Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Biogeochemical Stoichiometry Reveals P and N Limitation Across the Post-glacial Landscape of Denali National Park,Alaska

Global warming has accelerated glacial retreat in high-elevation and high-latitude ecosystems, exposing new terrain that can undergo predictable patterns of ecosystem succession, especially in coastal areas with relatively mild climates. However, little work has been done in harsher high-elevation and inland areas where the rate of plant and microbial succession may be greatly slowed by dryness and low temperatures. The present study is the first to address microbial succession at a major glacial foreland (the Middle Fork Toklat Glacier) in the interior of Alaska. We used a spatially nested sampling regime to reveal the landscape patterns in microbial activity and biogeochemical pools during the pre-plant stage of primary succession along this high-elevation and high-latitude chronosequence. Recently deglaciated soils (0–10 years) were colonized by a diverse microbial community that included many chemoautotrophs that likely subsist on high levels of un-weathered minerals (for example, pyrite) found at this site. Rates of N-fixation and extracellular enzyme activities were very low in the youngest soils sampled, but increased during the first 20 years of succession coinciding with a decrease in TOC and C:N levels. In older soils (20–54 years), TOC and TON increased and IN became undetectable perhaps indicating N limitation. Indicators of microbial activity stopped increasing 20 years post de-glaciation and remained at levels well below those seen at lower elevation and lower latitude sites, perhaps indicating severe nutrient limitations. Stoichiometric analyses also indicated phosphorus and nitrogen limitation across the entire chronosequence, with no indication of carbon limitation of microbial activity. These results indicate that nutrient limitation, rather than the constraints of a severe climate, may be the dominant factor slowing the rate of succession at high-latitude and high-altitude glacial forelands.     

Wind dispersal of soil invertebrates in the McMurdo Dry Valleys, Antarctica

Dispersal of soil organisms is crucial for their spatial distribution and adaptation to the prevailing conditions of the Antarctic Dry Valleys. This study investigated the possibility of wind dispersal of soil invertebrates within the dry valleys. Soil invertebrates were evaluated in (1) pockets of transported sediments to lake ice and glacier surfaces, (2) wind-transported dust particles in collection pans (Bundt pans) 100 cm above the soil surface, and (3) sediments transported closer to the surface (<50 cm) and collected in open top chambers (OTCs). Invertebrates were extracted and identified. Nematodes were identified to species and classified according to life stage and sex. Three species of nematodes were recovered and Scottnema lindsayae was the most dominant. There were more juveniles (∼71%) in the transported sediments than adults (29%). Tardigrades and rotifers were more abundant in sediments on lake and glacier surfaces while nematodes were more abundant in the dry sediment collections of Bundt pans and OTCs. The abundance of immobile (dead) nematodes in the Bundt pans and OTCs was three times greater than active (live) nematodes. Anhydrobiosis constitutes a survival mechanism that allows wind dispersal of nematodes in the McMurdo Dry Valleys. Our results show that soil invertebrates are dispersed by wind in the Dry Valleys and are viable in ice communities on lake surfaces and glaciers.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.