A New Species of Scutellospora with a Coiled Germination Shield

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1 Annals of Botany 86: 21±27, 2000 doi:10.6/anbo , available online at on A New Species of Scutellospora with a Coiled Germination Shield KARTINI KRAMADIBRATA{, CHRISTOPHER WALKER*{, DANIEL SCHWARZOTT} and ARTHUR SCHUÈ ûler} {`Herbarium Bogoriense', Research and Development Centre for Biology, The Indonesian Institute of Sciences, J1. Ir. H. Juanda 22, Bogor, 16122, Indonesia, {School of Conservation Sciences, Bournemouth University, Talbot Campus, Fern Barrow, Poole, Dorset BH12 5BB, UK and }TU Darmstadt, FB10 Botanik, Schnittspahnstrasse 10, Darmstadt, Federal Republic of Germany Received: 10 September 1999 Returned for revision: 14 January 2000 Accepted: 25 February 2000 During a survey of mycorrhizal fungi on the upper part of the Cisadane River, on the slopes of Mount Pangrango in Gede Pangrango National Park, West Java, an undescribed species of Scutellospora (Glomales) was discovered. This species has metallic golden to yellow to yellowish-brown spores that possess columnar protuberances. It is described and named Scutellospora projecturata sp. nov. The sequence of the nearly complete SSU rrna gene was analysed and phylogenetic trees constructed. # 2000 Annals of Botany Company Key words: Scutellospora projecturata, Glomales, new species, 18s SSU rrna, West Java, phylogenetic tree, phylogeny, arbuscular mycorrhizal fungi, AMF. INTRODUCTION The Gede Pangrango National Park, West Java, is an important area of tropical forest that has remained uncultivated by agronomists and unexploited for timber. It has been studied extensively for its ora and fauna, but there are few studies of its fungal components, and none of its mycorrhizal fungi. Because such fungi are integral parts of most terrestrial ecosystems, soil samples from the root zone of understorey plants were examined for the presence of arbuscular mycorrhizal fungi (AMF) (Kramadibrata, 1992). One spore type found had characteristic features of fungi in the genus Scutellospora, but bore peculiar ornamentation quite unlike any described species in the genus. Attempts were made to establish the species in pure pot culture in symbiosis with various plants, but although a few spores were produced from one such culture attempt, it was not possible to maintain it alive. Detailed morphological studies were carried out, and the fungus is described as Scutellospora projecturata sp. nov. To analyse the phylogenetic position of the new species, DNA of single spores formed in mixed species, open-pot culture was isolated and the SSU rrna gene sequenced to construct phylogenetic trees. MATERIALS AND METHODS Soil cores were taken at random from a research site on the upper part of the Cisadane River, on the slopes of Mount Pangrango of Gede Pangrango National Park in West Java. Samples were carefully removed from the root zones of Villebrunea rubescens (B1.) B1., Cyathea contaminans (Wall. * For correspondence. cwalker@bournemouth.ac.uk ex Hook.) Copel., Ficus binnendijkii (Miq.) Miq. and Syzygium pyrifolium (B1.) DC (Mirmanto, 1991). Part of the soil was used for nutrient analysis, and the remainder was subjected to a sieving, centrifugation and sugar- oatation extraction procedure to retrieve spores of mycorrhizal fungi (Walker et al., 1982). Extracted spores were examined in a dish of water under a dissecting microscope, and sorted into morphologically similar specimens. Colour and general appearance were assessed under incident illumination from a quartz±halogen bre optic source with a colour temperature of 3000 K (Walker et al., 1993). Some specimens were mounted on microscope slides under No. 1 cover glasses in polyvinyl alcohol lacto-glycerol (PVLG) and observed through a compound microscope with bright eld and Nomarski di erential interference contrast illumination. To test the reaction to Melzer's reagent, samples were mounted in PVLG with the addition of Melzer's reagent (5 : 1 v/v). Measurements were made on intact spores with a calibrated eyepiece