skip to main content


Title: Maximizing Molecular Data From Low-Quality Fluid-Preserved Specimens in Natural History Collections
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 ) urn:lsid:zoobank.org :pub:F2AA44 E2-D2EF-4747-972A-652C34C2C09D.  more » « less
Award ID(s):
2224119
NSF-PAR ID:
10392384
Author(s) / Creator(s):
;
Date Published:
Journal Name:
Frontiers in Ecology and Evolution
Volume:
10
ISSN:
2296-701X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    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.

     
    more » « less
  2. Abstract

    Although they are a valuable source of specimens, insect natural history collections continue to be under‐utilized in molecular systematics, mostly due to difficulties in obtaining DNA sequences. Old specimens or specimens stored under suboptimal conditions are intractable for traditional Sanger sequencing. In this study we use an inexpensive hybrid capture with in‐house generated baits to retrieve commonly utilized ribosomal and mitochondrial loci from old museum specimens and combine them with a Sanger‐generated dataset comprising recently collected material. We focus on theCorixideagenus group (Schizopteridae), which comprises rarely collected, small (1–2 mm) and primarily tropical insects of which onlyc. 10–20% of the species have been described. A molecular phylogeny is needed to resolve relationships and revise the genus‐level classification to correctly place thec. 150 yet to be described species. Applying this approach, we constructed a dataset, containing 101 taxa, 11 of which were preserved in low‐percentage ethanol, 48 are dry and point‐mounted, and 40 are > 20 years old at DNA extraction. The obtained data proved sufficient for reconstructing a well‐supported phylogeny withc. 50% of the predicted diversity, and for the oldest successfully sequenced specimen (95 years) to be unambiguously placed in that phylogeny. We confirmed monophyly of theCorixideagenus group, showed paraphyly of the genusCorixidea, and recovered nine well‐supported clades within the group. Ancestral character states of selected morphological features were inferred and used to re‐examine primary homology hypotheses and inform an upcoming taxonomic revision.

     
    more » « less
  3. Abstract

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

     
    more » « less
  4. Abstract

    The use of gDNAs isolated from museum specimens for high throughput sequencing, especially targeted sequencing in the context of phylogenetics, is a common practice. Yet, little understanding has been focused on comparing the quality of DNA and results of sequencing museum DNAs. Dragonflies and damselflies are ubiquitous in freshwater ecosystems and are commonly collected and preserved insects in museum collections hence their use in this study. However, the history of odonate preservation across time and museums has resulted in wide variability in the success of viable DNA extraction, necessitating an assessment of their usefulness in genetic studies. Using Anchored Hybrid Enrichment probes, we sequenced DNA from samples at 2 museums, 48 from the American Museum of Natural History (AMNH) in NYC, USA and 46 from the Naturalis Biodiversity Center (RMNH) in Leiden, Netherlands ranging from global collection localities and across a 120-year time span. We recovered at least 4 loci out of an >1,000 locus probe set for all samples, with the average capture being ~385 loci (539 loci on average when a clade of ambiguous taxa omitted). Neither specimen age nor size was a good predictor of locus capture, but recapture rates differed significantly between museums. Samples from the AMNH had lower overall locus capture than the RMNH, perhaps due to differences in specimen storage over time.

     
    more » « less
  5. null (Ed.)
    Species that went extinct prior to the genomic era are typically out-of-reach for modern phylogenetic studies. We refer to these as “Alexandrian” extinctions, after the lost library of the ancient world. This is particularly limiting for conservation studies, as genetic data for such taxa may be key to understand extinction threats and risks, the causes of declines, and inform management of related, extant populations. Fortunately, continual advances in biochemistry and DNA sequencing offer increasing ability to recover DNA from historical museum specimens, including fluid-preserved natural history collections. Here, we report on success in recovering nuclear and mitochondrial data from the apparently-extinct subspecies Desmognathus fuscus carri Neill, 1951, a plethodontid salamander from spring runs in central Florida. The two specimens are 50 years old and were likely preserved in unbuffered formalin, but application of a recently derived extraction procedure yielded usable DNA and partially successful Anchored Hybrid Enrichment sequencing. These data suggest that the populations of D. f. carri from peninsular Florida are conspecific with the D. auriculatus A lineage as suggested by previous authors, but likely represented an ecogeographically distinct genetic segment that has now been lost. Genetic data from this Alexandrian extinction thus confirm the geographic extent of population declines and extirpations as well as their ecological context, suggesting a possibly disproportionate loss from sandy-bottom clearwater streams compared to blackwater swamps. Success of these methods bodes well for large-scale application to fluid-preserved natural history specimens from relevant historical populations, but the possibility of significant DNA damage and related sequencing errors in additional hurdle to overcome. 
    more » « less