Journal of Applied Botany and Food Quality 86, 113 - 125 (2013), DOI:10.5073/JABFQ.2013.086.016 1Departamento de Micologia, CCB, Universidade Federal de Pernambuco, Cidade Universitaria, Recife, PE, Brazil 2Agroscope Reckenholz-Tänikon Research Station ART, Ecological Farming Systems, Zürich, Switzerland Paraglomus pernambucanum sp. nov. and Paraglomus bolivianum comb. nov., and biogeographic distribution of Paraglomus and Pacispora Catarina Maria Aragão de Mello1, Gladstone Alves da Silva1, Daniele Magna Azevedo de Assis1, Juliana Souza de Pontes1, Araeska Carenna de Almeida Ferreira1, Mariele Porto Carneiro Leão3, Helder Elísio Evangelista Vieira1, Leonor Costa Maia1, Fritz Oehl1, 2* (Received April 22, 2013) * Corresponding author Summary Paraglomus pernambucanum sp. nov. (Paraglomeromycetes) was found in a tropical dry forest in the semi-arid Caatinga biome of Pernambuco State (NE Brazil), in a cowpea and in two maize pro- duction sites. It was characterized by combined morphological and molecular analyses on the spores isolated from field soil samples. Another species, Pacispora boliviana (Glomeromycetes), first de- scribed only by spore morphology, had been known from another semi-arid biome in Southern America, the Gran Chaco in Bolivia. We detected this fungus now also at different locations in semi-arid to semi-humid NE Brazil. As for P. pernambucanum phylogenetic analyses were performed on nuclear ribosomal RNA gene sequences of the LSU region. For P. boliviana, the spores for these analyses originated from a trap culture inoculated with soils from the type location. The results now revealed that also P. boliviana belongs to Paraglomus. It grouped in a separate monophyletic cluster adjacent to P. pernambucanum, to P. brasilianum, P. laccatum and the type species P. occultum. Thus, P. boliviana is transferred to Paraglomus, as Paraglomus bolivianum comb. nov. Remarkably, it is the first species known in the Paraglomeromycetes with pigmented spores. Paraglomus pernambucanum and P. bolivianum have several fea- tures in common: e.g. bi-walled spores, and densely pitted surface ornamentations on the structural layer of the outer wall. Spores of the two species can be distinguished by color and the diagnostic nature of their pitted ornamentation. The current knowledge about the global distribution of Paraglomus and Pacispora species is sum- marized and discussed. Introduction Arbuscular mycorrhizal (AM) fungi play an important role in eco- system functioning since they are associated with the majority of plant species, deliver essential nutrients to their symbiotic partners, and are for instance also able to suppress harmful soil-borne patho- gens and pests, and may protect soils against erosion through stabi- lization of soil aggregates (Smith and Read 2008). They can also serve as bio-, soil and land use indicators (e.g. Oehl et al. 2005a, 2011a, 2012). We assume that especially Pacispora species might be characteristic for specific soils, land use intensity and different climates since they were found to be characteristic for soils with pH > 6.0, either in alpine areas (e.g. P. robigina; Oehl and SieveRding 2004, Błaszkowski et al. 2008), in cultivated soils of temperate areas with lower phosphorus levels (e.g. P. dominikii; Błaszkowski, 1993; Oehl et al., 2005b; Oehl et al., 2010), or in Mediterranean and subtropical areas under semi-arid conditions subjected to naturally elevated soil pH (e.g. P. scintillans and P. franciscana; e.g. ROSe and Trappe, 1980; BaShan et al., 2007; palenzuela et al., 2008). One fungus, P. boliviana, was never found in alpine, temperate, Mediterranean or subtropical areas of higher soil pH despite of ex- tensive AMF diversity studies performed in Europe and Northern America during the last decades (e.g. Błaszkowski, 1993; koske and Tews, 1987; daniell et al., 2001; JanSa et al., 2002; landiS et al., 2004), but so far only in the tropical, semi-arid Gran Chaco of Bolivia (pH 6.3; Oehl and sieverding, 2004), and recently also from a tropical Atlantic rainforest in NE Brazil (Silva et al., 2012). We have lastly detected it several times in the semi-arid Caatinga biome of NE Brazil, which focused again our attention to this fungus. In tropical areas, however, other Pacispora species were so far never reported, neither by morphological spore identifica- tions from field soils, long-term trap culture propagations (e.g. gOtO et al., 2010; StüRmeR and siqueira, 2011; tchaBi et al., 2009) nor through molecular root analyses (e.g. HusBand et al., 2002; Öpik et al., 2010), although species of this genus could be expected in the Tropics at least in semi-arid regions and soils with increased pH. Pacispora boliviana has a distinct pitted ornamentation on its spore surface (SieveRding and oeHl, 2004). The fungus was preliminary attributed to this genus, since it forms, like all Pacispora, bi-walled spores terminally on subtending hyphae. However, it was obvious from the beginning that its spore structure, and above all the struc- ture of the subtending hyphae and the staining reaction of the wall layers in Melzer’s reagent, does not fit with those characters typical for Pacispora species (Oehl and sieverding, 2004). However, at that time, molecular analyses of the fungus were not available, and morphologically its spores could not be attributed to any other genus