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.     

Diversity and distribution of reptiles in Romania

The reptile fauna of Romania comprises 23 species, out of which 12 species reach here the limit of their geographic range. We compiled and updated a national database of the reptile species occurrences from a variety of sources including our own field surveys, personal communication from specialists, museum collections and the scientific literature. The occurrence records were georeferenced and stored in a geodatabase for additional analysis of their spatial patterns. The spatial analysis revealed a biased sampling effort concentrated in various protected areas, and deficient in the vast agricultural areas of the southern part of Romania. The patterns of species richness showed a higher number of species in the warmer and drier regions, and a relatively low number of species in the rest of the country. Our database provides a starting point for further analyses, and represents a reliable tool for drafting conservation plans.     

Diversity and distribution of amphibians in Romania

Nineteen species of amphibians inhabit Romania, 9 of which reach their range limit on this territory. Based on published occurrence reports, museum collections and our own data we compiled a national database of amphibian occurrences. We georeferenced 26779 amphibian species occurrences, and performed an analysis of their spatial patterns, checking for hotspots and patterns of species richness. The results of spatial statistic analyses supported the idea of a biased sampling for Romania, with clear hotspots of increased sampling efforts. The sampling effort is biased towards species with high detectability, protected areas, and large cities. Future sampling efforts should be focused mostly on species with a high rarity score in order to accurately map their range. Our results are an important step in achieving the long-term goals of increasing the efficiency of conservation efforts and evaluating the species range shifts under climate change scenarios.     

Morphometric measurements of dragonfly?wings: the accuracy of pinned,scanned and detached?measurement methods

Large-scale digitization of museum specimens, particularly of insect collections, is becoming commonplace. Imaging increases the accessibility of collections and decreases the need to handle individual, often fragile, specimens. Another potential advantage of digitization is to make it easier to conduct morphometric analyses, but the accuracy of such methods needs to be tested. Here we compare morphometric measurements of scanned images of dragonfly wings to those obtained using other, more traditional, methods. We assume that the destructive method of removing and slide-mounting wings provides the most accurate method of measurement because it eliminates error due to wing curvature. We show that, for dragonfly wings, hand measurements of pinned specimens and digital measurements of scanned images are equally accurate relative to slide-mounted hand measurements. Since destructive slide-mounting is unsuitable for museum collections, and there is a risk of damage when hand measuring fragile pinned specimens, we suggest that the use of scanned images may also be an appropriate method to collect morphometric data from other collected insect species.     

An Analysis of Factors Affecting Genotyping Success from Museum Specimens Reveals an Increase of Genetic and Morphological Variation during a Historical Range Expansion of a European Spider

Natural history collections house an enormous amount of plant and animal specimens, which constitute a promising source for molecular analyses. Storage conditions differ among taxa and can have a dramatic effect on the success of DNA work. Here, we analyze the feasibility of DNA extraction from ethanol preserved spiders (Araneae). We tested genotyping success using several hundred specimens of the wasp spider, Argiope bruennichi, deposited in two large German natural history collections. We tested the influence of different factors on the utility of specimens for genotyping. Our results show that not the specimen’s age, but the museum collection is a major predictor of genotyping success. These results indicate that long term storage conditions should be optimized in natural history museums to assure the utility of collections for DNA work. Using historical material, we also traced historical genetic and morphological variation in the course of a poleward range expansion of A. bruennichi by comparing contemporary and historical specimens from a native and an invasive population in Germany. We show that the invasion of A. bruennichi is tightly correlated with an historical increase of genetic and phenotypic variation in the invasive population.     

