway. Coding sequences for other enzyme classes with likely involvement in downstream steps of the SalA pathway (BAHD acyl transferases, alcohol dehydrogenases, and O-methyl transferases) were scattered throughout the genome with no clear indication of clustering. Differential gene expression analysis suggests that most of these genes are not inducible by methyl jasmonate treatment. CONCLUSIONS: This genome sequence and associated gene annotation are among the highest resolution in Salvia, a genus well known for the medicinal properties of its members. Here we have identified the cohort of genes responsible for the remaining steps in the SalA pathway. This genome sequence and associated candidate genes will facilitate the elucidation of SalA biosynthesis and enable an exploration of its full clinical potential.

A novel method for identifying Chlamydomonas reinhardtii (Chlorophyta) and closely related species from nature.

Here, we introduce a new method for efficiently sampling Chlamydomonas reinhardtii and closely related species using a colony PCR-based screen with novel primer sets designed to specifically detect these important model microalgae. To demonstrate the utility of our new method, we collected 130 soil samples from a wide range of habitats in Ontario, Canada and identified 33 candidate algae, which were barcoded by sequencing a region of the rbcL plastid gene. For select isolates, 18S rRNA gene and YPT4 nuclear markers were also sequenced. Based on phylogenetic and haplotype network analyses of these three loci, seven novel isolates were identified as C. reinhardtii, and one additional isolate appeared to be more closely related to C. reinhardtii than any other known species. All seven new C. reinhardtii strains were interfertile with previously collected C. reinhardtii field isolates, validating the effectiveness of our molecular screen.

Habitat preference in the critically endangered yellow-tailed woolly monkey (Lagothrix flavicauda) at La Esperanza, Peru.

Habitat loss is one of the main threats to wildlife. Therefore, knowledge of habitat use and preference is essential for the design of conservation strategies and identification of priority sites for the protection of endangered species. The yellow-tailed woolly monkey (Lagothrix flavicauda Humboldt, 1812), categorized as Critically Endangered on the IUCN Red List, is endemic to montane forests in northern Peru where its habitat is greatly threatened. We assessed how habitat use and preference in L. flavicauda are linked to forest structure and composition. The study took place near La Esperanza, in the Amazonas region, Peru. Our objective was to identify characteristics of habitat most utilized by L. flavicauda to provide information that will be useful for the selection of priority sites for conservation measures. Using presence records collected from May 2013 to February 2014 for one group of L. flavicauda, we classified the study site into three different use zones: low-use, medium-use, and high-use. We assessed forest structure and composition for all use zones using 0.1 ha Gentry vegetation transects. Results show high levels of variation in plant species composition across the three use zones. Plants used as food resources had considerably greater density, dominance, and ecological importance in high-use zones. High-use zones presented similar structure to medium- and low-use zones; thus it remains difficult to assess the influence of forest structure on habitat preference. We recommend focusing conservation efforts on areas with a similar floristic composition to the high-use zones recorded in this study and suggest utilizing key alimentation species for reforestation efforts.

Long Nights, Airplanes, and Avian Surgery: A Tale of Working With Volunteers to Study Long-tailed Ducks (Clangula hyemalis) Wintering on Lake Michigan.

Evidence suggests that wintering populations of long-tailed ducks along the Atlantic and Pacific coasts are in decline, but little is known about wintering populations on Lake Michigan. Researchers seek answers to basic questions regarding habitat use and migration patterns (temporal and spatial) of long-tailed ducks that winter on Lake Michigan, by using surgically implanted satellite transmitters. The processes of locating the birds, capturing and implanting satellite transmitters, and interpreting the results were challenging, and efforts relied on dedicated researchers, veterinarians, resource managers, and many volunteers.

Cryptic diversity hides host and habitat specialization in a gorgonian-algal symbiosis.

