Natural history museums across the globe are an invaluable resource for education and research, housing millions to billions of preserved specimens. These specimens, along with their associated data, are snapshots in time providing evidence of past biodiversity, genetic diversity, and environmental conditions. Specimen-based research has been vital in our understanding of topics such as evolution, ecology, and climate change. Modern specimen preservation often includes the separate preservation of tissue samples for genetic analysis, but many museum specimens predate this approach. Including older specimens (including paleontological and archaeological contexts) in genetic studies is valuable because they potentially represent extinct species, extirpated populations, distinct demographic conditions, and holotypes. However, this can be difficult because the DNA is but often degraded and/or crosslinked. Rapid innovation and technological advancement (laboratory, bioinformatic, sequencing) have "unlocked the vault" (Bi et al. 2013) to allow for the use of older museum specimens and ancient DNA in genomic studies. Researchers in different organism-based disciplines (e.g., biology, archaeology, and paleontology) have made advances, and we hope to call attention to the similarities in approaches. The aim of this Research Topic is to highlight recent advances, demonstrate the importance of museum specimens in research, and further drive advancement in the field of museomics. We welcome original research, perspectives, and brief communications focusing on using preserved specimens for genetic studies. Studies on all organisms are acceptable. In particular, we hope to highlight studies using older specimens and genomic approaches. We encourage topics including but not limited to: • Ancient DNA • Paleontological and archaeological collections • Population genetics and phylogenetics • Taxonomic clarification • Description of new laboratory/analytical methods • Comparison of different approaches • Conservation genetics • Host-pathogen relationships and zoonotic diseases • Metagenomics

Lingnan University

Department of Zoology, Faculty of Science, Kasetsart University

University of California, Berkeley

Department of Biology, Faculty of Sciences, Chulalongkorn University

Museum of Natural History Berlin (MfN)

University of Florida

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Frontiers in Ecology and Evolution

