The family Ajellomycetaceae (Onygenales) includes mammal-associated pathogens within the genera Blastomyces, Em- monsia, Histoplasma and Paracoccidioides, as well as the recently described genera, Emergomyces that causes disease in immunocompromised hosts, and Emmonsiellopsis, known only from soil. To further assess the phylogenetic relationships among and between members of these genera and several previously undescribed species, we sequenced and analyzed the DNA-directed RNA polymerase II (rPB2), translation elongation factor 3-a (TEF3), b-tubulin (TUB2), 28S large subunit rDNA (LSU) and the internal transcribed spacer regions (ITS) in 68 strains, in addition to morphological and physiological investigations. To better understand the thermal dimorphism among these fungi, the dynamic process of transformation from mycelial to yeast-like or adiaspore-like forms was also assessed over a range of temperatures (6–42 C). Molecular data resolved the relationships and recognized ﬁve major well-supported lineages that correspond largely to the genus level. Emmonsia, typiﬁed by Emmonsia parva, is a synonym of Blastomyces that also accommodates Blastomyces helicus (formerly Emmonsia helica). Emmonsia crescens is phylogenetically distinct, and found closely related to a single strain from soil without known etiology. Blastomyces silverae, Emergomyces canadensis, Emergomyces europaeus and Em- monsia sola are newly described. Almost all of the taxa are associated with human and animal disease. Emmonsia crescens, B. dermatitidis and B. parvus are prevalently associated with pulmonary disease in humans or animals. Blastomyces helicus, B. percursus, Emergomyces africanus, Es. canadensis, Es. europaeus, Es. orientalis and Es. pasteurianus (for- merly Emmonsia pasteuriana) are predominantly found in human hosts with immune disorders; no animal hosts are known for these species except B. helicus. Keywords Ajellomycetaceae Onygenales Phylogeny Ecology Thermal dimorphism Systemic infection Taxonomy Introduction predilection and virulence, but in general the family is unique in the fungal kingdom for its large number of species The majority of known systemic, thermally dimorphic fungi exhibiting specialized invasive phases adapted to survival are classiﬁed in a single family of the order Onygenales, the and replication in tissues of mammal hosts. For the most part, Ajellomycetaceae. Members of the family differ in host the course of infection by these fungi shares the following key features: inhalation of infectious propagules from the environment, pulmonary and extrapulmonary disease, a signiﬁcant role of the acquired cellular immune system in the & Hongguang Lu control of the infection, and, in some cases, endogenous firstname.lastname@example.org reactivation upon severe immune impairment of the host (de & Sybren de Hoog Hoog et al. 2016b). Molecular ﬁndings have suggested that email@example.com fungi with the above life cycle and described in the genera Extended author information available on the last page of the article 123 246 Fungal Diversity (2018) 90:245–291 Blastomyces, Emmonsia, Histoplasma, and Paracoccid- Several other emmonsia-like agents have been reported ioides are phylogenetically coherent. Lacazia loboi, the sporadically but with increasing frequency from human cause of lobomycosis (which follows subcutaneous inocu- hosts (Schwartz et al. 2015b; Dukik et al. 2017b; Yang lation), shares a common ancestor with Paracoccidioides et al. 2017). However, little is known about the phylogeny (Taborda et al. 1999; Vilela and Mendoza 2018). Genera of these organisms and several have not been formally recently added to the Ajellomycetaceae include Emmon- described. To further assess relationships of these emerging siellopsis comprising only geophilic species (Marin-Felix fungi with pathogens in the Ajellomycetaceae, a phyloge- et al. 2015) and Emergomyces that comprises systemic netic study was undertaken. Prior molecular studies pathogens primarily of immunocompromised human hosts revealed that ribosomal DNA sequences generated well- (Dukik et al. 2017b). Nearly all species described within the supported clades, but species deﬁnitions were insufﬁciently Ajellomycetaceae share morphological and ecological fea- clariﬁed (Untereiner et al. 2004; Peterson and Sigler 1998). tures of thermal dimorphism (conversion from ﬁlamentous In this study, in addition to the analysis of the traditional to yeast-like or enlarged adiaspore-like morphotypes, loci, ITS and LSU, sequences of partial protein-coding expressed under saprobic and pathogenic conditions, genes were obtained, i.e. rPB2, TEF3 and TUB2.We respectively), a highly similar conidial apparatus, often combined multilocus phylogenetic analysis with extensive restricted geographic distribution and absence of kerati- analysis of morphogenetic processes over a series of tem- nolytic activity (Baumgardner and Paretsky 1999; Restrepo peratures to better understand the process of conversion et al. 2000; Untereiner et al. 2004; Sigler 2005; Bagagli et al. from saprobic (mycelial) to invasive (yeast-like or other) 2006, 2008). Sexual phases (previously known as Ajel- stages. Thermodependent conversion has been studied lomyces states) are known within the genera Emmonsia, extensively in species of Histoplasma, Blastomyces, and Blastomyces and Histoplasma and comprise heterothallism, Paracoccidioides, but data are lacking for lesser known complex ascomata (gymnothecia) with coiled appendages, and novel members of the Ajellomycetaceae. evanescent asci and oblate ascospores (McDonough and Lewis 1968; Kwon-Chung 1973; Sigler 1996). Several species of Ajellomycetaceae are etiologic agents Materials and methods of well-known diseases in otherwise healthy mammals. The endemic mycoses, i.e. blastomycosis, histoplasmosis, Morphology and physiology and paracoccidioidomycosis, have considerable impact on public health with a combined estimated burden of 32,000 Sixty-eight emmonsia-like strains were analyzed (Table 1). cases of life-threatening disease each year (Colombo et al. These were obtained from the Centraalbureau voor 2011). Disease caused by Emmonsia species, on the other Schimmelcultures (CBS, housed at Westerdijk Fungal hand, is less well understood. Until recently, it was con- Biodiversity Institute, Utrecht, The Netherlands), the sidered to be primarily a disease of mammals other than University of Alberta Microfungus Collection and Her- humans. Emmonsia parva and Ea. crescens are associated barium (UAMH), Edmonton, Canada; now UAMH Centre with adiaspiromycosis, a localized lung infection mainly in for Global Microfungual Biodiversity, University of Tor- wild rodents and occasionally in humans (Emmons and onto, Toronto, Canada) and from the National Collection of Jellison 1960; Huba ´lek et al. 1998; Sigler 2005). The Pathogenic Fungi (NCPF), Mycology Reference Labora- association of Emmonsia species with invasive human tory, Bristol, England), supplemented by kind donations of disease received little attention until Emmonsia pasteuri- individual researchers. ana (recently renamed Emergomyces pasteurianus; Dukik Conditions for conidial formation and thermal conver- et al. 2017b) was described as the cause of disseminated sion were pre-tested on several commercially available disease associated with advanced HIV infection in an media; Malt Extract Agar (MEA, Oxoid) was found to be individual from Europe (Drouhet et al. 1998). Recently optimal for both sporulation and conversion. Morphology Kenyon et al. (2013) reported a disease among HIV-posi- and growth rate of colonies were determined on MEA tive individuals in South Africa caused by another during 4 weeks in the dark at temperatures of 6, 15, 21, 24, emmonsia-like fungus, since described as Emergomyces 27, 30, 33, 36, 37, 40 and 42 C. Colony characters were africanus (Dukik et al. 2017b). To date, over 80 human assessed on MEA after 3 weeks at 24 C (saprobic phase), cases of disease caused by Es. africanus have been diag- 33 C (intermediary phase) and 37 C (thermotolerant nosed in South Africa (Maphanga et al. 2017). In a retro- phase). Production of urease was determined in Chris- spective review of the ﬁrst 52 cases from South Africa, all tensen’s urea broth (Oxoid) after incubation for 8 h, 24 h patients were immunocompromised, cutaneous disease and 7 days at 24 and 37 C, a positive response being a occurred in 95% of patients and the case-fatality rate of color change from yellow to fuchsia. Cycloheximide tol- infections was 50% (Schwartz et al. 2015b). erance was evaluated by comparing growth on plain 123 Fungal Diversity (2018) 90:245–291 247 Table 1 Species and strains examined arranged by multilocus subclades Genus and Former name Culture accession number(s) MLST Origin Genbank accession numbers species subclade CBS No. Other collection ITS TUB LSU rRP2 TEF3 no. Emmonsia I Ea. crescens Ea. crescens CBS 142606 NCPF 4268 I-1a Lung, water vole, UK KY710920 KY710949 KY711008 KY711050 KY711102 Ajellomyces crescens CBS 100376 UAMH 349(MT) I-1a Lung, mole Talpa AF038336 KY710944 KY710990 KY711049 KY711110 europaea,UK Ea. crescens CBS 508.78 ATCC 24951, I-1a Lung, Microtus arvalis, KT155930 KT155578 KT155275 KY711046 KT156243 IHEM 3818 USA Ea. crescens CBS 177.60(T) UAMH 3008, ATCC I-1a Lung, rodent Arvicola AF071864 KT155341 KY710989 KY711047 KT155994 13704 terrestris, Norway Ea. crescens CBS 510.78 ATCC 32540 I-1a Lung, dark polecat, KT155932 KY710941 KT155277 KY711045 KT156245 Czechoslovakia Ajellomyces crescens CBS 100377 UAMH 7365(MT) I-1a Lung, Trichosurus AF038335 KY710942 KY710991 KY711051 KY711111 vulpecula, New Zealand Ea. crescens CBS 139870 UAMH 7268 I-1a lung, Trichosurus AF038337 KY710943 KY711004 KY711048 KY711126 vulpecula, New Zealand Ea. crescens CBS 139859 UAMH 135 I-1a Lung, Peromyscus sp., KY710919 KY710945 KY710994 KY711063 KY711115 USA Ea. crescens CBS 139863 UAMH 393 I-1a Lung, Cleithrionomys sp., AF038339 KY710946 KY710998 KY711052 KY711119 Korea Ea. crescens CBS 139864 UAMH 394 I-1a Lung, Cleithrionomys sp., AF038340 KY710947 KY710999 KY711064 KY711120 Korea Ea. crescens CBS 139865 UAMH 395 I-1a Lung, Apodemus sp., Korea AF038338 KY710948 KY711000 KY711060 KY711121 Ea. crescens CBS 139860 UAMH 136 I-1b Lung, skunk, USA AF038341 KY710950 KY710995 KY711057 KY711116 Ea. crescens CBS 139861 UAMH 137 I-1b Lung, muskrat, USA AF038342 KY710952 KY710996 KY711058 KY711117 Ea. crescens CBS 139869 UAMH 4077 I-1b Moldy straw bales, AF038349 KY710951 KY711003 KY711059 KY711125 mushroom house, Canada Ea. crescens CBS 139857 UAMH 132 I-1b Lung, Peromyscus AF038351 KY710958 KY711006 KY711065 KY711114 maniculatus, Canada Ea. crescens CBS 191.55 UAMH 126 I-1b Lung,mouse, Canada AF038319 KT155506 KY711005 KY711066 KT156160 Ea. crescens CBS 139866 UAMH 1067 I-1b Lung, wild ﬁeld mouse, AF038346 KY710957 KY711001 KY711055 KY711122 Canada Ea. crescens CBS 139856 UAMH 129 I-1b Lung, Peromyscus AF038343 KY710956 KY710993 KY711053 KY711113 maniculatus borealis, Canada Ea. crescens CBS 139867 UAMH 1140 I-1b Lung, wild ﬁelddeer AF038347 KY710959 KY711002 KY711061 KY711123 mouse, Canada Ea. crescens CBS 475.77 UAMH 127 I-1b Lung, rodent, Canada AF038344 KT155573 KT155264 KY711044 KT156236 Ea. crescens CBS 139868 UAMH 4076 I-1b Greenhouse soil, Canada AF038348 KY710955 KY711007 KY711056 KY711124 Ea. crescens CBS 139862 UAMH 140 I-1b Lung, Peromyscus AF038350 KY710954 KY710997 KY711054 KY711118 maniculatus, Canada Ea. crescens CBS 139855 UAMH 128 I-1b Lung, mouse, Canada AF038345 KY710953 KY710992 KY711062 KY711112 248 Fungal Diversity (2018) 90:245–291 Table 1 (continued) Genus and Former name Culture accession number(s) MLST Origin Genbank accession numbers species subclade CBS No. Other collection ITS TUB LSU rRP2 TEF3 no. Ea. sola Ea. parva CBS 142607 NCPF 4289(T) I-2 Soil, USA KY710916 KY710960 KY711015 KY711082 KY711101 Emergomyces Es. canadensis Ea. sp. 2 CBS 139872(T) UAMH 7172 I-3a Skin, human,HIV,Canada AF038322 KY710965 KY710971 KY711078 KY711127 Ea. sp. 2 CBS 139873 UAMH 10370 I-3a Blood, human, renal EF592151 KY710966 KY710972 KY711077 KY711128 transplant, Canada Es.orientalis Ea. sp. 7 CBS 124587(T) Peng 5Z489, I-3b Skin, human, diabetic, KT155765 KT155457 KT155092 KY711076 KY711099 CGMCC 2.4011 China Es. pasteurianus Ea. pasteuriana CBS 139522 I-3c Skin, human, China KT155632 KY195934 KT155632 KY711080 KY195947 Ea. sp. 4 CBS 140361 I-3c Skin, human, HIV, South KY195962 KY195938 KY195969 KY711081 KY195946 Africa Ea. pasteuriana CBS 101426(T) UAMH 9510, I-3c Skin, human, HIV,Italy KT155671 KT155364 KT154983 KY711079 KT156020 NCPF 4236 Es. europaeus Ea. sp. 6 CBS 102456(T) UAMH 10427 I-3d Lung, human, Germany EF592164 KY710961 KT154987 KY711067 KT156022 Es. africanus Ea. sp. 5 CBS 136260(T) I-3e Skin, human, HIV, South KT155802 KY710962 KY710973 KY711068 KY711100 Africa Ea. sp. 5 CBS 136730 I-3e Skin, human, HIV, South KY195959 KT155489 KT155137 KY711069 KT156143 Africa Ea. sp. 5 CBS 139543 I-3e Skin, human, HIV, South KY195956 KY195935 KY195966 KY711070 KY195944 Africa Ea. sp. 5 CBS 142608 NCPF 4164 I-3e Skin, human, HIV, South KY195960 KY195941 KT155321 KY711073 KY195945 Africa Ea. sp. 5 CBS 140363 I-3e Skin, human, HIV, South KY195958 KY710964 KY195965 KY711075 NA Africa Ea. sp. 5 CBS 140362 I-3e Skin, human, HIV, South KY195961 KY195939 KY195968 KY711072 NA Africa Ea. sp. 5 CBS 140360 I-3e BM, human, HIV, South KY195957 KY195937 KY195967 KY711074 NA Africa Ea. sp. 5 CBS 140359 I-3e blood, human, HIV, South KY710921 KY710963 KY710975 KY711071 NA Africa Blastomyces II B. parvus Ea. parva CBS UAMH 130 II-1 Lung, rodent, USA AF038333 KY710938 KY710988 KY711038 KY711134 139881(ET) Ea. parva CBS 139880 UAMH 125 II-1 Lung, rodent, USA AF038331 KY710937 KY710977 KY711036 KY711133 Ea. parva CBS 139883 UAMH 434 II-1 soil AF038332 KY710939 KY710978 KY711037 KY711136 Ea. parva CBS 204.48 II-1 Lung, rodent, USA EX51306 KY710936 KT155163 KY711035 KT156162 Ea. parva CBS 139882 UAMH 134 II-1 Lung, Neotama micropus, AF038326 KY710940 KY710987 KY711039 KY711135 USA Ea. parva CBS 178.60 ATCC 14051 II-1 Lung, Thomomys sp., USA KY710911 KT155342 NA KY711041 KT155995 Ea. parva CBS 205.48 II-1 Lung, rodent, USA KY710918 KT155509 KT155166 KY711042 KT156164 Fungal Diversity (2018) 90:245–291 249 Table 1 (continued) Genus and Former name Culture accession number(s) MLST Origin Genbank accession numbers species subclade CBS No. Other collection ITS TUB LSU rRP2 TEF3 no. B. silverae Ea. sp. CBS 139886 UAMH 6312 II-2 Soil, Canada AF038330 KY710927 KY710980 KY711025 KY711140 Ea. sp. CBS 139885(T) UAMH 4770 II-2 coyote dung, Canada AF038325 KY710924 KY710979 KY711024 KY711139 Ea. sp. CBS 139887 UAMH 7045 II-2 bronchial washings, AF038329 KY710925 KY710981 KY711022 KY711141 human, Canada Ea. sp. CBS 139888 UAMH 10478 II-2 Lung, human, Canada EF592158 KY710926 KY711012 KY711023 KY711142 Ea. sp. CBS 139871 UAMH 4489 II-2 bird’s nest, USA AF038327 KY710922 KY710976 KY711020 KY711138 Ea. sp. CBS 139879 UAMH 139 II-2 Lung, weasel, USA AF038328 KY710923 KY711010 KY711021 KY711137 Ea. parva CBS 509.78 ATCC 32539 II-2 Lung, prairie polecat, KT155931 KT155579 KT155276 KY711043 KT156244 Czechoslovakia B. dermatitidis CBS 134.36 II-3 No data KT155797 KT155483 KT155130 KY711018 KT156137 CBS ATCC 18188, II-3 Human, USA KT155962 KT155601 KT155307 KY711017 KT156267 674.68(ET) UAMH 3539, IHEM 3783,RV 24701 CBS 642.77 II-3 Brain, human, USA KT155954 KT155594 KT155299 KY711019 KT156263 B. gilchristii CBS 134223(T) PHO TB00018/2005 Sputum, human, Kenora, KT155800 KT155485 KT155133 NA KT156139 Canada B. percursus Ea.sp. 3 CBS 139878(T) UAMH 7425, UAMH II-4 Skin, human, Israel KY195964 KY195936 KY195971 KY711033 KY195942 Ea.sp. 3 CBS 142605 NCPF 4091 II-4 Skin, human, HIV, South KY195963 KY195940 KY195972 KY711034 KY195943 Africa B. helicus Ea.sp. 1 CBS 140058 UAMH 11034 II-5 Lung, feline, USA KY710914 KY710930 KY711011 KY711032 KY711144 Ea.sp. 1 CBS 139874 UAMH 3398 II-5 CSF, human, Canada EF592153 KY710928 KY710982 KY711026 KY711129 Ea.sp. 1 CBS 139876 UAMH 11660 II-5 Lung, female cat, USA KY710913 KY710935 KY710984 KY711031 KY711131 Ea.sp. 1 CBS 140059 UAMH 10539 II-5 Lung, dog, USA EF592156 KY710934 KY710974 KY711030 KY711145 Ea.sp. 1 CBS 140056(T) UAMH 7101 II-5 sputum, BM, blood human, EF592154 KY710929 KY711009 KY711029 KY711143 Canada Ea.sp. 1 CBS 140060 UAMH 10593 II-5 BAL and blood, human, EF592157 KY710931 KY710986 KY711028 KY711146 Canada Ea.sp. 1 CBS 139877 UAMH 11718 II-5 CSF, human, USA KY710915 KY710933 KY710985 KY711040 KY711132 Ea.sp. 1 CBS 139875 UAMH 11294 II-5 Blood, pleural effusion KY710912 KY710932 KY710983 KY711027 KY711129 human, USA Histoplasma III H. duboisii CBS 215.53 No data KY711103 CBS 175.57 Skin, human, Senegal KY710917 NA KY711016 NA KY711105 250 Fungal Diversity (2018) 90:245–291 Table 1 (continued) Genus and Former name Culture accession number(s) MLST Origin Genbank accession numbers species subclade CBS No. Other collection ITS TUB LSU rRP2 TEF3 no. H. capsulatus H. capsulatum var. CBS 537.84 Horse, Egypt KT155937 KT155583 KT155282 KY711084 KT156250 farciminosum H. capsulatum var. CBS 478.64 Poland KT155921 KT155575 KT155266 KY711086 KT156238 farciminosum H. capsulatum var. CBS ATCC 58332,CDC Horse, Egypt KT155936 KT155582 KT155281 KY711085 KT156249 farciminosum 536.84(NT) B-3786 H. duboisii CBS 114388 