Museum metabarcoding: A novel method revealing gut helminth communities of small mammals across space and time

Natural history collections spanning multiple decades provide fundamental historical baselines to measure and understand changing biodiversity. New technologies such as next generation DNA sequencing have considerably increased the potential of museum specimens to address significant questions regarding the impact of environmental changes on host and parasite/pathogen dynamics. We developed a new technique to identify intestinal helminth parasites and applied it to shrews (Eulipotyphla: Soricidae) because they are ubiquitous, occupy diverse habitats, and host a diverse and abundant parasite fauna. Notably, we included museum specimens preserved in various ways to explore the efficacy of using metabarcoding analyses that may enable identification of helminth symbiont communities from historical archives. We successfully sequenced the parasite communities (using 12S mtDNA, 16S mtDNA, 28S rDNA) of 23 whole gastrointestinal tracts. All gastrointestinal tracts were obtained from the Museum of Southwestern Biology, USA, and from recent field collections, varying both in time since fixation (ranging from 4?months to 16?years) and preservation method (70% or 95% ethanol stored at room temperature, or flash frozen in liquid nitrogen and stored at ?80?°C). Our proof of concept demonstrates the feasibility of applying next generation DNA sequencing techniques to authoritatively identify the parasite/pathogen communities within whole gastrointestinal tracts from museum specimens of varying age and fixation, and the value of future preservation of host-associated whole gastrointestinal tracts in public research archives. This powerful approach facilitates future comparative examinations of the distributions and interactions among multiple associated groups of organisms through time and space.     

Exploiting formalin-preserved fish specimens for resources of DNA barcoding

With the development of the DNA barcoding project, a large number of specimens are required to establish the library of reference barcode. Formalin-fixed samples from museums provide a potential resource for it. However, recovery of DNA and amplification of the target gene from formalin-fixed samples are challenging. In this study, a hot alkali pre-treatment accompanied by the use of cetyltrimethylammonium bromide (CTAB) method was employed for DNA recovery from formalin-preserved samples, with the purpose of pursuing the optimal condition for high quantity and quality of DNA and minimizing PCR inhibition. Meanwhile, a semi-nested PCR-based method was developed to enhance the efficacy of amplification. This advanced protocol was demonstrated to be reliable and effective. Even for 23-year-old samples, genomic DNA could be extracted, and COI gene was correctly sequenced.     

硅胶填充昆虫幼虫标本制作方法

介绍一种利用硅胶填充制作昆虫幼虫标本的方法。将活体昆虫幼虫经过去内脏、填充硅胶、整形和干燥制成昆虫幼虫标本。该方法制作的标本形态生动,保存性与保色性好,并且制作简便、成本低,有利于观赏和教学。     

线粒体COⅠ基因在昆虫DNA条形码中的研究与应用

自2003年DNA条形码(DNA barcodes)概念出现以来,DNA条形码技术(DNA barcoding)受到生物分类学领域普遍关注,线粒体细胞色素氧化酶亚基I(mtDNACOⅠ)被用作动物类群的主要条形码序列,基于该基因片段的昆虫条形码研究在国内外广泛开展。本文在概述DNA条形码、条形码技术及已开展的昆虫条形码研究计划的基础上,总结了昆虫mtDNACOⅠ条形码及其技术在发现和描述隐种、种类分子鉴定以及系统发育等方面的研究进展,分析了细胞核线粒体假基因(Numts)对mtDNACOⅠ条形码扩增的影响,提出检测和避免Numts的方法,并对DNA条形码技术的进一步研究和应用进行了讨论和展望。     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

A multilocus sequence typing (MLST) approach to diminish the problems that are associated with DNA barcoding: A reply to Stahlhut et al. (2012)

In a recent Perspective, Stahlhut et al. (2012) argued that potential Wolbachia-induced effects on inheritance patterns of mitochondrial DNA do not significantly affect DNA barcoding efforts. Since this hypothesis can be readily tested, we suggest to do so by including multiple, nuclear markers in DNA barcoding studies.     