Evolutionary and Population Genetics

Phylogenetics, Phylogenomics, and Systematics

Frontiers in Genetics

Frontiers in Plant Science

Recent Advances in Museomics: Revolutionizing Biodiversity Research

Museomics, a term coined by Drs. Stephan Schuster and Webb Miller in ~2009, refers to “the large-scale analysis of the DNA content of museum collections” (<a href="http://mammoth.psu.edu/museomics.html">http://mammoth.psu.edu/museomics.html</a>). Although such DNA studies existed before the term was first used, “museomics” highlighted the importance of specimens in biological studies.Specimens in natural history collections (NHCs) have been collected for hundreds of years to document the spatial and temporal occurrences of species. It is estimated that NHCs worldwide house 3 billion specimens (<a href="#B22">Soberon, 1999</a>). These specimens preserve a wealth of information, such as morphological and genetic data on the identity and phylogenetics of species, biogeographic and ecological data, and even biographical information of the collectors, and the contributions of NHCs extend well-beyond organismal biology research to fields such as public health (<a href="#B23">Suarez and Tsutsui, 2004</a>; <a href="#B2">Cook et al., 2020</a>) and education (<a href="#B4">Ellwood et al., 2020</a>; <a href="#B12">Lendemer et al., 2020</a>; <a href="#B16">National Academies of Sciences Engineering and Medicine, 2020</a>). NHCs are valuable resources with unknown future potential, and there are countless examples of research made possible that was not the goal of the original collector (<a href="#B9">Heberling et al., 2019</a>; <a href="#B13">Miller et al., 2020</a>). We provide three examples. First, <a href="#B15">Moritz et al. (2008)</a> compared modern specimens of small mammals to those collected ~100 years prior to document how climate change caused the distributions of some species to shift in elevation. Second, bird egg collections in museums were instrumental in showing the role of DDT in causing egg-shell thinning that adversely affected raptor and pelican populations (<a href="#B18">Ratcliffe, 1967</a>; <a href="#B10">Hickey and Anderson, 1968</a>). Lastly, <a href="#B6">Freelance et al. (2022)</a> stress the importance of properly designing captive breeding programs, since the sensory organs of the endangered Lord Howe Island stick insect (Dryococelus australis) differed between wild specimens (&gt;100 years old) and individuals bred in captivity. Given the accelerated rate of biodiversity loss, the role of NHCs will increase in prominence by being an archive of genetic and phenotypic diversity across space and time for many species that have gone extinct or where populations have vanished.Similarly in terms of unexpected potential, the advent of DNA sequencing technology opened up new avenues for specimen-based research. Modern specimen preparation now includes special steps to preserve DNA/RNA in tissues (e.g., freezing or placing tissues in ethanol or other storage media) for genetic studies, while previously there were no special efforts to preserve the DNA. There are challenges working with these materials, such as DNA naturally degrading over time and the DNA of formalin-fixed specimens being cross-linked with proteins and other DNA (<a href="#B19">Raxworthy and Smith, 2021</a>). Advances in laboratory methods and new sequencing technologies (e.g., high throughput short-read sequencing) have facilitated improvements in our ability to recover and sequence DNA from museum specimens.There are four primary sources of DNA that we discuss here: ancient DNA (aDNA), historical DNA (hDNA), modern DNA, and archival DNA (<a href="#B19">Raxworthy and Smith, 2021</a>). DNA extracted from samples that died under natural circumstances and were later recovered from the field are referred to as aDNA. Familiar examples of aDNA include samples obtained from species such as mammoths and cave bears, which can be quite old and are often &gt;200 years in age. In contrast, DNA extracted from formalin-fixed or ethanol-fixed specimens that were preserved and stored in museum collections is referred to as hDNA (these specimens are usually &lt;200 years old). DNA extracted from tissue samples specifically prepared with genetic analysis in mind is referred to as modern DNA and is usually &lt;40 years old. Archival DNA refers to hDNA and modern DNA stored in museum specimens. The first studies from researchers using the word “museomics” sequenced mitochondrial genomes from the aDNA in hair of the extinct Siberian mammoth (<a href="#B7">Gilbert et al., 2008</a>) and Tasmanian tiger (<a href="#B14">Miller et al., 2009</a>).This Research Topic is a collection of studies highlighting advances in museomics, both in demonstrating applications and refining methodologies. Some applications demonstrated in this Research Topic include using DNA barcoding of a degraded whale sample to identify it to subspecies (<a href="https://doi.org/10.3389/fevo.2022.921106">Ren et al.</a>), obtaining data from a holotype to verify the existence of an undescribed rodent genus (<a href="https://doi.org/10.3389/fevo.2022.930356">Castañeda-Rico et al.</a>), obtaining DNA from hundreds of herbarium specimens to elucidate the phylogeography of the genus Dalbergia (<a href="https://doi.org/10.3389/fevo.2022.910250">Sotuyo et al.</a>), and using target capture to understand the phylogenetic placement of two rare shark species (<a href="https://doi.org/10.3389/fevo.2022.910009">Agne, Naylor et al.</a>). These studies are diverse in the DNA type used (hDNA and modern DNA), taxa studied, objectives, and approaches. A variety of factors have been identified that affect the performance of sequencing DNA from specimens, and a major goal of museomics is to develop a set of best practices to maximize success (<a href="#B19">Raxworthy and Smith, 2021</a>). Efforts are being made to document and understand these factors (e.g., <a href="#B11">Irestedt et al., 2022</a>), and this Research Topic was initiated to further this cause. As an overview of this Research Topic, we identify several factors being addressed across the articles (<a href="#F1">Figure 1</a>). Following the terminology of <a href="https://doi.org/10.3389/fevo.2022.931644">Roycroft et al.</a>, we organize these factors temporally in the research process as pre-sequencing and post-sequencing (<a href="#F1">Figure 1</a>). This list of factors is not exhaustive, but rather highlights those that are addressed in this Research Topic. We note that findings in different studies may contradict each other, highlighting the dynamic state of the field and the need for more exhaustive research on this topic.Pre-sequencing factors dealt with in these studies are either related to the specimen or methodological advances to improve our ability to obtain DNA from historical collections.Four specimen-related factors are addressed: taxa, tissue type, age, and preservation history. A diversity of taxa was targeted across studies (mammals, insects, gastropods, bony fish, cartilaginous fish, reptiles, sponges, polychaetes, crustaceans, amphibians, plants, arachnids, birds), with mammals being the most frequent focal group (six studies). <a href="https://doi.org/10.3389/fevo.2022.909846">Agne, Preick et al.</a> included samples from nine classes of animals and found lower success with crustaceans, insects, and cartilaginous fish, and higher success with sponges, gastropods, polychaetes, and amphibians. Another study on gastropods (<a href="https://doi.org/10.3389/fevo.2022.907889">Clewing et al.</a>) noted that mollusks can be difficult to work with because their tissues are high in mucopolysaccharides, which can hinder DNA extraction.Several studies compared the performance of different tissue types. In a study of wolf specimens comparing tissue types (jaw bone, nasal bone, skin), skin had the best performance and should be preferred because it is less destructive to the specimen (<a href="https://doi.org/10.3389/fevo.2022.970249">Pacheco et al.</a>). In contrast, <a href="https://doi.org/10.3389/fevo.2022.931644">Roycroft et al.</a> found in their mammal study that DNA extraction from toe pad and bone tissue performed better than with skin.The importance of the age of specimens was commonly explored in these studies, with both types of archival DNA (hDNA and modern DNA) investigated across studies. The oldest specimen included was 192 years old (<a href="https://doi.org/10.3389/fevo.2022.909846">Agne, Preick et al.</a>). Some studies found a negative correlation between age and DNA yield (<a href="https://doi.org/10.3389/fevo.2022.893088">Bernstein and Ruane</a>; <a href="https://doi.org/10.3389/fevo.2022.984056">Hawkins et al.</a>; <a href="https://doi.org/10.3389/fevo.2022.931644">Roycroft et al.</a>), while others found no relationship (<a href="https://doi.org/10.3389/fevo.2022.943361">Nunes et al.</a>; <a href="https://doi.org/10.3389/fevo.2022.970249">Pacheco et al.</a>; <a href="https://doi.org/10.3389/fevo.2022.910084">Pavlek et al.</a>).Preservation history is an important factor that can be difficult to evaluate because the entire preservation process is usually not fully documented. Frozen tissue, as expected, preserves DNA better than other methods (<a href="https://doi.org/10.3389/fevo.2022.953131">Speer et al.</a>). <a href="https://doi.org/10.3389/fevo.2022.909846">Agne, Preick et al.</a> found that dry specimens performed better than wet across a variety of taxa, while <a href="https://doi.org/10.3389/fevo.2022.943361">Nunes et al.</a> found the opposite for insects where ethanol-preserved specimens performed better than dry papered and pinned specimens. Variation within preservation types, obscuring trends, is potentially confounded by the time between euthanization and preservation (<a href="https://doi.org/10.3389/fevo.2022.953131">Speer et al.</a>).Three lab work-related factors are target loci, DNA extraction protocol, and method of library preparation.For target loci, four major approaches were used&#x2014;target capture, barcoding, shotgun sequencing, and cDNA sequencing. The approach used was largely determined by the objective of the study. One common theme is that the loci targeted are short in length, due to the tendency of DNA to fragment over time in historical and ancient tissues.For DNA extraction, <a href="https://doi.org/10.3389/fevo.2022.984056">Hawkins et al.</a> compared four methods (spin column, spin column with aDNA modifications, magnetic beads, and phenol chloroform) and found that the spin column and phenol chloroform methods outperformed magnetic beads. The spin column with aDNA modifications retained smaller fragments but took more time and was more expensive. Taking into consideration performance, cost, time, and toxicity, they recommended the spin column method.For library preparation, <a href="https://doi.org/10.3389/fevo.2022.931644">Roycroft et al.</a> compared the performance of single and dual barcoded library indexing strategies. They found that sequencing performance was better with dual barcoded libraries, having more reads and lower heterozygosity (=less cross contamination) compared to single barcoded libraries.Post-sequencing factors addressed in these studies are related to the bioinformatic approaches.Two bioinformatic approaches were addressed in these studies: database and mapping approach.Databases are important in genetic studies, especially when identifying an unknown sample or determining its evolutionary relationship with other taxa. Existing data in a database may affect the resolution of genetic analyses. <a href="https://doi.org/10.3389/fevo.2022.966605">Nakazato and Jinbo</a> compared two commonly used DNA databases (GenBank and BOLD) and found that data for barcode loci are not the same in each database, despite each database importing from each other. This finding highlights the need of researchers to cross reference databases for relevant data.To identify the genetic location of sequence reads and compare homologous loci, a mapping approach can be used. Erroneous read mapping can impact the results of a population genetics study, such as estimation of selection or genetic parameters. <a href="https://doi.org/10.3389/fevo.2022.931644">Roycroft et al.</a> compared the effect of two different mapping approaches (sample-specific historical de novo assembly vs. high-quality “closest sister” de novo assembly) and found that data quality was better when mapping to a high-quality “closest sister” de novo assembly.Lastly, we note one study that in the strict sense may not qualify as “museomics”, since it is not a genetic study. <a href="https://doi.org/10.3389/fevo.2022.924941">Balmaki et al.</a> studied plant-pollinator relationships by preparing pollen slides, taking photographs, and using an artificial neural network to help in identification. This approach, compared to metabarcoding, had greater resolution when identifying plant species. We include this study in the Research Topic because it exemplifies the spirit of developing novel research uses of specimens.In the early 1900s, natural history museums were recognized as an “indispensable feature of modern civilization” due to the growing public interest in nature, their recognition of evolutionary trends in nature, and concerns regarding disappearing biodiversity (<a href="#B5">Farrington, 1915</a>). Despite their popularity and importance (<a href="#B1">Allmon, 1994</a>; <a href="#B23">Suarez and Tsutsui, 2004</a>), NHCs are currently facing a survival crisis of their own due to shrinking budgets (<a href="#B3">Dalton, 2003</a>; <a href="#B8">Gropp, 2004</a>; <a href="#B17">Pennisi, 2020</a>). To survive, NHCs need to find creative ways to publicize and acknowledge the usefulness of specimens and their data (<a href="#B21">Schindel and Cook, 2018</a>; <a href="#B13">Miller et al., 2020</a>; <a href="#B16">National Academies of Sciences Engineering and Medicine, 2020</a>). Some ideas proposed are to develop an “extended specimen network” digitizing and linking all associated data to a specimen (<a href="#B12">Lendemer et al., 2020</a>) and to recognize NHCs as coauthors on research articles (<a href="#B20">Rouhan et al., 2017</a>). We are heartened to see museomics helping to expand interest in specimen-based research while showcasing the importance of natural history collections, and we look forward to seeing how newly developed technologies are used to study existing specimens.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.We would like to thank all the contributors to this Research Topic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Editorial: Recent advances in museomics: revolutionizing biodiversity research

