Bull. Natl. Mus. Nat. Sci., Ser. B, 43(2), pp. 33–44, May 22, 2017

DNA Extraction, PCR and Sequencing were largely Unsuccessful fromthe 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: khosaka@kahaku.go.jp(Received 16 March 2017; accepted 22 March 2017)

Abstract DNA extraction, PCR and sequencing of the nuclear ribosomal internal transcribedspacer region from a total of 293 type specimens of Agaricomycotina (Basidiomycota, Fungi) wereconducted. 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 discernable 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 thespecimens at F, only 2 sequences were from apparent contamination and the remaining 14 wereconsidered authentic. The oldest specimen from which authentic sequence has been obtained wasfrom 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. Alternatively, 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
Fungi in general have limited morphologicalcharacters and mushrooms with macroscopic
fruit bodies are no exception. It has often been
demonstrated that mushrooms with similar or
indistinguishable fruit body morphology represent phylogenetically distantly related taxa (e.g.,
Kasuya et al., 2012; Wilson et al., 2016). For thisreason, DNA sequence data, especially from thetype specimens, are essential to establish stabletaxonomic system of mushrooms.
It is generally appreciated that the nuclear
ribosomal internal transcribed spacer (ITS)
region is an ofﬁcial DNA barcode region forfungi (Schoch et al., 2012). Although the ITS
sequences cannot discriminate some closely
related species, especially among Ascomycota,

the presence of “universal” primers and high success rate of PCR and sequencing make thisregion widely applicable to many studies, such as
taxonomy and ecology (Dentinger et al., 2010;
Nagy et al., 2011; Schoch et al., 2012).
In contrast to the rapid accumulation of the
ITS sequences in GenBank, the number ofsequences from the type specimens is notincreasing at the same rate. The study by Nagy etal. (2011) showed that a number of unidentiﬁable
sequences from environmental sequencing projects matched with the sequences from the typestrains of Mortierella. Similarly, Brock et al.
(2009) estimated that ca. 70% of taxonomic
diversity at the fungal herbarium of Kew Gardens alone was not yet represented in GenBank.
These studies clearly demonstrated the need ofobtaining ITS sequences from the type speci-

34

Kentaro Hosaka

mens to appropriately link fungal names andDNA sequence data.
Molecular studies using the type specimens,
however, face a number of challenges. In general, DNA in specimens degrades with time
(Erkens et al., 2008; Willerslev and Cooper,
2005), but the majority of type specimens are“old”. In addition, storage conditions such astemperature, humidity, and type of fumigation allaffect the quality of DNA (Willerslev and Cooper, 2005). It has also been demonstrated thatolder specimens tend to be more contaminatedby other fungi (Brock et al., 2009).
In this study, the type specimens of diverse
groups of mushrooms housed at two different
herbaria collected in different times (from 1880ʼs
to 1990ʼs) were tested for assessing the DNA
quality. All specimens were extracted forgenomic DNA, PCR ampliﬁed using generalprimers of the ITS, and sequence data wereobtained by direct sequencing of PCR products.
The aforementioned challenges are further
emphasized, and a potential solution for futurestudies is presented.
Materials and Methods
Type specimens used in this study
A total of 235 specimens housed at the fungal
herbarium, Department of Botany, National
Museum of Nature and Science, Tsukuba, Japan(TNS), and a total of 58 specimens housed at theField Museum of Natural History, Chicago, USA(F) were studied. All of them were dried specimens with various ages (collection years ranged
from 1882–1992). Specimens collected more
recently (within 20 years) were intentionally
excluded from this study because one of the main
purposes was to assess the DNA quality from old
specimens.
All specimens belong to the subphylum Agaricomycotina (mushrooms) of various families andorders (Table 2, 3). Although the original specimen labels all indicate they are type specimens(including holotype, isotype and paratype), thenomenclatural status could not be conﬁrmed forsome specimens. For example, “Ganoderma
spathulatum” (TNS-F-201590) was indicated on
the specimen label as “Typus” but no such names
could be found either from Index Fungorum(http://www.indexfungorum.org/) or a cumulative list of Japanese fungi by Katsumoto (2010).
Such species of uncertain type status are indicated with quotation marks throughout the text
and tables (Table 2, 3).
All dried specimens, including the specimenlabels and packets, were photographed for later
assessing the quality of specimens, such as presence of fungal or insect damage. Some representative photographs and specimen data of typespecimens were uploaded at Type Specimen
Database of National Museum of Nature and Science, and they are publicly available from http://
www.type.kahaku.go.jp/TypeDB/.
DNA preparation, PCR, sequencing, and BLAST
searchDNA was extracted from hymenial tissues ofdried specimens (~200 mg) stored overnight in
DMSO buffer (Seutin et al., 1991; Hosaka,2009). Tissues were ﬁrst ground under liquid
nitrogen using a mortar and pestle. Ground tissues were then transferred to new 1.5 ml tubes
using clean spatulas, and CTAB buffer wasadded. DNA was extracted using the modiﬁed
CTAB extraction protocol (Doyle and Doyle,1987) followed by glass milk puriﬁcation methods as summarized by Hosaka (2009) andHosaka and Castellano (2008). Brieﬂy, groundsamples were incubated in CTAB buffer at 65°C
for 1 hour, and proteins were removed using the
mixture of chloroform: isoamylalcohol (24 : 1).
The materials were further puriﬁed using 6M
sodium iodine buffer with glass milk, washedwith ethanol/buffer solution, and ﬁnally eluted in
100 μl of TE buffer. All samples of extracted
genomic DNA were deposited in the molecularlaboratory, Department of Botany, National
Museum of Nature and Science, Tsukuba, Japan,
under the DNA voucher numbers denoted inTables 2 and 3.
DNA sequence data were obtained from theDNA from Type Specimens

