Acaulospora baetica, a new arbuscular mycorrhizal fungal species from two mountain ranges in Andalucía (Spain)

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1 CNova Hedwigia published online June 2015 PrePub Article Acaulospora baetica, a new arbuscular mycorrhizal fungal species from two mountain ranges in Andalucía (Spain) Javier Palenzuela 1, Concepción Azcón-Aguilar 1, José-Miguel Barea 1, Gladstone Alves da Silva 2 and Fritz Oehl 2,3 1 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, Granada, Spain 2 Departamento de Micologia, CCB, Universidade Federal de Pernambuco, Avenida da Engenharia s/n, Cidade Universitária, , Recife, PE, Brazil 3 Agroscope, Federal Research Institute of Sustainability Sciences, Reckenholzstrasse 191, Plant-Soil-Interactions, CH-8046 Zürich, Switzerland With 11 figures and 1 table Abstract: A new Acaulospora species, A. baetica, was found in two adjacent mountain ranges in Andalucía (southern Spain), i.e. in several mountainous plant communities of Sierra Nevada National Park at m asl around roots of the endangered and/or endemic plants Sorbus hybrida, Artemisia umbelliformis, Hippocrepis nevadensis, Laserpitium longiradium and Pinguicula nevadensis, and in two Mediterranean shrublands of Sierra de Baza Natural Park at m asl around roots of Prunus ramburii, Rosmarinus officinalis, Thymus mastichina and Lavandula latifolia among others. The fungus produced spores in single species cultures, using Sorghum vulgare or Trifolium pratense as bait plant. The spores are µm in diameter, brownish creamy to light brown, often appearing with a grayish tint in the dissecting microscope. They have a pitted surface (pit sizes about µm in diameter and µm deep), and are similar in size to several other Acaulospora species with pitted spore surfaces, such as A. nivalis, A. paulinae and A. sieverdingii, but smaller than A. minuta, A. puncatata and A. scrobiculata. The new species can be distinguished from all these fungi by the combination of surface ornamentation, spore color and spore size. Phylogenetic analyses of sequences obtained from the ITS and partial LSU of the ribosomal gene confirm the new species in a separate clade within the Acaulosporaceae, close to A. nivalis, A. paulinae and A. sieverdingii. The phylogenetically most closely related fungus to A. baetica, however, is A. ignota, a species that does not have any pits, but innumerous inconspicuous warts and flattened elevations on the spore surface. Key words: arbuscular mycorrhizal fungi, biodiversity, endangered plant species, endemisms, Glomeromycetes, high Mediterranean mountains, phylogeny. Corresponding author: fritz.oehl@agroscope.admin.ch 2015 J. Cramer in Gebr. Borntraeger Verlagsbuchhandlung, Stuttgart, Germany. DOI: /nova_hedwigia/2015/ /2015/0285 $