Next-generation sequencing (NGS) technologies have revolutionized phylogenomics by decreasing the cost and time required to generate sequence data from multiple markers or whole genomes. Further, the fragmented DNA of biological specimens collected decades ago can be sequenced with NGS, reducing the need for collecting fresh specimens. Sequence capture, also known as anchored hybrid enrichment, is a method to produce reduced representation libraries for NGS sequencing. The technique uses single-stranded oligonucleotide probes that hybridize with pre-selected regions of the genome that are sequenced via NGS, culminating in a dataset of numerous orthologous loci from multiple taxa. Phylogenetic analyses using these sequences have the potential to resolve deep and shallow phylogenetic relationships. Identifying the factors that affect sequence capture success could save time, money, and valuable specimens that might be destructively sampled despite low likelihood of sequencing success. We investigated the impacts of specimen age, preservation method, and DNA concentration on sequence capture (number of captured sequences and sequence quality) while accounting for taxonomy and extracted tissue type in a large-scale butterfly phylogenomics project. This project used two probe sets to extract 391 loci or a subset of 13 loci from over 6,000 butterfly specimens. We found that sequence capture is a resilient method capable of amplifying loci in samples of varying age (0–111 years), preservation method (alcohol, papered, pinned), and DNA concentration (0.020 ng/μl - 316 ng/ul). Regression analyses demonstrate that sequence capture is positively correlated with DNA concentration. However, sequence capture and DNA concentration are negatively correlated with sample age and preservation method. Our findings suggest that sequence capture projects should prioritize the use of alcohol-preserved samples younger than 20 years old when available. In the absence of such specimens, dried samples of any age can yield sequence data, albeit with returns that diminish with increasing age.

Next-generation sequencing (NGS) technologies have revolutionized phylogenomics by decreasing the cost and time required to generate sequence data from multiple markers or whole genomes. Further, the fragmented DNA of biological specimens collected decades ago can be sequenced with NGS, reducing the need for collecting fresh specimens. Sequence capture, also known as anchored hybrid enrichment, is a method to produce reduced representation libraries for NGS sequencing. The technique uses single-stranded oligonucleotide probes that hybridize with pre-selected regions of the genome that are sequenced via NGS, culminating in a dataset of numerous orthologous loci from multiple taxa. Phylogenetic analyses using these sequences have the potential to resolve deep and shallow phylogenetic relationships. Identifying the factors that affect sequence capture success could save time, money, and valuable specimens that might be destructively sampled despite low likelihood of sequencing success. We investigated the impacts of specimen age, preservation method, and DNA concentration on sequence capture (number of captured sequences and sequence quality) while accounting for taxonomy and extracted tissue type in a large-scale butterfly phylogenomics project. This project used two probe sets to extract 391 loci or a subset of 13 loci from over 6,000 butterfly specimens. We found that sequence capture is a resilient method capable of amplifying loci in samples of varying age (0–111 years), preservation method (alcohol, papered, pinned), and DNA concentration (0.020 ng/μl - 316 ng/ul). Regression analyses demonstrate that sequence capture is positively correlated with DNA concentration. However, sequence capture and DNA concentration are negatively correlated with sample age and preservation method. Our findings suggest that sequence capture projects should prioritize the use of alcohol-preserved samples younger than 20 years old when available. In the absence of such specimens, dried samples of any age can yield sequence data, albeit with returns that diminish with increasing age.

Predictors of sequence capture in a large-scale anchored phylogenomics project

Museomics is an approach to the DNA sequencing of museum specimens that can generate both biodiversity and sequence information. In this study, we surveyed both the biodiversity information-based database BOLD (Barcode of Life System) and the sequence information database GenBank, by using DNA barcoding data as an example, with the aim of integrating the data from these two databases. DNA barcoding is a method of identifying species from DNA sequences by using short genetic markers. We surveyed how many entries had biodiversity information (such as links to BOLD and specimen IDs) by downloading all fish, insect, and flowering plant data available from the GenBank Nucleotide, and BOLD ID was assigned to 26.2% of entries for insects. In the same way, we downloaded the respective BOLD data and checked the status of links to sequence information. We also investigated how many species do these databases cover, and 7,693 species were found to exist only in BOLD. In the future, as museomics develops as a field, the targeted sequences will be extended not only to DNA barcodes, but also to mitochondrial genomes, other genes, and genome sequences. Consequently, the value of the sequence data will increase. In addition, various species will be sequenced and, thus, biodiversity information such as the evidence specimen photographs used as a basis for species identification, will become even more indispensable. This study contributes to the acceleration of museomics-associated research by using databases in a cross-sectional manner.

Museomics is an approach to the DNA sequencing of museum specimens that can generate both biodiversity and sequence information. In this study, we surveyed both the biodiversity information-based database BOLD (Barcode of Life System) and the sequence information database GenBank, by using DNA barcoding data as an example, with the aim of integrating the data from these two databases. DNA barcoding is a method of identifying species from DNA sequences by using short genetic markers. We surveyed how many entries had biodiversity information (such as links to BOLD and specimen IDs) by downloading all fish, insect, and flowering plant data available from the GenBank Nucleotide, and BOLD ID was assigned to 26.2% of entries for insects. In the same way, we downloaded the respective BOLD data and checked the status of links to sequence information. We also investigated how many species do these databases cover, and 7,693 species were found to exist only in BOLD. In the future, as museomics develops as a field, the targeted sequences will be extended not only to DNA barcodes, but also to mitochondrial genomes, other genes, and genome sequences. Consequently, the value of the sequence data will increase. In addition, various species will be sequenced and, thus, biodiversity information such as the evidence specimen photographs used as a basis for species identification, will become even more indispensable. This study contributes to the acceleration of museomics-associated research by using databases in a cross-sectional manner.

