ARTICLE 213 ) of et al#$$K#$$' Key words: Acremonium Emericellopsis alkaline soils molecular phylogeny pH tolerance soda soils et al #$$Š 3 -clade et al. NJJŠ @ IMA FUNGUS · VOLUME 4 · NO 2: 213–228 NO 2: 4 · · VOLUME FUNGUS IMA et alNJ#*; Emericellopsis . 10), 34 acremonium-like ca. 10), 34 acremonium-like , and Elena N. Bilanenko , and Elena 1 such as high osmotic pressures, low water potentials, and, such as high osmotic pressures, low water Æ$ at pH above so-called alkaliphiles, with a growth optimum @ • 9, include prokaryotes (Duckworth 6$| `?`G is uncommon, while alkalitolerance, on the other hand, is far Alkalitolerant fungi, i.e. fungi that can grow more widespread. 6 interest for the molecular mechanisms of adaptation, but also in the search for potentially biotechnologically valuable enzymes. It has become more obvious that alkalitolerant fungi may be encountered in many neutral soils (Kladwang et al. 2003, Elíades of alkalitolerant fungi has facilitated studies on both their And yet, truly biodiversity and their enzymatic properties. @ 6 alkaliphilic fungi show a bias towards fungi with simple et al. sp. nov. Previous studies showed two distinct ecological Previous studies showed two distinct sp. nov. rans-Baikal regions (Russia), the Aral lake (Kazakhstan), Aral the rans-Baikal regions (Russia), , Alfons J.M. Debets , ). On an alkaline medium (pH ). On an alkaline medium 2 lineage (Bionectriaceae lineage Emericellopsis E. alkalina obtained from the same type of soils but which showed a narrower obtained from the same type of soils but which 6 4+` 1 Emericellopsis et alNJJ' , one consisting of terrestrial isolates and one predominantly marine. Remarkably, all predominantly marine. Remarkably, , one consisting of terrestrial isolates and one , described here as

lineage. We tested the capacities of all newly isolated strains, and the few available reference tested the capacities of all newly isolated lineage. We Emericellopsis et al. 2009). In Russia, alkaline soils are mostly Surveying the fungi of alkaline soils in Siberia, T alkaline soils in Siberia, Surveying the fungi of

Emericellopsis Eurotiales *PJM*KJ L+ Naturally, high salts concentration and high environmental Naturally, `‘|;›;4"##$JN#4› <x+4"_#?#N+`##$N*P4› + ` ~ " `  _ „ # ŠJ'~x  @ |> marine origin? marine Abstract: species representing the we report an abundance of alkalitolerant and Eastern Mongolia, ?I6G‚ cluster of fungi (order Hypocreales within the Acremonium †6 of the genus Emericellopsis clades within 6+"_""_„|;›~xN @œ<#?!?‘@"@ ? in the 6?@ Whereas every newly isolated strain from soda soils in growth rate as well as in pH preference. showed differences 6PM##NThe growth showed moderate and no alkalitolerance, respectively. reference marine-borne and terrestrial strains pattern of the alkalitolerant Article info:"IN¤NJ#*>;IN*†NJ#*>~IK|NJ#* unrelated alkaliphilic Sodiomyces alkalinus, preference towards high pH. © 2013 International Mycological Association © 2013 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: 2 3 1 ;6;`?`G Are alkalitolerant fungi of the fungi of the Are alkalitolerant Non-commercial:No derivative works:Any of the above conditions can be waived if you get For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. (Buchalo #$$'"66 Tanzania the Magadi Lake in Kenya and the Natron Lake in Seventy where the pH values of water are as high as 11–12. The Dead Sea in Israel, almost fungi have been isolated from half restricted to areas adjacent to saline lake basins in south- western Siberia (Sorokin Alkaline soils (or soda soils) and soda lakes represent a unique Alkaline soils (or soda soils) and soda lakes There are few studies available on the environmental niche. The eye-catching characteristic of fungal biodiversity therein. these soils is a high pH maintained mainly by the buffering capacities of soluble carbonates present. Soda accumulation is thought to be a common process associated with savannas, steppes and desert regions across the world (Jones INTRODUCTION I#JKK$'LNJ#*JPJNJ VOLUME 4 · NO. 2 pH impose a substantial amount of stress to any living organism. Some have adapted and therefore evolved metabolic pathways in order to thrive in such harsh conditions, Grum-Grzhimaylo et al. ARTICLE chloride-sulfate sulphate sulfate-soda chloride-sulfate chloride soda-chloride-sulfate soda soda chloride chloride-sulfate soda-chloride-sulfate chloride-sulfate soda-chloride chloride-sulfate soda soda chloride soda soda soda soda sulfate-soda soda soda soda-chloride sulfate-soda L. soda MB. soda - 139.4 - 47 100 33 57 #' 225 100 53 137 *' 310 73 ŠK ŠJ 7.1 1.9 1.9 139.4 73 310 137 139 10.1 11 $Š 9.5 taken from Salicornia europaea Kuchiger area 9 Sulfatnoe Lake 10.3 Cape AktumsykCape ' Bezimyannoe Lake 9.1 Mirabilit Lake $Š Burd Lake Solyonoe Lake 10 - Mirabilit Lake $Š Shukurtuz Lake 9.9 Zheltir’ Lake $Š Bezimyannoe Lake 10.1 Bezimyannoe Lake 9.9 Tanatar LakeTanatar 10.2 - - south, Berdabay 10.1 Nuhe-Nur Lake 10.1 Nuhe-Nur Lake 10.1 Nuhe-Nur Lake 10.1 Sulfatnoe Lake 10.3 Tanatar LakeTanatar 10.1 Bezimyannoe Lake 9.9 Zheltir’ Lake $Š Sulfatnoe Lake 10.3 Trans-Baikal, Russia Trans-Baikal, Trans-Baikal, Russia Trans-Baikal, Aral lake, Kazakhstan Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, North-East Mongolia Choibalsan area, North-East Mongolia - Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Kulunda steppe, Altai, Russia Kulunda steppe, Trans-Baikal, Russia Trans-Baikal, Trans-Baikal, Russia Trans-Baikal, Trans-Baikal, Russia Trans-Baikal, Trans-Baikal, Russia Trans-Baikal, ------CBS 120044Altai, Russia Kulunda steppe, -3040 - - Russia Trans-Baikal, Alla River near ' - sulfate - - Russia Trans-Baikal, Alla River near ' - sulfate - -Altai, Russia Kulunda steppe, - taken from Atriplex verrucifera - - Russia Trans-Baikal, Orongoyskoe Lake ' NŠ soda-sulfate - - Russia Trans-Baikal, Sulfatnoe Lake 'K 3.7 sulfate-soda - - Russia Trans-Baikal, Chedder Lake 9.1 - soda - - Aral lake, KazakhstanAktumsyk Cape taken from Sueda salsa ------<?#PŠ - FW-1473 - FW-1474 - - FW-1471 ------