Lung, human, Africa KY710910 NA NA KY711087 KY711104 H. capsulatum var. CBS 205.35 No data KT155827 KT155508 KT155165 KY711083 KT156163 farciminosum Paracoccidioides IV P. brasiliensis CBS 372.73 ATCC 32069 Human, Colombia KT155885 KT155554 KT155229 KY711088 KT156212 CBS 121.34 No data KT155747 KT155443 KT155074 KY711089 KT156098 CBS 109819 No data KT155685 NA KT155000 KY711090 KT156034 Emmonsiellopsis V Emms. terrestris CBS 273.77(T) UAMH 2304 V Soil, USA KT155850 KT155526 KT155190 KY711091 KY711106 CBS 139889 UAMH 141 V Soil, USA AF038321 KY710968 KY711013 KY711093 KY711108 CBS 137499 FMR 4023 V Soil, Spain KT155804 KT155491 KT155139 KY711092 KY711107 Emms. CBS 137500(T) FMR 4024 V Soil, Spain KT155631 KY710967 KY711014 KY711094 KY711109 coralliformis Arthroderma Art. ﬂavescens CBS 473.78(T) Kingﬁsher KT155916 KY710969 KT155261 KY711095 KY711098 CBS 474.78 No data KT155918 KY710970 KT155263 KY711096 KY711097 ATCC American Type Culture, Manassas, USA, CBS Centraalbureau voor Schimmelcultures, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, CDC Centers for Disease Control, Atlanta, USA, CGMCC The Chinese General Microbiological Culture Collection Center, Beijing, China, IHEM Institute of Hygiene and Microbiology, Brussels, Belgium, FMR Universitat Rovira i Virgili, Reus, Spain, NCPF National Collection of Pathogenic Fungi, Bristol, England, UAMH Centre for Global Microfungal Biodiversity, Toronto, Canada, Ea Emmonsia, ET epitype culture, MT mating type culture, NT Neotype culture, T Type culture, NA not available Names used in reference Schwartz et al. (2015b) Fungal Diversity (2018) 90:245–291 251 Sabouraud glucose agar (SGA) (Oxoid) with that on SGA base, and may bear short lateral branches (secondary supplemented with 0.2% cycloheximide (Sigma-Aldrich, conidiophores) arising at right angles. Conidia are sessile Zwijndrecht, The Netherlands) at 24 and 37 C for or borne on minute pedicels. Incubation at 33–37 C led to 21 days. Isolates were grown on blood agar (BioMe ´rieux, conversion to another growth form (thermotolerant Marcy-l’Etoile, France) for 14 days at 24 and 37 Cto phase) in nearly all strains, resembling the forms known in assess hemolysis (clearing) according to Kane et al. (1997). host tissues. This was initiated by moderate or pronounced Slide cultures, applied to examine microscopic features and swelling, then leading to giant cells. Large, spherical cells to assess the process of thermal conversion, were prepared in vitro measuring over 30 lm (up to 140 lm) without using the method of Dukik et al. (2017b), incubated at 21, budding or any other type of reproduction (terminal phase) 24, 27, 30, 33 and 37 C and examined after 7, 14, 21 and and with very thick walls are known as adiaspores. Cells 28 days. Slides were made using Shear’s mounting med- between 10–30 lm in diam, mostly produced at or beyond ium without pigments (Samson et al. 2010). Micrographs intermediate temperatures (33 C) and with thinner walls, were taken using a Nikon Eclipse 80i microscope and DS often more irregular in shape due to occasional broad- Camera Head DS-Fi1/DS-5m/DS-2Mv/DS-2MBW using based budding are referred to as adiaspore-like cells. Cells NIS-Element freeware package (Nikon Europe, Badho- measuring 5–10 lm in diam and producing daughter cells evedorp, The Netherlands). Measurements of conidia and from a broad base are large yeast, and cells below 5 lmin other structures were determined using the Nikon Eclipse diam budding at narrow bases are small yeast. Budding in 80i measurement module. any cell type may be unipolar, bipolar or multilateral. A At saprobic temperature strains were ﬁlamentous. Lat- diagrammatic overview of the main morphological features eral cells bearing conidial structures are referred to as is given in Fig. 1. conidiophores, which have or have not a septum at the Fig. 1 Descriptions and terms for the main morphological features of the fungi in this study 123 252 Fungal Diversity (2018) 90:245–291 Fig. 2 Phylogram (50% majority rule) resulting from ML analysis ofc Molecular analysis concatenated rPB2, TUB2, TEF3, ITS and LSU sequences with conﬁdence values of bootstrap proportions from the ML analysis and DNA was obtained from 68 strains grown for 7–14 days on posterior probabilities from the Bayesian analysis above branches MEA at 24 C in a class II biological safety cabinet. DNA ([ 50% for BS from ML analyses, [ 0.95 for PP from Bayesian). Branches with full statistical support (ML-BS C 99%; PP C 0.99) are extracts of 12 reference strains belonging to Blastomyces highlighted by thickened branches. Genus and species clades are (2), Histoplasma (7) and Paracoccidioides (3) were discriminated with boxes of different colours. The scale bar shows the obtained from the Centraalbureau voor Schimmelcultures expected number of changes per site (housed at Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands) because of their hazard level. About 10 mm of material was added to a screw-capped chains (one cold and three heated) and the STOPRULE option, tube containing 490 lL CTAB-buffer (2% cetyltrimethyl stopping analyses at an average standard deviation of split ammonium bromide, 100 mM Tris–HCl, 20 mM EDTA, frequencies of 0.01. The combined dataset was partitioned 1.4 M NaCl) and 6–10 acid-washed glass beads per locus and the analysis was done in MRBAYES v3.1.2. (* 1.5–2 mm diam). Ten microliters proteinase K were Two parallel runs with four Markov chain Monte Carlo added and the mixture was vortexed for 10 min. Tubes (MCMC) simulations for each run were set for 2,000,000 were incubated at 60 C for 1 h, again vortexed and 500 lL generations and stopped when average standard deviation of chloroform: isoamylalcohol (24:1) were added followed of split frequencies fell below 0.01. The sample frequency by shaking for 2 min. Tubes were spun at 14,000 r.p.m. in a was set to 100 and the ﬁrst 25% of trees were removed as microfuge for 10 min and the upper layer was collected in burn-in. The different loci within the combined data sets new sterile tubes with 0.55 volume ice-cold iso-propanol were analysed as separate partitions. Arthroderma ﬂa- and spun again. Finally, pellets were washed with 70% vescens, a member of family Arthrodermataceae (Onyge- ethanol, air-dried and suspended in 100 lL TE buffer. nales) was used as outgroup for all analyses. Phylograms Five genomic regions were ampliﬁed using primers are shown using FIGTREE v1.3.1 (Shapiro et al. 2010). using standard PCR conditions except for partial LSU with Bootstrap values C 80% and posterior probabilities C 0.99 a cycle extension of 90 s (Liu et al. 1999; Stielow et al. were considered as statistically supported and were indi- 2015; Dukik et al. 2017b). PCR products were visualized cated above thickened branches (Figs. 2, 3, 4). on 1% agarose gels and sequenced using ABI big dye APE and SPIDER and their associated R statistical terminator v3.1. Sequencing reaction was at 95 C for package were also used to evaluate sequence data (https:// 1 min, 30 cycles of 95 C for 10 s, 50 C for 5 s and 60 C cran.r-project.org/; http://spider.r-forge.r-project.org/; for 4 min. A capillary electrophoresis system (Life Tech- Brown et al. 2012; Popescu et al. 2012). A sliding window nologies 3730XL DNA analyser) was used for bidirectional and barcoding gap analysis was conducted to identify the sequencing. Sequences were those listed in a preceding gene section that provides the highest information content study (Dukik et al. 2017b), retrieved from GenBank, or in the identiﬁcation of members in Ajellomycetaceae. The were newly generated (291, including 12 ITS, 46 LSU, 80 sliding window was set to a length of 100 nucleotides as a rPB2, 50 TEF3 and 49 TUB2) and deposited in GenBank prior and the distance metric was set as Tamura’s ‘K2P’ (Table 1); in six of eight Es. africanaus TEF3 could not be (Popescu et al. 2012; Van den Brink et al. 2015). obtained. Consensus sequences for each locus were obtained and edited using SEQMAN in the Lasergene soft- ware (DNAStar, WI, U.S.A.). Alignment was done with Results MUSCLE (Edgar 2004) using MEGA v6.0 (Tamura et al. 2013) with minor manual editing. Missing data for partial Morphology and physiology or complete sequences were recoded. A concatenated alignment was made with DATACONVERT1 for ITS, partial Colony morphologies are similar among below-described LSU, TUB2, TEF3, and rPB2. species of Emmonsia, Blastomyces and Emergomyces. On Maximum likelihood (ML) and Bayesian inference MEA at 24 C the colonies have slow to moderate growth analyses (BI) were performed using RAxML (Stamatakis and are yellowish-white to buff colored, rather compact, 2014) and MRBAYES v3.1.2 (Ronquist and Huelsenbeck sometimes with grooves (sulcate) or slightly zonate and 2003). Robustness of tree topology for each ML analysis lack exudates. Colony types are similar to those distin- was evaluated by 1000 bootstrap replicates. Suitable sub- guished by Carmichael (1951), dependent on medium and stitution models of ML trees were determined using MEGA incubation temperature. Granular colonies are powdery, v6.0. ML in the CIPRES web server (https://www.phylo.org). with low aerial mycelium and with sometimes folded sur- Bayesian analyses were performed with two sets of four face. This colony type is common among species of 123 Fungal Diversity (2018) 90:245–291 253 123 254 Fungal Diversity (2018) 90:245–291 Fig. 3 Phylogram (50% majority rule) resulting from a ML of thec Emmonsia, Emergomyces and Blastomyces and is associ- partial rPB2 gene alignment, with the conﬁdence values BS and PP ated with heavy conidial sporulation. Cottony colonies are analysis above branches ([ 50% for BS from ML analyses,[ 0.95 for dense with felty aerial mycelium, while ﬂoccose colonies PP from Bayesian). Branches with full statistical support (ML-BS C have loose aerial mycelium giving the appearance of 99%; PP C 0.99) are highlighted by thickened branches. Figures in the column at the right demonstrate the main morphologic features at sheep’s wool. The cottony colony type may be associated 24, 33 and 37 C with lower sporulation or individual strain degeneration as is also the case with glabrous colonies that are smooth to slightly hairy, ﬂat or slightly elevated, sometimes with tufts of hyphae centrally. Glabrous colonies are often produced showing relative inhibition over 35%. Both Emmonsiel- when isolates are grown at temperatures above 30–33 C lopsis species were strongly tolerant with Emms. terrestris and occur commonly among Es. africanus strains at 24 C. even growing better on SGA with cycloheximide. Positive Among species of Emmonsia, Blastomyces and Emer- urease activity occurred within 24 h at 24 and 37 Cin gomyces, vegetative hyphae are hyaline, narrow Emmonsia species and some Emergomyces species but not (2.5–4.5 lm wide), regularly septate and branched. Coni- in Blastomyces or Emmonsiellopsis species. After 7 days diophores often have a septum at the base, are slightly to incubation at these temperatures, two Emergomyces spe- moderately swollen near the tip, and often bear short sec- cies (Es. canadensis and Es. orientalis) and Emmonsiel- ondary conidiophores arising at right angles (Fig. 1). lopsis species were urease negative and urease activity was Conidia are formed by holothallic conidiogenesis and are variable among some Blastomyces strains. Almost all sessile or borne on minute pedicels at the tip or on the sides strains showed hemolysis on blood agar after 2 weeks at of the conidiophores. Conidia are solitary or occur in 37 C; results varied among Emergomyces species at chains of two to three, are single-celled, often subspherical, 24 C. smooth to ﬁnely roughened and have rhexolytic dehis- cence. Conidial formation was absent in B. helicus and in Phylogeny and barcoding three other strains: B. percursus (NCPF4091) and B. parvus (CBS 178.60 and CBS 205.48). Thin- to thicker-walled In the combined phylogenetic analysis of ﬁve gene loci, the helically coiled hyphae are sometimes present. family Ajellomycetaceae formed a well-supported lineage Growth rates and the dynamic process of conversion (ML/BI 100/1.00), with the geophilic genus Emmonsiel- over a range of temperatures were evaluated for all species lopsis in ancestral position (Clade V) (Fig. 2). Five clades with particular attention to type strains (Fig. 5, Table 2). were strongly supported as follows: Clade I (ML/BI The temperature of 33 C was determined as optimum for 95/0.98), Clade II (ML/BI 100/1.00), Clade III (ML/BI elaboration of the intermediate transition stages for species 100/0.99), Clade IV (ML/BI 100/1.00) and CladeV (ML/BI that were able to grow at 37 C or higher. Swollen, adi- 100/1.00). The topology of the tree largely corresponds aspore-like cells between 10–30 lm wide were produced at with those of Marin-Felix et al. (2015), Dukik et al. or beyond 33 C, were somewhat irregular in shape and (2017b) and Mun ˜ oz et al. (2015). No topological conﬂicts had thinner walls. Conversion to non-ﬁlamentous growth were found when comparing the reciprocal tree topologies forms in vitro occurred in nearly all strains incubated at based on the rPB2, TUB2 and TEF3 datasets. The con- 37 C. In most cases, this thermotolerant phase produced in catenated alignment consisted of 3302 characters (includ- culture resembled forms that occur in infected host tissues ing alignment gaps) with 628, 663, 1088, 370 and 536 and can be hypothesized to represent a pathogenic phase, characters used in the ITS, LSU, rPB2, TEF3 and TUB2 although pathogenicity is unknown in some species (Em- partitions, respectively. For Bayesian inference, a monsia sola, Emmonsiellopsis species). Blastomyces der- SYM?I?G model was selected for rPB2, GTR?I?G matitidis is unable to produce conidia beyond 33 C. In Ea. model for ITS, TrN ?I?G model for LSU, a K81uf ?I?G sola NCPF 4289 conidia were produced until 33 C, model for TUB2 and TEF3. Analyses of the rPB2 gene leaving a narrower temperature range of the thermotolerant region demonstrated the same ﬁve well-supported clades as phase, and conversion was more difﬁcult. revealed with the combined data, except that Clade I had Physiological characteristics were analyzed for all spe- lower bootstrap support (Fig. 3). Most species were cies with a subset of isolates (Table 2). All tested isolates resolved in the rPB2 and ITS trees (Fig. 4). TEF3 and TUB were tolerant of cycloheximide at 24 C, but the degree of demonstrated species delimitation but with lower bootstap tolerance differed among species. Blastomyces species support (not shown). Generic relationships were better varied in tolerance with B. dermatitidis having lowest resolved in the rPB2 and combined analyses, but these tolerance. Emmonsia crescens was less tolerant than B. genes individually were too variable to show relationships parvus as noted previously (Sigler 1996). Species of over the entire family. In the multigene phylogram, Emergomyces had the lowest tolerance to cycloheximide, 123 Fungal Diversity (2018) 90:245–291 255 123 256 Fungal Diversity (2018) 90:245–291 Fig. 4 Phylogram (50% majority rule) resulting from a ML analysisc Histoplasma (Clade III) was paraphyletic to Blastomyces of the ITS alignment, with the conﬁdence values BS analysis above (Clade II) (ML/BI 100/0.99). Paracoccidioides brasiliensis branches ([ 50%). Branches with full statistical support (ML-BS C (Clade IV) was distant from other genera (ML/BI 99/0.99) 80%) are highlighted by thickened branches with strong statistical support (ML/BI 100/1.00). Clade I comprises two strongly-supported subclades including strains formerly classiﬁed as Emmonsia crescens maintained species limit and we therefore keep them as (Clade I-1; ML/BI 100/1.00) and the other Emergomyces separate species pending analysis of additional strains. species (Clade I-3; ML/BI 100/1.00) (Figs. 2, 3). While a Clade II is strongly supported (ML/BI 100/1.00) and single strain CBS 142607 (= NCPF 4289) (Clade I-2) is comprises ﬁve highly supported monophyletic lineages close to Ea. crescens, there is no statistical support for the (Figs. 2, 3). One lineage includes B. dermatitidis (Clade II- sister relationship and the strain deviated from its nearest 3; ML/BI 100/1.00), the type species of Blastomyces neighbors in at least 20 nucleotide positions in ITS, and 23 together with its relative B. gilchristii (Fig. 4). A second and 16 positions in rPB2 and TUB2, respectively. Most lineage (Clade II-1; ML/BI 100/1.00) comprises the gen- strains in the Ea. crescens clade are derived from lungs of eric type species of Emmonsia, Ea. parva represented by terrestrial animal hosts except for two soil-associated the type strain (CBS 139881 = UAMH 130) which is strains (Table 1). The clade is comprised of two closely designated herein as B. parvus. The B. parvus lineage related lineages as was previously noted by Peterson and contains subgroups each containing a few strains that show Sigler (1998) on the basis of ITS-partial LSU data. Their differences in growth rates or morphologies. The lower Eurasian lineage, shown here with high support (Clade subclade includes four sublineages having moderate boot- I-1a; ML/BI 100/0.99) includes the ex-type strain of Ea. strap support (ML/BI 77/0.91). One with strong support crescens and ten other strains, while their North