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DOI: 10.3852/mycologia.100.2.259
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Mycologia, 100(2), 2008, pp. 259-274.
© 2008 by The Mycological Society of America

Siepmannia, a new genus in the Mucoraceae


Hanna Kwasna 1

     Department of Forest Pathology, Agriculture University, ul. Wojska Polskiego 71 c, 60- 625 Poznan, Poland

Helgard I. Nirenberg

     Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biologial Safety, Königin-Luise-Straße 19, D-14195 Berlin (Dahlem), Germany

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METERIALS AND METHODS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 

A new genus Siepmannia of the Mucoraceae family is described. Siepmannia pineti sp. nov. and S. lariceti sp. nov. were isolated from forest soils in Poland. The species were differentiated on the basis of morphology and ITS1/2 rDNA sequencing. Siepmannia pineti and S. lariceti are identical in some aspects. Both show distinct morphological dimorphism in culture depending on conditions of growth and exhibit distinct mycotrophism manifested by sporulation in the presence of other fungi. Their morphological elements are minute. The phylogenetic grouping of Siepmannia with Absidia parricia and A. zychae leads to the creation of two new combinations, S. parricida and S. zychae.

Key words: ITS1/2 rDNA sequencing, new genus, new species, Siepmannia lariceti, Siepmannia parricida, Siepmannia pineti, Siepmannia zychae, taxonomy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METERIALS AND METHODS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
During an investigation on the mycobiota of forest soils from Poland two unknown mucoraceous fungi were recorded in soils of pine, birch and larch forest stands when the soil-particle plating method was used for isolation of fungi (Nirenberg and Metzler 1990Go). Both fungi, together with other soil mycobiota, grew only on and near the soil particles placed on a synthetic low nutrient agar (SNA). Their occurrence was detected by the presence of sporangia formed after 2 wk. All initial attempts to separate the new fungi from other components of the fungal community were unsuccessful. The species with oval spores was tentatively named Mucor laxorhizus Ling-Young (Kwasna and Nirenberg 1994Go). Both fungi were successfully separated from other members of soil mycobiota after incubation 6 mo at 4 C. In axenic culture they did not grow on commonly used media. Soon it was noticed that both fungi need a fungal partner to grow in vitro. All morphological elements produced by both fungi were smaller than those of the currently known Mucoraceae. The fungus with circinate conidiophores resembled Fennellomyces linderi (Hesseltine & Fennell) Benny & R.K. Benj. (=Circinella linderi Hesseltine & Fennell). The other one, with sympodially branched conidiophores, resembled Mucor circinelloides van Tiegh., disregarding its different size. Studies to determine the identity of both new fungi are reported here.


   METERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METERIALS AND METHODS
TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
Isolation of fungi.— – In summer 1992 and autumn 2002 four soil samples were collected from a nursery of Scots pine (Pinus sylvestris L.) from 17 y old Scots pine, 20 y old birch (Betula pendula L.) and 20 y old larch (Larix decidua L.) stands in Zielonka Forest District, near Poznan, West Poland (17°10'E, 52°50'N). In each stand the soil was sampled from a depth of 10–15 cm, from six different places that were 5–6 m apart. In the laboratory subsamples were mixed, dried, pressed through a 0.6 mm mesh sieve and mixed by rotating 6 h. Single particles, 0.5 mm diam, were placed individually on SNA (Nirenberg 1976Go; 1 g KH2PO4, 1 g KNO3, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 20 g agar, 10 mg chlorotetracycline, 50 mg dihydrostreptomycin sulphate, 100 mg penicillin G, 1 L distilled water) in the center of a 9 cm diam Petri dish. Incubation conditions included 5 d at 20 C in darkness followed by 7 d at 17 C under continuous near-ultraviolet light (Philips 40/80 W) and 30 d at 20–25 C under natural day/night light.

Separation and identification.— – The fungi were transferred to Czapek-Dox Agar (CDA; 30 g sucrose, 3 g NaNO3, 1 g K2HPO4, 0.5 g KCl, 0.5 g MgSO4·7H2O, 0.01 g FeSO4, 22 g agar, 1 L distilled water), malt-yeast peptone agar (MYPA; 7 g malt extract, 1 g soy peptone, 0.5 g yeast extract, 22 g agar, 1 L distilled water), oatmeal agar (OA; 30 g filtered oak flakes, 15 g agar, 1 L distilled water), potato-dextrose agar (PDA; 40 g filtered white potatoes, 20 g agar, 1 L distilled water, pH = 7 and 4), synthetic Mucor agar (SMA; 40 g dextrose, 2 g asparagine, 0.5 g KH2PO4, 0.25 g MgSO4·7H2O, 0.5 g thiamine chloride, 15 g agar, 1 L distilled water) and SNA (pH = 4.6 and 2.0). The color of colonies was determined from Kornerup and Wanscher (1978)Go. Growth rate of colonies on PDA was determined at 20 C under a natural day/night cycle. Measurements of 50 sporangiophores, sporangia and spores from SNA cultures were made in water for identification.