2 Introduction In recent years, the diversity of arbuscular mycorrhizal (AM) fungi has been extensively studied in the Baetic Cordillera of Andalucía (Southern Spain) and other mountain areas world-wide (e.g. Castillo et al. 2006, Sánchez-Castro et al. 2011, Azcón-Aguilar et al. 2012, Yang et al. 2013, Li et al. 2014). A surprisingly high diversity of these fungi was found in mountainous areas, especially of Acaulospora spp. (e.g. Oehl et al. 2006, 2011a, 2011d, 2012, Palenzuela et al. 2013a). In the Baetic Cordillera several very original and distinctive fungi concerning spore morphology, molecular phylogeny and their biogeography have been revealed so far (e.g. Palenzuela et al. 2008, 2010, 2013a, 2013b). During recent AM fungal diversity studies in the Baetic Cordillera another so far unknown AM fungal species was isolated from field soil samples, and it was propagated and cultured on several AM host plants. The objective of this study was to thoroughly investigate its morphology, its molecular phylogeny, and, if possible, also its biogeographic distribution. The fungus is hereafter described under the epithet Acaulospora baetica. Material and methods Study SiteS and Study plants: Soil samples were taken from several locations at two adjacent mountain ranges in Andalucía, Sierra Nevada and Sierra de Baza, as indicated in Table 1. plant and Soil Sampling: Five intact plant individuals and the soil attached to the roots were collected at the study sites to establish AM fungal pot cultures. In addition, three samples of the soil surrounding the roots of each plant (0.5 1 kg) were taken with a shovel from a depth of 5 25 cm; they were thoroughly mixed to prepare a composite soil sample per individual plant. These samples were used to isolate AM fungal spores and to determine soil ph (in a 1:2.5 w/v aqueous solution). am fungal pot cultures: To cultivate AM fungi, bait cultures were established with the collected native plants in 1500 ml pots (cylindric, 12 cm diam.) filled with the soil originally obtained from around the plant roots in the field. The pots were irrigated three times per week and fertilized every four weeks with Long-Aston nutrient solution (Hewitt 1966). The cultures were maintained in a greenhouse of the Estación Experimental del Zaidín (Granada) for more than three years. Single species cultures of the new fungus were established with Sorghum vulgare L. or Trifolium pratense L. as host plants in 350 ml pots as described in Palenzuela et al. (2010). Spores isolated from the bait cultures were stratified during two weeks at 4 C before inoculation. morphological analyses: AM fungal spores were obtained by a wet sieving process (Sieverding 1991). The spore morphological characteristics and their subcellular structures were described from specimens mounted in: (i) polyvinyl alcohol-lactic acid-glycerol (PVLG; Koske & Tessier 1983); (ii) a mixture of PVLG and Melzer s reagent (Brundrett et al. 1994); (iii) a mixture of lactic acid and water (1:1); (iv) Melzer s reagent; and (v) water (Spain 1990). The spore structure terminology follows Palenzuela et al. (2011) and Oehl et al. (2011c, 2012) for species with acaulosporoid spore formation. Photographs (Figs 1 9) were taken with a high-definition digital camera (Nikon DS-Fi1) on a compound microscope (Nikon eclipse 50i) or with a Leica DFC 290 digital camera on a Leitz Laborlux S compound microscope using Leica Application Suite Version V R1 software. Specimens mounted in PVLG and PVLG+Melzer s reagent were deposited at the herbaria Z+ZT (ETH Zurich, Switzerland) and GDA-GDAC (University of Granada, Spain). molecular analyses: Five spores were isolated from each of the two pot cultures of a single fungal species culture originating from Sorbus hybrida in Sierra Nevada and from Prunus ramburii in Sierra 2

3 Table 1. Geographical data, sampling time and selected soil characteristics at isolation sites of Acaulospora baetica. Plant species at mountain range Sierra Nevada Sorbus hybrida L. Artemisia umbelliformis Lam. Hippocrepis nevadensis (HrabeŠtová) Talavera & E.Domínguez Study site Upper part of the San Juan ravine Tajos del Goterón (Goterón icefalls) S. Jeronimo s pathway Altitude (m asl) Latitude Longitude Sampling time Soil type 'N 3 22'W June 2007 Haplic Regosol 'N 3 16'W October 'N 37 06'N 3 26'W 3 26'W November 2006 July 2007 Distric Regosol Calcaric Cambisol Calcaric Cambisol ph (H 2 O) Laserpitium longiradium Boiss. Pinguicula nevadensis (H.Lindb.) Casper Sierra de Baza Rosa pouzinii Tratt. Berberis hispanica Boiss. & Reut. Prunus ramburii Boiss. Crataegus monogyna Jacq. S. Jerónimo s pasture Hygrophilic high mountain pastures in the Campos de Otero Spiny Mediterranean shrubland Rosmarinus Rosemary officinalis L. Mediterranean Thymus mastichina L. shrubland Thymus zygis L. Lavandula latifolia Medik. Genista cinerea (Vill.) DC 'N 3 23'W July 'N 2 49'W March 'N 2 50'W March 2004 Humic Cambisol Calcaric Regosol Calcaric Regosol Soil types were classified according to the World Reference Base for soil resources (IUSS Working Group WRB 2014). de Baza. The five spores per site were surface-sterilized with chloramine T (2%) and streptomycin (0.02%; Mosse 1962) and crushed together with a sterile disposable micropestle in 23 µl milli-q water as described in Palenzuela et al. (2013b). DNA amplification of the crude extracts was obtained in an automated thermal cycler (Gene Amp PCR System 2400, Perkin-Elmer, Foster City, California) using a puretaq Ready-To-Go PCR Bead (Amersham Biosciences Europe GmbH, Germany) following manufacturer s instructions, with 0.4 µm concentration of each primer. A two-step PCR amplified the SSU end, ITS1, 5.8S, ITS2 and partial LSU rdna fragment using the SSUmAf/LSUmAr and 3