within the Glomeromycota, such as Glomus or Paraglomus (Oehl et al., 2011b). Glomus has only mono-walled spores, while the spore wall structure of Paraglomus has not yet been fully resolved, and the genus has so far had only species without substantial spore pig- mentation. When morphological analyses cannot (or not yet) re- solve the relationship between different taxa, molecular analyses are urgently needed to elucidate their phylogeny (gampeR et al., 2009; palenzuela et al., 2010; Oehl et al., 2011d; gOtO et al., 2012). In the present study, we performed first molecular analyses on P. boliviana in order to clarify its phylogenetic position in relation to the other glomeromycotan fungi. More recently, an undescribed fungus, with congruent overall spore and subtending hyphae morphology as P. boliviana, was found in another tropical, semi-arid biome of Southern America, i.e. in the Caatinga biome of NE Brazil. Thus, the second objective of the pre- sent study was to carefully elaborate both the spore morphology and the phylogenetic position of this new fungus. Based on the results obtained from concomitant morphological and molecular phylo- genetic analyses, the new fungus is formally described hereafter. Another objective was to discuss the biogeographic distribution of the two fungi, as far as this was possible at this early stage of spe- cies identification. Finally, we aimed at comprehensively presenting the biogeography of the two genera, Paraglomus and Pacispora, in general. 114 C.M. Aragão de Mello, G.A. Silva, D.M.A. de Assis, J.S. Pontes, A.C.A. Ferreira, M.P.C. Leão, H.E.E. Vieira, L. Costa Maia, F. Oehl Materials and methods Study sites The new AM fungus was found in Caruaru and Serra Talhada, Pernambuco State (NE Brazil), in maize (Zea mays L.) fields in the semi-arid tropical Caatinga biome. The sampling sites are located at 08º08’00”S and 36º02’00”W (550 m a.s.l.) and 7º59’00’’S and 38º19’16’’W (650 m a.s.l.), respectively. The climate is tropical- (semi-)arid (type Bs of Köppen-Geiger; koTTek et al., 2006) with six to eight months of dry season. Mean annual temperature is 24- 26°C, and annual rainfall is 650 and 550 mm, respectively. The maize variety sown was BR 5026 ‘São José’ (lemOS et al., 1995). At these two sites, the pasture plant species sudan grass (Sorghum sudanense (Piper) Stapf) and onion had previously been grown, respectively. More recently, the fungus was also found in a tropical Caatinga dry forest where Mimosa tenuifolia was the most dominant plant spe- cies and in an adjacent cowpea (Vigna unguiculata) field. Both loca- tions are near Petrolina, in Pernambuco State (6°28’20’’-6°30’00’’S; 34°55’50-34°57’10’’W, 380 m a.s.l., mean annual temperature 26°, mean annual rainfall 550 mm). At the natural Caatinga site Mimosa tenuifolia was the most dominant plant species. The type location of Pacispora boliviana was described in Oehl and SieveRding (2004). Briefly, it was isolated from a degraded pasture in the semi-arid Gran Chaco of tropical Bolivia (Departa- mento de Santa Cruz de la Sierra; 18°05’S; 63°20’W, 550 m a.s.l., mean annual temperature 24°, mean annual rainfall approximately 800 mm), and from trap cultures inoculated with soils from that pas- ture. The trap cultures were maintained under tropical temperatures in the greenhouse at the University of Basel (Switzerland) for eight months in 2001. In recent years, we found P. boliviana also in the semi-arid Caatinga of NE Brazil, e.g. in Triunfo (7°50’17’’s; 38°06’06’’w), Belém do São Francisco (8°45’28’’s; 38°57’52’’w) and, together with the herein described new fungus, also in Serra Talhada (7º59’00’’S; 38º19’16’’W). All these sites are also in Pernambuco State. Mean annual temperature in Belém do São Francisco (400 m a.s.l.) is 26°C, and the mean annual rainfall is 450 mm. These values are 22°C and approximately 1200 mm, respectively, in Triunfo. This isolation site has a more humid climate than it is common in the Caatinga biome, with typical vegetation of an Atlantic rainforest. This site profits from more frequent rainfalls due to its exposed higher altitude (650 m a.s.l.), when compared to the typical Caatinga dry forest in its surrounding. Soil sampling and chemical soil analyses Soil samples in Caruaru, Serra Talhada and Petrolina (NE Brazil) were taken in March 2011 as described by mellO et al. (2012). The soils in Belém do São Francisco and Triunfo were already taken in September and October 2008, respectively. Soil samples in the Gran Chaco (Bolivia) were taken in November 2000 as described in Oehl and SieveRding (2004). The soil in Caruaru had a pH (H2O) of 6.4, 1.1 g kg-1 organic C and 49.4 mg kg-1 available P extracted according to nelSOn et al. (1953). In Serra Talhada, the soil had a pH (H2O) of 6.2, 0.7 g kg-1 organic C and 58.0 mg kg-1 available P, and in Petrolina, pH was 5.2-6.0, organic C was 16.1 g kg-1, and available P was 22.0 mg kg-1. In Belém do São Francisco, pH was 6.8, organic C was 23.8 g kg-1, and available P was 16.8 mg kg-1, while pH was 6.8, organic C 39.4 g kg-1, and available P 195.0 mg kg-1 in Triunfo. The Bolivian soil had a pH (H2O) of 6.5; organic carbon was 26 mg kg-1, and available P (here extracted with Na-acetate, see Oehl et al., 2005b) was 2.3 mg kg-1. AM fungal trap cultures Trap cultures were established directly after sampling as described in Oehl et