Comparison.— – Morphological and molecular comparison were made with Absidia parricida Renner & Muskat ex Hesseltine & J.J. Ellis (CBS174.67), A. zychae Hesseltine & J.J. Ellis (CBS104.35), F. linderi (CBS158.54), M. circinelloides (IMI209778) and a group of Mucoraceae commonly occurring in forest soil in Poland (TABLE IGo).


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  		TABLE I. Species, collection information and GenBank accession numbers

 
DNA extraction and amplification.— – DNA was extracted from fungi grown in L-broth (10 g sodium chloride, 10 g tryptone, 5 g yeast extract, 1 L distilled water) 10 d at 25 C. The mycelium was separated by vacuum filtration and freeze-dried and ground. DNA extraction was based on the method of Lee and Taylor (1990)Go as described by Ward and Gray (1992)Go. DNA was amplified with primers ITS4 and ITS5 (White et al 1990Go). Each 25 µL PCR mixture contained 25 pmol of each primer, 0.25 units of MBI Taq polymerase (MBI Fermentas, St Leon-Rot, Germany), buffer (10 mM Tris-HCl pH 8.8, 50 mM KCl, 0.08% Nonidet P-40, 0.1 mg mL–1 BSA, 1.5 mM MgCl2), 0.2 mM deoxyribonucleoside triphosphates (dNTPs) and 100 ng fungal DNA. Cycling conditions included an initial denaturation step at 94 C for 10 min, followed by 30 cycles of 94 C for 30 s, 42 C for 1 min and 72 C for 2 min. This was followed by a final extension of 72 C for 10 min.

PCR-RFLP analysis.— – Aliquots of 8 µL of each amplicon were digested overnight at 37 C with 10 U of restriction endonuclease AluI, CfoI, DdeI, HaeIII, HincII, HinfI, HpaII, Sau3A1 and TaqI. Digested fragments were separated by electrophoresis in agarose gels (2.0% NuSieve GTG agarose supplemented with 1.0% FMC agarose) for 2 h at 5.5 V cm–1 in 1x TBE buffer and detected by ethidium bromide staining. Molecular weight markers, 1 kb {Phi}X-174 DNA digested with HaeIII (1 µg) and low DNA mass ladder (Invitrogen Ltd., Paisley, UK) (1 µg) were in first two lanes of the gel. The gel was photographed under UV light at 254 nm.

Sequencing of the ITS1/2 rDNA.— – Amplicons generated with ITS4 and ITS5 primers were purified with the MinElute PCR Purification Kit (QIAGEN, Crawley, UK). DNA sequences were determined with primers ITS4 and ITS5 using the ABI Prism Big Dye terminator cycle sequencing ready reaction kit (version 3.1, Applied Biosystems, Foster City, CA 94404, USA). Sequencing reactions were run at Oxford University, UK (http://polaris.bioch.ox.ac.uk/dnaseq/index.cfm). DNA sequences were assembled with the STADEN package (Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK). Sequence analyses were performed with the programs BLAST and FASTA in the GCG (Wisconsin) package (Genetic Computer Group 1994). The newly derived sequences were aligned with sequences from other fungi, partly obtained from EMBL/Genbank (TABLE IGo) using the GCG programs PILEUP and Clustal X (Thompson et al 1997Go), and edited manually with GENEDOC (Nicholas and Nicholas 1997Go). Phylogenetic analysis was carried out with program MEGA 3.1 (Kumar et al 2004Go).

Fungal interactions.— – Effects of fungi on sporulation of Siepmannia pineti and Siepmannia lariceti were evaluated in paired cultures on SNA in Petri dishes (9 cm). Siepmannia species and one of the soil fungi, Geomyces asperulatus Sigler & J.W. Carmich., Mortierella alpina Peyronel, Oidiodendron tenuissimum (Peck) S. Hughes, Penicillium adametzii Zaleski, P. daleae Zaleski, Trichoderma viride Pers., or Umbelopsis vinacea, grew from inocula placed centrally. In control dishes S. pineti or S. lariceti grew alone. The presence and intensity of sporulation of S. pineti and S. lariceti were checked microscopically after 60 d at 20–25 C, in a natural day/night cycle. Parasitism of A. parricida and A. zychae also was evaluated in paired cultures on SNA in Petri dishes (9 cm). Absidia and Cokeromyces recurvatus Poitras or Zygorhynchus moelleri Vuill. grew from inocula placed 2 cm apart. The presence of coiling around hyphae or invasion in mycelium of Z. moelleri or C. recurvatus by A. parricida or A. zychae hyphae was checked microscopically after 20–30 d at 20–25 C in a natural day/night cycle.