214 IMA FUNGUS Are alkalitolerant fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin?

6 Acremonium or ARTICLE Verticillium species, and typically, without the development 6 † et al. 1993, Kladwang et al. NJJ* " Acremonium species imposed by their simple morphology have stimulated @ array of fungi with acremonium-like conidiation has been shown to be highly polyphyletic, occupying several lineages throughout Ascomycota (Summerbell et al. 2011). However, most Acremonium species belong to Hypocreales (subphylum Hypocreomycetidae † ? within the hypocrealean acremonia is the Emericellopsis- clade (family Bionectriaceae), which includes isolates derived from various ecological niches. Notably, previous studies have shown a phylogenetic separation of marine-derived and terrestrial isolates within the Emericellopsis-clade (Zuccaro et al. 2004). The marine clade also contains fungi derived @ relationships between marine-borne and soda soil fungi of the genus Emericellopsis. Here, we analyse acremonium- like strains isolated from soda soils in western Siberia, the Trans-Baikal area (Russia), the Aral Sea (Kazakhstan) and `„4 relationships, with an emphasis on the Emericellopsis- clade. A new Emericellopsis species, E. alkalina sp. nov., is described. We also analysed the newly isolated strains for sea water - - - sea water - - - growth at various pH values, in comparison with reference 6? group within the known Emericellopsis isolates originated from the marine habitats. We discuss a possible origin of alkalitolerance in this particular lineage of mostly sea-borne fungi.

MATERIALS AND METHODS

Soil samples, strains and media Soil samples were collected from several locations on the Ukraine Ukraine edge of the soda lakes (Table 1). We used alkaline agar (AA) N L+ for alkalitolerant species isolation. For routine subculturing on AA of the newly isolated strains, the antibiotic was not used. The AA medium was prepared as described previously `?`G et al NJ#* " 6? Emericellopsis strains were obtained from the KNAW-CBS Fungal Biodiversity Centre (CBS) as well as from the All- Russian Collection of Microorganisms (VKM). For the colony morphology characterization we used several types of media: F-1057 /x"'#Š' ` - - - - - Aral lake, KazakhstanAktumsyk Cape '* - chloride-sulfate WA, CZ, MYA, PDA, OA and AA (Mueller et al. 2004). The elucidation of the pH optimum was performed in duplicate using race tubes with the media ranging in pH as described `?`Get al. 2013), with the following

T

Morphology We used light microscopy (LM) and scanning electron Table 1. (Continued). Table Strain VKM number CBS no. Isolation area Isolation place pH of the soil salts (g/kg) Total +" Emericellopsis maritima Emericellopsis minima Emericellopsis minima Emericellopsis pallida Sarocladium sp. A131 microscopy (SEM) for morphological characterization of the

VOLUME 4 · NO. 2 215 Grum-Grzhimaylo et al.

Table 2. Loci and substitution models used for the phylogenetic analyses. Phylogenetic analysis Locus Model for each partition Characters Informative Uninformative Invariable characters variable characters characters 1 LSU @‘4#őÅ``@›Å‘Å`Ê $ŠN #ŠN ŠN *' 2 ITS @‘4#Å``@›Å‘Å`Ê 503 101 ŠN 340

ARTICLE !? @|Å`“Å`Ê 333 'J 29 224 RPB2 @‘4*Å``@›Å`Ê 1070 73 134 'Š* @œ<#? @‘4*Å``@›Å`Ê 904 54 K' 792 * - for MrBayes.

