MYCOTAXON. Scutellospora pernambucana, a new fungal species in the Glomeromycetes with a diagnostic germination orb

MYCOTAXON Volume 106, pp. 361 370 October December 2008 Scutellospora pernambucana, a new fungal species in the Glomeromycetes with a diagnostic germination orb Danielle Karla Alves da Silva 1, Nicácio de Oliveira Freitas 1 Gisela Cuenca 2, Leonor Costa Maia 1* & Fritz Oehl 3 1 *leonorcmaia@yahoo.com.br 1 Departamento de Micologia, CCB, Universidade Federal de Pernambuco Av. Prof. Nelson Chaves s/n, Cidade Universitária, 50670-420, Recife, PE, Brazil 2 Instituto Venezuelano de Investigaciones Cientificas, IVIC, Centro de Ecologia Apdo. 21827, Caracas 1020 A, Venezuela 3 Zurich-Basel Plant Science Center, Institute of Botany, University of Basel Hebelstrasse 1, 4056 Basel, Switzerland Abstract A new species of the arbuscular mycorrhiza-forming Scutellosporaceae (Glomeromycetes), described here as Scutellospora pernambucana, forms globose to subglobose (rarely oval), yellow to brown-yellow glomerospores terminally on sporogenous cells. The 110 150 µm diameter spores have three unornamented spore walls: outer (3-layered), middle (2-layered), and inner (3-layered). The germination shield formed on the outer surface of the inner wall is circular, hyaline to (rarely) light yellow, and has a characteristic coiled, orb-like structure formed by a single lobe that surrounds the initial germ hole and terminally bears one germ tube initiation. This unique germ shield structure easily distinguishes the new species from all other Scutellospora species; S. projecturata, which forms a similarly coiled germ shield, is differentiated by conspicuous columnar projections on the outer spore wall. S. pernambucana, collected from the Mata Atlantica and Caatinga biomes in Pernambuco State and a reforestation area in Maranhão State, appears to be a common fungus in Northeastern Brazil. Key words Gigasporaceae, Glomeromycota, Diversisporales Introduction Arbuscular mycorrhizal fungal species that form spores on bulbous sporogenous cells have been reorganized into four families based on germination structure characteristics and phylogenies generated by independent analyses of 18S and 25S rrna gene sequences (Oehl et al. 2008). Species with only one spore wall containing a warty inner germinal wall layer remain in the revised family Gigasporaceae (Morton & Benny 1990, Oehl et al. 2008), while those producing spores with 2 4 walls and a discrete germination shield on the innermost wall

362... Silva & al. were transferred into three new families: a) species with a yellow-brown to brown germ shield were transferred into the Dentiscutataceae, b) species with a multiple-lobed, hyaline to subhyaline germ shield and multiple germ tube initiations were attributed to the Racocetraceae, and c) species with a simple, bilobed to (rarely) mono-lobed, hyaline to subhyaline germ shield with (1-)2 germ tube initiations were transferred to the Scutellosporaceae (Oehl et al. 2008). The newly established Scutellosporaceae is mono-generic with Scutellospora (Walker & Sanders 1986) as type genus. A new arbuscular mycorrhizal fungal species was found during diversity studies in Northeastern Brazil in Pernambuco State in a Tropical Atlantic forest fragment and in the Caatinga biome and in Maranhão State in a forest revegetation area. The species that forms spores terminally on sporogenous cells and a unique germination shield on the outer surface of the innermost spore wall is hereafter described under the epithet Scutellospora pernambucana. Material and methods Soil sampling and culturing of AM fungi Soils were sampled in a tropical forest fragment (i.e., Zambana ) situated among other native forest fragments and sugarcane plantations near the community of Igarassu, Pernambuco State, Northeastern Brazil. The forest site is located at 07 41 29 07 43 17 S and 35 00 09 34 58 38 W (20-110 m above sea level). In December 2007 samples were taken from the rhizospheres (0 20 cm depth) of several native plant species characteristic of the Mata Atlantica tropical forest [e.g., Coccoloba latifolia (Polygonaceae), Maytenus spp. (Celastraceae), Clusia nemorosa (Clusiaceae), Margaritaria nobilis (Euphorbiaceae), Guapira spp. (Nyctaginaceae), Calyptranthes brasiliensis (Myrtaceae), Alseis pickelii (Rubiaceae), Pouteria peduncularis (Sapotaceae)]. The ph (H 2 O) of the sandy soil (91% sand) was 4.8, and available P (extracted after Mehlich; Nelson et al. 1953) was low (3 mg kg -1 ). The spore material was studied from field samples that were also used for pot cultures with Panicum miliaceum as host. The pots (300 g) were filled with each soil sample and maintained at the greenhouse of the Department of Mycology, Universidade Federal de Pernambuco, Recife. No host colonization or spore formation has yet been observed. Morphological analyses Glomerospores were extracted from field soils by wet sieving (Gerdemann & Nicolson 1963) and sucrose centrifugation (Jenkins 1964). The spores were thereafter mounted in PVLG, PVLG + Melzer s reagent, and in water, respectively (Brundrett et al. 1994). For the species descriptions, the terminology recently applied for other genera of the Diversisporales (Oehl & Sieverding 2004, Oehl et