   TAXONOMY
TOP
 ABSTRACT
 INTRODUCTION
 METERIALS AND METHODS
 TAXONOMY
DISCUSSION
 LITERATURE CITED
 
Morphological and molecular studies of two new and other related species of fungi contributed to the erection of a new genus Siepmannia within Mucoraceae, with the following description.

Siepmannia Nirenberg & Kwasna gen. nov.

Coloniae in agaro PDA dicto moderate vel lentissime crescentes, paulo lanosae usque funiculosae vel gelatinosae postea pulverulosae. Mycelio aerio albo vel pallide ochraceo usque subfusco. Mycelium filamentosum e hyphis tenuitunicatis, ramosis vel cellulis inflatis, magnis, globosis compositum. Sporangiophora recta vel circinata, monopodialiter vel sympodialiter ramose. Rami unum sporangium terminale ferentes, septo subsporangiodivisi. Sporangia singularia, minuta, globosa, multispora; tunica partim deliquescentia. Columellae hemisphaericae vel globosae, collarium distinctum praebentes. Sporangiosporae minutae, unicellulares, hyalinae, leves, globosae vel ovulares vel subcylindricae. Stolones et rhizoides saepe presentes. Zygosporae etiam saepe presentes, in mycelio aerio formatae, brunneolae; suspensores aequales, nonnumquam appendicibus carentes. Gemmae absunt. Mycotrophicae.

Colonies on PDA moderately to slow growing, loosely cottony to funiculose or gelatinous, later powdery, with white to ochraceous aerial mycelium. Mycelium composed of filamentous, thin-walled and branched hyphae or of swollen hyphae and large, round cells. Sporangiophores erect or circinate, monopodially or sympodially branched, usually with a septum under the sporangium. Sporangia single, terminal, small, globose, many-spored with a partly deliquescent sporangial wall. Columella hemispherical to globose, mostly with a well-defined collar. Sporangiospores minute, one-celled, hyaline, smooth, globose to oval or cylindrical. Stolons and rhizoids may occur. Zygospores, if present, borne in the aerial mycelium, brownish, warty. Suspensors equal in size. Appendages usually prominent.

Genus Siepmannia includes two new species, S. pineti and S. lariceti, and two new combinations, S. parricida and S. zychae.

Siepmannia pineti Kwasna & Nirenberg sp. nov.

FIGS. 1–6Go


Figure 1
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  		FIGS. 1–6. Siepmannia pineti. 1. 30 d old colony on PDA at 25 C. 2–6. Morphological characters from S. pineti grown on SNA at 20–25 C. 2. Sporangium on sporangiophore with stolon. 3. Two young sporangia on a branched sporangiophore. 4. Sporangium with sporangiospores. 5. Sporangiophore branched monopodially. 6. Sporangiospores. Bars (2, 5) = 20 µm, (3, 4) = 10 µm, (6) = 5 µm.

 
Coloniae in agaro PDA dicto valde restricto crescentes; post 30 dies ad 25 C 2–10 mm diam, gelatinosae vel pulverulosae, rugosae vel convolutae, margine arachnoideae, albae vel aurantiaceae, reverso cremeo vel pallide brunneo; post 4 menses ad 4 C 5–30 mm diam. Mycelium aerium sparsum, lanosum, album vel griseo-ochraceum, vel cellulis inflatis, magnis, globosis compositum. Mycelium substratum hyalinum, tenue, ramosum. Sporulatio in cultura axenica rara, secundo fungo praesente copiosa. Sporangiophora uniformia, non ramosa vel 1–4 x monopodialiter vel sympodialiter ramosa, e stolonibus hyalinis, opposita rhizoidea in mycelio immersio. Rami plerumque circinati, raro erecti, hyalini vel brunnei, levi vel incrustati, 50–70(–80) µm longi, basis 5–8(–10) µm diam et apice 3–5 µm diam. Stolones hyalini vel brunnei, incrustates vel leves, ad 1 mm longi et (1–)2–5(–6) µm diam. Distantia inter rhizoidea et ramus basis (15–)20–25(–30) µm. Sporangia terminaliter formata, initio hyalina, deinde ochracea-brunnea, globosa, (16–)20–40 µm diam, tunica grisea vel ochracea, incrustata, deliquescentia vel persistentia. Columellae hyalineae vel pallide brunneae, globosae vel ovales, cum distinctis collariis, 14–20(–22) µm diam. Sporangiosporae pallide brunneae, ovales usque fere ellipticae, leves 2–2.5 x 1.5 µm. Chlamydosporae absentes. Zygosporas non visas.

