Bringing Laboulbeniales into the 21st century: enhanced techniques for extraction and PCR amplification of DNA from minute ectoparasitic fungi
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Citation Haelewaters, Danny, Michał Gorczak, Walter P. Pfliegler, András Tartally, Marta Tischer, Marta Wrzosek, and Donald H. Pfister. 2015. “Bringing Laboulbeniales into the 21st century: enhanced techniques for extraction and PCR amplification of DNA from minute ectoparasitic fungi.” IMA Fungus 6 (2): 363-372. doi:10.5598/imafungus.2015.06.02.08. http://dx.doi.org/10.5598/ imafungus.2015.06.02.08.
Published Version doi:10.5598/imafungus.2015.06.02.08
Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:24984019
Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ARTICLE 363 , was sp.1, , as the can be GLQJDXWKRU , Donald H. , Donald 3 Laboulbeniales VSHFL¿FSULPHUV FIXQJL Pyxidiophora Ascomycota Laboulbeniales Laboulbeniales ). Weir & Blackwell’s & Blackwell’s ). Weir RUVH\RXRU\RXUXVHRIWKHZRUN Key words: Ascomycota isolation DNA insect collections Laboulbeniales ribosomal DNA unculturable fungi , Marta Wrzosek Hesperomyces coccinelloides 3 , that includes both IMA FUNGUS IMA (2015) 363–372 · 6(2): (Weir & Blackwell 2001a). This & Blackwell 2001a). (Weir , . 1991). Useful advice about general . High bootstrap support for this hypothesis , Marta Tischer , Marta et al 7 Laboulbeniales Pyxidiophorales . DNA extractions . DNA . These fungi are . Zodiomyces vorticellarius Pyxidiophorales Laboulbeniomycetes , based on very few molecular systematics It was only recently that the order ivecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get ivecommons.org/licenses/by-nc-nd/3.0/legalcode. , sister to LGHQWL¿FDWLRQ RI D KRVW RIWHQ IDFLOLWDWHV of hosts occur, associated fungi, but since fortuitous infections LGHQWL¿FDWLRQ RI LWV their morphology or it is best to identify these fungi based on lists are available sequence comparisons. Host-parasite DNA for some countries (Scheloske 1969, Huldén 1983, Majewski 1994, De Kesel 1998, Santamaría 1998, 2003) and regions (Santamaría Stigmatomyces limnophorae PHWKRGRORJ\ DQG LGHQWL¿FDWLRQ RI and phylogenetic determination was based on four (partial) (rDNA) sequences ( SSU ribosomal DNA found in Thaxter (1896), Scheloske (1969), Benjamin (1971), found in Majewski (1994), and Santamaría (1998). recognized as a well-supported lineage in class and (2001a) phylogeny suggested a close relationship with Sordariomycetes , András Tartally Ascomycota XHQFHVIRUVSHFLHVLQWKHJHQHUD DWLRQVKDYHSURYHQGLI¿FXOW+HUH Polyandromyces HUVLW\'LYLQLW\$YHQXH&DPEULGJH0$86$ FRUUHVSRQ KRURUOLFHQVRU EXWQRWLQDQ\ZD\WKDWVXJJHVWVWKDWWKH\HQG FV 'HEUHFHQ(J\HWHPWpU'HEUHFHQ+XQJDU\ on Laboulbeniales GHQHZO\JHQHUDWHG differ differ , and Laboulbeniales Laboulbeniomycetes Ascomycota in that they do into the 21st century: enhanced techniques for techniques enhanced 21st century: into the Monoicomyces :DOWHU 3 3ÀLHJOHU , 3 Laboulbeniales $FFHSWHG 2FWREHU 3XEOLVKHG 1RYHPEHU Ascomycota Laboulbenia , is one of the most peculiar orders of 0LFKDá *RUF]DN 1,2 are obligate ectoparasitic Herpomyces Laboulbeniales Laboulbeniales ,
was later achieved by Schoch et al. (2009) based on a six- RQ&RQJHU )DLUFKLOG DQG/HH 7D\ORU 7KH gene phylogeny. The order Laboulbeniales was represented technique from Weir & Blackwell (2001b) was successful only in that dataset by only SSU and LSU sequences for two ZKHQKRVWVZHUHSUHVHUYHGLQ HWKDQROIRUQRWPRUHWKDQ species (Hesperomyces virescens and Stigmatomyces six months. Thalli taken from dried insect specimens have not protrudens). been available for molecular phylogenetic analyses because
ARTICLE Molecular studies of LaboulbenialesKDYHSURYHQGLI¿FXOW extractions have been unsuccessful with this type of material for several reasons. The thalli are microscopic, on average :HLU %ODFNZHOOE 7KLVWHFKQLFDOGLI¿FXOW\OLPLWVERWK 200-300 μm in length. Among the smallest species known the taxonomical and geographical diversity of species that are Rickia euxesti (total length 40–68 μm), R. lenoirii ± can be included in phylogenetic studies (e.g. Thaxter 1899, 67 μm), and Siemaszkoa annae ±P 7KD[WHU D E :HLU +DPPRQG 0DMHZVNL 6DQWDPDUtD (VSDGDOHU Haelewaters et alDE At the other end of the size spectrum are Zodiomyces Owing to the difficulties in DNA isolation and vorticellarius WR PP DQG Laboulbenia kunckelii amplification of phylogenetically informative genes, ± PP *LDUG 6XJL\DPD 3KDQLFKDSRO the molecular phylogenetic relationships within this Haelewaters unpubl.). For study and extraction of DNA, group have been understudied. Weir & Hughes thalli need to be removed from their host, which requires (2002) constructed a partial SSU rDNA phylogeny PLFURPDQLSXODWLRQ WHFKQLTXHV DQG VSHFL¿F WRROV +RVWV of ten species of Laboulbeniales, representing three may bear only a few thalli but certain hosts carry multiple, subfamilies (Ceratomycetoideae, Laboulbenioideae, RIWHQ SRVLWLRQVSHFL¿F