Rethinking the links between systematic studies and ex situ living collections as a contribution to the Global Strategy for Plant Conservation

The Muséum national d’histoire naturelle (MNHN) in Paris holds ca. 70 million specimens. The collections were in need of a strategy to ensure their long-term conservation. We discuss how the Department of Botanical and Zoological Gardens (DJBZ; tropical living collections), and the Department of Systematics and Evolution (DSE; herbarium) contribute to achieving GSPC’s Target 1 (‘a widely accessible working list of known plant species as a step towards a complete world flora’). The DJBZ started encouraging better management of the collections, evolving towards focused reference collections, where all specimens have well-documented collection data. The objective is to link all collections to a scientific referee. This has already been achieved for a number of taxa. The herbarium of the DSE (acronym P) is among the world’s largest (11 million specimens, including 400,000 types). The collection’s heterogeneity impedes access to its data, since P is a mix of recent well-documented collections and historical collections at various curational levels. P is currently under renovation, which started by mounting all ca. 2 million unmounted specimens. The project also includes databasing and imaging of every specimen. The database now holds around 1,000,000 records. For taxonomic studies, living collections are crucial, especially for plants that are not easily preserved as herbarium specimens. Living collections also enable studies impossible to forecast at the time of collecting. Herbaria and living collections should therefore be conceived as interoperable entities requiring common scientific curation. Through a combination of its assets and the expertise of its researchers, the MNHN is well prepared to tackle the new objectives of the GSPC beyond 2010.     

Museum epigenomics: Characterizing cytosine methylation in historic museum specimens

Museum genomics has transformed the field of collections‐based research, opening up a range of new research directions for paleontological specimens as well as natural history specimens collected over the past few centuries. Recent work demonstrates that it is possible to characterize epigenetic markers such as DNA methylation in well preserved ancient tissues. This approach has not yet been tested in traditionally prepared natural history specimens such as dried bones and skins, the most common specimen types in vertebrate collections. In this study, we developed and tested methods to characterize cytosine methylation in dried skulls up to 76 years old. Using a combination of ddRAD and bisulphite treatment, we characterized patterns of cytosine methylation in two species of deer mouse (Peromyscus spp.) collected in the same region in Michigan in 1940, 2003, and 2013–2016. We successfully estimated methylation in specimens of all age groups, although older specimens yielded less data and showed greater interindividual variation in data yield than newer specimens. Global methylation estimates were reduced in the oldest specimens (76 years old) relative to the newest specimens (1–3 years old), which may reflect post‐mortem hydrolytic deamination. Methylation was reduced in promoter regions relative to gene bodies and showed greater bimodality in autosomes relative to female X chromosomes, consistent with expectations for methylation in mammalian somatic cells. Our work demonstrates the utility of historic specimens for methylation analyses, as with genomic analyses; however, studies will need to accommodate the large variance in the quantity of data produced by older specimens.     

Biological collections in an ever changing world: Herbaria as tools for biogeographical and environmental studies

Plant specimens stored in herbaria are being used as never before to document the impacts of global change on humans and nature. However, published statistics on the use of biological collections are rare, and ecologists lack quantitative data demonstrating the relevance to science of herbarium specimens. I found 382 studies with original data that used herbarium specimens to document biogeographical patterns or environmental changes. Most studies are less than 10 years old, and only 1.4% of the herbarium specimens worldwide have been used to answer biogeographical or environmental questions. The vast majority (82%) of papers dealt with vascular plants, but some studies also used bryophytes, lichens, seaweeds and fungi. The herbarium specimens were collected from all continents, but most of the studies used specimens from North America (40% of studies) or Europe (28%). Many types of researches (conservation, plant disease, plant invasion, pollution, etc.) can be conducted using herbarium specimens. Climate change, and especially phenological reconstructions, are clearly emerging research topics. By group, small herbaria (<100,000 specimens) are consulted as often as very large herbaria (>1,000,000 specimens) for biogeographical and environmental research, but in most cases, only large facilities provide specimens collected worldwide. The median number of specimens per study in papers using computerized collections (15,295) was much higher than for papers that did not include electronic data (226). The use of molecular analyses to investigate herbarium specimens is still relatively unexplored, at least from biogeographical and environmental points of view. Combined with recently developed procedures to correct biases, herbarium specimens might provide in the near future exciting additional spatio-temporal insights that are currently unimaginable.     