Holotypus. – IMI 393564, cultura exsiccata ex terra in Polonia.

Colonies on potato-dextrose agar (PDA) in axenic cultures slow-growing; after 30 d at 25 C 2–10 mm diam, first gelatinous, later powdery, unevenly wrinkled or convoluted, cauliflower-like, margin uneven, white to cream, reverse cream to brownish; after 4 mo at 4 C, 5–30 mm diam. Aerial mycelium sparse, delicately lanose, white to grayish orange or composed of round, hyaline cells (10–)15–100 µm diam. Substrate mycelium hyaline, thin-walled, delicate and branched. Sporulation in axenic culture rare, after 6 mo incubation at 4 C, in the presence of a second fungus abundant. Sporangiophores uniform, unbranched to 1–4 times monopodially or sympodially branched with stolon and rhizoids submerged in the substrate. Branches mostly circinate, rarely erect, hyaline to brownish, incrusted when young and smooth-walled when older, 50–70(–80) µm long, 5–8(–10) µm diam at the base and 3–5 µm diam at the top, with one septum below the sporangium. Stolons hyaline to brownish, incrusted when young and smooth-walled when older, up to 1 mm long and (1–)2–5(–6) µm diam. The distance from rhizoids to the base of branch (15–)20–25(–30) µm. Sporangia single, first hyaline, later ochraceous-brownish, globose, (16–)20–40 µm diam. Sporangial wall gray-ochraceous, incrusted, deliquescent to persistent. Columella hyaline to light brown, globose to oval, with a distinct collar, 14–20(–22) µm diam. Sporangiospores light brown, oval to almost ellipsoid, smooth, thin-walled 2–2.5 x 1.5 µm. Chlamydospores absent. Zygospores not observed.

Holotype. – IMI 393564 CABI Bioscience UK Centre, dried culture of No KFL S11 on SNA. Isolated from soil of Betula pendula, Zielonka, Poznan, Poland, 17°10'E, 52°50'N, H. Kwasna, Nov 2002.

Isotype. – KFL S12, Department of Forest Pathology, Poznan, PL.

Ex-type. – IMI 391615, BBA 72521, RR 212 Rothamsted Research, Harpenden, UK.

Additional cultures examined. – KFL S21 from soil of 20 y old Betula pendula, KFL S22 from soil of 17 y old Scots pine stand, Zielonka, Poznan, Poland, 17°10'E, 52°50'N, H. Kwasna, Nov 2002, Nov 2003. EMBL No AJ748134.

Etymology. – pineti refers to "inhabiting pine" (Pinus) in the soil of which the fungus was first and most often found.

Known distribution. – Poland, Germany.

Habitat. – In soil of Scots pine and birch forests.

Siepmannia lariceti Nirenberg & Kwasna sp. nov.

FIGS. 7–15Go


Figure 2
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  		FIGS. 7–15. Siepmannia lariceti. 7. 30 d old colony on PDA at 25 C. 8–15. Morphological characters from S. lariceti grown on SNA at 20–25 C. 8. Sporangia in aerial mycelium. 9. Sympodially branched sporangiophore with sporangia. 10. Sympodially branched sporangiophore. 11. Zygospores. 12. Formation of zygospore from two gametangia. 13. Mature zygospore. 14–15. Sporangiospores. Bars (8) = 40 µm, (9, 10) = 20 µm, (11–13) = 10 µm, (14–15) = 5 µm.

 
Coloniae in agaro PDA dicto valde restrictae, post 30 dies ad 25 C 5–10 mm diam crescentes, rugosae vel convolutae, margine arachnoideae, albae vel cremeae, reverso pallide cremeae. Mycelium aerium sparsum, funiculosum, hyalinum aut gelatinosum et pulverulosum, cum globosis cellulis 10–(15–100) µm diam. Mycelium substratum hyalinum, tenerem, ramosum, 1–2 µm diam. Sporulation in cultura singulare raris, cum secundo fungo abundans. Sporangiophora ab mycelio immersio erecta, fortiter ramosa (1–13 rami), semper sympodialiter proliferata, usque 800 µm longa, incrustata, brunnea. Rami (40–)60–80(–100) x (2.5–)3–4 µm, cum septum 8–10 µm subsporangiis. Distantia inter rami (20–)40–70(–80) µm. Sporangia ochraceo-brunnea, globosa, 12–20(–24) µm diam, cum parietibus sporangii incrustatis, deliquescentis. Columellae hyalinae vel pallide brunneae, globosae vel applanatae, cum distinctis collariis (8–)10–12(–16) x 6–8(–10) µm. Sporangiosporae plus minusve aequales, pallide brunneae, globosae, angulariae, leves, 1.5–2 µm diam. Zygosporae fere, globosae (24–28) vel ovales (28–32) x (12–)16–20 µm diam, pallide-brunneae, asperulatae. Chlamydosporae absentes. Fungus homothallicus.