ITS region using the primers ITS5 and ITS4 foramplifying ca. 600 bp region (White et al., 1990).
PCR reactions were carried out using 20 μl reaction volumes each containing: 1 μl genomic
DNA, 1 μl dNTPs (4 mM), 1 μl of each primer(8 μM), 0.5 units of Taq polymerase (TaKaRa,
Tokyo, Japan), 2 μl MgCl2 (25 mM), 2 μl Bovine
Serum Albumin (BSA). Cycling parameters
were 1 cycle of 94°C for 3 min, 30 cycles of
94°C for 1 min, 51°C for 30 s and 72°C for 1 min,with a ﬁnal extension at 72°C for 15 min. PCR
products were electrophoresed in 1% agarosegels stained with ethidium bromide and visualized under UV light. When ampliﬁcation bands
were conﬁrmed, the results were scored either as“successful (O)” (with single, clear band) or“failed (X)” (no bands or only smears wereobserved) (Tables 2, 3). PCR products were thenpuriﬁed using the ExoSap-IT (Millipore, Molsheim, France) and directly sequenced using the
Big Dye Terminator Cycle Sequencing Kit on
ABI3500 (Applied Biosystems Inc., Foster City,
CA, USA), following the manufacturerʼs instructions.
Before editing raw sequence data, quality ofsequences were scored as “good (O)” (readilyeditable sequences with clear chromatograms),“hetero (H)” (chromatograms with hetero-peaks),or “messy (X)” (sequences that are not editablewith no discernable peaks), using Sequence
Scanner version 1.0 (Applied Biosystems Inc.,
Foster City, CA, USA) (Table 2, 3). Only thesequences scored as “good” were further editedusing ATGC version 7.1.0 (GENETYX Corporation, Tokyo, Japan).
Edited sequences were analyzed using the
GenBank BLAST search (Altschul et al., 1990).
Default settings of blastn option were used suchas “Database”＝“Others (nr etc.)” and “Program
Selection”＝“Highly similar sequences (megablast)”. No parameters, such as “Max target
sequences” and “Expect threshold”, werechanged from the default settings. The top hit(regardless of its taxonomic level) and the next
fully identiﬁed hit (to species level) wererecorded. When those hits could be considered

35

Table 1. Comparison of the results of PCR andsequencing from the specimens housed at two
herbaria
Herbarium
Numbers of specimens tested
Year collected (oldest)
Year collected (latest)
PCR successful
Sequences with low quality
Sequences with hetero-peaks
Sequences from apparent
contamination
Authentic sequences

TNS*1

F*2

235
1891
1992
169
114
12
43

58
1882
1991
42
21
5
2

0

14

*1 Department of Botany, National Museum of Natureand Science, Tsukuba, Japan.
*2 Department of Botany, Field Museum of Natural History, Chicago, USA.

erroneous (e.g., the top hit was ascomycetes but
all the other hits showed basidiomycetous taxa),
the “erroneous” hits were discarded and theresults of the remaining hits were recorded.
Because sequence data from the majority of
species tested in this study were expected not to
be in GenBank database yet, BLAST results
matched at generic level (or synonym of such)
were considered “authentic” (DNA sequenceswere truly obtained from the specimens, not fromthe other sources). All the other matches wereconsidered sequences from “apparent contamination” (Table 1, 2, 3).
Results and Discussion
Most specimens used in this study were in“good” condition macroscopically, without obvious insect damage (Fig. 1). However, some specimens had obvious fungal growth on the surfaceof fruit bodies, presumably by ascomycetous
molds (Fig. 1A, D). The results of BLAST search
indicated that sequences obtained from morethan 20 specimens at TNS herbarium were fromapparent contamination of ascomycetes (Table 1,2). The visible presence of molds on fruit bodies
was, however, not necessarily corresponding to
the BLAST results. For example, “molds” on“Ganoderma spathulatum” (Fig. 1A) and Gas-

36

Kentaro Hosaka

Table 2. Type specimens tested for this study housed at National Museum of Nature and Science (TNS) with theresults of PCR and sequencing
Herbarium

DNA

Voucher Nos.

Voucher
Nos.*1

Order

FamilyTNS-F-201590
TNS-F-180124
TNS-F-201999
TNS-F-206977
TNS-F-202252
TNS-F-202482
TNS-F-202550
TNS-F-202243
TNS-F-201565
TNS-F-201364
TNS-F-202551
TNS-F-203004
TNS-F-202818
TNS-F-202337
TNS-F-201423
TNS-F-202805
TNS-F-203033
TNS-F-201758
TNS-F-201801
TNS-F-202823
TNS-F-203077
TNS-F-203063
TNS-F-202071
TNS-F-202757
TNS-F-203006
TNS-F-203069
TNS-F-202783
TNS-F-201575
TNS-F-201448
TNS-F-207812
TNS-F-202077
TNS-F-195151
TNS-F-202751
TNS-F-202075
TNS-F-201771
TNS-F-204262
TNS-F-201309
TNS-F-201566
TNS-F-202941
TNS-F-202748
TNS-F-202774
TNS-F-202073
TNS-F-202754
TNS-F-202755
TNS-F-202756
TNS-F-201365
TNS-F-201366
TNS-F-203018
TNS-F-203017
TNS-F-201532
TNS-F-244375
TNS-F-201260
TNS-F-200234
TNS-F-200021
TNS-F-203362
TNS-F-200828
TNS-F-200762
TNS-F-200900
TNS-F-172699
TNS-F-193009
TNS-F-203372
TNS-F-206958
TNS-F-206965
TNS-F-208290
TNS-F-207357
TNS-F-207357
TNS-F-190105
TNS-F-208513
TNS-F-6563

A3016
A3056
A3023
A3057
A3025
A3001
A3037
A3061
A3006
A3031
A3033
A2336
A3028
A3026
A2998
A3027
A3040
A3002
A3000
A3034
A3029
A3035
A3011
A3067
A3007
A2271
A3017
A3032
A3036
A3018
A3060
A2215
A3063
A3059
A2218
A2272
A2995
A3012
A3070
A3062
A3069
A3024
A3064
A3065
A3066
A2996
A2997
A3039
A3038
A3010
A2179
A3004
A2261
A3058
A3030
A2236
A3003
A2306
A3019
A2197
A3021
A3005
A3013
A2278
A3071
A2262
A2200
A2294
A2199

Polyporales
Boletales
Thelephorales
Polyporales
Polyporales
Polyporales
Polyporales
Polyporales
Polyporales
Polyporales
Polyporales
Russulales
Thelephorales
Polyporales
Polyporales
Hymenochaetales
Russulales
Polyporales
Polyporales
Thelephorales
Polyporales
Polyporales
Polyporales
Russulales
Russulales
Polyporales
Russulales
Polyporales
Polyporales
Agaricales
Polyporales
Cantharellales
Russulales
Polyporales
Hymenochaetales
Polyporales
Polyporales
Polyporales
Russulales
Russulales
Russulales
Polyporales
Russulales
Russulales
Russulales
Polyporales
Polyporales
Russulales
Russulales
Polyporales
Agaricales
Polyporales
Hymenochaetales
Polyporales
Hymenochaetales
Polyporales
Polyporales
Phallales
Agaricales
Auriculariales
Polyporales
Polyporales
Hymenochaetales
Polyporales
Hymenochaetales
Hymenochaetales
Auriculariales
Agaricales
Auriculariales