Scutellospora pernambucana sp. nov. (Brazil)... 363 al. 2006, Sieverding & Oehl 2006, Palenzuela et al. 2008) were adopted, as well as the terminology of Walker & Sanders (1986) for the germ shield structures. For the spore denomination the terminology proposed by Goto & Maia (2006) was used. Description of the new species Scutellospora pernambucana Oehl, D.K Silva, N. Freitas, L.C. Maia, sp. nov. MycoBank MB 512130 figs. 1-10 Sporocarpia ignota. Sporae singillatim in solo efformatae anguste adiacetae ad cellulas sporogeneas subterminales vel intercalares, flavae ad bruneo-flavae ad flavo-bruneae, globosae (110 145 µm in diametro) vel subglobosae vel ovales (105 135 120 150 µm); sporae tunicis tribus: tunica exterior stratis tribus, in totum 3.5 5.5 µm crassa in aqua, expandens cum pressione (5.5 8.5 µm crassa) vel in PVLG (6.0 )9 14 µm crassa; stratum exterius tunicae exterioris hyalinum, evanescens ad semi-persistens, 1.0 2.1µm crassum; stratum medium laminatum, flavum ad brunneo-flavum, expandens cum pressione in aqua vel in PVLG; stratum interius tunicae exterioris flavum ad brunneo-flavum, subtile, 0.5 1.1 µm crassum; tunica media et tunica interior de novo formantes stratis hyalinibus; tunica media duobus stratis in totum 1.1 2.6( 3.3) µm crassa; tunica interior tribus stratis, 1.7 2.8( 3.5) µm crassa; solo stratum medium tunicae interioris purpureo colorans reagente Melzeri; scutellum germinale coniunctum ad tunicam interiorem, hyalinum vel rarum albo-flavum; circulare vel ovale, 50 80( 90) 60 80( 94) µm in diametro, cum lobo singulare, orbem formans, generaliter cum depressione singulare germinationis; structurae mycorrhizarum ignotae. Holotype: 83 8301 (URM No. 79239) from Zambana tropical forest fragment, Usina São José, Igarassu, Pernambuco State, Brazil. Etymology: pernambucana referring to the State in Northeastern Brazil where the new species was found first in two different biomes. Sporocarp formation unknown. Glomerospores are singly formed in soils terminally on a subterminal or intercalary bulbous suspensor cell (= sporogenous cell; fig. 1). Glomerospores are dark yellow to brown yellow to yellow brown, globose (110 145 µm in diameter) to subglobose (105 135 120 150 µm) to rarely oval, and have with three walls: an outer, a middle and an inner wall (ow, mw and iw; fig. 2). Outer wall is in total 3.6 5.5 µm thick in water, but expanding under pressure to 5.5 8.5 µm and to (6.0 )9.0 14 µm in PVLG based mountants; ow consists of three layers (figs. 3 7): outermost wall layer (owl1), hyaline to subhyaline to light yellow, evanescent to semi-persistent and about 1.0 2.1 µm thick (figs. 3, 4, 7). Under field conditions, owl1 is sometimes recognized only as fragmented remnants on the spore surface (fig. 6). The second layer (owl2) is dark yellow to brown-yellow, 2.0 3.6 µm thick in water, expanding to 5.0 7.5 µm under pressure and to (7.0 )8.5 12.0 µm in PVLG based mountants (figs. 1 6); owl3 is concolorous with owl2, or slightly lighter in color, thin and flexible (0.5 1.1 µm thick) and usually difficult to observe as closely adherent