Holotypus. – IMI 393565, cultura exsiccata ex terra in Polonia.

Colonies on potato-dextrose agar (PDA) slow-growing, restricted, 5–10 mm diam after 30 d at 25 C, unevenly wrinkled or convoluted, margin uneven, white to cream, reverse pale cream. Aerial mycelium sparse, delicately funiculose, hyaline or gelatinous to roughly powdery, often consisting of round 10–(15–100) µm diam cells. Substrate mycelium hyaline, delicate, thin-walled and strongly branched, 1–2 µm diam. Sporulation in axenic culture rare, in the presence of a second fungus abundant. Sporangiophores arising from the submerged mycelium, sympodially branched (1–13 times), <800 µm long, incrustate, brown. Side branch (40–)60–80(–100) x (2.5–)3–4 µm, with septum located 8–10 µm below the sporangium. Distance between branches (20–)40–70(–80) µm. Sporangia ochraceous-brown, globose, 12–20(–24) µm diam with wall slightly incrustate, deliquescent. Columella hyaline to pale brown, globose to applanate, with a distinct collar (8–)10–12 (–16) x 6–8(–10) µm. Sporangiospores more or less regular in size and shape, pale brown in mass, globose, angular, smooth-walled, 1.5–2 µm diam. Zygospores numerous, globose (24–28) to ovoid (28–32) x (12–)16–20 µm diam, golden-brown to dark brown, at first with many oil droplets. Zygospore wall with small, separate warts. Suspensors identical, 8–10 µm diam, straight, smooth-walled, hyaline. Zygophores rough-walled, bearing the zygospores just above or at the surface of the medium. Chlamydospores absent. Fungus homothallic.

Holotype. – IMI 393565 CABI Bioscience UK Centre, dried culture of No KFL S13 on SNA. Isolated from soil of Larix decidua, Zielonka, Poznan, Poland, 17°10'E, 52°50'N, H. Kwasna, Nov 2002. EMBL No AJ748857.

Isotype. – KFL S14, Department of Forest Pathology, Poznan, PL.

Ex-type. – IMI 391741, RR 392 Rothamsted Research, Harpenden, UK.

Additional cultures examined. – KFL S15 from soil of 20 y old Betula pendula, KFL S16 from soil of 20 y old Larix decidua, Zielonka, Poznan, Poland, 17°10'E, 52°50'N, H. Kwasna, Nov 2002.

Etymology. – lariceti refers to "inhabiting larch" (Larix) in the soil of which the fungus was first and most often found.

Known distribution. – Poland.

Habitat. – In soil of larch and birch forests.

Occurrence.— – One, nine and two isolates of S. pineti were recorded respectively on 20 soil particles from each of these sites: the Scots pine nursery, the 17 y old Scots pine and 20 y old birch. Two and nine isolates of S. lariceti were recorded respectively on 20 soil particles from each of the 20 y old birch and 20 y old larch stands in Zielonka Forest District, near Poznan (Kwasna and Nirenberg 2004Go).

DNA of S. pineti, S. lariceti, F. linderi and M. circinelloides were amplified successfully by ITS4 and ITS5 primers. AluI, CfoI, DdeI, HaeIII, HincII, HinfI, HpaII, Sau3A1 and TaqI generated distinctive, informative and species-specific RFLP patterns which clearly separated S. pineti from S. lariceti, and S. pineti from F. linderi as well as S. lariceti from M. circinelloides.

Sequences of ITS1/2 rDNA region were determined for S. pineti, S. lariceti and compared with another 30 ITS1/2 rDNA sequences of Mucorales, partly done by the first author and partly obtained from EMBL database (TABLE IGo). The alignment of rDNA included 385 nucleotide from the ITS1, 5.8S rDNA and ITS2 regions. Sequence alignment was used for phylogenetic analyses. (The phylogram obtained with the MEGA 3.1 program is shown in FIG. 16Go.)


