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Microbial generation of coal bed methane (CBM) represents a significant source of natural gas on Earth. While biostimulation has been demonstrated in batch cultures, environmental parameters such as overburden pressure and formation water flow need to be tested at the laboratory scale to understand in situ potential. We designed and constructed a high-pressure (HP) flow-through reactor system that simulates in situ conditions of underground coal seams. Two stainless-steel columns contained coal from the Powder River Basin (PRB), USA, or a coal/sand mixture to represent the interface of coal seams with sandstone layers, which are hypothesized to exhibit higher methanogenesis rates in situ. The system was filled with CBM formation water, inoculated with a methanogenic enrichment from PRB coal beds, and stimulated with algal biomass as a nutrient. The reactors were incubated under pressure (5.4 atm) and flow of CBM water (0.01 mL/min), and control batch cultures were incubated at ambient pressure and without flow (± amendment). Dissolved and headspace methane concentrations were analyzed over time by gas chromatography for 75 days. The pressurized reactors exhibited longer latency periods than ambient pressure controls, but methane production did not reach a plateau phase, which might reflect the impact of scale on the inoculum. The coal/sand reactor exhibited higher methane production than the coal-only reactor, a pattern also observed in the corresponding controls, suggesting an interface effect on methanogenesis. This study indicates that the HP flow test system we designed is well suited for the study of methanogenesis and provides a successful demonstration of CBM generation from the PRB in field-relevant laboratory conditions as a precursor to meso‑scale demonstrations.

Microbial ammonia oxidation is the first and usually rate limiting step in nitrification and is therefore an important step in the global nitrogen cycle. Ammonia-oxidizing archaea (AOA) play an important role in nitrification. Here, we report a comprehensive analysis of biomass productivity and the physiological response of Nitrososphaera viennensis to different ammonium and carbon dioxide (CO2) concentrations aiming to understand the interplay between ammonia oxidation and CO2 fixation of N. viennensis. The experiments were performed in closed batch in serum bottles as well as in batch, fed-batch, and continuous culture in bioreactors. A reduced specific growth rate (μ) of N. viennensis was observed in batch systems in bioreactors. By increasing CO2 gassing μ could be increased to rates comparable to that of closed batch systems. Furthermore, at a high dilution rate (D) in continuous culture (≥ 0.7 of μmax) the biomass to ammonium yield (Y(X/NH3)) increased up to 81.7% compared to batch cultures. In continuous culture, biofilm formation at higher D prevented the determination of Dcrit. Due to changes in Y(X/NH3) and due to biofilm, nitrite concentration becomes an unreliable proxy for the cell number in continuous cultures at D towards μmax. Furthermore, the obscure nature of the archaeal ammonia oxidation prevents an interpretation in the context of Monod kinetics and thus the determination of KS. Our findings indicate that the physiological response of N. viennensis might be regulated with different enzymatic make-ups, according to the ammonium catalysis rate. We reveal novel insights into the physiology of N. viennensis that are important for biomass production and the biomass yield of AOA. Moreover, our study has implications to the field of archaea biology and microbial ecology by showing that bioprocess technology and quantitative analysis can be applied to decipher environmental factors affecting the physiology and productivity of AOA.