Ganodermataceae
Boletaceae
Thelephoraceae
Ganodermataceae
Polyporaceae
Polyporaceae
Polyporaceae
Polyporaceae
Polyporaceae
Polyporaceae
Polyporaceae
Stereaceae
Thelephoraceae
Meruliaceae
Polyporaceae
Hymenochaetaceae
Stereaceae
Polyporaceae
Polyporaceae
Thelephoraceae
Meruliaceae
Meruliaceae
Polyporaceae
Stereaceae
Stereaceae
Meruliaceae
Stereaceae
Ganodermataceae
Polyporaceae
Pterulaceae
Polyporaceae
Cantharellaceae
Stereaceae
Polyporaceae
Hymenochaetaceae
Meruliaceae
Polyporaceae
Polyporaceae
Stereaceae
Stereaceae
Stereaceae
Polyporaceae
Stereaceae
Stereaceae
Stereaceae
Polyporaceae
Polyporaceae
Stereaceae
Stereaceae
Polyporaceae
Strophariaceae
Ganodermataceae
Hymenochaetaceae
Polyporaceae
Hymenochaetaceae
Fomitopsidaceae
Ganodermataceae
Protophallaceae
Agaricaceae
Auriculariaceae
Polyporaceae
Polyporaceae
Hymenochaetaceae
Polyporaceae
Hymenochaetaceae
Hymenochaetaceae
Auriculariaceae
Marasmiaceae
Auriculariaceae

Taxon names inYearthe original label*2collected“Ganoderma spathulatum”
Boletus hiratsukae
Thelephora komabensis
Ganoderma subumbraculum
Daedalea kusanoi
Fomes olivaceus
Fomes angularis
Daedalea dickinsii
Polyporus pubertatis
Polyporus sendaiensis
Fomes pusillus
Stereum roseum
Thelephora papillosa
Merulius castaneus
Polyporus versisporus
Hymenochaete boninensis
Stereum japonicum
Polystictus nipponicus
Polystictus glabratus
Thelephora japonica
Irpex iyoensis
Irpex purpureus
Trametes sensitivus
Aleurodiscus japonicus
Stereum sendaiense
Irpex tabacinoides
Stereum liratum
Ganoderma tsunodae
Polyporus proﬁssilis
Pterula fusispora
Trametes japonica
Cantharellus pallidus
Aleurodiscus orientalis
Trametes symploci
Coltriciella pusilla
Irpex tabacinoides
Polyporus greenii
Polyporus kanehirae
Coniophora matsuzawae
Aleurodiscus tsugae“Aleurodiscus suborientalis”
Trametes minutissima
Aleurodiscus reﬂexus
Aleurodiscus sendaiensis
Aleurodiscus stereoides
Polyporus sendaiensis
Polyporus sendaiensis
Stereum hamamelidis
Stereum kurilense
Polyporus liukiuensis
Agrocybe media
Ganoderma hainanense
Hymenochaete yasudai“Polyporus kinkazanensis”
Hymenochaete rufo-marginata“Fomitopsis laevigata”
Ganoderma neojaponicumProtubera nipponica
Cyathus badius
Auricularia leucochroma
Fomes mangrovicus
Daedaleopsis nipponica
Inonotus sciurinus
Lentinus palauensis
Hymenochaete yasudaiHymenochaete yasudai
Auricularia semipellucida
Marasmius aurantiacus
Auricularia polytricha

1891
1896
1898
1908
1909
1911
1912
1912
1912
1913
1913
1913
1914
1914
1914
1915
1915
1915
1915
1915
1916
1916
1916
1916
1916
1916
1916
1917
1917
1917
1917
1917
1918
1918
1918
1918
1918
1918
1918
1919
1919
1920
1920
1920
1920
1921
1921
1922
1923
1923
1925
1928
1929
1931
1932
1934
1935
1935
1936
1936
1937
1938
1938
1939
1939
1939
1940
1941
1941

PCR*3 Sequencing*4
X
O
O
O
O
X
O
O
X
O
X
X
O
X
X
O
O
O
O
O
O
O
O
O
O
X
X
O
O
O
O
X
O
O
X
X
X
X
X
O
O
O
O
O
O
O
X
O
O
O
O
O
X
O
O
X
O
O
O
X
X
O
X
O
O
X
O
O
X

–
H
X
X
X
–
H
X
–
H
–
–
H
–
–
H
H
X
X
X
H
X
X
X
X
–
–
H
H
X
O
–
X
O
–
–
–
–
–
X
X
X
X
X
X
X
–
H
H
O
X
X
–
X
H
–
X
O
O
–
–
O
–
X
O
–
X
X
–

BLAST results*5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
uncultured ascomycetes––
Rhodosporidium
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Psathyrella
–
–
–
–
–
–
–
Laccaria
Psathyrella––
Psathyrella––
Pleurotus––––

DNA from Type Specimens
Table 2.
Herbarium

DNA

Voucher Nos.

Voucher
Nos.*1

Order

FamilyTNS-F-212095
TNS-F-213570
TNS-F-237517
TNS-F-237667
TNS-F-234247
TNS-F-237485
TNS-F-237560
TNS-F-237621
TNS-F-237369
TNS-F-237379
TNS-F-237296
TNS-F-237333
TNS-F-237490
TNS-F-237518
TNS-F-237522
TNS-F-237523
TNS-F-237524
TNS-F-237307
TNS-F-237325
TNS-F-237319
TNS-F-237304
TNS-F-237328
TNS-F-237347
TNS-F-237373
TNS-F-237480
TNS-F-237491
TNS-F-237519
TNS-F-237375
TNS-F-237273
TNS-F-237278
TNS-F-237276
TNS-F-237281
TNS-F-237479
TNS-F-237619
TNS-F-237359
TNS-F-237229
TNS-F-237321
TNS-F-237385
TNS-F-237494
TNS-F-237301
TNS-F-237284
TNS-F-237367
TNS-F-237625
TNS-F-237309
TNS-F-237324
TNS-F-237352
TNS-F-237492
TNS-F-237332
TNS-F-237351
TNS-F-237365
TNS-F-237368
TNS-F-237495
TNS-F-237629
TNS-F-237322
TNS-F-237366
TNS-F-237487
TNS-F-237337
TNS-F-237354
TNS-F-237277
TNS-F-237508
TNS-F-237303
TNS-F-237329
TNS-F-237645
TNS-F-237647
TNS-F-237641
TNS-F-237310
TNS-F-237481
TNS-F-237338
TNS-F-237353
TNS-F-237637