Figure 3
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  		FIG. 16. Phylogenetic relationships among Mucorales obtained from analysis of the ITS1/2 rDNA. Bootstrap values, based on 1000 replicates, are indicated above the branches. The tree was rooted with Neocallimastix frontalis (Chytridiomycota, Neocallimasticales, AY429664 [GenBank] ) as outgroup.

 
Siepmannia pineti and S. lariceti grouped with A. parricida and A. zychae (subclade 1), which are currently the only slow-growing Absidia species with minute morphological characters. Two groupings were supported by a high bootstrap value of 96–98%. Subclade 1 joined subclade 2 that grouped Absidia glauca Hagem, A. coerulea Bainier, A. cylindrospora Hagem and A. repens Tiegh. Absidia corymbifera (Cohn) Saccardo & Trotter grouped with F. linderi in subclade 3 (100% boostrap support). The Mucor, Actinomucor, Zygorhynchus and Gilbertella species formed a separate distinct clade. The tree was rooted with Neocallimastix frontalis (R.A. Braune) Vavra & Joyon ex I.B. Heath (Chytridiomy-Chytridiomycota, Neocallimasticales, AY429664 [GenBank] ) as outgroup. Grouping of both Siepmannia species with A. parricida and A. zychae led to inclusion of the two latter species in the genus Siepmannia (FIGS. 16GoGo–27Go).


Figure 4
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  		FIGS. 17–22. Siepmannia parricida (CBS 174.67) on different media in darkness at 20 C. 17. Sporangiophore with rami branching almost at right angles on MEA after 36 d. 18. Aerial mycelium with emptying sporangium on MEA after 36 d. 19. Rhizoids with short stalk at the base of sporangiophore and stolon on MEA after 36 d. 20. Gametangia with developing zygospore on 5% CDA after 11 d. 21. Ramified substrate mycelium on SNA after 11 d. 22. Mature warty zygospore on 5% CDA after 11 d. Bars = 25 µm.

 

Figure 5
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  		FIGS. 23–27. Siepmannia zychae (CBS 104.35) on SNA under natural light conditions at 22 C. 23. Sporulation after 16 d. 24. Sympodial and verticillate ramification of sporangiophores ending in sporangia after 9 d. 25. Lower part of sporangiophore with stolon and rhizoides after 20 d. 26. Subglobose to turbinate, 20 d old sporangiospores. 27. Twenty d old knobbed substrate mycelium with sporangiospores. Bars = 25 µm.

 
In axenic cultures S. pineti and S. lariceti did not grow on CDA, MYPA, OA, PDA, SMA and SNA at 37 C. Both fungi had restricted growth on PDA and SNA at 25 C and sporulated sparsely after incubation for at least 6 mo at 4 C. Geomyces asperulatus, P. adametzii, P. daleae and U. vinacea stimulated sporulation of both fungi on SNA at 20–25 C in a day/night cycle. Usually single sporangia with sporangiospores were produced after 60 d of incubation. More intense sporulation appeared when S. pineti and S. lariceti grew in association with U. vinacea. Oidiodendron tenuissimum affected neither growth nor sporulation of either fungus, which as in the control cultures produced gelatinous, nonsporulating colonies. Mortierella alpina and T. viride inhibited growth of S. pineti and S. lariceti, which produced neither vegetative mycelium nor sporangia.

In the paired cultures on SNA, S. parricida and S. zychae grew mostly on the surface of the medium while C. recurvatus and Z. moelleri produced only mycelium submerged in the medium. No parasitism expressed as coiling or invasion of mycelium of Z. moelleri or C. recurvatus by S. parricida or S. zychae hyphae was observed in the centers of plates where the fungi met.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METERIALS AND METHODS
 TAXONOMY
 DISCUSSION
LITERATURE CITED
 
The new genus Siepmannia with two new species and two new combinations was separated from Absidia sensu stricto and A. corymbifera on the basis of morphological and molecular data. Two new species of Siepmannia, S. pineti and S. lariceti share the same characteristics: (i) they occur in the same habitat, which is sandy forest soil in the temperate zone; (ii) they show a distinct morphological dimorphism resulting from the condition of growth; (iii) in axenic culture, particularly at lower temperatures, produce restricted colonies composed mostly of giant, globose, hyaline cells; (iv) in the presence of at least one associate fungus show distinct mycotrophism manifested by an increase in growth, the formation of a filamentous mycelium and moderately abundant sporulation; (v) in the lower aerial mycelium produce specialized hyphae with numerous, short and swollen branches; (vi) sporulate rarely; (vii) produce minute sporulating elements; (viii) quickly lose the ability to grow and sporulate in the next few transfers.

Siepmannia lariceti differs from S. pineti in not producing stolons, rhizoids or apophyses, or forming sympodially branched sporangiophores and has the ability to form zygospores.