VSHFLHV HJ Chitonomyces spp., Peyritschielloideae). A combined dataset of the partial 'H .HVHO +DHOHZDWHUV *ROGPDQQ :HLU SSU and ITS rDNA regions was used to study the Hesperomyces coleomegillae and H. palustris *ROGPDQQ phenomenon of position specificity in 13 species of et al. 2013). Many species are heavily pigmented with Chitonomyces on Laccophilus maculosus (Coleoptera melanin in their cell walls, providing rigidity (Weir & Beakes Dytiscidae *ROGPDQQ :HLU *ROGPDQQ et 7KLV SLJPHQW LQWHUIHUHV ZLWK 3&5 DPSOL¿FDWLRQ E\ al. (2013) described two position specific species of binding to the DNA polymerase (Eckhart et al. 2000). Thalli Hesperomyces on Coleomegilla maculata (Coleoptera are relatively long-lasting and their form is such that they Coccinellidae), again based on partial SSU+ITS rDNA. absorb impacts and friction during their entire existence on All these studies used the extraction methodology of Weir the hosts’ integument. These tough and resilient cells are & Blackwell (2001b). GLI¿FXOW WR EUHDN %HFDXVH Laboulbeniales have not been We tested more generalized techniques that could be grown in culture to more than a few cells, obtaining DNA adapted to sample the thalli of Laboulbeniales. from cultured material has been impossible. Only Whisler (1968) was partly successful in this with Stigmatomyces ceratophorus REWDLQLQJ FHOOHG WKDOOL RQWR VWHULOH À\ MATERIAL AND METHODS wings on brain-heart infusion agar, but perithecia were not produced. Collection Laboulbeniales DUH D UHPDUNDEOH FODGH IRU WKHLU Insects were collected around the world by ourselves or REOLJDWH ELRWURSK\ VWULFWO\ GHWHUPLQDWH JURZWK ZLWK collaborators using standard entomological methods (sticky development from a two-celled ascospore to a thallus of up traps, light trap, entomological net, and hand collecting) or WRVHYHUDOWKRXVDQGFHOOV ELODWHUDOV\PPHWU\ DQG ORVV obtained from the pet store (Blatta lateralis). Insects were of germ tubes, hyphae, and conidia. Despite these special killed in 70–100 % ethanol, ethyl acetate vapors, or simply features, the order and the class were not included in studies by freezing. Screening for Laboulbeniales was done using a dealing with “major lineages in Ascomycota” (Prieto & Wedin GLVVHFWLQJPLFURVFRSHDW[ 2013) or the subphylum Pezizomycotina, to which they belong (Spatafora et al. 2006). Morphological studies Extraction of DNA using a variety of methods and Individual thalli were removed from the host using an protocols have given poor results or failed. These include HQWRPRORJLFDO SLQ VHOIPDGH VRPHWLPHV ÀDWWHQHG RU prolonged boiling of thalli (Henson 1992), microwave the tip of a scalpel. Slide mounts followed techniques for WUHDWPHQW *RRGZLQ /HH LPPHUVLRQ LQ OLTXLG permanent microscope slides (Benjamin 1971, Haelewaters nitrogen (Haugland et al. 1999), and direct addition of entire HW DO E ,GHQWL¿FDWLRQ RI Laboulbeniales followed thalli to PCR master mix (Haelewaters 2011). Also, the use Thaxter (1908, 1931), Majewski (1994), and De Kesel (2011). of commercial kits (Puregene Kit A, DNeasy Plant Mini Kit, Voucher slides are deposited at BP (Botanical Department, 4LDJHQ +DHOHZDWHUV KDVVRIDUSURYHQXQVXFFHVVIXO Hungarian Natural History Museum), FH (Farlow Herbarium, 7KH¿UVWVXFFHVVIXOSXEOLVKHGH[WUDFWLRQSURWRFROLQYROYHG Harvard University), and WA (Faculty of Biology, University of
transferring thalli to double distilled (dd) H20, air drying, and :DUVDZ +HUEDULXPDFURQ\PVDUHDFFRUGLQJWR7KLHUV manually crushing thalli between microscope slides (Weir & %ODFNZHOOD 7KHVXFFHVVUDWHIRUWKLVSURWRFROZDV DNA extraction protocols Weir & Blackwell (2001b) developed an improved technique Between one and 30 thalli were removed from each host in which thalli were manually crushed on a microscope slide VSHFLPHQ ,Q WKLV VWXG\ ZH ZDQWHG WR WHVW WKH HI¿FDF\ RI and picked up with a micropipette facilitated by the use of a different commercial and noncommercial DNA extraction EHGRIGU\LFHDPRGL¿FDWLRQIURPSUHYLRXVHQGHDYRUVEDVHG SURWRFROV7KHIROORZLQJZHUHXVHG 4,$DPS'1$0LFUR
364 IMA FUNGUS '1$H[WUDFWLRQDQGDPSOL¿FDWLRQLQLaboulbeniales
.LW 4LDJHQ 6WDQIRUG &$ ([WUDFW1$PS 3ODQW 3&5 the suggested protocol in the manufacturer’s instructions. ARTICLE .LW 6LJPD$OGULFK 6W /RXLV 02 D KHDWH[WUDFWLRQ When PCR reactions did not produce clear bands during SURWRFRO DQG ,62/$7( ,, 3ODQW '1$ .LW %LROLQH gel electrophoresis, conditions were optimized to include Reagents, London). D WZRVWHS & & ³WRXFKGRZQ´ DQQHDOLQJ SKDVH (1) QIAamp DNA Micro Kit '1$ZDVLVRODWHGIURPWZRWR (Sohrabi et al. 2010). In some cases, a semi-nested “touch- sixteen thalli for each extraction, following the manufacturer’s GRZQ´3&5ZDVSHUIRUPHGXVLQJWKHSURGXFWRIWKH¿UVW instructions. Some extracts received pre-treatment with liquid unsuccessful PCR reaction (e.g. PCR 1 using primers LR0R QLWURJHQRUWZRF\FOHVRIKHDWLQJWR &DQGIUHH]LQJRQ DQG/5VHPLQHVWHG3&5XVLQJWKHSURGXFWRI3&5ZLWK liquid nitrogen. primers LR0R and LR3). (2) 0RGL¿HG ([WUDFW1$PS 3ODQW 3&5 .LW 7KH Products that showed clear bands on agarose gel manufacturer’s instructions were followed but with 20 μL of ZHUH FOHDQHG ZLWK 4LDTXLFN 3&5 3XUL¿FDWLRQ .LW 4LDJHQ Extraction Solution (EX) and 60 μL of Dilution Solution. One 6WDQIRUG&$ RU([WUDFW0H'1$*HORXWNLW %OLUW*GDĔVN to 20 thalli were removed from the host with the help of a 3RODQG DQG VXEVHTXHQWO\ VHTXHQFHG :H SUHSDUHG ȝ/ tiny drop of Hoyer’s medium (30 g arabic gum, 200 g chloral sequencing reactions containing the same primer pairs and