A3014
A2237
A2414
A2318
A2217
A2303
A2339
A2344
A2284
A2291
A2216
A2257
A2309
A2329
A2332
A2333
A2334
A2229
A2249
A2244
A2223
A2252
A2263
A2286
A2297
A2311
A2330
A2287
A2178
A2186
A2183
A2189
A2296
A2343
A2275
A2228
A2245
A2288
A2316
A2220
A2201
A2282
A2345
A2231
A2248
A2267
A2312
A2256
A2266
A2280
A2283
A2317
A2347
A2246
A2281
A2305
A2259
A2269
A2184
A2323
A2222
A2253
A2270
A2349
A2361
A2232
A2298
A2260
A2268
A2356

Hymenochaetales
Polyporales
Russulales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Russulales
Russulales
Russulales
Russulales
Agaricales
Agaricales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Russulales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Russulales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales

Hymenochaetaceae
Fomitopsidaceae
Russulaceae
Entolomataceae
Omphalotaceae
Pleurotaceae
Strophariaceae
Tricholomataceae
Agaricaceae
Lyophyllaceae
Omphalotaceae
Hygrophoraceae
Tricholomataceae
Russulaceae
Russulaceae
Russulaceae
Russulaceae
Cortinariaceae
Hygrophoraceae
Hygrophoropsidaceae
Cortinariaceae
Hygrophoraceae
Inocybaceae
Agaricaceae
Mycenaceae
Hymenogastraceae
Russulaceae
Agaricaceae
Bolbitiaceae
Amanitaceae
Amanitaceae
Amanitaceae
Mycenaceae
Tricholomataceae
Russulaceae
Cortinariaceae
Hygrophoraceae
Agaricaceae
Entolomataceae
Cortinariaceae
Bolbitiaceae
Agaricaceae
Tricholomataceae
Cortinariaceae
Hygrophoraceae
Inocybaceae
Hymenogastraceae
Hygrophoraceae
Inocybaceae
Agaricaceae
Agaricaceae
Entolomataceae
Tricholomataceae
Hygrophoraceae
Agaricaceae
Boletaceae
Hygrophoraceae
Inocybaceae
Amanitaceae
Entolomataceae
Cortinariaceae
Hygrophoraceae
Inocybaceae
Boletaceae
Boletaceae
Cortinariaceae
Strophariaceae
Hygrophoraceae
Inocybaceae
Boletaceae

37

Continued
Taxon names inYearthe original label*2collected

Cryptoderma cercidiphyllum“Fomitopsis tenuis”
Russula kansaiensis
Rhodophyllus cyanoniger
Collybia ozeensis
Pleurotus incarnatus
Stropharia rugosoannulata
Tricholoma muscarium
Lepiota clypeolaria
Lyophyllum musashiense
Collybia neofusipes
Hygrophorus pseudococcineus
Pseudohiatula ohshimae
Russula metachroa
Russula niigatensis
Russula omiensis
Russula subnigricans
Cortinarius subalboviolaceus
Hygrophorus dichrous
Hygrophoropsis bicolor
Cortinarius claricolor
Hygrophorus imazekii
Inocybe ammophila
Lepiota subcitrophylla
Mycena umbilicata
Psilocybe fasciata
Russula nauseosa
Leucocoprinus denudatus
Agrocybe farinacea
Amanita lutescens
Amanita citrina
Amanita pseudoporphyria
Mycena roseomarginata
Tricholoma maculatipus
Lactarius circellatus
Cortinarius shigaensis
Hygrophorus cantharellus
Leucocoprinus otsuensis
Rhodophyllus ater
Cortinarius aurantiofulvus
Bolbitius incarnatus
Lepiota aurantioﬂava
Tricholomopsis bambusina
Cortinarius subdelibutus
Hygrophorus cruentus
Inocybe macrosperma
Psilocybe subcaerulipes
Hygrophorus pantoleucus
Inocybe kobayasii
Lepiota alborubescens
Lepiota cinnamomea
Rhodophyllus babingtonii
Tricholomopsis sasae
Hygrophorus conicus
Lepiota atrosquamulosa
Porphyrellus subvirens
Hygrophorus subcinnabarinus
Inocybe subvolvata
Amanita griseofarinosa
Rhodophyllus omiensis
Cortinarius cinnamomeoides
Hygrophorus lilacinogriseus
Inocybe subvolvata
Tylopilus areolatus
Xerocomus nigromaculatus
Cortinarius watamukiensis
Naematoloma gracile
Hygrophorus suzukaensis
Inocybe quercina
Tylopilus otsuensis

1941
1943
1945
1950
1950
1952
1952
1952
1953
1953
1953
1954
1954
1954
1954
1954
1954
1954
1954
1954
1954
1954
1954
1955
1955
1955
1955
1955
1956
1956
1956
1956
1956
1956
1956
1956
1956
1956
1956
1956
1957
1957
1957
1957
1957
1957
1957
1957
1957
1958
1958
1958
1958
1958
1958
1958
1958
1958
1959
1960
1960
1960
1960
1961
1961
1961
1961
1961
1961
1962

PCR*3 Sequencing*4
X
O
O
O
X
O
O
O
O
O
X
O
O
O
O
O
O
O
O
O
X
X
X
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
X
O
O
O
O
O
O
O
X
X
O
O
O
O
O
O
O
X
X
O
O
X
X
X
O
O
O
O
X
X
O

–
O
X
X
–
X
X
X
X
X
–
X
X
X
X
X
X
O
O
O
–
–
–
X
X
X
X
O
X
X
X
X
X
X
X
O
O
O
O
–
X
X
X
O
O
O
O
–
–
X
X
X
X
O
O
O
–
–
X
X
–
–
–
X
X
O
O
–
–
X

BLAST results*5–
uncultured ascomycetes
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
uncultured ascomycetes
uncultured ascomycetes
uncultured ascomycetes
–
–
–
–
–
–
–
Laccaria
–
–
–
–
–
–
–
uncultured ascomycetes
uncultured ascomycetes
Laccaria
uncultured ascomycetes––––
uncultured ascomycetes
uncultured ascomycetes
uncultured ascomycetes
Laccaria
–
–
–
–
–
–
uncultured ascomycetes
Laccaria
Laccaria
–
–
–
–
–
–
–
–
–
uncultured ascomycetes
Laccaria
–
–
–

38

Kentaro Hosaka
Table 2.

Herbarium

DNA

Voucher Nos.