Siepmannia pineti resembles S. parricida and S. zychae in the formation of (i) swollen hyphae and large, round cells; (ii) incrusted to smooth-walled sporangiophores and stolons growing from the same point, with a short supporting "stalk" opposite the rhizoids: (iii) branched, short, hyaline to brownish rhizoids; (iv) a single septum under the sporangium; (v) funnel-shaped apophyses; (vi) columellae without projections; (vii) tiny, oval to cylindrical spores with transparent walls (Renner and Muskat 1958Go; Hesseltine and Ellis 1964Go, 1966Go).

Siepmannia parricida and S. zychae produce (i) regular colonies with lanose, although very low, and in S. zychae restricted, aerial mycelium; (ii) well-developed substrate mycelium; (iii) abundant sporulation in axenic culture, even after many transfers; (iv) longer and straight sporangiophore branches; (v) deliquescent sporangial walls that leave only an indistinct collar. Moreover sporangiospores of S. zychae are irregular in shape and size, and zygospores in the homothallic S. parricida are produced easily and abundantly (TABLE IIGo).


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  		TABLE II. Morphological characteristics of Siepmannia pineti, S. lariceti, S. parricida and S. zychae

 
Siepmannia parricida and S. zychae are rare fungi. The former has been isolated twice, from soil collected in Tunis (Renner and Muskat 1958Go) and grassland soil in the UK (CBS 174.67 = ATCC 24168 = IMI 238607 = NRRL 2409) (Hesseltine and Ellis 1966Go). The latter was isolated once from rotten wood in Germany (CBS 104.35 = ATCC 24266 = NRRL 2806) (Zycha 1935). Both were reported to be able to parasitize other fungi. Siepmannia parricida was shown to parasitize 13 host strains of Mucorales and A. zychae appeared to be a potential parasite on species of Mucor but not on Absidia or Rhizopus species (Renner and Muskat 1958Go, Hesseltine and Ellis 1964Go).

Many morphological characters of S. pineti are similar to those of Fennellomyces species, particularly F. linderi. The latter also produces slow-growing, white to grayish-orange colonies, stolons and rhizoids, short, sympodially branched, circinate, incrusted sporangiophores on a "stalk" opposite the rhizoids, single, globose sporangia with apophyses and incrusted and persistent walls, septum below the sporangium, columella with a well-defined collar, oval sporangiospores and no zygospores (Hesseltine and Fennell 1955Go, Zycha et al 1969Go). All these morphological characters of F. linderi are however much larger.

Many morphological characters of S. lariceti are similar to those of M. circinelloides. The latter produces incrusted, brownish sporangiophores with repeatedly, sympodially branched, erect or slightly circinate branches and septum above the next branching, globose sporangia with incrusted, partly deliquescent and partly persistent walls, columella with a distinct collar, and brownish round to oval warty zygospores with short suspensors and no stolons, rhizoids or aphophyses. Mucor circinelloides can produce globose, angular, smooth-walled sporangiospores. It grows much faster, however, produces lanose and high colonies, much larger morphological structures and chlamydospores, which were not observed in S. lariceti.

Phylogenetic analysis of the ITS1/2 rDNA region placed S. pineti, S. lariceti, S. parricida and S. zychae in one subclade, suggesting their close relationship. It joined a subclade which grouped A. glauca, A. coerulea, A. repens and A. cylindrospora. Absidia corymbifera, which is known to be a human pathogen, grouped together with F. linderi and their subclade showed a much stronger relationship to other Mucorales than to Absidia.

The genus Siepmannia currently includes four species. Three shapes of spores are formed within the genus (i.e. irregular formed by S. zychae, cylindrical formed by. S. parricida and S. pineti, and round formed by S. lariceti). The architecture of the Siepmannia subclade suggests the possibility that there are missing links. They may be sought by further molecular studies of the Mucoraceae.

The genus Absidia sensu lato has been divided into three or two sections (Zycha et al 1969Go, Domsch et al 1980Go) on the basis of sporangiospore shape. Phylogenetic analysis of Absidia sensu lato suggests however that spore morphology, although helpful in morphological identification, is irrelevant for assessing phylogenetic relationships. Our differentiation of Absidia sensu lato into three groups is relevant for many morphological characters, including size of all organs, type, shape and branching of sporangiophore, presence of stolons, rhizoids, septum below the sporangium, columella projection, finger-like appendages attached to zygospore suspensors and distinctness of the collar. The separation is also consistent with temperature and nutritional requirements and sexual behavior.