al. (2003), tchaBi et al. (2009) and mellO et al. (2012). Pacispora boliviana produced abundantly spores only in one of 64 trap cultures, in the rhizosphere of Stylosanthes guianensis, Bracharia humicicola and Chromolaena odorata. The new fungus from Pernambuco did not form spores in the trap cultures. Using Sorghum bicolor as host plant, single species cultures were initiated, that were inoculated with single or multiple (10-20) spores isolated directly from the field for the new fungus, or obtained from the trap cultures for Pacispora boliviana. All these mono- or multiple spore cultures failed so far. Morphological analyses Spores of the two fungi were separated from the soil samples by wet sieving as described by SieveRding (1991). The described morphological characteristics of spores and their subcellular struc- tures are based on observations of specimens mounted in polyvinyl alcohol-lactic acid-glycerol (PVLG; koske and teSSieR, 1983), in a mixture of PVLG and Melzer’s reagent (BrundreTT et al., 1994), a mixture of lactic acid to water at 1:1, Melzer’s reagent, and in water (spain, 1990). The terminology of the spore structure follows Oehl and SieveRding (2004) and Oehl et al. (2011b, c) for species with glomoid (sensu lato and sensu stricto), paraglomoid and paci- sporoid spore formation. Specimens mounted in PVLG and the mix- ture of PVLG and Melzer’s reagent were deposited at Z+ZT (Zurich, Switzerland) and URM (Recife, Brazil) herbaria. Molecular analyses Before DNA extraction all spores isolated were first washed in ul- trapure water and sonicated three to four times. For P. boliviana (culture code: BOL 35), crude DNA extracts were obtained from three single isotype spores, extracted from the trap culture that had been inoculated with field soil samples from the type location in the Gran Chaco. For the new fungus from Pernambuco, the DNA ex- tracts were obtained from two single spores that were directly sepa- rated from the field samples of the type location in Caruaru. The spores were singly placed on a slide in a drop (5-10 μl) of ultrapure water, crushed with a sterile needle. Crude DNA extract was used as template for a semi-nested PCR using the primers ITS3 (wHiTe et al., 1990) 28G2 (Silva et al., 2006) and LR1 (van Tuinen et al., 1998) 28G2, consecutively. PCR reactions were carried out in a volume of 50 μl, containing 75 mM Tris-HCl pH 8.8, 200 mM (NH4)2SO4, 0.01% Tween 20, 2 mM MgCl2, 200 μM each dNTPs, 1 μM of each primer and 2 units of TaqTM DNA polymerase (Fer- mentas, Maryland, USA); cycling parameters were 5 min at 95°C (1 cycle), 45s at 94°C, 1 min at 55°C, 1 min at 72°C (40 cycles), and a final elongation of 7 min at 72°C followed the last cycle. The final amplicons (~690bp) were purified with the PureLink PCR Purification Kit (Invitrogen), sequenced directly or cloned with a CloneJETTM PCR Cloning kit (Fermentas; Carlsbad, USA) follow- ing the manufacturer’s instructions and sequenced. Sequencing was provided by the Human Genome Research Center (São Paulo, Brazil). Sequence data were compared to gene libraries (EMBL and Gen Bank) using BLASTn (alTscHul et al., 1990). The new sequences de- riving from the AM fungi from Bolivia and Pernambuco were deposited in the NCBI database under the accession numbers JX122769- JX122777. Phylogenetic analyses The phylogeny was reconstructed by partial sequences of the LSU rRNA gene. The AM fungal sequences were aligned in ClustalX (larkin et al., 2007) and edited with the BioEdit program (Hall, 1999). Boletus edulis Bull. and Neurospora crassa Shear & B.O. Dodge were included as outgroup. Prior to phylogenetic analysis, Paraglomus pernambucanum sp. nov. 115 the model of nucleotide substitution was estimated using Topali 2.5 (milne et al., 2004). Bayesian (two runs over 1 x 106 genera- tions with a burnin value of 2500) and maximum likelihood (1,000 bootstrap) analyses were performed, respectively, in MrBayes 3.1.2 (ronquisT and HuelsenBeck, 2003) and PhyML (guindon and gascuel, 2003), launched from Topali 2.5, using the GTR + G model. Neighbor-joining (established with the model cited above) and maximum parsimony analyses were performed using PAUP*- 4b10 (swofford, 2003) with 1,000 bootstrap replications. Biogeographic analyses A comprehensive literature and sequence data bank research was performed in order to study the biogeographic distribution of the genera Paraglomus and Pacispora on global scale. We included identifications performed either on the genus or on the species level, and considered for studies that were either based on spore morpho- logy, or on the generation of species and environmental sequences. Results Molecular phylogeny Phylogenetic analyses on the partial sequences of the LSU rRNA gene place the two fungi from Bolivia and Pernambuco firmly, with high support values, in a monophyletic clade within the order Paraglomerales next to Paraglomus occultum, P. laccatum and P. brasilianum (Fig. 1). Taxonomy Paraglomus pernambucanum Oehl, C.M. Mello, Magna & G.A. Silva sp. nov. (Figs. 2-9) Mycobank MB 800575 Diagnosis: Sporae albae ad flavo-albae, 66-95 × 62-75 mm in dia- metro, tunicis duabus. Stratum laminatum tunicae exterioris, album ad flavo-album, 2.9-4.2 mm crassum, cum depressionibus subtilibus, 0.5-1.1 mm latis et 0.5-1.0 mm profundis ornatum, 1.3-2.4 mm in distancia. Tunica interior hyalina; 1.9-3.0(-3.9) mm