4 SSUmCf/LSUmBr primers consecutively (Krüger et al. 2009). PCR products were analyzed by electrophoresis in 1.2% agarose gels stained with Gel Red (Biotium Inc., Hayward, CA, U.S.A.) and viewed by UV illumination. The expected amplicons were purified using the GFX PCR DNA kit and Gel Band Purification Illustra, cloned into the PCR 2.1 vector (Invitrogen, Carlsbad, CA, USA), and transformed into One shot TOP10 chemically competent Escherichia coli cells. After plasmid isolation from transformed cells, the cloned DNA fragments were sequenced with vector primers (White et al. 1990) in both directions by Taq polymerase cycle sequencing on an automated DNA sequencer (Perkin-Elmer ABI Prism 373). Sequence data were compared to gene libraries (EMBL and GenBank) using BLAST (Altschul et al. 1990). The percentage of identity among the Acaulospora sequences was calculated using the BLASTn analysis. The new sequences were deposited in the EMBL database under the accession numbers LN LN phylogenetic analyses: The phylogeny was reconstructed by independent analyses of the ITS region and partial LSU rdna. The AM fungal sequences obtained were aligned with other Acaulosporaceae sequences from GenBank in ClustalX (Larkin et al. 2007). Claroideoglomus etunicatum (W.N.Becker & Gerd.) C.Walker & A.Schüssler was included as outgroup. Prior to phylogenetic analysis, the model of nucleotide substitution was estimated using Topali 2.5 (Milne et al. 2004). Bayesian (two runs over generations with a sample frequency of 200 and a burn-in value of 25%) and maximum likelihood (1000 bootstrap) analyses were performed in MrBayes (Ronquist & Huelsenbeck 2003) and PhyML (Guindon & Gascuel 2003), respectively, launched from Topali 2.5, using the GTR + G model. Maximum parsimony analysis was performed using PAUP*4b10 (Swofford 2003) with 1000 bootstrap replications. Results Acaulospora baetica Palenz., Oehl, Azcón-Aguilar & G.A.Silva, sp. nov. Figs 1 9 Mycobank MB etymology: Latin, baetica, referring to the baetic mountain ranges in Andalucía where the fungus was detected first. Acaulospora baetica has creamy brown to light brown spores, µm. It differs from hyaline- to light yellow-spored A. sieverdingii especially by creamy brown to light brown spore color, from yellow-spored Acaulospora nivalis especially by finer, more regular pit ornamentation on the spore surface, and from yellow-spored A. ignota by having pits on the spores surface instead of warts and flattened projections. holotype: Spain, Andalucía, Granada, from single species pot culture generated at the Estación Experimental del Zaidín, on Sorghum vulgare. Culture was initially inoculated with spores isolated from pot bait cultures of Sorbus hybrida grown in the original soil substrate deriving from Sierra Nevada (37 07'N; 3 22'W; 1896 m asl). Specimens were deposited at Z + ZT (common mycological herbarium of the University and ETH of Zurich, Switzerland; ZT Myc 56317). Isotypes of the pot culture were deposited at Z+ZT (accession ZT Myc 56318) and GDA-GDAC (herbarium of the University of Granada, Spain). Paratypes deposited at Z+ZT (accession ZT Myc 56319) and GDA- GDAC were isolated from rhizospheric soils, trap and single species cultures representing other sites in Sierra Nevada and Sierra de Baza ( m asl; Table 1). description: Sporiferous saccules are hyaline and singly formed at the end of mycelial hyphae (Fig. 1). The saccule termini are globose to subglobose ( µm), with 1 2 wall layers that are in total µm thick. The saccule necks are µm broad at the saccule termini, about 8 16 µm at the point of spore formation, and taper to 6 12 µm in µm distance from the spore towards the mycelium. The saccule usually collapses after the spore wall has formed and usually is detached from mature spores. 4