Cross-sectional use of barcode of life data system and GenBank as DNA barcoding databases for the advancement of museomics

The subterranean ecosystem exerts strong selection pressures on the organisms that thrive in it. In response, obligate cave-dwellers have developed a series of morphological, physiological, and behavioral adaptations, such as eye reduction, appendage elongation, low metabolic rates or intermittent activity patterns, collectively referred to as troglomorphism. Traditionally, studies on cave organisms have been hampered by the difficulty of sampling (i.e., small population sizes, temporal heterogeneity in specimen occurrence, challenges imposed by the difficult-to-access nature of caves). Here, we circumvent this limitation by implementing a museomics approach. Specifically, we aim at comparing the genetic population structures of five cave spider species demonstrating contrasting life histories and levels of troglomorphism across different caves in the northern Dinarides (Balkans, Europe). We applied a genome-wide hybridization-capture approach (i.e., HyRAD) to capture DNA from 117 historical samples. By comparing the population genetic structures among five species and by studying isolation by distance, we identified deeper population structuring and more pronounced patterns of isolation by distance in the highly troglomorphic Parastalita stygia and Stalita pretneri ground dwellers, while the three web-building Troglohyphantes species, two of which can occasionally be found in surface habitats, showed less structured populations compatible with higher dispersal ability. The spatial distribution of genetic groups revealed common phylogeographic breaks among lineages across the studied species, which hint at the importance of environmental features in driving dispersal potential and shaping underground diversity.

The subterranean ecosystem exerts strong selection pressures on the organisms that thrive in it. In response, obligate cave-dwellers have developed a series of morphological, physiological, and behavioral adaptations, such as eye reduction, appendage elongation, low metabolic rates or intermittent activity patterns, collectively referred to as troglomorphism. Traditionally, studies on cave organisms have been hampered by the difficulty of sampling (i.e., small population sizes, temporal heterogeneity in specimen occurrence, challenges imposed by the difficult-to-access nature of caves). Here, we circumvent this limitation by implementing a museomics approach. Specifically, we aim at comparing the genetic population structures of five cave spider species demonstrating contrasting life histories and levels of troglomorphism across different caves in the northern Dinarides (Balkans, Europe). We applied a genome-wide hybridization-capture approach (i.e., HyRAD) to capture DNA from 117 historical samples. By comparing the population genetic structures among five species and by studying isolation by distance, we identified deeper population structuring and more pronounced patterns of isolation by distance in the highly troglomorphic Parastalita stygia and Stalita pretneri ground dwellers, while the three web-building Troglohyphantes species, two of which can occasionally be found in surface habitats, showed less structured populations compatible with higher dispersal ability. The spatial distribution of genetic groups revealed common phylogeographic breaks among lineages across the studied species, which hint at the importance of environmental features in driving dispersal potential and shaping underground diversity.

Life-history traits drive spatial genetic structuring in Dinaric cave spiders

Historical DNA obtained from voucher specimens housed in natural history museums worldwide have allowed the study of elusive, rare or even extinct species that in many cases are solely represented by museum holdings. This has resulted in the increase of taxonomic representation of many taxa, has led to the discovery of new species, and has yielded stunning novel insights into the evolutionary history of cryptic or even undescribed species. Peromyscus mekisturus, is a critically endangered cricetid rodent endemic to Mexico and is only known from two museum specimens collected in 1898 and 1947. Intensive field work efforts to attempt to determine if viable populations still exist have failed, suggesting that this species is extinct or is nearing extinction. In addition, a recent study using mitogenomes demonstrated that P. mekisturus forms a well-supported clade outside the genus Peromyscus and hypothesized that this taxon is the sister group of the genus Reithrodontomys. Here, we used target enrichment and high-throughput sequencing of several thousand nuclear ultraconserved elements and mitogenomes to reconstruct dated phylogenies to test the previous phylogenetic hypothesis. We analyzed the holotype and the only other known specimen of P. mekisturus and museum samples from other peromyscine rodents to test the phylogenetic position of the species. Our results confirm that the only two specimens known to science of P. mekisturus belong to the same species and support the hypothesis that this species belongs to an undescribed genus of cricetid rodents that is sister to the genus Reithrodontomys. We dated the origin of P. mekisturus together with other speciation events in peromyscines during the late Pliocene – early Pleistocene and related these events with the Pleistocene climatic cycles. In light of our results, we recommend a taxonomic re-evaluation of this enigmatic species to properly recognize its taxonomic status as a new genus. We also acknowledge the relevance of generating genomic data from type specimens and highlight the need and importance of continuing to build the scientific heritage of the collections to study and better understand past, present, and future biodiversity.

Historical DNA obtained from voucher specimens housed in natural history museums worldwide have allowed the study of elusive, rare or even extinct species that in many cases are solely represented by museum holdings. This has resulted in the increase of taxonomic representation of many taxa, has led to the discovery of new species, and has yielded stunning novel insights into the evolutionary history of cryptic or even undescribed species. Peromyscus mekisturus, is a critically endangered cricetid rodent endemic to Mexico and is only known from two museum specimens collected in 1898 and 1947. Intensive field work efforts to attempt to determine if viable populations still exist have failed, suggesting that this species is extinct or is nearing extinction. In addition, a recent study using mitogenomes demonstrated that P. mekisturus forms a well-supported clade outside the genus Peromyscus and hypothesized that this taxon is the sister group of the genus Reithrodontomys. Here, we used target enrichment and high-throughput sequencing of several thousand nuclear ultraconserved elements and mitogenomes to reconstruct dated phylogenies to test the previous phylogenetic hypothesis. We analyzed the holotype and the only other known specimen of P. mekisturus and museum samples from other peromyscine rodents to test the phylogenetic position of the species. Our results confirm that the only two specimens known to science of P. mekisturus belong to the same species and support the hypothesis that this species belongs to an undescribed genus of cricetid rodents that is sister to the genus Reithrodontomys. We dated the origin of P. mekisturus together with other speciation events in peromyscines during the late Pliocene – early Pleistocene and related these events with the Pleistocene climatic cycles. In light of our results, we recommend a taxonomic re-evaluation of this enigmatic species to properly recognize its taxonomic status as a new genus. We also acknowledge the relevance of generating genomic data from type specimens and highlight the need and importance of continuing to build the scientific heritage of the collections to study and better understand past, present, and future biodiversity.

Museomics and the holotype of a critically endangered cricetid rodent provide key evidence of an undescribed genus

Advances in the field of museomics have promoted a high sampling demand for natural history collections (NHCs), eventually resulting in damage to invaluable resources to understand historical biodiversity. It is thus essential to achieve a consensus about which historical tissues present the best sources of DNA. In this study, we evaluated the performance of different historical tissues from Iberian wolf NHCs in genome-wide assessments. We targeted three tissues—bone (jaw and femur), maxilloturbinal bone, and skin—that have been favored by traditional taxidermy practices for mammalian carnivores. Specifically, we performed shotgun sequencing and target capture enrichment for 100,000 single nucleotide polymorphisms (SNPs) selected from the commercial Canine HD BeadChip across 103 specimens from 1912 to 2005. The performance of the different tissues was assessed using metrics based on endogenous DNA content, uniquely high-quality mapped reads after capture, and enrichment proportions. All samples succeeded as DNA sources, regardless of their collection year or sample type. Skin samples yielded significantly higher amounts of endogenous DNA compared to both bone types, which yielded equivalent amounts. There was no evidence for a direct effect of tissue type on capture efficiency; however, the number of genotyped SNPs was strictly associated with the starting amount of endogenous DNA. Evaluation of genotyping accuracy for distinct minimum read depths across tissue types showed a consistent overall low genotyping error rate (<7%), even at low (3x) coverage. We recommend the use of skins as reliable and minimally destructive sources of endogenous DNA for whole-genome and target enrichment approaches in mammalian carnivores. In addition, we provide a new 100,000 SNP capture array validated for historical DNA (hDNA) compatible to the Canine HD BeadChip for high-quality DNA. The increasing demand for NHCs as DNA sources should encourage the generation of genomic datasets comparable among studies.