K\GUDWHP/JO\FHUROP/GG+20) or glycerine at the very ȝ/ RI SXUL¿HG 3&5 SURGXFW 7KH VHTXHQFLQJ UHDFWLRQV HQGRIDPLFURSLQDQGWKHQDGGHGWR(;¿OOHG/WXEHV were performed using the Big Dye® Terminator v3.1 Cycle When hosts were preserved in dried collections, 16–30 thalli Sequencing Kit (Life Technologies, Carlsbad, CA). were used. Again, the pre-treatment described above was Sequences were trimmed, edited and assembled in applied for some extracts. 6HTXHQFKHU *HQH &RGHV &RUSRUDWLRQ$QQ$UERU (3) +HDWH[WUDFWLRQ SURWRFRO 7KLV PHWKRG ZDV DGDSWHG MI). We performed BLAST searches on all of our sequences from a protocol for single-spore extractions and subsequent DW KWWS QFELQOPQLKJRYEODVW%ODVWFJL IRU VLPLODU 3&5 UHDFWLRQV )HUUHLUD *ODVV EDVHG RQ *RRGZLQ VHTXHQFHV)RUJHQHUDQRW\HWUHSUHVHQWHGLQ*HQ%DQNZH & Lee 1993). Thalli were removed from the host (3 thalli of compared sequences with our personal database, which Hesperomyces virescens, 20–30 thalli of Rickia wasmannii) or is accessible at the Harvard University Herbaria internal DaPPSRUWLRQRIDKHDYLO\LQIHFWHGBlatta lateralis antenna server. with Herpomyces stylopygae thalli was removed, placed in P/3&5WXEHVDQGPLFURZDYHWUHDWHG :IRUPLQ
7KHQ / GG+20 was added to the individual tubes, and RESULTS the thalli (or antennal parts) were manually crushed using a sterile pipette tip under a dissecting microscope. Some loss Our study shows that some simple, general DNA extraction of material did occur by capillary action, but it was minimal. protocols work. The commercial kits we tested are widely 7KH3&5WXEHVZHUHLQFXEDWHGDW&IRUPLQ6WURQJ available. pressure was applied to the ice inside the PCR tubes to Table 2 shows the success rates of the individual further break apart thalli using a sterile pipette tip. protocols, per genus extracted. Extractions using the (4) ,62/$7( ,, 3ODQW '1$ .LW 8S WR WZHQW\ WKDOOL ZHUH QIAamp DNA Micro Kit yielded the lowest rates of success UHPRYHGIURPWKHKRVWDQGWUDQVIHUUHGWRP/(SSHQGRUI among the tested protocols, with seemingly no effect of WXEHVZLWK±/ HWKDQRO$OWHUQDWLYHO\LQWKHFDVH pre-treatment. The overall success rate was 22 % (n = 27 of Herpomyces ectobiae on Blattella germanica, a piece of extractions total), for Hesperomyces virescens extractions DQDQWHQQDZDVLVRODWHGDQGWUDQVIHUUHGDOWRJHWKHU7KH WKHVXFFHVVUDWHZDV n = 17). Overall success of the mL tubes were vacuum-dried at room temperature. Thalli Extract-N-Amp Plant PCR Kit was 64 % (n = 66), with 92 were subsequently crushed in liquid nitrogen, using a sterile % success for Herpomyces spp. (n = 13) and 66 % for H. pipette with melted-closed tip. CTAB-based isolation buffer virescens (n )RU WKH WKLUG KHDWH[WUDFWLRQ SURWRFRO (PA1, ISOLATE II Plant DNA Kit) was added to the tubes and the success rate was 83 % for Herpomyces ectobiae (n incubated in liquid nitrogen for 3 min, followed by incubation in = 6) and 100 % for H. virescens (n = 3). The ISOLATE II DKHDWEORFNVHWDW± &IRUPLQ7KLVF\FOHRIIUHH]LQJ 3ODQW'1$.LWJDYHDQRYHUDOOVXFFHVVUDWHRI n = heating was repeated twice. Further steps were performed 34), with a 100 % success rate for H. ectobiae (n DQG following the ISOLATE II Plant DNA Kit manufacturer’s 86 % for H. virescens (n = 7). Interestingly, extracting DNA protocol. of Laboulbenia species was only successful 20 % of the time with the Extract-N-Am Plant PCR Kit and 10 % with 3&5DPSOL¿FDWLRQDQG'1$VHTXHQFLQJ the ISOLATE II Plant DNA Kit. Four extraction attempts of 7KUHHJHQHORFLZHUHDPSOL¿HG SDUWLDOU'1$668 ca 1100 Laboulbenia species with the QIAamp DNA Micro Kit were ES U'1$,76 LQFOXGLQJ,766DQG,76 caES unsuccessful. and partial rDNA LSU (ca ES 3&5 DPSOL¿FDWLRQ :H JHQHUDWHG VHTXHQFHV 668 ,76 DQG was performed using both previously published and RU /68 U'1$ IRU LVRODWHV RI WKH IROORZLQJ VSHFLHV newly designed primers (Table 1). LaboulbenialesVSHFL¿F Gloeandromyces nycteribiidarum, Herpomyces chaetophilus, primers were designed for the SSU region based on H. ectobiae, H. periplanetae, H. stylopygae, Hesperomyces H[LVWLQJ VHTXHQFHV LQ *HQ%DQN 7DEOH 3&5 UHDFWLRQV virescens, Laboulbenia diopsidis, Monoicomyces invisibilis, were performed according to the protocols listed in the Polyandromyces coptosomalis, Rhachomyces philonthinus, respective reference for mentioned primers, or, in the Rickia wasmannii, and Zodiomyces vorticellarius (Table 3). case of the Extract-N-Amp Plant PCR Kit, according to Rhachomyces philonthinus was removed from a specimen
VOLUME 6 · NO. 2 365 Haelewaters et al.