Shallow water anthozoans, the major builders of modern coral reefs, enhance their metabolic and calcification rates with algal symbionts. Controversy exists over whether these anthozoan-algae associations are flexible over the lifetimes of individual hosts, promoting acclimative plasticity, or are closely linked, such that hosts and symbionts co-evolve across generations. Given the diversity of algal symbionts and the morphological plasticity of many host species, cryptic variation within either partner could potentially confound studies of anthozoan-algal associations. Here, we used ribosomal, organelle and nuclear sequences, along with microsatellite variation, to study the relationship between lineages of a common Caribbean gorgonian and its algal symbionts. The gorgonian Eunicea flexuosa is a broadcast spawner, composed of two recently diverged, genetically distinct lineages largely segregated by depth. We sampled colonies of the two lineages across depth gradients at three Caribbean locations. We find that each host lineage is associated with a unique Symbiodinium B1/184 phylotype. This relationship between host and symbiont is maintained when host colonies are reciprocally transplanted, although cases of within phylotype switching were also observed. Even when the phylotypes of both partners are present at intermediate depths, the specificity between host and symbiont lineages remained absolute. Unrecognized cryptic diversity may mask host-symbiont specificity and change the inference of evolutionary processes in mutualistic associations. Symbiotic specificity thus likely contributes to the ecological divergence of the two partners, generating species diversity within coral reefs.

Tracking migrations of yellow-billed loons (Gavia adamsii) from the Canadian arctic.

Because the population of yellow-billed loons (Gavia adamsii) is declining, satellite telemetry was used to learn more about the needs of this species and the interdependencies of various populations that breed in isolated regions. In July 2013, the author accompanied a team of biologists to Victoria Island in the Canadian Arctic to surgically implant satellite transmitters in 14 yellow-billed loons. This article chronicles the trip with the objective of connecting the reader with the loons and the interesting people that strive to conserve them.

Rubisco supplies pyruvate for the 2-C-methyl-D-erythritol-4-phosphate pathway.

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (Rubisco) produces pyruvate in the chloroplast through ß-elimination of the aci-carbanion intermediate1. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid and branched-chain amino acid biosynthesis in photosynthetically active tissue. 13C labelling studies of intact Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate to serve as a precursor. Low oxygen stimulates Rubisco carboxylase activity and increases pyruvate production and flux through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis2,3. Metabolome analysis of mutants defective in phosphoenolpyruvate or pyruvate import and biochemical characterization of isolated chloroplasts further support Rubisco as the main source of pyruvate in chloroplasts. Seedlings incorporated exogenous,13C-labelled pyruvate into MEP pathway intermediates, while adult plants did not, underscoring the developmental transition in pyruvate sourcing. Rubisco ß-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin-Benson-Bassham cycle. These insights solve the "pyruvate paradox"4, improve the fit of metabolic models for central metabolism and connect the MEP pathway directly to carbon assimilation.

Salt stress alters the spectrum of de novo mutation available to selection during experimental adaptation of Chlamydomonas reinhardtii.

The genetic basis of adaptation is driven by both selection and the spectrum of available mutations. Given that the rate of mutation is not uniformly distributed across the genome and varies depending on the environment, understanding the signatures of selection across the genome is aided by first establishing what the expectations of genetic change are from mutation. To determine the interaction between salt stress, selection, and mutation across the genome, we compared mutations observed in a selection experiment for salt tolerance in Chlamydomonas reinhardtii to those observed in mutation accumulation (MA) experiments with and without salt exposure. MA lines evolved under salt stress had a single-nucleotide mutation rate of 1.1 × 10 - 9 $1.1 \times 10^{-9}$ , similar to that of MA lines under standard conditions ( 9.6 × 10 - 10 $9.6 \times 10^{-10}$ ). However, we found that salt stress led to an increased rate of indel mutations, but that many of these mutations were removed under selection. Finally, lines adapted to salt also showed excess clustering of mutations in the genome and the co-expression network, suggesting a role for positive selection in retaining mutations in particular compartments of the genome during the evolution of salt tolerance. Our study shows that characterizing mutation rates and spectra expected under stress helps disentangle the effects of environment and selection during adaptation.