American (Clade II-2; ML/BI 98/1.00) includes seven strains repre- group comprising strains from Canada and the United senting a new species, B. silverae. A small intra-lineage States, is less well supported (Clade I-1b; ML/BI 65/0.97) difference was noted for B. silverae strain (CBS 509.78 = (Fig. 2). The Eurasian and North American groups differed ATCC 32539) that was sister to the main bootstrap-sup- from each other at several positions both in protein-coding ported cluster and unusual in producing conidia over a genes and the rDNA operon: 1 position in rPB2,2in wide temperature range (21–37 C). The other three sub- TUB2, 1–2 in TEF3, ITS and LSU. One strain CBS 191.55 lineages are sister to B. dermatitidis (ML/BI 93/0.99) and deviated from other strains at ﬁve positions in rPB2 and represent the recently described species B. percursus two positions in TUB2 and phenotypically in lacking (Dukik et al. 2017b) (Clade II-4; ML/BI 100/1.00) and B. sporulation and a lower maximum growth temperature of helicus (formerly Emmonsia helica Sigler, Clade II-5) 33 C. The fungi in the Ea. crescens subclades differ from comprising eight strains that varied at no more than 2 those in the Emergomyces clade in host associations, nucleotide positions in ITS, and fewer than 5 in rPB2 and pathogenicity and pathogenic cell types. TUB2. The Emergomyces subclade (Clade I-3; ML/BI 100/1.00) The genus Emmonsiellopsis (Clade V; ML/BI 100/1.00) includes ﬁve well-supported lineages encompassing species occurs in an ancestral position within the Ajellomycetaceae having small yeast-like cells as invasive forms and associ- and is phylogenetically distant to other genera. The genus ated only with human infections (Dukik et al. 2017b). comprises two environmental species initially suggested as Included are the previously described species Es. pasteuri- closely related to the pathogenic dimorphic fungi in the anus from Europe (Clade I-3c; ML/BI 100/1.00) (Drouhet family (Marin-Felix et al. 2015). Emmonsiellopsis ter- et al. 1998)and Es. africanus from southern Africa (Clade restris (ML/BI 100/0.99) is represented by the type and two I-3e; ML/BI 100/1.00) (Dukik et al. 2017b), each of which other strains (CBS 273.77 = UAMH 2304; CBS 139889 = received strong support. Another strain (CBS 102426) from UAMH 141; CBS 137499 = FMR 4023) while Emms. Germany (Clade I-3d), here described as Es. europaeus,has coralliformis comprises a single isolate (CBS 137500 = a close relationship with these two species but differs from FMR 4024). The thermodependent form is produced in Es. pasteurianus and Es. africanus in 10 positions in ITS and culture with difﬁculty and resembles that of some Blasto- in 14 vs. 19 and 29 vs. 30 positions in rPB2 and TUB2, myces species. respectively. Emergomyces orientalis (Clade I-3b) (Wang We assessed information density of the different loci et al. 2017) is closely related to two strains from Canada individually to infer: (a) which marker shows the best here designated Es. canadensis (Clade I-3a). Comparison of potential alone to recognize the described taxa, thus max- sequences reveals differences in 7 positions in ITS, 8 in imizing afﬁliation to an existing taxon e.g. in the case of an rPB2, 17 in TUB2 and 7 in TEF3. Compared to other species unknown clinical sample, and (b) which sections of the in the Ajellomycetaceae these differences exceed the here studied genes are most informative and render taxa 123 Fungal Diversity (2018) 90:245–291 257 123 258 Fungal Diversity (2018) 90:245–291 Fig. 5 Summarized cladogram of the Ajellomycetaceae, compared determined by 3 biological replicates, each with 3 technical replicates. with growth and conversion characteristics. Bold branches represent Right panel summarizes conversion at different temperatures after bootstrap support ([ 80%) in the original phylogram based on 4 weeks incubation on MEA. 0 = saprobic phase, 1–3 = Thermotol- multilocus sequencing. Left panel represents growth at temperature erant phase, at three levels of conversion. Yellow circles represent ranges after 3 weeks incubation on MEA. Mean diameters were maximum temperatures of conidiation with the highest probability as monophyletic. The R exposed a higher non-conspeciﬁc distance (inter-speciﬁc packages SPIDER and APE were used to assess these distance) over a lower intra-speciﬁc distance between the density metrics over all loci. Figure 6 outlines subse- analysed taxa. However, clear qualitative differences quently the following plots for each locus: (a) ‘congruence where observable with particular attention to (a) the of NJ (neighbor joining) trees’, (b) ‘proportion of species informativeness of the individual sequence section anal- that are monophyletic’ and (c) ‘sum of diagnostic nucleo- ysed, and (b) information density over a particular section tides’. The ﬁrst metric describes the proportion/quantity of analysed. When referring to the informativeness of the neighbor joining trees being congruent (identical) to each individual sections (NJ tree congruency/Proportion of other for a given sliding window, and thus is a consistency monophyletic species), the ranking is the following: TUB2 metric for statistical resampling. It describes the robustness [ rPB2[ ITS/TEF3 (approximately equal)[ LSU. TUB2 of the given section for a given locus to retrieve the same here provides the highest consistency over the entire species afﬁliations. The second metric describes in a sim- sequence analysed, as revealed in Fig. 6 and the corre- ilar way to the ﬁrst metric, how consistent taxa could be sponding plots for ‘NJ tree congruency/Proportion of rendered as monophyletic entities over the statistical monophyletic species’, particularly since the latter values resampling process. The third metric is quantitatively are consistently[ 0.8 from * 90–450 bp of the sequence describing how many nucleotides per window position are matrix. A similar result was obtained for rPB2, which informative/diagnostic, thus describing the information scores high in its information content. Complete rPB2 was content numerically. Values for the ﬁrst and second metric determined as slightly superior over any other of the other range from 0 to 1, with 0 no NJ trees/no species being four partial gene sections for recognition of the here congruent/monophyletic, respectively, while 1 is inter- studied fungi. In contrast to these positive results, sur- preted as the opposite, and the third metric range is from 0 prisingly only the ITS-1 of the gold standard DNA barcode to inﬁnitive. Secondly, Fig. 7 shows the non-conspeciﬁc renders NJ-trees/taxa proportionally consistent, while the K2P distances over the intra-speciﬁc K2P distances over ITS-2 section performs poorly and indicates almost a each window, in other words the ‘classical’ barcoding gap complete drop-out as is revealed in Fig. 6, and in the (Meyer and Paulay 2005). corresponding plot ‘Sum of diagnostic nucleotides’ (sec- The informativeness of the analysed loci was relatively tion * 250–550 bp). While the fungal speciﬁc TEF3 also even between the individual loci, thus none of the genes exposes a relatively high consistency, it overall ranks lower 123 Fungal Diversity (2018) 90:245–291 259 Table 2 Key physiological features of fungi in this study a b Species Urease activity after 24 h and Hemolysis Cycloheximide Growth characteristics on MEA 7 days tolerance 3 weeks 24 C37 C24 C37 C24 C37 C24 C RI (%) Max. temp S-T at 6 C 40 C conidiation 33 C 3 weeks 3 weeks (C) 3 weeks Emmonsia crescens 11 ?? ?? ? ? ? 30 24 ?? 2 (n = 8) Ea. sola (n = 1) 11 ?? ?? ? ? ? 33.8 33 2 ? 2 Emergomyces ?? 11 ?? ?? - ? ? 61.6 27 ?? 2 africanus (n = 8) Es. canadensis 2 222 -? ? 42.4 33 ?? 2 (n = 2) Es. europaeus 11 ?? ?? (?) ?? 62.9 27 ?? 2 (n = 1) Es. orientalis 2 222 -? ? 46 21 ?? 2 (n = 1) Es. pasteurianus 11 ?? ?? - ? ? 34.9 24 ?/-? 2 (n = 3) Blastomyces 22 ?? ?? ? 35 30 ? 2 ? dermatitidis (n = 1) B. helicus (n = 8) 22 ?/-?/-? ? ? 11.2 / ? 2 ?/- B. parvus (n = 7) 22 ?/- ?/-? ?/-? 12.8 33 -? ?/- B. percursus (n = 2) 22 ?? ?? ? 133 ?? 2 B. silverae (n = 7) 22 ?/-?/-? ? ? 10.3 37 2 ? 2 Emmonsiellopsis 2 222 ?? ? 14.2 33 ? 22 coralliformis (n = 1) Emms. terrestris 2 222 ?? ? - 30.4 37 22 2 (n = 3) Reactions in urea broth were negative (straw yellow), weak (?, pink), or positive (??, fuchsia) Hemolysis on blood agar after 2 weeks: ? = positive, - = negative, (?) = scant positive ? Growth on Sabouraud glucose agar with cycloheximide; - growth on plain SGA; RI% refers to the rate of inhibition at 24 C, which can be negative Conversion from saprobic to thermotolerant forms (yeast-like, adiaspore-like or adiaspores) Urease activity occurred after 8 h Usually positive; occasionally negative Sporulation absent Positive at 33 C (or similarly equal to ITS) due to a lower ratio of diagnostic Taxonomy nucleotides, e.g. when compared to TUB2. The most uninformative locus, at least at the species level, was par- Emmonsia Cif. & Montemartini – Mycobank MB8151 tial LSU; all sliding window plots revealed that only a small section, approximately between 250 and 350 bp Colonies yellowish-white to ochraceous. Hyphae hyaline. contains a sufﬁciently variable number of nucleotides to Conidiophores arising at right angles from vegetative recognize the taxa studied here. However, maximum con- hyphae, with secondary conidiophores arising from a sistency between NJ trees/monophyletic taxa is approxi- swollen tip, Conidia forming at the tip or on the sides of mately half of the best two loci TUB2/rPB2. conidiophores, or sessile on vegetative hyphae. Conidia holothallic, with rhexolitic dehiscence. Adiaspores usually 123 260 Fungal Diversity (2018) 90:245–291 Fig. 8 Emmonsia crescens (CBS 177.60). A–C Colonies on MEAc produced at 37 C, spherical with thickened walls, over 3 weeks at 24, 33 and 37 C. D–F 24 C. Slightly swollen conidio- 100 lm in diam. No growth at 40 C. phore; secondary conidiophore present. G–I 33 C. Conidiophores Comments: The genus Emmonsia was proposed with and hyphae swelling and disarticulating to form giant cells. J– Haplosporangium parvum as type species (Ciferri and M 37 C. Swollen hyphae leading to adiaspores. Scale bar = 10 lm Montemartini 1959). The type species of Emmonsia, Ea. parva, is shown here and in prior reports to be a species of Emmonsia crescens C.W. Emmons & Jellison—Ann. Blastomyces (de Hoog et al. 2016a; Dukik et al. 2017b). A N. Y. Acad. Sci. 89: 98. 1960. MycoBank MB330349 second species, Ea. crescens, did not group in Blastomyces (Fig. 8) and its status is insufﬁciently resolved in the phylogenetic trees generated by the used barcoding genes. Ea. crescens = Ajellomyces crescens Sigler—J. Med. Vet. Mycol. 34(5): requires transfer to a different genus or, potentially, con- 305. 1996. servation of the genus Emmonsia with Ea. crescens as type Type culture: CBS 177.60 = ATCC 13704 = UAMH 3008, species. We therefore postpone a name change until Hamar, Norway, isolated from lungs of rodent Arvicola investigation of the most suitable option is completed. terrestris by C.W. Emmons and W.L. Jellison, January Fig. 6 Sliding window analysis conducted with the R package process and as deﬁned in this study. C Sum of diagnostic nucleotides SPIDER, covering a 100 bp sliding-window for the analyzed partial at each window position corresponding to the degree of informative- gene sequences (multiple alignment) respectively. A Congruency of ness given a particular alignment position. Note: scaling of Y-axis is neighbor-joining (NJ) trees, when re-sampled 1000 times over each not proportional, values range from 0 (lowest) to 1 (highest) for window. B Proportion of species that are monophyletic, indicating (A) and (B) respectively, for C, number indicates quantities of quantity of discrete species entities derived from NJ re-sampling diagnostic nucleotides for a given window Fig. 7 Pairwise distance sliding window analysis of ﬁve genetic loci (B). Hypervariable alignment sections were automatically excluded, alignments (analysed individually) showing closest inter-speciﬁc as indicated by ‘gaps’ for the plot ‘proportion of species that are (orange whiskers) and intra-speciﬁc (blue whiskers) distances (A) and monophyletic’ per gene (section w140e200 bp in BT2 and proportion of monophyletic species over a 100 bp sliding window w150e230 bp in ITS region) 123 Fungal Diversity (2018) 90:245–291 261 123 262 Fungal Diversity (2018) 90:245–291 Fig. 9 Emmonsia sola (CBS 142607 = NCPF4289). A–C Colonies onc Saprobic phase (24 C, MEA, 3 weeks): Colonies 35 mm MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Conidiophore slightly diam, dense, granular to felty, yellowish-white. Conidio- swollen, secondary conidiophore present. G–I 33 C. Hyphae and phores about 1.5–3.7 (2.2 ± 0.5) lm wide at the base, with conidiophores present J–M 37 C. Giant cells often liberated with a secondary conidiophores. Conidia solitary or rarely in short hyphal extension. Scale bars: D–M =10 lm chains of two, subspherical, 2.1–4.8 9 2.4–4.9 lm (3.0 ± 0.5 9 3.7 ± 0.5, n = 44), smooth to ﬁnely roughened. Intermediate phase (33 C, MEA, 3 weeks): Colonies Comments: Emmonsia sola, known only from the type 2 mm diam, glabrous, cream, without aerial tufts. Conidia strain NCPF 4289, was identiﬁed originally as Emmonsia absent. Hyphae densely septate and somewhat swollen, parva. It was isolated from soil in the U.S.A., but precise 2.9–7.1 lm wide. Swollen hyphal cells and conidiophores strain data are lacking. Our multilocus analysis demon- sometimes becoming giant cells, 5.9–25 lm (12.6 ± 3.9, strated that this strain was close to Ea. crescens with low n = 44) or adiaspore-like cells. Thermotolerant phase bootstrap support (ML/BI 68/–). It deviated from all other (37 C, MEA, 3 weeks): Colonies 4–5 mm, glabrous to strains in this study in at least 20 positions in ITS, and in 23 slightly hairy, yellowish white. Adiaspores abundant, and 16 positions in rPB2 and TUB2, respectively. The ovoidal to spherical, abundant, 16–117 lm (69.0 ± 39.2, species differs from Ea. crescens in its habitat from soil n = 30) in diam, with smooth walls about 10 lm thick. and from all other species by (1) producing giant cells less Comments: The species is the only one that produces large than 20 lm diam with thin walls and occasional broad- thick walled adiaspores (Sigler 2005; this study) requiring based budding and requiring 4 weeks of incubation at about 2 weeks at 37 C. Intermediate morphology includes 37 C to develop; (2) having smaller conidia and longer giant cells without budding; Optimal sporulation occurs at primary conidiophores (up to 74 lm in length); (3) pro- 21–24 C; conidia absent at 30 C. Grows at 6 C, no ducing conidia at 33 C; (4) having strong cycloheximide growth at 40 C. tolerance and the strongest hemolytic reaction at 24 C. The single strain of this species had the strongest hemolytic Emmonsia sola Y. Jiang, Borman & de Hoog, sp. nov.— MycoBank MB821087 (Fig. 9) reaction at 24 C. Due to insufﬁcient taxon sampling, with only a single strain available, phylogenetic resolution Etymology: referring to soil as the species’ habitat. remained poor. Strain NCPF 4289 is preliminarily pre- Holotype: Arizona, U.S.A., specimen of culture CBS served in Emmonsia as Ea. sola. Optimal sporulation 142607 (preserved in metabolically inactive condition in occurs at 24–27 C with conidia produced up to 33 C. liquid nitrogen) from soil, sent by A. Borman, December Conversion is slow, requiring about 4 weeks to produce 2012; living strain CBS 142607 = NCPF 4289. giant cells at 37 C. Grows at 6 C; no growth at 40 C. Saprobic phase (24 C, MEA, 3 weeks): Colonies 64 mm Blastomyces Gilchrist & W.R. Stokes—J. Exp. Med. 3: 76. diam, ﬂoccose, buff to pale buff at periphery, somewhat 1898; conserved name, non Blastomyces Costantin & sulcate centrally, margin thin. Coiled hyphae sometimes Rolland—Bull. Soc. Mycol. Fr. 4: 153. 1889. MycoBank present. Conidiophores 1.1–1.7 lm (1.3 ± 0.2) wide with a MB7389. septum at the base, sometimes multiseptate, erect, rather Colonies yellowish-white to ochraceous. Hyphae hyaline, long (18–74 lm); short secondary conidiophores arising at right angles. Conidia single or in chains of two to four, sometimes helically twisted. Conidiophores arising at right angles from the vegetative hyphae, narrow, unbranched, subspherical, 1.3–2.8 9 1.7–2.8 lm (1.9 ± 0.4 9 2.2 ± 0.3, n = 46), smooth-walled to ﬁnely roughened. Inter- sometimes with secondary conidiophores arising from a swollen tip, Conidia forming at the tip or on the sides of mediate phase (33 C, MEA, 3 weeks): Colonies 57 mm diam, dense, ﬂat, cottony, buff centrally, white peripher- conidiophores, or sessile on vegetative hyphae, holothallic with rhexolytic dehiscence. At 37 C producing large ally, radially sulcate with numerous ﬁssures. Conidia and yeast-like cells with budding at a broad base or giant cells conidiophores similar to those at 24 C; conidiophores swelling near the middle and distally, becoming chlamy- that may proliferate. Growth at 37 C, some species up to 40 C. dospore-like; no transformation to other forms. Thermo- tolerant phase (37 C, MEA, 3 weeks): Colonies 6 mm Type species: Blastomyces dermatitidis Gilchrist & W.R. diam, compact, white, moist to slightly hairy. Hyphal ele- Stokes. ments moniliform, variably shaped, 2.0–5.7 lm wide. Comments: The genus Blastomyces Gilchrist and W.R. Conidiophores mostly swollen, ampulliform; septa absent Stokes has recently been conserved (de Hoog et al. 2016a) or located at the base, forming giant cells 6. 