graticule through the compound microscope, and wall structures were assessed from spores crushed by application of pressure to the cover glass. To observe any secondary e ects of mounting media, some spores were crushed a second time after allowing a few minutes' reaction time. Attempts at producing cultures were made either by the open-pot culture method (Gilmore, 1968) or in a sealed bag system (Walker and Vestberg, 1994). The attempts were given numbers in a database system that allows complete records of origin and culture history to be maintained, along with details of voucher specimens. Attempts are coupled with culture number (e.g. Attempt 5-6 is subculture number 6 from attempt 5), and vouchers, in Walker's herbarium are given a unique number preceded by the letter /00/ $35.00/00 # 2000 Annals of Botany Company

2 22 Kramadibrata et al.ðscutellospora projecturata sp. nov. from Indonesia W (e.g. W400) (Walker and Vestberg, 1998). Two methods of isolation were attempted. In the rst, `soil traps' were made from samples of the soil mixed with disinfected substrate and sown or planted with Plantago lanceolata L. The other attempts were from spores extracted from soil or substrate from soil traps. These were placed individually, or grouped, on the roots of seedlings in a sterile substrate. DNA for PCR was extracted from single spores of attempt After cleaning three times by sonication (20 sec) and rinsing with sterile double-distilled water, DNA was extracted by crushing a spore in a 0.5-ml PCR-tube with a sterile pipette-tip in 2 ml double-distilled water. The resultant solution was frozen in liquid nitrogen and subsequently heated three times in a microwave oven for 15 sec at 600 W. The microwaving process results in more e cient PCR ampli cation. PCR-ampli cation of DNA was carried out on the resultant extract with the universal primers NS1 and the primer Geo10 (Gehrig et al., 1996) in a nal volume of 50 ml. PCR was optimized by the temperature gradient function with a Mastercycler Gradient (Eppendorf, Hamburg, Germany). The ampli cation reaction was performed as follows: 1 2 min at 948C; sec at 948C, 60 sec at 528C, 150 sec at 728C; 1 30 min at 728C. Control reactions were performed without adding the template DNA. The ampli cation products were separated electrophoretically on 1.2 % agarose gels and stained with ethidium bromide. Fragments were cloned into pcr TOPO vector from Invitrogen (Groningen, Netherlands). Plasmid DNA of clone pwd was prepared with a QIAprep Spin Miniprep Kit (Quiagen, Hilden, Germany) and the inserts were sequenced by SEQLAB (GoÈ ttingen, Germany). Manual sequence alignment was performed with the program ALIGN 4.0 of D. Hepperle (Neuglobsow, Germany), taking the secondary structure into account (De Rijk et al., 1992). The demonstration version of this program can be found at ftp:// /pub/ Herr.Hepperle/align.htm. An initial phylogenetic analysis (not shown) was carried out to con rm the glomalean origin of the sequences. The analysis was performed on a dataset containing representative sequences of all fungal divisions, including all Zygomycetes so far sequenced. Stylonychia pustulata (Protista) was used as the outgroup. Thraustochytridium kinnei (Chromista), Ulkenia profunda (Chromista), Zea mays (Planta), Homo sapiens (Animalia) and Aphrodita aculeata (Animalia) were included as controls for possible contamination by non-fungal organisms. After this analysis one sequence (S. castanea BEG1, clone rusc1-18s; accession AF038589) was excluded, since it is derived from a contaminating organism (SchuÈ ûler, 2000). Two more sequences are published from S. castanea BEG1, but one (accession U31997) is too short for satisfactory analysis and was excluded. The two known sequences of G. mosseae BEG12 (accession U31995 and U96139) are partial sequences, and were combined to obtain a near full-length sequence. We took 1585 sites that were certain to be in alignment for the construction of phylogenetic trees. Analyses were carried out with PHYLIP, version 3.572c (Felsenstein, 1982, 1989). Input order