5 Figs 1 9. Acaulospora baetica: 1. Spore formed laterally on the neck of a sporiferous saccule Spores with pitted spore surface in cross and plan view, respectively. 6. Crushed spore, pitted outer wall in cross-view Spore wall composition: Outer wall with three layers (OWL1-3), middle wall bi-layered (MWL1-2) and triple-layered inner wall (IWL1-3). IWL2 staining purple in Melzer s reagent (Fig. 7). 9. Cicatrix on outer spore wall without pits inside the ring-like structure. Spores form laterally on the neck of sporiferous saccules in µm distance from the saccule termini. They are globose to subglobose, µm in diameter and creamy brown to light brown (Figs 1 6), often appearing with a grayish tint when observed in water under the dissecting microscope. The spores have three walls. Outer spore wall consists of three layers (OWL1 OWL3; Fig. 7). Outer layer (OWL1) is hyaline to subhyaline, µm thick, evanescent. 5

6 Second layer (OWL2) is creamy brown to light brown, laminated, µm thick. OWL2 has a pitted surface, and the pits are about µm in diam., µm deep and about (1.5 ) µm apart. The inner layer of the outer wall (OWL3) is concolorous with OWL2, about µm thick and often difficult to observe, especially when the middle wall does not separate readily from OW. None of the OW layers stains in Melzer s reagent. Middle wall is hyaline, bi-layered and thin; µm thick in total (Fig. 7). Both layers (MWL1 and MWL2) are semi-flexible, tightly adherent to each other and thus often appear as being only one wall layer. None of the MW layers stains in Melzer s reagent. Inner wall is hyaline (Figs 7 8), with two to three layers (IWL1 IWL 3). The IWL1 is about µm thick with a 'beaded', granular structure. IWL2 is µm thick and regularly stains pinkish purple to dark purple in Melzer s reagent. IWL3 is very thin (<0.6 µm) and usually very difficult to detect since closely adherent to IWL2. Cicatrix (Fig. 9) remains after detachment of the connecting hypha, 8 12 µm wide. The pore is closed by inner laminae of OWL2 and by OWL3. mycorrhiza formation: forming arbuscular mycorhizal associations with Sorghum vulgare L. and Trifolium pratense L. as plant hosts in pot cultures. The mycorrhizal structures consisted of arbuscules, vesicles, and intra- and extraradical hyphae staining dark blue in 0.05% trypan blue. molecular analyses: The phylogenetic analysis from ITS region and LSU rdna sequences places the new fungus in a separated clade within the Acaulosporaceae, close to A. ignota and A. nivalis (Figs 10 11). Sequences corresponding to the spores originating from Sorbus hybrida in Sierra Nevada and from Prunus ramburii in Sierra de Baza grouped together confirming that they correspond to the same AM fungal species. No environmental sequence deposited in GenBank seems to correspond totally to the new fungus in the BLASTn analysis. The environmental LSU rdna sequences with closest match (97%) to A. baetica were found in roots or rhizosphere soil of Solanum tuberosum (HF970218, HF HF970284, HF970236, HG969357) sampled in the Peruvian Andes (Senes-Guerrero et al. 2014). For the ITS region, also sequences (HG969314, HG969318) from roots of Solanum tuberosum, sampled in the Peruvian Andes (Senes-Guerrero et al. 2014), were the closest to A. baetica (95 96%). However these sequences grouped close to A. nivalis, in another clade than that of A. baetica (results not shown). distribution of A. baetica: The new fungus was found in field soil samples from six sites in two mountain ranges, i.e. i) in Sierra Nevada National Park between 1580 and 2912m asl, growing in the rhizosphere of endangered to critically endangered plants species such as Sorbus hybrida, Artemisia umbelliformis, Hippocrepis nevadensis, Laserpitium longiradium and the non-mycorrhizal plant species Pinguicula nevadensis and ii) in Sierra de Baza Natural Park, at 1380 and 1855 m asl, in the rhizosphere of Mediterranean shrubland plant species like Rosmarinus officinalis, Thymus mastichina, Lavandula latifolia, Rosa pouzinii and Berberis hispanica (Table 1). Soils were of the Regosol and Cambisol type, with a ph range from slightly acidic to slightly alkaline ( ). 6