2–17.7 lm with B. dermatitidis as type species and CBS 674.68 (11.3 ± 2.4; n = 44) in diam with thin walls; sometimes (=ATCC 18187 = UAMH 3539) as epitype. Our multilocus liberated with broad-based budding. and rPB2 analyses (Figs. 2, 3) provided strong support for 123 Fungal Diversity (2018) 90:245–291 263 123 264 Fungal Diversity (2018) 90:245–291 Fig. 10 Blastomyces dermatitidis (CBS 642.72). A–C Colonies onc Clade II including Ea. parva, type species of Emmonsia, MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Swollen, ﬂexuose Ea. helica (Sigler 2015), the recently described species B. conidiophore without secondary conidiophores. G–I 33 C. Swollen, percursus (Dukik et al. 2017b) and a new species, B. sil- ﬂexuose, hyphae producing terminal yeast-like cells and giant cells verae. B. dermatitidis and B. percursus produce large yeast which may show multilateral budding. J–M 37 C. Large yeast-like cells produced multilaterally from irregularly swollen giant cells, cells with broad-based budding at 37 C. Blastomyces sil- large yeast cells abundant, showing uni- or bipolar budding at a broad verae and B. parvus mainly produce giant cells which base. Scale bars: D–M =10 lm could proliferate, as well as occasional large yeast cells with broad-based budding. Species of Blastomyces show a formed within 1 week. Optimal conidiation occurs at tolerance to higher temperature and to cycloheximide but 24–27 C. Growth occurs at 9 C, optimum 24 C, no urease activity is delayed and variably positive among growth at 42 C. strains of B. parvus and B. silverae (Table 2). No strains of Blastomyces helicus (Sigler) Y. Jiang, Sigler, Schwartz & the recently described species B. gilchristii were available de Hoog, comb. nov.—MycoBank MB821083 (Fig. 11) for study. Basionym: Emmonsia helica Sigler—Index Fungorum 237: Blastomyces dermatitidis Gilchrist & W.R. Stokes—J. 1. 2015. Exp. Med. 3: 76. 1898. MycoBank MB361754 (Fig. 10) = Emmonsia sp. 1, Schwartz et al. – PLoS Pathog. 11: = Ajellomyces dermatitidis McDonough & A.L. Lewis— e1005198, p. 2. 2015. Mycologia 60: 77. 1968. Type culture: CBS 140056 = UAMH 7101, Edmonton, Lectotype: plate VI, Gilchrist & W.R. Stokes – J. Exp. Canada, specimen of culture from sputum, blood from a Med. 3: 53–78, pls. IV–VIII. 1898; epitype culture: CBS male chronic leukemia with disseminated infection, P. 674.68 (= ATCC 18188 = UAMH 3539), U.S.A., isolated Kibsey, December 1991. from human patient, by E.S. McDonough, 1968 (de Hoog et al. 2016a). Saprobic phase (24 C, MEA, 3 weeks): Colonies 37 mm diam, yellowish white, cottony to felty, radially sulcate; Saprobic phase (24 C, MEA, 3 weeks): Colonies 41 mm reverse ochraceous-buff to warm buff peripherally. Hyphae diam, cottony to felty, white to buff at the centre, radially 1.0–2.0 lm wide, hyaline, septate, branched; yellowish sulcate, margin thin, reverse pale buff. Hyphae narrow with helically coiled hyphae often present (Fig. 11D). Conid- some spirally twisted hyphae present. Conidiophores sep- iophore-like stalks arising at right angles from hyphae, tate at the base and at conidial insertion, 1.6–4.1 (2.2 ± having a basal septum and sometimes with several septa 0.5) lm wide, unbranched slightly bent apically (Fig. 10E). (Fig. 11E, F) but no conidia are produced (Fig. 11E, F). Conidia borne singly, subspherical, 1.8–5.5 9 2.4–5.7 lm Intermediate phase (33 C, MEA, 3 weeks): Colonies (3.5 ± 0.7 9 3.6 ± 0.7, n = 45), ﬁnely roughened some- 45 mm diam, ﬂat, glabrous with radial ﬁssures, tufts of times adherent to the conidiophore. Intermediate phase hyphae occurring centrally. Hyphae somewhat swollen, (33 C, MEA, 3 weeks): Colonies 12 mm diam, cerebri- 2–4 lm, hyaline, slightly pigmented, septate at short form, slightly raised, yellowish white, dense, felty. Swol- intervals, constricted at the septa. Conidiophore-like len, bent, conidiophore-like structures emerge terminally structures swollen; giant cells 4.8–12.9 lm (7.5 ± 1.4, n = producing inﬂated yeast-like cells with mulitlateral 40) with multilateral budding, producing chains of thin- daughter cells. Some cells become giant cells with thin walled variably shaped yeast-like cells budding at a broad walls 10.6–29.2 lm (16.9 ± 4.7, n = 40) diam with some base. Thermotolerant phase (37 C, MEA, 3 weeks): multipolar budding. Yeast-like cells with broad-based Colonies 20 mm diam, folded, yeast-like, yellowish-tan. budding present (Fig. 10I) measuring 6.8–10.0 9 Large giant cells bearing spindle-shaped daughter cells 5.4–8.7 lm (9.1 ± 0.5 9 6.6 ± 0.7, n = 45). Thermotol- 4.2–9.8 9 1.3–3.2 lm (6.5 ± 1.7 9 2.1 ± 0.5, n = 40) erant phase (37 C, MEA, 3 weeks): Colonies 6 mm (Fig. 11L). Yeast cells varying in shape, budding multi- diam, glabrous, smooth, yellowish white. Swollen, bent, laterally, occurring in short branched chains, sometimes conidiophore-like structures producing large giant cells, liberating and measuring, 2.4–7.0 9 2.6–6.2 lm (5.3 ± 0.8 10.6–29.2 lm (16.9 ± 4.7, n = 40) in diam (Fig. 10L). 9 4.0 ± 0.7, n = 50). Yeast cells measuring 5.0–14.2 9 3.5–8.9 lm (8.0 ± 1.6 9 Comments: Blastomyces helicus is differentiated from all 5.8 ± 1.1, n = 40) with unipoloar, rarely bipolar budding at here described ajellomycetaceous fungi by absence of a broad base. conidia and by formation of variably shaped yeast-like Comments: B. dermatitidis is distinguished by its simple cells in short chains. The temperature for conversion is conidiophores without secondary conidiophores in contrast low, starting at 33 C, and conversion time is short, taking to other Blastomyces species, Ea. crescens and Emer- about 1–2 weeks to produce the yeast-like stage at 37 C. gomyces species. Giant cells are produced as initial stages, Grows at 9 C, optimum 30 C, grows at 40 C. while at 37 C abundant broad-based budding cells are 123 Fungal Diversity (2018) 90:245–291 265 123 266 Fungal Diversity (2018) 90:245–291 Fig. 11 Blastomyces helicus (CBS 139874). A–C Colonies on MEAc All strains of this species (n = 8) originate from North 3 weeks at 24, 33 and 37 C. D–F 24 C. Helically coiled and America. Our phylogenetic data show that B. helicus is swollen hyphae present, conidia absent. G–I 33 C. Structures similar sister to B. dermatitidis and the species have a number of and giant cells with thin walls and multilateral budding sometimes features in common. Both are thermophilic. All strains of present. J–M 37 C. Giant cells bearing spherical daughter cells over the surface, with spindle-shaped daughter cells. Yeast-like cells with B. helicus grow better at 33 C than at 24 C, and at 37 C, multilateral budding at a broad base. Scale bars: D–M =10 lm show the most proliﬁc growth of all species studied attaining 20 mm in diam on MEA in 3 weeks. Strains CBS peripherally. Hyphae 0.9–3.0 lm wide, with few spirally 140058 (= UAMH 11034), CBS 139874 (= UAMH 3398) twisted hyphae. Conidiophores arising at right angles from and CBS 139875 (= UAMH 11294) were able to grow at vegetative hyphae, with septa at the base; conidiophores 40 C and beyond. Blastomyces helicus is characterized by less than 1.5 lm in width, slightly swollen at the tip, absence of conidia in the ﬁlamentous phase, only spirally occasionally with secondary conidiophores on which coiled hyphae with some brownish pigmentation being conidia are formed at the tip or on the sides. Conidia present, and in the thermotolerant phase strains producing solitary, rarely in short chains. subspherical, 1.6–3.7 9 yeast-like cells (Fig. 11L). After prolonged incubation 1.9–4.1 lm (2.7 ± 0.42 9 2.8 ± 0.5, n = 44), smooth to (4–5 weeks) at 24–27 C some dumbbell-shaped, spherical ﬁnely roughened, sometimes adherent to the conidiophore. or ovoidal conidium-like cells were produced on MEA Intermediate phase (33 C, MEA, 3 weeks): Colonies (Fig. 11E), consistent with observations of Sekhon et al. 39 mm diam, in macro- and microscopic details nearly (1982) who mentioned dumbbell-shaped conidia after identical to 24 C. Conidia 1.5–3.8 9 1.5–4.0 lm (2.4 ± several weeks on cereal agar at 25 C. The latter author 0.6 9 2.6 ± 0.6, n = 50). Thermotolerant phase (37 C, reported the infection caused by strain CBS 139874 as a MEA, 3 weeks): Colonies 8 mm diam, tan, compact, moist case of blastomycosis. The strain CBS 139874 showed to slightly hairy. Hyphal elements short, swollen, neurotropism, being isolated from spinal ﬂuid; also a sec- 2.3–4.1 lm wide, hyaline, septate at short intervals, con- ond isolate, CBS 139877 (= UAMH 11718) originated stricted at the septa; some cells enlarging to become giant from human CSF from a patient with brain infection. cells with thin walls, 4.7–15.7 lm (9.3 ± 2.3, n = 44) wide Central nervous system involvement is known to occur in or forming occasional yeast-like cells with broad-based B. dermatitidis (Bariola et al. 2010) and B. percursus buds (Fig. 12M). (Dukik et al, 2017b). A further clinical similarity is the Comments: No type material was designated by Emmons occurrence of disease in animals. While B. dermatitidis and Ashburn so a lectotype and epitype are proposed disease predominates in dogs (Baumgardner et al. 1995), herein. The B. parvus lineage contains subgroups each canine infection with B. helicus also seems to occur: CBS containing a few strains that show differences in growth 140058 and CBS 140059 of B. helicus were isolated from rates or morphologies. Strains in one subcluster (CBS lungs of dogs. Feline infections caused by B. helicus have 139880, CBS 139883, CBS 204.48) produce conidia also been reported (Schwartz et al. 2017). abundantly at 21–33 C, while strains of a second (CBS 178.60, CBS 205.48) did not sporulate and grew faster at Blastomyces parvus (Emmons & Ashburn) Y. Jiang, Sigler 37 C. Strains demonstrate high tolerance to cyclohex- & de Hoog, comb. nov. – MycoBank MB821085 (Fig. 12) imide, with growth at 37 C, while other species of Blas- Basionym: Haplosporangium parvum Emmons & Ashburn – tomyces were inhibited at this temperature. The Publ. Health Rep., Wash. 57: 1719. 1942 : Emmonsia parva thermotolerant phase of B. parvus has traditionally been (Emmons & Ashburn) Cif. & Montemartini – Mycopath. interpreted as small adiaspores (Dowding 1947; Emmons Mycol. Appl. 10: 314. 1959 : Chrysosporium parvum and Jellison 1960; Dvor ˇak et al. 1973; Sigler 2005), but the (Emmons & Ashburn) Carmichael var. parvum (Emmons & cells produced at 37 C have occasional broad-based Ashburn) Carmichael – Can. J. Bot. 46: 1164. 1962. budding. In contrast, the thick-walled adiaspores of Ea. Lectotype: Fig. 1 in C.W. Emmons & L.L. Ashburn – Publ. crescens represent a terminal phase without further repro- Health Rep. 57: 1720. No. 46; epitype: Arizona, U.S.A., duction. Transfer temperature is high, at least 36 C, con- specimen of culture CBS 139881 (preserved in metaboli- version time is long, taking about 4 weeks. Optimal cally inactive condition in liquid nitrogen), isolated from sporulation temperature: 24–27 C; grows at 6 and 40 C. lungs of rodent, C.W. Emmons, November 1946; living Blastomyces percursus Dukik, Mun ˜ oz, Sigler & de Hoog – strain CBS 139881 = UAMH 130. Mycoses 60: 306. 2017. MycoBank MB817662 (Fig. 13) Saprobic phase (24 C, MEA, 3 weeks): Colonies 50 mm Type culture: CBS 139878 = Kemna 408-93 = UAMH diam, white, cottony to glabrous with hyphae tufts in 7425 = UAMH 7426, Israel, specimen of culture from centre, radially sulcate, sometimes cracking the agar, granulomatous lesion on lip of otherwise healthy patient margin ﬂat, thin, reverse warm-buff to light buff 123 Fungal Diversity (2018) 90:245–291 267 123 268 Fungal Diversity (2018) 90:245–291 Fig. 12 Blastomyces parvus (CBS 139881). A–C Colonies on MEAc with disseminated infection, isolated by I. Polachek, 3 weeks at 24, 33 and 37 C. D–F 24 C. Slightly swollen conidio- November 1993. phore with secondary conidiophore. G–I 33 C. Conidiophores and conidia. J–M 37 C. Swollen hyphae; some cells becoming giant Saprobic phase (24 C, MEA, 3 weeks): Colonies 51 mm cells with thin walls; large yeast-like cells with thick walls present, diam, white, sectoring, glabrous to ﬂoccose, with tufts of rarely showing broad-based budding. Scale bars: D–M =10 lm hyphae centrally, radially sulcate, reverse warm-buff to light buff periperally. Hyphae 1.1–2.8 lm wide, with few Currah, 3 May 1983; living strain CBS 139885 = UAMH spirally twisted hyphae. Conidiophores septate near the 4470. base and below the conidium, often with an intercalary Saprobic phase (24 C, MEA, 3 weeks): Colonies 68 mm swelling and measuring 1.6–4.1 lm (2.2 ± 0.6) in width; diam, yellowish-white, ﬂoccose, radially sulcate; margin secondary conidiophores present. Conidia solitary, forming thin 3–5 mm wide, reverse warm-buff to light buff in groups of 3–4 on swollen conidiophores, subspherical, peripherally. Few spirally twisted hyphae present. Conid- 1.5–4.4 9 1.7–4.6 lm (2.7 ± 0.6 9 2.6 ± 0.5, n = 45), iophores cylindrical, about 1 lm wide at the base, with smooth-walled to slightly roughened, adherent to the basal septum, often swollen near the tip, secondary coni- conidiophore. Yeast-like cells arising from vegetative diophores present. Conidia sessile, occasionally on pedicels hyphae (Fig. 13F), sessile, spherical, 3.0–8.0 lm (5.1 ± when alongside hyphae, spherical, 2.2–5.6 9 1.8–5.6 lm 1.1, n = 45), budding from narrow base. Intermediate (3.9 ± 0.6 9 3.8 ± 0.7, n = 50), smooth or slightly phase (33 C, MEA, 3 weeks): Colonies 20 mm diam, roughened, often adherent. Some broad-based budding velvety, cerebriform, tan to greyish-white. Hyphal ele- cells may be present. Intermediate phase (33 C, MEA, ments moniliform 3.5–5.5 lm wide. Conidiophore-like 3 weeks): Colonies restricted 16 mm diam, glabrous with structures (Fig. 13G, H) composed of swollen cells and central tufts of hyphae centrally; margin ﬁmbriate. Hyphal arising from intercalary swollen cells 3.7–6.6 lm, some- elements moniliform, 3.7–4.5 lm wide. Conidiophores times producing conidia multilaterally; conidia 1.9–4.7 9 cylindrical, sometimes swollen, 2.0–4.6 lm wide. Conidia 2.2–4.4 lm (3.2 ± 0.6 9 2.6 ± 0.4, n = 45), smooth-walled larger than at 24 C, subspherical, 3.0–5.6 9 2.4–5.7 lm to ﬁnely roughened, sessile. Some giant cells present, (4.4 lm ± 0.7 9 3.8 ± 0.7, n = 45), smooth-walled to 7.2–24.0 lm (12.8 ± 3.2, n = 40) diam. Thermotolerant slightly roughened, adherent to supporting structures. Some phase (37 C, MEA, 3 weeks): Colonies 6 mm, smooth, broad-based budding or giant cells may be present. Ther- yeast-like, yellowish-white. Hyphal elements swollen pro- motolerant phase (37 C, MEA, 3 weeks): Colonies ducing giant cells 7.2–24.0 lm (12.8 ± 3.2, n = 40) diam restricted, 4–5 mm, cerebriform, glabrous to slightly hairy, and hyphae fragmenting to become large subspherical yellowish-white to buff; hyphal elements partly swollen, yeast cells 5.2–12.2 9 2.4–6.5 lm (8.1 ± 1.7 9 4.8 ± 0.9, 2.3–4.1 lm wide, densely septate. Conidiophores simple or n = 45), with uni- or bipolar budding at a broad base. absent. Conidia identical to those at 33 C. Some thick- Comments: Dukik et al. (2017b) considered B. percursus as walled, broad-based budding cells or giant cells with thin being closely related to B. dermatitidis and that is veriﬁed walls measuring 9.2–28.7 lm (13.3 ± 3.3, n = 44) may be in our multilocus analyses. The two available strains, both present. from human sources, grouped with high support in all Comments: Some strains in B. silverae were identiﬁed analyses. One strain failed to sporulate (CBS 142605 = initially as Ea. parva (Sigler 1996) but analysis of partial NCPF 4091). The species is differentiated by producing ITS and LSU sequences (Peterson and Sigler 1998) and large yeasts (greater than 5 lm in length) mostly from results of our study determined that they cluster separately fragmenting hyphae within 2 weeks of incubation at 37 C. from Ea. parva. Blastomyces silverae produces conidia At lower temperatures (24–33 C), characteristic, spheri- over a temperature range of 21–37 C; giant cells and cal, sessile yeast-like cells are present along hyphae. The yeast-like cells are produced at 36–37 C but conversion optimal sporulation temperature is 21–24 C, conidia being time is slow, requiring about 3–4 weeks. One strain (CBS absent at 30 C. Growth occurs at 9 C, optimum 27 C, no 139871 = UAMH 4489) deviated by the producing dif- growth at 40 C. fusing brown pigment when grown at 24 C. A seventh Blastomyces silverae Y. Jiang, Sigler & de Hoog, sp. strain (CBS 509.78 = ATCC 32539) was sister to the main nov.—MycoBank MB821084 (Fig. 14) bootstrap-supported cluster, and it showed some pheno- typic differences. The strain did not grow at 6 C and Etymology: named after Canadian mycologist Eleanor formed giant cells more quickly at 37 C. Four strains Silver Keeping (nee Dowding). come from human and animal sources but pathogenicity Holotype: Alberta, Canada, specimen of culture CBS has not been demonstrated for this species. Optimum 139885 (preserved in metabolically inactive condition sporulation temperature is 24–27 C, with conidia present under liquid nitrogen) from coyote dung, isolated by R.S. until 37 C. Growth occurs at 6 C, no growth at 40 C. 123 Fungal Diversity (2018) 90:245–291 269 123 270 Fungal Diversity (2018) 90:245–291 Fig. 13 Blastomyces percursus (CBS 139878). A–C Colonies onc Emergomyces Dukik, Sigler & de Hoog—Mycoses 60: MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Conidiophores and 304. 