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USGS2022

Using biological responses to monitor freshwater post-spill conditions over 3 years in Blacktail Creek, North Dakota, USA

A pipeline carrying unconventional oil and gas (OG) wastewater spilled approximately 11 million liters of wastewater into Blacktail Creek, North Dakota, USA. Flow of the mix of stream water and wastewater down the channel resulted in storage of contaminants in the hyporheic zone and along the banks, providing a long-term source of wastewater constituents to the stream. A multi-level investigation was used to assess the potential effects of oil and brine spills on aquatic life. In this study, we used a combination of experiments using a native fish species, Fathead Minnow ( Pimephales promelas ), field sampling of the microbial community structure, and measures of estrogenicity. The fish investigation included in situ experiments and experiments with collected site water. Estrogenicity was measured in collected site water samples, and microbial community analyses were conducted on collected sediments. During the initial post-spill investigation, February 2015, performing in situ fish bioassays was impossible because of ice conditions. However, microbial community (e.g., the presence of members of the Halomonadaceae, a family that is indicative of elevated salinity) and estrogenicity differences were compared to reference sites and point to early biological effects of the spill. We noted water column effects on in situ fish survival 6 months post-spill during June 2015. At that time, total dissolved ammonium (sum of ammonium and ammonia, TAN) was 4.41 mg NH 4 /L with an associated NH 3 of 1.09 mg/L, a concentration greater than the water quality criteria established to protect aquatic life. Biological measurements in the sediment defined early and long-lasting effects of the spill on aquatic resources. The microbial community structure was affected during all sampling events. Therefore, sediment may act as a sink for constituents spilled and as such provide an indication of continued and cumulative effects post-spill. However, lack of later water column effects may reflect pulse hyporheic flow of ammonia from shallow ground water. Combining fish toxicological, microbial community structure and estrogenicity information provides a complete ecological investigation that defines potential influences of contaminants at organismal, population, and community levels. In general, in situ bioassays have implications for the individual survival and changes at the population level, microbial community structure defines potential changes at the community level, and estrogenicity measurements define changes at the individual and molecular level. By understanding effects at these various levels of biological organization, natural resource managers can interpret how a course of action, especially for remediation/restoration, might affect a larger group of organisms in the system. The current work also reviews potential effects of additional constituents defined during chemistry investigations on aquatic resources.

North Dakota↗

USGS2013

Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish ( Gambusia holbrooki )

Background The eastern mosquitofish ( Gambusia holbrooki ) has the potential to become a bioindicator organism of endocrine disrupting chemicals (EDCs) due to its androgen-driven secondary sexual characteristics. However, the lack of molecular information on G . holbrooki hinders its use as a bioindicator coupled with biomarker data. While traditional gene-by-gene approaches provide insight for biomarker development, a holistic analysis would provide more rapid and expansive determination of potential biomarkers. The objective of this study was to develop and utilize a mosquitofish microarray to determine potential biomarkers of subchronic androgen exposure. To achieve this objective, two specific aims were developed: 1) Sequence a G . holbrooki cDNA library, and 2) Use microarray analysis to determine genes that are differentially regulated by subchronic androgen exposure in hepatic tissues of 17β-trenbolone (TB) exposed adult female G . holbrooki . Results A normalized library of multiple organs of male and female G . holbrooki was prepared and sequenced by the Illumina GA IIx and Roche 454 XLR70. Over 30,000 genes with e-value ≤ 10 -4 were annotated and 14,758 of these genes were selected for inclusion on the microarray. Hepatic microarray analysis of adult female G . holbrooki exposed to the vehicle control or 1 μg/L of TB (a potent anabolic androgen) revealed 229 genes upregulated and 279 downregulated by TB (one-way ANOVA, p < 0.05, FDR α = 0.05, fold change > 1.5 and < −1.5). Fifteen gene ontology biological processes were enriched by TB exposure (Fisher’s Exact Test, p < 0.05). The expression levels of 17β - hydroxysteroid dehydrogenase 3 and zona pellucida glycoprotein 2 were validated by quantitative polymerase chain reaction (qPCR) (Student’s t-test, p < 0.05). Conclusions Coupling microarray data with phenotypic changes driven by androgen exposure in mosquitofish is key for developing this organism into a bioindicator for EDCs. Future studies using this array will enhance knowledge of the biology and toxicological response of this species. This work provides a foundation of molecular knowledge and tools that can be used to delve further into understanding the biology of G . holbrooki and how this organism can be used as a bioindicator organism for endocrine disrupting pollutants in the environment.