Advances in the field of museomics have promoted a high sampling demand for natural history collections (NHCs), eventually resulting in damage to invaluable resources to understand historical biodiversity. It is thus essential to achieve a consensus about which historical tissues present the best sources of DNA. In this study, we evaluated the performance of different historical tissues from Iberian wolf NHCs in genome-wide assessments. We targeted three tissues&#x2014;bone (jaw and femur), maxilloturbinal bone, and skin&#x2014;that have been favored by traditional taxidermy practices for mammalian carnivores. Specifically, we performed shotgun sequencing and target capture enrichment for 100,000 single nucleotide polymorphisms (SNPs) selected from the commercial Canine HD BeadChip across 103 specimens from 1912 to 2005. The performance of the different tissues was assessed using metrics based on endogenous DNA content, uniquely high-quality mapped reads after capture, and enrichment proportions. All samples succeeded as DNA sources, regardless of their collection year or sample type. Skin samples yielded significantly higher amounts of endogenous DNA compared to both bone types, which yielded equivalent amounts. There was no evidence for a direct effect of tissue type on capture efficiency; however, the number of genotyped SNPs was strictly associated with the starting amount of endogenous DNA. Evaluation of genotyping accuracy for distinct minimum read depths across tissue types showed a consistent overall low genotyping error rate (&lt;7%), even at low (3x) coverage. We recommend the use of skins as reliable and minimally destructive sources of endogenous DNA for whole-genome and target enrichment approaches in mammalian carnivores. In addition, we provide a new 100,000 SNP capture array validated for historical DNA (hDNA) compatible to the Canine HD BeadChip for high-quality DNA. The increasing demand for NHCs as DNA sources should encourage the generation of genomic datasets comparable among studies.

Assessing the performance of historical skins and bones for museomics using wolf specimens as a case study

Research on plant-pollinator interactions requires a diversity of perspectives and approaches, and documenting changing pollinator-plant interactions due to declining insect diversity and climate change is especially challenging. Natural history collections are increasingly important for such research and can provide ecological information across broad spatial and temporal scales. Here, we describe novel approaches that integrate museum specimens from insect and plant collections with field observations to quantify pollen networks over large spatial and temporal gradients. We present methodological strategies for evaluating insect-pollen network parameters based on pollen collected from museum insect specimens. These methods provide insight into spatial and temporal variation in pollen-insect interactions and complement other approaches to studying pollination, such as pollinator observation networks and flower enclosure experiments. We present example data from butterfly pollen networks over the past century in the Great Basin Desert and Sierra Nevada Mountains, United States. Complementary to these approaches, we describe rapid pollen identification methods that can increase speed and accuracy of taxonomic determinations, using pollen grains collected from herbarium specimens. As an example, we describe a convolutional neural network (CNN) to automate identification of pollen. We extracted images of pollen grains from 21 common species from herbarium specimens at the University of Nevada Reno (RENO). The CNN model achieved exceptional accuracy of identification, with a correct classification rate of 98.8%. These and similar approaches can transform the way we estimate pollination network parameters and greatly change inferences from existing networks, which have exploded over the past few decades. These techniques also allow us to address critical ecological questions related to mutualistic networks, community ecology, and conservation biology. Museum collections remain a bountiful source of data for biodiversity science and understanding global change.

Research on plant-pollinator interactions requires a diversity of perspectives and approaches, and documenting changing pollinator-plant interactions due to declining insect diversity and climate change is especially challenging. Natural history collections are increasingly important for such research and can provide ecological information across broad spatial and temporal scales. Here, we describe novel approaches that integrate museum specimens from insect and plant collections with field observations to quantify pollen networks over large spatial and temporal gradients. We present methodological strategies for evaluating insect-pollen network parameters based on pollen collected from museum insect specimens. These methods provide insight into spatial and temporal variation in pollen-insect interactions and complement other approaches to studying pollination, such as pollinator observation networks and flower enclosure experiments. We present example data from butterfly pollen networks over the past century in the Great Basin Desert and Sierra Nevada Mountains, United States. Complementary to these approaches, we describe rapid pollen identification methods that can increase speed and accuracy of taxonomic determinations, using pollen grains collected from herbarium specimens. As an example, we describe a convolutional neural network (CNN) to automate identification of pollen. We extracted images of pollen grains from 21 common species from herbarium specimens at the University of Nevada Reno (RENO). The CNN model achieved exceptional accuracy of identification, with a correct classification rate of 98.8%. These and similar approaches can transform the way we estimate pollination network parameters and greatly change inferences from existing networks, which have exploded over the past few decades. These techniques also allow us to address critical ecological questions related to mutualistic networks, community ecology, and conservation biology. Museum collections remain a bountiful source of data for biodiversity science and understanding global change.

Modern approaches for leveraging biodiversity collections to understand change in plant-insect interactions

The extraction of nucleic acids is one of the most routine procedures used in molecular biology laboratories, yet kit performance may influence the downstream processing of samples, particularly for samples which are degraded, and in low concentrations. Here we tested several commercial kits for specific use on commonly sampled mammalian museum specimens to evaluate the yield, size distribution, and endogenous content. Samples were weighed and had approximately equal input material for each extraction. These sample types are typical of natural history repositories ranged from 53 to 130 years old. The tested protocols spanned spin-column based extractions, magnetic bead purification, phenol/chloroform isolation, and specific modifications for ancient DNA. Diverse types of mammalian specimens were tested including adherent osteological material, bone and teeth, skin, and baleen. The concentration of DNA was quantified via fluorometry, and the size distributions of extracts visualized on an Agilent TapeStation. Overall, when DNA isolation was successful, all methods had quantifiable concentrations, albeit with variation across extracts. The length distributions varied based on the extraction protocol used. Shotgun sequencing was performed to evaluate if the extraction methods influenced the amount of endogenous versus exogenous content. The DNA content was similar across extraction methods indicating no obvious biases for DNA derived from different sources. Qiagen kits and phenol/chloroform isolation outperformed the Zymo magnetic bead isolations in these types of samples. Statistical analyses revealed that extraction method only explained 5% of the observed variation, and that specimen age explained variation (29%) more effectively.