2017. MycoBank MB818569. secondary conidiophores, some yeast cells arising from vegetative hyphae. G–I 33 C. Swollen, ampulliform conidiophores and conidia Colonies yellowish-white. Hyphae hyaline. Conidiophores present, some becoming giant cells with thin or thick walls. J– short, unbranched, arising at right angles from vegetative M 37 C. Swollen hyphae, septate at short intervals, constricted at the hyphae, slightly swollen at the top. Conidia holothallic, on septa, fragmenting to become large yeast-like cells with uni- or short stalks. At 37 C small yeast cells are produced, less bipolar budding at a broad base. Scale bars: D–M =10 lm than 5 lm in diam, budding at narrow bases; large, broad- based budding cells occasionally present. No growth at 40 C. adiaspore-like cells, 3.9–10.0 lm (5.7 ± 1.3, n = 44) Type species: Emergomyces pasteurianus (Drouhet, Gue ´ho (Fig. 15H, I) or from tip of conidiophore-like structures, & Gori) Dukik, Sigler & de Hoog ovoidal, 1.7–5.3 9 0.9–2.2 lm (2.9 ± 0.7 9 1.6 ± 0.3, n = Comments: Emergomyces was described recently for Em- 45). Thermotolerant phase (37 C, MEA, 3 weeks): monsia pasteurianus and Es. africanus (Dukik et al. Colonies 7 mm diam, yeast-like, cerebriform, yellowish- 2017b). Our data show these species grouping with two white. Hyphae scant. Yeast cells abundant, ovoidal to novel species in a well-supported lineage (Clade I-3, subspherical, 1.7–5.3 9 0.9–2.2 lm (2.9 ± 0.7 9 1.6 ± Fig. 2) (ML/BI 100/1.00). Strains of all species come from 0.3, n = 45) with unipolar budding at a narrow base. human specimens (Table 1) and produce small yeast cells Comments: The species is distinguished by development of as the thermotolerant phase. A second strongly supported secondary conidiophores which lead to a complex cluster subclade within Clade I comprises Emmonsia crescens of four to eight conidia and production of small-celled (Figs. 2, 3). These subclades are supported by ecological yeasts at 37 C within 1 week. Optimal sporulation tem- and phenotypic differences distinguishing Emergomyces perature is 21–24 C; conidia are absent at 30 C. Grows at from Emmonsia crescens, i.e. prevalence of human rather 6 C, optimum 24 C, no growth at 40 C. than animal (largely rodent) hosts and conversion to yeasts Emergomyces canadensis Y. Jiang, Sigler & de Hoog, sp. rather than adiaspores. Emergomyces species are associated nov.—MycoBank MB821102 (Fig. 16) with disease in immunocompromised hosts especially among those with HIV infection (Schwartz et al. 2015a, b). = Emmonsia sp., Sigler et al. – J. Clin. Microbiol. 36: 2920. Emergomyces africanus Dukik, Kenyon, Schwartz, = Emmonsia sp. 2, Schwartz et al. – PLoS Pathog. 11: Govender & de Hoog—Mycoses 60: 305, 2017. MycoBank e1005198, p. 2. 2015. MB818571 (Fig. 15) Etymology: referring to the country of origin of the type = Emmonsia sp., Kenyon et al. – N. Engl. J. Med. 369: specimen. 1416. 2013. Holotype: Saskatchewan, Canada, specimen of culture = Emmonsia sp. 5, Schwartz et al. – PLoS Pathog. 11: CBS 139872 (preserved in metabolically inactive condition e1005198, p. 2. 2015. under liquid nitrogen) from skin lesions of HIV-positive Type culture: CBS 136260, New Somerset Hospital, Cape patient, H. Congly, June 1992; living strain CBS 139872 = Town, South Africa, isolated from skin biopsy of an HIV- UAMH 7172. infected male, collected by N.P. Govender, 11 June 2010. Saprobic phase (24 C, MEA, 3 weeks): Colonies 30 mm Saprobic phase (24 C, MEA, 3 weeks): Colonies 26 mm diam, yellowish white, cottony to glabrous, with tufts of diam, glabrous to ﬂoccose centrally, yellowish white, hyphae centrally, radially sulcate, reverse ochraceous-buff radially sulcate, reverse warm-buff to light buff peripher- to warm-buff peripherally. Few helical hyphae present. ally. Few helical hyphae present. Conidiophores 0.9–3.3 Conidiophores 1.4–2.5 lm (1.7 ± 0.3) wide with septum at (1.6 ± 0.3) lm wide with a septum at the base and at the base, cylindrical or slightly swollen in the middle and at conidial insertion; moderately swollen at the tip with 4–8 the tip, bearing 1–2 conidia on narrow pedicels (\ 1 lm conidia borne on narrow pedicels. Conidia single or in long). Conidia subspherical, smooth to slightly roughened short chains (2–4), subspherical, 1.2–3.2 9 1.7–3.8 lm 2.1–3.8 9 1.8–3.4 lm (2.8 ± 0.4 9 2.7 ± 0.3, n = 45). (2.2 ± 0.5 9 2.7 ± 0.5, n = 45), smooth to ﬁnely rough- Intermediate phase (33C, MEA, 3 weeks): Colonies ened. Intermediate phase (33 C, MEA, 3 weeks): 9 mm diam, cerebriform, felty, yellowish-white. Hyphae Colonies 14 mm, yeast-like, smooth, yellowish-white. broader 2.8–5.8 lm wide. Conidiophores mostly swollen, Hyphae densely sepate, somewhat moniliform, 1.6–3.0 lm ampulliform; septa absent or present at the base, wide. Conidia absent. Conidiophore-like cells arising from 1.7–4.6 lm wide. Conidia spherical, 2.3–7.1 lm (4.4 ± hyphae, 5–15 9 1.5–2.2 lm (Fig. 15G). Small ovoidal 1.1, n = 45). Hyphal cells and conidia in part swelling and yeast-like cells produced from irregularly shaped becoming giant cells, 5.3–11.3 lm (7.8 ± 1.6, n = 40). 123 Fungal Diversity (2018) 90:245–291 271 123 272 Fungal Diversity (2018) 90:245–291 Fig. 14 Blastomyces silverae (CBS 139879). A–C Colonies on MEAc Small yeast cells present, some originating by budding 3 weeks at 24, 33 and 37 C. D–F 24 C. Slightly swollen conidio- from giant cells. Thermotolerant phase (37 C, MEA, phore with secondary conidiophore. G–I 33 C. Swollen conidio- 3 weeks): Colonies restricted, about 3 mm, yeast-like, phores and conidia. J–M 37 C. Conidia, giant cells with thin or thick smooth, yellowish-white. Yeast cells abundant, spherical, walls, large yeast-like cells with thick walls and broad-based budding. Scale bars: D–M =10 lm 2.2–4.8 lm (3.5 ± 0.63, n = 45) with uni- or bipolar budding at narrow base. Few short, swollen hyphal ele- ments and giant cells present. Colonies 10 mm diam, yeast-like, pasty, cerebriform, tan. Comments: Emergomyces canadensis is closely related to Hyphae scant, densely septated and somewhat inﬂated, Es. orientalis (Wang et al. 2017; Figs. 2, 3) and both 1.8–4.8 lm wide, forming slightly swollen conidiophore- species come from human sources (in Canada and China). like cells 1.2–3.8 lm wide. Conidia absent. Small yeast At 37 C they both produce small spherical yeast cells cells present with uni- or bipolar budding from narrow below 5 lm in diam. Urease broth test remained negative base, ovoidal, 2.6–5.9 9 1.7–3.8 lm (3.7 ± 0.6 9 2.6 ± even after a week of incubation, and a red pigment is 0.5, n = 45), developing from swollen conidiophore-like produced on BHI and TSA at 37 C. Their distinction as cells or from giant cells 4–10 lm (7.2 ± 1.3, n = 44). separate species is supported by differences between their Thermotolerant phase (37 C, MEA, 3 weeks): Colonies sequences, including 7 nucleotide positions in ITS, 8 in strongly inhibited, 4 mm diam, with features similar to rPB2 and 17 in TUB2,7 in TEF3, and by phenotypic dif- those at 33 C. Swollen hyphae and giant cells present; ferences. Optimal sporulation temperature for Es. yeast cells ovoidal to subspherical, 2.6–5.9 9 17–3.8 lm canadensis is 24–27 C, while Es. orientalis produced (3.7 ± 0.6 9 2.6 ± 0.5, n = 45), with uni- or bipolar conidia only at 21 C. Emergomyces canadensis produces budding at a narrow base. small budding yeast cells at 33 C and time for conversion Comments: This species is known from a single strain from at 37 C is fast (1 week), while Es. orientalis budding a human source and the isolate was thought initially to be occurs at lower temperature ([ 30 C) and conversion at Es. pasteurianus but its conidial phenotype was different 37 C is slow (2 weeks). Compared to remaining species in (Wellinghausen et al. 2003). In our multilocus analyses, Es. the Ajellomycetaceae these differences exceed the main- europaeus differs from Es. pasteurianus and Es. africanus tained species limit. Despite the near-absence of pheno- in 10 positions in ITS, and in (14, 19) and (29, 30) positions typic characters we therefore keep them as separate species in rPB2 and TUB2. The three species are similar at 37 C pending availability of more material. in producing ovoidal to subspherical yeast cells of less than 5 lm in diam, but the saprobic phases differ. Emergomyces Emergomyces europaeus Y. Jiang, Sigler & de Hoog, sp. europaeus has conidiophores without secondary branches nov. – MycoBank MB821103 (Fig. 17) and conidia are larger and more roughened than those of = Emmonsia sp., Wellinghausen et al. – Int. J. Med. other species. Yeast transformation occurs at a lower Microbiol. 293: 441–445. 2003. temperature (33 C). The Es. europaeus strain is the only = Emmonsia sp. 6, Schwartz et al. – PLoS Pathog. 11: one among all Emergomyces species in its source from an e1005198, p. 2. 2015. immunocompetent individual, although the patient had Etymology: referring to the origin of the type specimen. been on prolonged low-dose corticosteroid therapy. This Holotype: Ulm, Germany, specimen of culture CBS strain was isolated from a lung lesion rather than from skin, 102456 (preserved in metabolically inactive condition which is more common in the other two species. Transfer under liquid nitrogen) from transbronchial biopsy of time is short, only 1 week being needed for total conver- 64-year-old male farmer with chronic granulomatous lung sion at 37 C .The optimal sporulation temperature is infection, G. Haase, 2003; living culture CBS 102456 = 21–24 C; conidia are absent at 30 C. Grows at 6 C, UAMH 10427. optimum 24 C, no growth at 40 C. Saprobic phase (24 C, MEA, 3 weeks): Colonies 30 mm Emergomyces orientalis P. Wang, Y.C. Xu & H.W. Fan— diam, dense, white, felty to ﬂoccose, radially sulcate, Mycoses 60: 316. 2017. MycoBank MB517245 (Fig. 18) glabrous at the margin; reverse warm-buff to light buff = Emmonsia sp. 7, Schwartz et al. – PLoS Pathog. 11: periphally. Few helical hyphae present. Conidiophores e1005198, p. 2. 2015. unbranched, 0.9–2.3 lm (1.6 ± 0.2) wide, with septum at Type culture: CBS 124587 = Peng 5Z489 = the base, cylindrical to slightly swollen at the tip, bearing CGMCC2.4011, Shanxi, China, isolated from sputum of a one or two conidia. Conidia subspherical 2.9–5.7 9 64-year-old male with type-2 diabetes mellitus by P. Wang, 3.0–5.7 lm (3.9 ± 0.6 9 4.2 ± 0.7, n = 45) slightly roughened. Intermediate phase (33 C, MEA, 3 weeks): 123 Fungal Diversity (2018) 90:245–291 273 123 274 Fungal Diversity (2018) 90:245–291 Fig. 15 Emergomyces africanus (CBS 139543). A–C Colonies onc Saprobic phase (21 C, MEA, 3 weeks): Colonies 23 mm MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Slightly swollen diam, yellowish white, felty with hyphal tufts centrally, conidiophore without secondary conidiophores. G–I 33 C. Hyphae radially sulcate, reverse ochraceous-buff to warm-buff short and swollen, disarticulating to liberate giant cells. J–M 37 C. peripherally. Coiled hyphae frequently present (Fig. 18F). Scant, swollen hyphae, abundant small yeast cells with unipolar budding at narrow base. Scale bars: D–M =10 lm Conidiophores cylindrical or slightly swollen in the middle, with a septum at the base, 1.7–3.7 (2.8 ± 0.7) lm, with 1–2 thin secondary conidiophores. Conidia subsphaerical, 1.1–2.8 9 1.7–4.8 lm (1.9 ± 0.4 9 2.8 ± 0.6, n = 40), branches, with thickened walls and often with a median smooth to slightly roughened; absent at 24 C. Interme- septum. Intermediate phase (33 C, MEA, 3 weeks): diate phase (33 C, MEA, 3 weeks): Colonies 12 mm Colonies 21 mm diam, cerebriform, yellowish white. diam, yeast-like, cerebriform, yellowish-white. Hyphae Hyphae scant and ampulliform conidiophore-like cells somewhat inﬂated, 2.7–5.0 lm wide, constricted at septa. becoming somewhat inﬂated and disarticulating. Conidia Part of inﬂated hyphal cells and conidiophores swelling to absent. Thermotolerant phase (37 C, MEA, 3 weeks): become giant cells, 5.7–12.7 lm (8.5 ± 1.7, n = 26) diam, Colonies restricted, 8 mm diam, yeast-like, cerebriform, sometimes with narrow-based budding. Yeast cells spher- yellowish white. Hyphae scant, moniliform, some cells ical, 2.0–4.5 (3.4 ± 0.6, n = 45) diam. Thermotolerant becoming giant cells, 5.4–12.0 lm (8.4 ± 1.6, n = 42) wide phase (37 C, MEA, 3 weeks): Colonies restricted, 5 mm (Fig. 19K). Yeast cells arising from giant cells or from diam, yeast-like, cerebriform, yellowish white. Hyphal fragments of swollen conidiophores or hyphae; small elements scant. Yeast cells spherical, 2.0–4.5 (3.4 ± 0.64, yeasts with narrow-based budding, 2.1–5.1 9 1.6–4.2 lm n = 45) diam, with uni- or bipolar budding at a narrow base. (3.7 ± 0.7 9 2.9 ± 0.6, n = 44); larger yeasts 5.0–11.2 9 Few giant cells present. 2.4–6.3 lm (8.0 ± 1.79 4.7 ± 0.80, n = 40), with uni- or Comments: This species, known only from the type strain bipolar budding from narrow or broad bases, (CBS 124587), produced conidia at 21 C on MEA but not Comments: Emergomyces pasteurianus was ﬁrst reported at 24 C. Conversion to yeast stage occurs at lower tem- from a case of disseminated infection with secondary perature (33 C) and time for transformation to yeast at cutaneous lesions in a female with advanced HIV disease 37 C is short (1 week). See comments under Es. and a history of injection drug use in Italy in 1994 (Gori canadensis for comparisons between these two species. et al. 1998). The species produces small yeasts with narrow Grows at 6 C, optimum 24 C, no growth at 40 C. based budding and large yeasts with broad-based budding at 37 C, characteristic of Emergomyces species and Emergomyces pasteurianus (Drouhet, Gueho & Gori) Blastomyces species, respectively. Conversion to yeast is Dukik, Sigler & de Hoog – Mycoses 60: 304. 2017. slower (2–3 weeks) and occurs at higher temperature than MycoBank MB517245 (Fig. 19) in Es. africanus and Es. europaeus. Conidia occur at : Emmonsia pasteuriana Drouhet, Gue ´ho & Gori – J. 21–24 C but are absent at 30 C, except strain CBS Mycol. Med. 8: 70. 1998. 140361 that deviates by conidiation until 33 C and for- = Emmonsia sp. 4, Schwartz et al. – PLoS Pathog. 11: mation of a brown pigment. Growth at 6 C, optimum e1005198, p. 2. 2015. 24 C, no growth at 40 C. Type culture: CBS 101426 = UAM H9510 = NCPF 4236 = Emmonsiellopsis Marın, Stchigel, Guarro & Cano – IP 2310.95, Italy, isolated from disseminated cutaneous Mycoses 58: 455. 2015. MycoBank MB811334 disease in a 40-year-old HIV? female by E. Gue ´ho, April Type species: Emmonsiellopsis terrestris Marı ´n, Stchigel, Guarro & Cano. Saprobic phase (24 C, MEA, 3 weeks): Colonies 38 mm Colonies whitish to cream, pale brownish near the centre. diam, yellowish white, dense, felty to ﬂoccose, radially Hyphae hyaline, sometimes contorted to dendritic. Conidia sulcate, with thin margin, reverse ochraceous-buff to warm ovoidal to clavate, smooth walled and verrucose or spin- buff peripherally. Few helical hyphae present. Conidio- ulose born laterally or terminally on conidiophores, sessile phores with septa at the base and at conidial insertion, at on vegetative hyphae or on pedicels. base 0.6–1.7 lm (1.2 ± 0.3) wide, cylindrical or moder- Comments: The genus Emmonsiellopsis was differentiated ately swollen at the tip. Conidia formed singleton narrow from other members of Ajellomycetaceae in part by absence pedicels (ca. 1 lm long) or in short chains (2–4), sub- of thermal dimorphism (Marin-Felix et al. 2015). Its distinc- spherical, 0.9–2.8 9 1.8–3.2 lm (2.0 ± 0.4 9 2.5 ± 0.4, tion as a separate genus is supported in our multilocus anal- n = 45), smooth-walled to ﬁnely roughened. Some ysis (ML/BI 100/0.99). In the present study both species were chlamydospore-like cells arising terminally on short lateral found to transform to large yeast-like cells. 123 Fungal Diversity (2018) 90:245–291 275 123 276 Fungal Diversity (2018) 90:245–291 Fig. 16 Emergomyces canadensis (CBS 139872). A–C Colonies onc Emmonsiellopsis coralliformis Mar´ ın, Stchigel, Guarro & MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Conidiophores, Cano – Mycoses 58: 455. 