Voucher
Nos.*1TNS-F-237652
TNS-F-237525
TNS-F-193006
TNS-F-174776
TNS-F-237350
TNS-F-237362
TNS-F-237231
TNS-F-237382
TNS-F-237297
TNS-F-237355
TNS-F-237557
TNS-F-237558
TNS-F-174775
TNS-F-237228
TNS-F-244373
TNS-F-237348
TNS-F-237330
TNS-F-237489
TNS-F-174771
TNS-F-237305
TNS-F-237331
TNS-F-237287
TNS-F-237288
TNS-F-237289
TNS-F-244377
TNS-F-237510
TNS-F-237226
TNS-F-237293
TNS-F-237655
TNS-F-237372
TNS-F-237504
TNS-F-237651
TNS-F-50254
TNS-F-237306
TNS-F-237323
TNS-F-237274
TNS-F-237488
TNS-F-234519
TNS-F-172603
TNS-F-237283
TNS-F-244379
TNS-F-237302
TNS-F-237290
TNS-F-228004
TNS-F-237377
TNS-F-228005
TNS-F-228018
TNS-F-176271
TNS-F-228000
TNS-F-225511
TNS-F-228299
TNS-F-228001
TNS-F-225521
TNS-F-228002
TNS-F-225479
TNS-F-237513
TNS-F-228066
TNS-F-225485
TNS-F-237275
TNS-F-237364
TNS-F-237503
TNS-F-237313
TNS-F-237314
TNS-F-182277
TNS-F-228435
TNS-F-174773
TNS-F-237648
TNS-F-237285
TNS-F-198224
TNS-F-198225

A2363
A2335
A2235
A2355
A2265
A2276
A2230
A2293
A2219
A2273
A2337
A2338
A2365
A2224
A2191
A2264
A2254
A2308
A2354
A2225
A2255
A2207
A2209
A2210
A2192
A2326
A2205
A2214
A2213
A2285
A2320
A2348
A2315
A2226
A2247
A2181
A2307
A2196
A2353
A2193
A2208
A2221
A2211
A2194
A2289
A2295
A2299
A2304
A2346
A2195
A2301
A2279
A2274
A2300
A2328
A2327
A2241
A2203
A2182
A2277
A2319
A2234
A2238
A2212
A2310
A2360
A2352
A2206
A2187
A2188

Order
Boletales
Russulales
Agaricales
Boletales
Agaricales
Russulales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Agaricales
Agaricales
Boletales
Boletales
Boletales
Agaricales
Russulales
Agaricales
Boletales
Boletales
Agaricales
Agaricales
Boletales
Gomphales
Agaricales
Agaricales
Agaricales
Agaricales
Auriculariales
Boletales
Agaricales
Agaricales
Agaricales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Boletales
Polyporales
Russulales
Polyporales
Russulales
Russulales
Agaricales
Boletales
Agaricales
Boletales
Agaricales
Agaricales
Agaricales
Boletales
Agaricales
Boletales
Boletales
Boletales
Agaricales
Agaricales

FamilyBoletaceae
Russulaceae
Mycenaceae
Boletaceae
Inocybaceae
Russulaceae
Cortinariaceae
Marasmiaceae
Psathyrellaceae
Strophariaceae
Strophariaceae
Strophariaceae
Boletaceae
Cortinariaceae
Amanitaceae
Inocybaceae
Hygrophoraceae
Bolbitiaceae
Boletaceae
Cortinariaceae
Hygrophoraceae
Boletaceae
Boletaceae
Boletaceae
Amanitaceae
Russulaceae
Amanitaceae
Boletaceae
Boletaceae
Agaricaceae
Entolomataceae
Boletaceae
Gomphaceae
Cortinariaceae
Hygrophoraceae
Amanitaceae
Psathyrellaceae
Auriculariaceae
Boletaceae
Amanitaceae
Amanitaceae
Cortinariaceae
Boletaceae
Physalacriaceae
Agaricaceae
Omphalotaceae
Strophariaceae
Mycenaceae
Tricholomataceae
Boletaceae
Boletaceae
Polyporaceae
Russulaceae
Polyporaceae
Russulaceae
Russulaceae
Strophariaceae
Boletaceae
Amanitaceae
Boletaceae
Entolomataceae
Agaricaceae
Hymenogastraceae
Boletaceae
Hymenogastraceae
Boletaceae
Boletaceae
Boletaceae
Amanitaceae
Amanitaceae

Continued
Taxon names inYearthe original label*2collected

Xerocomus parvulus
Russula virescens
Dictyopanus orientalis
Tylopilus otsuensis
Inocybe kasugayamensis
Lactarius subpiperatus
Cortinarius subarmillatus
Marasmius aurantioferrugineus
Coprinus aokii
Kuehneromyces castaneus
Stropharia aeruginosa
Stropharia aeruginosa
Xerocomus nigromaculatus
Cortinarius galeroides
Amanita rufoferruginea
Inocybe atroumbonata
Hygrophorus olivaceoviridis
Pseudoconocybe nodulosospora
Tylopilus neofelleus
Cortinarius neoarmillatus
Hygrophorus olivaceoviridis
Boletus griseus
Boletus laetissimus
Boletus obscureumbrinus
Amanita sphaerobulbosa
Russula bella
Amanita neoovoidea
Boletus umbiriniporus
Boletus subfuscus
Lepiota neomastoidea
Rhodophyllus kansaiensis
Tylopilus alutaceoumbrinus
Ramaria zippelii
Cortinarius nigrosquamosus
Hygrophorus croceoluteus
Amanita alboﬂavescens
Psathyrella pennata
Auricularia ﬁbrillifera
Tylopilus hongoi
Amanita sychnopyramis
Amanita pseudogemmata
Cortinarius aureobrunneus
Boletus pseudocalopus
Armillariella fellea
Leucocoprinus subglobisporus
Micromphale paciﬁcum
Naematoloma papuanum
Poromycena rubra
Tricholomopsis elata
Aureoboletus novoguineensis
Boletus oksapminensis
Lentinus papuanus
Lactarius austrovolemus
Panus verruciceps
Russula eburneoareolata
Russula castanopsidis
Gymnopilus novoguineensis
Boletus erythropus
Amanita castanopsidis
Leccinum subradicatum
Rhodophyllus kansaiensis
Cystoderma neoamianthinum
Galerina fasciculata“Boletus subcinnamomeus”
Psilocybe argentipes
Tylopilus vinosobrunneus
Tylopilus eximus
Boletus fuscopunctatus
Amanita miculifera
Amanita miculifera

1962
1962
1962
1962
1962
1963
1963
1963
1963
1963
1964
1964
1964
1964
1965
1965
1965
1966
1966
1966
1966
1966
1966
1966
1967
1967
1967
1967
1967
1968
1968
1968
1968
1968
1968
1969
1969
1969
1969
1970
1970
1970
1970
1971
1971
1971
1971
1971
1971
1971
1971
1971
1971
1971
1971
1971
1971
1971
1972
1972
1973
1973
1973
1973
1975
1975
1975
1975
1976
1976