`?`G et al. MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001). Two 2013). independent searches and four chains were set to run for 10 M generations for both phylogenetic analyses sampling DNA extraction, PCR, and sequencing every 100th generation. The convergence of the runs was @ „|; „|; 6 checked in TRACER v. 1.5 (Rambaut & Drummond 2007). „|~4š//; @*J’KJ’? 6 trees was eliminated from the further analysis. The rest subunit rDNA, internal transcribed spacers 1 and 2, including were used to generate a 50 % majority rule consensus tree K'"„|;›~xN@œ<#?!?„|; and calculate posterior probabilities (PP). The consensus standard primers set. Primer sets, thermo cycling programs G @` NJP? and sequencing procedures were performed as described NJŠ"!4NJ#J;‘/"Š `?`Get alNJ#*@ (Adobe Systems, San Jose, CA). The node supports were ?*O!?P •4+Æ$J in Zuccaro et al. (2004). ~~ÆJ$P| `x Phylogenetic analyses Table 3. Phylogenetic analyses were deposited in TreeBase  I ‘„#P#$Š „|;+"_‘@"›~xN@œ<#?!? The gene for small subunit rRNA (SSU), although sequenced, was not included in our phylogenetic reconstructions since RESULTS it carried too little phylogenetic signal to contribute to clade differentiation. We constructed separate alignments for each Isolated strains of the analysed genes using the online MAFFT v. 7 service On the selective AA medium buffered at pH 10 and containing (Katoh & Standley 2013). Ambiguous regions were removed *P manually from the alignments with BioEdit v. 7.1.3.0 (Hall soda soils adjacent to the soda lake basins. Several of the 1999). Two data sets for different phylogenetic analyses isolated strains were deposited in CBS and VKM. All strains were constructed in order to achieve different degrees of 6 ? resolution within the studied groups. Appropriate reference 6 `x@ was found to be a new species of the Emericellopsis lineage included a single LSU gene in order to build a large-scale based on molecular, morphological and growth data (see 6@? below). ‘@"!?›~xN@œ<#?? 6 Molecular phylogenetic analyses relationships in the Emericellopsis-clade and our newly @ isolated alkalitolerant strains. The four-gene concatenated +"_ $ŠN #ŠN # ’ data set was constructed using Mesquite v. 2.75 (Maddison & being phylogenetically informative (Table 2). The negative Maddison 2011) and divided into four partitions corresponding log likelihoods (-Ln) of the ML and BI consensus trees @ ? PŠ$ŠJ* K###'N @ substitution for each partition was chosen according to the reconstruction based on LSU sequences of our isolates from corrected Akaike Information Criterion (AICc) as implemented soda lakes along with the pertinent reference sequences •4@ N## ` ! ` NJJ* „ from hypocrealean acremonia is consistent with the topology et al NJ#N @ N `;›+‘ NJ } NJJŠ described by Summerbell et al. (2011), hence we follow 46 + 4+ > the clade delineation outlined in that study. As seen in Fig. both phylogenetic analyses the number of searches was #6 NJJ ; KJ 6 $L#J % majority rule consensus trees were constructed using Emericellopsis-clade (Bionectriaceae). This clade is known SumTrees v. 3.3.1 application within DendroPy v. 3.11.0 to include marine-borne fungi such as Acremonium fuci, package (Sukumaran & Holder 2010) running under Python A. tubakii, E. maritima, as well as terrestrial isolates like NŠxx‘ E. terricola, some Stilbella species, and the Stanjemonium

216 IMA FUNGUS Are alkalitolerant fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin?

@G?6??A. exuviarum ARTICLE (UAMH 9995), producing chains of conidia, has been shown UAMH 6508 CBS 190.55 T Emericellopsis-clade (Sigler et al. CBS 491.71 T CBS 490.71 T 2004). Thirty of our new isolates in the Emericellopsis-clade CBS 379.70F Emericellopsis alkalina A103 ŠLJ$$ Acremonium sp. A104 Acremonium sp. A105 6?E. minima (CBS 190.55), Acremonium sp. A106 Acremonium sp. A107 E. maritima (CBS 491.71), and E. pallida (CBS 490.71), Acremonium sp. A108 as well as “A. potroniiP /x" *$J<> Acremonium sp. A110 Acremonium sp. A111 a single isolate of an undescribed species that has so far Emericellopsis alkalina A112 Emericellopsis alkalina A113 = FW-1476 only been isolated from a dolphin skin lesion, apparently not Emericellopsis alkalina A114 = FW-1473 Emericellopsis alkalina A115 = FW-1474 as an agent of infection (Zuccaro et al. 2004). The marine Emericellopsis alkalina A116 Emericellopsis alkalina A117 = FW-1471 species from Fucus, a brown seaweed, A. fuci_;4ŠKJ' Emericellopsis alkalina A118 Emericellopsis alkalina A119 also grouped with our isolates from soda soils. There is not Emericellopsis alkalina A120 Emericellopsis enough phylogenetic signal from our LSU-based phylogenetic Emericellopsis alkalina A121 -clade Emericellopsis alkalina A122 reconstruction to resolve the Emericellopsis-clade further. Emericellopsis alkalina A123 Emericellopsis alkalina A124 Four new isolates from soda soils appeared to be in the sister Emericellopsis alkalina A125 Emericellopsis alkalina A126 clades, namely, two in the sclerotigenum-clade, one in the Emericellopsis alkalina A127 Emericellopsis alkalina A128 Sarocladium-clade and one in the -clade. They are Emericellopsis alkalina M14 = CBS 120043 Emericellopsis alkalina M20 = CBS 120044 Emericellopsis alkalina M71 = CBS 120049 Emericellopsis alkalina E101 = CBS 127350 T The second phylogenetic analysis included partial Acremonium sp. E102 ‘@"!?›~xN@œ<#? NRRL 26548 CBS 721.71 T to have a higher mutation rate than LSU. We sampled a DAOM 229279 CBS 790.69 T 6?6@ CBS 824.69 CBS 120.40 T sequences for the Emericellopsis-clade had a high degree CBS 229.59 CBS 871.68 of similarity, and were easily aligned and edited. The most CCFC226707 UAMH 9995 T !? CBS 124.42 T CBS 786.69 A101 the reliability of the resulting tree. The alignment for this A130 -clade CBS 385.73 T N '#J *J' ## ’ CBS 407.66 T CBS 117.25 T phylogenetically informative (Table 2). The MCMC runs in CBS 682.71 T T Bayesian analysis reached stationary status with a deviation CBS 866.73 brachypenium CBS 189.70 -clade CBS 149.62 T JJJ'K4@ IFO 7729 ?+4+x‘'P''# IFO 31131 CBS 596.70 T 'ŠPK'K T CBS 144.62 ATCC 22087 -clade The tree that was generated for the Emericellopsis- ATCC 22102 ATCC 22107 <N AR 2750 CBS 346.85 the Emericellopsis-clade is deeply resolved, displaying CBS 201.35 CBS 397.67 several major clades consistent with the previous study by CBS 310.59 T Zuccaro et al. (2004). The basal group consists of a highly CBS 989.69 T persicinum CBS 102349 -clade 6 "tanjemonium 6 Stilbella CBS 101694 ATCC 36093 ?6? CBS 750.69 T CBS 794.69 T of E. synnematicola, /x"#ŠŠJE. salmosynnemata, CBS 157.72 CBS 134.56 T -clade /x"*'NŠN@6?Acremonium exuviarum, CBS 181.27 T CBS 396.66 T mentioned earlier, seems to be more distally basal to the rest CBS 425.67 T † CBS 796.69 T CBS 428.67 T the presence of the two ecological groups in the Emericellopsis CBS 749.69 T Sarocladium CBS 346.70 T lineage, both of which were supported by the molecular CBS 399.73 T -clade CBS 180.74 studies. The clades designated as marine (M) and terrestrial CBS 122.29 T CBS 801.69 T (T), outlined previously by Zuccaro et al. (2004), also appear Sarocladium sp. A131 T @ @ $'L#J CBS 150.62 breve CBS 142.62 T -clade CBS 726.71 T T CBS 430.66 curvulum-clade CBS 229.75 CBS 770.84 T CBS 212.69 CBS 439.70 -clade Fig. 1. Phylogenetic reconstruction of Acremonium species in CBS 289.62 T Acremonium sp. A109 Bionectriaceae as inferred from the partial LSU gene sequences. ATCC 18873 CBS 646.77 |6/ AR 2824 CBS 110115 delineation is from Summerbell et al. (2011). Bayesian topology with BBA 70749 4+L~~@ ATCC 9182 FAU 553 4+Æ$J~~ÆJ$PT – subs./site L6?