Table 1./LVWRISULPHUVXVHGIRU3&5DPSOL¿FDWLRQRIVPDOOVXEXQLW 668 LQWHUQDOWUDQVFULEHGVSDFHU ,76 DQGODUJHVXEXQLW /68 U'1$ Locus Primer name Sequence Reference SSU rDNA NS1 forward *7$*7&$7$7*&77*7&7& White et al. 1990 SSU rDNA NS2 reverse **&7*&7**&$&&$*$&77*& White et al. 1990 SSU rDNA NS4 reverse &77&&*7&$$77&&777$$* White et al. 1990 ARTICLE SSU rDNA SL344 forward **7&*&$$**&7*$$$&77$ Landvik et al. 1997 SSU rDNA NS6 reverse *&$7&$&$*$&&7*77$77*&&7& White et al. 1990 SSU rDNA SL122 forward $**&*&*&$$$77$&&&$$7 Landvik et al. 1997 SSU rDNA SR4 reverse $$$&&$$&$$$$7$*$$ R. Vilgalys unpublished SSU rDNA NSL1 forward *7$*7*7&&7&U&$7*&7777*$& present study SSU rDNA NSL2 reverse $$7&\$$*$$777&$&&7&7*$& present study SSU rDNA L forward $$&&7**77*$7&&7*&&$*7 Wrzosek 2000 SSU rDNA 402 forward *&7$&&$&$7&&$$**$$**&$ Wrzosek 2000 SSU rDNA 416 reverse $777*&*&*&&7*&7*&&77&& Wrzosek 2000 SSU rDNA forward *7&$*$**7*$$$77&77**$7 Wrzosek 2000 SSU rDNA 898 reverse 7$$$7&&$$*$$777&$&&7&7 Wrzosek 2000 SSU rDNA 1144 forward *&&7*&**&77$$777*$&7&$$&$ Wrzosek 2000 SSU rDNA 1308 reverse &7&*77&*77$$&**$$77$$&& Wrzosek 2000 SSU rDNA R reverse 7*$7&&77&7*&$**77&$&&7$&* Wrzosek 2000 ITS rDNA ITS1f forward &77**7&$777$*$**$$*7$$ *DUGHV %UXQV ITS rDNA ITS4 reverse 7&&7&&*&77$77*$7$7*& White et al. 1990 ITS rDNA ITS4_kyo1 reverse 7&&7&&*&77:77*:7:7*& Toju et al. 2012 ITS rDNA ,76 forward **$$*7$$$$*7&*7$$&$$** White et al. 1990 ITS rDNA ITS2 reverse *&7*&*77&77&$7&*$7*& White et al. 1990 LSU rDNA LR0R forward $&&&*&7*$$&77$$*& R. Vilgalys unpublished LSU rDNA LR1R forward $**$$$$*$$$&&$$&& Moncalvo et al. 1993 LSU rDNA LIC24R forward *$$$&&$$&$***$77* Miadlikowska & Lutzoni 2000 LSU rDNA LR3 reverse **7&&*7*777&$$*$& Vilgalys & Hester 1990 LSU rDNA /5 reverse $7&&7*$***$$$&77& Vilgalys & Hester 1990 LSU rDNA LR7 reverse 7$&7$&&$&&$$*$7&7 Vilgalys & Hester 1990 LSU rDNA NL1 forward *&$7$7&$$7$$*&**$**$$$$* Kurtzman & Robnett 1997 LSU rDNA NL4 reverse **7&&*7*777&$$*$&** Kurtzman & Robnett 1997
of Philonthus that had been collected by Tomasz Majewski in DISCUSSION August 2004. The host specimen was preserved for 11 years in 70 % ethanol. Micromanipulation practices We were able to extract DNA from thalli of Hesperomyces Laboulbeniales are more problematic to work with than many virescens from dried insect specimens (with the Extract-N- RWKHUJURXSVRIIXQJL2QHRIWKHPDLQGLI¿FXOWLHVLVWKHLUVPDOO $PS3ODQW3&5.LW RQCycloneda sanguinea sanguinea from size, which requires sterile micromanipulation with precise *XDWHPDODFROOHFWHGLQ0D\DQGRQHarmonia axyridis micropin handling. from Massachusetts collected in August 2006 (details in It is preferable to separate thalli from the host’s body, but Haelewaters et alE ([WUDFWLRQVZHUHSHUIRUPHGIURP minute thalli of Rickia, Herpomyces or Siemaszkoa are hard H. paranensis on a dried Archimandrita tessellata (Blattodea to detach. Using whole infected body parts in an extraction Blaberidae) collected in 2001 [deposited at the Harvard makes the procedure faster and easier. Most of the primers Museum of Comparative Zoology] and from Rodaucea sp. used in this study do not amplify the host insect’s DNA, on a dried Cholevinae sp. (Coleoptera Leiodidae) collected +RZHYHUDPSOL¿FDWLRQRILQVHFW'1$E\VRPHSULPHUVPD\ in 1991 [part of the collection of Invertebrate Zoology at the KDSSHQ DV ZLWK /55/5 DQG WKH VHWV RI 668 SULPHUV American Museum of Natural History], but no bands were used in Wrzosek 2000). Prominent appendages, such as noted on the agarose gel after PCR. those in many species of Laboulbenia or or Rhachomyces, SRVH DQRWKHU GLI¿FXOW\ GHEULV LV RIWHQ REVHUYHG WR VWLFN WR the appendages and is very hard to impossible to wash away. In this case contamination with fungal propagules may be inevitable. LaboulbenialesVSHFL¿F SULPHUV ZLOO VHUYH WR reduce the chance of amplifying non-target DNA. Another
366 IMA FUNGUS '1$H[WUDFWLRQDQGDPSOL¿FDWLRQLQLaboulbeniales
Table 2. Success rates per DNA extraction protocol used in this study, for all tested genera. Laboulbeniales from dried host insects were only extracted using the Extract-N-Amp Plant PCR Kit. ARTICLE QIAamp DNA Micro Kit Extract-N-Amp Plant PCR Kit # extractions # success # failed % success # extractions # success # failed % success Aphanandromyces Chitonomyces 4040 % Gloeandromyces 1 1 0 100 % Haplomyces 2020 %2020 % Herpomyces 13 12 1 92 % Hesperomyces 17 6 11 23 12 66 % Laboulbenia 4040 % 1 4 20 % Monoicomyces Polyandromyces 2 2 0 100 % Rhachomyces Rickia Zodiomyces
Herpomyces (dried) 2020 % Hesperomyces (dried) 3 2 60 % Rodaucea (dried) 1010 %