Sequence capture phylogenomics of historical ethanol‐preserved museum specimens: Unlocking the rest of the vault

Natural history collections play a crucial role in biodiversity research, and museum specimens are increasingly being incorporated into modern genetics‐based studies. Sequence capture methods have proven incredibly useful for phylogenomics, providing the additional ability to sequence historical museum specimens with highly degraded DNA, which until recently have been deemed less valuable for genetic work. The successful sequencing of ultraconserved elements (UCEs) from historical museum specimens has been demonstrated on multiple tissue types including dried bird skins, formalin‐fixed squamates and pinned insects. However, no study has thoroughly demonstrated this approach for historical ethanol‐preserved museum specimens. Alongside sequencing of “fresh” specimens preserved in >95% ethanol and stored at ?80°C, we used extraction techniques specifically designed for degraded DNA coupled with sequence capture protocols to sequence UCEs from historical museum specimens preserved in 70%–80% ethanol and stored at room temperature, the standard for such ethanol‐preserved museum collections. Across 35 fresh and 15 historical museum samples of the arachnid order Opiliones, an average of 345 UCE loci were included in phylogenomic matrices, with museum samples ranging from six to 495 loci. We successfully demonstrate the inclusion of historical ethanol‐preserved museum specimens in modern sequence capture phylogenomic studies, show a high frequency of variant bases at the species and population levels, and from off‐target reads successfully recover multiple loci traditionally sequenced in multilocus studies including mitochondrial loci and nuclear rRNA loci. The methods detailed in this study will allow researchers to potentially acquire genetic data from millions of ethanol‐preserved museum specimens held in collections worldwide.     

The contribution of small collections to species distribution modelling: A case study from Fuireneae (Cyperaceae)

The recent and rapid digitization of biodiversity data from natural history collection (NHC) archives has enriched collections based data repositories; this data continues to inform studies of species' geographic distributions. Here we investigate the relative impact of plant data from small natural history collections (collections with < 100,000 specimens) on species distributional models in an effort to document the potential of data from small NHCs to contribute to and inform biodiversity research. We modelled suitable habitat of five test case species from Fuireneae (Cyperaceae) in the United States using specimen records available via the Global Biodiversity Information Facility and that of data ready to mobilize from two regional small herbaria. Data were partitioned into three datasets based on their source: 1) collections-based records from large NHCs accessed GBIF, 2) collections-based records from small NHCs accessed from GBIF, and 3) collections-based records from two small regional herbaria not yet mobilized to GBIF. We extracted and evaluated the ecological niche represented for each of the three datasets by applying dataset occurrences to 14 environmental factors, and we modelled habitat suitability using Maxent to compare the represented distribution of the environmental values among the datasets. Our analyses indicate that the data from small NHCs contributed unique information in both geographic and environmental space. When data from small collections were combined with data from large collections, species models of the ecological niche resulted in more refined predictions of habitat suitability, indicating that small collections can contribute unique occurrence data which enhance species distribution models by bridging geographic collection gaps and shifting modelled predictions of suitable habitat. Inclusion of specimen records from small collections in ongoing digitization efforts is essential for generating informed models of a species' niche and distribution.     