The extraction of nucleic acids is one of the most routine procedures used in molecular biology laboratories, yet kit performance may influence the downstream processing of samples, particularly for samples which are degraded, and in low concentrations. Here we tested several commercial kits for specific use on commonly sampled mammalian museum specimens to evaluate the yield, size distribution, and endogenous content. Samples were weighed and had approximately equal input material for each extraction. These sample types are typical of natural history repositories ranged from 53 to 130 years old. The tested protocols spanned spin-column based extractions, magnetic bead purification, phenol/chloroform isolation, and specific modifications for ancient DNA. Diverse types of mammalian specimens were tested including adherent osteological material, bone and teeth, skin, and baleen. The concentration of DNA was quantified via fluorometry, and the size distributions of extracts visualized on an Agilent TapeStation. Overall, when DNA isolation was successful, all methods had quantifiable concentrations, albeit with variation across extracts. The length distributions varied based on the extraction protocol used. Shotgun sequencing was performed to evaluate if the extraction methods influenced the amount of endogenous versus exogenous content. The DNA content was similar across extraction methods indicating no obvious biases for DNA derived from different sources. Qiagen kits and phenol/chloroform isolation outperformed the Zymo magnetic bead isolations in these types of samples. Statistical analyses revealed that extraction method only explained 5% of the observed variation, and that specimen age explained variation (29%) more effectively.

A comparative analysis of extraction protocol performance on degraded mammalian museum specimens

Animal specimens in natural history collections are invaluable resources in examining the historical context of pathogen dynamics in wildlife and spillovers to humans. For example, natural history specimens may reveal new associations between bat species and coronaviruses. However, RNA viruses are difficult to study in historical specimens because protocols for extracting RNA from these specimens have not been optimized. Advances have been made in our ability to recover nucleic acids from formalin-fixed paraffin-embedded samples (FFPE) commonly used in human clinical studies, yet other types of formalin preserved samples have received less attention. Here, we optimize the recovery of RNA from formalin-fixed ethanol-preserved museum specimens in order to improve the usability of these specimens in surveys for zoonotic diseases. We provide RNA quality and quantity measures for replicate tissues subsamples of 22 bat specimens from five bat genera (Rhinolophus, Hipposideros, Megareops, Cynopterus, and Nyctalus) collected in China and Myanmar from 1886 to 2003. As tissues from a single bat specimen were preserved in a variety of ways, including formalin-fixed (8 bats), ethanol-preserved and frozen (13 bats), and flash frozen (2 bats), we were able to compare RNA quality and yield across different preservation methods. RNA extracted from historical museum specimens is highly fragmented, but usable for short-read sequencing and targeted amplification. Incubation of formalin-fixed samples with Proteinase-K following thorough homogenization improves RNA yield. This optimized protocol extends the types of data that can be derived from existing museum specimens and facilitates future examinations of host and pathogen RNA from specimens.

Animal specimens in natural history collections are invaluable resources in examining the historical context of pathogen dynamics in wildlife and spillovers to humans. For example, natural history specimens may reveal new associations between bat species and coronaviruses. However, RNA viruses are difficult to study in historical specimens because protocols for extracting RNA from these specimens have not been optimized. Advances have been made in our ability to recover nucleic acids from formalin-fixed paraffin-embedded samples (FFPE) commonly used in human clinical studies, yet other types of formalin preserved samples have received less attention. Here, we optimize the recovery of RNA from formalin-fixed ethanol-preserved museum specimens in order to improve the usability of these specimens in surveys for zoonotic diseases. We provide RNA quality and quantity measures for replicate tissues subsamples of 22 bat specimens from five bat genera (Rhinolophus, Hipposideros, Megareops, Cynopterus, and Nyctalus) collected in China and Myanmar from 1886 to 2003. As tissues from a single bat specimen were preserved in a variety of ways, including formalin-fixed (8 bats), ethanol-preserved and frozen (13 bats), and flash frozen (2 bats), we were able to compare RNA quality and yield across different preservation methods. RNA extracted from historical museum specimens is highly fragmented, but usable for short-read sequencing and targeted amplification. Incubation of formalin-fixed samples with Proteinase-K following thorough homogenization improves RNA yield. This optimized protocol extends the types of data that can be derived from existing museum specimens and facilitates future examinations of host and pathogen RNA from specimens.

A comparative study of RNA yields from museum specimens, including an optimized protocol for extracting RNA from formalin-fixed specimens

The pantropical genus Dalbergia includes more than 250 species. Phylogenetic studies of the group are scarce and have only included two or three species distributed in Mexico. We obtained herbarium samples of Mexican, Central American, and South American species (sourced from MEXU). In addition, sequences of GenBank accessions were used to complement the study. Using internal transcribed spacer (ITS), the matK and rbcL sequences from 384 accessions comprising species from America, Asia, and Africa were sampled to evaluate phylogenetic relationships of Mexican species and infrageneric classifications based on morphological data. Phylogenetic analyses suggest that the genus Dalbergia is monophyletic and originated in South America. The species distributed in Mexico are not a monophyletic clade but are divided into four clades with affinities to South American and Asian species clades. There is no correlation between geography and large-scale phylogeny. The estimated ages of the Mexican and Central American clades ranged from 11.32 Ma (Dalbergia granadillo clade) to 1.88 Ma (Dalbergia ecastaphyllum clade). Multiple long-distance dispersal events should be used to explain the current genus distribution.

The pantropical genus Dalbergia includes more than 250 species. Phylogenetic studies of the group are scarce and have only included two or three species distributed in Mexico. We obtained herbarium samples of Mexican, Central American, and South American species (sourced from MEXU). In addition, sequences of GenBank accessions were used to complement the study. Using internal transcribed spacer (ITS), the matK and rbcL sequences from 384 accessions comprising species from America, Asia, and Africa were sampled to evaluate phylogenetic relationships of Mexican species and infrageneric classifications based on morphological data. Phylogenetic analyses suggest that the genus Dalbergia is monophyletic and originated in South America. The species distributed in Mexico are not a monophyletic clade but are divided into four clades with affinities to South American and Asian species clades. There is no correlation between geography and large-scale phylogeny. The estimated ages of the Mexican and Central American clades ranged from 11.32 Ma (Dalbergia granadillo clade) to 1.88 Ma (Dalbergia ecastaphyllum clade). Multiple long-distance dispersal events should be used to explain the current genus distribution.

Insights into phylogenetic divergence of Dalbergia (Leguminosae: Dalbergiae) from Mexico and Central America

The application of high-throughput, short-read sequencing to degraded DNA has greatly increased the feasibility of generating genomic data from historical museum specimens. While many published studies report successful sequencing results from historical specimens; in reality, success and quality of sequence data can be highly variable. To examine predictors of sequencing quality, and methodological approaches to improving data accuracy, we generated and analyzed genomic sequence data from 115 historically collected museum specimens up to 180 years old. Data span both population genomic and phylogenomic scales, including historically collected specimens from 34 specimens of four species of Australian rock-wallabies (genus Petrogale) and 92 samples from 79 specimens of Australo-Papuan murine rodents (subfamily Murinae). For historical rodent specimens, where the focus was sampling for phylogenomics, we found that regardless of specimen age, DNA sequence libraries prepared from toe pad or bone subsamples performed significantly better than those taken from the skin (in terms of proportion of reads on target, number of loci captured, and data accuracy). In total, 93% of DNA libraries from toe pad or bone subsamples resulted in reliable data for phylogenetic inference, compared to 63% of skin subsamples. For skin subsamples, proportion of reads on target weakly correlated with collection year. Then using population genomic data from rock-wallaby skins as a test case, we found substantial improvement in final data quality by mapping to a high-quality “closest sister” de novo assembly from fresh tissues, compared to mapping to a sample-specific historical de novo assembly. Choice of mapping approach also affected final estimates of the number of segregating sites and Watterson's θ, both important parameters for population genomic inference. The incorporation of accurate and reliable sequence data from historical specimens has important outcomes for evolutionary studies at both population and phylogenomic scales. By assessing the outcomes of different approaches to specimen subsampling, library preparation and bioinformatic processing, our results provide a framework for increasing sequencing success for irreplaceable historical specimens.