Heat-extraction protocol ISOLATE II Plant DNA Kit # extractions # success # failed % success # extractions # success # failed % success Aphanandromyces 1010 % Chitonomyces Gloeandromyces Haplomyces Herpomyces 6 1 83 % 0 100 % Hesperomyces 3 3 0 100 % 7 6 1 86 % Laboulbenia 10 1 9 10 % Monoicomyces 6 4 2 67 % Polyandromyces Rhachomyces 211 Rickia 11 6 Zodiomyces 3 3 0 100 %
option is to simply excise the appendage system prior to larger species of Laboulbeniales, which provide ample DNA extraction. concentration even from a single thallus (e.g. Zodiomyces vorticellarius), and mature ascospore-containing thalli, Preservation techniques which provide a higher concentration of DNA compared to One of the most important concerns regarding successful immature or old thalli (always without ascospores). Many molecular research is the method employed for preservation entomological practices involve preservation methods that of material. The most effective option for extraction of interfere with successful DNA extraction of either the host Laboulbeniales DNA involves using freshly collected material RULWVDVVRFLDWHGIXQJL PRVWLQVHFWVSHFLPHQVDUHSLQQHGLQ SUHIHUDEO\ VWRUHG LQ HWKDQRO 7KHVH WZR IDFWRUV museum collections or preserved on 70 % ethanol. certainly contribute to most of our DNA isolation positive For morphological study of Laboulbeniales, researchers UHVXOWV 6WRUDJH LQ HWKDQRO JHQHUDOO\ SURYLGHV are able to make use of the many excellent systematic insect good DNA preservation for a prolonged period of time. Our collections in natural history museums around the world. DNA extraction protocols enabled us to amplify DNA and Such collections of dried pinned insects give relatively easily generate sequences from Laboulbeniales material that was access to data (e.g. Weir & Hammond 1997, Haelewaters on average 1–2 years old (one specimen was 11 years old), et al. 2014). However, to date, extracting DNA from dried which is a novel development. Conditions that consistently specimens has resulted in a 100 % failure rate (Weir & \LHOGHGJRRGUHVXOWVLQFOXGHG IUHVKO\FROOHFWHGVSHFLPHQVRI Blackwell 2001b). We present sequences obtained from two
VOLUME 6 · NO. 2 367 Haelewaters et al.
Table 3'HWDLOHGFROOHFWLQJGDWDPHWKRGRISUHVHUYDWLRQQXPEHURIWKDOOLXVHGLQH[WUDFWLRQ'1$H[WUDFWLRQSURWRFRODQG*HQ%DQNDFFHVVLRQ QXPEHUVIRU668,76DQG/68U'1$VHTXHQFHVRILVRODWHV1% 7DEOHLVEHVWYLHZHGLQDSDJHYLHZDVLWFRQWLQXHVRQWKHQH[WSDJH Genus Species Isolate Host COUNTRY: locality Collector
Gloeandromyces nycteribiidarum DH619a Streblidae sp. TRINIDAD -&&DPDFKR ARTICLE Herpomyces chaetophilus DH602b Periplaneta americana 86$ 0DVVDFKXVHWWV T.W. Wang Cambridge, Kirkland House
Herpomyces ectobiae 0* Blattella germanica 32/$1' :DUVDZ 0*RUF]DN
Herpomyces periplanetae DH602c Periplaneta americana 86$ 0DVVDFKXVHWWV T.W. Wang Cambridge, Kirkland House Herpomyces stylopygae DE_HerpBL1 Blatta lateralis +81*$5< :33ÀLHJOHU
Hesperomyces virescens DH167e Cycloneda sanguinea sanguinea *8$7(0$/$ R.S. Zack Huehuetenango Dept., La Laguna Hesperomyces virescens -3D 2OODYQLJUXP 86$ *HRUJLD3HDFK E. Brooks Thompson County, USDA-ARS Hesperomyces virescens DH486c Harmonia axyridis 86$ 0DVVDFKXVHWWV -5\NNHQ World’s End peninsula Hesperomyces virescens DH646c Harmonia axyridis *(50$1< %DYDULDVWDWH S. Tragust Bereuth Hesperomyces virescens HM497c Harmonia axyridis 86$ *HRUJLD3HDFK E. Brooks Thompson County, USDA-ARS
Hesperomyces virescens DE_HV01 Harmonia axyridis +81*$5< 'HEUHFHQ :33ÀLHJOHU Hesperomyces virescens MT001 Harmonia axyridis 32/$1' :DUVDZ M. Tischer
Laboulbenia diopsidis DH468a Diopsidae sp. 6,(55$/(21( (DVWHUQ W. Rossi Province, Nemahugoima Monoicomyces invisibilis MT004 Anotylus sculpturatus 32/$1' :DUVDZ M. Tischer
Polyandromyces coptosomalis DH313f Phoeacia sp. nov. (&8$'25 2UHOODQD D. Forero Province, Quito Rhachomyces philonthinus TM10446 Philonthus sp. 32/$1' T. Majewski Zachodniopomorskie, àREĪDQ\ Rickia wasmannii DE_Rak4 Myrmica scabrinodis +81*$5< 5DNDFD A. Tartally Zodiomyces vorticellarius 0* Helochares obscurus 32/$1' :DUVDZ 0*RUF]DN