crassa, de novo formans et porum basae sporarum occudens. Tunica hyphae con- fluentis cum stratis exterioribus tunicae externae, raro porum hyphae affixae occludens. In solutione Melzeri tunica interior non colorans. Holotypus # 37-3701: ZT Myc 24205. Etymology: Latin, pernambucanum, referring to the NE Brazilian State where the species was found first. Holotype: Brazil, Pernambuco State, Caruaru (37/3701, ZT Myc 24205). Isotypes (37-3702–37-3705 at ZT Myc 24206; 37-3711–37- 3713 at URM). Paratypes: Brazil, Pernambuco State, in the munici-Paratypes: Brazil, Pernambuco State, in the munici- palities of Serra Talhada and Petrolina (37-3751, 37-3752, 37-3761; as ZT Myc 24207, 24208, 24209 at Z+ZT). Spores are formed singly in soil. They are white to yellowish white, globose to subglobose to ovoid, 66-95 × 62-75 mm in diameter (Figs. 1-4) and have an outer and an inner wall (Figs. 2-3). Outer wall with three layers (Figs. 5-7): outer layer (OWL1) eva- nescent to semi-persistent, subhyaline to light yellow, 0.5-0.9 mm thick, and usually tightly adherent to second layer (OWL2); OWL2 finely laminated, brilliant white in young mature spores to yellow- ish white in older spores, 2.0-2.6 mm thick, with regular, round but shallow and inconspicuous pits that are 0.5-1.1 μm in diameter and 0.5-1.0 μm deep and 1.3-2.4 μm apart; inner layer (OWL3) hyaline and thin, 0.4-0.7 mm, separable under pressure but usually adherent to OWL2 and then extremely difficult to observe. OWL2 stains yel- low in Melzer’s reagent. Inner wall is hyaline and three-layered (Figs. 6-8). Outer layer (IWL1) is 0.5-0.8 mm thick. In crushed spores it sometimes sepa- rates under light pressure from the central layer (IWL2); the cen- tral layer (IWL2) is 1.0-2.4 mm thick and the inner layer (IWL3) is very thin, 0.4-0.7 mm, flexible and may show several folds in broken spores; IWL3 is usually adherent to IWL2 and thus very difficult to observe. None of the layers stains in Melzer’s reagent (Fig. 9). Subtending hypha (sh) is straight or recurved, 4.0-5.5 mm in diam at the spore base tapering to 3.5-4.5 mm within 6-15 mm distance from the base. It is generally slightly funnel-shaped to cylindrical (Figs. 2-6), or rarely slightly constricted (Fig. 9). The wall of the subtending hypha is of the same color as, and continuous with the spore wall layers OWL1 and OWL2, and of the same thickness, tapering to 0.5-1.1 mm within 25-90 mm distance. The length of the subtending hypha persistently remaining at the spore is often shorter (10-15 mm from the spore base), or sh is completely broken away. Pore of the subtending hypha usually is open at spore base, and IW generally functions as pore closure at spore base. Distribution: The new fungus was detected in maize production sites in Caruaru and Serra Talhada, and in a tropical dry forest and a cowpea field in Petrolina. All these sites are located in the semi- arid Caatinga biome of Pernambuco State, NE Brazil. Specimens examined: Brazil, Pernambuco, Caruaru (37-3701– 37-3750). Brazil, Pernambuco, Serra Talhada (37-3751–37-3760); Brazil, Pernambuco, Petrolina (37-3761–37-3770). Paraglomus bolivianum (Sieverd. & Oehl) Oehl & G.A. Silva comb. nov. BaSiOnym: Pacispora boliviana Sieverd. & Oehl. J. Appl. Bot. Food Qual. 78:79. 2004. Mycobank MB 800596 Emendation: The species was well described in Oehl and SieveRding (2004). Here it might be mentioned that the subtending hyphae sometimes have a constricted appearance when OWL2 (hw2 in Fig. 6A of Oehl and sieverding, 2004) is constricted and OWL1 already degraded. However, overall subtending hyphae shape, in- cluding OWL1 respective hw1, is cylindrical to slightly funnel- shaped since sh is regularly broader at spore base than at some dis- tance from the spore (Fig. 6A of Oehl and sieverding, 2004). Comments: The intra-specific variation between the LSU rRNA gene sequences for P. pernambucanum and for P. bolivianum was around 1-2%. The sequences from P. bolivianum presented 95% of identity with those from P. pernambucanum. In the BLASTn analy- sis, the closest related species to P. bolivianum and P. pernambuca- num was P. brasilianum with 93% and 92% of identity, respectively 116 C.M. Aragão de Mello, G.A. Silva, D.M.A. de Assis, J.S. Pontes, A.C.A. Ferreira, M.P.C. Leão, H.E.E. Vieira, L. Costa Maia, F. Oehl Fig. 1: Phylogenetic tree of the Paraglomeromycetes and Archaeosporomycetes based on analysis from partial sequences of the LSU rRNA gene. Boletus edulis and Neurospora crassa were used as outgroup. Sequences are labeled with database accession numbers. Support values (from top) are from neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses, respectively. Sequences obtained in this study are in bold. (Consistency Index = 0.70; Retention Index = 0.86). Paraglomus pernambucanum sp. nov. 117 Figs. 2-9: Paraglomus pernambucanum: Figs. 2-3. Crushed and uncrushed spores with outer and inner wall (OW, IW), cylindric to funnel-shaped subtending hyphae (sh) and minute pit ornamentation on OW surface. Fig. 4. Outer wall staining yellow in Melzer’s reagent. Figs. 5-6. OW and IW triple-layered (OWL1-3; IWL1-3); outer surface of OWL2 with minute pits. Fig. 7. OWL2 staining yellow in Melzer’s. Figs. 8-9. Pore at spore base regularly open but closed by IW. 118 C.M. Aragão de Mello, G.A. Silva, D.M.A. de Assis, J.S. Pontes, A.C.A. Ferreira, M.P.C. Leão, H.E.E. Vieira, L. Costa