BMC Genomics↗

USGS2006

Toxicogenomics in regulatory ecotoxicology

Recently, we have witnessed an explosion of different genomic approaches that, through a combination of advanced biological, instrumental, and bioinformatic techniques, can yield a previously unparalleled amount of data concerning the molecular and biochemical status of organisms. Fueled partially by large, well-publicized efforts such as the Human Genome Project, genomic research has become a rapidly growing topical area in multiple biological disciplines. Since 1999, when the term “toxicogenomics” was coined to describe the application of genomics to toxicology (1), a rapid increase in publications on the topic has occurred (Figure 1). The potential utility of toxicogenomics in toxicological research and regulatory activities has been the subject of scientific discussions and, as with any new technology, has evoked a wide range of opinion (2–6).

Environmental Science & Technology↗

USGS2009

Measurement of soil carbon oxidation state and oxidative ratio by 13 C nuclear magnetic resonance

The oxidative ratio (OR) of the net ecosystem carbon balance is the ratio of net O 2 and CO 2 fluxes resulting from photosynthesis, respiration, decomposition, and other lateral and vertical carbon flows. The OR of the terrestrial biosphere must be well characterized to accurately estimate the terrestrial CO 2 sink using atmospheric measurements of changing O 2 and CO 2 levels. To estimate the OR of the terrestrial biosphere, measurements are needed of changes in the OR of aboveground and belowground carbon pools associated with decadal timescale disturbances (e.g., land use change and fire). The OR of aboveground pools can be measured using conventional approaches including elemental analysis. However, measuring the OR of soil carbon pools is technically challenging, and few soil OR data are available. In this paper we test three solid-state nuclear magnetic resonance (NMR) techniques for measuring soil OR, all based on measurements of the closely related parameter, organic carbon oxidation state (C ox ). Two of the three techniques make use of a molecular mixing model which converts NMR spectra into concentrations of a standard suite of biological molecules of known C ox . The third technique assigns C ox values to each peak in the NMR spectrum. We assess error associated with each technique using pure chemical compounds and plant biomass standards whose C ox and OR values can be directly measured by elemental analyses. The most accurate technique, direct polarization solid-state 13 C NMR with the molecular mixing model, agrees with elemental analyses to ??0.036 C ox units (??0.009 OR units). Using this technique, we show a large natural variability in soil C ox and OR values. Soil C ox values have a mean of -0.26 and a range from -0.45 to 0.30, corresponding to OR values of 1.08 ?? 0.06 and a range from 0.96 to 1.22. We also estimate the OR of the carbon flux from a boreal forest fire. Analysis of soils from nearby intact soil profiles imply that soil carbon losses associated with the fire had an OR of 1.09 1 (??0.00 3 ). Fire appears to be a major factor driving the soil C pool to higher oxidation states and lower OR values. Episodic fluxes caused by disturbances like fire may have substantially different ORs from ecosystem respiration fluxes and therefore should be better quantified to reduce uncertainties associated with our understanding of the global atmospheric carbon budget. Copyright 2009 by the American Geophysical Union.

Journal of Geophysical Research G: Biogeosciences↗

USGS1998

Molecular contributions to conservation

Recent advances in molecular technology have opened a new chapter in species conservation efforts, as well as population biology. DNA sequencing, MHC (major histocompatibility complex), minisatellite, microsatellite, and RAPD (random amplified polymorphic DNA) procedures allow for identification of parentage, more distant relatives, founders to new populations, unidentified individuals, population structure, effective population size, population-specific markers, etc. PCR (polymerase chain reaction) amplification of mitochondrial DNA, nuclear DNA, ribosomal DNA, chloroplast DNA, and other systems provide for more sophisticated analyses of metapopulation structure, hybridization events, and delineation of species, subspecies, and races, all of which aid in setting species recovery priorities. Each technique can be powerful in its own right but is most credible when used in conjunction with other molecular techniques and, most importantly, with ecological and demographic data collected from the field. Surprisingly few taxa of concern have been assayed with any molecular technique. Thus, rather than showcasing exhaustive details from a few well-known examples, this paper attempts to present a broad range of cases in which molecular techniques have been used to provide insight into conservation efforts.

