MycoKeys 69: 33-52 (2020) A peer-reviewed open-access journal doi: 10.3897/mycokeys.69.53205 RESEARCH ARTICLE feo) Myco Keys http://mycokeys.pensoft.net Launched to accelerate biodiversity research Novel species of Huntiella from naturally-occurring forest trees in Greece and South Africa FeiFei Liu'?, Seonju Marincowitz', ShuaiFei Chen'”, Michael Mbenoun', Panaghiotis Tsopelas?, Nikoleta Soulioti?, Michael J. Wingfield’ | Department of Biochemistry, Genetics and Microbiology (BGM), Forestry and Agricultural Biotechnology In- stitute (FABI), University of Pretoria, Pretoria 0028, South Africa 2. China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), Zhanfiang, 524022, GuangDong Province, China 3 Institute of Mediter- ranean Forest Ecosystems, Terma Alkmanos, 11528 Athens, Greece Corresponding author: ShuaiFei Chen (shuaifei.chen@gmail.com) Academic editor: R. Phookamsak | Received 13 April 2020 | Accepted 4 June 2020 | Published 10 July 2020 Citation: Liu FE, Marincowitz S, Chen SF, Mbenoun M, Tsopelas P, Soulioti N, Wingfield MJ (2020) Novel species of Huntiella from naturally-occurring forest trees in Greece and South Africa. MycoKeys 69: 33-52. https://doi. org/10.3897/mycokeys.69.53205 Abstract Huntiella species are wood-infecting, filamentous ascomycetes that occur in fresh wounds on a wide va- riety of tree species. These fungi are mainly known as saprobes although some have been associated with disease symptoms. Six fungal isolates with typical culture characteristics of Huntiella spp. were collected from wounds on native forest trees in Greece and South Africa. The aim of this study was to identify these isolates, using morphological characters and multigene phylogenies of the rRNA internal transcribed spacer (ITS) region, portions of the 6-tubulin (BT 1) and translation elongation factor la (TEF-1a) genes. The mating strategies of these fungi were also determined through PCR amplification of mating type genes. The study revealed two new species; one from Platanus orientalis in Greece and one from Colophos- permum mopane and Senegalia nigrescens in South Africa. These novel taxa have been provided with the names, H. hellenica sp. nov. and H. krugeri sp. nov., respectively. The former species was found to have a homothallic and the latter a heterothallic mating system. Keywords Ceratocystidaceae, Ceratocystis moniliformis Complex, Colophospermum mopane, Huntiella, Platanus orien- talis, saprobes, Senegalia nigrescens Copyright FeiFei Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 34 FeiFei Liu et al. / MycoKeys 69: 33-52 (2020) Introduction Huntiella species are members of the family Ceratocystidaceae (Microascales, Sordari- omycetes) as defined by De Beer et al. (2014). This family includes 15 genera, namely Ambrosiella, Berkeleyomyces, Bretziella, Catunica, Ceratocystis, Chalaropsis, Davidsoniella, Endoconidiophora, Huntiella, Meredithiella, Phialophoropsis, Solaloca, Tielaviopsis, Toshi- olenlla and Wolfgangiella (De Beer et al. 2014, 2017; Mayers et al. 2015, 2020; Nel et al. 2018). The type species of Huntiella, H. moniliformis, was first isolated from a sweetgum (Liquidambar styraciflua) in Texas, USA (Von Schrenk 1903). It was initially described as Ceratostomella moniliformis (Hedgcock 1906) and later transferred to Ceratocystis (Moreau 1952). When the family Ceratocystidaceae was redefined (De Beer et al. 2014), Huntiella was established as a distinct genus, which can be distinguished from Cerato- cystis and other members of the Ceratocystidaceae, based on their unique morphological features (Davidson 1935; Van Wyk et al. 2006; Wingfield et al. 2013). Most Huntiel- la spp. are easily recognised by a relatively-thick collar plate connecting the ascomatal necks and bases and ascomatal bases that are rough and ornamented with conical spines (Hedgcock 1906). In addition, aleurioconidia are rarely found in Huntiella species un- like most species of Ceratocystis sensu stricto with which they were previously confused (Hedgcock 1906; De Beer et al. 2014; Mbenoun et al. 2016). Many species in the Ceratocystidaceae are important pathogens of woody plants, including agricultural, fruit