The genus Siepmannia (subclade 1, FIG. 16Go) includes species with restricted growth, maximum growth below 35 C, minute morphological structures, sporangiophores formed opposite the rhizoids, septum below sporangium, columella without projections, zygospores with equal suspensors, homothallism and mycotrophism. The genus Absidia sensu stricto (subclade 2) includes species with faster and more profuse growth, maximum growth at 30 C, larger morphological structures, sporangiophores formed in the raised part of stolons, septum below sporangium, columella with a single projection, zygospores with unequal suspensors and with finger-like appendages and heterothallism. Subclade 3 includes species with faster and profuse growth, maximum growth at 48–52 C, sporangiophores with or without septum below sporangium, columella with a single or few projections, zygospores without finger-like appendages, heterothallism, and pathogenicity to humans and mammals.

The proposed new arrangements of the genera support the findings of Hesseltine and Ellis (1964)Go, Voigt et al (1999)Go and O’Donnell et al (2001)Go, who also observed polyphyly in the former Absidia genus. Hesseltine and Ellis (1964)Go divided the genus Absidia into two subgenera, Absidia and Mycocladus Hesseltine and Ellis. The latter was represented by A. corymbifera and was separated on the basis of production of zygospore suspensors devoid of finger-like appendages. Voigt et al (1999)Go differentiated A. corymbifera from Absidia on the basis of nuclear rDNA sequences and showed that the former is a sister group of Rhizomucor Lucet & Cost. ex. Vuill. O’Donnell et al (2001)Go analyzed partial SSU 18S rDNA, nuclear LSU 28S rDNA and EF-1{alpha} gene exons in Mucorales and showed that A. corymbifera groups with Circinella umbellata Tieghem & G. Le Monnier, while A. repens groups with other Mucorales.

The formation of filamentous hyphae and sporulation in S. pineti and S. lariceti was usually stimulated by low temperature but was observed mostly when the fungi were accompanied by other forest-soil fungi. We assume that the stimulatory effect is initiated by diffusible metabolites produced by the associates. Most stimulation was observed when the accompanying fungus was U. vinacea. The latter is known to produce alpha-galactosidase and lipase enzymes (Gaspar et al 1999Go, Shibuya et al 1999Go), which may convert the constituents of the culture medium into products easily used by Siepmannia. The associates usually also stimulated the production of rhizoids and characteristic, strongly branched and swollen hyphae, which obviously participate in nutrient uptake and contribute to better colony development. Umbelopsis vinacea earlier was observed to stimulate the growth of Armillaria species (Kwasna et al 2001Go).

Siepmannia pineti and S. lariceti did not parasitize the accompanying Ascomycetes nor S. parricida and S. zychae parasitized Z. moelleri or C. recurvatus, which was reported to be a host of S. parricida (Hasseltine and Ellis 1966). Parasitism on other Mucorales reported to be hosts of S. parricida and S. zychae by Hesseltine and Ellis (1966)Go was not tested however.

Siepmannia pineti and S. lariceti occurred relatively frequently in soil particles sampled in 17–20 y old birch, larch and Scots pine forest and in a Scots pine nursery in Zielonka Forest District in Poland (Kwasna and Nirenberg 2004Go). Siepmannia pineti seems to be common also in Scots pine forest soils in Germany (Nirenberg, unpubl). Both fungi undoubtedly have been overlooked when the soil dilution method was used for isolation of forest-soil fungi (Chwalinski et al 1987Go, 1988Go; Manka et al 1987Go, 1989Go; Przezbórski and Kwasna 1989Go; Kwasna 1995Go). Both fungi can be detected by the soil-particle plating method used for isolation of fungi (Nirenberg and Metzler 1990Go). This requires the placing of small particles of soil onto SNA in Petri dishes and incubation for 42 d. Growing close to the soil particles, they are affected by the physical and chemical properties of the soil and associated microorganisms and their metabolites. Near-UV radiation applied during incubation (Nirenberg and Metzler 1990Go) may have induced their sporulation.


   ACKNOWLEDGMENTS
 
The authors thank the German Academic Exchange Service (DAAD) for financing the visit of the first author to the Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biological Safety, Berlin, Germany. We thank Rothamsted Research, UK, for making the molecular studies possible, CABI and CBS Fungus Collections for providing cultures for molecular comparison. Thanks are extended to Prof W. Gams for help with the Latin description, critical comments as well as much support in interpreting the results and rewriting the manuscript. We thank also Ms Heidrum Anders for her excellent technical assistance and Dr G.L. Bateman for correcting and improving the English.


   FOOTNOTES
 
Accepted for publication December 26, 2007.

1 Corresponding author. E-mail: kwasna{at}au.poznan.pl


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 METERIALS AND METHODS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
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