Ecology↗

USGS2025

A molecular specimen bank for contemporary and future study captures landscape-scale biodiversity baselines before Klamath River dam removal

Global restoration and conservation of freshwater biodiversity are represented in practice by works such as the Klamath River Renewal Project (KRRP), the largest dam removal and river restoration in the United States, which has reconnected 640 river kilometers. With dam removals, many biological outcomes remain understudied due to a lack of pre-impact data and complex ecosystem recovery timeframes. To avoid this, we created the KRRP molecular library, an environmental specimen bank, for long-term curation of environmental nucleic acids collected from the restoration project. We used these initial samples, environmental DNA metabarcoding, and generalized linear mixed-effects models to evaluate patterns of pre-dam removal fish richness and diversity. Demonstrating the suitability to resolve biological differences, the baseline shows that tributary and mainstem streams had greater native fish diversity and 2.3–10.7 times greater native fish species richness than reservoirs. These and future sampling efforts should, at a minimum, allow tracking of fish community response to ecosystem restoration. Anticipating the acceleration of omics innovation, we preserved samples for long-term storage and identified requisite phases for sustained function and adaptation of the molecular library: securing a physical storage facility for genetic material, establishing a governance structure, and confirming support for archive management.

California, Oregon↗

USGS1982

Hydrates of natural gas in continental margins

Natural gas hydrates in continental margin sediment can be inferred from the widespread occurrence of an anomalous seismic reflector which coincides with the predicted transition boundary at the base of the gas hydrate zone. Direct evidence of gas hydrates is provided by visual observations of sediments from the landward wall of the Mid-America Trench off Mexico and Guatemala, from the Blake Outer Ridge off the southeastern United States, and from the Black Sea in the U.S.S.R. Where solid gas hydrates have been sampled, the gas is composed mainly of methane accompanied by CO 2 and low concentrations of ethane and hydrocarbons of higher molecular weight. The molecular and isotopic composition of hydrocarbons indicates that most of the methane is of biolog cal origin. The gas was probably produced by the bacterial alteration of organic matter buried in the sediment. Organic carbon contents of the sediment containing sampled gas hydrates are higher than the average organic carbon content of marine sediments. The main economic importance of gas hydrates may reside in their ability to serve as a cap under which free gas can collect. To be producible, however, such trapped gas must occur in porous and permeable reservoirs. Although gas hydrates are common along continental margins, the degree to which they are associated with significant reservoirs remains to be investigated.

Book chapter↗

USGS2015

Biological response to climate change in the Arctic Ocean: The view from the past

The Arctic Ocean is undergoing rapid climatic changes including higher ocean temperatures, reduced sea ice, glacier and Greenland Ice Sheet melting, greater marine productivity, and altered carbon cycling. Until recently, the relationship between climate and Arctic biological systems was poorly known, but this has changed substantially as advances in paleoclimatology, micropaleontology, vertebrate paleontology, and molecular genetics show that Arctic ecosystem history reflects global and regional climatic changes over all timescales and climate states (10 3 –10 7 years). Arctic climatic extremes include 25°C hyperthermal periods during the Paleocene-Eocene (56–46 million years ago, Ma), Quaternary glacial periods when thick ice shelves and sea ice cover rendered the Arctic Ocean nearly uninhabitable, seasonally sea-ice-free interglacials and abrupt climate reversals. Climate-driven biological impacts included large changes in species diversity, primary productivity, species’ geographic range shifts into and out of the Arctic, community restructuring, and possible hybridization, but evidence is not sufficient to determine whether or when major episodes of extinction occurred.

arktos↗

USGS2018

Molecular and morphological data reveal non-monophyly and speciation in imperiled freshwater mussels (Anodontoides and Strophitus)