of species was randomized and the analyses were bootstrapped 0 times to estimate robustness of tree structure. Phylogenetic trees were computed by the neighbour-joining method (Saitou and Nei, 1987; Nei et al., 1995) with Kimura parameters (Kimura, 1980) and the parsimony method (Felsenstein, 1983), with Mortierella polycephala as the outgroup. In the phylogenetic tree presented only clades with bootstrap support of more than 60 % are shown. Others were collapsed to polytomies to prevent misinterpretation of such low bootstrap values. RESULTS Nineteen di erent types of glomalean spores were recovered from the soil samples, including nine from the genus Glomus, four from Acaulospora, three Sclerocystis (sensu lato) spp., Entrophospora infrequens (Hall) Ames and Schneider, and two undescribed species of Scutellospora (Kramadibrata, 1992). One of the spore types was particularly unusual (Figs 1±12). The spores were golden in colour and peculiarly ornamented (Figs 1±4). The rst spores examined had lost their attachment (Fig. 10), and were not recognized as a member of the Glomales, but specimens were then discovered that had characteristics of the genus Scutellospora (Fig. 3). Of 25 attempts to produce viable single species pot cultures of the undescribed fungus, none was successful. However, Attempt 10-0 resulted in establishment of mycorrhizas, but produced many di erent spore types. These included a few specimens of the putative new species that appeared to be in good condition. Ten further subculture attempts from this were almost all failures, but one, Attempt 10-5, produced a few spores. However, no more spores were found subsequently, and later staining did not reveal any mycorrhizas. Detailed examination showed the ornamented spores to possess a coiled pre-germination structure similar to that produced by some species of Acaulospora, but they also had a bulbous base (Figs 3±5) typical of members of the Gigaspora or Scutellospora (Walker and Sanders, 1986). These spores are the subjects of this protologue. SPECIES DESCRIPTION Scutellospora projecturata Kramadibrata and Walker sp. nov. (Figs 1±13) Sporae in solo singillatim enatae, juventute metallice aureae et nitidae, ochracentes vel siennentes, post maturitatim ochracentes, siennentes, xerampelinentes vel fuscentes, globosae vel subglobosae, raro late ellipsoideae vel irregulares, 102± ±181 mm, basi bulbosa terminale vel laterale a xa. Basis bulbosa sporae spora concolor, 32±60 29± 47 mm, cum vel sine una vel pluribus projecturis interdum ramosis. Sporae protuberationibus 2±4 mm longis. Tunicae sporae turmis tribus. Turma externa componento uno, 1±2 mm crasso, digitationibus prominentibus, rectis vel uncatis, interdum colliculoso. Turma media componento uno, exili, hyalino, 51 mm crasso. Turma interna componentis

3 Kramadibrata et al.ðscutellospora projecturata sp. nov. from Indonesia µm 500 µm µm b µm hp b 4 b µm 25 µm µm os p A B C µm gs gs gs FIGS 1±12. Scutellospora projecturata sp. nov. Diagnostic characteristics. Fig. 1. Spores from a freshly collected substrate from a mixed species pot culture. Spores are bright and have a metallic gold colour under re ected light. Fig. 2. Spores in water, extracted from stored eld soil. These spores are darker in colour and less re ective than those from the pot culture. Fig. 3. A spore mounted in polyvinyl alcohol lacto-glycerol (PVLG). This specimen has cracked open slightly under the weight of the cover glass, but is almost intact. The prominent protuberances that give the species its name are evident, and the bulbous base (b) can be seen at the upper right. Fig. 4. This specimen has been crushed gently after mounting in PVLG. Two of the wall groups are evident (the outermost and innermost). The bulbous base (b) is arrowed at bottom right. Fig. 5. In this crushed specimen, the peg-like hyphal protrusion (hp) from the bulbous base (b) is indicated. The subtending hypha is septate. Fig. 6. One of the prominent protuberances, formed by out-folding of the outer wall group, is detailed in this illustration. Fig. 7. In close