Maia, F. Oehl Tab. 1: Detection sites of Paraglomus and Pacispora species around the globe AMF isolate Ecosystem/plant species/(Isolate) 1 State/region, Country pH1 Detected Reference by2 Paraglomus spp. Paraglomus occultum Poplar plantations Iowa, USA NA M walker et al. (1982) P. occultum Ornamental plants in greenhouse Oregon, USA NA M walker et al. (1982) P. occultum NA Illinois, USA NA M walker et al. (1982) P. occultum NA The Netherlands NA M walker et al. (1982) P. occultum Agro-ecosystems; temperate Pennsylvania, USA NA M franke-snyder et al. (2001) North America P. occultum Sonoran desert Texas & Arizona, USA 7.7-7.9 M sTuTz and mORtOn (1996) P. occultum Boojum tree in a desert reserve Baja California, Mexico 7.0-8.0 M (BaShan et al. 2007) P. occultum Evergreen forests & pastures Nicaragua & Costa Rica 3.9-5.6 M picOne (2000) P. occultum Natural ecosystems; crops like Carimagua, Colombia 5.0-7.0 M SieveRding (1989) cassava, maize P. occultum Maize Pernambuco, Brazil 5.5-5.7 M maia and Trufem (1990) P. occultum Semi-arid copper mining area Bahia, Brazil 6.2-7.8 M Silva et al. (2005) P. occultum Evergreen and deciduous forests Araucanía Region, Chile 4.6-5.4 M caStillO et al. (2006) P. occultum Horticultural systems Araucanía Region, Chile 5.5-6.1 M caStillO et al. (2010) P. occultum Highest elevation of plant occurrence Valais, Switzerland ca. 5.0 M oeHl, in kÖrner (2011) in Europe, 4505 m asl! P. occultum Subnival siliceous scree, 2700 m asl Valais, Switzerland 5.0-5.4 M Oehl, unpublished P. occultum Poland M Błaszkowski (1989, 1993) P. occultum Grasslands and crop rotation systems Alsace, France; Baden, 4.0-8.0, M Oehl et al. (2005b, 2010) Germany; Basel, 5.3-7.7 Switzerland P. occultum Tropical forests and yam field sites Benin 6.1-6.9 M tchaBi et al. (2009) P. occultum Namib Desert Namibia 7.0-8.0 M sTuTz et al. (1999) P. occultum Forest and grassland India 7.8 M muTHukumar and udaiyan (2000) P. occultum Wheat Golestan, Iran NA M SadRavi (2006) P. occultum Natural habitats: date palm (oasis) Sultanate of Oman 8.2-8.4 M al-yaHya’ei et al. (2011) and native Prosopis cineraria (undisturbed desert habitat) P. occultum Desert ephemerals Xinjiang, China 8.2 M Shi et al. (2006) P. occultum (Isolates from INVAM; IA702, Iowa/Florida/Hawaii, USA NA SpSeq millneR et al. (2001), FL703, HA771) JameS et al. (2006), msiska and mORtOn (2009) P. occultum Cultivated Soils of the Saskatchewan, Canada NA SpSeq Unpublished (JX301683-4) Canadian Prairies P. occultum (FJ461883) (Isolate from INVAM, CR402) Costa Rica NA SpSeq Unpublished P. occultum (Isolates from INVAM; CL700, Colombia NA SpSeq millneR et al. ( 2001), CL700C, CL383) Turnau et al. (2001) P. occultum (Isolate from INVAM, GR582) Germany NA SpSeq millneR et al.( 2001) P. occultum (Isolate from BEG, BEG120) Alicante, Spain NA SpSeq ferrol et al. (2004) P. albidum Winter wheat Ohio, USA NA M walker and RhOdeS (1981) P. albidum Poplar plantations Iowa, USA NA M walker et al. (1982) P. albidum ‘Caatinga’ dry forest Bahia, Brazil 6.2 M Silva et al. (2005) P. albidum Maize mono-cropping, crop France, Germany, 5.3-7.7 M Oehl et al. (2009, 2010) rotations, grasslands Switzerland P. bolivianum Gran Chaco grasslands Santa Cruz, Bolivia 6.5 M&SpSeq Oehl and SieveRding (2004), present study P. bolivianum ‘Caatinga’ dry forest Pernambuco, Brazil 5.2-6.8 M Present study P. bolivianum coastal ‘restinga’ forest vegetation Pernambuco, Brazil 5.1 M Silva et al. (2012) P. brasilianum Greenhouse cultures on Allium porrum D.F., Brasilia, Brazil NA M Spain and miRanda (1996) P. brasilianum Desert ephemerals Xinjiang, China 8.2 M Shi et al. (2006) P. brasilianum (Isolate from INVAM; BR 105) D.F., Brasilia, Brazil NA SpSeq krüger et al. (2012), P. brasilianum (Isolates from INVAM; WV224, West Virginia, USA NA SpSeq millneR et al. (2001), WV219, WV215, WV215A) msiska and mORtOn (2009) Paraglomus pernambucanum sp. nov. 119 AMF isolate Ecosystem/plant species/(Isolate) 1 State/region, Country pH1 Detected Reference by2 P. laccatum Festuca sp. Jastrzębia Góra, Poland. NA M&SpSeq Błaszkowski (1988b), renker et al. (2007) P. laccatum Ammophila arenaria, Slowinski National Park, NA M&SpSeq tadych and Błaszkowski Helictotrichon pubescens Poland (2000), renker et al. (2007) P. laccatum Grassland Thuringia, Germany 7.0-7.5 EnSeq kÖnig et al. (2010) P. laccatum (Isolate Att960-3) UK NA SpSeq krüger et al. (2012) P. lacteum Central Oregon desert Oregon, USA 7.0-8.0 M ROSe and tRappe (1980) P. majewskii Zea mays Algarve, Portugal NA M Błaszkowski et al. (2012) P. majewskii Ammophila. arenaria Mallorca, Spain NA M Błaszkowski et al. (2012) P. majewskii Ammophila. arenaria Karabucak-Tuzla, Turkey NA M&SpSeq Błaszkowski et al. (2012) P. majewskii ‘Cultivated and uncultivated plants’ Lubuskie, Poland NA M Błaszkowski et al. (2012) P. majewskii Ammophila. arenaria Near Bornholm, Denmark NA M Błaszkowski et al. (2012) P. majewskii Weeds Asmara, Eritrea NA M Błaszkowski et al. (2012) P. majewskii Plantago lanceolata West Pomerian, Poland NA M&SpSeq Błaszkowski et al. (2012) P. pernambucanum Natural Caatinga, maize and cowpea Pernambuco, Brazil 5.2-6.4 M&SpSeq Present study Paraglomus sp. Isolated from Miscanthus Jeonbuk, South Korea NA EnSeq lee et al. (2008) sinensis Paraglomus sp. Roots of Panax japonicus Chungbuk, South Korea NA EnSeq lee et