and forest tree crops (Kile 1993; Roux and Wingfield 2009). These pathogens result in a multiplicity of symptoms, such as branch and stem cankers, vascular staining, wilt, root rot, die-back and fruit rot (Kile 1993; Harrington 2004; Roux and Wingfield 2009). Huntiella spp. are generally considered saprobes or weak pathogens associated with relatively-minor lesions or sap stain of timber (Van Wyk et al. 2004, 2006, 2011; Tarigan et al. 2010; Kamgan Nkuekam et al. 2012; Chen et al. 2013; Mbenoun et al. 2016; Liu et al. 2018). However, there have been a few reports of more severe disease symptoms and even mortality caused by Huntiella spp. (Cristobal and Hansen 1962; De Errasti et al. 2015) Huntiella species are most commonly isolated from freshly-made wounds on trees, to which they are vectored by insects, especially sap-feeding beetles in the Nitidulidae (Heath et al. 2009; Kamgan Nkuekam et al. 2012; Mbenoun et al. 2016, 2017). It has been suggested that the relationship between Huntiella species and sap beetle is symbiotic and mutually beneficial, as the insects benefit from essential nutritional sup- plementation from their fungal partners, while the fungi benefit from transportation and access to scanty and ephemeral substrates (Mbenoun et al. unpublished data). Moreover, one species (H. bhutanensis) is found in association with the bark beetle /ps schmutzenhoferi (Van Wyk et al. 2004), which is similar to various important species of Endoconidiophora (De Beer et al. 2014), although the nature of insect-fungus interac- tion in this association is unknown. Huntiella spp. are particularly interesting in terms of their mating biology. Huntiel- la fecunda and H. moniliformis were, for example, shown to exhibit a unisexual mat- ing system, unlike the many heterothallic species found in this genus (Wilson et al. 2015; Liu et al. 2018). More recent studies have revealed a diversity of mating systems Huntiella species in Greece and South Africa 35 in Huntiella spp., including those that are homothallic, heterothallic and unisexual (Wilson et al. 2015; Liu et al. 2018). Efforts are consequently being made to collect these fungi, providing a basis for future fungal genetics studies, but also, together with genomics data (Wingfield et al. 2016), to better understand their biology. Huntiella species are most commonly found in tropical and sub-tropical regions of the world (Van Wyk et al. 2004, 2006, 2011; Kamgan Nkuekam et al. 2012; De Errasti et al. 2015; Mbenoun et al. 2014, 2016; Liu et al. 2018). Twenty-nine species are currently recognised in the genus (Liu et al. 2018). These fungi are grouped in three well-supported genealogical lineages that correspond to geographic centres, where they appear to have radiated (Mbenoun et al. 2016; Liu et al. 2018). These include species in an African Clade known only from Africa, an Asian Clade distributed across Asia and an Indo-Pacific Clade found in Australia and Pacific Islands and some parts of Asia. However, the diversity of Huntiella in most regions, including especially Europe, North and South America, is largely unexplored. The objective of this study was to identify two fungal isolates collected from Pla- tanus orientalis L. in Greece and four isolates from Colophospermum mopane (Benth.) J. Léonard and Senegalia nigrescens (Oliv.) P. Hurter in South Africa. These fungi dis- played typical culture characteristics of Huntiella spp., including rapid growth on agar medium, white fluffy mycelia when young, as well as the production of fruity aroma. Identification was accomplished, based on morphology and multigene phylogenies for the ITS, BT1 and TEF-1a« gene regions. Furthermore, we considered the mating biol- ogy of these isolates in order to complement our taxonomic studies. Materials and methods Fungal isolations Three South African Huntiella isolates were collected from fresh wounds of Colophos- permum mopane in Kruger National Park in April 2009 and another one of the South African isolates was obtained from a broken branch of a Senegalia nigrescens tree dam- aged by elephants in Kruger National Park in June 2010. The isolates from Greece were obtained from the