Accurate taxonomic placement is vital to conservation efforts considering many intrinsic biological characteristics of understudied species are inferred from closely related taxa. The rayed creekshell, Anodontoides radiatus (Conrad, 1834), exists in the Gulf of Mexico drainages from western Florida to Louisiana and has been petitioned for listing under the Endangered Species Act. We set out to resolve the evolutionary history of A. radiatus, primarily generic placement and species boundaries, using phylogenetic, morphometric, and geographic information. Our molecular matrix contained 3 loci: cytochrome c oxidase subunit I, NADH dehydrogenase subunit I, and the nuclear-encoded ribosomal internal transcribed spacer I. We employed maximum likelihood and Bayesian inference to estimate a phylogeny and test the monophyly of Anodontoides and Strophitus. We implemented two coalescent-based species delimitation models to test seven species models and evaluate species boundaries within A. radiatus. Concomitant to molecular data, we also employed linear morphometrics and geographic information to further evaluate species boundaries. Molecular and morphological evidence supports the inclusion of A. radiatus in the genus Strophitus, and we resurrect the binomial Strophitus radiatus to reflect their shared common ancestry. We also found strong support for polyphyly in Strophitus and advocate the resurrection of the genus Pseudodontoideus to represent ‘Strophitus’ connasaugaensis and ‘Strophitus’ subvexus. Strophitus radiatus exists in six well-supported clades that were distinguished as evolutionary independent lineages using Bayesian inference, maximum likelihood, and coalescent-based species delimitation models. Our integrative approach found evidence for as many as 4 evolutionary divergent clades within S. radiatus. Therefore, we formally describe two new species from the S. radiatus species complex (Strophitus williamsi and Strophitus pascagoulaensis) and recognize the potential for a third putative species (Strophitus sp. cf. pascagoulaensis). Our findings aid stakeholders in establishing conservation and management strategies for the members of Anodontoides, Strophitus, and Pseudodontoideus.

Alabama, Florida, Louisiana, Mississippi↗

USGS2011

Gene expression changes in female zebrafish (Danio rerio) brain in response to acute exposure to methylmercury

Methylmercury (MeHg) is a potent neurotoxicant and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. The gene expression profile in adult female zebrafish whole brain induced by acute (96 h) MeHg exposure was investigated. Fish were exposed by injection to 0 or 0.5 µg MeHg/g. Gene expression changes in the brain were examined using a 22,000‐feature zebrafish microarray. At a significance level of p < 0.01, 79 genes were up‐regulated and 76 genes were down‐regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxicants and will investigate responsive genes as potential biomarkers of MeHg exposure.

Environmental Toxicology and Chemistry↗

USGS2018

Next‐generation conservation genetics and biodiversity monitoring

This special issue of Evolutionary Applications consists of 10 publications investigating the use of next‐generation tools and techniques in population genetic analyses and biodiversity assessment. The special issue stems from a 2016 Next Generation Genetic Monitoring Workshop, hosted by the National Institute for Mathematical and Biological Synthesis (NIMBioS) in Tennessee, USA. The improved accessibility of next‐generation sequencing platforms has allowed molecular ecologists to rapidly produce large amounts of data. However, with the increased availability of new genomic markers and mathematical techniques, care is needed in selecting appropriate study designs, interpreting results in light of conservation concerns, and determining appropriate management actions. This special issue identifies key attributes of successful genetic data analyses in biodiversity evaluation and suggests ways to improve analyses and their application in current population and conservation genetics research.

Evolutionary Applications↗

USGS2009

Biomineralization associated with microbial reduction of Fe 3+ and oxidation of Fe 2+ in solid minerals