al. (2008) Paraglomus sp. Isolate from INVAM - NI116B Nicaragua NA SpSeq Unpublished (FJ461884) Paraglomus sp. Pioneer grass species Miscanthus Hokkaido/Aichi/Okinawa, 2.7-6.8 EnSeq an et al. (2008) sinensis in acid sulfate soils Japan Paraglomus sp. Seminatural grasslands - Thuringia, Germany 6.2 EnSeq renker et al. (2003), Roots of Lolium multiforum BöRStleR et al. (2006) Paraglomus sp. Wetland, Roots of Dactylis glomerata Thuringia, Germany NA EnSeq wirsel et al. (2004) Paraglomus sp. Semi-natural grasslands – Thuringia, Germany 6.2 EnSeq BöRStleR et al. (2006) Roots of Plantago major Paraglomus sp. Semi-natural grasslands Thuringia, Germany 6.2 EnSeq BöRStleR et al. (2006), hempel et al. (2007) Paraglomus sp. Roots of Ajuga reptans Yorkshire, UK NA EnSeq Unpublished (AJ854100) Paraglomus sp. Onion (trap plant roots) England-UK NA EnSeq Unpublished (FN555262-92) Paraglomus sp. Arable soils – Roots of Zea mays Basel, Switzerland 4.8-5.4 EnSeq hiJRi et al. (2006) Paraglomus sp. Arable soils – Roots of Triticum Basel, Switzerland 5.4 EnSeq hiJRi et al. (2006) aestivum Paraglomus sp. Arable soils – colonized roots Basel, Switzerland 4.8 EnSeq hiJRi et al. (2006) Paraglomus sp. Aquatic macrophytes in oligotrophic The Netherlands NA EnSeq BaaR et al. (2011) and ultra-oligotrophic lakes Paraglomus sp. Organic and conventional farming The Netherlands 7.4 EnSeq galván et al. (2009) systems – Roots of Allium cepa Paraglomus sp. Geothermal soils – Roots of Iceland 4.0-4.5 EnSeq appOlOni et al. (2008) Agrostis stolonifera Paraglomus sp. Geothermal soils – colonized roots Yellowstone, USA 3.4-4.8 EnSeq appOlOni et al. (2008) Paraglomus sp. Geothermal soils– Roots from Yellowstone,USA 4.8 EnSeq Bunn et al. (2009) Dichanthelium lanuginosum Paraglomus sp. Colonized roots Yellowstone,USA 4.8-6.5 EnSeq lekBerg et al. (2011) Paraglomus sp. Grasslands dominated by exotic California, USA NA EnSeq Unpublished (GQ890654, GQ890656) plant species Paraglomus sp. Reforestation plots on degraded South of Ecuador 4.0-5.0 EnSeq Haug et al. (2010) pastures- Roots of Setaria sphacelata and Heliocarpus americanus Paraglomus sp. Roots of Retama sphaerocarpa, Murcia, Spain NA EnSeq alguacil et al. (2011a) Psoralea bituminosa and Lolium perenne Paraglomus sp. Seedlings grown in a heavy metal Murcia, Spain NA EnSeq alguacil et al. (2011b) polluted soil Paraglomus sp. Roots of Herniaria fruticosa and Murcia, Spain NA EnSeq alguacil et al. (2012b) Senecio auricula 120 C.M. Aragão de Mello, G.A. Silva, D.M.A. de Assis, J.S. Pontes, A.C.A. Ferreira, M.P.C. Leão, H.E.E. Vieira, L. Costa Maia, F. Oehl AMF isolate Ecosystem/plant species/(Isolate) 1 State/region, Country pH1 Detected Reference by2 Paraglomus sp. Galls and Roots of Prunus persica Aragua, Venezuela 5.18 EnSeq alguacil et al. (2011c) Paraglomus sp. Ricinus communis soil Guantanamo, Cuba 8.7 EnSeq alguacil et al. (2012a) Paraglomus sp. Roots of Dichanthium aristatum Guadeloupe, 7.8 EnSeq JalOnen et al. (2012) French Antilles Paraglomus sp. Roots of Zea mays Martonvasar, Hungary 5.8-6.2 EnSeq SaSváRi et al. (2011) Paraglomus sp. Roots of Tanacetum vulgare, Bohemia, Czech Republic NA EnSeq Unpublished (HE775341-50) Brachypodium pinnatum, and Knautia arvensis Paraglomus sp. Roots of Trifolium repens Lombardy, Italy 5.7-6.4 EnSeq lumini et al. (2011) Paraglomus sp. Roots of Zea mays Marche, Italy 8.3-8.5 EnSeq BORRiellO et al. (2012) Paraglomus sp. Roots of Larix decidua South Tyrol, Italy NA EnSeq Unpublished (HM044471) Paraglomus sp. Roots of Camellia japonica Piedmont, Italy 5.8 EnSeq Unpublished (JQ315319-36) Paraglomus sp. Pasture soil Sardinia, Italy 5.4-6.2 EnSeq orgiazzi et al. (2012) Paraglomus sp. Pasture soil Sardinia, Italy 5-6.5 EnSeq lumini et al. (2009) Paraglomus sp. NA Cameroon NA EnSeq Unpublished (HQ108153-9) Paraglomus sp. Chickpea rooting soil Canada NA EnSeq Unpublished (JF340036-39) Paraglomus sp. Rhizosphere of Tectona grandis Chiang Mai, Thailand NA EnSeq Unpublished (JQ864337) Paraglomus sp. Semiarid Mediterranean prairies Murcia, Spain NA EnSeq tORRecillaS et al. (2012) Pacispora spp. Pacispora scintillans High Mediterranean dessert Oregon, USA NA M ROSe and tRappe (1980) P. scintillans Pot culture with Plantago lanceolata Pomerania, Poland NA SpSeq walker et al. (2004) inoculated with field soil from beneath Triticum aestivum P. scintillans Sandy heathland, beneath Dactylis Hessen, Germany NA SpSeq walker et al. (2004), glomerata, Anthericum liliago and krüger et al. (2009) associated plants P. scintillans Ancient meadow, beneath Lolium Dorset, UK NA SpSeq walker et al. (2004) perenne and associated plants P. chimonobambusae Bamboo garden Nan-Tou, Taiwan NA M wu et al. (1995) P. chimonobambusae Trisetum grassland on serpentinite Grisons, Switzerland 6.1 M Oehl, unpublished P. coralloidea Subnival siliceous scree Valais, Switzerland 6.5 M Oehl and SieveRding (2004) P. dominikii Trifolium pratense NW Poland NA M Błaszkowski (1988a) P. dominikii Trisetum grasslands Grisons, Switzerland 7.8 M Oehl, unpublished P. dominikii Organic farming systems Basel, Switzerland 6.4-7.5 M Oehl et al. (2005b, 2010) P. dominikii Pterocephalus spathulatus, Andalusía, Spain 8.1 M&SpSeq palenzuela et al. (2008) Thymus granatensis P. franciscana Olive tree-grasslands Umbria, Italy 7.0-7.5 M Oehl and SieveRding (2004) P. franciscana Subnival calcareous screes Grisons, Switzerland 