VOLUME 4 · NO. 2 217 Grum-Grzhimaylo et al.

Table 3. +6"6 bold. Taxon Voucher Appearance in LSU ITS !%< RPB2 =#$'% SSU phylogenetic analysis (1,2) Acremonium acutatum T /x"Š'N# 1 šN*#$ŠK ARTICLE Acremonium alternatum T /x"PJŠŠ 1 šN*#$'' Acremonium biseptum T /x"KJŠ$ 1 šN*#$$' Acremonium brachypenium T /x"'ŠŠ* 1 šN*NJJP Acremonium breve T /x"#KJŠN 1 šN*NJJK Acremonium chrysogenum T /x"#PPŠN 1 šN*NJ# Acremonium curvulum T /x"P*JŠŠ 1 šN*NJNŠ Acremonium curvulum CBS 229.75 1 šN*NJN# Acremonium exuviarum T UAMH 9995 1,2 šN*NJ*Š ;''N$PŠ ;''N$P - - 3! T /x"K$ŠJ 1 šN*NJ* Acremonium fuci T /x"##N'Š' 2 ;Š*NŠK* ;Š*NŠ$J - - Acremonium fuci /x"##*''$ 2 ;Š*NŠKN - - - Acremonium fuci _;4ŠKJ' 1 šN*NJ*' Acremonium gamsii T /x"NŠ# 1 šN*NJPJ 3 T /x"N#NŠ$ 1 šN*NJKJ 3 CBS 439.70 1 šN*NJK# Acremonium persicinum T CBS 310.59 1 šN*NJ Acremonium persicinum /x"#J#Š$P 1 šN*NJ'K Acremonium persicinum CBS 102349 1 šN*NJ'Š “Acremonium potronii” /x"#'$J 1 šN*NJ$P “Acremonium potronii” CBS 379.70F 1,2 šN*NJ$Š ;Š*NŠKK ;Š*NŠ$# - - Acremonium radiatum T /x"#PNŠN 1 šN*N#JP Acremonium roseolum T /x"N'$ŠN 1 šN*N#N* Acremonium rutilum T /x"*$ŠŠŠ 1 šN*N#NP Acremonium salmoneum T CBS 721.71 1 šN*N#NK Acremonium salmoneum JS-NJ01 2 4P#ŠN - - - Acremonium sclerotigenum T CBS 124.42 1 šN*N#NŠ Acremonium sclerotigenum 1 KC987215 KC987139 KC987101 KC998999 KC998961 KC987177 A101 Acremonium sclerotigenum 1 KC987242 KC987166 KC987128 KC999024 KC998988 KC987204 A130 Acremonium sclerotigenum /x"'ŠŠ$ 1 šN*N#*J Acremonium sordidulum T /x"*'K* 1 šN*N#*Š Acremonium sp. A104 1,2 KC987217 KC987141 KC987103 KC999001 KC998963 KC987179 Acremonium sp. A105 1,2 KC987218 KC987142 KC987104 KC999002 KC998964 KC987180 Acremonium sp. A106 1,2 KC987219 KC987143 KC987105 KC999003 KC998965 KC987181 Acremonium sp. A107 1,2 KC987220 KC987144 KC987106 KC999004 KC998966 KC987182 Acremonium sp. A108 1,2 KC987221 KC987145 KC987107 KC999005 KC998967 KC987183 Acremonium sp. A109 1 KC987222 KC987146 KC987108 KC999006 KC998968 KC987184 Acremonium sp. A110 1,2 KC987223 KC987147 KC987109 KC999007 KC998969 KC987185 Acremonium sp. A111 1,2 KC987224 KC987148 KC987110 KC999008 KC998970 KC987186 Acremonium sp. E102 1,2 KC987248 KC987172 KC987134 KC999030 KC998994 KC987210 Acremonium tubakii T /x"$JŠ$ 1 šN*N#P' Acremonium tubakii /x"###*ŠJ 2 ;Š*NŠKP ;Š*NŠ'$ - - “Acremonium tubakii” /x"'NPŠ$ 1 šN*N#P$ Acremonium verruculosum T /x"$'$Š$ 1 šN*N#KJ “Cephalosporium malorum” T CBS 117.25 1 šN*NJ#K “Cephalosporium purpurascens” T /x"#P$ŠN 1 šN*NJ#