Numerous populations from six spruce taxa, including four relict endemics, Picea chihuahuana (Chihuahua spruce), P. martinezii (Martinez spruce), P. mexicana (Mexican spruce), and P. breweriana (Brewer spruce), and two widespread species, P. engelmannii (Engelmann spruce) and P. pungens (blue spruce), were compared at homologous isozyme loci to test various hypotheses about their affinities and origins. Each of the species was clearly separated, and Neighbor-joining and Unweighted Pair Group analyses of Nei's genetic distance grouped all populations within a taxon into their own clusters. Spruces from Flys Peak, Chiricahua Mountains, Arizona, joined a P. engelmannii cluster and were not a bridge to P. mexicana as previously believed. Spruces from Cerro Mohinora, Chihuahua, were clearly P. mexicana, not phantom hybrids of P. chihuahuana and R pungens. Nuclear random amplified polymorphic DNA and chloroplast simple sequence repeat and cleaved amplified polymorphic genetic markers were compared in a smaller sample of populations, using distance and parsimony approaches. DNA markers, like isozymes, clearly identified spruces from Cerro Mohinora as P. mexicana. In contradiction to the most recent taxonomic treatment, P. chihuahuana and P. martinezii were separated as distinct species by both isozyme and DNA markers, and formed a sister-species group. Picea engelmannii and P. mexicana formed a separate cluster, and the genetic distance between them was similar to values associated with closely related species but greater than distances typical of subspecies or varieties in conifers. Picea pungens, which is so similar to P. engelmannii that the two are frequently misidentified, was clearly distinguished from it, sometimes joining a P. chihuahuana-martinezii group and sometimes a P. engelmannii-mexicana group, depending on analysis. Picea breweriana was well isolated from all other taxa. Both DNA and isozyme phylogenies agreed with results from crossability studies and contradicted intrageneric relationships constructed largely on cone morphology.

Abstract Rare species present a challenge under changing environmental conditions as the genetic consequences of rarity may limit species ability to adapt to environmental change. To evaluate the evolutionary potential of a rare species, we assessed variation in traits important to plant fitness using multigenerational common garden experiments. Torrey pine, Pinus torreyana Parry, is one of the rarest pines in the world, restricted to one mainland and one island population. Morphological differentiation between island and mainland populations suggests adaptation to local environments may have contributed to trait variation. The distribution of phenotypic variances within the common garden suggests distinct population‐specific growth trajectories underlay genetic differences, with the island population exhibiting substantially reduced genetic variance for growth relative to the mainland population. Furthermore, F1 hybrids, representing a cross between mainland and island trees, exhibit increased height accumulation and fecundity relative to mainland and island parents. This may indicate genetic rescue via intraspecific hybridization could provide the necessary genetic variation to persist in environments modified as a result of climate change. Long‐term common garden experiments, such as these, provide invaluable resources to assess the distribution of genetic variance that may inform conservation strategies to preserve evolutionary potential of rare species, including genetic rescue.

We describe colour, hardness, grain size, geological type and surface modifications of ochre pieces excavated, first by Malan and later by Harper, from the Middle Stone Age (MSA) of Rose Cottage Cave, 96, 000 to 30, 000 years ago. Soft, bright-red shales are abundant, and most ochre has clayey or silty grain sizes. The post-Howiesons Poort layers contain the most ochre pieces, but the Howiesons Poort layers have the highest frequency of ochre per sediment volume. The pre-Howiesons Poort layers have the highest utilization rate. Use-traces include rubbing, grinding, combined grinding and rubbing, and rare instances of scoring. The processing techniques are proxies for ochre use. Rubbing transfers red ochre powder directly onto soft surfaces, such as human skin, or animal hide. This is appropriate when skin colouring and marking or skin protection (for example from sun, insects or bacteria) is the purpose. Grinding produces ochre powder that can be used for a variety of tasks. It can be mixed with water or other products to create paint, cosmetics or adhesives. Multiple uses of ochre powder and ochre pieces are therefore implied at Rose Cottage and changes through time are apparent.

Kaingo Sheep Rock Shelter was used by Later Stone Age (LSA) hunter-gatherers between 4370±180 and 170±30 BP. The site has rock art that includes a fine-line painting of a large, fat-tailed sheep, animal finger paintings, and geometric motifs. There are many microlithic end scrapers, a few backed tools, and more than 500 complete, incomplete and broken ostrich eggshell beads, as well as grooved stones and worked bone. By ~170 BP the density of material culture items reduced and the shelter may have been used only occasionally for ritual purposes like rain-making or initiations. Hunters, herders and farmers are represented in one way or another in the shelter, but it is unclear whether residential and non-residential 'time-share' is involved during the contact period. Since the shelter has contemporaneous LSA and Iron Age material culture signatures, there may have been sporadic interaction between the groups. Most ceramics belong to the Eiland facies, but a few fragments of one of the earliest ceramics found in southern Africa, the Bambata facies, were also discovered. Seed and charcoal identifications reveal bushveld vegetation similar to that of today, but possible evidence for mopane trees in the last 1 900 years implies greater diversity of plant life at that time. [ABSTRACT FROM AUTHOR]