7.5 M Oehl and SieveRding (2004) P. franciscana NA Pomerania, Poland NA SpSeq krüger et al. (2012) P. franciscana NA Lower Saxony, Germany NA SpSeq krüger et al. (2012) P. patagonica Nothofagus forest Santa Cruz, Argentina 5.5 M nOvaS et al. (2005) P. robigina Subnival calcareous and Grisons, Switzerland 7.0-7.8 M Oehl and SieveRding (2004) serpentinite screes P. robigina Subnival screes Valais Switzerland 6.5 M Oehl and SieveRding (2004) P. robigina Soldanella carpatica Tatra Mountains, Poland NA M Oehl and SieveRding (2004) Pacispora sp. McLaughlin Reserve on serpetinite California, USA NA EnSeq SchechteR and Bruns (2008) Pacispora sp. Alpine plant species (4500 m asl) Tibet, China NA EnSeq liu et al. (2011) Pacispora sp. Tibet Plateau China NA EnSeq Unpublished (JQ182767) Accession numbers are given in the left column for available sequences that so far have not yet been published in peer-reviewed journals. 1 No information Available. 2 M = Identified by morphological analyses, SpSeq identified by molecular analyses, M&SpSeq identified by both approaches, EnSeq = environ- mental sequences deposited in public data bases. Paraglomus pernambucanum sp. nov. 121 (Fig. 1). Environmental sequences that were closely related to the two fungi were not found in the public data bases. Distribution: Paraglomus bolivianum was found in a degraded pas- ture of the semi-arid Gran Chaco (Bolivia, Oehl and sieverding, 2004). In recent years, it was also found in the semi-arid Caatinga of NE Brazil, e.g. in Triunfo, Belém do São Francisco and Serra Talhada, all in Pernambuco State. Finally, P. bolivianum was also found in a coastal ‘restinga’ forest vegetation in Mataraca (Paraíba State; 6°28’20’’-6°30’00’’S; 34°55’50-34°57’10’’W (Silva et al., 2012). Biogeography of Paraglomus and Pacispora spp. In Tab. 1, a comprehensive summary of the biogeography of the genus Paraglomus and Pacispora, and their conclusively and non- conclusively identified species, is given. The identifications have been based on spore morphology, molecular analyses on formerly morphologically identified species, and on so-called environmen- tal sequences deposited in the public data bases. It can be deduced from these results that the genus Paraglomus has a worldwide distri- bution and occurs in many terrestrial ecosystems throughout the globe. It has been recorded from high alpine to nival areas, temperate to Mediterranean up to sub- to inner tropic, arid to humid areas, in different soil types covering a wide spectrum of soil pH and land use intensities (Tab. 1). There have been > 300 Paraglomus sequences from the ribosomal gene deposited in the public data bases, from 36 countries and from many quite different ecosystems. The most widespread fungus of this genus so far might be P. occultum, fol- lowed by P. albidum, since these two species were most often iden- tified. However, the phylogenetic tree shows at least two species identified as P. occultum, revealing current problems in the spe- cies identification of this small-spored genus. Only one P. occultum clade, fixed by the isolate from the type area in Iowa, can be P. oc- cultum, while the sequence AJ271713 obviously belongs to another Paraglomus species (Fig. 1). The genus Pacispora is biogeographically more restricted to spe- cific habitats and specific climatic zones than Paraglomus (Tab. 1), which confirms our assumption in the introduction. Based on mor- phological spore identification, the genus is characteristic for higher pH soils (> 6.0) and a more restricted to specific ecosystems when compared to Paraglomus. One exception might be P. patagonica which was found in soil with pH 5.5, but it was not given in nOvaS et al. (2005) in which medium the soil pH was measured. Also in the public data bases, sequences of Pacispora spp. have so far been rarely deposited: from the ribosomal gene, only nine environmental sequences and 23 sequences from formerly morphologically identi- fied species have been found. They are from USA, Poland, Germany, UK, Spain and China, all detected from soils with pH > 6.0 (Tab. 1). Nevertheless, our investigation shows that the genus occurs world- wide, but none of the Pacispora species appear to have a similar wide distribution as P. occultum. Discussion Paraglomus pernambucanum and P. bolivianum can easily be dis- tinguished by their spore wall ornamentations, and by spore color and size. The spores of P. bolivianum are yellow brown to brown and their pits are substantially larger and deeper (Oehl and sieverding, 2004) than those of P. pernambucanum, whose spores are hyaline to subhyaline. In Paraglomus, there is one other species known with ornamentation on the spore wall. This is P. brasilianum whose or- namentation is labyrinthiform (Spain and miranda, 1996; mORtOn and redecker, 2001). Within Paraglomus, six to seven of the eight species might have two spore walls. These are P. occultum (walker, 1982), P. brasilianum (Spain and miranda, 1996), P. bolivianum (Oehl and sieverding, 2004), P. pernambucanum, and, according to our analyses (Oehl, own observations), also P. lacteum (ROSe and Trappe, 1980) and P. laccatum (renker et al., 2007). Beneath its multi-laminated spore wall layer, P. laccatum has a separate inner wall which is difficult to observe (renker et al., 2007; Oehl et al., 2011c). This might be also true for P. albidum (walker and rHodes, 1981; Oehl et al., 2011c) but this need to be checked on newly isolated spores. Thus, only P. majewskii might have solely one, triple-layered spore wall with a relatively thin innermost layer, which is substantially thinner than those in the other Paraglomus species like P. occultum, P. brasi- lianum, and P. bolivianum. Interestingly, P. majewskii was re- ported as forming a relatively distant lineage within Paraglomus (Błaszkowski et al., 2012), which is confirmed by our analyses. Beside P. pernambucanum, there has been only one other recently described Paraglomus species, with phylogenetic analyses in the original description (P. majewskii, Błaszkowski et al., 2012). Like P. bolivianum, P. laccatum was transferred to the genus Paraglomus due to new phylogenetic analyses (renker et al., 2007) on type specimens of the former Glomus laccatum (Błaszkowski, 1988b). Sequences on the ribosomal gene of P. occultum and P. brasilianum (formerly G. occultum and G. brasilianum) were the base for the transfer of the later two species (walker, 1982; Spain and miranda, 1996) from the Glomerales and Glomeraceae to the Paraglomerales and Paraglomeraceae, respectively (mORtOn and redecker, 2001; SchüSSleR et al., 2001). Up to date, six Paraglomus species have been sequenced on the rRNA or other genes (Fig. 9), while for P. albidum and P. lacteum molecular phylogenetic evidence is still missing (Oehl et al., 2011c). Remarkably, P. bolivianum is the first species known in the Paraglomeromycetes with pigmented spores. In the Glomeromycota, there are currently two genera with bi-walled spores formed on subtending hyphae. This is true for all Pacispora species (Oehl and sieverding, 2004; Oehl et al., 2001b) and for most of the Paraglomus species. Pacispora species form charac- teristic constricted to cylindrical subtending hyphae that may bear one to a few hyphal pegs, and the outermost spore wall layer is semi-persistent, while the inner wall regularly stains purple to deep purple in Melzer’s reagent. In contrast, bi-walled Paraglomus spores generally have slightly funnel-shaped to cylindrical subtending hy- phae without hyphal pegs, and the outermost spore wall layer is rapidly degrading (‘short-lived’) and thus, can be called evanescent, while the inner wall never stains in Melzer’s. Remarkably, species of Pacispora with ornamented spore surfaces regularly have pro- jections on OWL1 (P. scintillans, P. dominikii, P. coralloidea, P. chimonobambusae, P. patagonia) (Oehl and sieverding, 2004; walker, 2008), while the Paraglomus species with ornamented spore surfaces have a pitted OWL2 (P. brasilianum, P. bolivianum and P. pernambucanum). Our literature and data bank research strongly suggests that Paraglomus species have a wider distribution than Pacispora spe- cies, since they were found in many different ecosystems from the warm to very cold climates and in soils of very different soil pH (Tab. 1). Pacispora species are characteristic for soils with pH > 6.0, either in high alpine areas (e.g. P. robigina, Oehl and sieverding, 2004; Błaszkowski et al., 2008), in cultivated soils of temperate areas (e.g. P. dominikii, Błaszkowski, 1993; Oehl et al., 2005b; Oehl et al., 2010) or in Mediterranean and subtropical areas un- 122 C.M. Aragão de Mello, G.A. Silva, D.M.A. de Assis, J.S. Pontes, A.C.A. Ferreira, M.P.C. Leão, H.E.E. Vieira, L. Costa Maia, F. Oehl der semi-arid conditions subjected to naturally elevated soil pH (e.g. P. scintillans and P. franciscana, ROSe and Trappe, 1980; Błaszkowski, 1993; Oehl and sieverding, 2004; BaShan et al., 2007). However, so far they were, to our knowledge, never found in tropical areas (e.g. sieverding, 1989; StüRmeR and siqueira, 2011; tchaBi et al., 2008). In contrast, Paraglomus bolivianum and P. pernambucanum were so far only found from tropical regions in South America, beside a single isolation site of P. bolivianum reported from southeast Tibet (wang and sHi, 2008). It has to be taken into account that many Paraglomus species, and especially P. pernambucanum, form rather small, rapidly degrading spores that in the past might have often been difficult to identify from field samples. Thus, we do not exclude that both species have, like P. occultum, a much larger distribution than known so far. Acknowledgements Catarina M. A. de Mello wishes to acknowledge the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for a Ph.D. scholarship. This study has been supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) that provided research grants to Leonor C. Maia (INCT- Herbário Virtual da Flora e dos Fungos, Proc.573.883/2008-4; Protax, Proc. 562.330/2010-2; Sisbiota, Proc. 563.342/2010-2), by FACEPE and UFPE which provided grants to F. Oehl as ‘visiting professor’ and by the Swiss National Science Foundation (SNSF) Project 315230_130764/1). References alguacil, m.m., Torres, M.P., Torrecillas, E., díaz, G., roldán, a., 2011a: Plant type differently promote the arbuscular mycorrhizal fungi biodiversity in the rhizosphere after revegetation of a degraded, semiarid land. Soil. Biol. Biochem. 43, 167-173. alguacil, m.m., Torrecillas, E., koHler, J., roldán, a., 2011b: Molecular approach to ascertain the success of “in situ” AM fungi in- oculation in the revegetation of a semiarid, degraded land. Sci. 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