Phylogenomics using formalin‐fixed and 100+ year‐old intractable natural history specimens

Museum specimens provide a wealth of information to biologists, but obtaining genetic data from formalin‐fixed and fluid‐preserved specimens remains challenging. While DNA sequences have been recovered from such specimens, most approaches are time‐consuming and produce low data quality and quantity. Here, we use a modified DNA extraction protocol combined with high‐throughput sequencing to recover DNA from formalin‐fixed and fluid‐preserved snakes that were collected over a century ago and for which little or no modern genetic materials exist in public collections. We successfully extracted DNA and sequenced ultraconserved elements ( = 2318 loci) from 10 fluid‐preserved snakes and included them in a phylogeny with modern samples. This phylogeny demonstrates the general use of such specimens in phylogenomic studies and provides evidence for the placement of enigmatic snakes, such as the rare and never‐before sequenced Indian Xylophis stenorhynchus. Our study emphasizes the relevance of museum collections in modern research and simultaneously provides a protocol that may prove useful for specimens that have been previously intractable for DNA sequencing.     

Biological collections and ecological/environmental research: a review,some observations and a look to the future

Housed worldwide, mostly in museums and herbaria, is a vast collection of biological specimens developed over centuries. These biological collections, and associated taxonomic and systematic research, have received considerable long‐term public support. The work remaining in systematics has been expanding as the estimated total number of species of organisms on Earth has risen over recent decades, as have estimated numbers of undescribed species. Despite this increasing task, support for taxonomic and systematic research, and biological collections upon which such research is based, has declined over the last 30‐40 years, while other areas of biological research have grown considerably, especially those that focus on environmental issues. Reflecting increases in research that deals with ecological questions (e.g. what determines species distribution and abundance) or environmental issues (e.g. toxic pollution), the level of research attempting to use biological collections in museums or herbaria in an ecological/environmental context has risen dramatically during about the last 20 years. The perceived relevance of biological collections, and hence the support they receive, should be enhanced if this trend continues and they are used prominently regarding such environmental issues as anthropogenic loss of biodiversity and associated ecosystem function, global climate change, and decay of the epidemiological environment. It is unclear, however, how best to use biological collections in the context of such ecological/environmental issues or how best to manage collections to facilitate such use. We demonstrate considerable and increasingly realized potential for research based on biological collections to contribute to ecological/environmental understanding. However, because biological collections were not originally intended for use regarding such issues and have inherent biases and limitations, they are proving more useful in some contexts than in others. Biological collections have, for example, been particularly useful as sources of information regarding variation in attributes of individuals (e.g. morphology, chemical composition) in relation to environmental variables, and provided important information in relation to species' distributions, but less useful in the contexts of habitat associations and population sizes. Changes to policies, strategies and procedures associated with biological collections could mitigate these biases and limitations, and hence make such collections more useful in the context of ecological/environmental issues. Haphazard and opportunistic collecting could be replaced with strategies for adding to existing collections that prioritize projects that use biological collections and include, besides taxonomy and systematics, a focus on significant environmental/ecological issues. Other potential changes include increased recording of the nature and extent of collecting effort and information associated with each specimen such as nearby habitat and other individuals observed but not collected. Such changes have begun to occur within some institutions. Institutions that house biological collections should, we think, pursue a mission of ‘understanding the life of the planet to inform its stewardship’ ( Krishtalka & Humphrey, 2000 ), as such a mission would facilitate increased use of biological collections in an ecological/environmental context and hence lead to increased appreciation, encouragement and support from the public for these collections, their associated research, and the institutions that house them.     

THE GREEN ALGAL GENUS CLONIOPHORA REPRESENTS A NOVEL LINEAGE IN THE ULVALES: A PROPOSAL FOR CLONIOPHORACEAE FAM. NOV.1

The green algal genus Cloniophora has been classified in the Chaetophorales (Chlorophyceae) based on morphological characters. This study uses DNA sequence data from the nucleus (SSU) and the chloroplast (tufA) from collections in the Hawaiian Islands and a culture originating from Portugal to test this classification. Taxonomic identities of contemporary collections were confirmed by sequencing small fragments of DNA (rbcL and SSU) from type specimens, including the generitype, Cloniophora willei L. H. Tiffany. These molecular data show that Cloniophora does not have close affinities to the Chaetophorales and belongs instead to the Ulvales (Ulvophyceae). The morphological features of eight or more reproductive products per cell and a pyrenoid with a traversing thylakoid membrane support the molecular data and confirm the placement of this group in the Ulvales. As this genus does not belong to any recognized family in the Ulvales, the family Cloniophoraceae, containing the single genus Cloniophora, is proposed.     