Coal bed methane (CBM) production has resulted in thousands of ponds in the Powder River Basin of low-quality water in a water-challenged region. A green alga isolate, PW95, was isolated from a CBM production pond, and analysis of a partial ribosomal gene sequence indicated the isolate belongs to the Chlorococcaceae family. Different combinations of macro- and micronutrients were evaluated for PW95 growth in CBM water compared to a defined medium. A small level of growth was observed in unamended CBM water (0.15 g/l), and biomass increased (2-fold) in amended CBM water or defined growth medium. The highest growth rate was observed in CBM water amended with both N and P, and the unamended CBM water displayed the lowest growth rate. The highest lipid content (27%) was observed in CBM water with nitrate, and a significant level of lipid accumulation was not observed in the defined growth medium. Growth analysis indicated that nitrate deprivation coincided with lipid accumulation in CBM production water, and lipid accumulation did not increase with additional phosphorus limitation. The presented results show that CBM production wastewater can be minimally amended and used for the cultivation of a native, lipid-accumulating alga. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine differences in muscular activation between males and females while walking in restricted postures. Restricted postures are evident in various industries, including mining, construction and agriculture. These postures are associated with musculoskeletal disorders and lower back pain. Studies generally focus on a male workforce; however, more females are entering industrial workplaces. Twelve male and 12 female subjects between the ages of 18 and 25 years participated in the study. Subjects walked on a treadmill at a speed of 3.5 km/h for four minutes under conditions of upright walking, and stooped walking under restrictions at 85% and 70% of stature. Electromyographic activity was measured on seven muscles (trapezius, latissimus dorsi, erector spinae, rectus femoris, biceps femoris, medial gastrocnemius and tibialus anterior). Ratings of Perceived Exertion (RPE) and Body Discomfort were also obtained. The extent of vertical restriction significantly altered levels of muscle activation. Female subjects had significantly lower levels of activation of the medial gastrocnemius than males. Local RPE was greatest under the lowest restriction, and body discomfort of the neck, lower back and hamstrings was evident during restricted walking. Work place design and interventions should consider these consequences. [ABSTRACT FROM AUTHOR]

The properties of ochre from the Middle Stone Age (MSA) layers of Sibudu, KwaZulu-Natal, South Africa, are described here. The assemblage comprises 5449 pieces (>8 mm), including 682 pieces with markings from use. Shale is the most common geological form. Ochre of medium hardness is common and the grain sizes are generally silty or clayey. Some change in ochre types occurs through time. The pre- Still Bay and Howiesons Poort layers have the highest percentage of utilised pieces. Clayey grain sizes are favoured during the Still Bay and Howiesons Poort, whereas silty grain sizes are preferred in the younger MSA occupations. Bright-red ochre was preferentially selected for use throughout the sequence, with the exception of the final MSA where a wider range of colours was utilised. High frequencies of red amongst the utilised pieces, coupled with high frequencies of yellow or orange pieces with no evidence of use, suggest that colour choices were deliberate and not a product of post-depositional heating. Chemical analysis on a sample of utilised pieces indicates that they all contain iron, silicon, aluminium and calcium. Many pieces contain hematite and some maghemite. There is evidence of preferential selection of specific types of ochre for use in the Sibudu assemblage. [ABSTRACT FROM AUTHOR]

A correction to the author's affiliation in the article "Comparisons of Muscular Activity in Males and Females While Walking in Restricted Postures", in the 2013 issue is presented .