364... Silva & al. to laminate owl2; when separating from owl2, owl3 often shows several folds (fig. 5). None of the layers stain in Melzer s reagent. The straight pore channel at the spore base (about 2.8 5.2 µm broad) is often closed by a plug formed by spore wall material of owl2, but sometimes appears to be open. Middle wall consists of two hyaline, flexible to semi-flexible layers (figs. 4 6). Outer layer (mwl1) is about 0.6 1.3 µm thick. Inner layer (mwl2) generally is more rigid as slightly thicker, 1.0 1.5( 2.0) µm. Both layers may slightly expand in PVLG based mountants. Inner wall is three-layered (fig. 4 6) bearing a germination shield on the outer surface (figs. 6 7). Outer layer of the inner wall (iwl1) is hyaline, semiflexible to sometimes finely laminate and 0.4 1.0 µm thick. Second layer (iwl2) is unite to finely laminate, 1.5 2.0( 2.7) µm thick. Innermost layer (iwl3) is thin (0.4 0.8 µm thick), flexible, and as usually tightly adherent to iwl2 often difficult to observe. The three layers may slightly expand in PVLG based mountants. Only iwl2 stains in Melzer s reagent become dark red purple within a few hours. Sporogenous cell is globose to elongate, concolorous with the spore, or slightly lighter or darker in color, and 31 48 µm long and 26 43 µm broad (figs. 1 3). The two wall layers generally visible on the sporogenous cell are continuous with owl1 and owl2. owl1 on the sporogenous cell is about 0.7 1.4 µm and evanescent to semi-persistent; persistent owl2 is about 2.0 3.5 µm thick. The sporogenous hypha attached is 8 11 µm broad and also bi-layered tapering to 5 7 µm within 200 350( 500) µm distance from the sporogenous cell. The sporogenous hyphal wall is concolorous with the spore wall, or sometimes darker yellow than the spore, and tapers from 1.7 2.8 µm to 0.9 1.5 µm within this distance. Several (3-7) septa originating from the inner layer (owl2) might be visible in the hypha. Germination shield is hyaline to subhyaline to rarely light yellow, circular (50 )62 85( 95) µm to subglobose to rarely oval (50 75( 85) 63 90( 98) µm). Shield with one initial germ hole (gh) in the shield center (fig. 6-7; and drawings of figs. 11 14), and one lobe which surrounds one to two folds the shield center and forms a circular, orb-like shield structure (figs. 6 14). One germ tube initiation (gti, 2.2 4.0 µm in diameter) is positioned towards the end of the lobe, from where one to rarely two dark yellow germination tubes emerge during germination and penetrate the middle and the outer wall, and branch in a short distance from the spore (fig. 9). Shield walls (0.8 1.5 µm thick) often appear sparsely dentate and generally wrinkling (figs. 7 10), since the shield wall respective the lobe wall is rather thin, 0.6 1.2 µm. Spore development The major spore developmental stages were deduced from clearly identified spores of S. pernambucana found in the field samples.

Scutellospora pernambucana sp. nov. (Brazil)... 365 Figs. 1 10: Scutellospora pernambucana Figs. 1 3. Spores with three spore walls (outer, middle and inner wall; OW, MW, IW), and formed terminally on sporogenous cells (sc). Sporogenous hyphae with several initial, but rudimentary branchings (pegs), and with several septa. 2 3. Germination shield visible in cross view on the inner wall. Figs. 3 7. Spore wall structure with three layered OW (OWL1-3), bi-layered MW (MWL1-2) and three-layered IW (IWL1-3). Fig. 6. IWL2 with purple reaction in PVLG + Melzer s reagent. Figs. 6 10. Orb-like germination shield formed on the outer surface of IW. The initial germ hole (gh; Fig. 6 7) and the terminal germ tube initiation (gti; Fig. 9) are not easily detectable. Fig. 8. Lobe apparently changed growth direction during shield formation, but remained tightly attached to the shield. Fig. 9. Germ tube (gt) emerging from the gti. Fig. 10. Germination shield isolated from IW.

366... Silva & al. Figs. 11 14. Scutellospora pernambucana: drawings of circular to oval germination shields with a central initial germ holes (gh), single lobes that terminally or subterminally bear one germ tube initiation (gti) from where the germ tubes emerge during germination. On a few spores, the lobes apparently changed the growth direction during shield formation, but remained tightly attached to the shield (Fig. 11). The gti might not be detectable in young, developing spores (Fig. 12). First the outer spore wall differentiates into one evanescent to semi-persistent outer layer (owl1), a laminate layer (owl2), and the adherent thin inner layer (owl3). The bi-layered middle wall (mw) and (subsequently) the three-layered inner wall (iw) develop de novo without visible connection with the outer wall. Finally, the orb-like germination shield develops on the outer surface of the inner wall. Auxiliary cells unknown. Mycorrhiza formation unknown. Distribution Brazil: so far found in the semi-humid Atlantic Forest or Mata Atlantica biome, in the Municipality of Igarassu and in the Caatinga biome, in the Municipality of Araripina (both in Pernambuco State) and in a Forest regeneration area in São Luis (Maranhão State), suggesting that the new fungus might be common in Northeastern Brazil. Other specimens or isolates examined: BRAZIL. Pernambuco State. Igarassu, Usina São José, Zambana fragment 83 8302 (URM79240) & 83 8303 (URM79241)