stump of a Platanus orientalis tree that was cut about two months before sampling, in a natural forest along the banks of the Spercheios River in Phthiotis Regional Unit during November 2018. Isolation from wood samples was performed using a trapping technique originally described by Grosclaude et al. (1988). This is a standard diagnostic protocol for the isolation of Ceratocystis platani (Walter) Engelbre- cht & Harrington using freshly-cut twigs of P orientalis as bait (OEPP/EPPO 2014). Isolates from Greece were made by transferring ascospore masses from the tips of the ascomata on the surface of Platanus twig baits, formed on infected wood surface, to 2% malt extract agar (MEA: 20 g Biolab malt extract, 20 g Difco agar, 1 litre water), using a sterile needle under a dissection microscope (Carl Zeiss Co. Ltd., Oberkochen, Germany). The South African isolate was obtained by transferring my- celial strands from infected wood on to MEA. Primary isolations were incubated 36 FeiFei Liu et al. / MycoKeys 69: 33-52 (2020) for 3-7 d at 25 °C. From these isolations, purified cultures from single hyphal tips were prepared for morphological characterisation, phylogenetic analyses and mating- type studies. All purified isolates were deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa and the living culture collection (PPRI) of the South African National Collection of Fungi (NCF), Roodeplaat, Pretoria, South Africa. The dried-down type specimens were deposited in the National Collection of Fungi (PREM), Roodeplaat, Pretoria, South Africa. DNA extraction, PCR and sequencing All the isolates obtained in this study were used for DNA sequence-based characterisa- tion. Total genomic DNA was extracted from the mycelium of isolates grown on 2% MEA for 3-4 d at 25 °C, using Prepman Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocols. ‘Three gene regions were amplified for sequencing and phylogenetic analyses. These included the Internal Transcribed Spacer (ITS) regions 1 and 2, including the 5.8S rRNA, a partial B-tubulin 1 gene (BT1) and a partial Translation Elongation factor-la gene (TEF-1«), amplified using the set of primers as described by Liu et al. (2018). A total volume of 25 pl PCR reaction mixture contained 1 yl of DNA template, 0.5 pl (10 pM) of each primer (Forward and Reverse), 5 ul MyTaq PCR buffer (Bio- line GmbH, Germany) and 0.3 ul of MyTaq DNA Polymerase (Bioline GmbH, Ger- many). Ihe PCR reactions were conducted using an Applied Biosystems ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The PCR programme for amplification of the ITS, BT'1 and TEF1-« gene regions was as follows: an initial de- naturation step at 95 °C for 5 min followed by 35 cycles of 30 s at 95 °C, 45 s at 56 °C and 60 s at 72 °C and a final extension step at 72 °C for 10 min. Amplified fragments were purified using ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher Sci- entific, Waltham, MA, USA) to remove excess primers and dNTPs. Amplicons were sequenced in both directions using an AB] PRISM™ 3100 DNA sequencer (Applied Biosystems, USA) at the Sequencing Facility of the Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, South Africa. Multi-gene phylogenetic analyses The programme Geneious v. 7.0 was used to edit and assemble raw sequence reads into contigs (Kearse et al. 2012). Sequence data for representative type isolates of all de- scribed Huntiella spp. (except H. decorticans) were downloaded from GenBank (http:// www.ncbi.nlm.nih.gov). The sequences were aligned using MAFFT v. 7 with an online FFT-NS-i strategy (https://maftt.cbrc.jp/alignment/server/; Katoh and Standley 2013) Huntiella species in Greece and South Africa 37 and confirmed visually. Sequences for the novel species discovered in this study were deposited in GenBank. Single gene sequence datasets of the ITS, BT1 and TEF-la and the combined dataset of the three gene regions were analysed using Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI). The appropriate substitution model for each dataset was obtained using the software package jModeltest v. 2.1.5 (Posada 2008). The ML phylogenetic analyses were conducted using PhyML v. 3.0 (Guindon