218 IMA FUNGUS Are alkalitolerant fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin?

Table 3. (Continued). ARTICLE Taxon Voucher Appearance in LSU ITS !%< RPB2 =#$'% SSU phylogenetic analysis (1,2) 4#'' ;›N'NP 1 ;P'$JŠ Emericellopsis alkalina A103 1,2 KC987216 KC987140 KC987102 KC999000 KC998962 KC987178 Emericellopsis alkalina A112 1,2 KC987225 KC987149 KC987111 KC999009 KC998971 KC987187 Emericellopsis alkalina A113 <?#PŠ 1,2 KC987226 KC987150 KC987112 KC999010 KC998972 KC987188 Emericellopsis alkalina A114 FW-1473 1,2 KC987227 KC987151 KC987113 KC999011 KC998973 KC987189 Emericellopsis alkalina A115 FW-1474 1,2 KC987228 KC987152 KC987114 KC999012 KC998974 KC987190 Emericellopsis alkalina A116 1,2 KC987229 KC987153 KC987115 - KC998975 KC987191 Emericellopsis alkalina A117 FW-1471 1,2 KC987230 KC987154 KC987116 KC999013 KC998976 KC987192 Emericellopsis alkalina A118 1,2 KC987231 KC987155 KC987117 KC999014 KC998977 KC987193 Emericellopsis alkalina A119 1,2 KC987232 KC987156 KC987118 KC999015 KC998978 KC987194 Emericellopsis alkalina A120 1,2 KC987233 KC987157 KC987119 KC999016 KC998979 KC987195 Emericellopsis alkalina A121 1,2 KC987234 KC987158 KC987120 KC999017 KC998980 KC987196 Emericellopsis alkalina A122 1,2 KC987235 KC987159 KC987121 KC999018 KC998981 KC987197 Emericellopsis alkalina A123 1,2 KC987236 KC987160 KC987122 KC999019 KC998982 KC987198 Emericellopsis alkalina A124 1,2 KC987237 KC987161 KC987123 KC999020 KC998983 KC987199 Emericellopsis alkalina A125 1,2 KC987238 KC987162 KC987124 KC999021 KC998984 KC987200 Emericellopsis alkalina A126 1,2 KC987239 KC987163 KC987125 KC999022 KC998985 KC987201 Emericellopsis alkalina A127 1,2 KC987240 KC987164 KC987126 - KC998986 KC987202 Emericellopsis alkalina A128 1,2 KC987241 KC987165 KC987127 KC999023 KC998987 KC987203 Emericellopsis alkalina E101 T CBS 127350 1,2 KC987247 KC987171 KC987133 KC999029 KC998993 KC987209 (=VKM

VOLUME 4 · NO. 2 219 Grum-Grzhimaylo et al.

Table 3. (Continued). Taxon Voucher Appearance in LSU ITS !%< RPB2 =#$'% SSU phylogenetic analysis (1,2) Emericellopsis stolkiae T CBS 159.71 2 ;Š*NŠŠ' ;Š*NŠ'P - -

ARTICLE Emericellopsis synnematicola T /x"#ŠŠJ 2 ;Š*NŠŠK ;Š*NŠ'# - - Emericellopsis terricola T CBS 120.40 1,2 _KJ'N _KŠŠ - - - Emericellopsis terricola CBS 229.59 1,2 AY305034 ;Š*NŠŠN ;Š*NŠ' - - Emericellopsis terricola //<*'#K 2 FJ430737 - - - Emericellopsis terricola NRRL 54109 2 šŠ$'K$N - - - Geosmithia lavendula IFO 7729 1 „''*NK Geosmithia putterillii IFO 31131 1 AB047215 Gliomastix masseei T /x"$PŠ$ 1 šN*NJŠJ Gliomastix murorum CBS 157.72 1 šN*NJŠ Gliomastix polychroma T /x"#'#N 1 šN*NJ$# Gliomastix roseogrisea T /x"#*PKŠ 1 šN*N#N# Glomerella cingulata FAU 553 1 ;Heleococcum japonense /x"*$Š 1 ¤#K'PP# Hydropisphaera erubescens ;@//*ŠJ$* 1 ;KPKNŠ Melanopsamma pomiformis ;@//#''* 1 ;P'$J$ Mycoarachis inversa ATCC 22107 1 `šKJK$$# Mycopepon smithii "4#ŠJ$ 1 AF279400 Myrothecium verrucaria BBA 70749 1 AJ301999 Nigrosabulum globosum ATCC 22102 1 ;Roumegueriella rufula /x"*PŠ'K 1 „šK#'Š Sarocladium attenuatum T CBS 399.73 1 šN*N#ŠK Sarocladium bacillisporum T /x"PNKŠ 1 šN*#$$N Sarocladium bactrocephalum T /x"P$Š$ 1 šN*#$$P Sarocladium glaucum T /x"$ŠŠ$ 1 šN*NJP# Sarocladium kiliense T CBS 122.29 1 šN*NJKN Sarocladium ochraceum T /x"PN'Š 1 šN*NJJ Sarocladium oryzae /x"#'JP 1 šN*N#ŠŠ Sarocladium sp. A131 1 KC987243 KC987167 KC987129 KC999025 KC998989 KC987205 Sarocladium strictum T /x"*PŠJ 1 šN*N#P# Sarocladium zeae T /x"'J#Š$ 1 šN*N#KN Scopinella solani /x"J'P 1 ;J#KŠ*N Selinia pulchra AR 2750 1 ;<#$*NPŠ Selinia pulchra ;›N'#N 2 4P'P'K$ 4P'P''P - 4P'P'P# Stachybotrys chartarum ;@//$#'N 1 ;P'$#P Stanjemonium grisellum |››+NŠKP' 1 AF049171 Stanjemonium grisellum T /x"ŠKK$ 2 ;Š*NŠ# ;Š*NŠ' - - Stanjemonium ochroroseum T /x"ŠKŠ$ 2 ;Š*NŠN ;Š*NŠ'' - - ' „$$JNŠ 2 ;$KNPŠ - - - ' DAOM 1 šN*N#Š 229279 ' 4#' 2 FJ430712 - - - ' SES201 2 FJ939394 - - - Verrucostoma freycinetiae T MAFF 2 4P'P'ŠŠ 4P'P''K - 4P'P'K* 240100