PCR*3 Sequencing*4
O
O
O
O
X
O
O
O
O
X
O
O
O
X
O
O
X
O
O
X
X
X
X
X
O
O
X
X
X
O
O
O
O
O
O
X
O
O
O
O
X
X
X
O
O
O
O
O
O
O
O
O
O
O
O
O
X
X
X
O
O
O
X
X
O
O
O
X
O
O

X
X
O
O
–
X
O
O
–
–
X
X
X
–
X
X
–
X
O
–
–
–
–
–
X
O
–
–
–
X
X
X
X
O
O
–
X
X
X
X
–
–
–
X
X
X
X
X
X
X
X
X
X
O
O
O
–
–
–
O
X
O
–
–
X
X
X
–
X
X

BLAST results*5––
uncultured ascomycetes
Laccaria––
uncultured ascomycetes
Rhodosporidium
–
–
–
–
–
–
–
–
–
–
Laccaria
–
–
–
–
–
–
Laccaria
–
–
–
–
–
–
–
uncultured ascomycetes
uncultured ascomycetes
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Laccaria
Laccaria
Laccaria–––
uncultured ascomycetes–
uncultured ascomycetes
–
–
–
–
–
–
–
–

DNA from Type Specimens
Table 2.
Herbarium

DNA

Voucher Nos.

Voucher
Nos.*1Order

TNS-F-237633
TNS-F-237326
TNS-F-237653
TNS-F-237657
TNS-F-237511
TNS-F-237311
TNS-F-237378
TNS-F-174748
TNS-F-237327
TNS-F-237505
TNS-F-174772
TNS-F-174774
TNS-F-237520
TNS-F-237512
TNS-F-237282
TNS-F-237650
TNS-F-237646
TNS-F-182343
TNS-F-237654
TNS-F-237506
TNS-F-237562
TNS-F-237381
TNS-F-237316
TNS-F-237670
TNS-F-200842
TNS-F-171953

A2359
A2250
A2362
A2364
A2324
A2233
A2290
A2258
A2251
A2321
A2358
A2357
A2331
A2325
A2190
A2202
A2350
A2242
A2351
A2322
A2342
A2292
A2243
A2180
A3009
A2240

Boletales
Agaricales
Boletales
Boletales
Russulales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Boletales
Russulales
Russulales
Agaricales
Boletales
Boletales
Agaricales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Polyporales
Boletales

FamilyBoletaceae
Hygrophoraceae
Boletaceae
Boletaceae
Russulaceae
Inocybaceae
Amanitaceae
Hygrophoraceae
Hygrophoraceae
Entolomataceae
Boletaceae
Boletaceae
Russulaceae
Russulaceae
Amanitaceae
Boletaceae
Boletaceae
Hymenogastraceae
Boletaceae
Entolomataceae
Tricholomataceae
Lyophyllaceae
Hygrophoraceae
Hymenogastraceae
Polyporaceae
Boletaceae

39

Continued
Taxon names inYearthe original label*2collected

Tylopilus ruguloso-reticulatus
Hygrophorus hahashimensis
Xerocomus obscurebrunneus
Xerocomus subcinnamomeus
Russula alboareolata
Crepidotus roseus
Limacella olivaceobrunnea
Hygrophorus subacutus
Hygrophorus hypohaemactus
Rhodophyllus kujuensis
Tylopilus ruguloso-reticulatus
Tylopilus rigens
Russula neoemetica
Russula alboareolata
Amanita pseudovaginata
Boletus aokii
Tylopilus argillaceus
Hebeloma radicosoides
Tylopilus castaneiceps
Rhodophyllus kujuensis
Tricholoma aurantiipes
Lyophyllum sykosporum
Hygrocybe miniatoaurantiaca
Alnicola lactariolens
Polyporus tuberaster
Gastroboletus doii

1976
1977
1977
1977
1977
1977
1977
1977
1977
1978
1978
1978
1978
1978
1980
1980
1981
1983
1983
1983
1985
1986
1987
1988
1992
1992

PCR*3 Sequencing*4
O
O
O
O
O
O
O
X
X
O
O
O
O
O
O
X
O
O
O
O
O
O
O
O
O
X

X
X
X
X
X
O
O
–
–
X
X
X
X
O
X
–
X
X
X
O
X
X
O
X
X
–

BLAST results*5
–
–
–
–
–
uncultured ascomycetes
Laccaria
–
–
–
–
–
–
Laccaria–––––
Laccaria––
uncultured ascomycetes–––*1 DNA sampes are stored under the DNA voucher numbers at Department of Botany, National Museum of Nature and Science, Tsukuba, Japan.
*2 Species names with uncertain nomenclatural status were indicated with quotation marks.
*3 PCR ampliﬁcation was condidered “successful” with clear single band (O) or “failed” with no bands or smears (X).
*4 Direct sequencing resulted in clear, readily editable sequences was considered “good” (O). Sequences were considered low quality or “messy” with nodiscernable peaks (X). Sequences with hetero-peaks are indicated with “H”.
*5 The results of the top hit are shown.

troboletus doii (Fig. 1D) were not ampliﬁed(PCR ampliﬁcation was negative for both cases).
On the other hand, some specimens without visible molds on the surface also resulted in “apparent contamination” (Table 1, 2) using BLAST.
Many ascomycetous molds can secondarily colonize on mushroom specimens and old specimens
have much higher risk of such contamination.
When dealing with old specimens, visual inspection of specimens by stereo microscope and PCRampliﬁcation using basidiomycetes speciﬁcprimers can minimize the risk of contamination,
but we always need to recognize that old specimens are always contaminated by other (micro-)organisms by certain extent.
Such “apparent contamination” was caused not
only by ascomycetes, but also by other basidiomycetous mushrooms. For example, thesequence obtained from Amanita aestivalis
F-1099363 (Amanitaceae) matched with Pholiota (Strophariaceae) (Table 3). Similarly, thesequence from Leccinum albellum F-1015573(Boletales) matched with Skeletocutis (Polyporales) (Table 3). It is noteworthy that the sequencesfrom various TNS specimens matched with unrelated mushroom Laccaria (Hydnangiaceae)(Table 2). During this study, the project on Laccaria phylogeography was on-going at the sametime, and a large number of Laccaria specimens
were processed in the same molecular lab. I
believe “apparent contamination” of Laccaria
was partially caused by genomic DNA accidentally mixed in some reagents commonly used in
our molecular lab. It should be emphasized thatno such Laccaria contamination has been
detected in any other studies conducted in ourmolecular lab ever since.
Although the contamination by ascomycetes
tends to be more frequent, mushroom contamination by other mushrooms is also widespread asshown here. Such contamination at molecular lab
can be avoided by always using fresh reagents,

