The cosmopolitan genus Botrytis contains 22 recognized species and one hybrid. The current classification is largely based on morphological characters and, to a minor extent, on physiology and host range. In this study, a classification of the genus was constructed based on DNA sequence data of three nuclear protein-coding genes (RPB2, G3PDH, and HSP60) and compared with the traditional classification. Sexual reproduction and the host range, important fitness traits, were traced in the tree and used for the identification of major evolutionary events during speciation. The phylogenetic analysis corroborated the classical species delineation. In addition, the hybrid status of B. allii (B. byssoidea x B. aclada) was confirmed. Both individual gene trees and combined trees show that the genus Botrytis can be divided into two clades, radiating after the separation of Botrytis from other Sclerotiniaceae genera. Clade 1 contains four species that all colonize exclusively eudicot hosts, whereas clade 2 contains 18 species that are pathogenic on either eudicot (3) or monocot (15) hosts. A comparison of Botrytis and angiosperm phylogenies shows that cospeciation of pathogens and their hosts have not occurred during their respective evolution. Rather, we propose that host shifts have occurred during Botrytis speciation, possibly by the acquisition of novel pathogenicity factors. Loss of sexual reproduction has occurred at least three times and is supposed to be a consequence of negative selection.

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.

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4–3.8% of fresh control DNA and 1.0–1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL (UAA) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10–143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200–300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.

Herbarium genomics is proving promising as next‐generation sequencing approaches are well suited to deal with the usually fragmented nature of archival DNA. We show that routine assembly of partial plastome sequences from herbarium specimens is feasible, from total DNA extracts and with specimens up to 146 years old. We use genome skimming and an automated assembly pipeline, Iterative Organelle Genome Assembly, that assembles paired‐end reads into a series of candidate assemblies, the best one of which is selected based on likelihood estimation. We used 93 specimens from 12 different Angiosperm families, 73 of which were from herbarium material with ages up to 146 years old. For 84 specimens, a sufficient number of paired‐end reads were generated (in total 9.4 × 1012 nucleotides), yielding successful plastome assemblies for 74 specimens. Those derived from herbarium specimens have lower fractions of plastome‐derived reads compared with those from fresh and silica‐gel‐dried specimens, but total herbarium assembly lengths are only slightly shorter. Specimens from wet‐tropical conditions appear to have a higher number of contigs per assembly and lower N50 values. We find no significant correlation between plastome coverage and nuclear genome size (C value) in our samples, but the range of C values included is limited. Finally, we conclude that routine plastome sequencing from herbarium specimens is feasible and cost‐effective (compared with Sanger sequencing or plastome‐enrichment approaches), and can be performed with limited sample destruction.