detail, a curved prominent protuberance (p) and the collicular ornamentation on the surface of the outer wall group can be seen (os). Fig. 8. When mounted on a microscope slide in water, and crushed, the three wall groups (A, B and C) can be seen. Wall group C appears to be single, coriaceous component when treated in this way. Fig. 9. This specimen has been mounted in PVLG, crushed by gentle pressure on the cover glass, left for a few minutes, then crushed again with more force. The wall groups have separated, and their individual components as seen through a compound microscope are indicated by arrows. The complex nature (3, 4 and 5) of wall group C is revealed by this treatment. Fig. 10. The purple reaction of the inner wall components to PVLG containing Melzer's reagent is illustrated. An arrow (upper right) indicates the colourless, delicate germination shield (gs). This specimen has lost its bulbous base. Fig. 11. The coiled germination shield (gs) is illustrated in lateral view in this image. This specimen was mounted in PVLG with Melzer's reagent, but there is little reaction. Only two small areas of the inner wall group have become purple (middle, right and lower, left). Fig. 12. In this specimen, the spore has been crushed and broken open to reveal the germination shield (gs). Part of the broken, outer wall component is evident at upper left. 12

4 24 Kramadibrata et al.ðscutellospora projecturata sp. nov. from Indonesia usually wrinkling considerably (Fig. 9) after crushing in mounting medium. Wall structure in water similar to that in PVLG (Fig. 13), except group C appearing as a single coriaceous component, 1.5±2 mm thick (Fig. 8). Germination shield coiled, formed by protrusion of wall components in Group C, and coiling against the main structural wall group through which a germ tube emerges (Figs 11 and 12). Auxiliary cells unknown. (O) A B C A B C Wall component appearance in PVLG Wall component appearance in water F IG. 13. Alternative murographs (Walker, 1983) of Scutellospora projecturata sp. nov. Wall components indicated by ll: laminated, vertical dotted lines; exible, single hatching; amorphous, parallel arcs; coriaceous, cross-hatching. tribus: componentum externum 1.5±2 mm crassum; componentum medium circa 1 mm crassum, in solutione Melzeri purpureum; componentum internum 1 mm crassum, in solutione Melzeri purpureum. Cellulae auxiliares ignotae. Spores formed singly in the soil. Metallic gold and shiny when fresh (no suitable colour chart match found), becoming duller, and ochre to sienna (colour on the chart ) with time. Ochraceous to ochre or sienna to bay to fuscous black ( ) when past maturity or moribund (Figs 1 and 2): globose to subglobose (rarely broadly ellipsoid or irregular), 102± ±181 mm (mean mm, n ˆ 94) with a terminally or laterallyattached bulbous base (Figs 3±5), produced from a coenocytic to septate subtending hypha. Bulbous spore base concolorous with the spore, 32±60 29±47 mm, with or without one or more sometimes branched peg-like projections. Spores with long protuberances 2.0 to 4.0 mm long (Figs 1±4) formed by outfolding and whole or partial fusion of a digitation of the main structural wall component (Fig. 6). Spore wall structure by light microscopy of ve components (Walker, 1983; Walker and Vestberg, 1998) in three groups. In PVLG and PVLG/Melzer's reagent, outer wall group (Group A, Fig. 8) a laminated element (component 1), 1±2 mm thick, with prominent straight or hooked digitations (Figs 3, 4, 6 and 7), sometimes ornamented with low dense rounded bumps or collicles (Fig. 7) approx 1±3 mm across and separated by approx. 0.5 mm. Component 2 (Group B) exible, hyaline, 51 mm thick (Fig. 8), di cult or impossible to see in some older specimens. Group C (Fig. 8) exible, of three components (3, 4 and 5); component 3, 1.5±2.0 mm thick, attached tightly to components 4 and 5, approx. 