Bull. Natl. Mus. Nat. Sci., Ser. B, 43(2), pp. 33–44, May 22, 2017 DNA Extraction, PCR and Sequencing were largely Unsuccessful from the Type Specimens of Mushrooms but Some 50-year-old or Older Specimens produced Authentic Sequences Kentaro Hosaka Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan E-mail: [email protected] (Received 16 March 2017; accepted 22 March 2017) Abstract DNA extraction, PCR and sequencing of the nuclear ribosomal internal transcribed spacer region from a total of 293 type specimens of Agaricomycotina (Basidiomycota, Fungi) were conducted. Although more than 50% of those specimens produced positive PCR bands, a total of 114 specimens from National Museum of Nature and Science (TNS) and 21 specimens from Field Museum of Natural History (F), respectively, produced sequences of low quality with no discern- able peaks. All of the 43 good sequences obtained from the specimens at TNS were demonstrated to be from apparent contamination. In contrast, out of the 16 good sequences obtained from the specimens at F, only 2 sequences were from apparent contamination and the remaining 14 were considered authentic. The oldest specimen from which authentic sequence has been obtained was from 1940, but the PCR and sequencing success rate from older specimens was general low. This study clearly demonstrates the need of immediate DNA extraction from type specimens. Alterna- tively, small pieces of type specimens should be preserved separately from the herbarium for future DNA studies. Key words : BLAST, herbarium, ITS, mushrooms, taxonomy, type specimens. Introduction the presence of “universal” primers and high suc- cess rate of PCR and sequencing make this Fungi in general have limited morphological region widely applicable to many studies, such as characters and mushrooms with macroscopic taxonomy and ecology (Dentinger et al., 2010; fruit bodies are no exception. It has often been Nagy et al., 2011; Schoch et al., 2012). demonstrated that mushrooms with similar or In contrast to the rapid accumulation of the indistinguishable fruit body morphology repre- ITS sequences in GenBank, the number of sent phylogenetically distantly related taxa (e.g., sequences from the type specimens is not Kasuya et al., 2012; Wilson et al., 2016). For this increasing at the same rate. The study by Nagy et reason, DNA sequence data, especially from the al. (2011) showed that a number of unidentifiable type specimens, are essential to establish stable sequences from environmental sequencing proj- taxonomic system of mushrooms. ects matched with the sequences from the type It is generally appreciated that the nuclear strains of Mortierella. Similarly, Brock et al. ribosomal internal transcribed spacer (ITS) (2009) estimated that ca. 70% of taxonomic region is an official DNA barcode region for diversity at the fungal herbarium of Kew Gar- fungi (Schoch et al., 2012). Although the ITS dens alone was not yet represented in GenBank. sequences cannot discriminate some closely These studies clearly demonstrated the need of related species, especially among Ascomycota, obtaining ITS sequences from the type speci- 34 Kentaro Hosaka mens to appropriately link fungal names and some specimens. For example, “Ganoderma DNA sequence data. spathulatum” (TNS-F-201590) was indicated on Molecular studies using the type specimens, the specimen label as “Typus” but no such names however, face a number of challenges. In gen- could be found either from Index Fungorum eral, DNA in specimens degrades with time (http://www.indexfungorum.org/) or a cumula- (Erkens et al., 2008; Willerslev and Cooper, tive list of Japanese fungi by Katsumoto (2010). 2005), but the majority of type specimens are Such species of uncertain type status are indi- “old”. In addition, storage conditions such as cated with quotation marks throughout the text temperature, humidity, and type of fumigation all and tables (Table 2, 3). affect the quality of DNA (Willerslev and Coo- All dried specimens, including the specimen per, 2005). It has also been demonstrated that labels and packets, were photographed for later older specimens tend to be more contaminated assessing the quality of specimens, such as pres- by other fungi (Brock et al., 2009). ence of fungal or insect damage. Some represen- In this study, the type specimens of diverse tative photographs and specimen data of type groups of mushrooms housed at two different specimens were uploaded at Type Specimen herbaria collected in different times (from 1880’s Database of National Museum of Nature and Sci- to 1990’s) were tested for assessing the DNA ence, and they are publicly available from http:// quality. All specimens were extracted for www.type.kahaku.go.jp/TypeDB/. genomic DNA, PCR amplified using general primers of the ITS, and sequence data were DNA preparation, PCR, sequencing, and BLAST obtained by direct sequencing of PCR products. search The aforementioned challenges are