2015. MycoBank MB811335 conidia and secondary conidiophores. G–I 33 C. Swollen conidio- (Fig. 20) phores and subspherical conidia; hyphal cells and conidia in part swell and become giant cells with thin walls. J–M 37 C. Scant, Type culture: CBS 137500 = FMR 4024 (holotype: CBS swollen hyphae, abundant yeast cells with budding at narrow base. H-21624), Girona, Spain, ex ﬂuvial sediment, P. Vidal, Scale bars: D–M =10 lm June 1991. cerebriform, glabrous to hairy, greyish-white. Hyphal ele- Saprobic phase (24 C, MEA, 3 weeks): Colonies 35 mm ments moniliform. Conidia 3.0–7.7 9 2.8–6.9 lm (5.3 ± diam, ﬂoccose, somewhat cerebriform to sulcate, yellowish 1.1 9 4.3 ± 1.0, n = 45), enlarging to form giant cells white, reverse warm-buff. Conidiophores cylindrical, about 5.7–23.0 lm (14.4 ± 4.3, n = 44) diam; with uni- or 1 lm wide, somewhat ﬂexuose (20F), lacking secondary bipolar, sometimes multilateral, budding at a broad base. conidophores. Conidia solitary, mostly sessile, subspheri- Conversion reluctant, taking almost 4–5 weeks on MEA. cal, 2.9–4.9 9 2.3–4.6 lm (3.9 ± 0.5 9 3.6 ± 0.60, n = Comments: Emmonsiellopsis species are similar in lacking 45), roughened to spinulose. Intermediate phase (30 C, secondary conidiophores. Emmonsiellopsis terrestris dif- MEA, 3 weeks): Colonies 21 mm diam, yellowish white, fers from Emms. coralliformis in conversion temperature at yeast-like to slightly hairy, cerebriform. Conidia-like cells or beyond 37 C but time of conversion is slow in both swollen, 3.1–6.5 9 2.5–5.4 lm (4.6 ± 0.8 9 3.7 ± 0.7, n = species (4–5 weeks). Optimal sporulation temperature: 45); some with broad-based budding. Thermotolerant 24–30 C. Growth occurs at 9 C, optimum 21 C, no phase (33 C, MEA, 3 weeks): Colonies restricted 5 mm growth at 40 C. diam, yellowish white, yeast-like, cerebriform. Hyphal cells and conidia becoming swollen, forming giant cells, 7.1–17.3 lm (10.7 ± 2.2, n = 45) diam, with some broad- Discussion based budding, uni- or bipolar, occasionally multilateral. Conidia spherical, 2.0–3.8 lm (3.0 ± 0.4, n = 45) diam Over the last two decades, several new genera and species often sessile on supporting structures (Fig. 20K). have been added to the family Ajellomycetaceae suggest- Comments: This species is known only from the type strain ing the need for a comprehensive review of phylogenetic which has a lower transformation temperature, starting at relationships and morphological features among taxa in 30 C with formation of giant cells, which develop into this important family of vertebrate pathogens. In particular, thicker-walled adiaspore-like cells (less than 20 lm diam) several fungi having emmonsia-like morphologies have that may bud at a broad base (33 C). Conversion time is emerged as causative agents of human disease in case slow (4–5 weeks). Grows at 9 C, optimum 24 C, no reports globally (Schwartz et al. 2015a, b; Dukik et al. growth above 33 C. 2017b) (Fig. 22). In the present paper we resolved phylo- Emmonsiellopsis terrestris Marı ´n, Stchigel, Guarro & genetic relationships among Emmonsia and emmonsia-like fungi, which proved to be closely similar to Blastomyces Cano – Mycoses 58: 456. 2015. MycoBank MB811598 (Fig. 21) and Emergomyces and to a less extent to other members of Ajellomycetaceae, using concatenated sequence data of the Type culture: CBS 273.77 = FMR 13882 = UAMH 2304 loci LSU, ITS, TUB2, TEF3, and rPB2. (holotype CBS H-22118), Phillips County, Kansas, U.S.A., As shown in earlier phylogenetic overviews (Peterson ex barnyard, C.W. Emmons, Jan. 1965. and Sigler 1998; Brown et al. 2013, Schwartz et al. 2015b), the generic type species Emmonsia parva, with epitype Saprobic phase (24 C, MEA, 3 weeks): Colonies 58 mm strain CBS 139881 (= UAMH 130) clusters in the Blasto- diam, cottony-ﬂoccose, yellowish white; reverse warm- buff to pale buff peripherally. Conidiophores cylindrical, myces clade (Clade II). The genus Blastomyces has recently been validated (de Hoog et al. 2016a) with B. dermatitidis about 1 lm wide at the base, sometimes slightly swollen or ﬂexuous, producing 1–4 (up to 7) solitary conidia; sec- as type species and strain CBS 674.68 (= ATCC 18188 = UAMH 3539) as epitype of B. dermatitidis. Emmonsia is ondary conidiophores lacking. Conidia spherical, 2.8–7.3 9 2.2–4.9 lm (4.9 ± 0.9 9 3.8 ± 0.70, n = 45), roughened now a younger synonym of this genus. As circumscribed in the present study, Blastomyces now comprises ﬁve species to spinulose, sessile, often adherent to the conidiophore, having strong support: B. dermatitidis/B. gilchristii (ML/BI some budding at a broad base. Intermediate phase (33 C, 100/1.00), B. helicus (ML/BI 100/1.00), B. parvus (ML/BI MEA, 3 weeks): Colonies 30 mm diam, cottony-ﬂoccose, 100/1.00), B. percursus (ML/BI 100/1.00) and B. silverae yellowish white, sulcate. Conidiation moderate; conidia (ML/BI 98/1.00). While B. gilchristii (ML/BI 100/1.00) slightly larger than at 24 C. Thermotolerant phase could not be distinguished from B. dermatitidis in our study (37 C, MEA, 3 weeks): Colonies restricted 7 mm, 123 Fungal Diversity (2018) 90:245–291 277 123 278 Fungal Diversity (2018) 90:245–291 Fig. 17 Emergomyces europaeus (CBS 102456). A–C Colonies onc with standard barcoding by ITS (Fig. 4), the species has MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Slightly swollen been segregated on the basis of genealogical concordance conidiophore without secondary conidiophores; conidia slightly phylogenetic species recognition (GCPSR; Brown et al. roughened. G–I 33 C. Swollen hyphae disarticulating to liberate 2013) using seven partial genes, some of which are not giant cells; small yeasts produced. J–M 37 C. Scant, irregularly swollen hyphae, small yeast cells with budding at narrow base. Scale commonly used in phylogenetic studies (e.g. histidine bars: D–M =10 lm kinase, orotidine 5’-phosphate decarboxylase). Blasto- myces gilchristii was found to be reproductively and genetically isolated from B. dermatitidis and had a smaller, with mammal vectoring and leading to a gradual separation overlapping geographic distribution. Similar diversity had of Eurasian and American genotypes. been noted by Meece et al. (2011). Whole genome sequencing of strains of B. dermatitidis (ER-3, ATCC Emmonsia crescens occurs in animal and, less com- monly, in human hosts in which it forms large non-repli- 18188 and ATCC 26199) and B. gilchristii (SLH14081) provided additional evidence for separation of these spe- cating, thick-walled adiaspores in lung and occasionally other tissues. Almost all data on this species’ host and cies (Mun ˜ oz et al. 2015). Species of the genus Blastomyces are associated with geographic distribution come from animal surveys in which lungs and other tissues of trapped animals have been human and animal disease. Blastomyces dermatitidis and dissected and examined for presence of the large adias- B. gilchristii cause disease pulmonary or disseminated pores (up to 400 lm diam) characteristic of this species disease in immunocompetent humans, companion animals (Dvor ˇak et al. 1973; Emmons and Jellison 1960; Hubalek and other mammals (Baumgardner et al. 1995; Bariola et al. 2010; Saccente and Woods 2010; Brown et al. 2013). 1999; Hubalek et al. 1995, 1998; Jellison 1969; Sigler 2005; Borman et al. 2009, 2018). These adiaspores are B. helicus is associated with disease in predominantly immunocompromised humans and in companion animals pathognomonic and can be recognized in published reports even in the absence of preserved material. Over 120 spe- (Kappagoda et al. 2017; Rofael et al. 2018; Schwartz et al. 2017) whereas known cases of B. percursus infection cies of mammal hosts have been identiﬁed and include members of the orders Insectivora, Edentata, Lagomorpha, include immunocompetent and immunocompromised hosts (Kemna et al. 1994; Heys et al. 2014; Dukik et al. 2017b). Rodentia and Carnivora. Terrestrial rodents and burrowing animals are most commonly represented. Rare reports B. parvus causes pulmonary disease only in rodents and concern domestic or farm animals including two dogs (Al- terrestrial mammals; no human cases are substantiated Doory et al. 1971; Koller et al. 1976), a goat (Koller and (Sigler 2005; Anstead et al. 2012). All species are shown Helfer 1978), a horse (Pusterla et al. 2002) and a deer here to produce moderate to large yeast cells at higher (Matsuda et al. 2015), although no voucher material is temperatures in culture that replicate by broad-based bud- available to conﬁrm the diagnoses. Approximately 50 cases ding, including, to a lesser extent, B. parvus in which giant cells may propagate as broad based yeast cells. of human pulmonary adiaspiromycosis have been reported from the Americas, Europe, and North Africa (England and With the transfer of the type species, Ea. parva to Blastomyces, as proposed herein, the disposition of Ea. Hochholzer 1993; Sigler 1998; Anstead et al. 2012). Dvor ˇak et al. (1973) suggested that growth of the saprobic crescens is not resolved. Strains of Ea. cresens clustered phase is more important as determinant of the life cycle with strong support in a single, moderately variable clade than the host itself. They suggested that localization of Ea. in both ML and rPB2 analyses. The clade was paraphyletic crescens in the biotope of the animal burrow in temperate to clade I-3 comprising the genus Emergomyces but Ea. to subboreal climates is a signiﬁcant ecological prerequi- crescens differs from all members of the Emergomyces site. The limited number of conidia produced may con- clade in not demonstrating budding, in host associations and pathogenicity. Herein we continue to use the current taminate the air inside the burrow sufﬁciently to provide an effective inoculum, thus enabling the animal to transfer name to avoid nomenclatural instability. Emmonsia cres- cens has been recorded from all ﬁve continents, yet our conidia to another site having the same ecological param- eters. The species has a wide range of temperature toler- study conﬁrms data of Peterson and Sigler (1998)in showing two molecular subgroups within the species: a ance, from 6 C which is comparable to winter temperatures of burrows at around 3–5 C, and up to 40 C panboreal Eurasian population, and a North American population. Sequence differences are small, and there are at which it grows restrictedly (Fig. 5). At higher tempera- tures, fast-growing saprobes may suppress the growth of no signiﬁcant phenotypic differences between the two Ea. crescens, which explains that highest incidence of populations and Sigler (1996) showed interfertility among infection occurs in winter and early spring (Dvorak et al. several strains of the two populations. The observed 1973). In accordance with this hypothesis, we found that structuring of the two populations may indicate that the vector of dispersal of Ea. crescens is slow and local, in line conidiation in Ea. crescens occurred at 21 C and below. 123 Fungal Diversity (2018) 90:245–291 279 123 280 Fungal Diversity (2018) 90:245–291 Fig. 18 Emergomyces orientalis (CBS 124587). A–C Colonies onc The recently-described genus Emergomyces (Clade I-3) MEA 3 weeks at 24, 33 and 37 C. D–F 21 C. Slightly swollen is here conﬁrmed to include taxa exclusively associated conidiophores and coiled hyphae; conidia present (absent at 24 C); with human disease, including the previously described G–I 33 C. Swollen conidiophores and hyphae forming giant cells species Es. pasteurianus [formerly Ea. pasteuriana] (Gori with thin walls. J–M 37 C. Hyphae scant, swollen; small spherical yeast cells with budding at narrow base. Scale bars: D–M =10 lm et al. 1998; Dukik et al. 2017b)], Es. africanus (Dukik et al. 2017b), Es. orientalis (Wang et al. 2017) and the new species Es. canadensis and Es. europaeus. The species with fungal meningitis, tolerated 40 C, required 1 week to differ slightly with respect to maximum growth tempera- convert at 37 C, and was noted to have already converted tures and sizes of yeast cells as well geographic distribu- at 33 C. Mammals have different body temperatures, tion. Both Es. canadensis (Clade I-3a) and Es. orientalis ranging from 35 to 40 C, and signiﬁcant differences are (Clade 1-3b; Wang et al. 2017) have been isolated from observed between species e.g. armadillo, rodent and feline human patients (North America and China respectively), hosts (Fig. 23). Emmonsia crescens and B. parvus are both produce small, spherical yeast cells at 37 C and a unique associated with rodents. In Ea. crescens, these are small red pigment on BHI and TSA at 37 C. Differences occur terrestrial animals and rodents: moles (Talpa europaea), with respect to mold-to-yeast transitions: in Es. canadensis dark polecats (Trichosurus vulpecula), European water small yeasts are produced at 33 C which reproduce by voles (Arvicola amphibius), common voles (Microtus budding (Fig. 16H), while in Es.orientalis beyond 30 C arvalis), red-backed voles (Myodes sp.), ﬁeld mice yeast-like cells originate from swollen hyphal cells (Apodemus sp.) and deer mice (Peromyscus sp.) (Dvor ˇak (Fig. 18I). Additional isolates are needed to gain a better et al. 1973), among others, which have relatively low body understanding of the relationship between these two temperatures of 35.0–37.5 C. Hosts of B. parvus have species. somewhat higher body temperatures, e.g. Neotama micro- According to the above, a range is observed from the pus (CBS 139882 = UAMH 134) and pocket gopher (CBS adiaspore of Ea. crescens to the small yeast of Emer- 178.60 = ATCC14051), that are 36–40 C. Similarly B. gomyces, and from reluctant budding in Ea. sola (NCPF silverae (CBS 139879 = UAMH 139), originated from a 4289) to abundant budding in Emergomyces. Although we weasel with a body temperature of 37.8–40 C. No infec- could not satisfactorily reconstruct the evolutionary pro- tions caused by members of the Ajellomycetaceae are cess, in line with the different forms of the parasitic phase, known to occur in cold-blooded hosts. the species seem to acquire certain ecologically relevant Inferring the evolutionary histories on traits of thermal capacities, e.g. to infect and colonize mammal hosts; this conversion, fungal pathogens could have evolved with their situation seems to open new opportunities in terms of speciﬁc animal and human hosts. Emmonsiellopsis, found expanding its traditional ecological niche and exerting in an ancestral position, has no known animal hosts but has pressures for speciation (Coyne and Orr 2004). The sig- a predominantly ﬁlamentous life cycle, and reluctant con- niﬁcance of Ea. crescens colonizing lungs of burrowing version. The prime host of Paracoccidioides is the arma- animals is unknown. From a longer evolutionary perspec- dillo, a terrestrial mammal with a primitive immune system tive, what is its role in the fungus’s adaptation and speci- and a low body temperature of 34 C (Bagagli et al. ation? The adiaspore is so unique that it is unlikely to be 2006, 2008; Hrycyk et al. 2018). The prime host of coincidental. Ea. crescens might be an interesting model Histoplasma is the bat, with a very low winter body tem- for study that may serve as a useful comparator to other perature which rises to 37.8–40.6 C during ﬂight. Most dimorphic fungi. rodents that are hosts for Ea. crescens have temperatures The morphological features of thermal transformation ca. 35–37 C. Blastomyces dermatitidis is commonly found among members of the Ajellomycetaceae have been central in felines and canines, which have slightly higher body to the taxonomy and ecology of the family. Among the temperatures (38–40 C) and a more advanced immune species examined here, those having a long saprobic phase, system. Emergomyces clade comprises species in humans i.e. expressing conidiation over a broad temperature range (body temperature 36.1–37.2 C) and as yet no known and having a short thermotolerant phase take more time to animal or environmental alternative cycle. convert (Fig. 5, Table 2). It is unknown whether this can be It should be noted that diverse types of giant cells occur linked to virulence (Medoff et al. 1986). Strains with rel- at elevated temperatures across most taxa in Ajellomyc- atively long saprobic phases almost invariably originated etaceae, even the genus Emmonsiellopsis described by from rodents and their burrows. Species that were regularly Marin-Felix et al. (2015). Two strains of Emms. terrestris found in human hosts were more often able to grow at (CBS 139889 = UAMH 141; CBS 273.77 = UAMH 2304) 40 C or higher, had a broader temperature range of the examined by Sigler (1996) and reexamined in the present thermotolerant phase, and required less time to convert. For study were able to produce giant cells at 37 C. Already in example, B. helicus CBS 139874, from CSF of a patient 123 Fungal Diversity (2018) 90:245–291 281 123 282 Fungal Diversity (2018) 90:245–291 Fig. 19 Emergomyces pasteurianus (CBS 101426). A–C Colonies onc 1947, Dowding suggested that Emmonsia parva was more MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Slightly swollen close to Blastomyces than to Ea. crescens, as was sub- conidiophores and secondary conidiophores. G–I 33 C. Conidio- stantiated with molecular data by Perterson and Sigler phores and hyphal fragments becoming swollen and disarticulating. (1998) and in the present paper. In line with this, the giant J–M 37 C. Hyphae producing giant cells; small and large yeast cells with uni- or bipolar budding. Scale bars: D–M =10 lm cells of B. parvus (Emmonsia parva) appeared not to be a terminal (i.e., non-replicating) structure in the fungus’ pathogenic phase, as they are in Ea. crescens, but are able the resulting phylogeny. The phylogenies of the taxa to propagate as broad-based yeast cells, very similar to compared in this study were inferred from analyses using those of classical Blastomyces species (Fig. 3). Wu et al. different data sets and algorithms. The topologies of the (2005) reviewed ﬁve cases of blastomycosis that presented trees constructed using different algorithms performed on histopathologically with very large budding cells different data sets were largely congruent, which make the (20–40 lm diam). Paracoccidioides brasiliensis appears as delimitation of major lineages and clades more clear and spherical budding cells with multiple buds; younger cells conﬁdent. A