Scutellospora pernambucana sp. nov. (Brazil)... 367 deposited in Recife, Brazil; 83 8304 & 83 8305 deposited at OSC (No. OSC# 134503; Corvallis, Oregon, USA); 83 8306 & 83 8307 deposited at Z+ZT (No. ZT Myc 641; Zurich, Switzerland); 83 8308, 83 8309 & 83 8310 (Oehl collection). Araripina specimens deposited at URM. Maranhão State. São Luis specimens deposited at URM. Discussion Scutellospora pernambucana can easily be distinguished from all other known species in Scutellospora by the spore wall structure and in particular by the unique structure and shape of the mono-lobed germination shield resembling a germination orb. Of all known Scutellospora species, only S. projecturata forms a similar, mono-lobed, coiled germ shield, but the later species has distinctive, prominent columnar protuberances on the spore surface (Kramadibrata et al. 2000) while S. pernambucana has no ornamentation on any of the spore walls. Moreover, the S. projecturata shield has a smooth but firm shield wall (fig. 12 in Kramadibrata et al. 2000), while the thin S. pernambucana shield wall is conspicuously wrinkled. The pronounced expansion of the laminate owl2 in lactic acid-based mountants or under pressure and the evanescent to semipersistent nature of owl1, an extremely rare unique feature for Scutellospora, are two further helpful diagnostic characters. Observation of these last features is particularly helpful when the shield cannot be observed in planar view or has not yet differentiated in young, developing spores. However, it is also possible that S. projecturata has an evanescent outermost wall layer on the surface of the laminated, structural wall layer that was not reported in the protologue. In contrast to the two aforementioned species, most other Scutellospora species (e.g. S. calospora and S. pellucida) have a unit, semi-persistent to persistent layer on the outer surface of the structural, laminate layer (here owl2), (Koske & Walker 1986). We interpret the S. pernambucana germination shield type as simplestructured and mono-lobed, since obviously only one lobe develops during shield formation. The single lobe surrounds the germ shield initiation, i.e. the initial germ hole (gh; Walker & Sanders 1986) in the shield center. In all shields observed, the lobe surrounded the shield center one to two times and was thus responsible for the characteristic orb-like shield structure (figs. 6 14). In a few specimens it appeared that the lobe started to cover the shield center in one direction but obviously changed the direction and thus did not form a typical orb (figs. 8, 13). Kramadibrata et al. (2000) described the S. projecturata germ shield as coiled but did not refer to the shield structure as an orb or orb-like. In that paper, the illustrated shield of a crushed spore somehow resembles the S. pernambucana shield structure presented here in fig. 13 for, so we assume that the shield structures of both species are indeed closely related.

368... Silva & al. There are several other Scutellospora species with simple and hyaline to subhyaline germ-shields that are, however, bi-lobed (Silva et al. 2006b, Oehl et al. 2008): e.g. S. calospora (Koske & Walker 1986), S. tricalypta (Ferrer & Herrera 1981), S. dipapillosa (Koske & Walker 1985), S. arenicola (Koske & Halvorson 1990), and S. aurigloba (Walker & Hall 1991). In spores of these species, fully developed shields are violin-shaped, oval to ovoid, or (rarely) cardioid, each generally with one germ tube initiation (gti) on the terminal ends of the two lobes (see Walker & Sanders 1986). Other species such as S. castanea (Walker et al. 1993), S. heterogama sensu Franke & Morton (1994) and Jeffries et al. (2007), and S. reticulata (de Souza et al. 2005) have more complex, multiple-lobed shields with multiple gti or yellow-brown to brown shields that species-specifically may have multiple small compartments and multiple gti, and a conspicuously dentate shield periphery. Such species have been transferred due to their shield characteristics and for phylogenetic reasons to newly described genera in the Racocetraceae and Dentiscutaceae (Oehl et al. 2008). So far, germination orbs have been described for only a few Acaulospora and Kuklospora species of the Acaulosporaceae (Spain 1992, Sieverding & Oehl 2006) but not for Scutellospora. Oehl & Sieverding (2004) did not correctly interpret the circular characteristics of germination orbs (Spain 1992) when they called the Pacispora spore germination structures germination orbs. The germination structures in Pacispora species generally form first during germination and not, as in Scutellospora, during late spore formation, and thus should not be even called shields. Their germ structures are not orb-like but multiple-lobed, and the germ tubes emerge, as in acaulosporoid spores of Ambispora (Spain et al. 2006, Goto et al. 2008), from the germination center between the lobes (Oehl unpublished). Interestingly, the germ tubes in the phylogenetically related Scutellospora, Acaulospora and Kuklospora (e.g. Schüßler et al. 2001, Silva et al. 2006a, Palenzuela et al. 2008) emerge from the germ tube initiations positioned at the terminal ends of the lobed germination shields ( gti in Walker & Sanders 1986, germ tube loci in Spain 1992), and their germination structures generally persist once formed during spore formation. After analyzing recently published phylogenetic trees for Glomeromycota species (e.g. Santos et al. 2006, Silva et al. 2006a, Palenzuela et al 2008), we concluded that formation of a persistent germ structure (= germ shield) might be an evolutionary advance in Scutellospora and Acaulospora over other genera (e.g. Ambispora and Pacispora) that also form an inner, so-called germinal wall. We further assume that formation of a typical germ orb in S. pernambucana like that found in Kuklospora colombiana (Spain et al. 1992, Sieverding & Oehl 2006) and K. kentinensis (Oehl, pers. obs.) might be ancestral in Scutellospora, as is inferred for K. colombiana in the Acaulosporaceae (e.g. Santos et al. 2006, Palenzuela et al. 2008). This later