Sugar pine, Pinus lambertiana Douglas, is a keystone species of montane forests from Baja California to southern Oregon. Like other North American white pines, populations of sugar pine have been greatly reduced by the disease white pine blister rust (WPBR) caused by a fungal pathogen, Cronartium ribicola , that was introduced into North America early in the twentieth century. Major gene resistance to WPBR segregating in natural populations has been documented in sugar pine. Indeed, the dominant resistance gene in this species, Cr1 , was genetically mapped, although not precisely. Genomic single nucleotide polymorphisms (SNPs) placed in a large scaffold were reported to be associated with the allele for this major gene resistance ( Cr1 R ). Forest restoration efforts often include sugar pine seed derived from the rare resistant individuals (typically Cr1 R / Cr1 r ) identified through an expensive 2-year phenotypic testing program. To validate and geographically characterize the variation in this association and investigate its potential to expedite genetic improvement in forest restoration, we developed a simple PCR-based, diploid genotyping of DNA from needle tissue. By applying this to range-wide samples of susceptible and resistant ( Cr1 R ) trees, we show that the SNPs exhibit a strong, though not complete, association with Cr1 R . Paralleling earlier studies of the geographic distribution of Cr1 R and the inferred demographic history of sugar pine, the resistance-associated SNPs are marginally more common in southern populations, as is the frequency of Cr1 R . Although the strength of the association of the SNPs with Cr1 R and thus, their predictive value, also varies with geography, the potential value of this new tool in quickly and efficiently identifying candidate WPBR-resistant seed trees is clear.

Identifying and quantifying the importance of environmental variables in structuring population genetic variation can help inform management decisions for conservation, restoration, or reforestation purposes, in both current and future environmental conditions. Landscape genomics offers a powerful approach for understanding the environmental factors that currently associate with genetic variation, and given those associations, where populations may be most vulnerable under future environmental change. Here, we applied genotyping by sequencing to generate over 11,000 single nucleotide polymorphisms from 311 trees and then used nonlinear, multivariate environmental association methods to examine spatial genetic structure and its association with environmental variation in an ecologically and economically important tree species endemic to Hawaii, Acacia koa . Admixture and principal components analyses showed that trees from different islands are genetically distinct in general, with the exception of some genotypes that match other islands, likely as the result of recent translocations. Gradient forest and generalized dissimilarity models both revealed a strong association between genetic structure and mean annual rainfall. Utilizing a model for projected future climate on the island of Hawaii, we show that predicted changes in rainfall patterns may result in genetic offset, such that trees no longer may be genetically matched to their environment. These findings indicate that knowledge of current and future rainfall gradients can provide valuable information for the conservation of existing populations and also help refine seed transfer guidelines for reforestation or replanting of koa throughout the state.

Rare species present a challenge under changing environmental conditions as the genetic consequences of rarity may limit species ability to adapt to environmental change. To evaluate the evolutionary potential of a rare species, we assessed variation in traits important to plant fitness using multigenerational common garden experiments. Torrey pine, Pinus torreyana Parry, is one of the rarest pines in the world, restricted to one mainland and one island population. Morphological differentiation between island and mainland populations suggests adaptation to local environments may have contributed to trait variation. The distribution of phenotypic variances within the common garden suggests distinct population-specific growth trajectories underlay genetic differences, with the island population exhibiting substantially reduced genetic variance for growth relative to the mainland population. Furthermore, F1 hybrids, representing a cross between mainland and island trees, exhibit increased height accumulation and fecundity relative to mainland and island parents. This may indicate genetic rescue via intraspecific hybridization could provide the necessary genetic variation to persist in environments modified as a result of climate change. Long-term common garden experiments, such as these, provide invaluable resources to assess the distribution of genetic variance that may inform conservation strategies to preserve evolutionary potential of rare species, including genetic rescue.