Phylogenomics using low‐depth whole genome sequencing: A case study with the olive tribe

Species trees have traditionally been inferred from a few selected markers, and genome‐wide investigations remain largely restricted to model organisms or small groups of species for which sampling of fresh material is available, leaving out most of the existing and historical species diversity. The genomes of an increasing number of species, including specimens extracted from natural history collections, are being sequenced at low depth. While these data sets are widely used to analyse organelle genomes, the nuclear fraction is generally ignored. Here we evaluate different reference‐based methods to infer phylogenies of large taxonomic groups from such data sets. Using the example of the Oleeae tribe, a worldwide‐distributed group, we build phylogenies based on single nucleotide polymorphisms (SNPs) obtained using two reference genomes (the olive and ash trees). The inferred phylogenies are overall congruent, yet present differences that might reflect the effect of distance to the reference on the amount of missing data. To limit this issue, genome complexity was reduced by using pairs of orthologous coding sequences as the reference, thus allowing us to combine SNPs obtained using two distinct references. Concatenated and coalescence trees based on these combined SNPs suggest events of incomplete lineage sorting and/or hybridization during the diversification of this large phylogenetic group. Our results show that genome‐wide phylogenetic trees can be inferred from low‐depth sequence data sets for eukaryote groups with complex genomes, and histories of reticulate evolution. This opens new avenues for large‐scale phylogenomics and biogeographical analyses covering both the extant and the historical diversity stored in museum collections.     

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.     

The relevance of time series in molecular ecology and conservation biology

The genetic structure of a species is shaped by the interaction of contemporary and historical factors. Analyses of individuals from the same population sampled at different points in time can help to disentangle the effects of current and historical forces and facilitate the understanding of the forces driving the differentiation of populations. The use of such time series allows for the exploration of changes at the population and intraspecific levels over time. Material from museum collections plays a key role in understanding and evaluating observed population structures, especially if large numbers of individuals have been sampled from the same locations at multiple time points. In these cases, changes in population structure can be assessed empirically. The development of new molecular markers relying on short DNA fragments (such as microsatellites or single nucleotide polymorphisms) allows for the analysis of long‐preserved and partially degraded samples. Recently developed techniques to construct genome libraries with a reduced complexity and next generation sequencing and their associated analysis pipelines have the potential to facilitate marker development and genotyping in non‐model species. In this review, we discuss the problems with sampling and available marker systems for historical specimens and demonstrate that temporal comparative studies are crucial for the estimation of important population genetic parameters and to measure empirically the effects of recent habitat alteration. While many of these analyses can be performed with samples taken at a single point in time, the measurements are more robust if multiple points in time are studied. Furthermore, examining the effects of habitat alteration, population declines, and population bottlenecks is only possible if samples before and after the respective events are included.     

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.     

Using Museum Collections to Estimate Diversity Patterns along Geographical Gradients

Quantifying species-richness patterns along geographical gradients (typically latitude and elevation) has a long history in ecology and can be based on more-or-less complete censuses from a specified area (plot sampling), selective collection within a specified area (e.g. museum collections), or general information about species distributions (e.g. observations of extremes along the gradient, distribution maps). All these approaches require complete sampling to give the true richness in an area, but the richness pattern (i.e., the relative changes in richness along the gradient) may be estimated without complete sampling, although equal sampling between areas is necessary. This is relatively easy to do for fine-scale plot sampling, but rarely easy for other types of data. For data extracted from museum collections, a correct perception of the species richness pattern therefore depends on post-sampling treatment of data. Two commonly applied techniques for quantifying species richness patterns with these types of data are discussed, namely interpolation of species ranges and rarefaction. Such treatment may correct for unequal sampling in some instances, but may in other cases introduce artificial patterns. With incomplete sampling interpolation introduces an artificial humped pattern and rarefaction requires similar species abundance distributions to make unbiased comparisons among samples. One must therefore be cautious when applying these methods for estimating species richness patterns along geographical gradients.     

长江春大豆核心种质构建及分析

利用长江春大豆初选核心种质SSR(simple sequence repeat）标记和农艺性状表型等基础数据,对用不同个体取样方法以及不同数据类型建立的核心种质进行评价,目的是确定中国大豆（Glycine max）核心种质的最佳取样策略提供依据,结果表明,根据SSR分子数据聚类,采用类内随机取样,类内以遗传相似性系数取样以及仅依据遗传相似性系数取样都可用于大豆核心种质构建,但是综合不同评价参数发现,以类内随机取样最佳,类内按遗传相似性系数取样次之,单独以遗传相似性系数取样较差。分析不同SSR等位变异保留比例的遗传多样性指数发现,当保留90%和80%的SSR等位变异时,核心种质具有更高的遗传多样性,由于与SSR分子数据种质遗传关系评价的不一致性,农艺性状等基础数据虽然可用来构建核心种质,但其SSR分子水平代表性相对较低,本研究结果还表明,用不同方法或同一方法不同重复次数取样建立的核心种质具有异质性,且这种异质性随核心种质取样比例的降低而增大,因此,虽然可依据不同数据类型确定相应的方法建立核心种质,但综合表型和分子数据建立的核心种质更具有代表性。     