The application of high-throughput, short-read sequencing to degraded DNA has greatly increased the feasibility of generating genomic data from historical museum specimens. While many published studies report successful sequencing results from historical specimens; in reality, success and quality of sequence data can be highly variable. To examine predictors of sequencing quality, and methodological approaches to improving data accuracy, we generated and analyzed genomic sequence data from 115 historically collected museum specimens up to 180 years old. Data span both population genomic and phylogenomic scales, including historically collected specimens from 34 specimens of four species of Australian rock-wallabies (genus Petrogale) and 92 samples from 79 specimens of Australo-Papuan murine rodents (subfamily Murinae). For historical rodent specimens, where the focus was sampling for phylogenomics, we found that regardless of specimen age, DNA sequence libraries prepared from toe pad or bone subsamples performed significantly better than those taken from the skin (in terms of proportion of reads on target, number of loci captured, and data accuracy). In total, 93% of DNA libraries from toe pad or bone subsamples resulted in reliable data for phylogenetic inference, compared to 63% of skin subsamples. For skin subsamples, proportion of reads on target weakly correlated with collection year. Then using population genomic data from rock-wallaby skins as a test case, we found substantial improvement in final data quality by mapping to a high-quality “closest sister” de novo assembly from fresh tissues, compared to mapping to a sample-specific historical de novo assembly. Choice of mapping approach also affected final estimates of the number of segregating sites and Watterson's θ, both important parameters for population genomic inference. The incorporation of accurate and reliable sequence data from historical specimens has important outcomes for evolutionary studies at both population and phylogenomic scales. By assessing the outcomes of different approaches to specimen subsampling, library preparation and bioinformatic processing, our results provide a framework for increasing sequencing success for irreplaceable historical specimens.

Sequence Capture From Historical Museum Specimens: Maximizing Value for Population and Phylogenomic Studies

DNA barcoding technology is becoming an increasingly powerful tool in resolving issues of detailed species identification based on morphology, as commonly employed by museums. In the present study, we aimed to identify a stranded Bryde’s whale on Hainan Island, China by extracting DNA from a vertebra pre-treated by physical and/or chemical processes. Based on morphological characteristics, this Bryde’s whale was initially determined as Balaenoptera edeni. Then, DNA was efficiently extracted using ancient DNA techniques. The mitochondrial gene (COI) phylogenetic analysis further revealed that this museum whale specimen belonged to the sub-species B. e. edeni. This study provides a testable and rapid method for museum species verification, by using ancient DNA extraction methods to compensate the disadvantage of traditional DNA extraction methods that are difficult to extract valid DNA.

DNA barcoding technology is becoming an increasingly powerful tool in resolving issues of detailed species identification based on morphology, as commonly employed by museums. In the present study, we aimed to identify a stranded Bryde’s whale on Hainan Island, China by extracting DNA from a vertebra pre-treated by physical and/or chemical processes. Based on morphological characteristics, this Bryde’s whale was initially determined as Balaenoptera edeni. Then, DNA was efficiently extracted using ancient DNA techniques. The mitochondrial gene (COI) phylogenetic analysis further revealed that this museum whale specimen belonged to the sub-species B. e. edeni. This study provides a testable and rapid method for museum species verification, by using ancient DNA extraction methods to compensate the disadvantage of traditional DNA extraction methods that are difficult to extract valid DNA.

DNA Barcoding Technology Used to Successfully Sub-Classify a Museum Whale Specimen as Balaenoptera edeni edeni

Over the past decade, museum genomics studies have focused on obtaining DNA of sufficient quality and quantity for sequencing from fluid-preserved natural history specimens, primarily to be used in systematic studies. While these studies have opened windows to evolutionary and biodiversity knowledge of many species worldwide, published works often focus on the success of these DNA sequencing efforts, which is undoubtedly less common than obtaining minimal or sometimes no DNA or unusable sequence data from specimens in natural history collections. Here, we attempt to obtain and sequence DNA extracts from 115 fresh and 41 degraded samples of homalopsid snakes, as well as from two degraded samples of a poorly known snake, Hydrablabes periops. Hydrablabes has been suggested to belong to at least two different families (Natricidae and Homalopsidae) and with no fresh tissues known to be available, intractable museum specimens currently provide the only opportunity to determine this snake’s taxonomic affinity. Although our aim was to generate a target-capture dataset for these samples, to be included in a broader phylogenetic study, results were less than ideal due to large amounts of missing data, especially using the same downstream methods as with standard, high-quality samples. However, rather than discount results entirely, we used mapping methods with references and pseudoreferences, along with phylogenetic analyses, to maximize any usable molecular data from our sequencing efforts, identify the taxonomic affinity of H. periops, and compare sequencing success between fresh and degraded tissue samples. This resulted in largely complete mitochondrial genomes for five specimens and hundreds to thousands of nuclear loci (ultra-conserved loci, anchored-hybrid enrichment loci, and a variety of loci frequently used in squamate phylogenetic studies) from fluid-preserved snakes, including a specimen of H. periops from the Field Museum of Natural History collection. We combined our H. periops data with previously published genomic and Sanger-sequenced datasets to confirm the familial designation of this taxon, reject previous taxonomic hypotheses, and make biogeographic inferences for Hydrablabes. A second H. periops specimen, despite being seemingly similar for initial raw sequencing results and after being put through the same protocols, resulted in little usable molecular data. We discuss the successes and failures of using different pipelines and methods to maximize the products from these data and provide expectations for others who are looking to use DNA sequencing efforts on specimens that likely have degraded DNA.Life Science Identifier (Hydrablabes periops)<ext-link ext-link-type="uri" xlink:href="https://zoobank.org/" xmlns:xlink="http://www.w3.org/1999/xlink">urn:lsid:zoobank.org</ext-link>:pub:F2AA44 E2-D2EF-4747-972A-652C34C2C09D.</sec>