220 IMA FUNGUS Are alkalitolerant fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin?

AR 2812

((% ARTICLE +('' T pH 10 Emericellopsis alkalina A103 Emericellopsis alkalina A116 Emericellopsis alkalina A117 = FW-1471 Emericellopsis alkalina A119 Emericellopsis alkalina A120 Emericellopsis alkalina A121 Emericellopsis alkalina A122 Emericellopsis alkalina A123 Emericellopsis alkalina A124 Emericellopsis alkalina E101 = CBS 127350 T Emericellopsis alkalina A112 82/1.0 Soda soils clade Emericellopsis alkalina A113 = FW-1476 %R Emericellopsis alkalina A114 = FW-1473 Emericellopsis alkalina A115 = FW-1474 Emericellopsis alkalina A125 Emericellopsis alkalina A126

-/0.5 Emericellopsis alkalina A127 Emericellopsis alkalina A128 Emericellopsis alkalina M14 = CBS 120043 Emericellopsis alkalina M71 = CBS 120049 98/0.83 Emericellopsis alkalina A118 Emericellopsis alkalina M20 = CBS 120044 Acremonium sp. A111 Acremonium sp. A105

CBS 113889 76/1.0 $%& T

63/1.0 $%& T T 81/1.0 $%& CBS 190.55 T 59/0.98 Acremonium sp. A108

“ ” $%&' Marine (M) clade 89/1.0 %R 57/1.0 Acremonium sp. A104 Acremonium sp. E102

$%& T

57/1.0 $%& Acremonium sp. A106 Acremonium sp. A107 Acremonium sp. A110

CBS 120.40 T 65/0.92 $$' CBS 229.59 -/0.53 $%& $%& T $%& 88/1.0 $%& -/0.58 Terrestrial (T) clade NRRL 54109 % 53/0.7 $%& () CBS 119.40 T $%& -clade $%& T T 56/1.0 $%& $%& T $%& T $%& T

Emericellopsis 59/0.87 $%& * + SES201

0.0 0.1 UAMH 9995 T subs./site ,&R-,

Fig. 2. Four-gene phylogeny of the new alkalitolerant isolates within the Emericellopsis? ‘@"K'" „|;!?›~xN@œ<#?;x4+L~~ @4+Æ$J~~ÆJ$PTML6?› delineated clade are shown on AA medium plates (11-d-old).

VOLUME 4 · NO. 2 221 Grum-Grzhimaylo et al.

6 A

5 E. alkalina E101 = CBS 127350 T

E. minima CBS 190.55 T 4 CBS 491.71 T

Acremonium sp. A105 3

ARTICLE Acremonium sp. A111 2 E. pallida CBS 490.71 T

linear growth rate (mm/d)linear growth 1

E. minima CBS 871.68 0 4567891011 pH

6 B

5

4

3 Fig. 3.` Sarocladium . sp A131 strains at pH 4 through 11.2 based on MYA 2 A101 A130 medium. A. strains from the T, M and soda Acremonium sp. A109 linear growth rate (mm/d) rate growth linear soils clades within the Emericellopsis 1 lineage including intermediate Acremonium 0 sp. ;#JK ;###> B. isolated 4567891011 alkalitolerant strains from the sister clade of pH the Emericellopsis lineage.

