The origins of Clifford's herbarium

The development of the Hartekamp, George Clifford's estate, into one of the finest private Dutch botanical gardens was strongly influenced by the Hortus Botanicus of Leiden and its directors Boerhaave and Van Royen. Boerhaave's Index alter plantarum was used as the taxonomic framework for Clifford's herbarium until Linnaeus rearranged it.     

Amplification of DNA of Xanthomonas axonopodis pv. citri from historic citrus canker herbarium specimens

Herbaria are important resources for the study of the origins and dispersal of plant pathogens, particularly bacterial plant pathogens that incite local lesions in which large numbers of pathogen genomes are concentrated. Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus bacterial canker disease, is a notable example of such a pathogen. The appearance of novel strains of the pathogen in Florida and elsewhere make it increasingly important to understand the relationships among strains of this pathogen. USDA-ARS at Beltsville, Maryland maintains approximately 700 herbarium specimens with citrus canker disease lesions up to 90 years old, originally collected from all over the world, and so is an important resource for phytogeographic studies of this bacterium. Unfortunately, DNA in herbarium specimens is degraded and may contain high levels of inhibitors of PCR. In this study, we compared a total of 23 DNA isolation techniques in combination with 31 novel primer pairs in order to develop an efficient protocol for the analysis of Xac DNA in herbarium specimens. We identified the most reliable extraction method, identified in terms of successful amplification by our panel of 31 primer pairs. We also identified the most robust primer pairs, identified as successful in the largest number of extracts prepared by different methods. We amplified Xac genomic sequences up to 542 bp long from herbarium samples up to 89 years old. Primers varied in effectiveness, with some primer pairs amplifying Xac DNA from a 1/10,000 dilution of extract from a single lesion from a citrus canker herbarium specimen. Our methodology will be useful to identify pathogens and perform molecular analyses of bacterial and possibly fungal genomes from herbarium specimens.     

The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement

During the last few years we have been confronted with the need to use herbarium specimens in the molecular phylogeny studies, since it is generally difficult to obtain living material of some rare species. Ancient DNA has been sequenced, and there are also reports on successful DNA amplification from herbarium specimens. However, it is not easy to obtain amplified DNA from the first herbarium sample tested. In this paper, experiments are described about trials of DNA amplification from two to 151-year-old herbarium specimens of plant species we needed for our projects. Of the 17 herbarium samples tested only two allowed DNA amplification under standard DNA isolation conditions. Different types of PCR inhibiting activities were demonstrated in DNA extracts. In some of the extracts there was extremely low concentration of template with satisfactory quality. In some instances, PCR inhibiting activities were successfully removed by treating them either with insoluble polyvinylpyrrolidone or by adding bovine serum albumin (BSA) to the amplification mixture. However, some PCR-inhibiting activities were resistant to the treatments described above. When the concentration of template was very low, a second PCR amplification with internal primers was necessary to increase the amount of DNA for sequencing. Nevertheless, contamination of either DNA extract or amplification mixture were sometimes observed, and consequently precautions were taken to minimize them. Finally, successful amplification was obtained in eight samples out of the 17 examined.     

Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves

Journal of Plant Research - Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of...     

植物腊叶标本的消毒方法

植物腊叶标本消毒是腊叶标本制作的关键技术之一,是腊叶标本发挥其应有作用和长久保存的保障。对腊叶标本的消毒方法进行了总结．将目前所采用的方法分为物理方法和化学方法两大类,并对各种消毒方法的优缺点及注意事项进行了分析,以期对提高植物腊叶标本的制作质量提供技术支持。     

Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue

This report summarizes major changes in previously published protocols for DNA extraction to improve the quality of DNA extracted from plants. Here, we highlight the critical modifications in the original protocols. The efficiency of these changes results in high-quality DNA ready to use in a variety of phytogenetically distant plant families, in particular species with mucopolysaccharides. The DNA obtained can be used without further purification in various molecular biology assays, including direct sequencing and AFLP and RAPD (random-amplified polymorphic DNA) analyses. The effectiveness of this method is proven by the amplification and sequencing of PCR products of up to 1 kb with DNA extracted from herbarium tissue ≥60 years old. This versatility is not usually found in DNA extraction protocols. In addition, this method is quick, adaptable to standard laboratories, and most important, safer and more cost-effective.     