1 mm, and 51 mm thick, respectively. Component 4 becoming plastic and expanding when crushed after a few minutes in the mounting medium. Components 4 and 5 becoming purple in Melzer's reagent (Fig. 10) others not reacting. Component 5 (O) Mycorrhizal associations Unknown. Attempts to establish a single-species, mycorrhizal symbiosis failed, although the species has produced low numbers of spores in mixed pot culture with other members of the Glomales. Distribution and habitat Known only from an area in or around the Gede Pangrango National Park, or from the Cibodas Botanical Garden, Indonesia. Spores of this species have been collected several times beneath various hosts, but in particular under bamboo in Cibodas Botanical Garden, Indonesia. Collections examined TYPE Indonesia, West Java, Cianjur District, Cibodas Botanical Gardens from beneath cultivated bamboo (Holotype, W1717 BO). OTHER COLLECTIONS Indonesia, West Java, Cianjur District, Cibodas Botanical Gardens from beneath cultivated bamboo from soil W2700, 12 May 1996; W1730 from pot culture Attempt 10-0; W2223 from Attempt 10-5; W2228 from Attempt 35-0; W2232 from Attempt 34-0; W2405 from Attempt 10-0; W2833 from Attempt 625-0; W2873 from Attempt (all from Cibodas Botanical Gardens), and Indonesia, West Java, Sukabumi District, Gede Pangrango National Park beneath riparian tropical trees and associated plants. Etymology Latin, projecturata: in reference to the prominent digitations formed as part of the spore. Sequence analysis The results of the sequence analysis are shown as a phylogenetic tree (Fig. 14). The sequence is deposited in the EMBL database (accession number AJ242729). The almost full-length SSU rrna gene sequence of S. projecturata clusters rmly within the % bootstrap supported Gigaspora/Scutellospora clade. However, although it was expected to belong to the Scutellospora clade, it actually tends, with relatively strong bootstrap support, to t the Gigaspora branch.

5 Kramadibrata et al.ðscutellospora projecturata sp. nov. from Indonesia Gi. albida FL927 (Z14009) Gi. margarita DAOM (X58726) Gi. gigantea WV932 (Z14010) Gi. decipiens BEG45 (U96146) S. projecturata W 3254 (AJ24272) S. castanea BEG1 (AF038590) S. heterogama BR154 (U36593) S. heterogama WV858 (Z14013) S. pellucida WV873 (Z14012) A. spinosa WV860 (Z14004) A. rugosa WV949 (Z14005) E. colombiana FL356 (Z14006) E. contigua WV201 (Z14011) G. mosseae DAOM (U96143) G. mosseae DAOM (U96145) 86 G. mosseae BEG25 (U96140) G. mosseae DAOM (U96142) G. mosseae BEG69 (U96141) G. mosseae BEG12 (U96139+U31995) G. mosseae FL156 (Z14007) 95 G. vesiculiferum (L20824) 64 G. intraradices DAOM (X58725) G. manihotis (clarum) FL879 (U36590) G. versiforme BEG47 (X86687) G. microaggregatum DAOM (U96144) G. etunicatum UT316 (Z14008) G. luteum SA101 (U36591) G. sp. BR212 (U36592) Geosiphon pyriforme (X86686) A. gerdemannii MAFF520055; (AB015052) Endogone pisiformis CRBF#0001 (X58724) 0 Mortierella polycephala NRRL22890 (X89436). 01 FIG. 14. Neighbour-joining consensus tree of glomalean SSU rrna gene sequences. 0 bootstraps. Bootstrap values of the neighbour-joining analysis are shown above, and of a parsimony analysis below the branches. Branches supported by bootstrap values below 60 % are reduced to polytomies. The analysis shows other results of signi cance to glomalean phylogenetics. There is a statistically well supported close phylogenetic relationship between the dimorphic glomalean fungus A. gerdemannii (Sawaki et al., 1999; Redecker et al., 2000) and the endocyanosis forming fungus Geosiphon pyriforme (Gehrig et al., 1996; SchuÈ ûler and Kluge, 1999). The genus Glomus sensu lato forms at least two clades that may be paraphyletic, and G. versiforme might be a member of a third clade for which no other sequences are yet known. There is also an anomalous sequence of a fungus, erroneously identi ed as G. mosseae that groups with other fungi in the second clade, rather than with the cluster that contains all other G. mosseae cultures. A voucher specimen of this culture was deposited in DAOM as Glomus microaggregatum (Y. DalpeÂ, pers. comm.) and its inclusion as G. mosseae (Vandenkoornhuyse and Leyval, 1998) is the result of an error. DISCUSSION The pre-germination structure is di erent from the germination shield, reported from all currently described species of the genus Scutellospora. Its coiled nature is similar to the germination shield found in some species of the genus Acaulospora (Spain, 1992). This originally led us to believe that it may be closely related to some members of the genus Acaulospora, but it belongs rmly in the Scutellospora/ Gigaspora clade. In the phylogenetic tree constructed with the sequences known so far, Scutellospora projecturata

