Genetic variation in natural populations of Anthurium sinuatum and A. pentaphyllum var. pentaphyllum (Araceae) from north‐east Brazil using AFLP molecular markers

Genetic variation was investigated using AFLP markers in 12 populations of Anthurium sinuatum and A. pentaphyllum var. pentaphyllum (Araceae) in north‐east Brazil, Amazonia and the Brazilian Atlantic forest. Two unique genetic patterns characterized the populations of A. sinuatum as a group, but no correlation between genetic and geographical interpopulation distance was found; the Amazonian population was not separated from that in Ceará. The isolated Ceará brejo populations of A. sinuatum were genetically distinct, but genetic diversity levels were similar to populations elsewhere, with no evidence of genetic erosion. Anthurium pentaphyllum populations were significantly different from each other; Bayesian genetic structural analysis found no common genetic pattern, but revealed genetic clusters unique to subgroups and individual populations in the Atlantic forest and French Guiana. Anthurium pentaphyllum and A. sinuatum can be distinguished genetically, but individuals of both species formed intermediate genetic clusters that blurred their distinction. We suggest that genetic mixing of A. sinuatum and A. pentaphyllum has occurred in north‐east Brazil, possibly connected with cycles of humid forest expansion. The weak genetic structure in A. sinuatum is consistent with the natural fragmentation of continuous forest areas, possibly during the Holocene. This study highlights the scientific importance of the highly threatened brejo forests for tropical American biogeography. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 88–105.     

固定核空间法和最小凸多边形法估计藏狐家域的比较

最小凸多边形法(MCP)和固定核空间法(FKE)是目前最常用的家域计算方法,但受空间自相关性、偏远位点等问题的影响,两种方法均存在明显的局限性。本文根据2006 年和2007 年在四川省石渠县和青海省都兰县的7 只藏狐352 个活动位点数据,分析MCP 和FKE 家域估计的效果和存在的问题。结果显示:(1)利用概率百分比≤95% 时,MCP 计算结果和FKE 没有显著差异;(2)极端点对高百分比(85% ～ 100% ) 下MCP影响显著,而FKE 对极端点影响控制较好;(3)FKE 家域外形复杂,计算结果受平滑度系数设置影响显著。因此,研究领域行为时,应同时使用FKE 和95% MCP 两种方法。当数据分布较理想时,FKE 能够给出更为准确的面积估计,而MCP 则因其通用性,使得研究数据与其他研究的结果更具可比性。
     

Density,home range,seasonal movements and habitat use of the mountain pygmy‐possum Burramys parvus (Marsupialia: Burramyidae) at Mount Blue Cow,Kosciuszko National Park

A population of mountain pygmy‐possums Burramys parvus was studied at the Mount Blue Cow ski resort in Kosciuszko National Park between 1986 and 1989. Forty‐eight individuals were radiotracked during the snow‐free months and 21 individuals were tracked during winter over the 3 years of study. Trapping and radiotracking showed that the density, population structure, movements and home range sizes of B. parvus on Mount Blue Cow were strongly correlated with elevation and changed with the season. Female densities were greatest in habitats characterized by deep boulderfields, at high elevations with an abundance of Bogong moths. Males visited the areas where females were located to breed in November–December and then by February, the majority migrated to lower elevations or north and westerly aspects. Females that nested at lower elevations also visited high‐elevation habitats to access the high concentrations of Bogong moths, which were the main food source in summer. A high proportion of the juvenile males and some juvenile females dispersed to lower elevations in March and April. The resulting sexual segregation during autumn and winter may be a result of female aggression or scramble competition, but is also explainable by differences in energy requirements, seed availability and hibernation strategies between the sexes. The extraordinarily large nightly and seasonal movements between habitat patches of up to 2 km for females and 3 km for males, sexual segregation and the use of different hibernation sites have important implications for the management of this species. These include the need for movement and dispersal corridors and the conservation of boulder‐heath habitats outside the main boulderfields.     

High‐efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system

The base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of ?17 to ?12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.     

Identification and analysis of residues contained on β → α loops of the dual‐substrate (βα)8 phosphoribosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity

A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient‐like dual‐substrate (βα)₈ phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis–Menten enzyme kinetics for both isomerase activities in wild‐type PriA from S. coelicolor and in selected single‐residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg¹³⁹, which was postulated on structural grounds to be important for the dual‐substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser⁸¹Thr and PriA_Arg¹³⁹Asn showed that these residues, which are contained on β → α loops and in close proximity to the N‐terminal phosphate‐binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X‐ray crystallographic structure of PriA_Arg¹³⁹Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α loop as a major structural feature related to the evolution of the dual‐substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates—within a bifunctional and thus highly constrained active site—without compromising its structural stability.