Continental-scale patterns of Cecropia reproductive phenology: evidence from herbarium specimens

Plant phenology is concerned with the timing of recurring biological events. Though phenology has traditionally been studied using intensive surveys of a local flora, results from such surveys are difficult to generalize to broader spatial scales. In this study, contrastingly, we assembled a continental-scale dataset of herbarium specimens for the emblematic genus of Neotropical pioneer trees, Cecropia, and applied Fourier spectral and cospectral analyses to investigate the reproductive phenology of 35 species. We detected significant annual, sub-annual and continuous patterns, and discuss the variation in patterns within and among climatic regions. Although previous studies have suggested that pioneer species generally produce flowers continually throughout the year, we found that at least one third of Cecropia species are characterized by clear annual flowering behaviour. We further investigated the relationships between phenology and climate seasonality, showing strong associations between phenology and seasonal variations in precipitation and temperature. We also verified our results against field survey data gathered from the literature. Our findings indicate that herbarium material is a reliable resource for use in the investigation of large-scale patterns in plant phenology, offering a promising complement to local intensive field studies.     

DNA sequence analysis of herbarium specimens facilitates the revival of Botrytis mali, a postharvest pathogen of apple

The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931) describing the apple pathogen B. mali as a unique species.     

Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of juncaceae

Seven DNA extraction protocols were used to obtain DNA from herbarium specimens ofJuncus andLuzula (Juncaceae) of various ages. DNA of historical samples is difficult to extract, and the extracts are seldom of good quality. The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260/280 absorbance ratio. The total DNA yield was measured by a fluorometer. The results indicate the success of using both mixer mill grinding and a DNeasy Plant Kit. Another extraction protocol (grinding with mortar and pestle, using liquid nitrogen) yielded DNA from many samples. Modified CTAB extraction, with a lengthy precipitation, usually provided good amounts of DNA. Other protocols did not give satisfactory results.     

Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia

Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species.     

Metagenomic analysis of historical herbarium specimens reveals a postmortem microbial community

Advances in DNA extraction and next‐generation sequencing have made a vast number of historical herbarium specimens available for genomic investigation. These specimens contain not only genomic information from the individual plants themselves, but also from associated microorganisms such as bacteria and fungi. These microorganisms may have colonized the living plant (e.g., pathogens or host‐associated commensal taxa) or may result from postmortem colonization that may include decomposition processes or contamination during sample handling. Here we characterize the metagenomic profile from shotgun sequencing data from herbarium specimens of two widespread plant species (Ambrosia artemisiifolia and Arabidopsis thaliana) collected up to 180 years ago. We used blast searching in combination with megan and were able to infer the metagenomic community even from the oldest herbarium sample. Through comparison with contemporary plant collections, we identify three microbial species that are nearly exclusive to herbarium specimens, including the fungus Alternaria alternata, which can comprise up to 7% of the total sequencing reads. This species probably colonizes the herbarium specimens during preparation for mounting or during storage. By removing the probable contaminating taxa, we observe a temporal shift in the metagenomic composition of the invasive weed Am. artemisiifolia. Our findings demonstrate that it is generally possible to use herbarium specimens for metagenomic analyses, but that the results should be treated with caution, as some of the identified species may be herbarium contaminants rather than representing the natural metagenomic community of the host plant.     

Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques

A well‐covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self‐primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four‐sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400–500 bp without bringing or inducing any sequence errors. About one‐third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well‐covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA‐labelled next‐generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality.     

Preservation of secondary fungal metabolites in herbarium lichen specimens

Fresh picked and herbarium thalli of Cladonia stellaris, C. rangiferina, Allocetraria nivalis, A. cucullata, Cetraria islandica, Peltigera canina, and Nephroma articum epigene lichens were studied using the immune-enzyme analysis. No big difference was observed in the contents of mycotoxin secondary metabolites, i.e., deoxynivalenol, diacetoxyscirpenol, zearalenone, alternariol, citrinin, sterigmatocystin, cyclopiazonic acid, mycophenolic acid, emodin, and PR-toxin. The discovery of these substances in the specimens preserved for several decades shows that lichens have an effective system of conservation of metabolic exchange products.     