6 Emericellopsis, but were able to tolerate higher pH values. Identical growth such as E. robusta, E. terricola, and E. microspora. There patterns were seen in our strains Acremonium sp. A104, 6 E. donezkii /x" P'$# E. ;#JK;#JŠ;#J;#J';##J;### œ#JN minima/x"###*Š#A. tubakii/x"###*ŠJ shown) which also fall into the M clade. Two strains (A105 found in aquatic environments. The very weakly supported M and A111) seem to be paraphyletic to the M clade, but based KL#J on their growth patterns they belong to the M clade (dashed 6E. pallida CBS line). Members of the M clade grew faster than E. minima ŠNP* 6? E. minima, CBS 190.55. /x"'#Š'@;E. alkalina Interestingly, eight of our new isolates from the soda soils fall 6;## into the M clade while the majority (22 strains) form a well- preference) showed a higher growth rate than that seen in 'NL#JO the members of the M and T clades. They had a broad pH P‘NN optimum in the 7–11 range, and displayed a wide tolerance represent a new species named E. alkalina sp. nov. here. Of across the pH scale. those 22 strains, one formed ascomata, while the others only Isolates Acremonium sp. A109, A. sclerotigenum A101, 6@ A130 and Sarocladium sp. A131, which fall into a sister-clade /x"#N*KJ6 to the Emericellopsis-clade, had an overall slow growth rate we derived the type of E. alkalina. with a slight preference for neutral pH combined with the SSU sequences showed almost no variation among our ability to tolerate higher pH values. This pattern somewhat newly isolated strains in the Emericellopsis-clade. We found resembled that seen in the M clade (Fig. 3B). #Š*

Growth patterns TAXONOMY In order to link our phylogenetic data to ecological 6 Emericellopsis alkalinax!`sp. growth ability of all studied strains at different ambient pH nov. values. As seen in Fig. 3A, the pH preferences vary among 4x4x'JPKN the members of the different clades within the Emericellopsis (Figs 4–5) lineage. A reference member of the T clade, E. minima (CBS '#Š' Š Etymology: Epithet taken from the ability to grow at high with no ability to cope with both lower and higher pH values. ambient pH. @46? of E. maritima (CBS 491.71), E. minima (CBS 190.55), and Diagnosis: Asci saccate, 12–15 μm long, unitunicate. E. pallida/x"P$J#ŠM Ascospores ellipsoid, pale brown, with uneven surfaces,

222 IMA FUNGUS Are alkalitolerant fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin?

4.5–5.5 × 2.5–3.0 μm, surrounded by 3, but frequently 5 ARTICLE longitudinal, subhyaline, smooth-edged alar appendages, \@E. pallida6?/x" width up to 1.0 μm. Asexual morph acremonium-like. 490.71 is thinner, 1–2 layered. The ascospore morphology of the type of E. alkalina (CBS 127350) looks similar to that of Type: Russia: Altai, Kulunda steppe, soda soil (total salts E. pallida and E. minima. However, E. alkalina ascospores 73 g kg-1, pH 10.1) on the edge of the basin of Tanatar Lake, have an uneven surface with (3–)5 alar appendages, while E. August 2002, D. Sorokin/x"?N#P#NM> pallida6?/x"P$J# 6?œ#J#£/x"#N*KJ£D“4 x ! † #$Š# D x #$*$MPJ Culture characteristics: Colonies on alkaline agar (AA, pH E. terricola based on an isolate from soil collected near the #JJM#JN ? JM'J #J town of Baarn in The Netherlands. The generic name came NK /†4œ;ŠK*NM from the close morphological resemblance of the ascospore *' #J / ?? ornamentation to that of Emericella nidulans, which was darkening in centre due to the formation of ascomata with 6" tufted aerial mycelium sometimes forming concentric zones studies described additional soil-borne Emericellopsis 6›œ6 "#$KK` Decumbent vegetative hyphae thin-walled, hyaline, 0.5–2.0 #$K4!@#$ŠJ#$ŠNx!† ) 4 ? #$Š#;#$ŠJ #M*) ; G constituted the major criteria used to distinguish species Additional specimens examined: A103, A112, A113 (= VKM FW- (Durrell 1959). #PŠ;##P£D“4<?#P*;##K£D“4<?#PP;##Š;## The beginning of the 1970s marked a new period in the £ D“4 <?#P# ;##' ;##$ ;#NJ ;#N# ;#NN ;#N* ;#NP study of Emericellopsis with the establishment of marine ;#NK ;#NŠ ;#N ;#N' 4#P £ D“4

VOLUME 4 · NO. 2 223 Grum-Grzhimaylo et al. ARTICLE

Fig. 4. Emericellopsis alkalina (CBS 127350). A–E.##??N' /$~;;/G /}6~„;†;64;F–G. Hyphal bundles with acremonium-like conidiation (SEM). H. Conidiogeous cells emerging from single hypha (SEM). I. Conidial head on a single conidiogenous cell emerging from the hyphal bundle (SEM). J. Matured conidial heads (SEM). K. Single conidiogenous cell with young conidial head (SEM). L. Conidial head (LM). M. Conidia "œ4x¤+£#J)>‘“£K)>4£N)

224 IMA FUNGUS Are alkalitolerant fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin? ARTICLE

Fig. 5. Emericellopsis alkalina (CBS 127350). A. Cleistothecia (SEM). B./6"œ4C. Open cleistothecium (SEM). D.4"œ4E. Open cleistothecium (LM). F. Young asci (LM). G. Young asci (SEM). H–J. Lysing asci (SEM). K–M.;"œ4xI;œ£#JJ)>x£NJ)>/£#J)>„‘4£N)>“M+£#)

VOLUME 4 · NO. 2 225 Grum-Grzhimaylo et al.