Richard Meinertzhagen—a case of fraud examined

Museum collections are rich repositories of information. The specimens and the data they bear continue to provide new insights into ornithology and biological processes decades or even centuries after they were collected. The benefits to be gained from museum collections depend implicitly upon the accuracy of the information associated with the specimens and the correct interpretation of those data (Parkes 1989, Knox & Walters 1992). Collectors and dealers have often been suspected of fabricating data for a variety of reasons, but proven cases have been documented only rarely (e.g. Nicholson & Ferguson-Lees 1962). This paper examines one such case.     

Regionalization of the avifauna of the Baja California Peninsula, Mexico: a parsimony analysis of endemicity and distributional modelling approach

Aim To analyse the distributional patterns of the Baja California Peninsula's resident avifauna, and to generate a regionalization based on a method that uses a parsimony analysis (parsimony analysis of endemicity, PAE) of point data and modelled potential distributions. Location The Baja California Peninsula, Mexico. Methods A data base was constructed containing records of 113 species of resident terrestrial birds present in the Baja California Peninsula. Records and localities were obtained from the literature and from specimens housed in scientific collections world‐wide. Raw data points and potential distribution maps obtained using the software Genetic Algorithms for Rule‐set Prediction (GARP), were analysed with PAE. Results The data base consisted of 4164 unique records (only one combination of species/locality) belonging to 113 terrestrial resident bird species, in a total of 809 localities. From the point distribution matrix, the analysis generated 500 equally parsimonious trees, from which a strict consensus cladogram with 967 steps was obtained. The cladogram shows a basal polytomy and some geographical correspondence of a few resolved groups obtained in the analysis. These results do not allow the recognition of areas defined by avifaunistic associations. From the potential distribution matrix, the analysis generated 501 equally parsimonious trees, and a strict consensus cladogram of 516 steps was obtained. The cladogram shows a higher resolution because of the number of resolved groups with better geographical correspondence and therefore regions are well‐defined. Main conclusions The correspondence of some groupings of species suggest their validity as areas with biogeographical (historical and/or ecological) meaning. This regionalization in the Baja California avifauna seems to be consistent with previous regionalizations for other groups. Hence, PAE is a useful tool for area categorization if reliable point records and prediction tools are available. Our results suggest that the geographical definition is much better using potential data generated by GARP, particularly when they are contrasted with the results from point data. Thus, this is an excellent alternative for developing biogeographical studies, as well as for improving the use of data from scientific collections and other sources of biodiversity information.     

Diversity biases in terrestrial mammalian assemblages and quantifying the differences between museum collections and published accounts: A case study from the Miocene of Nevada

Understanding diversity through time in the fossil record has primarily relied on the raw count of species within a given time interval, or species richness. These estimates are often derived from published fossil data, and standardized for sample size or geographic area. However, most methods that standardize richness by sample size are sensitive to changes in evenness, which introduces a potential problem with relying on published records: published accounts could be more even than the museum collections from which they are drawn. We address this bias in the context of mammalian paleodiversity, comparing published and museum collections of the Hemphillian Thousand Creek fauna to those of the Barstovian Virgin Valley fauna. We rarified specimen data, both number of identified specimens (NISP) and minimum number of individuals (MNI), and presence/absence data to compare published and museum data within and between faunas. Within faunas, published numbers of specimens are more even than museum samples, but the difference for localities in Virgin Valley is not significant. Neither published nor museum numbers of specimens indicate a significant difference between faunas, but the diversity pattern is reversed between the two data sets. Presence/absence rarefactions show no differences between sources; here, published data adequately sample the underlying museum records. Specimen-based evenness is not accurate in the published sample, and therefore we suggest that future studies of diversity in terrestrial mammalian assemblages must assess unpublished collections. Additionally, NISP data for Thousand Creek are more even than MNI data, suggesting that relying solely on NISP for assessing species diversity can also be misleading. Because publication bias alters richness and evenness, diversity estimates using published data must be circumspect about data sources.     