6 26 Kramadibrata et al.ðscutellospora projecturata sp. nov. from Indonesia tends more to the clade comprising the Gigaspora species than to Scutellospora species. There are other ( presently undescribed) Scutellospora-like organisms with similar germination characteristics, but suitable material for molecular analysis has not yet been recovered (G. Cuenca, Venezuela, unpubl. res.). Scutellospora projecturata belongs to a Scutellospora-group distinct from those sequenced so far. Di erent Scutellospora clades seem to arise sequentially on a branch, which eventually leads to the Gigaspora clade, but until more sequences and other phylogenetic characteristics have been comparatively analysed, the evolutionary trends of this complex group will remain unclear. Assessing the number of wall components is di cult. Most spores of Scutellospora species seem to have a tightly adherent, outermost, unit wall component. We found it impossible to determine if such a component exists. Microscopic observation of spores is di cult because of their large and robust nature and, although on occasions it appeared that the outermost wall group might have been double, it was never possible to be quite certain that this was not an artefact of microscopy. Certainly, for the majority of specimens examined, there was no evidence of such a component. The exible wall component comprising group B seems to be common to all species in the genus Scutellospora (Walker et al., 1998). In S. projecturata, it is extraordinarily thin, and on many spores remains fairly tightly adherent to the laminated component. In specimens such as this, it is very di cult to see, and on others, particularly those that appear older due to darker pigmentation and complete development of the germination shield, it cannot be seen at all. When it adheres to other wall groups, it usually can be seen, at least in places, to be slightly separated often where the spore wall breaks on crushing. Because of the reactive nature of the innermost group, we have presented two alternative murographs (Fig. 13), showing either a coriaceous component [which can also appear as a rigid or semi-rigid unit wall (Morton 1986a; Morton and Koske, 1988) or an amorphous component (Morton, 1986b)]. In some preparations, wall components 4, 5 and 6 can be di cult to resolve, but they can usually be rendered more evident, after a short period of immersion in PVLG, by crushing for a second time. After a few weeks in PVLG/Melzer's, the colour reaction begins to fade, and the specimens photographed for Fig. 10 were completely colourless when re-examined after a couple of years. On other samples, there was no amorphous reaction and no change of colour in Melzer's reagent. We believe these spores were moribund or dead. The wall structure determinations, including murographs, are for guidance only, and the latter are not intended to be other than graphical representations useful for drawing the observer's attention to their appearance under a light microscope. Transmission electron microscopy would reveal a more complex structure. We do not know why our attempts at producing viable pot cultures of this fungus failed. By analogy with other members of the Glomales, it is likely that it forms mycorrhizal associations with plants in nature. Two culture attempts with Sorghum sp. (in Indonesia) and P. lanceolata (in Britain) did result in spore production for a time, but they later failed. It is commonly believed that members of the Glomales form mycorrhizas, and that they are generally non-host-speci c. 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