Large Differences in Livelihood Responses and Outcomes to Increased Conservation Enforcement in a Protected Area.

Despite the popularity of integrated conservation and development approaches to protected area management, adjacent communities increasingly face livelihood dilemmas. Yet understanding of how market processes and conservation enforcement interact to influence livelihood responses remains limited. Targeting eight villages in Nam Et-Phou Louey (NEPL) National Park in northern Lao PDR, we draw on survey data with 255 households, 93 semi-structured interviews, and meso-level data on village conditions to examine how residents navigate associated livelihood dilemmas. A cluster analysis reveals five livelihood types with divergent capacities to engage in market development and cope with enforcement pressures. We show how market linkages, historical conservation interventions, and local access conditions shape livelihoods and differences between villages. Our approach yields a nuanced picture of how global conservation efforts result in an uneven distribution of costs and benefits at local scales. Conservation measures must account for highly divergent capacities to cope with access loss and diversify livelihoods. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10745-021-00267-4.

Searching for nests of the invasive Asian hornet (Vespa velutina) using radio-telemetry.

Asian hornets (Vespa velutina) are voracious predators of bees, and are the latest emerging threat to managed and wild pollinator populations in Europe. To prevent establishment or reduce the rate of spread of V. velutina, early detection and destruction of nests is considered the only option. Detection is difficult as their nests are well hidden and flying hornets are difficult to follow over long distances. We address this challenge by tracking individual V. velutina workers flying back to their nests using radio telemetry for the first time, finding five previously undiscovered nests, up to 1.33 km from hornet release points. Hornets can fly with 0.28 g tags if the tag:hornet ratio is less than 0.8. This method offers a step-change in options to tackle the spread of this invader, providing an efficient means of finding V. velutina nests in complex environments to manage this emerging threat to pollinators.

The influence of hydrous ferric oxide, earthworms, and a hypertolerant plant on arsenic and iron bioavailability, fate, and transport in soils.

Historic applications of lead arsenate pesticides and smelting activities have resulted in elevated concentrations of arsenic in Washington State soils. For example, old orchard topsoils in Washington have concentrations reaching upwards of 350 mg As/kg soil with an estimated 187,590 acres of arsenic contamination from pesticide application alone. Iron oxides have been indicated as a key factor in modulating the fate and transport of arsenic in the soil environment. We employed a factorial design to investigate the role of a specific iron oxide, hydrous ferric oxide (HFO), and terrestrial organisms on the mobility, bioavailability, and fate of arsenic and iron in locally collected soils. Earthworms in soils amended with both arsenic and HFO had 47.2 % lower arsenic tissue concentrations compared to those in soils only amended with arsenic. Similarly, arsenic leachate concentrations and plant tissue concentrations were lower when HFO was present, although this was with a reduced magnitude and was not consistently significant. A lack of significance of HFO in three of the linear models for leachate and plant bioavailability, however, indicates that the role of HFO in arsenic mobility, bioavailability, and fate is more complicated than can be explained by the simple addition or not of HFO. For example, our analyses showed that earthworms decreased pH and increased bioavailability for both arsenic and iron as demonstrated by increases in leachate and plant tissue concentrations. The mechanisms for this could include a biotransformation of earthworm-ingested arsenic combined with an earthworm-induced change in pH. We also found that arsenic amendments increased the mobility and bioavailability of iron, evidenced by increased iron concentrations in earthworms, plants, and leachate. A mechanistic explanation for this change in bioavailability is not readily apparent but does support a need for more work on bioavailability when mixtures are present. From these results, it is clear that a combination of biotic and abiotic factors influences metal/metalloid fate and transport in soils, with earthworms being one of the most important factors in our work. Study designs such as the factorial analysis can help to address the role each factor plays while efficiently generating new hypotheses and areas of inquiry; this approach can also bridge knowledge generated through reductionist and holistic approaches to complex environmental problems.