Accessibility maps as a tool to predict sampling bias in historical biodiversity occurrence records

Historical biodiversity occurrence records are often discarded in spatial modeling analyses because of a lack of a method to quantify their sampling bias. Here we propose a new approach for predicting sampling bias in historical written records of occurrence, using a South African example as proof of concept. We modelled and mapped accessibility of the study area as the mean of proximity to freshwater and European settlements. We tested the model's ability to predict the location of historical biodiversity records from a dataset of 2612 large mammal occurrence records collected from historical written sources in South Africa in the period 1497–1920. We investigated temporal, spatial and environmental biases in these historical records and examined if the model prediction and occurrence dataset share similar environmental bias. We find a good agreement between the accessibility map and the distribution of sampling effort in the early historical period in South Africa. Environmental biases in the empirical data are identified, showing a preference for lower maximum temperature of the warmest month, higher mean monthly precipitation, higher net primary productivity and less arid biomes than expected by a uniform use of the study area. We find that the model prediction shares similar environmental bias as the empirical data. Accessibility maps, built with very simple statistical rules and in the absence of empirical data, can thus predict the spatial and environmental biases observed in historical biodiversity occurrence records. We recommend that this approach be used as a tool to estimate sampling bias in small datasets of occurrence and to improve the use of these data in spatial analyses in ecological and conservation studies.     

Anthropic modifications on megafauna bones in the paleontological collections of the Museum national d’Histoire naturelle de Paris: Historical aspects and implications for the Pampean Pleistocene peopling

The most abundant evidence of Pampean Pleistocene human presence are modified bones, as lithic procurement sites located farther than 300 km away. Therefore, we focused on the study of bone modifications, in particular cut and percussion marks. We studied Pampean paleontological collections of Argentine and European museums as an alternative resource of previously unnoticed human modification evidences. We compared marks characteristics with those of archaeological collections from diverse climatic and cultural adaptations, from middle Pleistocene sites (Vallonet, Atapuerca, Lazaret, Arago and Terra Amata) to terminal Pleistocene ones (Abri Pataud, Isturiz and La Vache). Marks typologies were defined, leading to the identification of a particular cutmark that we named double parallel considered as highly diagnostic of anthropic use of lithic artifacts. We also propose that hafted artifacts were used on carcass processing. Perimortem bone modifications are classified in relation with mark morphology (e.g., percussion striae), inferred gesture or action (e.g., breakage of diaphysis), and inferred objective of that action (e.g., marrow extraction). The megafauna specimens analyzed from the historical collections (d’Angelis-Vilardebó, 1847, Muñiz-Dupotet, 1842 and Breton-Bonnement, 1881) present modifications compatible with dismemberment, defleshing, tongue and masseter extraction, and utilization as anvils. The lack of contextual data (location, stratigraphy) and the loss due to museum selection/collecting of accompanying material prevent paleoenvironmental and paleoecological inferences. Regional geology indicates that most of the historical collections were exhumed in riverbank cuts with ages between 70 and 13 ky BP. Preliminary direct dating presents evidence of a Pampean human occupation, at least, since OIS 2.     

Using carbon isotopes to track dietary change in modern,historical, and ancient primates

Stable isotope analysis can be used to document dietary changes within the lifetimes of individuals and may prove useful for investigating fallback food consumption in modern, historical, and ancient primates. Feces, hair, and enamel are all suitable materials for such analysis, and each has its own benefits and limitations. Feces provide highly resolved temporal dietary data, but are generally limited to providing dietary information about modern individuals and require labor-intensive sample collection and analysis. Hair provides less well-resolved data, but has the advantage that one or a few hair strands can provide evidence of dietary change over months or years. Hair is also available in museum collections, making it possible to investigate the diets of historical specimens. Enamel provides the poorest temporal resolution of these materials, but is often preserved for millions of years, allowing examination of dietary change in deep time. We briefly discuss the use of carbon isotope data as it pertains to recent thinking about fallback food consumption in ancient hominins and suggest that we may need to rethink the functional significance of the australopith masticatory package. Am J Phys Anthropol 140:661–670, 2009. © 2009 Wiley-Liss, Inc.     

Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.     

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.     

Quantifying regional biodiversity in the tropics: A case study of freshwater fish in Trinidad and Tobago

Extinction rates are predicted to accelerate during the Anthropocene. Quantifying and mitigating these extinctions demands robust data on distributions of species and the diversity of taxa in regional biotas. However, many assemblages, particularly those in the tropics, are poorly characterized. Targeted surveys and historical museum collections are increasingly being used to meet the urgent need for accurate information, but the extent to which these contrasting data sources support meaningful inferences about biodiversity change in regional assemblages remains unclear. Here, we seek to elucidate uncertainty surrounding regional biodiversity estimates by evaluating the performance of these alternative methods in estimating the species richness and assemblage composition of the freshwater fish of Trinidad & Tobago. We compared estimates of regional species richness derived from two freshwater fish datasets: a targeted two year survey of Trinidad & Tobago rivers and historical museum collection records submitted to The University of the West Indies Zoology Museum. Richness was estimated using rarefaction and extrapolation, and assemblage composition was benchmarked against a recent literature review. Both datasets provided similar estimates of regional freshwater fish species richness (50 and 46 species, respectively), with a large overlap (85%) in species identities. Regional species richness estimates based on survey and museum data are thus comparable, and consistent in the species they include. Our results suggest that museum collection data are a viable option for setting reliable baselines in many tropical systems, thereby widening options for meaningful monitoring and evaluation of temporal trends. Abstract in Spanish is available with online material.     

Genomic Treasure Troves: Complete Genome Sequencing of Herbarium and Insect Museum Specimens

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today''s next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.     

Field sampling bias, museum collections and completeness of the fossil record

Museum specimens, particularly old collections, typically lack comprehensive field data and determination of substrate, sampling biases, etc., is problematic. Diversity at the generic level of all identifiable latest Cretaceous (Campanian–Maastrichtian) echinoderm remains in major museum collections from the Mons (southern Belgium) and Danish (Jylland (Jutland) and Sjælland (Zealand)) basins were compared to those of the Liège-Limburg Basin. The last-named has been studied in detail, including microscopical analysis of ossicles picked from bulk samples. Echinoids of the Mons Basin show similarities to those of the Liège-Limburg Basin, but crinoids, asteroids and ophiuroids remain poorly known from the former. Echinoderms of the Danish Basin resemble those of similar chalk lithofacies in the Liège-Limburg Basin, despite significant geographical separation. These disparities can be explained, at least in part, by collector bias in sampling methodology, although differences in substrate presumably also had an influence.     

How Global Is the Global Biodiversity Information Facility?

There is a concerted global effort to digitize biodiversity occurrence data from herbarium and museum collections that together offer an unparalleled archive of life on Earth over the past few centuries. The Global Biodiversity Information Facility provides the largest single gateway to these data. Since 2004 it has provided a single point of access to specimen data from databases of biological surveys and collections. Biologists now have rapid access to more than 120 million observations, for use in many biological analyses. We investigate the quality and coverage of data digitally available, from the perspective of a biologist seeking distribution data for spatial analysis on a global scale. We present an example of automatic verification of geographic data using distributions from the International Legume Database and Information Service to test empirically, issues of geographic coverage and accuracy. There are over 1/2 million records covering 31% of all Legume species, and 84% of these records pass geographic validation. These data are not yet a global biodiversity resource for all species, or all countries. A user will encounter many biases and gaps in these data which should be understood before data are used or analyzed. The data are notably deficient in many of the world's biodiversity hotspots. The deficiencies in data coverage can be resolved by an increased application of resources to digitize and publish data throughout these most diverse regions. But in the push to provide ever more data online, we should not forget that consistent data quality is of paramount importance if the data are to be useful in capturing a meaningful picture of life on Earth.