Over the past decade, museum genomics studies have focused on obtaining DNA of sufficient quality and quantity for sequencing from fluid-preserved natural history specimens, primarily to be used in systematic studies. While these studies have opened windows to evolutionary and biodiversity knowledge of many species worldwide, published works often focus on the success of these DNA sequencing efforts, which is undoubtedly less common than obtaining minimal or sometimes no DNA or unusable sequence data from specimens in natural history collections. Here, we attempt to obtain and sequence DNA extracts from 115 fresh and 41 degraded samples of homalopsid snakes, as well as from two degraded samples of a poorly known snake, Hydrablabes periops. Hydrablabes has been suggested to belong to at least two different families (Natricidae and Homalopsidae) and with no fresh tissues known to be available, intractable museum specimens currently provide the only opportunity to determine this snake’s taxonomic affinity. Although our aim was to generate a target-capture dataset for these samples, to be included in a broader phylogenetic study, results were less than ideal due to large amounts of missing data, especially using the same downstream methods as with standard, high-quality samples. However, rather than discount results entirely, we used mapping methods with references and pseudoreferences, along with phylogenetic analyses, to maximize any usable molecular data from our sequencing efforts, identify the taxonomic affinity of H. periops, and compare sequencing success between fresh and degraded tissue samples. This resulted in largely complete mitochondrial genomes for five specimens and hundreds to thousands of nuclear loci (ultra-conserved loci, anchored-hybrid enrichment loci, and a variety of loci frequently used in squamate phylogenetic studies) from fluid-preserved snakes, including a specimen of H. periops from the Field Museum of Natural History collection. We combined our H. periops data with previously published genomic and Sanger-sequenced datasets to confirm the familial designation of this taxon, reject previous taxonomic hypotheses, and make biogeographic inferences for Hydrablabes. A second H. periops specimen, despite being seemingly similar for initial raw sequencing results and after being put through the same protocols, resulted in little usable molecular data. We discuss the successes and failures of using different pipelines and methods to maximize the products from these data and provide expectations for others who are looking to use DNA sequencing efforts on specimens that likely have degraded DNA.Life Science Identifier (Hydrablabes periops): <a href="https://zoobank.org/" style="color:#D54449;">urn:lsid:zoobank.org</a>:pub:F2AA44 E2-D2EF-4747-972A-652C34C2C09D.

Maximizing Molecular Data From Low-Quality Fluid-Preserved Specimens in Natural History Collections

Etmopteridae (lantern sharks) is the most species-rich family of sharks, comprising more than 50 species. Many species are described from few individuals, and re-collection of specimens is often hindered by the remoteness of their sampling sites. For taxonomic studies, comparative morphological analysis of type specimens housed in natural history collections has been the main source of evidence. In contrast, DNA sequence information has rarely been used. Most lantern shark collection specimens, including the types, were formalin fixed before long-term storage in ethanol solutions. The DNA damage caused by both fixation and preservation of specimens has excluded these specimens from DNA sequence-based phylogenetic analyses so far. However, recent advances in the field of ancient DNA have allowed recovery of wet-collection specimen DNA sequence data. Here we analyse archival mitochondrial DNA sequences, obtained using ancient DNA approaches, of two wet-collection lantern shark paratype specimens, namely Etmopterus litvinovi and E. pycnolepis, for which the type series represent the only known individuals. Target capture of mitochondrial markers from single-stranded DNA libraries allows for phylogenetic placement of both species. Our results suggest synonymy of E. benchleyi with E. litvinovi but support the species status of E. pycnolepis. This revised taxonomy is helpful for future conservation and management efforts, as our results indicate a larger distribution range of E. litvinovi. This study further demonstrates the importance of wet-collection type specimens as genetic resource for taxonomic research.

Etmopteridae (lantern sharks) is the most species-rich family of sharks, comprising more than 50 species. Many species are described from few individuals, and re-collection of specimens is often hindered by the remoteness of their sampling sites. For taxonomic studies, comparative morphological analysis of type specimens housed in natural history collections has been the main source of evidence. In contrast, DNA sequence information has rarely been used. Most lantern shark collection specimens, including the types, were formalin fixed before long-term storage in ethanol solutions. The DNA damage caused by both fixation and preservation of specimens has excluded these specimens from DNA sequence-based phylogenetic analyses so far. However, recent advances in the field of ancient DNA have allowed recovery of wet-collection specimen DNA sequence data. Here we analyse archival mitochondrial DNA sequences, obtained using ancient DNA approaches, of two wet-collection lantern shark paratype specimens, namely Etmopterus litvinovi and E. pycnolepis, for which the type series represent the only known individuals. Target capture of mitochondrial markers from single-stranded DNA libraries allows for phylogenetic placement of both species. Our results suggest synonymy of E. benchleyi with E. litvinovi but support the species status of E. pycnolepis. This revised taxonomy is helpful for future conservation and management efforts, as our results indicate a larger distribution range of E. litvinovi. This study further demonstrates the importance of wet-collection type specimens as genetic resource for taxonomic research.

Taxonomic Identification of Two Poorly Known Lantern Shark Species Based on Mitochondrial DNA From Wet-Collection Paratypes

A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.

A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.

Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set

Museum material is an important source of metadata for past and recent biological events. With current sequencing technologies, it is possible to obtain historical DNA (hDNA) from older material and/or endangered species to answer taxonomic, systematic, and biogeographical questions. However, hDNA from museum collections is often highly degraded, making it difficult to assess relationships at or above the species level. We therefore studied two probably extinct gastropod species of the genus Laevicaspia, which were collected ∼140 years ago in the Caspian Sea, to map “standard” mitochondrial and nuclear markers and assess both the sequencing depth and the proportion of ambiguous sites as an indicator for the phylogenetic quality of the NGS data. Our study resulted in the first phylogenetically informative mitochondrial and nuclear markers for L. caspia. Assessment of both sequencing depth (mean coverage) and proportion of ambiguous sites suggests that our assembled consensus sequences are reliable for this species. In contrast, no informative gastropod-specific DNA was obtained for L. conus, likely due to a high degree of tissue digestion and contamination with non-gastropod DNA. Nevertheless, our results show that hDNA may in principle yield high-quality sequences for species-level phylogenetic analyses, which underlines the importance of museum collections as valuable archives of the biological past.

Museum material is an important source of metadata for past and recent biological events. With current sequencing technologies, it is possible to obtain historical DNA (hDNA) from older material and/or endangered species to answer taxonomic, systematic, and biogeographical questions. However, hDNA from museum collections is often highly degraded, making it difficult to assess relationships at or above the species level. We therefore studied two probably extinct gastropod species of the genus Laevicaspia, which were collected ∼140 years ago in the Caspian Sea, to map “standard” mitochondrial and nuclear markers and assess both the sequencing depth and the proportion of ambiguous sites as an indicator for the phylogenetic quality of the NGS data. Our study resulted in the first phylogenetically informative mitochondrial and nuclear markers for L. caspia. Assessment of both sequencing depth (mean coverage) and proportion of ambiguous sites suggests that our assembled consensus sequences are reliable for this species. In contrast, no informative gastropod-specific DNA was obtained for L. conus, likely due to a high degree of tissue digestion and contamination with non-gastropod DNA. Nevertheless, our results show that hDNA may in principle yield high-quality sequences for species-level phylogenetic analyses, which underlines the importance of museum collections as valuable archives of the biological past.