Limited sampling hampers “big data” estimation of species richness in a tropical biodiversity hotspot

Macro‐scale species richness studies often use museum specimens as their main source of information. However, such datasets are often strongly biased due to variation in sampling effort in space and time. These biases may strongly affect diversity estimates and may, thereby, obstruct solid inference on the underlying diversity drivers, as well as mislead conservation prioritization. In recent years, this has resulted in an increased focus on developing methods to correct for sampling bias. In this study, we use sample‐size‐correcting methods to examine patterns of tropical plant diversity in Ecuador, one of the most species‐rich and climatically heterogeneous biodiversity hotspots. Species richness estimates were calculated based on 205,735 georeferenced specimens of 15,788 species using the Margalef diversity index, the Chao estimator, the second‐order Jackknife and Bootstrapping resampling methods, and Hill numbers and rarefaction. Species richness was heavily correlated with sampling effort, and only rarefaction was able to remove this effect, and we recommend this method for estimation of species richness with “big data” collections.     

How old can we go? Evaluating the age limit for effective DNA recovery from historical insect specimens

Historical museum specimens are valuable for exploring population genetics and evolutionary questions because they can provide snapshots of morphological and genetic characteristics from populations over space and time. Unfortunately, DNA found in older museum specimens is frequently degraded, so obtaining genotypes from many individual samples necessary for rigorous molecular population genetic studies is challenging. Previous studies have varied greatly in their success at obtaining genotypes from older preserved insect material. Many well-intentioned collection curators have used research results showing poor preservation of DNA preserved in museum specimens to inform curatorial best practices, in some cases choosing not to allow DNA extraction by destructive sampling because, in their estimation, the likelihood of success would be low. Recent methodological advances in DNA extraction, amplification, and genotyping have allowed some researchers to include mid-19th century samples in molecular genetic analyses. Here we present a robust, high-throughput, and low-cost DNA extraction and genotyping protocol for historical insect specimens employing restriction digests of PCR products followed by high sensitivity electrophoresis. Using this technique, we obtained mitochondrial haplotypes for 100% of 48 New World Junonia butterfly specimens (Nymphalidae) ranging in age from pre-1813 to 1909 and show that the haplotype frequencies obtained are statistically indistinguishable from 20th-century and contemporary reference populations of Junonia (1632 specimens) matched by geographic region. As most extant insect specimens were collected after 1813, based on our findings we would expect that many or even most pinned specimens preserved in museum collections contain usable DNA for mitochondrial haplotyping.     

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.     

Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades‐old dried museum specimens

Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high‐throughput laboratory workflows. The strategy uses hemi‐nested, degenerate, M13‐tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard‐compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, ‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.     

Strategies for agrobiodiversity conservation and promotion: a case from Yunnan, China

This paper deals with strategies for agrobiodiversity conservation and promotion based on studies on four plant groups (selected from 50 plant groups) occurring in the Yunnan Province of China. These plants are edible konjac (Amorphophallus), medicinal Paris, Musella lasiocarpa and wild tea (Camellia sinensis and its wild relatives), including their cultivars and wild populations. After analyzing the target plants, we conclude that the following strategies should be adopted to conserve and promote agrobiodiversity: (1) in situ conservation of agrobiodiversity, including habitat protection of wild populations, maintenance of native species and varieties in traditional agroecosystems, and relevant environmental education; (2) ex situ conservation and promotion of agrobiodiversity, including establishment of living collections and germplasm banks, and introduction of species and varieties into agroecosystems for agricultural practice; and (3) promotion and conservation of agrobiodiversity through sustainable uses, including technique development of propagation, cultivation, pest and disease control, on farm and off farm management, and other activities such as new variety breeding and scientific studies. Strategies developed here will be helpful to conserve and promote agrobiodiversity at agroecosystem, species, variety or landrace, and management system levels.