recent study of fewer representatives by whole measure 2–10 lm in diam, and mature cells often genome sequencing data constructed a tree of similar [ 30 lm, or even reach 60 lm in diam (Brummer et al. topology to our multilocus data (Dukik et al. 2017b), 1993). The biological coherence of taxa in Ajellomyc- conﬁrming that the main topological traits of the present etaceae is underlined by teleomorphs. Ajellomyces-type study are robust. sexuality has been obtained after matings in H. capsulatum, Bayesian analysis is usually believed to be more reliable in B. dermatitidis, and in Ea. crescens and the morpholo- compared to parsimony and neighbour-joining methods, gies of the ascomata and ascospores is relatively homo- especially for an extensive sampling with a high diver- geneous over the species. All these features show gence occurring among the sequences (Alfaro et al. 2003; signiﬁcant overlap and apparent atavisms, suggesting that Holder and Lewis 2003; James et al. 2006). In line with all species might be attributed to a single evolutionary expectations, the Bayesian analysis of the ﬁve-gene data set series, i.e. classiﬁed in a single genus. However, such a showed a robust phylogeny. Bayesian and ML supports taxonomic decision resulting in many name changes of showed signiﬁcant correlation of PP and BP values. In clinically important pathogens is undesirable because it Eurotiomycetes, ribosomal ITS is often considered to be an could sow confusion among clinicians, clinical laboratories inadequate marker for species distinction (Skouboe et al. and patients about these fungi and the diseases that they 1999; Seifert et al. 2007; Visagie et al. 2014). Based on cause. previous and present phylogenetic studies, it was demon- With sequence data of the loci LSU, ITS, TUB2, TEF3, strated that the rDNA operon is sufﬁciently variable to and rPB2, 80 strains were analyzed, of which 44 clustered distinguish most species of Ajellomycetaceae (Peterson and in Blastomyces/Emergomyces, and with representative Kurtzman 1991; Berres et al. 1995; Kretzer et al. 1996; isolates from classical genera of dimorphic fungi, i.e. Peterson and Sigler 1998; Untereiner et al. 2004; this study; Histoplasma (n = 7) and Paracoccidioides (n = 3). Fig. 4), but phylogenetic relationships among genera Despite our best effort to obtain a complete sequence remained largely unresolved (Untereiner et al. 2004). In dataset for all the genes and strains employed, the sequence our ITS analysis (Fig. 4) the genera Blastomyces, Em- of some genes, especially the nuclear protein-coding gene monsia, Emmonsiellopsis, Emergomyces, Helicocarpus, TEF3, could not be determined for a small percentage of Histoplasma, Lacazia and Paracoccidioides were included strains because of failure in the PCR ampliﬁcation or in the Ajellomycetaceae, but the backbone of the tree sequencing reactions. Interestingly, all unsuccessful reac- showed low support. Histoplasma was nested in Blasto- tions in TEF3 originated from Es. africanus. Speciﬁcally, myces, and some taxa lacked strong bootstrap support, 2.5, 1.25, 2.5, 7.5 and 5.0% of the total 80 strains employed especially B. parvus (ML/BI 10/–) and B. silverae (ML/BI failed in the sequence determination of the LSU, ITS, 65/–). The family Ajellomycetaceae had the geophilic rPB2, TEF3 and TUB2 genes, respectively. This problem is genus Emmonsiellopsis in an ancestral position (Clade V). known from all groups of fungi (Schoch et al. 2012). Paracoccidioides (Clade IV) was remote from remaining Another study has shown that an inferred phylogeny is not genera, and Histoplasma (Clade III) was paraphyletic to sensitive to 25% or even 50% missing data for sufﬁciently Blastomyces (Clade II). Emergomyces (Clade I-3) was large alignments (e.g., * 30,000 positions and 36 species; paraphyletic to Ea. crescens (Clade I-1). With multilocus Philippe et al. 2004). Though the length of the ﬁve-gene analysis, strains CBS 178.60 and CBS 205.48 formed a alignment in this study comprises only about 3302 posi- bootstrap-supported subclade (ML/BI 100%/1.00), para- tions, the amount of missing data is much less. Thus, we phyletic to B. parvus, matching with a difference of 4 bases assume that the relative minor amount of missing data in in ITS, 4 in TUB2,0in TEF3 and 14 in rPB2. The strains our study will not signiﬁcantly inﬂuence the reliability of 123 Fungal Diversity (2018) 90:245–291 283 123 284 Fungal Diversity (2018) 90:245–291 Fig. 20 Emmonsiellopsis coralliformis (CBS 139500). A–C Coloniesc originated from lungs of rodents and were indistinguishable on MEA 3 weeks at 24, 30 and 33 C. D–F 24 C. Conidiophores from B. parvus strains in all phenotypic and ecological lacking secondary conidiophores; conidia roughened. G–I (30 C). markers. We suggest that at this level of molecular diver- Conidia, swollen cells andbudding cells present. J–M 33 C. Swollen sity of the currently used markers, strains may still belong hyphae and conidia becoming giant cells with rather thick walls, some showing broad-based budding. Scale bars: D–M =10 lm to the same species. Markers may have different functions. In addition to Usually shorter fragments coincide with higher PCR ﬁde- barcodes for species distinction, less variable genes are needed for the development of pan-family diagnostic pri- lity, shorter PCR cycles (= shorter time-to-result) and higher capillary sequencing success, a key criterion to mers. Also, markers for phylogenetic purposes may be different from those used for diagnostic purposes, where successful application in the clinics, and faster patient barcoding gaps should preferably be present. TUB2 has treatment. We consequently conclude that (1) rPB2 and TUB2 can both be recommended for routine identiﬁcation, successfully been used species and variety delimitation in Histoplasma, Blastomyces and Paracoccidioides (Matute (2) TEF3, in combination with ITS, TUB2, and rPB2 yield stable topologies for multilocus sequence typing and et al. 2006; de Teixeira et al. 2009, 2016); this gene was sequenced over our entire set of strains. TUB2 is easy to GCPSR, and (3) for the development of a pan-Ajellomyc- etaceae primer to detect hitherto unknown species, from amplify, with a success rate in our strains of 95%. The gene has been reported to vary in the number of introns, and animal burrows or in archived lung specimens of terrestrial animals, rDNA ITS is probably sufﬁcient. Whole genome sometimes PCR results in the ampliﬁcation of paralogous genes (Peterson 2008; Hubka and Kolarik 2012), but these trees usually show excellent resolution (Mun ˜ oz et al. 2015; problems were not encountered in our data set. Other Dukik et al. 2017b), but as yet relatively low numbers of options for secondary markers include TEF3, which was genomes are available and trees suffer from signiﬁcant recently introduced by Stielow et al. (2015) as an excellent strain and taxon sampling effects. marker, but the success rate of sequencing for each strain is lower (92.5%). rPB2 was shown to have similar discrimi- Biosafety considerations natory power as TUB2 (Liu et al. 1999). PCR ampliﬁcation in our dataset was signiﬁcant (97.5%). In this study, the Data presented in this paper including detailed species TEF3 and TUB2 sequences showed less parsimony-infor- mative characters for the inference of phylogenetic rela- descriptions and reports of associated human and animal infections provides a foundation for assessment of biosaf- tionships in different taxa compared to rPB2. The TEF3 and TUB2 data sets generated lower resolution across the ety consideration. Risk assessment should include the capacity of the organism to cause human disease, severity Bayesian and ML trees, in which only 3 strongly supported of the disease, and availability of effective treatment clades were resolved with high BP and PP values, respectively. Among the three protein-coding genes, rPB2- (Anon. 2009). Formal biosafety risk assessments by national level committees exist for only some members of based phylogeny drawn from Bayesian and ML analyses not only resolved the same number of clades in the family the Ajellomycetacaeae, described in this report i.e. B. dermatitidis, B. parvus, Ea. crescens, and Es. pasteurianus, Ajellomycetaceae, but also had an equivalent resolution power, and topologies of trees were comparable to multi- Emergomyces species have only been reported to cause disease among persons with immunocompromising condi- locus trees in Blastomyces (Clade II) (ML/BI 100/0.99), Histoplasma (Clade III) (ML/BI 100/0.99), Paracoccid- tions (Gori et al. 1998, Pelegrin et al. 2011, Feng et al. 2015, Malik et al. 2016, Dukik et al. 2017b, Schwartz et al. ioides (Clade IV) (ML/BI 100/0.99), Emmonsiellopsis (Clade V) (ML/BI 100/0.99). Emergomyces (Clade I-3) 2018). In vitro antifungal susceptibility testing results (ML/BI 99/0.99), leaving only the relationship between suggested susceptible to available antifungals, including polyenes and most triazoles (Maphanga et al. 2017). Bel- genera Emmonsia and Emergomyces unresolved with lower bootstrap support (ML/BI 46/–). gium’s Scientiﬁc Institute of Public Health (www.biosaf ety.be), the Swiss Agency for the Environment, Forests and Barcoding analysis conﬁrms that rPB2 and TUB2 with the current primer pairs allow recognition of entities. There Landscape (http://www.uab.cat/doc/fongs), and the Amer- ican Type Culture Collection have classiﬁed Es. pasteuri- is still room for improvement of primer design considering that not all parts of the sequences are equally informative. anus as a biosafety level 2 organism. We propose that other species in the genus be handled similarly. This aspect is particularly useful for the design of pan- Ajellomycetaceae primers or genus-speciﬁc barcode iden- Emmonsia crescens is a rodent pathogen and has occa- sionally been implicated in pulmonary disease in immune tiﬁers (Heinrichs et al. 2012). For example, the ITS-1 section is useful, as it provides essential information to competent persons based on size of adiapores in lung delimit entities, while the ITS-2 region contributes less. (Anstead et al. 2012). In vitro antifungal susceptibility 123 Fungal Diversity (2018) 90:245–291 285 123 286 Fungal Diversity (2018) 90:245–291 123 Fungal Diversity (2018) 90:245–291 287 b Fig. 21 Emmonsiellopsis terrestris (CBS 273.77). A–C Colonies on safety level 2 pathogen and the mycelial phase as a MEA 3 weeks at 24, 33 and 37 C. D–F 24 C. Conidiophores short, biosafety level 3 pathogen (Anon. 2009). In contrast, slightly swollen, without secondary conidiophores conidia sometimes ACDP considers both phase as hazard group 3 pathogens sessile, roughened. G–I 33 C. Conidia. J–M 37 C. Conidia, some (ACDP 2013). becoming giant cells with occasional budding at a broad base. Scale bars: D–M =10 lm Blastomyces parvus has been stated as the cause of human disease in two immune compromised patients (Echavarria et al. 1993; Turner et al. 1999), but the iden- tiﬁcation of the putative pathogen is disputed (Sigler 2005; results showed susceptibility to amphotericin B, itracona- Anstead et al. 2012). There is no reliable report of B. zole, voriconazole and caspofungin (Borman et al. 2009; parvus human infection. In vitro antifungal susceptibilities Dukik et al. 2017a). However, the role of antifungal ther- suggest susceptibility to commonly used antifungals. apy is controversial, because in vivo replication does not Health risks of this fungus for humans are low, but its close occur (Anstead et al. 2012). The species has been classiﬁed afﬁnity to B. dermatitidis, as established in this paper, as a hazard group 2 pathogen by the Advisory Committee support its classiﬁcation as a hazard group 2 pathogen by for Dangerous Pathogens in the United Kingdom (ACDP ACDP (ACDP 2013). 2013). Blastomyces helicus infection appears to primarily cause Blastomyces dermatitidis can cause severe disease in disease in immune compromised persons (Sekhon et al. immune competent hosts. In vitro susceptibility studies 1982; Kappagoda et al. 2017; Schwartz et al. 2017; Rofael have reported susceptibility to amphotericin B, itracona- et al. 2018). Conidia are not produced in vitro, and thus the zole, and other triazoles (Dukik et al. 2017a). The Biosafety risk of occupational inhalational exposure is considered in Microbiological and Biomedical Laboratories 5th edi- low, although primary inoculation disease, may be possi- tion classiﬁes the yeast phase of B. dermatitidis as a bio- Fig. 22 Distribution map of infections caused by new Blastomyces and Emergomyces species during the last decades 123 288 Fungal Diversity (2018) 90:245–291 Fig. 23 Approximate relationships between optimal conversion tem- Several novel species are represented by very few isolates so that only peratures of ajellomycetaceous species and their prevalent host scant information on host ranges is available animal, with average body temperatures listed in the right column. ble. In vitro antifungal susceptibility results suggested References susceptibility to amphotericin B and extended azoles ACDP (2013) Approved list of biological agents. Advisory Commit- (Dukik et al. 2017a). National level biosafety risk assess- tee on Dangerous Pathogens (ACDP). Health and Safety ments for B. helicus do not yet exist. We propose that B. Executive (HSE), London. http://www.hse.gov.uk/pubns/ helicus be handled as a biosafety level 2 pathogen. misc208.pdf Al-Doory Y, Vice TE, Mainster ME (1971) Adiaspiromycosis in a Acknowledgements Yanping Jiang is indebted to Prof. Li Zuo, dog. J Am Vet Med Assoc 159(1):87–90 director of Department of Immunology, Basic Medical School, Alfaro ME, Zoller S, Lutzoni F (2003) Bayes or bootstrap? A Guizhou Medical University and director of the Department of Der- simulation study comparing the performance of Bayesian matology, The Afﬁliated Hospital, Guizhou Medical University for Markov chain Monte Carlo sampling and bootstrapping in supporting her studies performed at Westerdijk Institute. Yanping assessing phylogenetic conﬁdence. Mol Biol Evol 20:255–266. also gratefully acknowledges the China Scholarship Council (CSC) https://doi.org/10.1093/molbev/msg028 Fund support for the Advancement of Scholarship. Andy Borman is Anon (2009) Biosafety in microbiological and biomedical laborato- acknowledged for kindly making strains available for study. ries, 5th edn. US Department of Health and Human Services, Centers for Disease Control and Prevention & National Institutes Open Access This article is distributed under the terms of the Creative of Health, Atlanta Commons Attribution 4.0 International License (http://creative Anstead GM, Sutton DA, Graybill JR (2012) Adiaspiromycosis commons.org/licenses/by/4.0/), which permits unrestricted use, dis- causing respiratory failure and a review of human infections due tribution, and reproduction in any medium, provided you give to Emmonsia and Chrysosporium spp. J Clin Microbiol appropriate credit to the original author(s) and the source, provide a 50(4):1346–1354. https://doi.org/10.1128/JCM.00226-11 link to the Creative Commons license, and indicate if changes were Bagagli E, Bosco SMG, Theodoro RC, Franco M (2006) Phylogenetic made. and evolutionary aspects of Paracoccidioides brasiliensis reveal a long coexistence with animal hosts that explain several 123 Fungal Diversity (2018) 90:245–291 289 biological features of the pathogen. Infect Genet Evol Drouhet E, Gueho E, Gori S et al (1998) Mycological, ultrastructural 6(5):344–351. https://doi.org/10.1016/j.meegid.2005.12.002 and experimental aspects of a new dimorphic fungus Emmonsia Bagagli E, Theodoro RC, Bosco SM, McEwen JG (2008) Paracoc- pasteuriana sp. nov. isolated from a cutaneous disseminated cidioides brasiliensis: phylogenetic and ecological aspects. mycosis in AIDS. J Mycol Med 8:64–77 Mycopathologia 165(4–5):197–207. https://doi.org/10.1007/ Dukik K, Al-Hatmi AMS, Curfs-Breuker I et al (2017a) Antifungal s11046-007-9050-7 susceptibility of emerging dimorphic pathogens in the family Bariola J, Perry P, Pappas PG et al (2010) Blastomycosis of the Ajellomycetaceae. Antimicrob Agents Chemother. https://doi. central nervous system: a multicenter review of diagnosis and org/10.1128/AAC.01886-17 treatment in the modern era. Clin Infect Dis 50(6):797–804. Dukik K, Mun ˜ oz JF, Jiang Y et al (2017b) Novel taxa of thermally https://doi.org/10.1086/650579 dimorphic systemic pathogens in the Ajellomycetaceae (Onyge- Baumgardner DJ, Paretsky DP (1999) The in vitro isolation of nales). Mycoses 60(5):296–309. https://doi.org/10.1111/myc. Blastomyces dermatitidis from a woodpile in north central 12601 Wisconsin, USA. Med Mycol 37(3):163–168 Dvor ˇak J, Otc ˇenas ˇek M, Rosicky B (1973) Adiaspiromycosis caused Baumgardner DJ, Paretsky DP, Yopp AC (1995) The epidemiology of by Emmonsia crescens, Emmons and Jellison 1960. Studie blastomycosis in dogs: north 473 central Wisconsin, USA. J Med CSAV (Acad Praha) 14:1–120 Vet Mycol 33:171–176 Echavarria E, Cano EL, Restrepo A (1993) Disseminated adi- Berres ME, Szabo LJ, McLaughlin DJ (1995) Phylogenetic relation- aspiromycosis in a patient with AIDS. J Med Vet Mycol ships in auriculariaceous basidiomycetes based on 25S ribosomal 31(1):91–97 DNA sequences. Mycologia 87:821–840 Edgar RC (2004) MUSCLE: Multiple sequence alignment with high Borman