and Gascuel 2003). Confidence levels for the nodes were determined using 1000 bootstrap replicates. MP analyses were performed using PAUP v. 4.0b10 (Swof- ford 2003). Gaps were treated as a fifth character. BI analyses were conducted using MrBayes v. 3.2.6 (Ronquist et al. 2012) on the CIPRES Science Gateway v. 3.3. Four Markov Chain Monte Carlo (MCMC) chains were run from a random starting tree for five million generations and trees were sampled every 100 generations. Twenty-five percent of the trees sampled were discarded as burn-in and the remaining trees were used to construct 50% majority rule consensus trees. Ceratocystis cercfabiensis (isolate CMW 43029) was used as the outgroup taxon for all the phylogenetic analyses. The resulting trees were visualised using MEGA v. 7. Microscopy, growth study and mating-type assignment Morphological features were studied on the isolates grown on 2% MEA. The fruiting structures were initially mounted in water and this was later replaced with 85% lactic acid and in which measurements were made and images captured. Nikon microscopes (Eclipse Nz, SMZ 18, Nikon, Tokyo, Japan) mounted with a camera (Nikon DS Ri-2) were used for all observations. Fifty measurements of each relevant microscopic struc- ture were made when available and these are presented as minimum—maximum and average + standard deviation. A study of growth in culture was conducted at temperatures from 5—35 °C at 5 °C intervals on the 90 mm Petri dishes containing 2% MEA. A mycelial plug (5 mm diam.) taken from an actively-growing colony was placed at the centres of Petri dishes. Four replicates per isolate were used to study growth rate and the experiment was repeated once. Colony diameters were assessed by taking two measurements perpen- dicular to each other for all isolates daily and growth rates were calculated. Colony characteristics were described on the same medium used for the growth studies and colours were assessed using the colour charts of Rayner (1970). The mating type (MAT) of the studied Huntiella spp. was determined, based on the results of the mating type PCR reactions (Wilson et al. 2015). Primers, to see which of the MAT genes, Oman_111_F and Oman_111_R were thus used to amplify a 335 bp fragment of the MAT1-1-1 gene and Om_Mo_121_F and Om_ Mo_121_R to amplify a 572 bp fragment of the MAT1-2-1 gene, as described by Wilson et al. (2015). 38 FeiFei Liu et al. / MycoKeys 69: 33-52 (2020) Results Fungal isolations Six isolates resembling Huntiella spp. were included in this study. Two isolates had asco- mata with long necks, conical spines on the ascomatal bases and hat-shaped ascospores and four isolates had only thielaviopsis-like asexual state (Van Wyk et al. 1991). Four iso- lates were collected from Colophospermum mopane and Senegalia nigrescens in the Kruger National Park of South Africa and two isolates were from Platanus orientalis in Greece. Ascomata resembling Huntiella spp. were observed on twig baits from P orientalis sam- ples from Greece and two isolates were obtained in pure culture. All isolates obtained in this study have been preserved in the culture collections described above (Table 1). Multi-gene phylogenetic analyses All six isolates, included in this study, were successfully sequenced at all three selected gene regions for phylogenetic analyses, resulting in DNA sequence data of approxi- mately 614, 574 and 830 bp for the ITS, BT1 and TEF-1a gene regions, respectively. These newly-generated sequences were deposited in GenBank (Table 1). Comparisons with reference sequences of previously-described Huntiella spp. produced a concat- enated sequence alignment which was deposited in TreeBASE (no. 26341). The three tree topologies resulting from ML, MP and BI were concordant and showed similar phylogenetic relationships amongst taxa (Fig. 1, Suppl. materials 1-3: Figs S1-S3). Based on the phylogenetic analyses of the BT'1 (Suppl. materil 2, Fig. $2), TEF-1a (Suppl. material 3: Fig. S3) and the combined gene regions (Fig. 1), the six isolates clustered in two well-supported clades, clearly separated from each other and from previously described Huntiella spp. The ITS tree (Suppl. material 1: Fig. $1) provided a poor resolution to separate the species. All the isolates grouped in the African Clade of Huntiella spp. (Fig. 1). Taxonomy Huntiella hellenica ¥F.F. Liu. Marinc. & M.J. Wingf., sp. nov. MycoBank No: 835637 Fig, 2 Etymology. The name refers to the country, Greece where this fungus was collected. Mating strategy. Homothallic, with sexually complementary isolates having both the MAT 1-1-1 and MAT 1-2-1 genes. Sexual state. 