A century of genetic change and metapopulation dynamics in the Galápagos warbler finches (Certhidea)

Populations that are connected by immigrants play an important role in evolutionary and conservation biology, yet we have little direct evidence of how such metapopulations change genetically over evolutionary time. We compared historic (1894–1906) to modern (1988–2006) genetic variation in 11 populations of warbler finches at 14 microsatellite loci. Although several lines of evidence suggest that Darwin's finches may be in decline, we found that the genetic diversity of warbler finches has not generally declined, and broad‐scale patterns of variation remained similar over time. Contrary to expectations, inferred population sizes have generally increased over time (6–8%) as have immigration rates (8–16%), which may reflect a recent increase in the frequency and intensity of El Niño events. Individual island populations showed significant declines (18–19%) and also substantial gains (18–20%) in allelic richness over time. Changes in genetic diversity were correlated with changes in immigration rates, but did not correspond to population size or human disturbance. These results reflect the expected stabilizing properties of whole metapopulations over time. However, the dramatic and unpredictable changes observed in individual populations during this short time interval suggests that care should be taken when monitoring individual population fragments with snapshots of genetic variation.     

新世纪的中国昆虫系统学

对未来我国昆虫系统学在能力建设、物种编目、理论研究、技术创新和国际合作等方面提出一系列建议。在昆虫系统学能力建设方面,政府和科学家应该在生物分类学能力评估、基础硬件建设、各级生物标本馆中建立伙伴关系（包括标本采集、标本馆管理、 科学研究、 知识共享和标本与资料交换）等方面重点开展工作。在物种编目方面,我国的昆虫物种编目有赖于各级政府和机构继续关注标本的收集和保藏,继续启动一些考察项目,以满足发现和认识昆虫物种的实际需求。在物种水平上研究以往昆虫系统学家的工作,进行地区性和世界性的昆虫类群的订正更是非常必要的。在理论研究方面,我国昆虫系统应该在下列方面积极探索：物种概念、进化理论、比较生物学理论和高级分类系统研究。在技术创新方面,我国的昆虫系统学家应该在数据库与网络技术应用、图形图像处理技术、专家鉴定系统技术、分类性状分析技术、分子生物学技术、系统发育推断程序、信息统一管理技术和知识传播技术等方面进行深入研究,以满足昆虫系统学的发展需求。在国际合作方面,要进一步推动我国昆虫系统学研究机构加入生物分类学全球战略联盟、加入各种相关国际相关组织,要促进物种信息管理系统的建立与共享,要推动研究项目国际化。     

Multiple gains and losses of Wolbachia symbionts across a tribe of fungus‐growing ants

Although the intracellular bacterium Wolbachia is ubiquitous in insects, it has a unique relationship with New World ants on which particular bacterial strains have specialized. However, data are from distantly related hosts and detailed phylogenetic information which could reveal transmission dynamics are lacking. Here, we investigate host–Wolbachia relationships in the monophyletic fungus‐growing ant tribe Attini, screening 23 species and using multilocus sequence typing to reliably identify Wolbachia strains. This technique reduces the significant problem of recombination seen using traditional single gene techniques. The relationship between Wolbachia and the fungus‐growing ants appears complex and dynamic. There is evidence of co‐cladogenesis, supporting vertical transmission; however, this is incomplete, demonstrating that horizontal transmission has also occurred. Importantly, the infection prevalence is frequently different between closely related taxa, with the Acromyrmex leaf‐cutting ants appearing particularly prone to infection and there being no consistent relationship with any of the major life history transitions. We suggest that infection loss and horizontal transmission have driven epidemics or selective sweeps of Wolbachia, resulting in multiple gains and losses of infection across the fungus‐growing ants.     

The orthopteroid insects described by Linnaeus,with notes on the Linnaean collection

A list is given of the recently recurated Orthoptera s.l. held by the Linnean Society of London. The status of specimens in the collection is evaluated, eight lectotype designations are made, and the orthopteroid species described by Linnaeus are listed alphabetically.