7 Fig. 10. Phylogenetic tree of the Acaulosporaceae obtained by analysis from ITS1, 5.8S rdna and ITS2 sequences. Sequences are labeled with their database accession numbers. Support values (from top) are from maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses, respectively. Sequences obtained in this study are in boldface. Only support values of at least 50% are shown. Thick branches represent clades with more than 90% of support in all analyses. The tree was rooted by Claroideoglomus etunicatum. (Consistency Index = 0.50; Retention Index = 0.85). 7

8 Fig. 11. Phylogenetic tree of the Acaulosporaceae obtained by analysis from partial LSU rdna sequences of different Acaulospora spp. Sequences are labeled with their database accession numbers. Support values (from top) are from maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses, respectively. Sequences obtained in this study are in boldface. Only support values of at least 50% are shown. Thick branches represent clades with more than 90% of support in all analyses. The tree was rooted by Claroideoglomus etunicatum. (Consistency Index = 0.51; Retention Index = 0.86). 8

9 In Sierra Nevada, the fungus co-occurred with the following AM fungal species: Acaulospora laevis, A. paulinae, Ambispora gerdemannii, Claroideoglomus etunicatum, Diversispora versiformis, Funneliformis caledonius, Rhizoglomus intraradices, Sclerocystis rubiformis, Septoglomus altomontanum and Pacispora scintillans. In Sierra de Baza, Funneliformis mosseae, Septoglomus constrictum, Rhizoglomus intraradices, Entrophospora infrequens, Scutellospora calospora and the recently described Acaulospora viridis (Palenzuela et al. 2014) were the most frequent AM fungal species accompanying the new fungus. Specimen examined: Holotype, isotype and paratype specimen, deposited at Z+ZT and GDA-GDAC, and in private collections of J.Palenzuela and F.Oehl. Discussion Acaulospora baetica can easily be distinguished from all other Acaulospora species by the combination of surface ornamentation, spore color and spore size. Morphologically, A. baetica is most similar to Acaulospora sieverdingii, whose spores are hyaline to rarely light yellow (Oehl et al. 2011b). Acaulospora nivalis (Oehl et al. 2012) has bright yellow to dark yellow spores with larger and more irregular pits than A. baetica, while A. alpina has yellow to orange yellow spores with highly regular, conical pits (Oehl et al. 2006), and A. herrerae (Furrazola et al. 2013) has a much higher pit density than A. baetica. All other Acaulospora spp. with pitted surfaces have either substantially larger pits (e.g. A. paulinae; Błaszkowski 1988) or substantially larger spores (A. lacunosa, A. minuta, A. punctata and A. sieverdingii; Morton 1986, Oehl et al. 2011b, d, e), or both (e.g. A. cavernata, A. foveata and A. scrobiculata; Trappe 1977, Janos & Trappe 1982, Błaszkowski 1989). Other Acaulospora spp. of similar size as A. baetica have either spore surface projections instead of pitted surfaces (e.g. A. ignota, A. pustulata and A. tortuosa; Palenzuela et al. 2013a, Błaszkowski et al. 2015) or smooth surfaces (e.g. A. longula, A. mellea and A. morrowiae; Schenck et al. 1984). Phylogenetically, A. baetica was closest to A. ignota and clearly more distant to morphologically more similar species like A. nivalis, A. paulinae, A. sieverdingii, A. alpina and A. herrerae. So far, the new fungus has only been found in two neighboring mountain ranges of the Granada province in Andalucía, Spain, in Regolsols and Cambisols with a wide range of soil ph ( ). The inventory of environmental sequences in public data bases suggests that A. baetica has hitherto never been found in other parts of the world. Environmental sequences closest to those of A. baetica were found in the Peruvian Andes. However, these sequences cannot be attributed to A. baetica since in the phylogenetic analyses they grouped in a different clade (data not shown) and exhibit significant differences to the A. baetica sequences (97% of similarity for the LSU region analyzed). Also, it has never been reported in morphological surveys from other mountainous regions or lowlands of the Iberian peninsula or detected in other parts of the world (e.g. Castillo et al. 2006, Oehl et al. 2011a). Nevertheless, since AM fungal diversity studies have been intensified only during the last two decades, a clearer biogeographical conclusion for this fungus or many other rather rare species cannot yet be drawn at this stage of AM fungal research. 9

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