further DNA was extracted from hymenial tissues of emphasized, and a potential solution for future dried specimens (~200 mg) stored overnight in studies is presented. DMSO buffer (Seutin et al., 1991; Hosaka, 2009). Tissues were first ground under liquid nitrogen using a mortar and pestle. Ground tis- Materials and Methods sues were then transferred to new 1.5 ml tubes Type specimens used in this study using clean spatulas, and CTAB buffer was A total of 235 specimens housed at the fungal added. DNA was extracted using the modified herbarium, Department of Botany, National CTAB extraction protocol (Doyle and Doyle, Museum of Nature and Science, Tsukuba, Japan 1987) followed by glass milk purification meth- (TNS), and a total of 58 specimens housed at the ods as summarized by Hosaka (2009) and Field Museum of Natural History, Chicago, USA Hosaka and Castellano (2008). Briefly, ground (F) were studied. All of them were dried speci- samples were incubated in CTAB buffer at 65°C mens with various ages (collection years ranged for 1 hour, and proteins were removed using the from 1882–1992). Specimens collected more mixture of chloroform: isoamylalcohol (24 : 1). recently (within 20 years) were intentionally The materials were further purified using 6M excluded from this study because one of the main sodium iodine buffer with glass milk, washed purposes was to assess the DNA quality from old with ethanol/buffer solution, and finally eluted in specimens. 100 μl of TE buffer. All samples of extracted All specimens belong to the subphylum Agari- genomic DNA were deposited in the molecular comycotina (mushrooms) of various families and laboratory, Department of Botany, National orders (Table 2, 3). Although the original speci- Museum of Nature and Science, Tsukuba, Japan, men labels all indicate they are type specimens under the DNA voucher numbers denoted in (including holotype, isotype and paratype), the Tables 2 and 3. nomenclatural status could not be confirmed for DNA sequence data were obtained from the DNA from Type Specimens 35 ITS region using the primers ITS5 and ITS4 for Table 1. Comparison of the results of PCR and sequencing from the specimens housed at two amplifying ca. 600 bp region (White et al., 1990). herbaria PCR reactions were carried out using 20 μl reac- Herbarium tion volumes each containing: 1 μl genomic TNS*1 F*2 DNA, 1 μl dNTPs (4 mM), 1 μl of each primer (8 μM), 0.5 units of Taq polymerase (TaKaRa, Numbers of specimens tested 235 58 Year collected (oldest) 1891 1882 Tokyo, Japan), 2 μl MgCl2 (25 mM), 2 μl Bovine Year collected (latest) 1992 1991 Serum Albumin (BSA). Cycling parameters PCR successful 169 42 Sequences with low quality 114 21 were 1 cycle of 94°C for 3 min, 30 cycles of Sequences with hetero-peaks 12 5 94°C for 1 min, 51°C for 30 s and 72°C for 1 min, Sequences from apparent 43 2 with a final extension at 72°C for 15 min. PCR contamination Authentic sequences 0 14 products were electrophoresed in 1% agarose gels stained with ethidium bromide and visual- *1 Department of Botany, National Museum of Nature ized under UV light. When amplification bands and Science, Tsukuba, Japan. 2 were confirmed, the results were scored either as * Department of Botany, Field Museum of Natural His- tory, Chicago, USA. “successful (O)” (with single, clear band) or “failed (X)” (no bands or only smears were observed) (Tables 2, 3). PCR products were then erroneous (e.g., the top hit was ascomycetes but purified using the ExoSap-IT (Millipore, Mol- all the other hits showed basidiomycetous taxa), sheim, France) and directly sequenced using the the “erroneous” hits were discarded and the Big Dye Terminator Cycle Sequencing Kit on results of the remaining hits were recorded. ABI3500 (Applied Biosystems Inc., Foster City, Because sequence data from the majority of CA, USA), following the manufacturer’s instruc- species tested in this study were expected not to tions. be in GenBank database yet, BLAST results Before editing raw sequence data, quality of matched at generic level (or synonym of such) sequences were scored as “good (O)” (readily were considered “authentic” (DNA sequences editable sequences with clear chromatograms), were truly obtained from the specimens, not from “hetero (H)” (chromatograms with hetero-peaks), the other sources). All the other matches were or “messy (X)” (sequences that are not editable considered sequences from “apparent contamina- with no discernable peaks), using Sequence tion” (Table 1, 2, 3). Scanner version 1.0 (Applied Biosystems Inc., Foster City, CA, USA) (Table 2, 3). Only the Results and Discussion sequences scored as “good” were further edited using ATGC version 7.1.0 (GENETYX Corpora- Most specimens used in this study were in tion, Tokyo, Japan). “good” condition macroscopically, without obvi- Edited sequences were analyzed using the ous insect damage (Fig. 1). However, some spec- GenBank BLAST search (Altschul et al., 1990).