The genetic structure of Cycas seemannii A.Br. (Cycadaceae), sampled throughout its range in Vanuatu, New Caledonia, Fiji, and Tonga, was studied using starch-gel electrophoresis. Twenty enzyme loci in 13 enzyme systems were examined. Low genetic diversity within populations (A = 1.2, P = 21.3, Ho = 0.047, and He = 0.057) and a high degree of differentiation among populations (F(ST) = 0.594) were found. This, together with low gene flow estimates, suggests genetic drift by isolation to have been most critical to the current genetic structure of the species. Inbreeding may occur to some extent (F(IS) = 0.165). The decline in abundance of C. seemannii, coupled with the low level of genetic diversity, suggest that conservation strategies are urgently needed.

Weeping piñon (Pinus pinceana) has a restricted and fragmented range, trees are widely scattered within populations, and reproduction is limited. Nevertheless, genetic diversity was high; based on 27 isozyme loci in 18 enzyme systems, unbiased expected heterozygosity averaged 0.174. Differentiation also was high (F(ST) = 0.152), reflecting isolation between southern, central, and northern fragments of the range. Among populations in the northern fragment, F(ST) was only 0.056, and the number of migrants per generation (Nm) was 4.21, which should preclude fixation. Nm between central and southern populations or between them and populations in the northern fragment was lower, 0.99-1.66, indicating a degree of genetic isolation. Multilocus outcrossing rates (t(m)) ranged from 0.836 in the south to 0.897 in the north. Therefore, selfing is low but statistically significant. The equilibrium inbreeding coefficient (F(e)) calculated from t(m) was in good agreement with observed inbreeding coefficients, suggesting that weeping piñon may be near equilibrium with respect to inbreeding and selection against selfed trees. Weeping piñon was variable at all loci polymorphic in maxipiñon (Pinus maximartinezii) and, therefore, qualifies as a possible progenitor of maxipiñon. Because of the high level of diversity, reasonable levels of gene flow within the northern fragment of weeping piñon's range, high rates of outcrossing, and, perhaps, only weak selection against inbred trees, protection in reserves would be a viable option for conservation.

Stands of canyon live oak (Quercus chrysolepis, Fagaceae) are maintained for fuelwood, fire management, recreation, and as habitat for wildlife. Information about the link between the oak's reproductive ecology and its extent of genetic diversity is important in developing land management policies that will maintain the long-term viability of populations. Basal sprouting is the primary means of reproduction following fire or cutting, and stands frequently include groups of visibly connected trees in a clustered distribution that suggests cloning. We determined the extent to which clusters of trees were clonal and defined the spatial pattern and diversity of genotypes for six populations across nearly the entire east-west extent of the San Bernardino Mountains in southern California. We mapped over 100 trees at each of five sites and genotyped each tree for allozymes at seven polymorphic loci. We identified clones using these multilocus genotypes and detected an average of 34.4 ± 7.3 (SD) clones per site, most of which had unique genotypes. In general, clustered trees belong to single clones and most clones consist of few trees (mean = 3.4 ± 0.6 trees per clone). However, clone size increased significantly with increased individual heterozygosity, suggesting that selection may favor highly heterozygous clones. Clonal diversity and evenness were high relative to reports for most other clonal species; an average of 97% of clones had distinct genotypes, and Simpson's index of diversity averaged 0.95 ± 0.02. Population genetic analyses of 319 clones from six sites revealed high genetic diversity within sites (mean HS = 0.443). Only a small proportion of the total genetic diversity was explained by variation among sites (mean GST = 0.018), which is consistent with high gene flow among sites (Nm = 9.5). We found no significant substructure among plots within sites, and fixation indices within sites were generally small, suggesting that either little inbreeding occurs, and/or few inbred progeny survive. However, spatial autocorrelation analysis of clones indicated fine-scale genetic structure at distances under 4 m, possibly due to limited seed dispersal. Our data suggest that guidelines for seed collection of canyon live oak for use in restoration can be specified in a manner similar to that recommended for conifer species within the region studied.