40

Kentaro Hosaka

Table 3. Type specimens tested for this study housed at Field Museum of Natural History (F) with the results of
PCR and sequencing
Herbarium

DNA

Voucher
Voucher Nos.
Nos.*1F-1037060
F-332202
F-1295888
F-1174531
F-1247332
F-497510
F-1001353
F-1001210
F-1201600
F-1152706
F-1001332
F-1330205
F-1330212
F-1099363
F-1020987
F-1037071
F-1014447
F-1016880
F-1013501
F-1015825
F-1018786
F-1080297
F-1080296
F-1018284
F-1015573
F-1016998
F-1017120
F-1015677
F-1016843
F-1018209
F-1019126
F-1018595
F-1021123
F-1030847
F-1030894
F-1037055
F-1097232
F-1037054
F-1031387
F-1030749
F-1059109
F-1036989
F-1037041
F-1052251
F-1052236
F-1051926
F-1073280
F-1053721
F-1073299
F-1160662
F-1091979
F-1315642
F-1068563
F-1074629
F-1075916
F-1097223
F-1100021
F-1097225

A2771
A2768
A2785
A2739
A2760
A2795
A2777
A2764
A2737
A2784
A2736
A2782
A2790
A2747
A2767
A2772
A2786
A2773
A2794
A2765
A2758
A2799
A2803
A2734
A2759
A2801
A2743
A2752
A2762
A2738
A2774
A2778
A2757
A2766
A2791
A2749
A2754
A2761
A2742
A2770
A2744
A2781
A2792
A2769
A2800
A2793
A2740
A2796
A2763
A2751
A2735
A2802
A2797
A2745
A2798
A2787
A2741
A2746

Order

FamilyRussulales
Boletales
Agaricales
Agaricales
Russulales
Agaricales
Russulales
Russulales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Russulales
Agaricales
Agaricales
Agaricales
Boletales
Agaricales
Boletales
Boletales
Boletales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Russulales
Boletales
Russulales
Agaricales
Russulales
Agaricales
Polyporales
Russulales
Russulales
Boletales
Agaricales
Agaricales
Agaricales
Boletales
Agaricales
Agaricales
Agaricales
Agaricales
Boletales
Boletales
Boletales
Boletales
Boletales

Russulaceae
Boletaceae
Agaricaceae
Agaricaceae
Russulaceae
Amanitaceae
Stereaceae
Russulaceae
Agaricaceae
Agaricaceae
Marasmiaceae
Agaricaceae
Amanitaceae
Amanitaceae
Boletaceae
Russulaceae
Agaricaceae
Hydnangiaceae
Hygrophoraceae
Boletaceae
Hydnangiaceae
Boletaceae
Boletaceae
Boletaceae
Boletaceae
Hydnangiaceae
Hydnangiaceae
Strophariaceae
Hydnangiaceae
Bolbitiaceae
Hydnangiaceae
Hydnangiaceae
Hydnangiaceae
Bolbitiaceae
Boletaceae
Russulaceae
Boletaceae
Russulaceae
Physalacriaceae
Russulaceae
Hydnangiaceae
Polyporaceae
Russulaceae
Russulaceae
Boletaceae
Hydnangiaceae
Amanitaceae
Amanitaceae
Boletaceae
Amanitaceae
Hydnangiaceae
Agaricaceae
Physalacriaceae
Boletaceae
Paxillaceae
Boletaceae
Boletaceae
Boletaceae

Taxon names inYearthe original label*2collected

Lactarius mamorensis
Boletus debeauxii
Lepiota denudata
Lepiota naucinoides
Lactarius rubrifulvus
Amanita protecta
Aleurodiscus scutellatus
Arcangeliella scissilis
Lepiota ﬁscheri
Lepiota cystidiosa
Lactocollybia angiospermarum
Lepiota aurora
Amanita tenuifolia
Amanita aestivalis
Boletus guadelupae
Lactarius paulensis
Lepiota xanthophylloides“Laccaria carbonophila”
Botrydina lobata
Boletus fuligineotomentosus
Laccaria laccata var. vulcanica“Boletus ixoﬂavus”
Boletus subluridellus
Boletus michoacanus
Leccinum albellum“Laccaria moelleri”
Laccaria pussilla
Agrocybe amara
Laccaria montana
Bolbitius mesosporus
Laccaria laccata
Laccaria laccata var. pusilla
Laccaria laccata var. tatrensis
Boletellus sp.
Boletellus ananas var. crassotunicatus
Lactarius amazonensis
Boletellus ananas
Lactarius subpallidipes
Armillariella viridiﬂava
Lactarius igapoensis“Laccaria kauaiensis”
Lentinus prancei
Lactarius costaricensis
Lactarius guanacastensis
Boletellus ananas
Laccaria gomezii
Amanita ﬂavoconia
Amanita nauseosa
Boletus heterodermus
Agaricus pleropus
Laccaria longipes
Agaricus yucatanensis
Armillariella afﬁnis
Leccinum andinum
Austrogaster marthae
Laccinum roseoscabrum“Boletus pini-oocarpae”
Leccinum roseoscabrum

1882
1884
1892
1893
1904
1920
1926
1933
1938
1940
1942
1948
1948
1953
1957
1964
1966
1967
1968
1968
1968
1969
1969
1969
1969
1970
1970
1970
1971
1973
1974
1974
1974
1976
1976
1977
1978
1978
1979
1980
1981
1981
1981
1982
1982
1982
1983
1983
1983
1984
1984
1985
1986
1986
1988
1988
1991
1991

PCR*3 Sequencing*4
O
O
X
O
X
O
X
X
O
O
O
O
O
O
O
X
X
O
O
O
O
X
O
O
O
O
O
O
X
X
X
O
O
O
O
O
O
O
X
O
X
O
O
X
X
O
O
O
O
X
O
O
O
O
X
O
O
O

X
X
–
X
–
X
–
–
X
O
X
X
X
O
O
–
–
O
H
X
O
–
H
X
O
O
O
O
–
–
–
X
O
X
X
H
X
X
–
X
–
H
O
–
–
O
X
X
X
–
X
X
H
O
–
O
O
O

BLAST results*5
–
–
–
–
–
–
–
–
–
Cystolepiota cystidiosa–––Pholiota
Boletus erythropus––
Laccaria––
Laccaria–––
Skeletocutis
Laccaria
Laccaria
Agrocybe praecox––––
Laccaria
–
–
–
–
–
–
–
–
–
Lactarius fumosus––
Laccaria
–
–
–
–
–
–
–
Boletus bicolor–
Leccinum talamancae
Boletus subvelutipes
Leccinum talamancae*1 DNA sampes are stored under the DNA voucher numbers at Department of Botany, National Museum of Nature and Science, Tsukuba, Japan.
*2 Species names with uncertain nomenclatural status were indicated with quotation marks.
*3 PCR ampliﬁcation was condidered “successful” with clear single band (O) or “failed” with no bands or smears (X).
*4 Direct sequencing resulted in clear, readily editable sequences was considered “good” (O). Sequences were considered low quality or “messy” with no discernable peaks (X). Sequences with hetero-peaks are indicated with “H”.
*5 The results of the top hit are shown.