AM, Simpson VR, Palmer MD et al (2009) Adiaspiromy- accuracy and high throughput. Nucleic Acids Res cosis due to Emmonsia crescens is widespread in native British 32(5):1792–1797 mammals. Mycopathologia 168(4):153–163. https://doi.org/10. Emmons CW, Ashburn LL (1942) The isolation of Haplosporangium 1007/s11046-009-9216-6 parvum n. sp. and Coccidioides immitis from wild rodents. Publ Borman AM, Jiang Y, Dukik K, Sigler L, Schwartz IS, de Hoog S. Health Rep 57:1715–1727 (2018) Adiaspiromycosis and diseases caused by related fungi in Emmons CW, Jellison WL (1960) Emmonsia crescens sp. n. and Ajellomycetaceae. Springer International Publishing AG (in adiaspiromycosis (haplomycosis in mammals). Ann NY Acad press) Sci 89:91–101 Brown SD, Collins RA, Boyer S et al (2012) Spider: an R package for England DM, Hochholzer L (1993) Adiaspiromycosis: an unusual the analysis of species identity and evolution, with particular fungal infection of the lung. Am J Surg Pathol 17(9):876–886 reference to DNA barcoding. Molec Ecol Res 12(3):562–565. Feng P, Yin S, Zhu G et al (2015) Disseminated infection caused by https://doi.org/10.1111/j.1755-0998.2011.03108.x Emmonsia pasteuriana in a renal transplant recipient. J Dermatol Brown EM, McTaggart LR, Zhang SX et al (2013) Phylogenetic 42(12):1179–1182. https://doi.org/10.1111/1346-8138.12975 analysis reveals a cryptic species Blastomyces gilchristii, sp. Gori S, Drouhet E, Gue ´ho E et al (1998) Cutaneous disseminated nov. within the human pathogenic fungus Blastomyces dermati- mycosis in a patient with AIDS due to a new dimorphic fungus. tidis. PLoS ONE 8(3):e59237. https://doi.org/10.1371/journal. J Mycol Me ´d 8:57–63 pone.0059237 Heinrichs G, de Hoog GS, Haase G (2012) Barcode identiﬁers as a Brummer E, Castaneda E, Restrepo A (1993) Paracoccidioidomyco- practical tool for reliable species assignment of medically sis: an update. Clin Microbiol Rev 6(2):89–117 important black yeast species. J Clin Microbiol Carmichael JW (1951) The pulmonary fungus Haplosporangium 50(9):3023–3030. https://doi.org/10.1128/JCM.00574-12 parvum II. Strain and generic relationships. Mycologia Heys I, Taljaard J, Orth H (2014) An Emmonsia species causing 43:605–624 disseminated infection in South Africa. N Engl J Med Ciferri R, Montemartini A (1959) Taxonomy of Haplosporangium 370(3):283–284. https://doi.org/10.1056/NEJMc1314277#SA1 parvum. Mycopath Mycol Appl 10:303–316 Holder M, Lewis PO (2003) Phylogeny estimation: traditional and Colombo AL, Tobo ´ n A, Restrepo A et al (2011) Epidemiology of Bayesian approaches. Nat Rev 4(4):275–284. https://doi.org/10. endemic systemic fungal infections in Latin America. Med 1038/nrg1044 Mycol 49(8):785–798. https://doi.org/10.3109/13693786.2011. Hrycyk MF, Garcia Garces H, Bosco SMG et al (2018) Ecology of 577821 Paracoccidioides brasiliensis, P. lutzii and related species: Coyne JA, Orr HA (2004) Speciation. Sinauer Associates, Sunderland infection in armadillos, soil occurrence and mycological aspects. De Hoog GS, Redhead SA, Feng P et al (2016a) Proposals to conserve Med Mycol. https://doi.org/10.1093/mmy/myx142 Blastomyces Gilchrist & W.R. Stokes against Blastomyces Hubalek Z (1999) Emmonsiosis of wild rodents and insectivores in Costantin & Rolland and Ajellomycetaceae against Paracoccid- Czechland. J Wildl Dis 35:243–249 ´ ´ ioidaceae (Ascomycota: Onygenales). Taxon 65:1167–1169 Hubalek Z, Nesvadbova J, Rychnovsky B et al (1995) A heteroge- De Hoog GS, Guarro J, Gene J, Figueras MJ (2016b). Atlas of clinical neous distribution of Emmonsia parva var. crescens in an agro- fungi, E-version 4.1.4. CBS-KNAW Fungal Biodiversity Centre, ecosystem. J Med Vet Mycol 33(3):197–200 ´ ´ Utrecht Hubalek Z, Nesvadbova J, Halouzka J (1998) Emmonsiosis of rodents de Teixeira M, Theodoro RC, de Carvalho MJ et al (2009) in an agroecosystem. Med Mycol 36(6):387–390 Phylogenetic analysis reveals a high level of speciation in the Hubka V, Kolarik M (2012) b-Tubulin paralogue tubC is frequently misidentiﬁed as the benA gene in Aspergillus section Nigri Paracoccidioides genus. Mol Phylogenet Evol 52(2):273–283. https://doi.org/10.1016/j.ympev.2009.04.005 taxonomy: primer speciﬁcity testing and taxonomic conse- de Teixeira M, Patane ´ JS, Taylor ML et al (2016) Worldwide quences. Persoonia 29:1–10. https://doi.org/10.3767/ phylogenetic distributions and population dynamics of the genus 003158512X658123 Histoplasma. PLoS Neglect Trop Dis 10(6):e0004732. https:// James TY, Kauff F, Schoch CL et al (2006) Reconstructing the early doi.org/10.1371/journal.pntd.0004732 evolution of fungi using a six-gene phylogeny. Nature Dowding ES (1947) The pulmonary fungus Haplosporangium 443:818–822. https://doi.org/10.1038/nature05110 parvum, and its relationship with some human pathogens. Can Jellison WL (1969) Adiaspiromycosis (=haplomycosis). Mountain J Res Sect E Med Sci 25:195–206 Press Publishers, Montana 123 290 Fungal Diversity (2018) 90:245–291 Kane J, Summerbell RC, Sigler L et al (1997) Laboratory handbook Blastomyces and close relative Emmonsia. PLoS Genet of dermatophytes: a clinical guide and laboratory manual of 11(10):e1005493. https://doi.org/10.1371/journal.pgen.1005493 dermatophytes and other ﬁlamentous fungi from skin, hair, and Pelegrin I, Ayats J, Xiol X et al (2011) Disseminated adiaspiromy- nails. Star Publishing Co., Belmont cosis: case report of a liver transplant patient with human Kappagoda S, Adams JY, Luo R et al (2017) Fatal Emmonsia sp. immunodeﬁciency infection, and literature review. Transpl infection and fungemia after orthotopic liver transplantation. Infect Dis 13(5):507–514. https://doi.org/10.1111/j.1399-3062. Emerg Infect Dis 23(2):346–349. https://doi.org/10.3201/ 2011.00611.x eid2302.160799 Peterson SW (2008) Phylogenetic analyses of Aspergillus species Kemna ME, Weinberger M, Sigler L et al (1994) A primary oral using DNA sequences from four loci. Mycologia blastomycosis-like infection in Israel. In: 94th General Meeting 100(2):205–226 of the American Society for Microbiology, Washington, DC, Peterson SW, Kurtzman CP (1991) Ribosomal RNA sequence Abstract F-75, p 601 divergence among sibling species of yeasts. Syst Appl Microbiol Kenyon C, Bonorchis K, Corcoran C et al (2013) A dimorphic fungus 14:124–129 causing disseminated infection in South Africa. N Engl J Med Peterson SW, Sigler L (1998) Molecular genetic variation in 369(15):1416–1424. https://doi.org/10.1056/NEJMoa1215460 Emmonsia crescens and Emmonsia parva, etiologic agents of Koller LD, Helfer DH (1978) Adiaspiromycosis in the lungs of a goat. adiaspiromycosis, and their phylogenetic relationship to Blasto- J Am Vet Med Assoc 173:80–81 myces dermatitidis (Ajellomyces dermatitidis) and other systemic Koller LD, Patton NM, Whitsett DK (1976) Adiaspiromycosis in the fungal pathogens. J Clin Microbiol 36(10):2918–2925 lungs of a dog. J Am Vet Med Assoc 169:1316–1317 Philippe H, Snell EA, Bapteste E et al (2004) Phylogenomics of Kretzer A, Li Y, Szaro T et al (1996) Internal transcribed spacer Eukaryotes: impact of missing data on large alignments. Mol sequences from 38 recognized species of Suillus sensu lato: Biol Evol 21(9):1740–1752. https://doi.org/10.1093/molbev/ phylogenetic and taxonomic implications. Mycologia msh182 88:776–785 Popescu AA, Huber KT, Paradis E (2012) ape 3.0: new tools for Kwon-Chung KJ (1973) Studies on Emmonsiella capsulata I. distance-based phylogenetics and evolutionary analysis in R. Heterothallism and development of the ascocarp. Mycologia Bioinformatics 28(11):1536–1537. https://doi.org/10.1093/bioin 65(1):109–121 formatics/bts184 Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among Pusterla N, Pesavento PA, Leutenegger CM et al (2002) Disseminated ascomycetes: evidence from an RNA polymerse II subunit. Mol pulmonary adiaspiromycosis caused by Emmonsia crescens in a Biol Evol 16(12):1799–1808. https://doi.org/10.1093/oxfordjour horse. Equine Vet J 34(7):749–752 nals.molbev.a026092 Restrepo A, Baumgardner DJ, Bagagli E et al (2000) Clues to the Malik R, Capoor MR, Vanidassane I et al (2016) Disseminated presence of pathogenic fungi in certain environments. Med Emmonsia pasteuriana infection in India: a case report and a Mycol 38:67–77 review. Mycoses 59(2):127–132. https://doi.org/10.1111/myc. Rofael M, Schwartz IS, Sigler L et al (2018) Emmonsia helica 12437 infection in HIV-infected man, California, USA. Emerg Infect Maphanga TG, Britz E, Zulu TG et al (2017) In vitro antifungal Dis 24(1):166–168. https://doi.org/10.3201/eid2401.170558 susceptibility of yeast and mold phases of isolates of dimorphic Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phyloge- fungal pathogen Emergomyces africanus (formerly Emmonsia netic inference under mixed models. Bioinformatics sp.) from HIV-infected South African patients. J Clin Microbiol 19(12):1572–1574 55(6):1812–1820. https://doi.org/10.1128/JCM.02524-16 Saccente M, Woods GL (2010) Clinical and laboratory update on Marin-Felix Y, Stchigel AM, Cano-Lira JF et al (2015) Emmonsiel- blastomycosis. Clin Microbiol Rev 23(2):367–381. https://doi. lopsis, a new genus related to the thermally dimorphic fungi of org/10.1128/CMR.00056-09 the family Ajellomycetaceae. Mycoses 58(8):451–460. https:// Samson RA, Houbraken J, Thrane U et al (2010) Food and indoor doi.org/10.1111/myc.12336 fungi. CBS KNAW Biodiversity Center, Utrecht Matsuda K, Niki H, Yukawa A et al (2015) First detection of Schoch CL, Seifert KA, Huhndorf S et al (2012) Nuclear ribosomal adiaspiromycosis in the lungs of a deer. J Vet Med Sci internal transcribed spacer (ITS) region as a universal DNA 77(8):981–983. https://doi.org/10.1292/jvms.15-0083 barcode marker for Fungi. Proc Natl Acad Sci USA Matute DR, McEwen JG, Puccia R et al (2006) Cryptic speciation and 109(16):6241–6246. https://doi.org/10.1073/pnas.1117018109 recombination in the fungus Paracoccidioides brasiliensis as Schwartz IS, Govender NP, Corcoran C et al (2015a) Clinical revealed by gene genealogies. Mol Biol Evol 23(1):65–73. characteristics, diagnosis, management and outcomes of dissem- https://doi.org/10.1093/molbev/msj008 inated emmonsiosis: a retrospective case series. Clin Infect Dis McDonough ES, Lewis AL (1968) The ascigerous stage of Blasto- 61(6):1004–1012. https://doi.org/10.1093/cid/civ439 myces dermatitidis. Mycologia 60:76–83 Schwartz IS, Kenyon C, Feng P et al (2015b) 50 Years of Emmonsia Medoff G, Maresca B, Lambowitz AM et al (1986) Correlation disease in humans: the dramatic emergence of a cluster of novel between pathogenicity and temperature sensitivity in different fungal pathogens. PLoS Pathog 11(11):e1005198. https://doi. strains of Histoplasma capsulatum. J Clin Invest org/10.1371/journal.ppat.1005198 78:16381–16647 Schwartz IS, Wiederhold NP, Patterson TF, Sigler L (2017) Meece JK, Anderson JL, Fisher MC et al (2011) Population genetic Blastomyces helicus, an emerging dimorphic fungal pathogen structure of clinical and environmental isolates of Blastomyces causing fatal pulmonary and disseminated disease in humans and dermatitidis, based on 27 polymorphic microsatellite markers. animals in western Canada and United States. IDWeek, San Appl Environ Microbiol 77(15):5123–5131. https://doi.org/10. Diego, CA, 4–8 October 2017 1128/AEM.00258-11 Schwartz IS, Sanche S, Wiederhold NP, Patterson TF, Sigler L (2018) Meyer CP, Paulay G (2005) DNA barcoding: error rates based on Emergomyces canadensis, a dimorphic fungus causing fatal comprehensive sampling. PLoS Biol 3(12):e422. https://doi.org/ systemic human disease in North America. Emerg Infect Dis 10.1371/journal.pbio.0030422 24(4):758–761. https://doi.org/10.3201/eid2404.171765 Mun ˜ oz JF, Gauthier GM, Desjardins CA et al (2015) The dynamic Seifert KA, Hughes SJ, Boulay H et al (2007) Taxonomy, nomen- genome and transcriptome of the human fungal pathogen clature and phylogeny of three cladosporium-like hyphomycetes, 123 Fungal Diversity (2018) 90:245–291 291 Sorocybe resinae, Seifertia azaleae and the Hormoconis Tamura K, Stecher G, Peterson D et al (2013) MEGA6: Molecular anamorph of Amorphotheca resinae. Stud Mycol 58:235–245. Evolutionary Genetics Analysis version 6.0. Mol Biol Evol https://doi.org/10.3114/sim 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197 Sekhon AS, Jackson FL, Jacobs HJ (1982) Blastomycosis: report of Turner D, Burke M, Bashe E et al (1999) Pulmonary adiaspiromy- the ﬁrst case from Alberta, Canada. Mycopathologia 79:65–69 cosis in a patient with acquired immunodeﬁciency syndrome. Shapiro B, Ho SY, Drummond AJ et al (2010) A Bayesian Eur J Clin Microbiol Infect Dis 18(12):893–895 phylogenetic method to estimate unknown sequence ages. Mol Untereiner WA, Scott JA, Naveau FA et al (2004) The Ajellomyc- Biol Evol 28(2):879–887. https://doi.org/10.1093/molbev/ etaceae, a new family of vertebrate-associated Onygenales. msq262 Mycologia 96(4):812–821 Sigler L (1996) Ajellomyces crescens sp. nov., taxonomy of Van den Brink J, Facun K, de Vries M et al (2015) Thermophilic Emmonsia spp., and relatedness with Blastomyces dermatitidis growth and enzymatic thermostability are polyphyletic traits (teleomorph Ajellomyces dermatitidis). J Med Vet Mycol within Chaetomiaceae. Fung Biol 119(12):1255–1266. https:// 34(5):303–314 doi.org/10.1016/j.funbio.2015.09.011 Sigler L (1998) Agents of adiaspiromycosis. In: Ajello L, Hay R (eds) Vilela R, Mendoza L (2018) Paracoccidioidomycosis ceti (lacaziosis/ Topley & Wilson’s microbiology and microbial infections, vol 4, lobomycosis) in dolphins. In Seyedmousavi S et al. (eds) 9th edn. Arnold, London, pp 571–583 Emerging and epizootic fungal infections in animals (in press) Sigler L (2005) Adiaspiromycosis and other infections caused by Visagie CM, Hirooka Y, Tanney JB et al (2014) Aspergillus, Emmonsia species. In: Hodder A (ed) Topley & Wilson’s Penicillium and Talaromyces isolated from in house dust microbiology and microbial infections, 10th edn. Wiley, London, samples collected around the world. Stud Mycol 78:63–139. pp 809–824 https://doi.org/10.1016/j.simyco.2014.07.002 Sigler L (2015) Emmonsia helica Sigler. Index Fungorum 237:1. Wang P, Kenyon C, de Hoog GS et al (2017) A novel dimorphic http://www.speciesfungorum.org/Names/SynSpecies.asp?Recor pathogen, Emergomyces orientalis (Onygenales), agent of dis- dID=551157 seminated infection. Mycoses 60(5):310–319. https://doi.org/10. Skouboe P, Frisvad JC, Taylor JW et al (1999) Phylogenetic analysis 1111/myc.12583 of nucleotide sequences from the ITS region of terverticillate Wellinghausen N, Kern WV, Haase G et al (2003) Chronic Penicillium species. Mycol Res 103:873–881 granulomatous lung infection caused by the dimorphic fungus Stamatakis A (2014) RAxML version 8: a tool for phylogenetic Emmonsia sp. Int J Med Microbiol 293(6):441–445. https://doi. analysis and post-analysis of large phylogenies. Bioinformatics org/10.1078/1438-4221-00281 30(9):1312–1313. https://doi.org/10.1093/bioinformatics/btu033 Wu SJ, Valyi-Nagy T, Engelhard HH et al (2005) Secondary Stielow JB, Levesque CA, Seifert KA et al (2015) One fungus, which intracerebral blastomycosis with giant yeast forms. Myco- genes? Development and assessment of universal primers for pathologia 160(3):253–257 potential secondary fungal DNA barcodes. Persoonia Yang Y, Ye Q, Li K et al (2017) Genomics and comparative genomic 35:242–263. https://doi.org/10.3767/003158515X689135 analyses provide insight into the taxonomy and pathogenic Taborda PR, Taborda VA, McGinnis MR (1999) Lacazia loboi gen. potential of novel Emmonsia pathogens. Front Cell Infect nov., comb. nov., the etiologic agent of lobomycosis. J Clin Microbiol 7:105. https://doi.org/10.3389/fcimb.2017.00105 Microbiol 37(6):2031–2033 Afﬁliations 1,2,3 2,4 5 6 7,8 9 • • • • • • Yanping Jiang Karolina Dukik Jose F. Mun˜ oz Lynne Sigler Ilan S. Schwartz Nelesh P. Govender 10 11 2 2,12 13 • • • • • Chris Kenyon Peiying Feng Bert Gerrits van den Ende J. Benjamin Stielow Alberto M. Stchigel 1 2,3,4 Hongguang Lu Sybren de Hoog 1 8 Department of Dermatology, The Afﬁliated Hospital, Global Health Institute, Faculty of Medicine and Health Guizhou Medical University, Guiyang, China Sciences, University of Antwerp, Antwerp, Belgium 2 9 Westerdijk Fungal Biodiversity Institute, Utrecht, The National Institute for Communicable Diseases [Centre for Netherlands Healthcare-Associated Infections, Antimicrobial Resistance and Mycoses], A Division of the National Health Laboratory Center of Expertise in Mycology of RadboudUMC/Canisius Service, Johannesburg, South Africa Wilhelmina Hospital, Nijmegen, The Netherlands Sexually Transmitted Infection Unit, Institute of Tropical Institute of Biodiversity and Ecosystem Dynamics, Medicine, Antwerp, Belgium University of Amsterdam, Amsterdam, The Netherlands Third Afﬁliated Hospital, Sun Yat-Sen University, Broad Institute of MIT and Harvard, Cambridge, MA, USA Guangzhou, China University of Alberta Microfungus Collection and Herbarium Thermo Fisher Scientiﬁc, Landsmeer, The Netherlands [now UAMH Centre for Global Microfungal Diversity] and Biological Sciences, Edmonton, AB, Canada Mycology Unit and IISPV, University Rovira i Virgili, Reus, Tarragona, Spain Division of Infectious Diseases, Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
Fungal Diversity – Springer Journals
Published: Jun 5, 2018
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