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AWD 60TTT AWO VVVCC AAWO 6YVCT AWD L6CLT MWO 00EZT AWO €£0Z0E AWD T16Sc MWD O169€ AWD 9T69E AWD 008€ AWD 8V0IT AWD TO8ET MWD £087 MIND VICOL AWO 9866 AWO VITY MAWO ¢°N AWO my “LE mye “Ey SIULAOLYIIGIAL “Ey saudofiytgra “Ty DUDAIDULNS ‘TT DUDAIDULNS ‘TT suaavqqns “Hy suaavqqns “Ey aDUUDADS “TY IDUUDADS "TY DIADUYIDS "ET DIADUYIDS "ET sgauvurkd “Ey sgzuvurkd “Ey SISUIUDULO “TT SISUIUDULO “TT Y5U0]GO “Ey Y5U0]GO “Ey stsdowusofyrtuout “Fy ssdowusofy1tuoue “Fy SIULLOfNTUOUL "FT ysarsadg¢ 42 FeiFei Liu et al. / MycoKeys 69: 33-52 (2020) ITS+BT1+TEF 90/96/19 H. inquinans CMW21106 93/89/1] © H. inquinans CMW21107 H. glaber CMW43436 soe bol" H. glaber CMW49299 A! H. microbasis CMW21115 gael | H. microbasis CMW21117 59/76/1 lta) H. chinaeucensis CMW24658 63/67/0.96 H. chinaeucensis CMW24661 o2/or a! H. sumatrana CMW21109 9100/1) 1". sumatrana CMW21111 /2/68/4.93L. 1 H. confusa CMW43452 9700/1" H. confusa CMW43453 H. omanensis CMW11048 100/100/1 — H. omanensis CMW3800 99/99/1) H. eucalypti CMW44692 55/54*4 H. eucalypti CMW44693 H. ani CMW44684 H. ani CMW44686 100/1 0/1 H. bellula CMW49312 672" ly. bellula CMW49314 100/100) LH! fabiensis CMW49307 69/65/* H. fabiensis CMW49309 78/45/0.99 H. bhutanensis CMW8217 96/98/1" H. bhutanensis CMW8242 9900/1) H. inaequabilis CMW44372 H. inaequabilis CMW49306 89/80/11 H. meiensis CMW44374 gg/99/1 H. meiensis CMW44376 86/72/1 100/100/1) H. moniliformopsis CMW10214 H. moniliformopsis CMW9986 98/100/4 1O0/100/1) H. tribiliformis CMW13011 2 H. tribiliformis CMW13012 iw LOOMOOU 94/86/14) H. tyalla CMW28917 [exe soanoon |: tyalla CMWw28920 5 H. fecunda CMW49303 oe 76/7210.99" 11 fecunda CMW49302 m © 00/100/4 H. sublaevis CMW22444 a ris, H. sublaevis CMW22449 oO H. moniliformis CMW4114 74l79!"= H. moniliformis CMW9590 98/100/1) H. chlamydoformis CMW36932 H. chlamydoformis CMW37102 190/1 00/1 H. decipiens CMW25914 100/100/1 al ae By H. decipiens CMW25918 bay H. salinaria CMW25911 59/92/* H. salinaria CMW30703 H. ceramica CMW15245 > 98]140/1 " H. ceramica CMW15248 TI 30/65). CMW55933 ZY) 53/74/- 72/87/--) CMW55935 QO 7087/7] - CMW36849 H. krugeri sp. nov 5 CMW55934 95/97/0197) CMW54801 ; (2) —-t* cmws4soo H. hellenica sp. nov S 1#)/100/1) H. oblonga CMW23803 0 H. oblonga CMW23802 m 999/11 H. cryptoformis CMW36826 H. cryptoformis CMW36828 H. pycnanthi CMW36910 1o0/qpo/1'— H. pycnanthi CMW36916 aaa H. savannae CMW17300 H. savannae CMW17297 C. cercfabiensis CMW43029 0.02 Figure |. ML tree of Huntiella species generated from the combined DNA sequence data of ITS, BT1 and TEF-1« DNA. Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values > 0.9. Bootstrap values and posterior probabilities values are presented above branches as ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lack- ing the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup. Huntiella species in Greece and South Africa 43 157-493 um wide (avg. 218.2 um), ornamented with spine-like structures, dark brown, conical, 12—29 um long, 4-9 um wide at base becoming attenuated; ostiolar necks upright, straight, occasionally situated at off-centre of base, darker than base when young, 344— 616 um long (avg. 515.5 um), 34-60 pm wide (avg. 46.6 um) at base, gradually tapering towards apex; ostiolar hyphae hyaline, straight to divergent, 15—39 um long, 1-3 um wide, tapering towards apex. Asci evanescent. Ascospores hyaline, subglobose, aseptate, covered with sheath giving a hat-like feature in side view, 4-5.5 x 3-4.5 um (5 = 0.23 x 4 + 0.28 um) excluding sheath. Asexual state. Thielaviopsis-like Conidiophores macronematous, simple or branched; when branched radiating from basal cell once, often reduced to con- idiogenous cells. Conidiogenus cells endoblastic, hyaline, varying from lageniform to cylindrical depending spore shape; in case of thick barrel-shaped conidia, apex often becoming wider than base. Conidia hyaline, 1-celled, in two recognisable shapes; majority ellipsoidal to barrel-shaped (side swollen, ends round), typical fat barrel-shaped 5-8 x 4.5-7.5 um (5.9 + 0.61 x 5.3 + 0.55 um), width of some barrel-shaped ranging 2.5—4 um wide; rectangular-shaped (side straight, ends truncated), not commonly found, 5—9 x 1-3 um (6.9 + 1.18 x 2.3 + 0.38 um). Aleurioconidia not observed. Culture characteristics. Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium flat, superficial, medium dense and texture becom- ing pelt-like with age, colour above not uniform, salmon (11f’) to ochreous (15b’) with inner half irregularly umber (13m), below ochreous (15b’) with inner half irregularly umber (137’) at centre. Optimum growth temperatures at 30 °C at 9.6 mm/d, followed by at 25 °C (7.6 mm/d), 35 °C (7.2 mm/d), 20 °C (4.7 mm/d), 15 °C (3.2 mm/d), 10*°@:G Asmim/d)rand:5 °C (0:2 mm/d): Specimens examined. GREECE, Phthiotis, near the village Kastri, occurring on freshly-cut stumps of Platanus orientalis in a natural forest along the banks of the Spercheios River, Nov. 2018, P. Tsopelas & N. Soulioti, PREM 62889, holotype (dried culture of CMW 54800), culture ex-holotype CMW 54800 = PPRI 27982, other cultures CMW 54801 = PPRI 27983. Notes. ‘The sexual state of H. hellenica developed at temperatures over 25 °C. Cultures incubated at 20 °C and below produced only the asexual state. Huntiella hellenica is closely related to H. savannae (Kamgan Nkuekam et al. 2008), H. pyc- nanthi (Mbenoun et al. 2016) and H. krugeri. It can, however, be distinguished from these two species by the dimensions of ascomatal necks and barrel-shaped conidia and growth rate. Huntiella hellenica produced shorter (average 515.5 um long) ascomatal necks than H. savannae (average 579 um long) and H. pycnanthi (average 673 um long). Huntiella hellenica had larger (average 6.9 x 2.3 um) barrel- shaped conidia than H. savannae (average 4.8 x 3 um) and H. pycnanthi (average 6 x 3 um). Optimal temperature for growth of H. hellenica was 30 °C, similar to H. savannae and H. pycnanthi, but H. hellenica differed from H. pycnanthi in growing minimally at 10 °C and below. 44 FeiFei Liu et al. / MycoKeys 69: 33-52 (2020) Figure 2. Micrographs of Huntiella hellenica sp. nov. (ex-holotype CMW 54800 = PPRI 27982) A cul- ture grown on 2% MEA at 30 °C (optimum growth temperature) in the dark for 34 d B, C colony with ascomatal base embedded in mycelia with ascospore mass at the tip of ostiolar neck D-F young ascoma showing development of ostiolar neck and less-pigmented base G, H mature ascoma ornamented with spines I close-up of ascomatal wall showing spines J—-L close up of ornament (spin-like) M, N Ostiolar hyphae O Ascospores P Ascospores covered with sheath appearing like a hat Q, R Germinating ascospores S Lageniform conidiogenous cell T Cylindrical-shape conidiogenous cell U Conidia in various shapes from diverse barrel-shaped to rectangular-shaped V rectangular-shaped conidia W chains of conidia. Scale bars: 1 mm (B, C); 50 um (D-H); 10 um (I-W). Huntiella species in Greece and South Africa 45 Huntiella krugeri F.F. Liu. Marinc. & M.J. Wingf., sp. nov. MycoBank No: 835638 Fig. 3 Etymology. The name refers to the Kruger National Park in South Africa, where this fungus was collected. Mating strategy. Heterothallic with isolates having either a MAT 1-1-1 gene or a MAT 1-2-1 gene. Sexual state. Not observed. Asexual state. Produced on 2% MEA in 3 weeks. Thielaviopsis-like. Conidio- phores macronematous, upright, simple or branched in one tier, 29-37 um in length, often reduced to conidiogenous cells; Conidiogenous cells enteroblastic, lageniform, 10-20 um long, 1.5—3 um wide, tapering towards apex. Conidia hyaline, rectangular- shaped, usually straight, with top-end conidium often club-shaped, 4-11 x 1-2 um (avg. 6.2 x 1.7 um). Aleurioconidia hyaline, holoblastic, mostly terminal, ellipsoidal to subglobose with an extended tube-like base, club-shaped, 4—7 x 2—3 um (5.6 + 0.76 x 2.5 + 0.24 um). Culture characteristics. Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium superficial, flat, dense, colour above uniformly white, below luteous (19). Optimum growth temperatures were at 30 °C at 9 mm/d, followed by at 25 °C (8.2 mm/d), 35 °C (6.2 mm/d), 20 °C (6 mm/d), 15 °C (3.4 mm/d), 10 °C (0.9 mm/d) and 5 °C (0.3 mm/d). Specimens examined. SoutH Arrica, Mpumalanga, Kruger National Park, Sa- tara rest camp, Senegalia nigrescens, June 2010, M. Mbenoun, PREM 62883, holotype (dried culture of CMW 36849), culture ex-holotype CMW 36849 = CBS 131676 = PPRI27952. Other cultures. SourH Africa, Mpumalanga, Kruger National Park, Punda Ma- ria, Colophospermum mopane, April 2009, M. Mbenoun, CMW 55933, CMW 55934, CMW 55935. Figure 3. Micrographs of Huntiella krugeri sp. nov. (ex-holotype CMW 36849 = CBS 131676 = PPRI 27952). A Culture grown on 2% MEA in the dark for 34 d B, C Conidiogenous cell D Conidia in vari- ous shapes E Chain of conidia in different shapes F Chain of rectangular-shaped conidia with top-end of club-shaped G, H Aleurioconidia. Scale bars: 10 um (B=H). 46 FeiFei Liu et al. / MycoKeys 69: 33-52 (2020) Notes. Huntiella krugeri is closely related to H. hellenica described in the present study, 1. cryptoformis (Mbenoun et al. 2014) and H. savannae (Kamgan Nkuekam et al. 2008). Due to its heterothallic nature, H. krugeri produced only the asexual state in this study. The bacilliform conidia of H. krugeri (average 6.2 x 1.7 um) were longer than those of H. hellenica (average 5.9 x 5.3 um) and H. cryptoformis (average 5.5 x 2.5 um). In addition, H. krugeri produced hyaline aleurioconidia, which are absent in other closely-related species in the genus. Discussion This study led to the discovery of two novel Huntiella species isolated from Platanus orientalis in Greece, Colophospermum mopane and Senegalia nigrescens in the Kruger National Park of South Africa. These two species, provided with the name H. hellenica and H. krugeri, respectively, were shown to reside in the African Clade of Huntiella (Mbenoun et al. 2014; Liu et al. 2018). The identity of H. hellenica and H. krugeri emerged from a phylogenetic analysis of DNA sequence data for three gene regions (ITS, BT1 and TEF-1«), as well as their distinct morphological characteristics. Mating studies showed that Huntiella hellenica and H. krugeri were homothallic and heter- othallic, respectively. All indications were that these two species are saprobes that grow on the freshly-exposed surfaces of trees. The stump of P orientalis, from which H. hellenica emerged, was sampled approxi- mately two months after tree felling and it was also infected by the pathogen Ceratocys- tis platani, which causes a devastating disease in natural stands of P orientalis in Greece. Colonszation of the stump with H. /ellenica could have occurred on the freshly-cut surface with a contaminated tool as occurs for C. platani (Tsopelas et al. 2017) or was transferred by insect vectors. The novel species described in this study showed typical characteristics of Huntiella spp. [hey grew rapidly in culture; their mycelium was white when young and turned dark with age. The one species that displayed a sexual state - H. hellenica, produced hat-shaped ascospores and had short conical spines on the ascomatal bases. Temperature is known to influence the ability of Huntiella spp. to produce a sexual state (Wilson et al. 2015) and this was also true for H. hellenica, which did not produce acomata below 25 °C. Comparison of DNA sequence data for multiple gene regions is essential when seeking to identify species in Huntiella (Mbenoun et al. 2014; Liu et al. 2018). The three gene regions, selected for this purpose, have been used in previous studies show- ing that they can be collectively used to delineate species boundaries in the genus (Van Wyk et al. 2004, 2006, 2011; Kamgan Nkuekam et al. 2012; Mbenoun et al. 2014, 2016; De Errasti et al. 2015; Liu et al. 2018). However, analyses of individual gene re- gions revealed different levels of resolution, consistent with the results of previous stud- ies on this group of fungi (Mbenoun et al. 2014; Liu et al. 