DNA from Type Specimens

Fig. 1. Some representatives of the type specimens tested in this study.
A. “Ganoderma spathulatum” (TNS-F-201590) from 1891. B. Boletus hiratsukae (TNS-F-180124) from 1896.
C. Cystoderma neoamianthinum (TNS-F-237313) from 1973. D. Gastroboletus doii (TNS-F-171953) from1992. E. Lactarius mamorensis (F-1037060) from 1882. F. Lepiota denudata (F-1295888) from 1892. G. Lepiota cystidiosa (F-1152706) from 1940. H. Agrocybe amara (F-1015677) from 1970. Bars＝2.5 cm.

41

42

Kentaro Hosaka

Fig. 2. Success rate of PCR ampliﬁcation from thespecimens stored at TNS herbarium.

Fig. 3. Success rate of PCR ampliﬁcation from thespecimens stored at F herbarium.

but the contamination in the herbarium (e.g.,mushroom specimens covered with spores ofother mushrooms) is usually unavoidable.
Researchers attempting to obtain DNA from oldmushroom specimens should always be carefulwhen interpreting the sequence data.
PCR ampliﬁcation from more than 50% of
specimens housed at TNS and F herbaria was
successful (Table 1, Fig. 2, 3). Some specimens ofmore than 50 year old also had positive ampliﬁcation (Fig. 2, 3). Although all successfully obtained
sequences from TNS specimens were demonstrated to be from “apparent contamination”, themajority of direct sequencing was unsuccessful

due to low quality (114) and hetero-peaks (12),which may contain authentic sequences (Table
1). Similarly, out of 42 PCR products obtained
from F specimens, only 14 produced authentic
sequences and the remaining ones (21 low quality sequences and 5 hetero-peaks) may containauthentic sequences as well (Table 1). This study
is an initial screening of old type specimens andthe next obvious step is to use taxon-speciﬁcprimers and/or to sequence by cloning.
It is well documented from the previous studies that many specimens at TNS have very fragmented DNA due to fumigation (Hosaka andUno, 2013). Most specimens of 30 year old orolder have been fumigated by methyl bromide
and the average lengths of DNA fragments were
demonstrated to be 150 bp. or shorter (Hosaka
and Uno, 2013). More recently, TNS herbarium
has been fumigated by sulphuryl ﬂuoride, which
is claimed not to affect DNA molecules (Kigawa
et al., 2003; Whitten et al., 1999). Althoughsome positive PCR products with “failed”
sequencing may contain authentic sequences, thepossibility of obtaining ca. 600 bp. sequencesusing the primers ITS5 and ITS4 (White et al.,1990) is apparently low. The clear contrast in
success rate of obtaining authentic sequences
between TNS (0) and F (14) herbaria indicatesthat the effect of fumigation is not trivial.
Although no detailed records of fumigation at F
herbarium are currently available, the results ofthis study indicate that major fungal herbaria in
the USA and Japan may have different fumigation history.
It is obvious that DNA in specimens degradeswith time (Erkens et al., 2008; Willerslev andCooper, 2005). The current study shows that noauthentic sequences were obtained from thespecimens collected in 1930ʼs or before (Fig. 4).
The oldest specimen from which we could PCRamplify the ITS region (ca. 600 bp.) and successfully sequence was from 1940 (Lepiota cystidiosa F-1152706) (Table 3, Fig. 1G, 4). Althoughother factors such as humidity, temperature andfumigation also affect the quality of DNA (Willerslev and Cooper, 2005), this study clearly dem-DNA from Type Specimens

43

in this study may contain authentic sequences,which can be obtained by cloning.
As initially expected, an attempt to obtain
sequence data from the type specimens, especially from old specimens, faced a lot of challenges. As long as the fungal nomenclature isbased on the principle of typiﬁcation, however,further attempts should be made to obtain“authentic” sequence data from most, if not all,type specimens. If no such attempts are successful or type specimens are apparently absent, epitypiﬁcation from more recently collected specimens accompanied with good sequence data
should be conducted (Hyde and Zhang, 2008).
Fig. 4. Success rate of direct sequencing from thespecimens stored at F herbarium.
Sequencing reactions producing “authentic”
sequences were scored as success, and all theother sequences (apparent contamination, hetero-peaks, and “messy” sequences) were scoredas failed.

onstrates the need of immediate DNA extraction
from historically/taxonomically important specimens, i.e., type specimens. Historical records offumigation in major fungal herbaria should be
investigated for higher success rate of PCR andsequencing from such specimens. Alternatively,
as suggested by Hosaka and Uno (2013), small
pieces of type specimens should be preserved indeep-freezer, organic solvent (Fukatsu, 1999) or
DNA preservation buffer (Dawson et al., 1998;
Laulier et al., 1995; Nagy, 2010) for future
molecular studies.
For future studies, the protocol for each step ofDNA extraction, PCR and sequencing need to be
re-examined. For example, fragmented DNA
molecules are known to less effectively bind tosilica particles (glass milk), but the binding efﬁciency can be improved by lowering the pH of
the binding buffer (GENECLEAN Kit®, MP Biomedicals, Solon, OH, USA; protocol available
from https://www.funakoshi.co.jp/data/datasheet/
GEN/1001-200.pdf). The PCR efﬁciency cancertainly be improved by designing taxon-speciﬁc primers and targeting shorter regions ofDNA. As noted above, some “failed” sequences

Acknowledgments
The author thanks Kunihiko Uno and KyungOk Nam for assisting my molecular work. I also
thank Dr. Robert Luecking and other herbarium
staffs for facilitating the loan of the type specimens from Field Museum of Natural History.
This study was ﬁnanced in part by a JSPS Grantin-Aid for Young Scientists (A) (no. 24680085)
and JSPS Grant-in-Aid for Scientiﬁc Research(B) (no. 40414362).
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