collections of H. virescens from dried ladybirds (DH167e and al. 2013, Wang et al. 2014). If 70 % ethanol was used to DH486c) collected in 2013 and 2006, respectively. Often preserve insect hosts, it comes as no surprise that the DNA thalli acquired from dried hosts are in poor condition and both of Laboulbeniales harvested from them is adversely affected. LGHQWL¿FDWLRQ EDVHG RQ PRUSKRORJLFDO FKDUDFWHUV DQG '1$ When working with Laboulbeniales from dried collections, extraction may be a challenge. another challenge is that information about the habitat or Many insects in entomological collections are preserved methods of collection and preservation is typically sparse. in 70 % ethanol. This decreases the DNA quality of the insect The extraction of DNA from insects can be drastically affected and its associates – especially after an extended period by using certain media (such as killing agents in pitfall traps) of storage (e.g. A’Hara et al. 1998). Some studies have that degrade DNA. Some commonly used materials such as generated short segments of mitochondrial DNA (< 300 bp) ethylene glycol or formalin have been linked to considerable from material in 70 % ethanol (e.g. Colgan et al. 2002). For DNA degradation (e.g. Dillon et al. 1996, Stoeckle et al. 2010). phylogenetic studies, however, longer segments are needed, and these need to be acquired from non-degraded DNA. Negative results 1RQGHJUDGHG'1$LVDOVRUHTXLUHGIRU3&5DPSOL¿FDWLRQRI Our negative results can be explained based on protocols low copy-number nuclear genes commonly used in modern HPSOR\HG DQGRU WKH QDWXUH RI WKH IXQJL WKDW ZHUH XQGHU fungal phylogenies (e.g. Hibbett et al. 2007, Hansen et investigation. The 100 % failure rate of the QIAamp DNA
368 IMA FUNGUS '1$H[WUDFWLRQDQGDPSOL¿FDWLRQLQLaboulbeniales ARTICLE
Year of Preservation Number of thalli used Extraction protocol SSU ITS LSU collection 2014 (W2+ 12 thalli Extract-N-Amp (with KT800008 glycerine) 2014 (W2+ 10 female thalli Extract-N-Amp (with KT800023 KT800039 KT800009 glycerine)
2014 (W2+ piece of antenna with ± 20 adult ISOLATE II Plant DNA KT800024 KT800040 thalli .LWZLWKRXWIUHH]HWKDZ 2014 (W2+ 11 female thalli Extract-N-Amp (with .7 KT800041 KT800010 glycerine) 2014 80 % EtOH piece of antenna with ± 30 adult Heat-extraction KT800026 KT800042 KT800011 thalli 2013 dried 18 adult thalli Extract-N-Amp KT800027 KT800012
2014 (W2+ 10 adult thalli QIAamp DNA Micro Kit KT800028 KT800043 KT800013
2006 dried 16 adult thalli Extract-N-Amp (with KT800029 KT800044 KT800014 glycerine) 2013 (W2+ 2 adult thalli Extract-N-Amp .7 .7
2014 (W2+ DGXOWWKDOOL Extract-N-Amp (with KT800030 KT800046 KT800016 Hoyer’s medium)
2014 80 % EtOH 9 adult thalli Heat-extraction KT800031 KT800047 KT800017 (W2+ 1 adult thallus ISOLATE II Plant DNA KT800032 KT800048 KT800018 Kit 2013 100 % EtOH 12 adult thalli Extract-N-Amp KT800033 KT800049 KT800019
(W2+ 1 adult thallus ISOLATE II Plant DNA KT800034 Kit 2009 (W2+ 7 female and 2 male thalli Extract-N-Amp .7 KT800020
2004 70 % EtOH DGXOWWKDOOL ISOLATE II Plant DNA KT800036 Kit
2014 80 % EtOH 30 adult thalli Heat-extraction KT800037 .7 KT800021 (W2+ ISOLATE II Plant DNA 1 adult thallus Kit KT800038 KT800022
Micro Kit for Chitonomyces, Haplomyces, and Laboulbenia is Micro Kit and the Extract-N-Amp Plant PCR Kit probably is largely due to the fact that no pre-treatments were carried out GXHWRWKHFRPELQDWLRQRIWZRIDFWRUV WKHH[WUDFWUHFHLYHG for these extracts. However, for one Laboulbenia extraction QR SUHWUHDWPHQW DQG KRVW LQVHFWV ZHUH FROOHFWHG DQG using this protocol a pre-treatment was done involving two SUHVHUYHG IRUIRXUWR¿YH\HDUV LQHWKDQRO7KHUHODWLYHO\ F\FOHV RI KHDWLQJ WR & DQG IUHH]LQJ RQ OLTXLG QLWURJHQ low success rate with Rickia, with the heat-extraction protocol, Then why was this extraction unsuccessful? Laboulbenia may be explained by the fact that these small but very rigid species are generally heavily melanized, and the melanin WKDOOL DUH GLI¿FXOW WR EUHDN GXULQJ WKH WUHDWPHQWV WKDW ZHUH SLJPHQW VHHPV WR KLQGHU 3&5 DPSOL¿FDWLRQ UHDFWLRQV DSSOLHG YLVXDOLQVSHFWLRQDIWHUSHUIRUPLQJWKHHQWLUHSURWRFRO (Eckhart et al. 2000). Also in the Extract-N-Amp Plant PCR Kit shows many intact thalli. Thus, the amount of DNA available and the ISOLATE II Plant DNA Kit the success of extracting for the Taq polymerase during PCR was limited, despite the '1$ DQG VXEVHTXHQW 3&5 DPSOL¿FDWLRQ RI Laboulbenia high number of thalli (20–30) per reaction. species is considerably lower compared to other genera. We can only hint at the low success rate of extractions This observation shows that variables other than isolation from dried material. The extraction of Rodaucea sp. received techniques, such as the presence of pigments, are important no pre-treatment and the thalli were removed from a WR WKH VXFFHVV RI '1$ H[WUDFWLRQ DQG DPSOL¿FDWLRQ 7KH cholevine specimen collected in 1991. It might have been % success rate of Haplomyces using both the QIAamp DNA too old for successful DNA extraction. The same may be true
VOLUME 6 · NO. 2 369 Haelewaters et al.