Iron-reducing and oxidizing microorganisms gain energy through reduction or oxidation of iron, and by doing so play an important role in the geochemical cycling of iron. This study was undertaken to investigate mineral transformations associated with microbial reduction of Fe 3+ and oxidation of Fe 2+ in solid minerals. A fluid sample from the 2450 m depth of the Chinese Continental Scientific Drilling project was collected, and Fe 3+ -reducing and Fe 2+ -oxidizing microorganisms were enriched. The enrichment cultures displayed reduction of Fe 3+ in nontronite and ferric citrate, and oxidation of Fe 2+ in vivianite, siderite, and monosulfide (FeS). Additional experiments verified that the iron reduction and oxidation was biological. Oxidation of FeS resulted in the formation of goethite, lepidocrocite, and ferrihydrite as products. Although our molecular microbiological analyses detected Thermoan-aerobacter ethanolicus as a predominant organism in the enrichment culture, Fe 3+ reduction and Fe 2+ oxidation may be accomplished by a consortia of organisms. Our results have important environmental and ecological implications for iron redox cycling in solid minerals in natural environments, where iron mineral transformations may be related to the mobility and solubility of inorganic and organic contaminants.

American Mineralogist↗

USGS2017

Uptake and disposition of select pharmaceuticals by bluegill exposed at constant concentrations in a flow-through aquatic exposure system

The increasing use of pharmaceuticals has led to their subsequent input into and release from wastewater treatment plants, with corresponding discharge into surface waters that may subsequently exert adverse effects upon aquatic organisms. Although the distribution of pharmaceuticals in surface water has been extensively studied, the details of uptake, internal distribution, and kinetic processing of pharmaceuticals in exposed fish have received less attention. For this research, we investigated the uptake, disposition, and toxicokinetics of five pharmaceuticals (diclofenac, methocarbamol, rosuvastatin, sulfamethoxazole, and temazepam) in bluegill sunfish ( Lepomis macrochirus ) exposed to environmentally relevant concentrations (1000–4000 ng L –1 ) in a flow-through exposure system. Temazepam and methocarbamol were consistently detected in bluegill biological samples with the highest concentrations in bile of 4, 940, and 180 ng g –1 , respectively, while sulfamethoxazole, diclofenac, and rosuvastatin were only infrequently detected. Over 30-day exposures, the relative magnitude of mean concentrations of temazepam and methocarbamol in biological samples generally followed the order: bile ≫ gut > liver and brain > muscle, plasma, and gill. Ranges of bioconcentration factors (BCFs) in different biological samples were 0.71–3960 and 0.13–48.6 for temazepam and methocarbamol, respectively. Log BCFs were statistically positively correlated to pH adjusted log K ow (that is, log D ow ), with the strongest relations for liver and brain ( r 2 = 0.92 and 0.99, respectively), implying that bioconcentration patterns of ionizable pharmaceuticals depend on molecular status, that is, whether a pharmaceutical is un-ionized or ionized at ambient tissue pH. Methocarbamol and temazepam underwent rapid uptake and elimination in bluegill biological compartments with uptake rate constants ( K u ) and elimination rate constants ( K e ) at 0.0066–0.0330 h –1 and 0.0075–0.0384 h –1 , respectively, and half-lives at 18.1–92.4 h. Exposure to mixtures of diclofenac, methocarbamol, sulfamethoxazole, and temazepam had little or no influence on the uptake and elimination rates, suggesting independent multiple uptake and disposition behaviors of pharmaceuticals by fish would occur when exposed to effluent-influenced surface waters.

Environmental Science & Technology↗

USGS2021

Evaluation of a modified rapid viability-polymerase chain reaction method for Bacillus atrophaeus spores in water matrices

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viability-polymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non‑chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods.

Journal of Microbiological Methods↗

USGS2019

Successful molecular detection studies require clear communication among diverse research partners

Molecular detection techniques are powerful tools used in ecological applications ranging from diet analyses to pathogen surveillance. Research partnerships that use these tools often involve collaboration among professionals with expertise in field biology, laboratory techniques, quantitative modeling, wildlife disease, and natural resource management. However, in many cases, each of these collaborators lacks specific knowledge about the approaches, decisions, methods, and terminology used by their research partners, which can impede effective communication and act as a barrier to the efficient use of molecular data for ecological inferences and subsequent conservation decision making. We outline a collaborative framework to assist colleagues with diverse types of expertise to effectively translate their scientific and management needs to research partners from other specialties. The molecular techniques used to detect organisms will continue to advance both in sophistication and in the breadth of ecological applications. Our objective is to enable ecologists to harness the full utility of these methods by developing effective collaborative partnerships.