2018). Thus protein coding genes, in this case BT] and TEF-1a, provided the best resolution for species identifica- tion of Huntiella, while ITS sequences provided little information or no support. Huntiella species in Greece and South Africa 47 Primers, developed to identify the mating type idiomorphs in Huntiella spp. (Wil- son et al. 2015), were effective for this purpose in the present study. The results showed that H. hellenica has both mating-type idiomorphs and this explains the presence of sexual structures in all isolates derived from single hyphal tips. In contrast, the iso- late of H. krugeri contained one mating gene and is clearly a heterothallic species of Huntiella, also consistent with the fact that the isolate produced only an asexual state. This result is also consistent with those of Liu et al. (2018) who showed that closely- related Huntiella spp. can have different mating strategies. Collectively, Huntiella spp. have a remarkable range of mating strategies, including homothallic and heterothallic species, as well as those exhibiting unisexuality (Wilson et al. 2015; Liu et al. 2018). The new species described here will contribute to future studies considering the evolu- tion of mating in Huntiella. The two new species of Huntiella, discovered in this study, bring the total num- ber of species in the genus to 31. These are found in many different regions of the world and on a wide variety of woody substrates (Van Wyk et al. 2004, 2006, 2011; Kamgan Nkuekam et al. 2012; De Beer et al. 2014; Mbenoun et al. 2014, 2016; De Errasti et al. 2015; Liu et al. 2018). The renewed interest that these fungi have received during the course of the past decade has revealed unexpected complexity in their ecological interactions (Mbenoun et al. unpublished data), evolutionary his- tory (Mbenoun et al. 2014; Liu et al. 2018) and reproductive biology (Wilson et al. 2015). The description of novel taxa, as reported in this study and the growing accessibility of whole genome sequencing (Wingfield et al. 2016), should enable new avenues of research that will contribute to a considerably better understanding of Huntiella species in the future. Acknowledgements This study was initiated through the bilateral agreement between the Govern- ments of South Africa and China and supported by The National Key R&D Pro- gram of China (China-South Africa Forestry Joint Research Centre Project; pro- ject No. 2018YFE0120900), the National Ten-thousand Talents Program (Project No. W03070115) and the GuangDong Top Young Talents Program (Project No. 20171172). 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Wingfield Data type: phylogenetic tree Explanation note: Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values > 0.9. Bootstrap values and pos- terior probabilities value are presented above branches as ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lacking the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.69.53205.suppl1 Supplementary material 2 Figure S2. ML tree of Huntiella species generated from the BT 1 DNA sequence data Authors: FeiFei Liu, Seonju Marincowitz, ShuaiFei Chen, Michael Mbenoun, Panaghiotis Tsopelas, Nikoleta Soulioti, Michael J. Wingfield Data type: phylogenetic tree Explanation note: Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values > 0.9. Bootstrap values and pos- terior probabilities values are presented above branches as ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lacking the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.69.53205.suppl2 52 FeiFei Liu et al. / MycoKeys 69: 33-52 (2020) Supplementary material 3 Figure S3. ML tree of Huntiella species generated from the TEF-1~ DNA sequence data Authors: FeiFei Liu, Seonju Marincowitz, ShuaiFei Chen, Michael Mbenoun, Panaghiotis Tsopelas, Nikoleta Soulioti, Michael J. Wingfield Data type: phylogenetic tree Explanation note: Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values > 0.9. Bootstrap values and pos- terior probabilities values are presented above branches as ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lacking the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.69.53205.suppl3