for the unsuccessful attempts to extract DNA of Herpomyces Kesel, István Pócsi, Matthias Sipiczki, and Tomasz Majewski. For paranensis from a pinned specimen of Archimandrita UHYLHZLQJ HDUOLHU GUDIWV RI WKH PDQXVFULSW 3HGUR : &URXV$QGUp tessallata from 2001. De Kesel, David L. Hawksworth, Rosanne A. Healy, Monica Hughes, 7RPDV]0DMHZVNL DQG :DOWHU 5RVVL '+ ZRXOG OLNH WR VSHFL¿FDOO\ thank Lee H. Herman at the American Museum of Natural History
ARTICLE CONCLUSIONS and Brian D. Farrell and Philip Perkins at the Harvard Museum of Comparative Zoology for curatorial support. Even with fresh thalli available, successful extraction of DNA has been one of the greatest obstacles in applying molecular methods to research on Laboulbeniales. Their minute size, REFERENCES WKH GLI¿FXOW\ LQ IUDFWXULQJ WKDOOL WR UHOHDVH '1$ DQG WKH IDFW that (to date) they remain resistant to isolation into culture $¶+DUD6+DUOLQJ50F.LQOD\5*7RSSLQJ&- 5$3'SUR¿OLQJ makes molecular protocols applied to LaboulbenialesGLI¿FXOW of spider (Araneae) DNA. Journal of Arachnology 26 ± 7KLV LV WKH UHDVRQ ³ODERXOEHQLRORJLVWV´ QHHG FROOHDJXHV &ROJDQ '- %URZQ 6 0DMRU 5( &KULVWLH ) *UD\ 05 &DVVLV * (entomologists) or museums to provide high-quality, properly (2002) Population genetics of wolf spiders of fragmented habitat SUHSDUHGVDPSOHV DQG '1$LVRODWLRQSURWRFROVWKDWIRFXV in the wheat belt of New South Wales. 0ROHFXODU (FRORJ\ 11 heavily on deep homogenization of the material. Microwave ± KHDWLQJVXEPHUVLRQLQOLTXLGQLWURJHQIUHH]HWKDZF\FOHVDQG &RQJHU$')DLUFKLOG/0 $TXLFNIUHH]HPHWKRGIRUPDNLQJ simple yet effective crushing with pipette tips are all means smear slides permanent. Stain Technology 28 ± of destroying the tough cell walls without damaging the DNA. De Kesel A (1996a) Relative importance of direct and indirect infection As stated in previous studies, both the SSU and ITS in the transmission of Laboulbenia slackensis (Ascomycetes, portions of rDNA are suited for molecular phylogenetics of Laboulbeniales). Belgian Journal of Botany 128 ± the Laboulbeniales and universal fungal primers for these 'H .HVHO $ E +RVW VSHFL¿FLW\ DQG KDELWDW SUHIHUHQFH RI regions work well for most of the species (Weir & Blackwell Laboulbenia slackensis. Mycologia 88 ± E *ROGPDQQ :HLU :HLU +XJKHV 'H .HVHO $ ,GHQWL¿FDWLRQ DQG KRVWUDQJH RI WKH JHQXV *ROGPDQQet al. 2013)). We have found that LSU sequences Laboulbenia in Belgium. Sterbeeckia 18 ± DUH DOVR HDVLO\ WR REWDLQ 'HVLJQLQJ VSHFL¿F SULPHUV RIWHQ De Kesel A (2011) Hesperomyces (Laboulbeniales) and coccinellid IDFLOLWDWHV WKH ZRUN :HOOGHVLJQHG SULPHUV VSHFL¿F IRU hosts. Sterbeeckia 30 ± LaboulbenialesPD\SHUIRUPEHWWHUDQGWKHLUVSHFL¿FLW\KHOSV De Kesel A, Haelewaters D (2012) Belgian records of Laboulbeniales to avoid contamination. As the number of genes being used from aquatic insects (2) – Chitonomyces aculeifer. 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*RRGZLQ'&/HH6% 0LFURZDYHPLQLSUHSRIWRWDOJHQRPLF 3ULHWR 0 :HGLQ 0 'DWLQJ WKH GLYHUVL¿FDWLRQ RI WKH PDMRU ARTICLE DNA from fungi, plants, protists and animals for PCR. lineages of Ascomycota (Fungi). PLoS One 8 H BioTechniques 15 ± Santamaría S (1998) Laboulbeniales, I. Laboulbenia. Flora Haelewaters D (2011) Laboulbeniales ([SORULQJ DQG WHVWLQJ '1$ Mycologica Iberica 4 ± extraction protocols, carrion beetle hosts and species in ‘De Santamaría S (2003) Laboulbeniales, II. Acompsomyces-Ilyomyces. Kaaistoep’ (Netherlands). Master thesis, Department of Biology, Flora Mycologica Iberica 5 ± *KHQW8QLYHUVLW\%HOJLXP 6DQWDPDUtD6(VSDGDOHU; Rickia lenoirii, a new ectoparasitic +DHOHZDWHUV ' &RPRQW 5) =KDR 6< 3¿VWHU '+ species, with comments on world Laboulbeniales associated Hesperomyces virescens (Fungi, Ascomycota, Laboulbeniales) with ants. Mycoscience 56 ± attacking Harmonia axyridis (Coleoptera, Coccinellidae) in its Scheloske H-W (1969) Beiträge zur Biologie, Ökologie und native range. 