At the moment, Emericellopsis comprises homothallic elevated ambient pH values. As far as we know, however, saprobic cleistothecial species with acremonium-like such an ecological overlap has not been demonstrated for >E. synnematicola, also forms stilbella- other marine fungal lineages. To address this issue, we need like synnemata. However, different authors accept different systematic biodiversity research on the fungi from soda lakes. /#Š @!?6

ARTICLE four varieties are listed in the MycoBank database (Crous et to the phylogenetic signal in our study suggests a relatively al. 2004). All authors have so far supported the opinion that the recent divergence of E. alkalina from the M clade. Our main distinguishing features among species are the morphology Acremonium sp. strains A105 and A111 seem to be of the ascospores and their alar appendages. Molecular intermediate isolates situated in a statistically ambiguous studies conducted in the late 1990s placed Emericellopsis in position between the alkaline and marine lineages. The Hypocreales ` et al #$$Š; ""_ +"_ revealed it as a member of the family Hypocreaceae (Ogawa et our decision to include them within the M clade. al#$$ Emericellopsis alkalina grew well at pHs from 4 to 11.2, assigned to Bionectriaceae (Rossman et al. 1999, 2001). The with a slight preference towards 7–11. However, a few isolates genus appears to be monophyletic, with strong support values ;##*;##';#NN;#NŠ;#N obtained in the analysis of the ITS and beta-tubulin sequences 4NJ (Zuccaro et al. 2004). The Emericellopsis lineage s. lat. also values (data not shown). This feature could be seen as a 6 Stilbella and Stanjemonium, physiological trade-off that has evolved in some strains of E. along with the marine species Acremonium tubakii and A. fuci alkalina that thrive along with alkalitolerant strains from the M (Summerbell et al. 2011). ‘;#N';##J The accumulated knowledge on the genus Emericellopsis were isolated from the same soil sample at Sulfatnoe Lake. suggests a wide ecological amplitude and worldwide ;6E. alkalina strains distribution. This includes typical species of soils undergoing that were jointly isolated with M clade strains. It is unclear \ what makes the majority of E. alkalina strains grow more in bogs, the sediments of freshwater and seawater basins, vigorously than the M clade members essentially at every pH and even the soils around subterranean wasp nests where value we tested. That E. alkalina performs well along a large humidity and alkalinity are elevated (Batra et al. 1973, Tubaki 1973, Domsch et al. 2007). Some species have a broad of this species in conventional terms. It is technically not ecological distribution, such as E. terricola, which has been correct to label it an ‘alkaliphile’, since it is capable of growth isolated from alkaline soils at the Mono Lake in California as at low pH as well as at high pH. Nor is the term ‘alkalitolerant’ well as from both acidic and saline soils in the Czech National entirely true, since the optimal growth pH is above neutral. Park (Steiman et al. 2004, Hujslová et al. 2010). A survey of The term ‘pH-tolerant’ with the preference towards alkaline ascomycetous fungi in limestone soils in Argentina formed by conditions might be suitable. As opposed to the soda soils mollusc shells yielded E. minima, with its ability to grow from clade, members of the M clade can be appropriately called pH 5 to 11 (Elíades et al.NJJŠ@ ‘alkalitolerant’, while E. minima/x"'#Š'@ other salt-associated isolations has suggested that marine can safely be termed a ‘neutrophile’. habitats might harbour a large number of the Emericellopsis A link between marine and soda soil inhabitants has species. The ability to survive in high salinity and pH does previously been observed in bacteria. In metabolic studies not always coincide with the ability to develop the full life- Fusarium oxysporum, it has been shown cycle in those conditions, making the salts-adapted species 6ena1 encoding P-type NaÅ- OP ATPase, which is believed to be an important player in the efforts to estimate their ecological contribution (Kohlmeyer & halotolerance adaptation cascade response, is up-regulated Volkmann-Kohlmeyer 2003). A study by Zuccaro et al. (2004) as the ambient pH goes up (Caracuel et al. 2003). Therefore, revealed the presence of distinct marine and terrestrial clades halophilic or halotolerant species may hold a clue towards within Emericellopsis, as noted above. The M clade contained elucidating the mechanisms of the ability to thrive at high pH. isolates from saline habitats, including the recently described The molecular aspects of the ability to cope with high ambient A. fuci from the thalli of the seaweed Fucus serratus and F. < distichus. Members of the marine clade within Emericellopsis aimed at revealing these molecular properties could be showed an ability to utilize sugars present in seaborne brown carried out by contrasting the genomics of neutrophiles and algae (e.g. fucoidan, fucose). The presence of marine water alkaliphiles. Such a project might provide answers to the appeared to be necessary for conidial germination in A. fuci. intriguing questions inherent in the alkaliphily phenomenon. Involvement of additional loci in our phylogenetic 4@< N † 6 ACKNOWLEDGEMENTS the M clade, with our 22 E. alkalina isolates displaying an 6x We are grateful to Dmitry Sorokin for providing soil samples, Bertha molecular data suggest that the E. alkalina group originated Koopmanschap and Marijke Slakhorst for technical assistance, and from the marine isolates of the M clade, linking evolutionary Denis Landin for processing the images. The work was funded by the development in the marine habitat with that of the soda +`_@| soils. Clearly, these environments share high salinity and ›

226 IMA FUNGUS Are alkalitolerant fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin?

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"›/`/"¤„`""4 } ; " ›/ `  " ¤ 4 ›;`¤"¤;NJ##Acremonium phylogenetic JI (2004) A new Acremonium species associated with Fucus overview and revision of Gliomastix, Sarocladium, and Trichothecium. Studies in Mycology 68: 139#ŠN Emericellopsis clade. Studies in Mycology 50IN'*MN$ Tubaki K (1973) Aquatic sediment as a habitat of Emericellopsis, }„¤NJJŠGenetic algorithm approaches for the phylogenetic

ARTICLE with a description of an undescribed species of Cephalosporium. analysis of large biological sequence datasets under the Mycologia 65I$*'M$P# maximum likelihood criterion. PhD dissertation, University of van Beyma thoe Kingma FH (1939-40) Beschreibung einiger neuer @6; pilzarten aus dem Centraalbureau voor Schimmelcultures, Baarn (Nederland). Antonie van Leeuwenhoek 6INŠ*MN$J

228 IMA FUNGUS