Explaining invasiveness from the extent of native range: new insights from plant atlases and herbarium specimens

Aim

We tested the relationship between the extent of the native range and the success (number of occurrences) in the introduced range of European vascular plant species naturalized in the province of Québec (Canada). We hypothesized that the performance of models linking native range size and species invasiveness can be improved if residence time and climate tolerance are taken into account.

Methods

The extent of the native range (Europe, Asia) was estimated using plant atlases. The number of occurrences in the introduced range (Québec) was estimated using the number of herbarium specimens stored in herbaria. Herbarium specimens were also used to obtain residence time. Plant hardiness was used as an indicator of the suitability of a species to the climate of the introduced range. Multiple linear regression models, corrected to take into account phylogenetic biases, were used to calculate correlations between the extent of the native range and the number of occurrences in the introduced range.

Results

The larger the native distribution area in Eurasia, the greater the number of occurrences (herbarium specimens) in Québec. The shorter the residence time and the less hardy the plant, the fewer the number of occurrences. In all models tested, the phylogenetic structure explained a significant proportion of the variance, but its influence decreased as the number of species or area studied (Europe versus Eurasia) increased.

Main conclusions

The extent of the native range is a good explanatory variable for the invasion success of vascular plants, especially once other factors (residence time, climate tolerance, phylogeny) are taken into account. Thus, a model using these variables could be used by environmental managers to flag species warranting further investigation. With the emergence of online databases, gathering the required information is becoming easier and cheaper. As online databases continue to improve and new analytical tools are developed, this approach will become even more powerful.

     

Application of glycol methacrylate embedding to herbarium specimens of fruits

Aldehyde fixation and glycol methacrylate embedding were applied to herbarium specimens of fruits of the Compositae. Sections 1-2 micron thick were cut with glass knives. Softening was unnecessary and the hydrophilic properties of the resin permitted staining with a number of dyes. Specimens were examined with bright field and polarized light microscopy. The technique gives good structural preservation and resolution even with 81-year-old herbarium material.     

Developing integrated workflows for the digitisation of herbarium specimens using a modular and scalable approach

Digitisation programmes in many institutes frequently involve disparate and irregular funding, diverse selection criteria and scope, with different members of staff managing and operating the processes. These factors have influenced the decision at the Royal Botanic Garden Edinburgh to develop an integrated workflow for the digitisation of herbarium specimens which is modular and scalable to enable a single overall workflow to be used for all digitisation projects. This integrated workflow is comprised of three principal elements: a specimen workflow, a data workflow and an image workflow.The specimen workflow is strongly linked to curatorial processes which will impact on the prioritisation, selection and preparation of the specimens. The importance of including a conservation element within the digitisation workflow is highlighted. The data workflow includes the concept of three main categories of collection data: label data, curatorial data and supplementary data. It is shown that each category of data has its own properties which influence the timing of data capture within the workflow. Development of software has been carried out for the rapid capture of curatorial data, and optical character recognition (OCR) software is being used to increase the efficiency of capturing label data and supplementary data. The large number and size of the images has necessitated the inclusion of automated systems within the image workflow.     

Human origins of DNA replication selected from a library of nascent DNA

The identification of metazoan origins of DNA replication has so far been hampered by the lack of a suitable genetic screening and by the cumbersomeness of the currently available mapping procedures. Here we describe the construction of a library of nascent DNA, representative of all cellular origin sequences, and its utilization as a screening probe for origin identification in large genomic regions. The procedure developed was successfully applied to the human 5q31.1 locus, encoding for the IL-3 and GM-CSF genes. Two novel origins were identified and subsequently characterized by competitive PCR mapping, located approximately 3.5 kb downstream of the GM-CSF gene. The two origins (GM-CSF Ori1 and Ori2) were shown to interact with different members of the DNA prereplication complex. This observation reinforces the universal paradigm that initiation of DNA replication takes place at, or in close proximity to, the binding sites of the trans-acting initiator proteins.