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Haelewaters D, Zhao SY, De Kesel A, Royer IR, Handlin RE, Farrell Sohrabi M, Myllys L, Stenroos S (2010) Successful DNA sequencing %' 3¿VWHU '+ E Laboulbeniales (Ascomycota) of the RID\HDUROGKHUEDULXPVSHFLPHQRIAspicilia aschabadensis %RVWRQ+DUERU,VODQGV, VSHFLHVSDUDVLWL]LQJCoccinellidae and -6WHLQHU 0HUHVFKNLichenologist 42 ± Staphylinidae. Northeastern Naturalist 22 ± 6SDWDIRUD-:6XQJ*+-RKQVRQ'+HVVH&2¶5RXUNH%et al. +DQVHQ.3HUU\%$'UDQJLQLV$:3¿VWHU'+ $SK\ORJHQ\ $¿YHJHQHSK\ORJHQ\RIPezizomycotina. Mycologia 98 of the highly diverse cup-fungus family Pyronemataceae 1018–1028. (Pezizomycetes, Ascomycota FODUL¿HV UHODWLRQVKLSV DQG 6WRHFNOH%&'ZRUVFKDN.*RVVQHU00.XHKQ5 ,QÀXHQFH evolution of selected life history traits. Molecular Phylogenetics of arthropod sampling solutions on insect genotyping reliability. DQG(YROXWLRQ 67 ± (QWRPRORJLD([SHULPHQWDOLVHW$SSOLFDWD 135 ± +DXJODQG5$+HFNPDQ-/:\PHU/- (YDOXDWLRQRIGLIIHUHQW Sugiyama K, Phanichapol D (1984) Laboulbeniomycetes methods for the extraction of DNA from fungal conidia by (Ascomycotina) in Thailand, I. Natural History Bulletin of the quantitative competitive PCR analysis. Journal of Microbiological Siam Society 31 ± Methods 37 ± Thaxter R (1896) Contribution towards a monograph of the +HQVRQ-0 '1$K\EULGL]DWLRQDQGSRO\PHUDVHFKDLQUHDFWLRQ Laboulbeniaceae. Memoirs of the American Academy of Arts and 3&5 WHVWVIRULGHQWL¿FDWLRQRIGaeumannomyces, Phialophora Sciences 12 ± and Magnaporthe isolates. Mycological Research 96 ± Thaxter R (1899) Diagnosis of new species of Laboulbeniaceae. I. +LEEHWW'6%LQGHU0%LVFKRII-)%ODFNZHOO0&DQQRQ3)et al. 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Thaxter R (1902) Preliminary diagnosis of new species of /HH 6% 7D\ORU -0 ,VRODWLRQ RI WRWDO '1$ IURP IXQJL IRU Laboulbeniaceae. V. Proceedings of the American Academy of DPSOL¿FDWLRQ E\ WKH SRO\PHUDVH FKDLQ UHDFWLRQ ,Q PCR Arts and Sciences 38 ± Protocols: a guide to methods and applications (Innis MA, 7KD[WHU 5 3UHOLPLQDU\ GLDJQRVHV RI QHZ VSHFLHV RI *HOIDQG'+6QLQVN\--:KLWH7-HGV ±6DQ'LHJR Laboulbeniaceae. VI. Proceedings of the American Academy of Academic Press. Arts and Sciences 41 ± Majewski T (1994) The Laboulbeniales of Poland. Polish Botanical Thaxter R (1926) Contribution towards a monograph of the Studies 7 ± Laboulbeniaceae. Part IV. Memoirs of the American Academy of 0LDGOLNRZVND-/XW]RQL) 3K\ORJHQHWLFUHYLVLRQRIWKHJHQXV Arts and Sciences 15 ± Peltigera (lichen-forming Ascomycota) based on morphological, 7KLHUV % ,QGH[ +HUEDULRUXP D JOREDO GLUHFWRU\ RI SXEOLF chemical, and large subunit nuclear ribosomal DNA data. KHUEDULD DQG DVVRFLDWHG VWDII 1HZ VOLUME 6 · NO. 2 371 Haelewaters et al. 9LOJDO\V 5 +HVWHU 0 5DSLG JHQHWLF LGHQWL¿FDWLRQ DQG Weir A, Hughes M (2002) The taxonomic status of Corethromyces PDSSLQJRIHQ]\PDWLFDOO\DPSOL¿HGULERVRPDO'1$IURPVHYHUDO bicolor from New Zealand, as inferred from morphological, Cryptococcus species. Journal of Bacteriology 172 ± developmental, and molecular studies. Mycologia 94 ± :DQJ<7UHWWHU('-RKQVRQ(0.DQGHO3/LFKWZDUGW5:1RYDN Whisler HC (1968) Experimental studies with a new species of 6- 6PLWK -) :KLWH 00 8VLQJ D ¿YHJHQH SK\ORJHQ\ Stigmatomyces (Laboulbeniales). Mycologia 60 ± ARTICLE to test morphology-based hypotheses of Smittium and allies, :KLWH 7- %UXQV 7' /HH 6% 7D\ORU -: $QDO\VLV RI endosymbiotic gut fungi (Harpellales) associated with arthropods. SK\ORJHQHWLFUHODWLRQVKLSVE\DPSOL¿FDWLRQDQGGLUHFWVHTXHQFLQJ 0ROHFXODU3K\ORJHQHWLFVDQG(YROXWLRQ 79 ± RIULERVRPDO51$JHQHV,Q PCR Protocols: a guide to methods :HLU$%HDNHV*: &RUUHODWLYHOLJKWDQGVFDQQLQJHOHFWURQ and applications ,QQLV0$*HOIDQG'+6QLQVN\--:KLWH7- microscope studies on the developmental morphology of HGV ±6DQ'LHJR $FDGHPLF3UHVV Hesperomyces virescens. Mycologia 88 ± Wrzosek M (2000) 7DNVRQRPLDL¿ORJHQH]D0XFRUDOHV =\JRP\FHWHV Weir A, Blackwell M (2001a) Molecular data support the Laboulbeniales Z ĞZLHWOH DQDOL] PRUIRPHWU\F]Q\FK RUD] Z\EUDQ\FK PDUNHUyZ as a separate class of Ascomycota, Laboulbeniomycetes. molekularnych. [Taxonomy and phylogeny of Mucorales Mycological Research 105 ± =\JRP\FHWHV LQ WKH OLJKW RI PRUSKRPHWULFDO DQG VHOHFWHG :HLU$ %ODFNZHOO 0 E ([WUDFWLRQ DQG 3&5 DPSOL¿FDWLRQ RI molecular markers analyses]. PhD thesis, University of Warsaw. DNA from minute ectoparasitic fungi. Mycologia 93 ± Weir A, Hammond PM (1997) Laboulbeniales RQ EHHWOHV KRVW utilization patterns and species richness of the parasites. Biodiversity and Conservation 6 ± 372 IMA FUNGUS