DNA Sequencing Historical Lichen Specimens

Abstract

Biological specimens in natural history collections worldwide are increasingly being used in biogeographical, environmental, and taxonomic studies. For their meaningful use, correct species identification is crucial. For example, clarifying if a species is new to science requires an overview of what has already been described. This includes comparisons with existing authoritative specimens (types). Most type specimens are rather old and their DNA expected to be degraded to various extents. Comparative DNA sequence analysis is in regular use in taxonomic research of today and is essential for identifying and delimiting species. In this study, we focus on lichenized fungi (lichens), in which many species groups are highly inconspicuous and impossible to identify to species based on morphology alone. Our aim was to test the non-mutually exclusive hypotheses that DNA quality of lichens depends on (1) time since collection, (2) taxonomic affinity, and/or (3) habitat/ecology. We included two species from each of four different lichen genera (i.e., Cladonia, Nephroma, Peltigera, and Ramalina), each species pair with a different autecology. For each species, we included samples from approximately every 25 years from present to about 150 years back in time. We used a two-step PCR-based approach followed by sequencing on an Ion Torrent PGM to produce target sequences (mtSSU) of degraded DNA. We received satisfactory DNA sequence information for 54 of 56 specimens. We recovered full-length sequences for several more than 100-years-old specimens, including a 127-years-old specimen, and retrieved enough sequence information for species identification of a 150-years-old specimen. As expected, sequencing success was negatively correlated with age of the specimens. It also varied with taxonomic affinity. We found no significant correlation between sequencing success and habitat ecology of the investigated specimens. The herein tested Ion Torrent sequencing approach outperformed Sanger sequencing with regard to sequencing success and efficiency. We find the protocol used herein highly suitable for obtaining sequences from both young and old lichen specimens and discuss potential improvements to it.

Shotgun sequencing decades-old lichen specimens to resolve phylogenomic placement of type material

“…Similarly, shotgun sequencing approaches for generating genome-scale data may not be necessary in all cases. Standard PCR amplification and Sanger sequencing may be sufficient to accurately assign name-bearing types; and high-throughput sequencing of PCR amplicons has also been shown to effectively generate sequence data from historical lichen collections (Kistenich et al 2019). As sequencing technologies continue to change and the cost of generating genome-scale data decreases, researchers must carefully evaluate the most appropriate data for effectively addressing specific questions.…”

“…Natural history collections have also played a key role in phylogenetic research efforts involving lichen-forming fungi, with DNA routinely extracted from herbarium specimens (Sohrabi et al 2010;Gueidan et al 2015;Kistenich et al 2019), including a 151-year-old museum specimen (Redchenko et al 2012). However, in other cases, generating sequence data from some lichens can be problematic with the passing of even short amounts of time, on the scale of months to years, without careful, intentional preparation and storage to maximize the probability of extracting useable DNA.…”

“…While high-throughput sequencing of PCR amplicons has been shown to effectively generate sequence data from historical lichen collections (Kistenich et al 2019), to our knowledge this is the first attempt to use high-throughput whole-genome shotgun sequencing of historical lichen type specimens within a taxonomic context.…”

New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes

“…Specimens and related data can be explored for a wide array of research including, for instance, taxonomical (e.g., Martin & Alexopoulos, 1969;Bebber et al, 2010;Petersen & Hughes, 2010), biogeographic (e.g., Wollan et al, 2008Lavoie, 2013;Ronikier & Ronikier, 2010), global change (Meineke, Davis & Davies, 2018) and conservation biology studies (Greve et al, 2016). Over last years, with an increasing development of DNA-based molecular methods in ecology and evolution, herbarium specimens have appeared as an invaluable material for phylogenetic analyses (e.g., Moncalvo et al, 2002;Fiore-Donno et al, 2012;Fiore-Donno et al, 2013;Kistenich et al, 2019). In particular, type collections provide key reference for molecular barcoding databases used in biodiversity and environmental studies and provide irreplaceable source of specimens based on which the species' name can be correctly applied (e.g., Larsson & Jacobsson, 2004;Lehtonen & Christenhusz, 2010;Chomicki & Renner, 2015;Dormontt et al, 2018).…”

“…Two main obstacles hinder the use of herbarium collections as a source for DNA isolation. One, affecting all types of collections, is DNA degradation mostly related to collection age, its preparation, treatment and storage conditions (Lehtonen & Christenhusz, 2010;Staats et al, 2011;Weißet al, 2016;Kistenich et al, 2019). The second limitation is quantity of available biological material, especially relevant in the case of organisms with small dimensions and those characterized by ephemeral emergence substantially limiting the possibility of sampling in the field.…”