Low genetic diversity and population connectivity fuel vulnerability to climate change for the Tertiary relict pine Pinus bungeana

Endemic species are important components of regional biodiversity and hold the key to understanding local adaptation and evolutionary processes that shape species distributions. This study investigated the biogeographic history of a relict conifer Pinus bungeana Zucc. ex Endl. confined to central China. We examined genetic diversity in P. bungeana using genotyping-by-sequencing and chloroplast and mitochondrial DNA markers. We performed spatial and temporal inference of recent genetic and demographic changes, and dissected the impacts of geography and environmental gradients on population differentiation. We then projected P. bungeana's risk of decline under future climates. We found extremely low nucleotide diversity (average π 0.0014), and strong population structure (global F_ST 0.234) even at regional scales, reflecting long-term isolation in small populations. The species experienced severe bottlenecks in the early Pliocene and continued to decline in the Pleistocene in the western distribution, whereas the east expanded recently. Local adaptation played a small (8%) but significant role in population diversity. Low genetic diversity in fragmented populations makes the species highly vulnerable to climate change, particularly in marginal and relict populations. We suggest that conservation efforts should focus on enhancing gene pool and population growth through assisted migration within each genetic cluster to reduce the risk of further genetic drift and extinction.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

In vitro oxygen consumption and motility of Cryptobia salmositica, Cryptobia bullocki, and Cryptobia catostomi (Sarcomastigophora: Kinetoplastida).

Cryptobia salmositica (pathogenic and vaccine strains), Cryptobia bullocki (pathogenic), and Cryptobia catostomi (nonpathogenic) have similar oxygen consumption rates (0.17 +/- 0.01 nm O2/10(6) parasites). Incubation with sodium azide (5 microliters of a 1-M solution to 1 ml of parasite suspension, i.e., a 5-mM final concentration) reduced the oxygen consumption by approximately 4.5-fold. Motility of the parasites was also greatly reduced in sodium azide. The oxygen consumption and motility of the parasites returned to preazide treatment levels when the azide was removed even after 24 hr of incubation in sodium azide. The activities of hexokinase, pyruvate kinase, and cytochrome C oxidase were not detected in the 3 species of Cryptobia.     

Vector specificity of Trypanosoma catostomi and its infectivity to freshwater fishes.

Trypanosoma catostomi was found in 36.2% of 558 white suckers (Catostomus commersoni) from Ontario, Canada. The abundance of Actinobdella inequiannulata was 35% (68 leeches/197 suckers examined for leeches). The susceptibility of 3 species of leeches (Hemiclepsis marginata, Desserobdella phalera, and A. inequiannulata) and 7 species of fishes (C. commersoni, Amia calva, Anguilla rostrata, Ictalurus nebulosus, Oncorhynchus mykiss, Perca flavescens, and Esox lucius) to infection with T. catostomi was examined. Metatrypanosomes were found in the crop and proboscis sheath of 13 of 21 A. inequiannulata and in the crop of 10 of 12 H. marginata and 1 of 21 D. phalera. Only flagellates from A. inequiannulata were infective to C. commersoni. Cultured T. catostomi infected C. commersoni and A. calva but not any other fish species. Laboratory-reared C. commersoni were more susceptible than wild-caught specimens. Cultured Trypanosoma phaleri did not infect its natural host, A. calva. Host specificity should be established experimentally before a specific diagnosis is made. Cultures may be useful in simulating factors that affect development in the vector.     

Screening of Korean herbal medicines used to improve cognitive function for anti-cholinesterase activity.

Methanolic extracts of seven herbs (Acorus calamus, Acorus gramineus, Bupleurm facaltum, Dioscorea batatas, Epimedium koreanum, Poria cocos and Zizyphi jujuba) used in traditional Korean medicine for improvement of memory and cognition in old age were tested for cholinesterase inhibitory properties using the Ellman colorimetric method. Significant inhibition of the enzyme at 200 microg/ml was observed for extracts from A. calamus and E. koreanum. The possible bases for the reputation of these and the other herbs tested are discussed in the light of previous investigations into their chemistry and biological activity.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Cell separation from high cell density broths of Alcaligenes eutrophus by using a coagulant

Summary Alcaligenes eutrophus was successfully recovered from high cell density broths by pre-treatment with polyaluminium hydroxide chloride silicate as a coagulant at 36–90 mg Al/l. The optimum pH range for cell coagulation was 10–12. Subsequent centrifugation (45×g) and filtration (pore size 0.5 mm) gave a cell recovery of higher than 90%. The energy demand for cell recovery with the coagulant was only 3–11% of that without it.     

Gene identification in a complex chromosomal continuum by local genomic cross-referencing

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.