Frontiers in Ecology and the Environment↗

USGS2010

A population genetic analysis of the midget faded rattlesnake in Wyoming

Little is known about the population biology of midget faded rattlesnakes, a sensitive subspecies of the Western Rattlesnake, despite conservation efforts to protect them. We conducted a molecular genetic study of midget faded rattlesnakes in southwestern Wyoming to investigate population genetic structure in this area, particularly with reference to Flaming Gorge Reservoir and its associated human activities, and to document levels of genetic diversity. We genotyped 229 snakes from 11 sampling sites using 9 microsatellite loci. We found significant levels of genetic structure among sites that were better explained by geographic region and isolation by distance than by position relative to waterways. Sites on either side of the reservoir at its widest point were not significantly different. Six of the sites showed signatures of a population bottleneck using an alpha value of 0.05. Three of these bottlenecked sites (the three most northern) were the most genetically distinct and occur in areas of greatest impact from human activity.

Wyoming↗

USGS2017

Biocrust ecology: Unifying micro- and macro-scales to confront global change

Biological soil crusts (biocrusts) are communities of microbes, lichens and bryophytes living at the soil surface in drylands (Fig. 1; Belnap et al ., 2016). Biocrusts occur on all continents and can comprise a majority of cover in some systems (Belnap et al ., 2016). While species diversity and distributions have long been a research focus, interest in controls on community composition and cover has expanded as biocrusts are increasingly recognized for their roles in ecosystem functioning (Deane-Coe and Stanton, 2017). For example, biocrust organisms can stabilize soils (Belnap et al ., 2016; Faist et al ., 2017), fix atmospheric carbon (C) (Sancho et al ., 2016), and serve as the foremost source of ‘new’ soil nitrogen (N) in drylands, via N 2 fixation (Barger et al ., 2016) These contributions to gross primary production and soil fertility could be quite large, as high-end estimates suggest biocrusts and similar communities of bryophytes and lichens might account for 10% of terrestrial C- and 50% of N-fixation globally (Elbert et al ., 2012). Yet verifying these and other biocrust roles in ecosystem functioning is complicated by limited knowledge of biocrust cover and composition across the vast dryland biome (Ferrenberg et al ., 2017). It was against this backdrop that ‘Biocrust3: the 3 rd International workshop on biological soil crusts’ was held in Moab, UT, USA, on 26-30 September 2016. The workshop brought together over 50 scientists from 21 countries and six continents, and included numerous biocrust science pioneers (Fig. 2). The meeting was notable for its cross-scale focus, discussion of novel molecular and imaging techniques, and sessions on mapping and restoring biocrusts in a changing world. Here, we synthesize a central theme that emerged from Biocrust3, namely the potential for combining cutting edge tools with studies focused on organismal traits, ecosystem functions, and global change biology to advance the frontier of biocrust ecology.

New Phytologist↗

USGS2011

Experimental reintroduction of the federally endangered Santa Cruz Island bush mallow (Malacothamnus fasciculatus var. nestioticus)

Studies of Malacothamnus fasciculatus var. nesioticus were begun in 1995 to understand its distribution, reproductive biology and ecological requirements. After 100+ years of depredation by sheep, two known populations of fewer than 20 plants each survived in 1995. Molecular studies showed that each of the two populations was composed of 1–3 genets. During our study, two additional populations of similar size were discovered. Plants are self-compatible but require insect visitation to augment pollination. Based on seed set, viable embryos, and germination rates, we found no evidence for inbreeding depression. Bush mallow also reproduces vegetatively by rhizomes, the primary means of establishment and persistence in natural populations, and a key feature for maximizing recovery success. Ex situ observations and trial in situ outplantings suggested that supplemental watering was critical to initial survival. We developed a recovery strategy composed of four plots located at varying elevations and aspects. Each plot was enclosed to exclude feral pigs, which posed a continuing threat. Each plot was planted with twelve rooted plants derived from each of three natural populations. Plants were provided supplemental watering for four months. Survivorship after one year ranged from 46% to 91%. Significant differences in survivorship were observed relative to source of plants. After twelve months some plants were flowering and reproducing vegetatively.

California↗