Guided growth for tibia vara (Blount's disease).

Blount's disease is commonly attributed to an intrinsic, idiopathic defect in the posteromedial proximal tibial physis resulting in progressive bowing of the leg, intoeing, and lateral knee thrust. Treatment has historically included bracing, physeal stapling, or corrective osteotomy, and was determined primarily by age at presentation. As we feel the pathology is not necessarily age dependent, we have elected to use the technique of guided growth using a lateral tension band plate to correct limb alignment as a first-line treatment in all patients presenting to our clinic as long as they had growth remaining and no evidence of a physeal bar.We identified 17 patients with tibia vara (27 limbs) who were managed by means of guided growth of the proximal tibia, from age 1.8 years to 15.1 years. Clinical and radiographic parameters were followed pre- and postoperatively. The response to guided growth was documented as were any related complications.Twenty-one (78%) limbs had complete normalization of their mechanical axis (middle 50% of knee). Time to correction averaged 13.5 months (8-19 months). There were no peri-operative complications. We observed hardware failure in 3 patients; 2 with screw breakage and 1 patient with hardware migration, none requiring subsequent osteotomy or further treatment. Two patients had rebound varus: one is being observed and another has undergone a repeat procedure.Patients with pathologic tibia vara present at various ages and have historically undergone various treatments ranging from bracing to tibial osteotomy based on age at presentation. We have found that guided growth utilizing tethering plates can be used effectively as first-line treatment in all patients with growth remaining. This minimally invasive method is predictable and well tolerated. Recurrent deformity, though unlikely, is easily remedied by repeating the process and does not preclude osteotomy if eventually needed. Concomitant resolution of ligamentous laxity and inward torsion can be anticipated as the mechanical axis is restored to neutral. The only contraindications for guided growth include an unresectable physeal bar or skeletal maturity.

Use of Rib-Based Distraction in the Treatment of Early-Onset Scoliosis Associated With Neurofibromatosis Type 1 in the Young Child.

STUDY DESIGN: This study was an institutional review board-approved retrospective review of a prospectively collected multicenter database for rib-based distraction systems used in the treatment of young children with early-onset scoliosis associated with neurofibromatosis type 1 (NF-1). OBJECTIVE: To evaluate the effectiveness of rib-based distraction and associated complications in managing scoliosis in the growing child with NF-1. SUMMARY OF BACKGROUND DATA: Patients with NF-1 commonly have scoliosis with curves that can be dysplastic and progressive and respond poorly to bracing. Rib-based distraction systems have recently been described in the management of these complex patients. The efficacy and complication rate using these systems have not previously been reported. METHODS: Twelve children with NF-1 and scoliosis who were treated with rib-based distraction systems were identified from a prospectively collected multicenter registry. Preoperative and postoperative Cobb angle and T1-S1 spine height were measured from posteroanterior radiographs. The number of lengthenings, age at implantation, years of follow-up, and complications were also acquired from the registry or patient charts. RESULTS: Mean age at implantation of the Vertical Expandable Prosthetic Titanium Rib device was 6.34 years. Mean preoperative Cobb angle was 66.3°. Average follow-up was 5.2 years. Mean postoperative Cobb angle was 60.8° after an average of 7.75 lengthening procedures (range, 2-16 procedures). T1-S1 height increased in all patients. There were 17 complications in 8 patients (device migration in 6, wound dehiscence in 3, rod breakage in 2, medical issues in 5, and 1 revision for progression of curve). Of the 17 complications, 10 were grade I, 1 was grade II, and 6 were grade IIA; there were no grade III complications. CONCLUSIONS: The use of rib-based distraction is an effective and relatively safe method of stabilizing curve progression through growth in severe dysplastic scoliosis associated with NF-1.