Scutellospora pernambucana sp. nov. (Brazil)... 369 hypothesis can be tested for S. pernambucana only after DNA is extracted from viable spores isolated from the field or even better successful pure cultures of the new fungus. Acknowledgements The authors acknowledge Marta Cabello (Instituto Spegazzini, La Plata, Argentina) and Mauritz Vestberg (MTT Agrifood Research, Vihtavuori, Finland) for reviewing the manuscript and making helpful comments and suggestions. Thanks are also due to Prof. Ana Carolina Lins e Silva, who provided information regarding the plant species in the Forest area. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) that provided, respectively, a Master scholarship to Danielle Alves da Silva, a PhD scholarship to Nicacio de Oliveira Freitas and a research grant to Leonor C. Maia. This work was also supported by the Universidade Federal de Pernambuco, which provided a grant to F. Oehl as visiting professor. This is a contribution to the project Sustainability of remnants of the Atlantic rainforest in Pernambuco and its implications for conservation local development, a Brazilian- German scientific cooperation within the program Science and Technology for the Atlantic Rainforest funded by CNPq (590039/2006-7) and BMBF (01 LB 0203 A1), permitted and logistically supported by Usina São José S.A/Grupo Cavalcanti Petribú. Literature cited Brundrett M, Melville L, Peterson L. 1994. Practical Methods in Mycorrhizal Research. Mycologue Publications, University of Guelph, Guelph, Ontario. De Souza FA, Declerck S, Smit E, Kowalchuk GA. 2005. Morphological, ontogenetic and molecular characterization of Scutellospora reticulata (Glomeromycota). Mycol. Res. 109: 697 706. Ferrer RL, Herrera RA. 1981. El genero Gigaspora Gerdemann et Trappe (Endogonaceae) en Cuba. Rev. Jardin. Bot. Nacional Habana 1: 43 66. Franke M, Morton JB. 1994. Ontogenetic comparisons of arbuscular mycorrhizal fungi Scutellospora heterogama and Scutellospora pellucida: revision of taxonomic character concepts, species descriptions, and phylogenetic hypotheses. Can. J. Bot. 72: 122 134. Gerdemann JW, Nicolson TH. 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46: 235 244. Goto BT, Maia LC. 2006. Glomerospores: a new denomination for the spores of Glomeromycota, a group molecularly distinct from Zygomycota. Mycotaxon 96: 129 132. Goto BT, Maia LC, Oehl F. 2008. Ambispora brasiliensis, a new ornamented species in the arbuscular mycorrhiza-forming Glomeromycetes. Mycotaxon 105: in press. Jeffries P, Robinson-Boyer L, Rice P, Newsam RJ, Dodd JC. 2007. Ultrastructure of development in Scutellospora heterogama. Mycorrhiza 17: 395 403. Jenkins WR. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Dis. Rep. 48: 692. Koske RE, Halvorson WL. 1990. Scutellospora arenicola and Glomus trimurales: Two new species in the Endogonaceae. Mycologia 81: 927 933. Koske RE, Walker C. 1985. Species of Gigaspora (Endogonaceae) with roughened walls. Mycologia 77: 702 720.

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